WO2016050890A2 - Methods and materials for biosynthesis of mogroside compounds - Google Patents

Methods and materials for biosynthesis of mogroside compounds Download PDF

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WO2016050890A2
WO2016050890A2 PCT/EP2015/072645 EP2015072645W WO2016050890A2 WO 2016050890 A2 WO2016050890 A2 WO 2016050890A2 EP 2015072645 W EP2015072645 W EP 2015072645W WO 2016050890 A2 WO2016050890 A2 WO 2016050890A2
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leu
seq
glu
gly
val
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WO2016050890A3 (en
Inventor
Jens Houghton-Larsen
Katarzyna KRZYSTANEK
Angelika SEMMLER
Iver Klavs Riishede HANSEN
Soren DAMKIAER
Gary Liu
Yaoquan Liu
Jorgen Hansen
Sathish Kumar
Muthuswamy Panchapagesa MURALI
Nina Nicoline Rasmussen
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Evolva Holding SA
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Evolva AG
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Priority claimed from US14/504,109 external-priority patent/US10011859B2/en
Application filed by Evolva AG filed Critical Evolva AG
Priority to AU2015326892A priority Critical patent/AU2015326892B2/en
Priority to CA2963300A priority patent/CA2963300C/en
Priority to CN201580062037.XA priority patent/CN107466320B/en
Priority to JP2017517783A priority patent/JP2017529860A/en
Priority to SG11201702123SA priority patent/SG11201702123SA/en
Priority to US15/511,565 priority patent/US10633685B2/en
Priority to EP15797018.7A priority patent/EP3201315A2/en
Publication of WO2016050890A2 publication Critical patent/WO2016050890A2/en
Publication of WO2016050890A3 publication Critical patent/WO2016050890A3/en
Priority to IL251380A priority patent/IL251380B/en
Anticipated expiration legal-status Critical
Priority to US16/806,812 priority patent/US11091787B2/en
Priority to IL276781A priority patent/IL276781A/en
Priority to AU2022204012A priority patent/AU2022204012C1/en
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    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
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    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to methods and materials for biosynthesis of mogroi precursors, mogroi, and/or mogrosides. More particularly, the present invention relates to methods of using of cucurbitadienol synthase, cytochrome P450, cytochrome P450 reductase, and/or epoxide hydrolase enzymes to produce mogroi precursors and/or mogroi. The present invention also relates to methods of using of uridine-5'-diphospho (UDP) dependent glucosyltransferase (UGT) enzymes to glycosylate mogroi and produce various mogrosides.
  • UDP uridine-5'-diphospho
  • UTT uridine-5'-diphospho
  • Mogrosides are a family of triterpene glycosides isolated from fruit of Siraitia grosvenorii (S. grosvenorii, Swingle), also known as Momordica grosvenori. Fruit extracts are commercially used as natural sweeteners.
  • Four major compounds, mogroside V, mogroside IV, siamenoside I, and 11-oxomogroside V have been identified from S. grosvenorii as being responsible for the fruit's sweetness.
  • Mogroside V is the most abundant of these four compounds, at approximately 0.57% (w/w) of the dry fruit, followed by mogroside IV and siamenoside I, each of which contains four glucose moieties.
  • 11 -oxomogroside V has a ketone group instead of a hydroxyl at C11. See, e.g., Takemoto et al., 1983, Yakugaku Zasshi 103: 1151-4; 1 155-66; 1167-73; Kasai et al., 1989, Agric. Biol. Chem. 53:3347-9; Matsumoto Chem. Pharm. Bull., 1990, 38:2030-2; and Prakash er a/., 2011 , J. Carbohydrate Chem. 30:16-26.
  • mogrosides share the same mogroi triterpene core.
  • the aglycone mogroi is glycosylated with different numbers of glucose moieties to form various mogroside compounds.
  • Mogrosides can be synthesized in the following manner: synthesis of cucurbitadienol from the common triterpene precursor oxidosqualene, oxidation of cucurbitadienol to produce mogroi, and giycosylation of mogroi to produce various mogrosides. See, Tang et al., BMC Genomics 12: 343 (2011 ).
  • Tang ef al., 201 1 , BMC Genomics 12:343 describes seven cytochrome P450s and five UGTs as potential candidates involved in mogroside biosynthesis. However, Tang et al. does not specifically identify any cytochrome P450s or UGTs involved in mogroside biosynthesis. Thus, there remains the need to identify cytochrome P450s and UGTs capable of acting on any S. grosvenorii metabolites. Additionally, although mogrosides can be extracted from S. grosvenorii, there remains a need for improved production of mogrosides in recombinant hosts for commercial uses.
  • the present invention provides methods and materials for biosynthesis of mogroside compounds and provides enzymes involved in mogroside biosynthesis.
  • the invention disclosed herein is not limited to specific advantages or functionalities, the invention provides a recombinant host comprising one or more of:
  • At least one of the genes is a recombinant gene; wherein the host is capable of producing a mogrol precursor, a mogroside precursor, and/or a mogroside compound.
  • the squalene epoxidase polypeptide comprises a polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:54;
  • the cucurbitadienol synthase polypeptide comprises a polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:43;
  • the cytochrome P450 polypeptide comprises a CYP5491 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:44 and/or a CYP1798 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:44
  • the cytochrome P450 reductase polypeptide comprises a CPR4497 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:46;
  • the epoxide hydrolase polypeptide comprises an epoxide hydrolase 1 polypeptide having 75% or greater identity to an amino acid sequence set forth in SEQ ID NO:38 or an epoxide hydrolase 2 polypeptide having 65% or greater identity to an amino acid sequence set forth in SEQ ID NO:40.
  • the invention further provides a recombinant host comprising one or more of:
  • At least one of the genes is a recombinant gene.
  • the recombinant host further comprises a gene encoding squalene epoxidase polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO: 54.
  • the recombinant host has been modified to reduce expression of a lanosterol synthase (ERG7) polypeptide.
  • ESG7 lanosterol synthase
  • the ERG7 polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:55.
  • the invention further provides a method of producing a mogroside precursor and/or a mogroside compound, comprising:
  • mogroside precursor and/or the mogroside compound is synthesized by the recombinant host
  • the mogroside precursor is mogrol synthesized by epoxidation of 1 1-hydroxy-cucurbitadienol to synthesize 1 1-hydroxy- 24,25 epoxy cucurbitadienol and hydrolysis of 11-hydroxy-24,25 epoxy cucurbitadienol to synthesize mogrol.
  • the epoxidation of 1 1-hydroxy- cucurbitadienol to synthesize 11-hydroxy-24,25 epoxy cucurbitadienol is catalyzed by the CYP1798 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:74.
  • the invention further provides a method of producing a mogroi precursor in vitro, comprising:
  • the invention further provides a method of producing a mogroi in vitro, comprising contacting 11-hydroxy-24,25 epoxy cucurbitadienoi with one or more enzymes capable of catalyzing conversion of 11-hydroxy-24,25 epoxy cucurbitadienoi to produce mogroi.
  • the invention further provides a method of producing a mogroside compound in vitro, comprising contacting a mogroside precursor with one or more enzymes capable of catalyzing glycosylation of the mogroside precursor to produce a mogroside compound.
  • the method further comprises isolating the mogroi precursor, mogroi or the mogroside compound.
  • the one or more enzymes capable of catalyzing conversion of dioxidosqualene to produce 24,25 epoxy cucurbitadienoi comprise a cucurbitadienoi synthase having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:43;
  • the one or more enzymes capable of catalyzing conversion of oxidosqualene to produce cucurbitadieno! comprise a cucurbitadienol synthase having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:43;
  • the one or more enzymes capable of catalyzing conversion of 24,25 epoxy cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol comprise CYP5491 having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:44;
  • the one or more enzymes capable of catalyzing conversion of cucurbitadienol to produce 11 -hydroxy-cucurbitadienol comprise CYP5491 having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:44;
  • the one or more enzymes capable of catalyzing epoxidation of cucurbitadienol to produce 24,25 epoxy cucurbitadieno! comprise CYP1798 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO: 74;
  • the one or more enzymes capable of catalyzing epoxidation of 11 -hydroxy- cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol comprise CYP1798 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:74;
  • the one or more enzymes capable of catalyzing conversion of 11-hydroxy-24,25 epoxy cucurbitadienol to produce mogrol comprise a polypeptide comprising epoxide hydrolase 1 having 75% or greater identity to an amino acid sequence set forth in SEQ ID NO:38 or epoxide hydrolase 2 having 65% or greater identity to an amino acid sequence set forth in SEQ ID NO:40; and/or
  • the one or more enzymes capable of catalyzing conversion of the mogroside precursor to a mogroside compound comprise UGT1576 having 60% or greater identity to an amino acid sequence set forth in SEQ ID NO:48; UGT98 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:53; UGTSK98 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:50; UGT430 having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:62; UGT1697 having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:68; or UGT11789 having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:72.
  • the invention further provides a method of producing a mogroside compound, comprising contacting a recombinant host expressing one or more of: (a) a UGT1576 polypeptide having 60% or greater identity to an amino acid sequence set forth in SEQ ID N0.48;
  • the mogroside precursor is plant- derived or synthetic.
  • the method further comprises isolating the mogroside compound.
  • the mogroside compound is:
  • the mogroside compound is one or more of mogroside I A1 , mogroside I E1 , mogroside II A, mogroside II A1 , mogroside II A2, mogroside II E, mogroside ill A1 , mogroside III A2, mogroside III, mogroside III E, mogroside IV, mogroside IV A, mogroside V or siamenoside.
  • the mogrol precursor is one or more of squalene, dioxidosqualene, oxidosqualene, 24,25 epoxy cucurbitadienol, cucurbitadienol, 11-hydroxy-cucurbitadienol, 11 -hydroxy 24, 25 epoxy cucurbitadienol or 11-oxo-mogrol.
  • the mogroside precursor is one or more of mogroS, glycosylated mogrol, di-glycosylated mogrol or tri-glycosylated mogrol.
  • the recombinant host comprises a microorganism that is a yeast cell, a plant cell, a mammalian cell, an insect cell, a fungal cell, or a bacterial cell.
  • the bacterial cell comprises Escherichia bacteria celis, Lactobacillus bacteria cells, Lactococcus bacteria cells, Cornebacterium bacteria cells, Acetobacter bacteria celis, Acinetobacter bacteria cells, or Pseudomonas bacterial cells.
  • the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces iactis, Hansenula polymorpha, Candida boidinii, Arxuia adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species.
  • the yeast cell is a Saccharomycete.
  • the yeast cell is a cell from the Saccharomyces cerevisiae species.
  • one or more of the genes further comprise a nucleotide sequence coding a fusion tag.
  • the fusion tag is a protein or polypeptide.
  • the fusion tag is green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), a polyhistidine-tag (HIS tag), and a FLAG-tag, a chloroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, a signal peptide, or a secretion tag.
  • one or more of the genes are expressed as fusion proteins.
  • the invention further provides a mogroside composition produced by the recombinant host or the methods disclosed herein, wherein the composition comprises one or more of mogroside I A1 , mogroside I E1, mogroside II A, mogroside II E, mogroside III A1 , mogroside II! A2, mogroside III, mogroside III E, mogroside IV, mogroside V, and siamenoside.
  • the invention further provides a food or drink product comprising the mogroside composition disclosed herein.
  • Figure 1 shows chemical structures of mogroside V, mogroside IV, siamenoside I, and 11 -oxomogroside V.
  • Figure 2A is a schematic diagram of a pathway for producing mogrosides from glucose.
  • Figure 2B shows a pathway for production of mogro! precursors, mogro!, and mogrosides.
  • Figure 2B shows production of cucurbitadienol from oxidosqualene using a cucurbitadienol synthase (step A), production of 24,25 epoxy cucurbitadienol from dioxidosqualene using a cucurbitadienol synthase (step B), production of 1 1-hydroxy- cucurbitadienol from cucurbitadienol using a cytochrome P450 (step C), production of 11- hydroxy 24,25 epoxy cucurbitadienol from 24,25 epoxy cucurbitadienol using a cytochrome P450 (step D), production of 24,25 epoxy cucurbitadienol from cucurbitadienol using a cytochrome P450 (step E), production of 11 -hydroxy 24,25 epoxy cucurbitadienol from 1 1- hydroxy-cucurbitadienoi using
  • Figure 2C shows representative enzymes capable of catalyzing the reactions of steps A-H in Figure 2B.
  • Figure 2C shows production of cucurbitadienol from oxidosqualene using an S. grosvenorii cucurbitadienol synthase of SEQ ID NO:43 (step A), production of 24,25 epoxy cucurbitadienol from dioxidosqualene using an S.
  • step B grosvenorii cucurbitadienol synthase of SEQ ID NO:43
  • step C production of 1 1-hydroxy-cucurbitadienol from cucurbitadienol using CYP5491 of SEQ ID NO:44
  • step D production 11 -hydroxy 24,25 epoxy cucurbitadienol from 24,25 epoxy cucurbitadienol using CYP5491 of SEQ ID NO:44
  • step E production of 24,25 epoxy cucurbitadienol from cucurbitadienol using CYP1798 of SEQ ID NO:74
  • step F production of 11 -hydroxy 24,25 epoxy cucurbitadienol from 11-hydroxy-cucurbitadienol using CYP1798 of SEQ ID NO:74
  • step G production of mogrol from 11 -hydroxy 24,25 epoxy cucurbitadienol from using epoxide hydrolase 1 of SEQ ID NO:38 or epoxide hydrolase 2 of SEQ ID NO:40
  • Figure 3A shows a representative pathway for production of mogrol from cucurbitadienol, as disclosed herein.
  • Figure 3B is a schematic diagram of a pathway for production of mogrol from cucurbitadienol, as proposed in Tang et a/., 2011 , BMC Genomics 12:343.
  • FIG 4 is schematic diagram of pathways for the biosynthesis of mogroside I E1 , mogroside I A1 , mogroside II E, mogroside III A2, mogroside III, mogroside IV, and mogroside V from mogrol using UGTs.
  • UGTa of Figure 4 can be, for example, UGT1576 (SEQ ID NO:48) or UGT1697 (SEQ ID NO:68).
  • UGTb of Figure 4 can be, for example, UGT430 (SEQ ID NO:62) or UGT1697 (SEQ ID NO:68).
  • UGTc of Figure 4 can be, for example, UGT430 (SEQ ID NO:62) or UGT1697 (SEQ ID NO:68).
  • UGTd of Figure 4 can be, for example, UGT1576 (SEQ ID NO:48) or UGT1697 (SEQ ID NO:68).
  • UGTe of Figure 4 can be, for example, UGT98 (SEQ ID NO:53) or UGT11789 (SEQ ID NO:72).
  • UGTf of Figure 4 can be, for example, UGT98 (SEQ ID NO:53) or UGT11789 (SEQ ID NO:72).
  • UGTg of Figure 4 can be, for example, UGT98 (SEQ ID NO:53) or UGT11789 (SEQ ID NO;72).
  • Figure 5 is a schematic diagram showing enzymatic production of mogroside IV A, mogroside III, mogroside I E1 , mogroside I A1 , mogroside II E, mogroside II A1 , and mogrol from mogroside V.
  • Figure 6 shows the LC-MS mass peak 501 corresponding to the proton pius Na+ adduct of tetrahydroxysqualene in a sample from a yeast strain transformed with a piasmid expressing S. grosvenorii epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO:40), as described in Example 8.
  • Figure 7 A show an LC-MS chromatogram indicating lanosterol production in a yeast strain that does not express a cucurbitadienol synthase.
  • Figure 7B shows an LC-MS chromatogram indicating cucurbitadienol and lanosterol production in a yeast strain expressing cucurbitadienol synthase (SEQ ID NO:42, SEQ ID NO:43), as described in Example 9.
  • Figure 8 shows an LC-MS chromatogram with three peaks resulting upon expression of CYP5491 (SEQ ID NO:14, SEQ ID NO:44) and CPR4497 (SEQ ID NO:45, SEQ ID NO:46) in yeast (upper panel), as described in Example 10; the three lower panels show the fragmentation spectrum of these three peaks.
  • the masses of the 3 peaks (443.38, 441.37 and 457.36) correspond in weight to proton adducts of hydroxylated cucurbitadienol, oxo cucurbitadienol, and hydroxy plus oxo cucurbitadienol, respectively.
  • Figures 9A and 9B show biosynthetic routes from cucurbitadienol to mogrol and 1- oxo-mogrol with S. grosvenorii CYP5491 (SEQ ID NO: 14, SEQ ID NO:44), S. grosvenorii CYP1798 (SEQ ID NO:5, SEQ ID NO:73, SEQ ID NO:74), and S. grosvenorii epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO:40).
  • Figure 9C shows a potential biosynthetic route from oxidosqualene to mogrol and 11 -oxo-mogrol with S.
  • SEQ ID NO:54 cerevisiae squalene epoxidase ERG1
  • S. grosvenorii CYP1798 SEQ ID NO:5, SEQ ID NO:73, SEQ ID NO:74
  • S. grosvenorii cucurbitadienol synthase SEQ ID NO:42, SEQ ID NO:43
  • S. grosvenorii CYP5491 SEQ ID NO:14, SEQ ID NO:44
  • S. grosvenorii epoxide hydrolase 2 SEQ ID NO:39, SEQ ID NO:40. See Examples 9 and 15.
  • Figure 10A shows an LC-MS chromatogram of reference mogroside I A1.
  • Figure 10B shows an LC-MS chromatogram of a sample of yeast strain expressing UGT1576 (SEQ ID NO:47, SEQ ID NO:48) in a culture fed 50 ⁇ mogrol, as described in Example 1 1.
  • Figure 11A shows LC-MS chromatograms of samples from a yeast strain co- expressing UGT SK98 with UGT1576 and shows production of di-glycosylated mogrol (mogroside II A) as described in Example 11.
  • Figure 11 B shows LC-MS chromatograms of samples from a yeast strain co-expressing UGT98 with UGT1576 and shows production of di- and tri-g!ycosyiated mogrol (middle and lower frames), as described in Example 11.
  • Figure 12 shows a biosynthetic route from mogroi to mogroside ill A1 provided herein, as described in Example 11.
  • Figure 13A shows elution of a mogroside I E1 standard.
  • Figure 13B shows mogroside I E1 produced by UGT430 (SEQ ID NO:61 , SEQ ID NO:62), as described in Example 12.
  • Figure 14A shows elution of mogroside II E1 , mogroside II A, mogroside I E1 , and mogroside I A1 standards.
  • Figure 14B shows mogroside I A1 , mogroside If A, and mogroside II E1 produced by UGT1697 (SEQ ID NO:67, SEQ ID NO:68), as described in Example 13.
  • Figure 15A shows elution of reference compounds mogroside V (top panel) and mogroside II E (bottom panel).
  • Figure 15B shows production of mogroside V (top panel) and mogroside II E (bottom panel) in a yeast cell co-expressing UGT1576, UGT430, and UGT98.
  • Figure 15C shows production of mogroside V (top panel) and mogroside II E (bottom panel) in a yeast cell co-expressing UGT 576, UGT430, UGT98, and UGT11789, as described in Example 14.
  • Figure 15D shows production of a tri-glycosylated mogroside in a yeast cell co-expressing UGT1576, UGT430, and UGT11789, as described in Example 14.
  • Figure 16A shows elution of a mogroi standard.
  • Figure 16B shows mogroi produced in a cucurbitadienol-producing host expressing CYP5491 (SEQ ID NO:14, SEQ ID NO:44), CPR4497 (SEQ ID NO:45, SEQ ID NO:46), CYP1798 (SEQ ID NO:5, SEQ ID NO:73, SEQ ID NO:74), and an epoxide hydrolase, as described in Example 15.
  • Figure 17 shows a representative LC- S chromatogram of a crude isolate of a mogroside V-producing S. cerevisiae strain, as described in Example 16.
  • Figures 18A, 18B, and 18C show an NMR-elucidated structure, 1 H NMR spectrum, and 1 H and 13 C NMR chemical shifts (in ppm) for mogroside V, mogroside II A2, and mogroside IV A, respectively, as described in Example 16.
  • Figure 18D shows an NMR-elucidated structure, 1 H NMR spectrum, and 1 H NMR chemical shifts (in ppm) for mogroside I E1 , as described in Example 16.
  • nucleic acid means one or more nucleic acids.
  • nucleic acid can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof.
  • the term “and/or” is utilized to describe multiple components in combination or exclusive of one another.
  • x, y, and/or z can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x and (y or z),” or “x or y or z.”
  • "and/or” is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group.
  • "and/or” is used to refer to production of mogrosides, wherein one or more mogrosides is produced. In some embodiments, “and/or” is used to refer to production of mogrosides, wherein one or more mogrosides is produced through one or more of the following steps: culturing a recombinant microorganism, synthesizing one or more mogrosides in a recombinant microorganism, and isolating one or more mogrosides.
  • mogroside and “mogroside compound” can be used interchangeably to describe mogrol glycosylated at one or more positions.
  • a mogroside compound can be mogrol glycosylated with one or more glucose moieties at the positions 1 , 3, 1 1 , 24, and 25.
  • Mogrol is a compound of formula I provided below, wherein both
  • Mogrosides can be of the following formula I:
  • Ri and R 2 independently are -H, mono-glucoside, di-glucoside, tri- glucoside, and wherein at least one of and R 2 is not -H.
  • the mogroside can be one of the mogrosides described in Table 1.
  • "Glc” represents glucose
  • the 1 ,6- and ,2-bonds are indicated.
  • the R 2 group of mogroside V comprises 3 glucose molecules linked by one 1 ,6-bond and one 1 ,2-bond, a conformation represented as "Glc6- Glc2-Glc-”. See Figure 1 for the structures of mogroside IV, mogroside V, 11-oxo-mogroside V, and siamenoside.
  • Mogrosides can be produced from a number of mogroside precursors.
  • a mogroside precursor is mogrol, glycosylated mogrol, di-glycosylated mogrol or tri-giycosylated mogrol.
  • Mogrol precursors include squalene, dioxidosqualene, oxidosqualene, 24,25 epoxy cucurbitadienol, cucurbitadienol, 11 -hydroxy-cucurbitadienol, 1 1- hydroxy 24, 25 epoxy cucurbitadienol, 11-oxo-mogrol. See, e.g., Figures 2 and 9.
  • mogroside I A1 is a precursor to the products, mogroside II A and mogroside III A1. See, Figure 12.
  • mogroside II E is converted to mogroside V by three enzymatic glycosylations.
  • two glucose moieties are first attached through 1 ,6-bonds to the two glucose molecules of mogroside II E by a UGT not limited to UGT98 (SEQ ID NO:53) or UGT11789 (SEQ ID NO:72).
  • a third glucose moiety is added to the C24-bound glucose moiety with a 1 ,2 bond by a UGT not limited to UGT98 (SEQ ID NO:53) or UGT11789 (SEQ ID NO:72). See, Figure 4.
  • a pathway from cucurbitadienol to mogrol was proposed by Tang et a/., 2011 , BMC Genomics 12:343.
  • the precursors, cucurbitadienol and mogrol, have been isolated from S. grosvenorii. See Ukiya, et a!., 2002, J. Agric. Food Chem. 50: 6710-5.
  • Glycoside intermediates exist in both 11 -hydroxy and 11-oxo series and gradually change from mogroside I to mogroside V as fruits ripen, indicating that P450 enzymes fully oxidize the triterpene core of a mogrol precursor, such as cucurbitadienol, prior to subsequent glycosylations.
  • one or more mogrol precursors are produced.
  • Mogrol precursors, mogrol, and/or mogrosides can be produced in vivo ⁇ i.e., in a recombinant host), in vitro (i.e., enzymatica!ly), or by whole cell bioconversion, as described below.
  • the terms "detectable amount,” “detectable concentration,” “measurable amount,” and “measurable concentration” refer to a level of mogrosides and mogroside precursors measured in AUC, pM/OD 6 oo.
  • Mogroside production i.e. , total, supernatant, and/or intracellular steviol glycoside levels
  • LC-MS liquid chromatography-mass spectrometry
  • TLC thin layer chromatography
  • HPLC high-performance liquid chromatography
  • UV-Vis ultraviolet-visible spectroscopy/ spectrophotometry
  • MS mass spectrometry
  • NMR nuclear magnetic resonance spectroscopy
  • a mogrol precursor e.g., squalene or oxidosqualene
  • mogrol mogrol
  • mogroside is produced, as described herein.
  • Squalene can be produced from famesyl pyrophosphate using a squalene synthase
  • oxidosqualene can be produced from squalene using a squalene epoxidase.
  • the squalene synthase can be any enzyme classified under EC 2.5.1.21.
  • Squalene production can comprise a step of catalyzing conversion of famesyl pyrophosphate by a squalene synthase in the presence of NADPH.
  • the recombinant host can thus comprise a heterologous nucleic acid encoding a squalene synthase.
  • the squalene synthase can be endogenous.
  • the squalene synthase can be, for example, squalene synthase from Gynostemma pentaphyllum (protein accession number C4P9M2), a cucurbitaceae family plant.
  • the squalene synthase can also comprise a squalene synthase from Arabidopsis thaliana (protein accession number C4P9M3), Brassica napus, Citrus macrophylla, Euphorbia tirucalli (protein accession number B9WZW7), Glycine max, Glycyrrhiza glabra (protein accession number Q42760, Q42761 ), Glycrrhiza uralensis (protein accession number D6QX40, D6QX41 , D6QX42, D6QX43, D6QX44, D6QX45, D6QX47, D6QX39, D6QX55, D6QX38, D6QX53, D6QX37, D6QX35, B5AID5, B5AID4, B5AID3, C7EDD0, C6KE07, C6KE08, C7EDC9), Lotusjaponicas (protein accession number Q84LE3), Medic
  • Oxidosqualene can be produced from squalene by squalene epoxidase (also referred to as squalene monoxygenase. See, e.g., Leber et a/,, 1998, Mo! Biol Cell. 9(2):375- 86.
  • the squalene epoxidase can be any enzyme classified under EC 1.4.99.7.
  • Oxidosqualene production can comprise a step of catalyzing conversion of squalene by a squalene epoxidase in the presence of NADPH. See, e.g., Example 8.
  • the squalene epoxidase can also be the product of the ERG1 gene from S. cerevisiae.
  • the squalene epoxidase can be a polypeptide of SEQ ID NO:54 or a functional homolog thereof sharing at least 45% sequence identity therewith.
  • ERG1 is overexpressed.
  • the squalene epoxidase can be, for example, squalene epoxidase from Gynostemma pentaphyllum (protein accession number C4P9M2; SEQ ID NO: 88).
  • the squalene epoxidase can comprise a squalene epoxidase from Arabidopsis thaliana (protein accession number Q9S 02 (SEQ ID NO: 89), 065403 (SEQ ID NO: 90), 065402 (SEQ ID NO: 91 ), 065404 (SEQ ID NO: 92), 081000 (SEQ ID NO: 93), or Q9T064 (SEQ ID NO: 94)), Brassica napus (protein accession number 065727 (SEQ ID NO: 95), 065726 (SEQ ID NO: 96)), Euphorbia tirucalli (protein accession number A7VJN1 (SEQ ID NO: 97)), Medicago truncatula (protein accession number Q8GSM8 (SEQ ID NO: 98), Q8GSM9 (SEQ ID NO: 99)), Pisum sativum, and Ricinus communis (protein accession number B9R6V0 (SEQ ID NO:
  • One or more enzymes capable of catalyzing conversion of oxidosqualene to form cucurbitadienol comprise a cucurbitadienol synthase. See step A of Figures 2B and 2C and Example 9.
  • the cucurbitadienol synthase can be, for example, a cucurbitadienol synthase, which has been classified as an oxidosqualene cyclase, such as the oxidosqualene cyclase described by Shibuya, Tetrahedron, 60: 6995-7003 (2004).
  • the amino acid sequence of a cucurbitadienol synthase from Cucurbita pepo is provided herein as SEQ ID NO:1.
  • the cucurbitadienol synthase is a polypeptide of SEQ ID NO:1 or a functional homolog thereof sharing at least 70% sequence identity therewith, in some embodiments, a polypeptide having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:1 includes, but is not limited to, a polypeptide from Lotus japonicas (BAE53431 ), Populus trichocarpa (XP_002310905), Actaea racemosa (ADC84219), Betula platyphylla (BAB83085), Glycyrrhiza glabra (BAA76902), Vitis vinifera (XP_002264289), Centella asiatica (AAS01524), Panax ginseng (BAA33460), and Betula platyphy
  • the cucurbitadienol synthase from monk fruit was identified herein, and the sequence of the C-terminal portion of the polypeptide determined.
  • the amino acid sequence of the C-terminal portion of the monk fruit polypeptide is provided herein as SEQ ID NO:2.
  • the cucurbitadienol synthase is a polypeptide having an amino acid sequence set forth in SEQ ID NO:2.
  • the cucurbitadienol synthase is the polypeptide of SEQ ID NO:43 or a functional homolog thereof sharing at least 70% identity therewith.
  • 24,25 epoxy cucurbitadienol is produced from dioxidosqualene using one or more enzymes capable of catalyzing conversion of oxidosqualene to form cucurbitadienol.
  • One or more enzymes capable of catalyzing conversion of dioxidosqualene to 24,25 epoxy cucurbitadienol preferably comprises a cucurbitadienol synthase. See step B of Figures 2B and 2C and Example 9.
  • the cucurbitadienol synthase can be, for example, a cucurbitadienol synthase as described by Shibuya, Tetrahedron 60:6995- 7003 (2004) or a cucurbitadienol synthase as described above.
  • the cucurbitadienol synthase catalyzing conversion of dioxidosqualene to 24,25 epoxy cucurbitadienol is a polypeptide of SEQ ID NO:1 or a functional homolog thereof sharing at least 70% identity therewith.
  • 11-hydroxy-cucurbitadienol is produced from cucurbitadienol.
  • a cytochrome P450 enzyme catalyzes hydroxylation of cucurbitadienol to form 11 -hydroxy-cucurbitadienol.
  • CYP5491 (SEQ ID NO: 14, SEQ ID NO:44) catalyzes conversion of cucurbitadienol to 11-hydroxy-cucurbitadienol. See step C of Figures 2B and 2C and Example 10.
  • CYP533, CYP937, CYP1798, CYP 994, CYP2048, CYP2740, CYP3404, CYP3968, CYP4112, CYP4149, CYP4491 , CYP5491 , CYP6479, CYP7604, CYP8224, CYP8728, CYP10020, or CYP10285 can be used to produce mogrol.
  • eYAC technology can be used to assess activity of the cytochrome P450 enzymes, as set forth in Example 8.
  • an in vitro reaction can be used to assess the activity.
  • At least one cytochrome P450 enzyme comprises a polypeptide encoded by the nucleic acid sequence SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO:20 or a functional homolog thereof sharing at least 70% identity therewith.
  • 11-hydroxy-24,25 epoxy cucurbitadienol is produced from 24,25 epoxy cucurbitadienol using one or more enzymes capable of catalyzing hydroxylation of 24,25 epoxy cucurbitadienol to form 1 1-hydroxy-24,25 epoxy cucurbitadienol.
  • a cytochrome P450 enzyme catalyzes hydroxylation of 24,25 epoxy cucurbitadienol to form 11-hydroxy-24,25 epoxy cucurbitadienol.
  • the enzyme capable of catalyzing hydroxylation of 24,25 epoxy cucurbitadienol to form 1 1-hydroxy- 24,25 epoxy cucurbitadienol is CYP5491 (SEQ ID NO:14, SEQ ID NO:44) or a functional homolog sharing at least 50% sequence identity with SEQ ID NO:44. See step D of Figures 2B and 2C and Example 9.
  • 24,25 epoxy cucurbitadienol is produced from cucurbitadienol.
  • a cytochrome P450 catalyzes conversion of cucurbitadienol to 24,25 epoxy cucurbitadienol.
  • the cytochrome P450 can be CYP1798 of SEQ ID NO:74. See step E of Figures 2B and 2C.
  • 1 -hydroxy 24,25 epoxy cucurbitadienol is produced from 11 -hydroxy-cucurbitadienol.
  • a cytochrome P450 catalyzes conversion of 1 1 - hydroxy-cucurbitadienol to produce 11 -hydroxy 24,25 epoxy cucurbitadienol.
  • the cytochrome P450 can be CYP1798 of SEQ ID NO:74. See step F of Figures 2B and 2C.
  • mogrol is produced from 11 -hydroxy-cucurbitadienol using enzymes capable of catalyzing conversion of 11 -hydroxy-cucurbitadienol to form mogrol.
  • Enzymes having cytochrome P450 activity and epoxide hydrolase activity catalyze conversion of 11 -hydroxy-cucurbitadienol to mogrol. See steps F and G of Figures 2B and 2C.
  • Enzymes with cytochrome P450 activity include polypeptides encoded by the nucleic acid sequence set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or a functional homolog thereof sharing at least 70% sequence identity therewith.
  • an enzyme having epoxide hydrolase activity preferably catalyzes production of glycol from epoxide and water.
  • Non-limiting examples of enzymes with epoxide hydrolase activity include S. grosvenorii epoxide hydrolase 1 and S. grosvenorii epoxide hydrolase 2.
  • an enzyme with epoxide hydrolase activity can comprise polypeptides having at least 75% sequence identity with the amino acid sequence set forth in SEQ ID NO:38, having at least 65% sequence identity with the amino acid sequence set forth in SEQ ID NO:40, and functional homoiogs thereof.
  • mogrol is produced from 1 1-hydroxy-24,25 epoxy cucurbitadienol.
  • One or more enzymes capable of catalyzing conversion of 11-hydroxy-24,25 epoxy cucurbitadienol to form mogrol preferably comprise an enzyme with epoxide hydrolase activity. See step G of Figures 2B and 2C, Examples of enzymes with epoxide hydrolase activity include $. grosvenorii epoxide hydrolase 1 and S. grosvenorii epoxide hydrolase 2, as described above.
  • an enzyme capable of catalyzing conversion of 1 1- hydroxy-24,25 epoxy cucurbitadienol to produce mogrol comprises a polypeptide having at least 75% sequence identity with the amino acid sequence set forth in SEQ ID NO:38, having at least 65% sequence identity with the amino acid sequence set forth in SEQ ID NO:40, and functional homoiogs thereof.
  • CYP1798 catalyzes the epoxidation of the 24-25 carbon double bonds of cucurbitadienol, 1 1-hydroxy- cucurbitadienol, or 11-oxo cucurbitadienol.
  • Figures 9A and 9B are schematics of mogrol and 11 -oxo-mogrol production from cucurbitadienol
  • Figure 9C is a schematic of mogrol and 11- oxo-mogrol production from oxidosquaiene. See, also, Example 15.
  • One or more enzymes capable of catalyzing glycosylation of mogrol preferably comprise a Uridtne-5'-diphospho (UDP) dependent glucosyltransferase (UGT).
  • UGT can catalyze production of a mogroside not limited to mogroside I A1 , mogroside I E1 , mogroside II A, mogroside II A1 , mogroside II A2, mogroside II E, mogroside III A1 , mogroside III A2, mogroside III, mogroside III E, mogroside IV, mogroside IV A, or siamenoside.
  • Such UGT can comprise, for example, Arabidopsis thaliana UGT73C3 of SEQ ID NO:21 , Arabidopsis thaliana UGT73C6 of SEQ ID NO:23, Stevia rebaudiana UGT85C2 of SEQ ID NO:25, Arabidopsis thaliana UGT73C5 of SEQ ID NO:22, Stevia rebaudiana UGT73E1 of SEQ ID NO:24, or a functional homolog sharing at least 70% identity with a UGT described above.
  • a UGT can also comprise UGT98 of SEQ ID NO:53, UGT1495 encoded by SEQ ID NO:27, UGT1817 encoded by SEQ ID NO:28, UGT5914 encoded by SEQ ID NO:30, UGT8468 encoded by SEQ ID NO:31 , UGT10391 encoded by SEQ ID NO:32, or a functional homolog of any of the UGTs described above. See Examples 4 and 7.
  • UGT73C3, UGT73C6, UGT85C2, and UGT73E1 are capable of catalyzing giycosylation at the C24 position of mogrol or mogroside. Accordingly, in methods of the invention wherein the mogroside to be produced comprises a giycosylation at the C24 position, at least one UGT can be UGT73C3 of SEQ ID NO:21 , UGT73C6 of SEQ ID NO:23, UGT85C2 of SEQ ID NO:25, UGT73E1 of SEQ ID NO:24 or a functional homolog functional homolog sharing at least 70% identity with a UGT described above. See Example 4.
  • UGT73C5 is capable of catalyzing giycosylation at both the C3-OH of mogrol and mogroside and C24 position. Accordingly, in methods of the invention wherein the mogroside to be produced comprises a giycosylation at the C24 position and/or a giycosylation at the C3-OH position, at ieast one UGT can be UGT73C5 of SEQ ID NO:22 or a functional homolog sharing at Ieast 60% sequence identity therewith. See Example 4.
  • a UGT is UGT1576 of SEQ ID NO:48 or a UGT sharing at Ieast 60% sequence identity with UGT1576 of SEQ ID NO:48.
  • UGT1576 possesses mogrol C24-OH UDP-glycosyltransferase activity. See Example 11.
  • a UGT is UGT98 of SEQ ID NO:53 or a functional homolog thereof sharing at Ieast 70% sequence identity therewith.
  • the mogroside to be produced comprises a 1 ,2- glycosylation and a 1 ,6-giycosy!ation of the glucose at position C-24 to form mogroside III A1.
  • UGT98 SEQ ID NO:53
  • UGT98 can be used to convert mogroside II E to mogroside IV, mogroside V, 11-oxo-mogroside V, and/or siamenoside I. See Example 7.
  • a UGT is UGTSK98 of SEQ ID NO:50 or UGT sharing at ieast 70% identity with UGTS 98 of SEQ ID NO:50. See Example 11. In some aspects, UGT98 catalyzes 1 ,2 and 1 ,6 glucose attachments to convert mogroside II E to mogroside V. See Example 14.
  • a UGT is S. grosvenorii UGT430 (SEQ ID NO:61 , SEQ ID NO:62).
  • UGT430 is a member of UGT family 85A and glycosylates the 3C position of mogrol and particular mogrosides. See Example 12.
  • a UGT is S. grosvenorii UGT1697 (SEQ ID NO:67, SEQ ID NO:68).
  • UGT1697 is a member of UGT family 85A and glycosylates the 3C and 24C positions of mogrol and particular mogrosides. See Example 13.
  • a UGT is S. grosvenorii UGT11789 (SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 , SEQ ID NO:72).
  • UGT11789 catalyzes 1 ,2 and/or 1 ,6 glucose attachments on the 24-O-g!ucose and/or the 3-O-glucose of mogroside compounds.
  • UGT1 1789 glycosylates mogroside I E1 , mogroside I A1 , mogroside II E, mogroside II A, mogroside III E, mogroside III A2, mogroside III, mogroside IV, or siamenoside.
  • contacting UGT11789 with mogroside I E1 , mogroside I A1 , mogroside II E, mogroside II A, mogroside III E, mogroside II) A2, mogroside III, mogroside IV, or siamenoside produces mogroside II A1 , mogroside II A2, mogroside III, mogroside III A1 , mogroside ill A2, mogroside IV, mogroside IV A, siamenoside, or mogroside V. See Example 14.
  • a mogrol precursor, mogrol, or mogroside is produced in vivo by a host expressing of one or more nucleic acid molecules encoding one or more enzymes involved in the mogroside pathway.
  • a host expressing of one or more nucleic acid molecules encoding one or more enzymes involved in the mogroside pathway.
  • an oxidosqualene-producing recombinant host expressing one or more of a gene encoding a cucurbitadienol synthase polypeptide, a gene encoding a cytochrome P450 polypeptide, a gene encoding a cytochrome P450 reductase polypeptide, a gene encoding an epoxide hydrolase polypeptide, and a gene encoding a UGT polypeptide can produce a mogrol precursor, mogrol, or mogroside in vivo. See Examples 15 and 16.
  • more than one host is used to produce a mogrol precursor, mogrol, or mogroside.
  • a host capable of producing mogrol and a host expressing a UGT can be used to produce a mogroside.
  • the methods can also employ a mixture of a recombinant and a non-recombinant host.
  • the hosts can be co-cultivated or cultured separately. If the hosts are cultivated separately, the intermediate products can be recovered and optionaily purified or partially purified and fed to recombinant hosts using the intermediate products as substrates. Suitable recombinant hosts are described below.
  • production of a mogrol precursor, mogrol, or mogroside can be performed in vivo and a mogrol precursor, mogrol, or mogroside product can be used as a substrate for subsequent reactions to be performed in vitro, as described below. See WO 2013/076577 and WO 2014/086842.
  • a host produces oxidosqualene from glucose via the ergosterol pathway. See, e.g., WO 2014/00271 18.
  • host expressing a nucleic acid molecule encoding a squalene synthase polypeptide can produce squalene.
  • the squalene synthase is ERG9, and the amino acid sequence of ERG9 is set forth in SEQ ID NO:87.
  • squalene synthase is endogenous to the host.
  • increased copy numbers of an endogenous squalene synthase and/or squalene epoxidase, expression of a heterologous nucleic acid molecule encoding a squalene synthase and/or squalene epoxidase, or increased expression of an endogenous squalene synthase and/or squalene epoxidase can improve levels of mogrosides produced in a recombinant host.
  • the recombinant host comprises a heterologous nucleic acid encoding a squalene epoxidase operably linked to sequence directing high expression of the squalene epoxidase in the host.
  • the squalene epoxidase can be endogenous to the recombinant host, but the expression level can be increased by additional copies of nucleic acids encoding the squalene epoxidase and/or by use of stronger promoters.
  • Oxidosqualene serves as a substrate for production of lanosterol.
  • the level of oxidosqualene can be increased by reducing lanosterol synthase activity.
  • this can be achieved by substituting the endogenous promoter-directed expression of lanosterol synthase with a weaker promoter directing expression of a lower level of lanosterol synthase.
  • yeast the ERG7 gene encodes lanosterol synthase.
  • the ERG7 gene promoter can be substituted for another promoter, which directs a level of expression, which is lower than the endogenous expression level of ERG7.
  • the lanosterol synthase can thus be the product of the ERG7 gene of S. cerevisiae, the sequence of which is provided herein as SEQ ID NO:55, or a functional homolog thereof sharing at least 50% sequence identity therewith. See Examples 8 and 15.
  • tHMG1 3-hydroxy-3- methylglutaryl-CoA reductase
  • SEQ ID NO:77 SEQ ID NO:78
  • tHMG1 3-hydroxy-3- methylglutaryl-CoA reductase
  • tHMG1 A useful truncated form of yeast HMG reductase (tHMG1) is described in Donald er a/., 1997, Appl. Environ. Microbiol. 63:3341-4.
  • Dioxidosqualene ievels can be enhanced by high expression of a squaiene epoxidase.
  • the squaiene epoxidase can be the product of the S. cerevisiae ERG1 gene.
  • the squaiene epoxidase can be a polypeptide of SEQ ID NO:54 or a functional homolog thereof sharing at least 45% sequence identity therewith.
  • the Ievels of dioxidosqualene can also be enhanced by reducing lanosterol synthase activity.
  • Dioxidosqualene Ievels can also be enhanced by expression of a truncated form of 3-hydroxy-3-methylglutary!-CoA reductase (tHMG1 , SEQ ID N0.77, SEQ ID NO:78). See Examples 8 and 15.
  • hydroxylation of cucurbitadienol to form 11-hydroxy- cucurbitadienol or hydroxylation of 24,25 epoxy cucurbitadienol to form 11-hydroxy-24,25 epoxy cucurbitadienol can be aided by at least one CYP activator.
  • a recombinant host can co-express heterologous nucleic acids encoding one or more cytochrome P450 enzymes and a heterologous nucleic acid encoding a CYP activator.
  • the CYP activator can be, for example, CPR4497 (SEQ ID NO:45, SEQ ID NO:46) or a functional homolog sharing at least 50% sequence identity with SEQ ID NO:46. See Examples 10, 15, and 16.
  • the epoxide hydrolase is epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO:40).
  • cerevisiae strain further overexpresses squaiene epoxidase encoded by ERG1 (SEQ ID NO:54), expresses a truncated HMG reductase (tHMG1 , SEQ ID NO:77, SEQ ID NO:78), expresses S. grosvenorii cucurbitadienol synthase (SEQ ID NO:42, SEQ ID NO:43), is deleted of the TRP1 gene, and comprises a disrupted promoter of the endogenous ERG7 gene (SEQ ID NO:55). See Example 15.
  • a mogrol precursor, mogrol, or mogroside is produced in a recombinant host comprising one or more of a gene encoding a squaiene epoxidase polypeptide, a gene encoding a cucurbitadienol synthase polypeptide, a gene encoding a cytochrome P450 polypeptide, a gene encoding a cytochrome P450 reductase polypeptide, a gene encoding an epoxide hydrolase polypeptide, and/or a gene encoding a glycosyltransferase.
  • the gene encoding the glycosyltransferase comprises a gene encoding a UGT1576 polypeptide having 60% or greater identity to an amino acid sequence set forth in SEQ ID NO:48, a gene encoding a UGT430 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:62, a gene encoding a UGT1697 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:68, a gene encoding a UGT11789 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:72, and/or a gene encoding a UGT98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:53. See Example 16.
  • mogroside V is produced in an S. cerevisiae strain comprising S. grosvenorii cucurbitadienol synthase (SEQ ID NO:42, SEQ ID NO:43), CYP5491 (SEQ ID NO:81 , SEQ ID NO:44), CYP1798 (SEQ ID NO:5, SEQ ID NO:74), CYP1798-II (SEQ ID NO:86, SEQ ID NO:74), CPR4497 (SEQ ID NO:82, SEQ ID NO:46), epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO:40), UGT1576 (SEQ ID NO:83, SEQ ID NO:48), UGT430 (SEQ ID NO:84, SEQ ID NO:62), UGT1697 (SEQ ID NO:85, SEQ ID NO:68), UGT98 (SEQ ID NO:52, SEQ ID NO:53), and UGT11789 (SEQ ID NO:71 , SEQ ID NO:42, SEQ ID NO
  • the strain is a Mat alpha derivative of S. cerevisiae 288C with a deletion of the S. cerevisiae EXG1 gene.
  • the host further produces mogroside IV A, mogroside II A2, mogroside I E1 , and mogrol. See Example 16.
  • a mogroside is produced through contact of a mogrol precursor, mogrol, or glycosylated mogrol with one or more enzymes involved in the mogroside pathway in vitro.
  • a mogrol precursor is produced through contact of an upstream mogroside precursor with one or more enzymes involved in the mogroside pathway in vitro.
  • contact of cucurbitadienol with a cytochrome P450 polypeptide and an epoxide hydrolase can result in production of mogrol in vitro.
  • a mogrol precursor is produced by one or more of the following steps:
  • a cucurbitadienol synthase such as, but not limited to, a cucurbitadienol synthase having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:43, to produce cucurbitadienol (see step A of Figures 2B and 2C); or
  • a cucurbitadienol synthase such as, but not limited to, a cucurbitadienol synthase having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:43, to produce 24,25 epoxy cucurbitadienol (see step B of Figures 2B and 2C); or
  • cytochrome P450 such as, but not limited to, CYP5491 having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:44, to produce 11 -hydroxy-cucurbitadienol (see step C of Figures 2B and 2C); or
  • cytochrome P450 such as, but not limited to, CYP5491 having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:44, to produce 11-hydroxy-24,25 epoxy cucurbitadienol (see step D of Figures 2B and 2C); or
  • cytochrome P450 such as, but not limited to, CYP1798 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:74, to produce 24,25 epoxy cucurbitadienol (see step E of Figures 2B and 2C); or
  • cytochrome P450 such as, but not limited to, CYP1 98 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:74, to produce 1 1-hydroxy-24,25 epoxy cucurbitadienol (see step F of Figures 2B and 2C).
  • mogrol is produced in vitro by contacting H -hydroxy-24,25 epoxy cucurbitadienol with an epoxide hydrolase, such as, but not limited to, epoxide hydrolase 1 having 75% or greater identity to an amino acid sequence set forth in SEQ ID NO:38 or epoxide hydrolase 2 having 65% or greater identity to an amino acid sequence set forth in SEQ ID NO:40 (see step G of Figures 2B and 2C).
  • an epoxide hydrolase such as, but not limited to, epoxide hydrolase 1 having 75% or greater identity to an amino acid sequence set forth in SEQ ID NO:38 or epoxide hydrolase 2 having 65% or greater identity to an amino acid sequence set forth in SEQ ID NO:40 (see step G of Figures 2B and 2C).
  • a mogroside (see step H of Figures 2B and 2C) is produced in vitro by:
  • each of the steps described above can be performed separately.
  • a product of a step can be purified or partially purified before performing a subsequent step.
  • one or more of the steps can be performed simultaneously within the same mixture.
  • a cell lysate is prepared from a host expressing one or more of a gene encoding a squalene epoxidase polypeptide, a gene encoding a cucurbitadienol synthase polypeptide, a gene encoding a cytochrome P450 polypeptide, a gene encoding an epoxide hydrolase polypeptide, and a gene encoding a UGT polypeptide.
  • a cell lysate can be prepared from a host expressing one or more UGTs and used to contact mogrol, such that a mogroside can be produced in vitro.
  • a mogrol precursor, mogrol, or mogroside is produced by whole cell bioconversion.
  • a host expressing one or more enzymes involved in the mogroside pathway takes up and modifies a mogrol or mogroside precursor in the cell; following modification in vivo, a mogroside is excreted into the culture medium. See Examples 1 1-14.
  • the mogrol precursor is oxidosqualene, dioxidosqualene, cucurbitadienol, 24,25 epoxy cucurbitadienol and the mogroside precursor is mogrol.
  • a host expressing a gene encoding a UGT polypeptide can take up mogrol and glycosylate mogrol in the cell; following glycosylation in vivo, a mogroside is excreted into the culture medium.
  • a cell can be fed a mogrol precursor or mogroside precursor during cell growth or after cell growth.
  • the cell can be in suspension or immobilized.
  • the cell can be in fermentation broth or in a reaction buffer.
  • a permeabilizing agent is used for transfer of a mogrol precursor or mogroside precursor into a cell.
  • a mogrol precursor or mogroside precursor can be provided in a purified form or as part of a composition or an extract.
  • a mogrol precursor or mogroside precursor is produced in vitro; thereafter, the mogrol precursor or mogroside precursor is provided to a host capable of catalyzing conversion of the mogrol precursor or mogroside precursor.
  • a recombinant host expressing UGT98, UGT1576, and UGT430 converts fed mogrol to mogroside V. See Example 14.
  • a host expressing UGT1 789 catalyzes the conversion of mogroside II E to a tri-g!ycosylated mogroside.
  • a host expressing UGT11789, UGT1576, and UGT430 catalyzes the conversion of mogrol to a trig!ycosy!ated mogroside.
  • a recombinant host co-expressing UGT11789, UGT98, UGT1576, and UGT430 converts fed mogrol to mogroside V more efficiently than a recombinant host expressing UGT98, UGT1576, and UGT430. See Example 14. Recombinant Genes and Functional Homologs
  • recombinant gene refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence can already be present in such a host. "Introduced” or “augmented” in this context is known in the art to mean introduced or augmented by the hand of man.
  • a recombinant gene can be a DNA sequence from another species, or can be a DNA sequence that originated from or is present in the same species, but has been incorporated into a host by recombinant methods to form a recombinant host.
  • a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA.
  • the DNA is a cDNA copy of an mRNA transcript of a gene produced in a cell.
  • the coding sequence of a polypeptide described herein is a heterologous sequence.
  • the phrases "heterologous sequence” and “heterologous coding sequence” are used to describe a sequence derived from a species other than the recombinant host.
  • the recombinant host is an S. cerevisiae cell, and a heterologous sequence is derived from an organism other than S. cerevisiae.
  • a heterologous coding sequence for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant host expressing the heterologous sequence.
  • a coding sequence is a sequence that is native to the host.
  • a squalene epoxidase polypeptide, cucurbitadienol synthase polypeptide, cytochrome P450 polypeptide, cytochrome P450 reductase polypeptide, epoxide hydrolase polypeptide, and/or glycosyitransferase polypeptide is a fusion protein.
  • a squalene epoxidase polypeptide including, but not limited to, the squalene epoxidase polypeptide of SEQ ID NO:54, a cucurbitadienol synthase polypeptide (including, but not limited to, the cucurbitadienol synthase polypeptide of SEQ ID NO:43), a cytochrome P450 polypeptide (including, but not limited to, the CYP5491 polypeptide of SEQ ID NO:44), a cytochrome P450 reductase polypeptide (including, but not limited to, the CPR4497 polypeptide of SEQ ID NO:46), an epoxide hydrolase polypeptide (including, but not limited to, the EH1 polypeptide of SEQ ID NO:38 or the EH2 polypeptide of SEQ ID NO:40), and/or a UGT polypeptide (including, but not limited to, UGT1576 of SEQ ID NO:
  • chimera fusion polypeptide
  • fusion protein fusion protein
  • chimeric enzyme chimeric enzyme
  • a nucleic acid sequence encoding a squalene epoxidase polypeptide, cucurbitadienol synthase polypeptide, cytochrome P450 polypeptide, cytochrome P450 reductase polypeptide, epoxide hydrolase polypeptide, and/or glycosyltransferase polypeptide polypeptide include a tag sequence that encodes a "tag" designed to facilitate subsequent manipulation (e.g., to facilitate purification or detection), secretion, or localization of the encoded polypeptide.
  • Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide.
  • encoded tags include green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), polyhistidine-tag (HIS tag), and FlagTM tag (Kodak, New Haven, CT).
  • GFP green fluorescent protein
  • HA human influenza hemagglutinin
  • GST glutathione S transferase
  • HIS tag polyhistidine-tag
  • FlagTM tag Kodak, New Haven, CT.
  • Other examples of tags include a ch!oroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, signal peptide, or a secretion tag.
  • a fusion protein is a protein altered by domain swapping.
  • domain swapping is used to describe the process of replacing a domain of a first protein with a domain of a second protein.
  • the domain of the first protein and the domain of the second protein are functionally identical or functionally similar.
  • the structure and/or sequence of the domain of the second protein differs from the structure and/or sequence of the domain of the first protein.
  • a cytochrome P450 reductase polypeptide is altered by domain swapping.
  • the cytochrome P450 domain or reductase domain of CPR4497 (SEQ ID NO:46) is replaced by the cytochrome P450 domain or reductase domain of a cytochrome P450 reductase other than CPR4497 (SEQ ID NO:46).
  • a UGT polypeptide is altered by domain swapping.
  • a functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide.
  • a functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homoiogs, or orthologs, or paralogs.
  • Variants of a naturally occurring functional homolog can themselves be functional homologs.
  • Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally-occurring polypeptides ("domain swapping").
  • Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs.
  • the term "functional homolog” is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
  • Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of steviol glycoside biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PS I -BLAST analysis of non- redundant databases using a UGT amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a steviol glycoside biosynthesis polypeptide.
  • Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in steviol glycoside biosynthesis polypeptides, e.g., conserved functional domains.
  • conserveed regions can be identified by locating a region within the primary amino acid sequence of a steviol glycoside biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. The information included at the Pfam database is described in Sonnhammer et al., Nucl.
  • conserveed regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.
  • polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions.
  • conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity).
  • a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
  • Recombinant hosts described herein below can be used in methods to produce a mogrol precursor, mogrol, or mogroside.
  • the method can include growing the recombinant microorganism In a culture medium under conditions in which one or more of the enzymes catalyzing step(s) of the methods of the invention, e.g., synthases, hydrolases, CYP450s and/or UGTs are expressed.
  • the terms "microorganism” and "microorganism host” and “recombinant host” can be used interchangeably to refer to microscopic organisms, including bacteria or microscopic fungi, including yeast.
  • the microorganism can be, but not iimited to, a eukaryotic cell or immortalized cell.
  • prokaryotic and eukaryotic species are described in more detail below. However, it will be appreciated that other species can be suitable.
  • suitable species can be in a genus including Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Escherichia, Fusarium/Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces and Yarrowia.
  • Exemplary species from such genera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Physcomitrella patens, Rhodoturula glutinis 32, Rhodoturula mucilaginosa, Phaffia rhodozyma UBV-AX, Xanthophyllomyces dendrorhous, Fusarium fujikuroi/Gibberella fujikuroi, Candida utilis and Yarrowia lipolytica.
  • a microorganism can be an Ascomycete such as Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Aspergillus niger, or Saccharomyces cerevisiae.
  • a microorganism can be a prokaryote such as Escherichia coli, Rhodobacter sphaeroides, or Rhodobacter capsulatus. It will be appreciated that certain microorganisms can be used to screen and test genes of interest in a high throughput manner, while other microorganisms with desired productivity or growth characteristics can be used for large-scale production of mogro! precursor, mogrol, or mogroside.
  • microorganisms include, but are not limited to, S. cerevisiae, A. niger, A. oryzae, E. coli, L. lactis and B. subtilis.
  • the constructed and genetically engineered microorganisms provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, continuous perfusion fermentation, and continuous perfusion cell culture.
  • Exemplary embodiments comprising bacterial cells include, but are not limited to, cells of species, belonging to the genus Bacillus, the genus Escherichia, the genus Lactobacillus, the genus Lactobacillus, the genus Corynebaclerium, the genus Acetobacler, the genus Acinetobacler, or the genus Pseudomonas.
  • the microorganism can be a fungus, and more specifically, a filamentous fungus belonging to the genus of Aspergillus, e.g., A. niger, A. awamori, A. oryzae, or A. nidulans, a yeast belonging to the genus of Saccharomyces, e.g., S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, or S. uvarum, a yeast belonging to the genus Kluyveromyces, e.g., K. laclis, K. marxianus var. marxianus, or K.
  • a filamentous fungus belonging to the genus of Aspergillus e.g., A. niger, A. awamori, A. oryzae, or A. nidulans
  • yeast belonging to the genus of Saccharomyces
  • thermololerans a yeast belonging to the genus Candida, e.g., C. ulilis, C. Iropicalis, C. albicans, C. lipolylica, or C. versalilis, a yeast belonging to the genus Pichia, e.g., R. slipidis, R. pasloris, or P. sorbilophila, or other yeast genera, e.g., Cryplococcus, Debaromyces, Hansenula, Pichia, Yarrowia, Zygosaccharomyces, or Schizosaccharomyces.
  • Candida e.g., C. ulilis, C. Iropicalis, C. albicans, C. lipolylica, or C. versalilis
  • a yeast belonging to the genus Pichia e.g., R. slipidis, R. pasloris, or P. sorbilophila
  • other yeast genera e.g
  • filamentous fungi a species belonging to the genus Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Morlierella, and Trichoderma.
  • Saccharomyces cerevisiae is a widely used chassis organism in synthetic biology, and can be used as the recombinant microorganism platform. There are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for S. cerevisiae, allowing for rational design of various modules to enhance product yield. Methods are known for making recombinant microorganisms.
  • genes described herein can be expressed in yeast using any of a number of known promoters. Strains that overproduce phenylpropanoids are known and can be used as acceptor molecules in the production of a mogrol precursor, mogrol, or mogroside. Aspergillus spp.
  • Aspergillus species such as A. oryzae, A. niger and A. sojae are widely used microorganisms in food production, and can also be used as the recombinant microorganism platform. Nucleotide sequences are available for genomes of A. nidulans, A. fumigatus, A. oryzae, A. clavatus, A. flavus, A. niger, and A. terreus, allowing rational design and modification of endogenous pathways to enhance flux and increase product yield. Metabolic models have been developed for Aspergillus, as well as transcriptomic studies and proteomics studies. A. niger is cultured for the industrial production of a number of food ingredients such as citric acid and gluconic acid, and thus species such as A. niger are generally suitable for the production of a mogroi precursor, mogro!, or mogroside.
  • Escherichia coli another widely used platform organism in synthetic biology, can also be used as the recombinant microorganism platform. Similar to Saccharomyces, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for Saccharomyces can be used to make recombinant E. coli microorganisms.
  • Agaricus, Gibberella, and Phanerochaete spp. can be useful because they are known to produce large amounts of gibbere!lin in culture.
  • the precursors of terpenes used as acceptor molecules in the production of a mogroi precursor, mogroi, or mogroside are already produced by endogenous genes.
  • modules containing recombinant genes for biosynthesis of terpenes can be introduced into species from such genera without the necessity of introducing other compounds or pathway genes.
  • Arxula adeninivorans (Blastobotrvs adeninivorans)
  • Arxula adeninivorans is dimorphic yeast (it grows as budding yeast like the baker's yeast up to a temperature of 42°C, above this threshold it grows in a filamentous form) with unusual biochemical characteristics. It can grow on a wide range of substrates and can assimilate nitrate. It has successfully been applied to the generation of strains that can produce natural plastics or the development of a biosensor for estrogens in environmental samples.
  • Yarrowia lipolytica is dimorphic yeast (see Arxula adeninivorans) and belongs to the family Hemiascomycetes. The entire genome of Yarrowia lipolytica is known. Yarrowia species is aerobic and considered to be non-pathogenic. Yarrowia is efficient in using hydrophobic substrates (e.g. alkanes, fatty acids, oils) and can grow on sugars. It has a high potential for industrial applications and is an oleaginous microorganism. Yarrowia lipolyptica can accumulate lipid content to approximately 40% of its dry cell weight and is a model organism for lipid accumulation and remobilization.
  • hydrophobic substrates e.g. alkanes, fatty acids, oils
  • Rhodotorula is unicellular, pigmented yeast.
  • the oleaginous red yeast, Rhodotorula glutinis has been shown to produce lipids and carotenoids from crude glycerol (Saenge et al., 2011 , Process Biochemistry 46(1 ):210-8).
  • Rhodotorula toruloides strains have been shown to be an efficient fed-batch fermentation system for improved biomass and lipid productivity (Li et al., 2007, Enzyme and Microbial Technology 41 :312-7).
  • Rhodosporidium toruloides is oleaginous yeast and useful for engineering lipid- production pathways (See, e.g., Zhu et al., 2013, Nature Commun. 3:1112; Ageitos et al. , 201 , Applied Microbiology and Biotechnology 90(4): 1219-27).
  • Candida boidinii is methylotrophic yeast (it can grow on methanol). Like other methylotrophic species such as Hansenula polymorpha and Pichia pastoris, it provides an excellent platform for producing heterologous proteins. Yields in a multigram range of a secreted foreign protein have been reported.
  • a computational method, IPRO recently predicted mutations that experimentally switched the cofactor specificity of Candida boidinii xylose reductase from NADPH to NADH. See, e.g., attanovich et al., 2012, Methods Mol Biol. 824:329-58; Khoury et al., 2009, Protein Sci. 18(10):2125-38.
  • Hansenula polymorpha is methylotrophic yeast (see Candida boidinii). It can furthermore grow on a wide range of other substrates; it is therm o-tolerant and can assimilate nitrate (see also Kluyveromyces lactis). It has been applied to producing hepatitis B vaccines, insulin and interferon alpha-2a for the treatment of hepatitis C, furthermore to a range of technical enzymes. See, e.g., Xu ef a/., 2014, Virol Sin. 29(6):403-9.
  • Kluyveromyces lactis is yeast regularly applied to the production of kefir. It can grow on several sugars, most importantly on lactose which is present in milk and whey. It has successfully been applied among others for producing chymosin (an enzyme that is usually present in the stomach of calves) for producing cheese. Production takes place in fermenters on a 40,000 L scale. See, e.g., van Ooyen ef a/., 2006, FEMS Yeast Res. 6(3):381-92.
  • Pichia pastoris is methylotrophic yeast (see Candida boidinii and Hansenula polymorpha). It provides an efficient platform for producing foreign proteins. Platform elements are available as a kit and it is worldwide used in academia for producing proteins. Strains have been engineered that can produce complex human N-glycan (yeast glycans are similar but not identical to those found in humans). See, e.g., Piirainen er a/., 2014, N Biotechnol. 31 (6):532-7.
  • Physcomitrella mosses when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera can be used for producing plant secondary metabolites, which can be difficult to produce in other types of cells.
  • the particulars of the selection process for specific UGTs capable of glycosylating mogroi and mogrosides depend on the identities of selectable markers. Selection in all cases promotes or permits proliferation of ceils comprising the marker while inhibiting or preventing proliferation of cells lacking the marker. If a selectable marker is an antibiotic resistance gene, the transfected host population can be cultured in the presence of an antibiotic to which resistance is conferred by the selectable marker. If a selectable marker is a gene that complements an auxotrophy of the hosts, the transfected host population can be cultivated in the absence of the compound for which the hosts are auxotrophic.
  • recombinant hosts can be cloned according to any appropriate method known in the art.
  • recombinant microbial hosts can be plated on solid media under selection conditions, after which single clones can be selected for further selection, characterization, or use. This process can be repeated one or more times to enhance stability of the expression construct within the host.
  • recombinant hosts comprising one or more expression vectors can be cultured to expand cell numbers in any appropriate culturing apparatus known in the art, such as a shaken culture flask or a fermenter.
  • Culture media used for various recombinant hosts are well known in the art. Culture media used to culture recombinant bacterial cells will depend on the identity of the bacteria. Culture media used to culture recombinant yeast cells will depend on the identity of the yeast. Culture media generally comprise inorganic salts and compounds, amino acids, carbohydrates, vitamins and other compounds that are either necessary for the growth of the hosts or improve health or growth or both of the hosts. In particular, culture media typically comprise manganese (Mn 2+ ) and magnesium (Mg 2+ ) ions, which are co-factors for many, but not all, glycosyltransferases.
  • Mn 2+ manganese
  • Mg 2+ magnesium
  • fed-batch culture or “semi-batch culture” are used interchangeably to refer to as an operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation and in which the product(s) remain in the bioreactor until the end of the run. In some embodiments, all the nutrients are fed into the bioreactor.
  • a recombinant host can be modified in order to reduce giucanase activity, in particular giucanase activity, which can result in deglycosylation of mogrosides.
  • the recombinant host can for example be modified to reduce of even abolish exo-1 ,3-beta-Glucanase activity, in embodiments of the invention when the recombinant host is yeast, this can be accomplished by deletion of the EXG1 gene (SEQ ID NO:63, SEQ ID NO:64) and/or of the EXG2 gene (SEQ ID NO:65, SEQ ID NO:66), both of which are encoding an exo- 1 ,3-beta-glucanase.
  • Table 2 indicates the identities of the sequences utilized herein.
  • SEQ ID NO: 1 Nucleotide sequence encoding CYP4112
  • SEQ ID NO: 19 Nucleotide sequence encoding CY 10020
  • SEQ ID NO:28 Nucleotide sequence encoding S. grosvenorii UGT1817 SEQ ID NO:29 Partial nucleotide sequence encoding fragment of S.
  • SEQ ID NO:33 Partial nucleotide sequence encoding fragment of S.
  • SEQ ID NO:34 Partial nucleotide sequence encoding fragment of S.
  • SEQ ID NO:35 Partial nucleotide sequence encoding fragment of S.
  • SEQ ID NO:36 Partial nucleotide sequence encoding fragment of S.
  • SEQ ID NO:38 Amino acid sequence of S. grosvenorii Epoxide hydrolase 1
  • SEQ ID NO:40 Amino acid sequence of S. grosvenorii Epoxide hydrolase 2
  • SEQ ID NO:42 Codon-optimized nucleotide sequence encoding S. grosvenorii cucurbitadienol synthase
  • SEQ ID NO:50 Amino acid sequence of S. grosvenorii UGT SK98
  • SEQ ID NO:54 Amino acid sequence of S. cerevisiae squalene epoxidase encoded by the ERG1 gene
  • SEQ ID NO:55 Amino acid sequence of S. cerevisiae lanosterol synthase encoded by the ERG7 gene
  • SEQ ID NO:62 Amino acid sequence of S. grosvenorii UGT430
  • SEQ ID NO:66 Amino acid sequence of S. cerevisiae EXG2
  • SEQ ID NO:68 Amino acid sequence of S. grosvenorii UGT 1697
  • SEQ ID NO:70 Codon-optimized nucleotide sequence "A" of full-length S.
  • SEQ ID NO:71 Codon-optimized nucleotide sequence "B" of full-length S.
  • SEQ ID NO:76 Amino acid sequence of S. cerevisiae TRP1
  • SEQ ID NO:78 Amino acid sequence of S. cerevisiae tH G1
  • SEQ ID NO:79 Nucleotide sequence encoding S. grosvenorii Epoxide hydrolase 2
  • SEQ ID NO:80 Nucleotide sequence encoding S. grosvenorii cucurbitadienoi synthase
  • Mogroside V was purified from commercially available monk fruit extracts (PureLo®, Swanson). Three bottles of PureLo® (240 g) were dissolved in water (900 mL) and loaded on a column of HP-20 resin (400 g resin). The column was washed with water (2.5 liters) and further washed with 20% methanol in water. The product was eluted with methanol. After solvent evaporation and drying under high vacuum, mogroside V (2.5 g) was obtained. The product was approximately 80% pure, with 11-oxomogroside V being the largest impurity.
  • Mogroside V 300 mg was dissolved in 0.1 M sodium acetate buffer (pH 4.5, 100 mL), and crude pectinase from Aspergillus niger (25 mL, Sigma P2736) was added. The mixture was stirred at 50°C for 48 h. The reaction mixture was extracted with ethyl acetate (2x100 mL). The organic extract was dried under vacuum and subsequently purified with preparative HPLC. Pure mogrol (40 mg) was obtained, and its structure was confirmed by NMR and mass spectroscopy. See Figure 5.
  • Example 3 Enzymatic synthesis of mogrol 3-O-glucoside (mogroside I E1) and mogrol 24-O-glucoside (mogroside I A1) from mogroside V
  • Mogroside V (300 mg) was dissolved in 0.1 M sodium acetate buffer (pH 4.5, 100 mL), and crude pectinase from Aspergillus niger (25 mL, Sigma P2736) was added. The mixture was stirred at 50°C for 6.5 h and subsequently extracted with ethyl acetate (2x100 mL). The organic extract was dried under vacuum and purified with preparative HPLC. Pure mogroside I E1 (11.0 mg) and mogroside I A1 (8.0 mg) were obtained. Their structures were confirmed by N R and mass spectroscopy. See Figure 5.
  • UGT73C3 (SEQ ID NO:21), UGT73C5 (SEQ ID NO:22), UGT73C6 (SEQ ID NO:23), UGT73E1 (SEQ ID NO:24), and UGT85C2 (SEQ ID NO:25) were found to glycosylate mogrol in vitro.
  • the reaction mixtures included 4X Tris buffer, mogrol (250 ⁇ ), UDP-glucose (750 ⁇ ), and 1 % alkaline phosphatase.
  • 5 ⁇ 1_ of each partially purified UGT enzyme or crude enzyme extract was added to the reaction, and the reaction volume brought to 50 ⁇ _ with water.
  • the reactions were incubated overnight at 30°C and performed in sterilized 96 well plates. 25 ⁇ _ of DMSO were subsequently added into each reaction, and the reaction plates were centrifuged for 5 min. 40 ⁇ _ samples were taken from each well and filtered to be used for LC- MS analysis.
  • UGT73C3 (SEQ ID NO:21 ), UGT73C6 (SEQ ID NO:23) and UGT85C2 (SEQ ID NO:25) were found to convert the entire mogrol substrate to mogroside I A1.
  • UGT73C5 (SEQ ID NO:22) produced both mogroside I E1 and mogroside I A1.
  • UGT73E1 (SEQ ID NO:24) converted mogrol to mogroside 1 A1 (major product) and a glycosylated mogrol that was neither mogroside I E1 nor mogroside I A1. The product was caused by a glycosy!ation event on C11- OH; the exact mass was shown as a mogroside I.
  • CirCS gene codes for cucurbitadienol synthase in monk fruit, and the partial gene sequence covering 338 of the supposedly 764 amino acid sequence was identified by doing a tBLASTn (translated nucleotide database) analysis of the assembled data with a query cucurbitadienol synthase from Cucurbita pepo (accession number BAD34645.1 , SEQ ID NO:1 ).
  • the partial CirCS is 97.5% identical to the C. pepo gene at the protein level (SEQ ID NO:2; from residues 515 to 764 of SEQ ID NO:1 ).
  • Example 6 Monk fruit genes encoding P450 enzymes catalyzing formation of mogrol from cucurbitadienol [00162] To identify P450 enzymes catalyzing formation of mogrol from cucurbitadienol, a tBLASTn (translated nucleotide database) analysis was performed using reassembled sequencing reads of an S. grosvenorii transcriptome ⁇ see Tang et a/., BMC Genomics 12: 343 (201 1 )). E values of 10E-10 or lower were used to identify sequences homologous to the database query sequences.
  • tBLASTn translated nucleotide database
  • Example 7 Monk fruit genes encoding enzymes catalyzing glycosylation of mogroside II
  • the genes identified were UGT98 (SEQ ID NO:26), UGT1495 (SEQ ID NO:27), UGT1817 (SEQ ID NO:28), UGT3494 (SEQ ID NO:29), UGT5914 (SEQ ID NO:30), UGT8468 (SEQ ID NO:31 ), UGT10391 (SEQ ID NO:32), UGT11789 (SEQ ID NO:33), UGT1 999 (SEQ ID NO:34), UGT13679 (SEQ ID NO:35), and UGT15423 (SEQ ID NO:36).
  • UGT98 SEQ ID NO:26
  • UGT1495 SEQ ID NO:27
  • UGT1817 SEQ ID NO:28
  • UGT5914 SEQ ID NO:30
  • UGT8468 SEQ ID NO:31
  • UGT10391 SEQ ID NO:32
  • ERG7 expression was thereby decreased when the yeast strain was grown in normal SC medium.
  • ERG7 encodes lanosterol synthase and lowered expression is known to result in accumulation of oxidosqualene and dioxidosqualene in baker's yeast.
  • Oxidosqualene is generally the precursor of triterpenoids.
  • the squalene epoxidase encoded by ERG1 was overexpressed, and a truncated copy of the yeast HMG reductase (tHMG1 , SEQ ID NO:77, SEQ ID NO:78) was expressed.
  • Figure 6 shows the LC-MS mass peak 501 corresponding to the proton plus Na+ adduct of tetrahydroxysqualene in a sample from a yeast strain transformed with a plasmid expressing S. grosvenorii epoxide hydrolase 2.
  • Tetrahydroxysqualene is produced by hydrolysis of 2,3- and 22,23- epoxide bonds of dioxidosqualene. No accumulation of tetrahydroxysqualene was detected in the background yeast strain. Samples were made by boiling culture aliquots in 50% DMSO and then pelleting of cell material by centrifugation. Supernatants were then measured by ESI LC-MS.
  • Figures 7A and 7B show LC-MS chromatograms of samples of yeast expressing the cucurbitadienol synthase set forth in SEQ ID NO:42 and SEQ ID NO:43.
  • Figure 7A shows lanosterol peaks
  • Figure 7B shows cucurbitadienol and lanosterol peaks.
  • the peak corresponding to lanosterol shows a retention time of -8.05
  • the peak corresponding to cucurbitadienol has a retention time of 7.85.
  • Both lanosterol and cucurbitadienol show a mass in the LC-MS chromatogram of 409.4 (proton adduct minus mass of one H 2 0 molecule).
  • Example 11 Glycosylation of mogroi in S. cerevisiae by expression of S. grosvenorii UGT98, UGTSK98, and UGT1576
  • UGT98, UGTSK98 and UGT1576 genes were synthesized based on contigs made from publically-available sequence reads (Tang et a/., 2011 , BMC Genomics 12:343).
  • the nucleotide and amino acid sequences of UGT98 are set forth herein as SEQ ID NO:51 and SEQ ID NO:53, respectively, whereas SEQ ID NO:52 corresponds to a codon-optimized version of UGT98.
  • nucleotide and amino acid sequences of UGTSK98 are set forth herein as SEQ ID NO:49 and SEQ ID NO:50, respectively, and the nucleotide and amino acid sequences of UGT1576 are set forth herein as SEQ ID NO:47 and SEQ ID NO:48, respectively.
  • a yeast strain deleted of the exo-1,3-beta g!ucanases EXG1 and EXG2 was fed mogrol (10-100 ⁇ ) and transformed with a plasmid expressing UGT1576 (SEQ ID NO:47 and SEQ ID NO:48), mogroside I A1 was formed (Fig 11B).
  • FIG. 10A shows the LC-MS chromatogram of reference mogroside I A1
  • Figure 10B shows the peak from a yeast sample expressing UGT1576 in a culture fed with 50 ⁇ mogrol.
  • Example 12 Glycosyiation of mogrol in S. cerevisiae by expression of S. grosvenorii UGT430
  • UGT430 (SEQ ID NO:61 , SEQ ID NO:62) of the 85A UGT family was cloned from synthetic DNA to obtain a sequence identical to that of S. grosvenorii UGT430.
  • the cloned gene was transformed into a yeast strain deleted of EXG1 and EXG2 (to prevent de- g!ycosylation of produced mogrosides).
  • the yeast strain was grown in SC medium minus tryptophan for selection of plasmid maintenance, and comprising 10 ⁇ mogrol. Cells were grown for 2 days at 30°C with shaking at 140 rpm. After 2 days, 300 ⁇ _ culture samples were mixed with 300 pl_ of 96% ethanol and incubated for 10 min at 80°C. Then, samples were centrifuged, and the supernatant was analyzed by LC-MS.
  • LC-MS analyses were performed using a Waters Acquity l-Class UPLC (Waters Corporation, Milford, MA) with Waters Acquity UPLC ®BEH C18 column (2.1 x 50 mm, 1.7 ⁇ particles, 130 A pore size) coupled to a Waters Xevo TQD triple quadropole mass spectrometer with electrospray ionization (ESI) in negative mode.
  • Waters Acquity l-Class UPLC Waters Corporation, Milford, MA
  • Waters Acquity UPLC ®BEH C18 column 2.1 x 50 mm, 1.7 ⁇ particles, 130 A pore size
  • ESI electrospray ionization
  • Compound separation was achieved by a gradient of the two mobile phases A (water with 0.1 % formic acid) and B (MeCN with 0.1 % formic acid) by increasing from 20% to 50% B between 0.3 to 2.0 min, increasing to 100% B at 2.01 min, holding 100% B for 0.6 min and re-equilibrating for another 0.6 min.
  • the flow rate was 0.6 mL/min, and the column temperature 55°C.
  • Mogroside I E1 m/z 683.5; [M+FA] "
  • SIR Single Ion Recording
  • Example 13 Glycosylation of mogrol in S. cerevisiae by expression of S, grosvenorii UGT1697
  • UGT 697 (SEQ ID NO:67, SEQ ID NO:68) of the 85A UGT family was cloned from synthetic DNA to obtain a sequence identical to that of S. grosvenorii UGT1697.
  • the cloned gene was transformed into a yeast strain deleted of EXG1 and EXG2 (to prevent de- glycosylation of produced mogrosides.
  • the yeast strain was grown in SC medium minus histidine for selection of plasmid maintenance, and comprising 10 ⁇ mogrol. Cells were grown for 2 days at 30°C with shaking at 140 rpm. After 2 days, 300 pL culture samples were mixed with 300 ⁇ [_ of 96% ethanol and incubated for 10 min at 80°C. Then, samples were centrifuged, and the supernatant was analyzed by LC-MS.
  • LC-MS analyses were performed using a Waters Acquity !-Class UPLC (Waters Corporation, Milford, MA) with Waters Acquity UPLC ®BEH C18 column (2.1 x 50 mm, 1.7 pm particles, 130 A pore size) coupled to a Waters Xevo TQD triple quadropole mass spectrometer with e!ectrospray ionization (ESI) in negative mode.
  • ESI Waters Xevo TQD triple quadropole mass spectrometer with e!ectrospray ionization
  • Compound separation was achieved by a gradient of the two mobile phases A (water with 0.1 % formic acid) and B (MeCN with 0.1 % formic acid) by increasing from 20% to 50% B between 0.3 to 2.0 min, increasing to 100% B at 2.01 min, holding 100% B for 0.6 min and re-equilibrating for another 0.6 min.
  • the flow rate was 0.6 mL/min, and the column temperature 55°C.
  • Mogroside I m/z 683.5; [M+FA] " ) was monitored using SIR (Single Ion Recording) and compared with a standard.
  • UGT1697 acts on the C-3 position as well, since the presence of mogroside II E (containing one glucose on position C-24 and one on C-3) was detected, as depicted in Figure 14B (retention time of 2.22 min). Thus, UGT1697 glycosylates the C-3 and C-24 position on mogrol and is part of the S. grosvenorii mogroside biosynthetic pathway.
  • Example 14 Glycosylation of mogrol and mogrosides in S. cerevisiae by expression of S. grosvenor// UGT11789, UGT98, UGT430, and UGT1576
  • UGT1 789 The full-length sequence for UGT1 789 (SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 , SEQ ID NO:72) was cloned from synthetic DNA to obtain a sequence identical to that of S. grosvenorii UGT11789.
  • a yeast strain deleted of EXG1 and EXG2 was co-transformed with UGT11789 (SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 , SEQ ID NO:72), UGT430 (SEQ ID NO:61 , SEQ ID NO:62), UGT1576 (SEQ ID NO:47, SEQ ID NO:48), and UGT98 (SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53).
  • a yeast strain deleted of EXG1 and EXG2 was co-transformed with UGT430 (SEQ ID NO:61 , SEQ ID NO:62), UGT1576 (SEQ ID NO:47, SEQ ID NO:48), and UGT98 (SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53).
  • the yeast strains were grown in SC medium minus histidine, uracil, tryptophan, and leucine for selection of plasmid maintenance and comprising 10 ⁇ mogrol. Cells were grown for 2 days at 30°C with shaking at 140 rpm. After 2 days, 300 ⁇ !_ culture samples were mixed with 300 ⁇ _ of 96% ethanol and incubated for 10 min at 80°C. Then, samples were centrifuged, and the supernatant was analyzed by LC- S.
  • LC-MS analyses were performed using a Waters Acquity !-Class UPLC (Waters Corporation, Milford, MA) with Waters Acquity UPLC ®BEH C18 column (2.1 x 50 mm, 1.7 pm particles, 130 A pore size) coupled to a Waters Xevo TQD triple quadropole mass spectrometer with electrospray ionization (ESI) in negative mode. Compound separation was achieved by gradient I or gradient II.
  • ESI electrospray ionization
  • the initial buffer concentration of 80% mobile phase A (water with 0.1 % formic acid) and 20% mobile phase B (MeCN with 0.1 % formic acid) was increased from to 20% to 40% B between 0.3 to 2.0 min, increased to 100% B at 2.01 min, held at 100% B for 0.6 min, and re-equilibrated for another 0.6 min.
  • the initial buffer concentration of 80% mobile phase A (water with 0.1 % formic acid) and 20% mobile phase B (MeCN with 0.1 % formic acid) was increased from to 20% to 50% B between 0.3 to 2.0 min, increased to 100% B at 2.01 min, held at 100% B for 0.6 min, and re-equilibrated for another 0.6 min.
  • Figure 15A shows mogroside reference standards and indicates peaks corresponding to mogroside V and mogroside II E.
  • Comparison of Figure 15B and Figure 15C demonstrates the effect of expression of the UGT11789 codon-optimized sequence A (SEQ ID NO:70, SEQ ID NO.72).
  • Figure 15B shows that mogroside II E produced upon co-expression of S. grosvenorii UGT1576 (SEQ ID NO:47, SEQ ID NO:48) and UGT430 (SEQ ID NO:61 , SEQ ID NO:62) in an S. cerevisiae strain that was fed mogrol was converted to mogroside V by co-expression of the multifunctional UGT98 (SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53).
  • the intensity of the mogroside V peak in Figure 15B was measured to be 8.65E3 (peak ion intensity in an LC-MS chromatogram).
  • Co-expression of S, grosvenorii UGT1576 (SEQ ID NO:47, SEQ ID NO:48), UGT430 (SEQ ID NO:61 , SEQ ID NO:62), UGT98 (SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53) and UGT11789 (SEQ ID NO:70, SEQ ID NO:72) in an S. cerevisiae strain more efficiently converts fed mogrol to mogroside V, as shown in Figure 15C.
  • the intensity of the mogroside V peak in Figure 15C was measured to be 2.22E5 (peak ion intensity in an LC-MS chromatogram).
  • Example 15 Production of mogrol in S. cerevisiae by expression of S. grosvenorii CYP1798
  • CYP1798 was cloned from synthetic DNA to obtain sequence identical to that of S. grosvenorii CYP1798 (SEQ ID NO:5, SEQ ID NO: 74).
  • the nucleotide sequence was codon- optimized for expression in S. cerevisiae (SEQ ID NO:5).
  • the promoter of the endogenous ERG7 gene (SEQ ID NO:55) was disrupted to lower lanosterol synthase expression in an S. cerevisiae strain deleted of the TRP1 gene.
  • SEQ ID NO:55 the promoter of the endogenous ERG7 gene
  • the cucurbitadienol-producing S. cerevisiae strain was transformed with plasmids carrying S. grosvenorii CYP5491 (SEQ ID NO:14, SEQ ID NO:44), S. grosvenorii CYP 798 (SEQ ID NO:5, SEQ ID NO:73, SEQ ID NO:74), and S. grosvenorii epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO:40) and grown in SC medium minus uracil, leucin, histidine, and tryptophan for plasmid maintenance. Cells were grown for 4 days at 30°C with shaking at 140 rpm.
  • LC-MS analyses were performed using a Waters Acquity l-Class UPLC (Waters Corporation, ilford, MA) with Waters Acquity UPLC ®BEH C18 column (2.1 x 50 mm, 1.7 pm particles, 130 A pore size) coupled to a Waters Xevo TQD triple quadropole mass spectrometer with electrospray ionization (ESI) in negative mode.
  • a Waters Acquity l-Class UPLC Waters Corporation, ilford, MA
  • Waters Acquity UPLC ®BEH C18 column 2.1 x 50 mm, 1.7 pm particles, 130 A pore size
  • Compound separation was achieved by a gradient of the two mobile phases A (water with 0.1 % formic acid) and B (MeCN with 0.1 % formic acid) by increasing from 20% to 40% B between 0.3 to 3.5 min, increasing to 100% B within 1 .0 min, holding 100% B for 1 .0 min, and re-equilibrating for another 0.6 min.
  • the flow rate was 0.6 mL/min, and the column temperature 55°C.
  • Mogrol m/z 521.4; [ +FA- Hj " ) was monitored using SIR (Single Ion Recording) and compared with a standard.
  • CYP1798 catalyzes the epoxidation of the 24-25 carbon double bonds of cucurbitadienol and/or 11 -hydroxy-cucurbitadienol.
  • Mogroside V was produced in an EXG1 (SEQ ID NO:63, SEQ ID NO:64) knockout, Mat alpha derivative of S. cerevisiae S288C. S. grosvenorii cucurbitadienol synthase (SEQ ID NO:42, SEQ ID NO:43), CYP5491 (SEQ ID NO:81 , SEQ ID NO:44), CYP1798 (SEQ ID NO:5, SEQ ID NO:74), CYP1798-II (SEQ ID NO:86, SEQ ID NO:74), CPR4497 (SEQ ID NO:82, SEQ ID NO:46), epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO;40), UGT1576 (SEQ ID NO:83, SEQ ID NO-.48), UGT430 (SEQ ID NO:84, SEQ ID NO:62), UGT1697 (SEQ ID NO:85, SEQ ID NO:68), UGT98 (SEQ ID NO:52
  • Codon-optimized S. grosvenorii cucurbitadienol synthase (SEQ ID NO:42, SEQ ID NO:43), CYP1798 (SEQ ID NO:5, SEQ ID NO:74), CPR4497 (SEQ ID NO:81 , SEQ ID NO:46), and UGT98 (SEQ ID NO:52, SEQ ID NO:53) were synthesized by Genscript.
  • Codon-optimized CYP5491 (SEQ ID NO:81 , SEQ ID NO:44), UGT1576 (SEQ ID NO:83, SEQ ID NO:48), UGT430 (SEQ ID NO:84, SEQ ID NO:62), and UGT11789 (SEQ ID NO:71 , SEQ ID NO:72) were synthesized as S. cerevisiae gBlocks ® gene fragments (Integrated DNA Technologies).
  • Codon-optimized CYP1798-II (SEQ ID NO:86, SEQ ID N0.74) and UGT1697 (SEQ ID NO:85, SEQ ID NO:68) and native CPR4497 (SEQ ID NO:45, SEQ ID NO:46) were synthesized as GeneArt® StringsTM DNA Fragments (Life Technologies). Codon-optimized epoxide hydrolase 1 (SEQ ID NO:37, SEQ ID NO:38) and epoxide hydroase 2 (SEQ ID NO:39, SEQ ID NO:40) were synthesized by DNA2.0.
  • the system was equipped with a Synergi 4u Hydro RP 80A column (Phenomenex: column dimension 250 x 21.2 mm, 4 micron). Elution was carried out using a mobile phase of eluent B (Acetonitrile with 0.02% trifluoroacetic acid) and eluent A (water with 0.02 % trifluoroacetic acid) by increasing the gradient linearly from 5% to 8% B from min 0.0 to 2.0, increasing linearly from 8% to 25% B from min 2.0 to 12.0, 25% to 50% B from min 12.0 to 20.0, 50% to 100 % B from min 20.0 to 32.0, and finally washing with 100% B and re-equilibrating. A flow rate of 15 mL/min was used for the separation, which was conducted at room temperature. All fractions were analyzed by LC-MS, and fractions comprising a single mogroside compound were pooled and dried under vacuum.
  • eluent B Alcohol with 0.02% trifluoroacetic acid
  • Figure 18A shows an NMR-elucidated structure, 1 H NMR spectrum, and 1 H and 13 C NMR chemical shifts (in ppm) for mogroside V.
  • Figure 18B shows an NMR-elucidated structure, 1 H NMR spectrum, and 1 H and 13 C NMR chemical shifts (in ppm) for mogroside II A2.
  • Figure 18C shows an NMR-eiucidated structure, 1 H NMR spectrum, and 1 H and 13 C N R chemical shifts (in ppm) for mogroside IV A.
  • Figure 18D shows shows an NMR-eiucidated structure, 1 H NMR spectrum, and 1 H chemical shifts (in ppm) for mogroside I E1.
  • Table 3 Sequences disclosed herein (see also Table 2).
  • Trp Phe Gly lie Lys Gly Leu Val Ala Ala Gly Arg Thr Tyr Asn Asn
  • Gly Asp Phe Pro Gin Gin Glu lie Met Gly Val Phe Asn Lys Asn Cys 210 215 220
  • gatccacttg catgggagga tccatgtgac tttcgaccgg agagatttct gacaactcat 1320 aaggatttcg atcttagagg acatagtcct caattgatac catttgggag tggtcgaaga
  • gaggagctag ataacgtggt tgggagtaca cgcgccgtcg cggaatccga cattccgtcg 1140

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Abstract

Methods for recombinant and enzymatic production of mogroside compounds and compositions containing mogroside compounds are provided by this invention.

Description

METHODS AND MATERIALS FOR BIOSYNTHESIS OF MOGROSIDE COMPOUNDS
BACKGROUND OF THE INVENTION
Field of Invention
[0001] The present invention relates to methods and materials for biosynthesis of mogroi precursors, mogroi, and/or mogrosides. More particularly, the present invention relates to methods of using of cucurbitadienol synthase, cytochrome P450, cytochrome P450 reductase, and/or epoxide hydrolase enzymes to produce mogroi precursors and/or mogroi. The present invention also relates to methods of using of uridine-5'-diphospho (UDP) dependent glucosyltransferase (UGT) enzymes to glycosylate mogroi and produce various mogrosides.
Description of Related Art
[0002] Mogrosides are a family of triterpene glycosides isolated from fruit of Siraitia grosvenorii (S. grosvenorii, Swingle), also known as Momordica grosvenori. Fruit extracts are commercially used as natural sweeteners. Four major compounds, mogroside V, mogroside IV, siamenoside I, and 11-oxomogroside V (see Figure 1 ) have been identified from S. grosvenorii as being responsible for the fruit's sweetness. Mogroside V is the most abundant of these four compounds, at approximately 0.57% (w/w) of the dry fruit, followed by mogroside IV and siamenoside I, each of which contains four glucose moieties. 11 -oxomogroside V has a ketone group instead of a hydroxyl at C11. See, e.g., Takemoto et al., 1983, Yakugaku Zasshi 103: 1151-4; 1 155-66; 1167-73; Kasai et al., 1989, Agric. Biol. Chem. 53:3347-9; Matsumoto Chem. Pharm. Bull., 1990, 38:2030-2; and Prakash er a/., 2011 , J. Carbohydrate Chem. 30:16-26.
[0003] All mogrosides share the same mogroi triterpene core. The aglycone mogroi is glycosylated with different numbers of glucose moieties to form various mogroside compounds. Mogrosides can be synthesized in the following manner: synthesis of cucurbitadienol from the common triterpene precursor oxidosqualene, oxidation of cucurbitadienol to produce mogroi, and giycosylation of mogroi to produce various mogrosides. See, Tang et al., BMC Genomics 12: 343 (2011 ). Tang ef al., 201 1 , BMC Genomics 12:343 describes seven cytochrome P450s and five UGTs as potential candidates involved in mogroside biosynthesis. However, Tang et al. does not specifically identify any cytochrome P450s or UGTs involved in mogroside biosynthesis. Thus, there remains the need to identify cytochrome P450s and UGTs capable of acting on any S. grosvenorii metabolites. Additionally, although mogrosides can be extracted from S. grosvenorii, there remains a need for improved production of mogrosides in recombinant hosts for commercial uses.
SUMMARY OF THE INVENTION
[0004] It is against the above background that the present invention provides certain advantages and advancements over the prior art.
[0005] The present invention provides methods and materials for biosynthesis of mogroside compounds and provides enzymes involved in mogroside biosynthesis.
[0006] Although the invention disclosed herein is not limited to specific advantages or functionalities, the invention provides a recombinant host comprising one or more of:
(a) a gene encoding a squalene epoxidase polypeptide;
(b) a gene encoding a cucurbitadienol synthase polypeptide;
(c) a gene encoding a cytochrome P450 polypeptide;
(d) a gene encoding a cytochrome P450 reductase polypeptide;
(e) a gene encoding an epoxide hydrolase polypeptide;
(f) a gene encoding a UGT1576 polypeptide having 60% or greater identity to an amino acid sequence set forth in SEQ ID NO:48;
(g) a gene encoding a UGT430 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:62;
(h) a gene encoding a UGT1697 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:68;
(i) a gene encoding a UGT11789 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:72;
(j) a gene encoding a UGT98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:53;
(k) a gene encoding a UGTSK98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:50;
wherein at least one of the genes is a recombinant gene; wherein the host is capable of producing a mogrol precursor, a mogroside precursor, and/or a mogroside compound.
[0007] In some aspects of the recombinant host disclosed herein:
(a) the squalene epoxidase polypeptide comprises a polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:54;
(b) the cucurbitadienol synthase polypeptide comprises a polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:43;
(c) the cytochrome P450 polypeptide comprises a CYP5491 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:44 and/or a CYP1798 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID
NO:74;
(d) the cytochrome P450 reductase polypeptide comprises a CPR4497 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:46; and/or
(e) the epoxide hydrolase polypeptide comprises an epoxide hydrolase 1 polypeptide having 75% or greater identity to an amino acid sequence set forth in SEQ ID NO:38 or an epoxide hydrolase 2 polypeptide having 65% or greater identity to an amino acid sequence set forth in SEQ ID NO:40.
[0008] The invention further provides a recombinant host comprising one or more of:
(a) one or more genes encoding one or more enzymes capable of catalyzing conversion of dioxidosqualene to produce 24,25 epoxy cucurbitadienol;
(b) one or more genes encoding one or more enzymes capable of catalyzing conversion of oxidosqualene to produce cucurbitadienol;
(c) one or more genes encoding one or more enzymes capable of catalyzing hydroxylation of 24,25 epoxy cucurbitadienol to produce 1 1-hydroxy-24,25 epoxy cucurbitadienol;
(d) one or more genes encoding one or more enzymes capable of catalyzing hydroxylation of cucurbitadienol to produce 11-hydroxy-cucurbitadienol;
(e) one or more genes encoding one or more enzymes capable of catalyzing epoxidation of cucurbitadienol to produce 24,25 epoxy cucurbitadienol; or (f) one or more genes encoding one or more enzymes capable of catalyzing epoxidation of 11 -hydroxy-cucurbitadieno! to produce 11-hydroxy-24,25 epoxy cucurbitadienol;
(g) one or more genes encoding one or more enzymes capable of catalyzing conversion of H-hydroxy-24,25 epoxy cucurbitadienol to produce mogrol; or
(h) one or more genes encoding one or more enzymes capable of catalyzing glycosylation of a mogroside precursor to produce a mogroside compound;
wherein at least one of the genes is a recombinant gene.
[0009] In one aspect of the recombinant hosts disclosed herein, the recombinant host further comprises a gene encoding squalene epoxidase polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO: 54.
[0010] In one aspect of the recombinant hosts disclosed herein, the recombinant host has been modified to reduce expression of a lanosterol synthase (ERG7) polypeptide.
[0011] In one aspect of the recombinant hosts disclosed herein, the ERG7 polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:55.
[0012] The invention further provides a method of producing a mogroside precursor and/or a mogroside compound, comprising:
(a) growing the recombinant host disclosed herein in a culture medium, under conditions in which the genes disclosed herein are expressed;
wherein the mogroside precursor and/or the mogroside compound is synthesized by the recombinant host; and
(b) optionally isolating the mogroside precursor and/or the mogroside compound.
[0013] In some aspects of the methods disclosed herein, the mogroside precursor is mogrol synthesized by epoxidation of 1 1-hydroxy-cucurbitadienol to synthesize 1 1-hydroxy- 24,25 epoxy cucurbitadienol and hydrolysis of 11-hydroxy-24,25 epoxy cucurbitadienol to synthesize mogrol.
[00 4] In some aspects of the methods disclosed herein, the epoxidation of 1 1-hydroxy- cucurbitadienol to synthesize 11-hydroxy-24,25 epoxy cucurbitadienol is catalyzed by the CYP1798 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:74. [0015] The invention further provides a method of producing a mogroi precursor in vitro, comprising:
(a) contacting dioxidosqualene with one or more enzymes capable of catalyzing conversion of dioxidosqualene to produce 24,25 epoxy cucurbitadienoi; or
(b) contacting oxidosqualene with one or more enzymes capable of catalyzing conversion of oxidosqualene to produce cucurbitadienoi; or
(c) contacting 24,25 epoxy cucurbitadienoi with one or more enzymes capable of catalyzing hydroxylation of 24,25 epoxy cucurbitadienoi to produce 11 -hydroxy-24,25 epoxy cucurbitadienoi; or
(d) contacting cucurbitadienoi with one or more enzymes capable of catalyzing hydroxylation of cucurbitadienoi to produce 1 1-hydroxy-cucurbitadienol; or
(e) contacting cucurbitadienoi with one or more enzymes capable of catalyzing epoxidation of cucurbitadienoi to produce 24,25 epoxy cucurbitadienoi; or
(f) contacting 11-hydroxy-cucurbitadienol with one or more enzymes capable of catalyzing epoxidation of 11-hydroxy-cucurbitadieno! to produce 11-hydroxy-24,25 epoxy cucurbitadienoi.
[0016] The invention further provides a method of producing a mogroi in vitro, comprising contacting 11-hydroxy-24,25 epoxy cucurbitadienoi with one or more enzymes capable of catalyzing conversion of 11-hydroxy-24,25 epoxy cucurbitadienoi to produce mogroi.
[0017] The invention further provides a method of producing a mogroside compound in vitro, comprising contacting a mogroside precursor with one or more enzymes capable of catalyzing glycosylation of the mogroside precursor to produce a mogroside compound.
[0018] In one aspect of the methods disclosed herein, the method further comprises isolating the mogroi precursor, mogroi or the mogroside compound.
[0019] In some aspects of the recombinant hosts and methods disclosed herein:
(a) the one or more enzymes capable of catalyzing conversion of dioxidosqualene to produce 24,25 epoxy cucurbitadienoi comprise a cucurbitadienoi synthase having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:43; (b) the one or more enzymes capable of catalyzing conversion of oxidosqualene to produce cucurbitadieno! comprise a cucurbitadienol synthase having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:43;
(c) the one or more enzymes capable of catalyzing conversion of 24,25 epoxy cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol comprise CYP5491 having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:44;
(d) the one or more enzymes capable of catalyzing conversion of cucurbitadienol to produce 11 -hydroxy-cucurbitadienol comprise CYP5491 having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:44;
(e) the one or more enzymes capable of catalyzing epoxidation of cucurbitadienol to produce 24,25 epoxy cucurbitadieno! comprise CYP1798 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO: 74;
(f) the one or more enzymes capable of catalyzing epoxidation of 11 -hydroxy- cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol comprise CYP1798 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:74;
(g) the one or more enzymes capable of catalyzing conversion of 11-hydroxy-24,25 epoxy cucurbitadienol to produce mogrol comprise a polypeptide comprising epoxide hydrolase 1 having 75% or greater identity to an amino acid sequence set forth in SEQ ID NO:38 or epoxide hydrolase 2 having 65% or greater identity to an amino acid sequence set forth in SEQ ID NO:40; and/or
(h) the one or more enzymes capable of catalyzing conversion of the mogroside precursor to a mogroside compound comprise UGT1576 having 60% or greater identity to an amino acid sequence set forth in SEQ ID NO:48; UGT98 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:53; UGTSK98 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:50; UGT430 having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:62; UGT1697 having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:68; or UGT11789 having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:72.
[0020] The invention further provides a method of producing a mogroside compound, comprising contacting a recombinant host expressing one or more of: (a) a UGT1576 polypeptide having 60% or greater identity to an amino acid sequence set forth in SEQ ID N0.48;
(b) a UGT430 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:62;
(c) a UGT1697 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:68;
(d) a UGT11789 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:72;
(e) a UGT98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:53; or
(f) a UGTSK98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:50
with a mogroside precursor.
[0021] In one aspect of the methods disclosed herein, the mogroside precursor is plant- derived or synthetic.
[0022] In one aspect of the methods disclosed herein, the method further comprises isolating the mogroside compound.
[0023] In some aspects of the recombinant hosts and methods disclosed herein, the mogroside compound is:
(a) mogrol glycosylated at C3 position; or
(b) mogrol glycosylated at C24 position; or
(c) mogrol glycosylated at C3 position and C24 position.
[0024] In some aspects of the recombinant hosts and methods disclosed herein, the mogroside compound is one or more of mogroside I A1 , mogroside I E1 , mogroside II A, mogroside II A1 , mogroside II A2, mogroside II E, mogroside ill A1 , mogroside III A2, mogroside III, mogroside III E, mogroside IV, mogroside IV A, mogroside V or siamenoside.
[0025] In some aspects of the recombinant hosts and methods disclosed herein, the mogrol precursor is one or more of squalene, dioxidosqualene, oxidosqualene, 24,25 epoxy cucurbitadienol, cucurbitadienol, 11-hydroxy-cucurbitadienol, 11 -hydroxy 24, 25 epoxy cucurbitadienol or 11-oxo-mogrol. [0026] In some aspects of the recombinant hosts and methods disclosed herein, the mogroside precursor is one or more of mogroS, glycosylated mogrol, di-glycosylated mogrol or tri-glycosylated mogrol.
[0027] In some aspects of the recombinant hosts and methods disclosed herein, the recombinant host comprises a microorganism that is a yeast cell, a plant cell, a mammalian cell, an insect cell, a fungal cell, or a bacterial cell.
[0028] In some aspects of the recombinant hosts and methods disclosed herein, the bacterial cell comprises Escherichia bacteria celis, Lactobacillus bacteria cells, Lactococcus bacteria cells, Cornebacterium bacteria cells, Acetobacter bacteria celis, Acinetobacter bacteria cells, or Pseudomonas bacterial cells.
[0029] In some aspects of the recombinant hosts and methods disclosed herein, the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces iactis, Hansenula polymorpha, Candida boidinii, Arxuia adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species.
[0030] In some aspects of the recombinant hosts and methods disclosed herein, the yeast cell is a Saccharomycete.
[0031] In some aspects of the recombinant hosts and methods disclosed herein, the yeast cell is a cell from the Saccharomyces cerevisiae species.
[0032] In some aspects of the recombinant hosts disclosed herein, one or more of the genes further comprise a nucleotide sequence coding a fusion tag.
[0033] In one aspect of the recombinant hosts disclosed herein, the fusion tag is a protein or polypeptide.
[0034] In one aspect of the recombinant hosts disclosed herein, the fusion tag is green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), a polyhistidine-tag (HIS tag), and a FLAG-tag, a chloroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, a signal peptide, or a secretion tag.
[0035] In one aspect of the recombinant hosts disclosed herein, one or more of the genes are expressed as fusion proteins.
[0036] The invention further provides a mogroside composition produced by the recombinant host or the methods disclosed herein, wherein the composition comprises one or more of mogroside I A1 , mogroside I E1, mogroside II A, mogroside II E, mogroside III A1 , mogroside II! A2, mogroside III, mogroside III E, mogroside IV, mogroside V, and siamenoside.
[0037] The invention further provides a food or drink product comprising the mogroside composition disclosed herein.
[0038] These and other features and advantages of the present invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:
[0040] Figure 1 shows chemical structures of mogroside V, mogroside IV, siamenoside I, and 11 -oxomogroside V.
[0041] Figure 2A is a schematic diagram of a pathway for producing mogrosides from glucose.
[0042] Figure 2B shows a pathway for production of mogro! precursors, mogro!, and mogrosides. Figure 2B shows production of cucurbitadienol from oxidosqualene using a cucurbitadienol synthase (step A), production of 24,25 epoxy cucurbitadienol from dioxidosqualene using a cucurbitadienol synthase (step B), production of 1 1-hydroxy- cucurbitadienol from cucurbitadienol using a cytochrome P450 (step C), production of 11- hydroxy 24,25 epoxy cucurbitadienol from 24,25 epoxy cucurbitadienol using a cytochrome P450 (step D), production of 24,25 epoxy cucurbitadienol from cucurbitadienol using a cytochrome P450 (step E), production of 11 -hydroxy 24,25 epoxy cucurbitadienol from 1 1- hydroxy-cucurbitadienoi using a cytochrome P450 (step F), production of mogrol from 11- hydroxy 24,25 epoxy cucurbitadienol from using an epoxide hydrolase (step G), production of mogrol from 1 1-hydroxy-cucurbitadienol using a cytochrome P450 and an epoxide hydrolase (steps F and G), and production of one or more mogroside compounds using one or more UGTs (step H). [0043] Figure 2C shows representative enzymes capable of catalyzing the reactions of steps A-H in Figure 2B. Figure 2C shows production of cucurbitadienol from oxidosqualene using an S. grosvenorii cucurbitadienol synthase of SEQ ID NO:43 (step A), production of 24,25 epoxy cucurbitadienol from dioxidosqualene using an S. grosvenorii cucurbitadienol synthase of SEQ ID NO:43 (step B), production of 1 1-hydroxy-cucurbitadienol from cucurbitadienol using CYP5491 of SEQ ID NO:44 (step C), production 11 -hydroxy 24,25 epoxy cucurbitadienol from 24,25 epoxy cucurbitadienol using CYP5491 of SEQ ID NO:44 (step D), production of 24,25 epoxy cucurbitadienol from cucurbitadienol using CYP1798 of SEQ ID NO:74 (step E), production of 11 -hydroxy 24,25 epoxy cucurbitadienol from 11-hydroxy-cucurbitadienol using CYP1798 of SEQ ID NO:74 (step F), production of mogrol from 11 -hydroxy 24,25 epoxy cucurbitadienol from using epoxide hydrolase 1 of SEQ ID NO:38 or epoxide hydrolase 2 of SEQ ID NO:40 (step G), production of mogrol from 11-hydroxy-cucurbitadienol using CYP1798 of SEQ ID NO:74 and epoxide hydrolase 1 of SEQ ID NO:38 or epoxide hydrolase 2 of SEQ ID NO:40 (steps F and G), and production of mogroside compounds using UGT1576 of SEQ ID NO:48, UGT430 of SEQ ID NO:62, UGT1697 of SEQ ID NO:68, UGT98 of SEQ ID NO:53, and/or UGT11789 of SEQ ID NO:72 (step H).
[0044] Figure 3A shows a representative pathway for production of mogrol from cucurbitadienol, as disclosed herein. Figure 3B is a schematic diagram of a pathway for production of mogrol from cucurbitadienol, as proposed in Tang et a/., 2011 , BMC Genomics 12:343.
[0045] Figure 4 is schematic diagram of pathways for the biosynthesis of mogroside I E1 , mogroside I A1 , mogroside II E, mogroside III A2, mogroside III, mogroside IV, and mogroside V from mogrol using UGTs. UGTa of Figure 4 can be, for example, UGT1576 (SEQ ID NO:48) or UGT1697 (SEQ ID NO:68). UGTb of Figure 4 can be, for example, UGT430 (SEQ ID NO:62) or UGT1697 (SEQ ID NO:68). UGTc of Figure 4 can be, for example, UGT430 (SEQ ID NO:62) or UGT1697 (SEQ ID NO:68). UGTd of Figure 4 can be, for example, UGT1576 (SEQ ID NO:48) or UGT1697 (SEQ ID NO:68). UGTe of Figure 4 can be, for example, UGT98 (SEQ ID NO:53) or UGT11789 (SEQ ID NO:72). UGTf of Figure 4 can be, for example, UGT98 (SEQ ID NO:53) or UGT11789 (SEQ ID NO:72). UGTg of Figure 4 can be, for example, UGT98 (SEQ ID NO:53) or UGT11789 (SEQ ID NO;72).
[0046] Figure 5 is a schematic diagram showing enzymatic production of mogroside IV A, mogroside III, mogroside I E1 , mogroside I A1 , mogroside II E, mogroside II A1 , and mogrol from mogroside V. [0047] Figure 6 shows the LC-MS mass peak 501 corresponding to the proton pius Na+ adduct of tetrahydroxysqualene in a sample from a yeast strain transformed with a piasmid expressing S. grosvenorii epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO:40), as described in Example 8.
[0048] Figure 7 A show an LC-MS chromatogram indicating lanosterol production in a yeast strain that does not express a cucurbitadienol synthase. Figure 7B shows an LC-MS chromatogram indicating cucurbitadienol and lanosterol production in a yeast strain expressing cucurbitadienol synthase (SEQ ID NO:42, SEQ ID NO:43), as described in Example 9.
[0049] Figure 8 shows an LC-MS chromatogram with three peaks resulting upon expression of CYP5491 (SEQ ID NO:14, SEQ ID NO:44) and CPR4497 (SEQ ID NO:45, SEQ ID NO:46) in yeast (upper panel), as described in Example 10; the three lower panels show the fragmentation spectrum of these three peaks. The masses of the 3 peaks (443.38, 441.37 and 457.36) correspond in weight to proton adducts of hydroxylated cucurbitadienol, oxo cucurbitadienol, and hydroxy plus oxo cucurbitadienol, respectively.
[0050] Figures 9A and 9B show biosynthetic routes from cucurbitadienol to mogrol and 1- oxo-mogrol with S. grosvenorii CYP5491 (SEQ ID NO: 14, SEQ ID NO:44), S. grosvenorii CYP1798 (SEQ ID NO:5, SEQ ID NO:73, SEQ ID NO:74), and S. grosvenorii epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO:40). Figure 9C shows a potential biosynthetic route from oxidosqualene to mogrol and 11 -oxo-mogrol with S. cerevisiae squalene epoxidase ERG1 (SEQ ID NO:54), S. grosvenorii CYP1798 (SEQ ID NO:5, SEQ ID NO:73, SEQ ID NO:74), S. grosvenorii cucurbitadienol synthase (SEQ ID NO:42, SEQ ID NO:43), S. grosvenorii CYP5491 (SEQ ID NO:14, SEQ ID NO:44), and S. grosvenorii epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO:40). See Examples 9 and 15.
[0051] Figure 10A shows an LC-MS chromatogram of reference mogroside I A1. Figure 10B shows an LC-MS chromatogram of a sample of yeast strain expressing UGT1576 (SEQ ID NO:47, SEQ ID NO:48) in a culture fed 50 μΜ mogrol, as described in Example 1 1.
[0052] Figure 11A shows LC-MS chromatograms of samples from a yeast strain co- expressing UGT SK98 with UGT1576 and shows production of di-glycosylated mogrol (mogroside II A) as described in Example 11. Figure 11 B shows LC-MS chromatograms of samples from a yeast strain co-expressing UGT98 with UGT1576 and shows production of di- and tri-g!ycosyiated mogrol (middle and lower frames), as described in Example 11. [0053] Figure 12 shows a biosynthetic route from mogroi to mogroside ill A1 provided herein, as described in Example 11.
[0054] Figure 13A shows elution of a mogroside I E1 standard. Figure 13B shows mogroside I E1 produced by UGT430 (SEQ ID NO:61 , SEQ ID NO:62), as described in Example 12.
[0055] Figure 14A shows elution of mogroside II E1 , mogroside II A, mogroside I E1 , and mogroside I A1 standards. Figure 14B shows mogroside I A1 , mogroside If A, and mogroside II E1 produced by UGT1697 (SEQ ID NO:67, SEQ ID NO:68), as described in Example 13.
[0056] Figure 15A shows elution of reference compounds mogroside V (top panel) and mogroside II E (bottom panel). Figure 15B shows production of mogroside V (top panel) and mogroside II E (bottom panel) in a yeast cell co-expressing UGT1576, UGT430, and UGT98. Figure 15C shows production of mogroside V (top panel) and mogroside II E (bottom panel) in a yeast cell co-expressing UGT 576, UGT430, UGT98, and UGT11789, as described in Example 14. Figure 15D shows production of a tri-glycosylated mogroside in a yeast cell co-expressing UGT1576, UGT430, and UGT11789, as described in Example 14.
[0057] Figure 16A shows elution of a mogroi standard. Figure 16B shows mogroi produced in a cucurbitadienol-producing host expressing CYP5491 (SEQ ID NO:14, SEQ ID NO:44), CPR4497 (SEQ ID NO:45, SEQ ID NO:46), CYP1798 (SEQ ID NO:5, SEQ ID NO:73, SEQ ID NO:74), and an epoxide hydrolase, as described in Example 15.
[0058] Figure 17 shows a representative LC- S chromatogram of a crude isolate of a mogroside V-producing S. cerevisiae strain, as described in Example 16.
[0059] Figures 18A, 18B, and 18C show an NMR-elucidated structure, 1H NMR spectrum, and 1H and 13C NMR chemical shifts (in ppm) for mogroside V, mogroside II A2, and mogroside IV A, respectively, as described in Example 16. Figure 18D shows an NMR-elucidated structure, 1H NMR spectrum, and 1H NMR chemical shifts (in ppm) for mogroside I E1 , as described in Example 16.
DETAILED DESCRIPTION OF THE INVENTION
[0060] Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a "nucleic acid" means one or more nucleic acids.
[0061] It is noted that terms like "preferably," "commonly," and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
[0062] For the purposes of describing and defining the present invention it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
[0063] As used herein, the terms "polynucleotide," "nucleotide," "oligonucleotide," and "nucleic acid" can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof.
[0064] As used herein, the term "and/or" is utilized to describe multiple components in combination or exclusive of one another. For example, "x, y, and/or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x and (y or z)," or "x or y or z." In some embodiments, "and/or" is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group. In some embodiments, "and/or" is used to refer to production of mogrosides, wherein one or more mogrosides is produced. In some embodiments, "and/or" is used to refer to production of mogrosides, wherein one or more mogrosides is produced through one or more of the following steps: culturing a recombinant microorganism, synthesizing one or more mogrosides in a recombinant microorganism, and isolating one or more mogrosides.
Mogrosides and Mogroside Production
[0065] As used herein, the terms "mogroside" and "mogroside compound" can be used interchangeably to describe mogrol glycosylated at one or more positions. In particular, a mogroside compound can be mogrol glycosylated with one or more glucose moieties at the positions 1 , 3, 1 1 , 24, and 25. Mogrol is a compound of formula I provided below, wherein both
Figure imgf000014_0001
[0066] Mogrosides can be of the following formula I:
Figure imgf000015_0001
[0067] wherein Ri and R2 independently are -H, mono-glucoside, di-glucoside, tri- glucoside, and wherein at least one of and R2 is not -H. In particular, the mogroside can be one of the mogrosides described in Table 1. In Table 1 , "Glc" represents glucose, and the 1 ,6- and ,2-bonds are indicated. For example, the R2 group of mogroside V comprises 3 glucose molecules linked by one 1 ,6-bond and one 1 ,2-bond, a conformation represented as "Glc6- Glc2-Glc-". See Figure 1 for the structures of mogroside IV, mogroside V, 11-oxo-mogroside V, and siamenoside.
0068] Table 1. Mogrosides of formula i. (Glc=g!ucose)
Figure imgf000015_0002
Figure imgf000016_0001
mogroside I E1 (mogroside la) Glc- H
[0069] Mogrosides can be produced from a number of mogroside precursors. In some embodiments, a mogroside precursor is mogrol, glycosylated mogrol, di-glycosylated mogrol or tri-giycosylated mogrol. Mogrol precursors, in turn, include squalene, dioxidosqualene, oxidosqualene, 24,25 epoxy cucurbitadienol, cucurbitadienol, 11 -hydroxy-cucurbitadienol, 1 1- hydroxy 24, 25 epoxy cucurbitadienol, 11-oxo-mogrol. See, e.g., Figures 2 and 9. For example, mogroside I A1 is a precursor to the products, mogroside II A and mogroside III A1. See, Figure 12. In another example, mogroside II E is converted to mogroside V by three enzymatic glycosylations. In one possible route, two glucose moieties are first attached through 1 ,6-bonds to the two glucose molecules of mogroside II E by a UGT not limited to UGT98 (SEQ ID NO:53) or UGT11789 (SEQ ID NO:72). A third glucose moiety is added to the C24-bound glucose moiety with a 1 ,2 bond by a UGT not limited to UGT98 (SEQ ID NO:53) or UGT11789 (SEQ ID NO:72). See, Figure 4.
[0070] A pathway from cucurbitadienol to mogrol was proposed by Tang et a/., 2011 , BMC Genomics 12:343. The precursors, cucurbitadienol and mogrol, have been isolated from S. grosvenorii. See Ukiya, et a!., 2002, J. Agric. Food Chem. 50: 6710-5. Glycoside intermediates exist in both 11 -hydroxy and 11-oxo series and gradually change from mogroside I to mogroside V as fruits ripen, indicating that P450 enzymes fully oxidize the triterpene core of a mogrol precursor, such as cucurbitadienol, prior to subsequent glycosylations. According to the scheme proposed by Tang et a/., three independent cytochrome P450 enzyme-catalyzed oxidations result in mogrol formation from cucurbitadienol (Figure 3B). The proposed primary reaction, however, is unlikely, as saturation of the 24-25 double bond would be required prior to two hydroxylation reactions by cytochrome P450 enzymes. As shown in Figure 3A, epoxidation of cucurbitadienol by one cytochrome P450 enzyme, followed by a spontaneous or enzyme catalyzed hydration, and a second P450 enzyme-catalyzed oxidation can result in production of mogrol. Additional pathways for production of mogrol or 11-oxo-mogrol, as described in Example 1 1 , are shown in Figure 9. [0071] In some embodiments, one or more mogrol precursors are produced. Mogrol precursors, mogrol, and/or mogrosides can be produced in vivo {i.e., in a recombinant host), in vitro (i.e., enzymatica!ly), or by whole cell bioconversion, as described below. As used herein, the terms "detectable amount," "detectable concentration," "measurable amount," and "measurable concentration" refer to a level of mogrosides and mogroside precursors measured in AUC, pM/OD6oo. mg/L, μ , or m . Mogroside production (i.e. , total, supernatant, and/or intracellular steviol glycoside levels) can be detected and/or analyzed by techniques generally available to one skilled in the art, for example, but not limited to, liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), ultraviolet-visible spectroscopy/ spectrophotometry (UV-Vis), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR). As used herein, the term "relative abundance" is used to refer to the concentration of a particular ion measured by MS or LC-MS, where the most intense ion is assigned a relative abundance score of 100 and is referred to as the base peak.
Mogroside Production Pathway
[0072] In some embodiments, a mogrol precursor (e.g., squalene or oxidosqualene), mogrol, or mogroside is produced, as described herein. Squalene can be produced from famesyl pyrophosphate using a squalene synthase, and oxidosqualene can be produced from squalene using a squalene epoxidase. The squalene synthase can be any enzyme classified under EC 2.5.1.21. Squalene production can comprise a step of catalyzing conversion of famesyl pyrophosphate by a squalene synthase in the presence of NADPH. In embodiments of the invention wherein the methods are performed in vivo, the recombinant host can thus comprise a heterologous nucleic acid encoding a squalene synthase. In other aspects, the squalene synthase can be endogenous.
[0073] The squalene synthase can be, for example, squalene synthase from Gynostemma pentaphyllum (protein accession number C4P9M2), a cucurbitaceae family plant. The squalene synthase can also comprise a squalene synthase from Arabidopsis thaliana (protein accession number C4P9M3), Brassica napus, Citrus macrophylla, Euphorbia tirucalli (protein accession number B9WZW7), Glycine max, Glycyrrhiza glabra (protein accession number Q42760, Q42761 ), Glycrrhiza uralensis (protein accession number D6QX40, D6QX41 , D6QX42, D6QX43, D6QX44, D6QX45, D6QX47, D6QX39, D6QX55, D6QX38, D6QX53, D6QX37, D6QX35, B5AID5, B5AID4, B5AID3, C7EDD0, C6KE07, C6KE08, C7EDC9), Lotusjaponicas (protein accession number Q84LE3), Medicago truncatula (protein accessionnumber Q8GSL6), Pisum sativum, Ricinus communis (protein accession number B9RHC3), Prunus mume, or functional homoiogs sharing at least 70% identity with any of the squalene synthases described above.
[0074] Oxidosqualene can be produced from squalene by squalene epoxidase (also referred to as squalene monoxygenase. See, e.g., Leber et a/,, 1998, Mo! Biol Cell. 9(2):375- 86. The squalene epoxidase can be any enzyme classified under EC 1.4.99.7. Oxidosqualene production can comprise a step of catalyzing conversion of squalene by a squalene epoxidase in the presence of NADPH. See, e.g., Example 8.
[0075] The squalene epoxidase can also be the product of the ERG1 gene from S. cerevisiae. Thus, the squalene epoxidase can be a polypeptide of SEQ ID NO:54 or a functional homolog thereof sharing at least 45% sequence identity therewith. In some aspects, ERG1 is overexpressed.
[0076] The squalene epoxidase can be, for example, squalene epoxidase from Gynostemma pentaphyllum (protein accession number C4P9M2; SEQ ID NO: 88). The squalene epoxidase can comprise a squalene epoxidase from Arabidopsis thaliana (protein accession number Q9S 02 (SEQ ID NO: 89), 065403 (SEQ ID NO: 90), 065402 (SEQ ID NO: 91 ), 065404 (SEQ ID NO: 92), 081000 (SEQ ID NO: 93), or Q9T064 (SEQ ID NO: 94)), Brassica napus (protein accession number 065727 (SEQ ID NO: 95), 065726 (SEQ ID NO: 96)), Euphorbia tirucalli (protein accession number A7VJN1 (SEQ ID NO: 97)), Medicago truncatula (protein accession number Q8GSM8 (SEQ ID NO: 98), Q8GSM9 (SEQ ID NO: 99)), Pisum sativum, and Ricinus communis (protein accession number B9R6V0 (SEQ ID NO: 100), B9S7W5 (SEQ ID NO: 101 ), B9S6Y2 (SEQ ID NO: 102), B9T0Y3 (SEQ ID NO: 103), B9S7T0 (SEQ ID NO: 104), B9SX91 (SEQ ID NO: 105)), or functional homologs sharing at least 70% identity with any of the squalene epoxidases described above.
[0077] One or more enzymes capable of catalyzing conversion of oxidosqualene to form cucurbitadienol comprise a cucurbitadienol synthase. See step A of Figures 2B and 2C and Example 9. The cucurbitadienol synthase can be, for example, a cucurbitadienol synthase, which has been classified as an oxidosqualene cyclase, such as the oxidosqualene cyclase described by Shibuya, Tetrahedron, 60: 6995-7003 (2004).
[0078] The amino acid sequence of a cucurbitadienol synthase from Cucurbita pepo is provided herein as SEQ ID NO:1. In some embodiments, the cucurbitadienol synthase is a polypeptide of SEQ ID NO:1 or a functional homolog thereof sharing at least 70% sequence identity therewith, in some embodiments, a polypeptide having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:1 includes, but is not limited to, a polypeptide from Lotus japonicas (BAE53431 ), Populus trichocarpa (XP_002310905), Actaea racemosa (ADC84219), Betula platyphylla (BAB83085), Glycyrrhiza glabra (BAA76902), Vitis vinifera (XP_002264289), Centella asiatica (AAS01524), Panax ginseng (BAA33460), and Betula platyphylla (BAB83086). The cucurbitadienol synthase can be any cucurbitadienol synthase sharing at least 70% identity to a cucurbitadienol synthase described above.
(0079] As described in Example 5, the cucurbitadienol synthase from monk fruit was identified herein, and the sequence of the C-terminal portion of the polypeptide determined. The amino acid sequence of the C-terminal portion of the monk fruit polypeptide is provided herein as SEQ ID NO:2. Thus, in some embodiments, the cucurbitadienol synthase is a polypeptide having an amino acid sequence set forth in SEQ ID NO:2.
[00801 In other embodiments, the cucurbitadienol synthase is the polypeptide of SEQ ID NO:43 or a functional homolog thereof sharing at least 70% identity therewith.
[0081] In some embodiments, 24,25 epoxy cucurbitadienol is produced from dioxidosqualene using one or more enzymes capable of catalyzing conversion of oxidosqualene to form cucurbitadienol. One or more enzymes capable of catalyzing conversion of dioxidosqualene to 24,25 epoxy cucurbitadienol preferably comprises a cucurbitadienol synthase. See step B of Figures 2B and 2C and Example 9. The cucurbitadienol synthase can be, for example, a cucurbitadienol synthase as described by Shibuya, Tetrahedron 60:6995- 7003 (2004) or a cucurbitadienol synthase as described above. In some embodiments, the cucurbitadienol synthase catalyzing conversion of dioxidosqualene to 24,25 epoxy cucurbitadienol is a polypeptide of SEQ ID NO:1 or a functional homolog thereof sharing at least 70% identity therewith.
[0082] In some embodiments, 11-hydroxy-cucurbitadienol is produced from cucurbitadienol. In some embodiments, a cytochrome P450 enzyme catalyzes hydroxylation of cucurbitadienol to form 11 -hydroxy-cucurbitadienol. In some embodiments, CYP5491 (SEQ ID NO: 14, SEQ ID NO:44) catalyzes conversion of cucurbitadienol to 11-hydroxy-cucurbitadienol. See step C of Figures 2B and 2C and Example 10.
[0083] As indicated in Examples 6 and 15, one or more of CYP533, CYP937, CYP1798, CYP 994, CYP2048, CYP2740, CYP3404, CYP3968, CYP4112, CYP4149, CYP4491 , CYP5491 , CYP6479, CYP7604, CYP8224, CYP8728, CYP10020, or CYP10285 (encoded by SEQ ID NOs: 3-20, respectively) can be used to produce mogrol. eYAC technology can be used to assess activity of the cytochrome P450 enzymes, as set forth in Example 8. Alternatively, an in vitro reaction can be used to assess the activity. Thus, in one embodiment of the invention, at least one cytochrome P450 enzyme comprises a polypeptide encoded by the nucleic acid sequence SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO:20 or a functional homolog thereof sharing at least 70% identity therewith.
[0084] In some embodiments, 11-hydroxy-24,25 epoxy cucurbitadienol is produced from 24,25 epoxy cucurbitadienol using one or more enzymes capable of catalyzing hydroxylation of 24,25 epoxy cucurbitadienol to form 1 1-hydroxy-24,25 epoxy cucurbitadienol. In some embodiments, a cytochrome P450 enzyme catalyzes hydroxylation of 24,25 epoxy cucurbitadienol to form 11-hydroxy-24,25 epoxy cucurbitadienol. In some embodiments, the enzyme capable of catalyzing hydroxylation of 24,25 epoxy cucurbitadienol to form 1 1-hydroxy- 24,25 epoxy cucurbitadienol is CYP5491 (SEQ ID NO:14, SEQ ID NO:44) or a functional homolog sharing at least 50% sequence identity with SEQ ID NO:44. See step D of Figures 2B and 2C and Example 9.
[0085] In some aspects, 24,25 epoxy cucurbitadienol is produced from cucurbitadienol. In some aspects, a cytochrome P450 catalyzes conversion of cucurbitadienol to 24,25 epoxy cucurbitadienol. The cytochrome P450 can be CYP1798 of SEQ ID NO:74. See step E of Figures 2B and 2C. In some aspects, 1 -hydroxy 24,25 epoxy cucurbitadienol is produced from 11 -hydroxy-cucurbitadienol. In some aspects, a cytochrome P450 catalyzes conversion of 1 1 - hydroxy-cucurbitadienol to produce 11 -hydroxy 24,25 epoxy cucurbitadienol. The cytochrome P450 can be CYP1798 of SEQ ID NO:74. See step F of Figures 2B and 2C.
[0086] In some aspects, mogrol is produced from 11 -hydroxy-cucurbitadienol using enzymes capable of catalyzing conversion of 11 -hydroxy-cucurbitadienol to form mogrol. Enzymes having cytochrome P450 activity and epoxide hydrolase activity catalyze conversion of 11 -hydroxy-cucurbitadienol to mogrol. See steps F and G of Figures 2B and 2C. Enzymes with cytochrome P450 activity include polypeptides encoded by the nucleic acid sequence set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or a functional homolog thereof sharing at least 70% sequence identity therewith. An enzyme having epoxide hydrolase activity preferably catalyzes production of glycol from epoxide and water. Non-limiting examples of enzymes with epoxide hydrolase activity include S. grosvenorii epoxide hydrolase 1 and S. grosvenorii epoxide hydrolase 2. Thus, an enzyme with epoxide hydrolase activity can comprise polypeptides having at least 75% sequence identity with the amino acid sequence set forth in SEQ ID NO:38, having at least 65% sequence identity with the amino acid sequence set forth in SEQ ID NO:40, and functional homoiogs thereof.
[0087] In some embodiments, mogrol is produced from 1 1-hydroxy-24,25 epoxy cucurbitadienol. One or more enzymes capable of catalyzing conversion of 11-hydroxy-24,25 epoxy cucurbitadienol to form mogrol preferably comprise an enzyme with epoxide hydrolase activity. See step G of Figures 2B and 2C, Examples of enzymes with epoxide hydrolase activity include $. grosvenorii epoxide hydrolase 1 and S. grosvenorii epoxide hydrolase 2, as described above. In some embodiments, an enzyme capable of catalyzing conversion of 1 1- hydroxy-24,25 epoxy cucurbitadienol to produce mogrol comprises a polypeptide having at least 75% sequence identity with the amino acid sequence set forth in SEQ ID NO:38, having at least 65% sequence identity with the amino acid sequence set forth in SEQ ID NO:40, and functional homoiogs thereof.
[0088] In some embodiments, CYP1798 (SEQ ID NO:5, SEQ ID N0.73, SEQ ID NO:74) catalyzes the epoxidation of the 24-25 carbon double bonds of cucurbitadienol, 1 1-hydroxy- cucurbitadienol, or 11-oxo cucurbitadienol. Figures 9A and 9B are schematics of mogrol and 11 -oxo-mogrol production from cucurbitadienol, and Figure 9C is a schematic of mogrol and 11- oxo-mogrol production from oxidosquaiene. See, also, Example 15.
[0089] One or more enzymes capable of catalyzing glycosylation of mogrol preferably comprise a Uridtne-5'-diphospho (UDP) dependent glucosyltransferase (UGT). A UGT can catalyze production of a mogroside not limited to mogroside I A1 , mogroside I E1 , mogroside II A, mogroside II A1 , mogroside II A2, mogroside II E, mogroside III A1 , mogroside III A2, mogroside III, mogroside III E, mogroside IV, mogroside IV A, or siamenoside. Such UGT can comprise, for example, Arabidopsis thaliana UGT73C3 of SEQ ID NO:21 , Arabidopsis thaliana UGT73C6 of SEQ ID NO:23, Stevia rebaudiana UGT85C2 of SEQ ID NO:25, Arabidopsis thaliana UGT73C5 of SEQ ID NO:22, Stevia rebaudiana UGT73E1 of SEQ ID NO:24, or a functional homolog sharing at least 70% identity with a UGT described above. A UGT can also comprise UGT98 of SEQ ID NO:53, UGT1495 encoded by SEQ ID NO:27, UGT1817 encoded by SEQ ID NO:28, UGT5914 encoded by SEQ ID NO:30, UGT8468 encoded by SEQ ID NO:31 , UGT10391 encoded by SEQ ID NO:32, or a functional homolog of any of the UGTs described above. See Examples 4 and 7.
[0090] UGT73C3, UGT73C6, UGT85C2, and UGT73E1 are capable of catalyzing giycosylation at the C24 position of mogrol or mogroside. Accordingly, in methods of the invention wherein the mogroside to be produced comprises a giycosylation at the C24 position, at least one UGT can be UGT73C3 of SEQ ID NO:21 , UGT73C6 of SEQ ID NO:23, UGT85C2 of SEQ ID NO:25, UGT73E1 of SEQ ID NO:24 or a functional homolog functional homolog sharing at least 70% identity with a UGT described above. See Example 4.
[0091] UGT73C5 is capable of catalyzing giycosylation at both the C3-OH of mogrol and mogroside and C24 position. Accordingly, in methods of the invention wherein the mogroside to be produced comprises a giycosylation at the C24 position and/or a giycosylation at the C3-OH position, at ieast one UGT can be UGT73C5 of SEQ ID NO:22 or a functional homolog sharing at Ieast 60% sequence identity therewith. See Example 4.
[0092] In some embodiments, a UGT is UGT1576 of SEQ ID NO:48 or a UGT sharing at Ieast 60% sequence identity with UGT1576 of SEQ ID NO:48. In some embodiments, UGT1576 possesses mogrol C24-OH UDP-glycosyltransferase activity. See Example 11.
[0093] In some embodiments, a UGT is UGT98 of SEQ ID NO:53 or a functional homolog thereof sharing at Ieast 70% sequence identity therewith. This is in particular the case in embodiments of the invention wherein the mogroside to be produced comprises a 1 ,2- glycosylation and a 1 ,6-giycosy!ation of the glucose at position C-24 to form mogroside III A1. See Example 11. In some embodiments, UGT98 (SEQ ID NO:53) can be used to convert mogroside II E to mogroside IV, mogroside V, 11-oxo-mogroside V, and/or siamenoside I. See Example 7.
[0094] In some embodiments, for example in embodiments wherein the mogroside to be produced comprises a 1 ,2 giycosylation of the glucose at position C-24 to form mogroside II A, a UGT is UGTSK98 of SEQ ID NO:50 or UGT sharing at ieast 70% identity with UGTS 98 of SEQ ID NO:50. See Example 11. In some aspects, UGT98 catalyzes 1 ,2 and 1 ,6 glucose attachments to convert mogroside II E to mogroside V. See Example 14.
[0095] In some embodiments, a UGT is S. grosvenorii UGT430 (SEQ ID NO:61 , SEQ ID NO:62). UGT430 is a member of UGT family 85A and glycosylates the 3C position of mogrol and particular mogrosides. See Example 12. [0096] In some embodiments, a UGT is S. grosvenorii UGT1697 (SEQ ID NO:67, SEQ ID NO:68). UGT1697 is a member of UGT family 85A and glycosylates the 3C and 24C positions of mogrol and particular mogrosides. See Example 13.
[0097] In some embodiments, a UGT is S. grosvenorii UGT11789 (SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 , SEQ ID NO:72). UGT11789 catalyzes 1 ,2 and/or 1 ,6 glucose attachments on the 24-O-g!ucose and/or the 3-O-glucose of mogroside compounds. In some embodiments, UGT1 1789 glycosylates mogroside I E1 , mogroside I A1 , mogroside II E, mogroside II A, mogroside III E, mogroside III A2, mogroside III, mogroside IV, or siamenoside. In some embodiments, contacting UGT11789 with mogroside I E1 , mogroside I A1 , mogroside II E, mogroside II A, mogroside III E, mogroside II) A2, mogroside III, mogroside IV, or siamenoside produces mogroside II A1 , mogroside II A2, mogroside III, mogroside III A1 , mogroside ill A2, mogroside IV, mogroside IV A, siamenoside, or mogroside V. See Example 14.
Methods of Producing Mogrosides In Vivo
[0098] In some embodiments, a mogrol precursor, mogrol, or mogroside is produced in vivo by a host expressing of one or more nucleic acid molecules encoding one or more enzymes involved in the mogroside pathway. For example, an oxidosqualene-producing recombinant host expressing one or more of a gene encoding a cucurbitadienol synthase polypeptide, a gene encoding a cytochrome P450 polypeptide, a gene encoding a cytochrome P450 reductase polypeptide, a gene encoding an epoxide hydrolase polypeptide, and a gene encoding a UGT polypeptide can produce a mogrol precursor, mogrol, or mogroside in vivo. See Examples 15 and 16.
[0099] In some embodiments, more than one host is used to produce a mogrol precursor, mogrol, or mogroside. In a non-limiting example, a host capable of producing mogrol and a host expressing a UGT can be used to produce a mogroside. The methods can also employ a mixture of a recombinant and a non-recombinant host. In embodiments comprising use of two or more hosts, the hosts can be co-cultivated or cultured separately. If the hosts are cultivated separately, the intermediate products can be recovered and optionaily purified or partially purified and fed to recombinant hosts using the intermediate products as substrates. Suitable recombinant hosts are described below.
[00100] In some aspects, production of a mogrol precursor, mogrol, or mogroside can be performed in vivo and a mogrol precursor, mogrol, or mogroside product can be used as a substrate for subsequent reactions to be performed in vitro, as described below. See WO 2013/076577 and WO 2014/086842.
[00101] In some embodiments, a host produces oxidosqualene from glucose via the ergosterol pathway. See, e.g., WO 2014/00271 18. In some aspects, host expressing a nucleic acid molecule encoding a squalene synthase polypeptide can produce squalene. In some embodiments, the squalene synthase is ERG9, and the amino acid sequence of ERG9 is set forth in SEQ ID NO:87. In some embodiments, squalene synthase is endogenous to the host. In some embodiments, increased copy numbers of an endogenous squalene synthase and/or squalene epoxidase, expression of a heterologous nucleic acid molecule encoding a squalene synthase and/or squalene epoxidase, or increased expression of an endogenous squalene synthase and/or squalene epoxidase can improve levels of mogrosides produced in a recombinant host.
[00102] In one embodiment, the recombinant host comprises a heterologous nucleic acid encoding a squalene epoxidase operably linked to sequence directing high expression of the squalene epoxidase in the host. Thus, the squalene epoxidase can be endogenous to the recombinant host, but the expression level can be increased by additional copies of nucleic acids encoding the squalene epoxidase and/or by use of stronger promoters.
[00103] Oxidosqualene serves as a substrate for production of lanosterol. Thus, in some embodiments, the level of oxidosqualene can be increased by reducing lanosterol synthase activity. In recombinant hosts expressing an endogenous lanosterol synthase, this can be achieved by substituting the endogenous promoter-directed expression of lanosterol synthase with a weaker promoter directing expression of a lower level of lanosterol synthase. In yeast, the ERG7 gene encodes lanosterol synthase. Thus, when the recombinant host is yeast, the ERG7 gene promoter can be substituted for another promoter, which directs a level of expression, which is lower than the endogenous expression level of ERG7. The lanosterol synthase can thus be the product of the ERG7 gene of S. cerevisiae, the sequence of which is provided herein as SEQ ID NO:55, or a functional homolog thereof sharing at least 50% sequence identity therewith. See Examples 8 and 15.
[00104] In addition, expression of a truncated form of the enzyme 3-hydroxy-3- methylglutaryl-CoA reductase (tHMG1 , SEQ ID NO:77, SEQ ID NO:78) can also lead enhanced levels of oxidosqualene. A useful truncated form of yeast HMG reductase (tHMG1) is described in Donald er a/., 1997, Appl. Environ. Microbiol. 63:3341-4. [00105] Dioxidosqualene ievels can be enhanced by high expression of a squaiene epoxidase. The squaiene epoxidase can be the product of the S. cerevisiae ERG1 gene. Thus, the squaiene epoxidase can be a polypeptide of SEQ ID NO:54 or a functional homolog thereof sharing at least 45% sequence identity therewith. The Ievels of dioxidosqualene can also be enhanced by reducing lanosterol synthase activity. Dioxidosqualene Ievels can also be enhanced by expression of a truncated form of 3-hydroxy-3-methylglutary!-CoA reductase (tHMG1 , SEQ ID N0.77, SEQ ID NO:78). See Examples 8 and 15.
[00106] in some embodiments, hydroxylation of cucurbitadienol to form 11-hydroxy- cucurbitadienol or hydroxylation of 24,25 epoxy cucurbitadienol to form 11-hydroxy-24,25 epoxy cucurbitadienol can be aided by at least one CYP activator. A recombinant host can co-express heterologous nucleic acids encoding one or more cytochrome P450 enzymes and a heterologous nucleic acid encoding a CYP activator. The CYP activator can be, for example, CPR4497 (SEQ ID NO:45, SEQ ID NO:46) or a functional homolog sharing at least 50% sequence identity with SEQ ID NO:46. See Examples 10, 15, and 16.
[00107] In some embodiments, a cucurbitadienol-producing S. cerevisiae strain co- expressing S. grosvenorii CYP5491 (SEQ ID NO:14, SEQ ID NO:44), S. grosvenorii CYP1798 (SEQ ID NO:5, SEQ ID NO:73, SEQ ID NO:74), S. grosvenorii CPR4497 (SEQ ID NO:45, SEQ ID NO:46), and an epoxide hydrolase produces mogrol. In some embodiments, the epoxide hydrolase is epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO:40). In some embodiments, the cucurbitadienol-producing S. cerevisiae strain further overexpresses squaiene epoxidase encoded by ERG1 (SEQ ID NO:54), expresses a truncated HMG reductase (tHMG1 , SEQ ID NO:77, SEQ ID NO:78), expresses S. grosvenorii cucurbitadienol synthase (SEQ ID NO:42, SEQ ID NO:43), is deleted of the TRP1 gene, and comprises a disrupted promoter of the endogenous ERG7 gene (SEQ ID NO:55). See Example 15.
[00108] In some embodiments, a mogrol precursor, mogrol, or mogroside is produced in a recombinant host comprising one or more of a gene encoding a squaiene epoxidase polypeptide, a gene encoding a cucurbitadienol synthase polypeptide, a gene encoding a cytochrome P450 polypeptide, a gene encoding a cytochrome P450 reductase polypeptide, a gene encoding an epoxide hydrolase polypeptide, and/or a gene encoding a glycosyltransferase. In some aspects, the gene encoding the glycosyltransferase comprises a gene encoding a UGT1576 polypeptide having 60% or greater identity to an amino acid sequence set forth in SEQ ID NO:48, a gene encoding a UGT430 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:62, a gene encoding a UGT1697 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:68, a gene encoding a UGT11789 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:72, and/or a gene encoding a UGT98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:53. See Example 16.
[00109] In some embodiments, mogroside V is produced in an S. cerevisiae strain comprising S. grosvenorii cucurbitadienol synthase (SEQ ID NO:42, SEQ ID NO:43), CYP5491 (SEQ ID NO:81 , SEQ ID NO:44), CYP1798 (SEQ ID NO:5, SEQ ID NO:74), CYP1798-II (SEQ ID NO:86, SEQ ID NO:74), CPR4497 (SEQ ID NO:82, SEQ ID NO:46), epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO:40), UGT1576 (SEQ ID NO:83, SEQ ID NO:48), UGT430 (SEQ ID NO:84, SEQ ID NO:62), UGT1697 (SEQ ID NO:85, SEQ ID NO:68), UGT98 (SEQ ID NO:52, SEQ ID NO:53), and UGT11789 (SEQ ID NO:71 , SEQ ID NO:72). In some embodiments, the strain is a Mat alpha derivative of S. cerevisiae 288C with a deletion of the S. cerevisiae EXG1 gene. In some embodiments, the host further produces mogroside IV A, mogroside II A2, mogroside I E1 , and mogrol. See Example 16.
Methods of Producing Mogrosides In vitro
[00110] In some embodiments, a mogroside is produced through contact of a mogrol precursor, mogrol, or glycosylated mogrol with one or more enzymes involved in the mogroside pathway in vitro. For example, contact of mogrol with a UGT polypeptide can result in production of a mogroside in vitro. In some embodiments, a mogrol precursor is produced through contact of an upstream mogroside precursor with one or more enzymes involved in the mogroside pathway in vitro. For example, contact of cucurbitadienol with a cytochrome P450 polypeptide and an epoxide hydrolase can result in production of mogrol in vitro.
[00111] In some embodiments, a mogrol precursor is produced by one or more of the following steps:
a. Contacting oxidosqualene with a cucurbitadienol synthase, such as, but not limited to, a cucurbitadienol synthase having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:43, to produce cucurbitadienol (see step A of Figures 2B and 2C); or
b. Contacting dioxidosqualene with a cucurbitadienol synthase, such as, but not limited to, a cucurbitadienol synthase having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:43, to produce 24,25 epoxy cucurbitadienol (see step B of Figures 2B and 2C); or
c. Contacting cucurbitadienol with a cytochrome P450, such as, but not limited to, CYP5491 having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:44, to produce 11 -hydroxy-cucurbitadienol (see step C of Figures 2B and 2C); or
d. Contacting 24,25 epoxy cucurbitadienol with a cytochrome P450, such as, but not limited to, CYP5491 having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:44, to produce 11-hydroxy-24,25 epoxy cucurbitadienol (see step D of Figures 2B and 2C); or
e. Contacting cucurbitadienol with a cytochrome P450, such as, but not limited to, CYP1798 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:74, to produce 24,25 epoxy cucurbitadienol (see step E of Figures 2B and 2C); or
f. Contacting 1 1 -hydroxy-cucurbitadienol with a cytochrome P450, such as, but not limited to, CYP1 98 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:74, to produce 1 1-hydroxy-24,25 epoxy cucurbitadienol (see step F of Figures 2B and 2C).
[00112] In some embodiments, mogrol is produced in vitro by contacting H -hydroxy-24,25 epoxy cucurbitadienol with an epoxide hydrolase, such as, but not limited to, epoxide hydrolase 1 having 75% or greater identity to an amino acid sequence set forth in SEQ ID NO:38 or epoxide hydrolase 2 having 65% or greater identity to an amino acid sequence set forth in SEQ ID NO:40 (see step G of Figures 2B and 2C).
[00113] In some embodiments, a mogroside (see step H of Figures 2B and 2C) is produced in vitro by:
a. Contacting mogrol with UGT73C3 (SEQ ID NO:21 ), UGT73C6 (SEQ ID NO:23), UGT85C2 (SEQ ID N0.25), and/or UGT1576 (SEQ ID NO:48) to produce mogroside I A1 ; or
b. Contacting mogrol with UGT73C5 (SEQ ID NO:22) to produce mogroside I E1 and/or mogroside I A ; or c. Contacting mogroi with UGT73E1 (SEQ ID NO:24) to produce mogroside 1 A1 and/or a mogroside glycosylated on C11-OH; or
d. Contacting mogroi with UGT430 (SEQ ID NO:62) to produce mogroside I E1 ; or e. Contacting mogroi with UGT1697 (SEQ ID NO:68) to produce mogroside II E1 and/or mogroside I A1 ; or
f. Contacting mogroside I A1 with UGT98 (SEQ ID NO:53), UGTSK98 (SEQ ID NO:50), and/or UGT11789 (SEQ ID NO:72) to produce mogroside II A; or g. Contacting mogroside I A1 with UGT430 (SEQ ID NO:62) to produce mogroside II E; or
h. Contacting mogroside I A1 with UGT98 (SEQ ID NO:53) and/or UGT11789 (SEQ ID NO:72) to produce mogroside III A1 ; or
i. Contacting mogroside I E1 with UGT1576 (SEQ ID N0.48) and/or UGT1697 (SEQ ID NO:68) to produce mogroside II E; or
j. Contacting mogroside II A with UGT98 (SEQ ID NO:53) and/or UGT1 1789 (SEQ ID N0.72) to produce mogroside III A1 ; or
k. Contacting mogroside II E with UGT98 (SEQ ID NO:62) and/or UGT11789 (SEQ ID NO:72) to produce mogroside III A1 , mogroside III A2, mogroside III E, mogroside III, mogroside IV A, mogroside IV, siamenoside, or mogroside V; or
I. Contacting mogroside III A1 with UGT73C5 (SEQ ID NO:22) to produce siamenoside 1 ; or
m. Contacting siamenoside 1 with UGT98 (SEQ ID NO:53) and/or UGT1 1789 (SEQ ID NO:72) to produce mogroside V.
[00114] Each of the steps described above can be performed separately. In embodiments wherein at least two steps are performed separately, a product of a step can be purified or partially purified before performing a subsequent step. Alternatively, one or more of the steps can be performed simultaneously within the same mixture.
[00115] In some embodiments, a cell lysate is prepared from a host expressing one or more of a gene encoding a squalene epoxidase polypeptide, a gene encoding a cucurbitadienol synthase polypeptide, a gene encoding a cytochrome P450 polypeptide, a gene encoding an epoxide hydrolase polypeptide, and a gene encoding a UGT polypeptide. For example, a cell lysate can be prepared from a host expressing one or more UGTs and used to contact mogrol, such that a mogroside can be produced in vitro.
Methods of Producing Mogrosides by Whole Cell Bioconversion
[00116] In some embodiments, a mogrol precursor, mogrol, or mogroside is produced by whole cell bioconversion. For whole cell bioconversion to occur, a host expressing one or more enzymes involved in the mogroside pathway takes up and modifies a mogrol or mogroside precursor in the cell; following modification in vivo, a mogroside is excreted into the culture medium. See Examples 1 1-14.
[00117] In some embodiments, the mogrol precursor is oxidosqualene, dioxidosqualene, cucurbitadienol, 24,25 epoxy cucurbitadienol and the mogroside precursor is mogrol. In a non- limiting example of whole cell bioconversion, a host expressing a gene encoding a UGT polypeptide can take up mogrol and glycosylate mogrol in the cell; following glycosylation in vivo, a mogroside is excreted into the culture medium.
[00118] A cell can be fed a mogrol precursor or mogroside precursor during cell growth or after cell growth. The cell can be in suspension or immobilized. The cell can be in fermentation broth or in a reaction buffer. In some embodiments, a permeabilizing agent is used for transfer of a mogrol precursor or mogroside precursor into a cell. In some embodiments, a mogrol precursor or mogroside precursor can be provided in a purified form or as part of a composition or an extract.
[00119] In some aspects, a mogrol precursor or mogroside precursor is produced in vitro; thereafter, the mogrol precursor or mogroside precursor is provided to a host capable of catalyzing conversion of the mogrol precursor or mogroside precursor.
[00120] In some embodiments, a recombinant host expressing UGT98, UGT1576, and UGT430 converts fed mogrol to mogroside V. See Example 14. In some embodiments, a host expressing UGT1 789 catalyzes the conversion of mogroside II E to a tri-g!ycosylated mogroside. In some embodiments, a host expressing UGT11789, UGT1576, and UGT430 catalyzes the conversion of mogrol to a trig!ycosy!ated mogroside. In some embodiments, a recombinant host co-expressing UGT11789, UGT98, UGT1576, and UGT430 converts fed mogrol to mogroside V more efficiently than a recombinant host expressing UGT98, UGT1576, and UGT430. See Example 14. Recombinant Genes and Functional Homologs
[00121] The term "recombinant gene" refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence can already be present in such a host. "Introduced" or "augmented" in this context is known in the art to mean introduced or augmented by the hand of man. Thus, a recombinant gene can be a DNA sequence from another species, or can be a DNA sequence that originated from or is present in the same species, but has been incorporated into a host by recombinant methods to form a recombinant host. It will be appreciated that a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA. In a preferred embodiment, the DNA is a cDNA copy of an mRNA transcript of a gene produced in a cell.
[00122] In some embodiments, the coding sequence of a polypeptide described herein, such as the coding sequence of a UGT polypeptide, is a heterologous sequence. The phrases "heterologous sequence" and "heterologous coding sequence" are used to describe a sequence derived from a species other than the recombinant host. In some embodiments, the recombinant host is an S. cerevisiae cell, and a heterologous sequence is derived from an organism other than S. cerevisiae. A heterologous coding sequence, for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant host expressing the heterologous sequence. In some embodiments, a coding sequence is a sequence that is native to the host.
[00123] In some aspects of the invention, a squalene epoxidase polypeptide, cucurbitadienol synthase polypeptide, cytochrome P450 polypeptide, cytochrome P450 reductase polypeptide, epoxide hydrolase polypeptide, and/or glycosyitransferase polypeptide is a fusion protein. In some embodiments, a squalene epoxidase polypeptide (including, but not limited to, the squalene epoxidase polypeptide of SEQ ID NO:54, a cucurbitadienol synthase polypeptide (including, but not limited to, the cucurbitadienol synthase polypeptide of SEQ ID NO:43), a cytochrome P450 polypeptide (including, but not limited to, the CYP5491 polypeptide of SEQ ID NO:44), a cytochrome P450 reductase polypeptide (including, but not limited to, the CPR4497 polypeptide of SEQ ID NO:46), an epoxide hydrolase polypeptide (including, but not limited to, the EH1 polypeptide of SEQ ID NO:38 or the EH2 polypeptide of SEQ ID NO:40), and/or a UGT polypeptide (including, but not limited to, UGT1576 of SEQ ID NO:48, UGT430 of SEQ ID NO:62, UGT1697 of SEQ ID NO:68, UGT11789 of SEQ ID NO:72, UGT98 of SEQ ID NO:53, or UGTSK98 of SEQ ID NO:50) is a fusion polypeptide. The terms "chimera," "fusion polypeptide," "fusion protein," "fusion enzyme," "chimeric protein," "chimeric polypeptide," and "chimeric enzyme" can be used interchangeably herein to refer to proteins engineered through the joining of two or more genes that code for different proteins. In some embodiments, a nucleic acid sequence encoding a squalene epoxidase polypeptide, cucurbitadienol synthase polypeptide, cytochrome P450 polypeptide, cytochrome P450 reductase polypeptide, epoxide hydrolase polypeptide, and/or glycosyltransferase polypeptide polypeptide include a tag sequence that encodes a "tag" designed to facilitate subsequent manipulation (e.g., to facilitate purification or detection), secretion, or localization of the encoded polypeptide. Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), polyhistidine-tag (HIS tag), and Flag™ tag (Kodak, New Haven, CT). Other examples of tags include a ch!oroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, signal peptide, or a secretion tag.
[00124] In some embodiments, a fusion protein is a protein altered by domain swapping. As used herein, the term "domain swapping" is used to describe the process of replacing a domain of a first protein with a domain of a second protein. In some embodiments, the domain of the first protein and the domain of the second protein are functionally identical or functionally similar. In some embodiments, the structure and/or sequence of the domain of the second protein differs from the structure and/or sequence of the domain of the first protein. In some embodiments, a cytochrome P450 reductase polypeptide is altered by domain swapping. For example, in some aspects, the cytochrome P450 domain or reductase domain of CPR4497 (SEQ ID NO:46) is replaced by the cytochrome P450 domain or reductase domain of a cytochrome P450 reductase other than CPR4497 (SEQ ID NO:46). In other aspects, a UGT polypeptide is altered by domain swapping.
[00125] Functional homologs of the polypeptides described above are also suitable for use in producing steviol glycosides in a recombinant host. A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide. A functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homoiogs, or orthologs, or paralogs. Variants of a naturally occurring functional homolog, such as polypeptides encoded by mutants of a wild type coding sequence, can themselves be functional homologs. Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally-occurring polypeptides ("domain swapping"). Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term "functional homolog" is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
[00126] Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of steviol glycoside biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PS I -BLAST analysis of non- redundant databases using a UGT amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a steviol glycoside biosynthesis polypeptide. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in steviol glycoside biosynthesis polypeptides, e.g., conserved functional domains.
[00127] Conserved regions can be identified by locating a region within the primary amino acid sequence of a steviol glycoside biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. The information included at the Pfam database is described in Sonnhammer et al., Nucl. Acids Res., 26:320-322 (1998); Sonnhammer et al., Proteins, 28:405-420 (1997); and Bateman et al., Nucl. Acids Res., 27:260- 262 (1999). Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.
[00128] Typically, polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions. Conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). In some embodiments, a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
Recombinant Hosts
[00129] Recombinant hosts described herein below can be used in methods to produce a mogrol precursor, mogrol, or mogroside. For example, if the recombinant host is a microorganism, the method can include growing the recombinant microorganism In a culture medium under conditions in which one or more of the enzymes catalyzing step(s) of the methods of the invention, e.g., synthases, hydrolases, CYP450s and/or UGTs are expressed. In the present context the terms "microorganism" and "microorganism host" and "recombinant host" can be used interchangeably to refer to microscopic organisms, including bacteria or microscopic fungi, including yeast. The microorganism can be, but not iimited to, a eukaryotic cell or immortalized cell.
[00130] Exemplary prokaryotic and eukaryotic species are described in more detail below. However, it will be appreciated that other species can be suitable. For example, suitable species can be in a genus including Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Escherichia, Fusarium/Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces and Yarrowia. Exemplary species from such genera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Physcomitrella patens, Rhodoturula glutinis 32, Rhodoturula mucilaginosa, Phaffia rhodozyma UBV-AX, Xanthophyllomyces dendrorhous, Fusarium fujikuroi/Gibberella fujikuroi, Candida utilis and Yarrowia lipolytica. In some embodiments, a microorganism can be an Ascomycete such as Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Aspergillus niger, or Saccharomyces cerevisiae. In some embodiments, a microorganism can be a prokaryote such as Escherichia coli, Rhodobacter sphaeroides, or Rhodobacter capsulatus. It will be appreciated that certain microorganisms can be used to screen and test genes of interest in a high throughput manner, while other microorganisms with desired productivity or growth characteristics can be used for large-scale production of mogro! precursor, mogrol, or mogroside.
[00131] In certain embodiments of this invention, microorganisms include, but are not limited to, S. cerevisiae, A. niger, A. oryzae, E. coli, L. lactis and B. subtilis. The constructed and genetically engineered microorganisms provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, continuous perfusion fermentation, and continuous perfusion cell culture.
[00132] Exemplary embodiments comprising bacterial cells include, but are not limited to, cells of species, belonging to the genus Bacillus, the genus Escherichia, the genus Lactobacillus, the genus Lactobacillus, the genus Corynebaclerium, the genus Acetobacler, the genus Acinetobacler, or the genus Pseudomonas.
[00133] The microorganism can be a fungus, and more specifically, a filamentous fungus belonging to the genus of Aspergillus, e.g., A. niger, A. awamori, A. oryzae, or A. nidulans, a yeast belonging to the genus of Saccharomyces, e.g., S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, or S. uvarum, a yeast belonging to the genus Kluyveromyces, e.g., K. laclis, K. marxianus var. marxianus, or K. thermololerans, a yeast belonging to the genus Candida, e.g., C. ulilis, C. Iropicalis, C. albicans, C. lipolylica, or C. versalilis, a yeast belonging to the genus Pichia, e.g., R. slipidis, R. pasloris, or P. sorbilophila, or other yeast genera, e.g., Cryplococcus, Debaromyces, Hansenula, Pichia, Yarrowia, Zygosaccharomyces, or Schizosaccharomyces. Concerning other microorganisms a non-exhaustive list of suitable filamentous fungi is supplied: a species belonging to the genus Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Morlierella, and Trichoderma.
Saccharomyces cerevisiae
[00134] Saccharomyces cerevisiae is a widely used chassis organism in synthetic biology, and can be used as the recombinant microorganism platform. There are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for S. cerevisiae, allowing for rational design of various modules to enhance product yield. Methods are known for making recombinant microorganisms.
[00135] The genes described herein can be expressed in yeast using any of a number of known promoters. Strains that overproduce phenylpropanoids are known and can be used as acceptor molecules in the production of a mogrol precursor, mogrol, or mogroside. Aspergillus spp.
[00136] Aspergillus species such as A. oryzae, A. niger and A. sojae are widely used microorganisms in food production, and can also be used as the recombinant microorganism platform. Nucleotide sequences are available for genomes of A. nidulans, A. fumigatus, A. oryzae, A. clavatus, A. flavus, A. niger, and A. terreus, allowing rational design and modification of endogenous pathways to enhance flux and increase product yield. Metabolic models have been developed for Aspergillus, as well as transcriptomic studies and proteomics studies. A. niger is cultured for the industrial production of a number of food ingredients such as citric acid and gluconic acid, and thus species such as A. niger are generally suitable for the production of a mogroi precursor, mogro!, or mogroside.
Escherichia coli
[00137] Escherichia coli, another widely used platform organism in synthetic biology, can also be used as the recombinant microorganism platform. Similar to Saccharomyces, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for Saccharomyces can be used to make recombinant E. coli microorganisms.
Agaricus. Gibberella, and Phanerochaete spp.
[00138] Agaricus, Gibberella, and Phanerochaete spp. can be useful because they are known to produce large amounts of gibbere!lin in culture. Thus, the precursors of terpenes used as acceptor molecules in the production of a mogroi precursor, mogroi, or mogroside are already produced by endogenous genes. Thus, modules containing recombinant genes for biosynthesis of terpenes can be introduced into species from such genera without the necessity of introducing other compounds or pathway genes.
Arxula adeninivorans (Blastobotrvs adeninivorans)
[00139] Arxula adeninivorans is dimorphic yeast (it grows as budding yeast like the baker's yeast up to a temperature of 42°C, above this threshold it grows in a filamentous form) with unusual biochemical characteristics. It can grow on a wide range of substrates and can assimilate nitrate. It has successfully been applied to the generation of strains that can produce natural plastics or the development of a biosensor for estrogens in environmental samples.
Yarrowia lipolytica. [00140] Yarrowia lipolytics is dimorphic yeast (see Arxula adeninivorans) and belongs to the family Hemiascomycetes. The entire genome of Yarrowia lipolytica is known. Yarrowia species is aerobic and considered to be non-pathogenic. Yarrowia is efficient in using hydrophobic substrates (e.g. alkanes, fatty acids, oils) and can grow on sugars. It has a high potential for industrial applications and is an oleaginous microorganism. Yarrowia lipolyptica can accumulate lipid content to approximately 40% of its dry cell weight and is a model organism for lipid accumulation and remobilization. See e.g., Nicaud, 2012, Yeast 29(10):409-18; Beopoulos et al., 2009, Biochimie 91 (6):692-6; Bankar et a/., 2009, Appl Microbiol Biotechnol. 84(5):847- 65.
Rhodotorula so.
[00141] Rhodotorula is unicellular, pigmented yeast. The oleaginous red yeast, Rhodotorula glutinis, has been shown to produce lipids and carotenoids from crude glycerol (Saenge et al., 2011 , Process Biochemistry 46(1 ):210-8). Rhodotorula toruloides strains have been shown to be an efficient fed-batch fermentation system for improved biomass and lipid productivity (Li et al., 2007, Enzyme and Microbial Technology 41 :312-7).
Rhodosporidium toruloides
[00142] Rhodosporidium toruloides is oleaginous yeast and useful for engineering lipid- production pathways (See, e.g., Zhu et al., 2013, Nature Commun. 3:1112; Ageitos et al. , 201 , Applied Microbiology and Biotechnology 90(4): 1219-27).
Candida boidinii
[00143] Candida boidinii is methylotrophic yeast (it can grow on methanol). Like other methylotrophic species such as Hansenula polymorpha and Pichia pastoris, it provides an excellent platform for producing heterologous proteins. Yields in a multigram range of a secreted foreign protein have been reported. A computational method, IPRO, recently predicted mutations that experimentally switched the cofactor specificity of Candida boidinii xylose reductase from NADPH to NADH. See, e.g., attanovich et al., 2012, Methods Mol Biol. 824:329-58; Khoury et al., 2009, Protein Sci. 18(10):2125-38.
Hansenula polymorpha (Pichia angusta)
[00144] Hansenula polymorpha is methylotrophic yeast (see Candida boidinii). It can furthermore grow on a wide range of other substrates; it is therm o-tolerant and can assimilate nitrate (see also Kluyveromyces lactis). It has been applied to producing hepatitis B vaccines, insulin and interferon alpha-2a for the treatment of hepatitis C, furthermore to a range of technical enzymes. See, e.g., Xu ef a/., 2014, Virol Sin. 29(6):403-9.
Kluweromvces lactis
[00145] Kluyveromyces lactis is yeast regularly applied to the production of kefir. It can grow on several sugars, most importantly on lactose which is present in milk and whey. It has successfully been applied among others for producing chymosin (an enzyme that is usually present in the stomach of calves) for producing cheese. Production takes place in fermenters on a 40,000 L scale. See, e.g., van Ooyen ef a/., 2006, FEMS Yeast Res. 6(3):381-92.
Pichia pastoris
[00146] Pichia pastoris is methylotrophic yeast (see Candida boidinii and Hansenula polymorpha). It provides an efficient platform for producing foreign proteins. Platform elements are available as a kit and it is worldwide used in academia for producing proteins. Strains have been engineered that can produce complex human N-glycan (yeast glycans are similar but not identical to those found in humans). See, e.g., Piirainen er a/., 2014, N Biotechnol. 31 (6):532-7.
Phvscomitrella soo.
[00147] Physcomitrella mosses, when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera can be used for producing plant secondary metabolites, which can be difficult to produce in other types of cells.
[00148] As will be apparent to one skilled in the art, the particulars of the selection process for specific UGTs capable of glycosylating mogroi and mogrosides depend on the identities of selectable markers. Selection in all cases promotes or permits proliferation of ceils comprising the marker while inhibiting or preventing proliferation of cells lacking the marker. If a selectable marker is an antibiotic resistance gene, the transfected host population can be cultured in the presence of an antibiotic to which resistance is conferred by the selectable marker. If a selectable marker is a gene that complements an auxotrophy of the hosts, the transfected host population can be cultivated in the absence of the compound for which the hosts are auxotrophic.
[00149] After selection, recombinant hosts can be cloned according to any appropriate method known in the art. For example, recombinant microbial hosts can be plated on solid media under selection conditions, after which single clones can be selected for further selection, characterization, or use. This process can be repeated one or more times to enhance stability of the expression construct within the host. To produce a mogroside pathway polypeptide, recombinant hosts comprising one or more expression vectors can be cultured to expand cell numbers in any appropriate culturing apparatus known in the art, such as a shaken culture flask or a fermenter.
[00150] Culture media used for various recombinant hosts are well known in the art. Culture media used to culture recombinant bacterial cells will depend on the identity of the bacteria. Culture media used to culture recombinant yeast cells will depend on the identity of the yeast. Culture media generally comprise inorganic salts and compounds, amino acids, carbohydrates, vitamins and other compounds that are either necessary for the growth of the hosts or improve health or growth or both of the hosts. In particular, culture media typically comprise manganese (Mn2+) and magnesium (Mg2+) ions, which are co-factors for many, but not all, glycosyltransferases.
[00151] As used herein, the term "fed-batch culture" or "semi-batch culture" are used interchangeably to refer to as an operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation and in which the product(s) remain in the bioreactor until the end of the run. In some embodiments, all the nutrients are fed into the bioreactor.
[00152] In some embodiments, a recombinant host can be modified in order to reduce giucanase activity, in particular giucanase activity, which can result in deglycosylation of mogrosides. Thus, the recombinant host can for example be modified to reduce of even abolish exo-1 ,3-beta-Glucanase activity, in embodiments of the invention when the recombinant host is yeast, this can be accomplished by deletion of the EXG1 gene (SEQ ID NO:63, SEQ ID NO:64) and/or of the EXG2 gene (SEQ ID NO:65, SEQ ID NO:66), both of which are encoding an exo- 1 ,3-beta-glucanase.
[00153] Table 2 indicates the identities of the sequences utilized herein.
Table 2. Sequences used herein.
Figure imgf000038_0001
SEQ ID NO:4 Nucleotide sequence encoding CYP937
SEQ ID NO:5 Codon-optimized DNA sequence encoding CYP1798
SEQ ID NO:6 Nucleotide sequence encoding CYP1994
SEQ ID N0.7 Nucleotide sequence encoding CYP2048
SEQ ID N0:8 Nucleotide sequence encoding CYP2740
SEQ ID N0:9 Nucleotide sequence encoding CYP3404
SEQ ID NO: 10 Nucleotide sequence encoding CYP3968
SEQ ID NO: 1 1 Nucleotide sequence encoding CYP4112
SEQ ID NO:12 Nucleotide sequence encoding CYP4149
SEQ ID NO: 13 Nucleotide sequence encoding CYP4491
SEQ ID NO:14 Nucleotide sequence encoding CYP5491
SEQ ID NO: 15 Nucleotide sequence encoding CYP6479
SEQ ID NO:16 Nucleotide sequence encoding CYP7604
SEQ ID O:17 Nucleotide sequence encoding CYP8224
SEQ ID NO:18 Nucleotide sequence encoding CYP8728
SEQ ID NO: 19 Nucleotide sequence encoding CY 10020
SEQ ID NO:20 Nucleotide sequence encoding CYP10285
SEQ ID NO:21 Amino acid sequence of UGT73C3
SEQ ID NO:22 Amino acid sequence of UGT73C5
SEQ ID NO:23 Amino acid sequence of UGT73C6
SEQ ID NO:24 Amino acid sequence of UGT73E1
SEQ ID NO:25 Amino acid sequence of UGT85C2
SEQ ID NO:26 Nucleotide sequence encoding S. grosvenorii UGT98
SEQ ID NO:27 Nucleotide sequence encoding S. grosvenorii UGT1495
SEQ ID NO:28 Nucleotide sequence encoding S. grosvenorii UGT1817 SEQ ID NO:29 Partial nucleotide sequence encoding fragment of S.
grosvenorii UGT3494
SEQ ID NO:30 Nucleotide sequence encoding S. grosvenorii UGT5914
SEQ ID NO:31 Nucleotide sequence encoding S. grosvenorii UGT8468
SEQ ID NO:32 Nucleotide sequence encoding S. grosvenorii UGT10391
SEQ ID NO:33 Partial nucleotide sequence encoding fragment of S.
grosvenorii UGT11789
SEQ ID NO:34 Partial nucleotide sequence encoding fragment of S.
grosvenorii UGT1 1999
SEQ ID NO:35 Partial nucleotide sequence encoding fragment of S.
grosvenorii UGT13679
SEQ ID NO:36 Partial nucleotide sequence encoding fragment of S.
grosvenorii UGT15423
SEQ ID NO:37 Codon-optimized nucleotide sequence encoding S. grosvenorii
Epoxide hydrolase 1
SEQ ID NO:38 Amino acid sequence of S. grosvenorii Epoxide hydrolase 1
SEQ ID NO:39 Codon-optimized nucleotide sequence encoding S. grosvenorii
Epoxide hydrolase 2
SEQ ID NO:40 Amino acid sequence of S. grosvenorii Epoxide hydrolase 2
SEQ ID N0:41 Nucleotide sequence encoding CYP 10969
SEQ ID NO:42 Codon-optimized nucleotide sequence encoding S. grosvenorii cucurbitadienol synthase
SEQ ID NO:43 Amino acid sequence of S. grosvenorii cucurbitadienol synthase
SEQ ID NO:44 Amino acid sequence of S. grosvenorii CYP5491
SEQ ID NO:45 Nucleotide sequence encoding S. grosvenorii CPR4497
SEQ ID NO:46 Amino acid sequence of S. grosvenorii CPR4497 SEQ SD NO:47 Nucleotide sequence encoding S. grosvenorii UGT1576
SEQ ID NO:48 Amino acid sequence of S. grosvenorii UGT1576
SEQ ID NO:49 Nucleotide sequence encoding S. grosvenorii UGT SK98
SEQ ID NO:50 Amino acid sequence of S. grosvenorii UGT SK98
SEQ ID N0:51 Nucleotide sequence encoding S. grosvenorii UGT98
SEQ ID NO:52 Codon-optimized nucleotide sequence encoding S. grosvenorii
UGT98
SEQ ID NO:53 Amino acid sequence of S. grosvenorii UGT98
SEQ ID NO:54 Amino acid sequence of S. cerevisiae squalene epoxidase encoded by the ERG1 gene
SEQ ID NO:55 Amino acid sequence of S. cerevisiae lanosterol synthase encoded by the ERG7 gene
SEQ ID N0:61 Nucleotide sequence of S. grosvenorii UGT430
SEQ ID NO:62 Amino acid sequence of S. grosvenorii UGT430
SEQ ID NO:63 Nucleotide sequence of S. cerevisiae EXG1
SEQ ID NO:64 Amino acid sequence of $. cerevisiae EXG1
SEQ ID NO:65 Nucleotide sequence of S. cerevisiae EXG2
SEQ ID NO:66 Amino acid sequence of S. cerevisiae EXG2
SEQ ID NO:67 Nucleotide sequence of S. grosvenorii UGT1697
SEQ ID NO:68 Amino acid sequence of S. grosvenorii UGT 1697
SEQ ID NO:69 Nucleotide sequence encoding S. grosvenorii UGT1 1789 (full- length)
SEQ ID NO:70 Codon-optimized nucleotide sequence "A" of full-length S.
grosvenor/ UGT1 1789
SEQ ID NO:71 Codon-optimized nucleotide sequence "B" of full-length S.
grosvenorii UGT11789 SEQ ID NO:72 Amino acid sequence of S. grosvenorii UGT11789 (full-length)
SEQ ID NO:73 Nucleotide sequence encoding S, grosvenorii CYP1798
SEQ ID NO:74 Amino acid sequence of S. grosvenorii CYP1798
SEQ ID NO:75 Nucleotide sequence encoding S. cerevisiae TRP1
SEQ ID NO:76 Amino acid sequence of S. cerevisiae TRP1
SEQ ID NO:77 Nucleotide sequence encoding S, cerevisiae tH G1
SEQ ID NO:78 Amino acid sequence of S. cerevisiae tH G1
SEQ ID NO:79 Nucleotide sequence encoding S. grosvenorii Epoxide hydrolase 2
SEQ ID NO:80 Nucleotide sequence encoding S. grosvenorii cucurbitadienoi synthase
SEQ ID 0:81 Codon-optimized nucleotide sequence encoding CYP5491
SEQ ID NO:82 Codon-optimized nucleotide sequence encoding CYP4497
SEQ ID NO:83 Codon-optimized nucleotide sequence encoding UGT1576
SEQ ID NO:84 Codon-optimized nucleotide sequence encoding UGT430
SEQ ID O:85 Codon-optimized nucleotide sequence encoding CYP1697
SEQ ID NO:86 Codon-optimized nucleotide sequence encoding CYP1798-II
SEQ ID NO:87 Amino acid sequence of S. cerevisiae ERG9
SEQ ID NO:88 Amino acid sequence of Gynostemma pentaphyllum Squaiene epoxidase
SEQ ID NO:89 Amino acid sequence of Arabidopsis thaliana Squaiene epoxidase 1
SEQ ID NO:90 Amino acid sequence of Arabidopsis thaliana Squaiene epoxidase 4
SEQ ID N0:91 Amino acid sequence of Arabidopsis thaliana Squaiene epoxidase 6 SEQ ID NO:92 Amino acid sequence of Arabidopsis thaliana Squaiene epoxidase 5
SEQ ID NO:93 Amino acid sequence of Arabidopsis thaliana Squaiene epoxidase 2
SEQ ID NO:94 Amino acid sequence of Arabidopsis thaliana Squaiene epoxidase 3
SEQ ID NO:95 Amino acid sequence of Brassica napus Squaiene monooxygenase 1 ,1
SEQ ID NO:96 Amino acid sequence of Brassica napus Squaiene monooxygenase 1 ,2
SEQ ID NO:97 Amino acid sequence of Euphorbia tirucalli Squaiene epoxidase
SEQ ID NO:98 Amino acid sequence of Medicago truncatula Squaiene epoxidase
SEQ ID N0.99 Amino acid sequence of Medicago truncatula Squaiene monooxygenase
SEQ ID NO:100 Amino acid sequence of Ricinus communis Squaiene monooxygenase
SEQ ID NO:101 Amino acid sequence of Ricinus communis Squaiene monooxygenase
SEQ ID NO: 102 Amino acid sequence of Ricinus communis Squaiene monooxygenase
SEQ ID NO:103 Amino acid sequence of Ricinus communis Squaiene monooxygenase
SEQ ID NO: 104 Amino acid sequence of Ricinus communis Squaiene monooxygenase
SEQ ID NO:105 Amino acid sequence of Ricinus communis Squaiene monooxygenase [00154] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
[00155] The Examples that follow are illustrative of specific embodiments of the invention and various uses thereof. They are set forth for explanatory purposes only and are not to be taken as limiting the invention.
Example 1: Purification of mogroside V
[00156] Mogroside V was purified from commercially available monk fruit extracts (PureLo®, Swanson). Three bottles of PureLo® (240 g) were dissolved in water (900 mL) and loaded on a column of HP-20 resin (400 g resin). The column was washed with water (2.5 liters) and further washed with 20% methanol in water. The product was eluted with methanol. After solvent evaporation and drying under high vacuum, mogroside V (2.5 g) was obtained. The product was approximately 80% pure, with 11-oxomogroside V being the largest impurity.
Example 2: Enzymatic synthesis of mogrol from mogroside V
[00157] Mogroside V (300 mg) was dissolved in 0.1 M sodium acetate buffer (pH 4.5, 100 mL), and crude pectinase from Aspergillus niger (25 mL, Sigma P2736) was added. The mixture was stirred at 50°C for 48 h. The reaction mixture was extracted with ethyl acetate (2x100 mL). The organic extract was dried under vacuum and subsequently purified with preparative HPLC. Pure mogrol (40 mg) was obtained, and its structure was confirmed by NMR and mass spectroscopy. See Figure 5.
Example 3: Enzymatic synthesis of mogrol 3-O-glucoside (mogroside I E1) and mogrol 24-O-glucoside (mogroside I A1) from mogroside V
[00158] Mogroside V (300 mg) was dissolved in 0.1 M sodium acetate buffer (pH 4.5, 100 mL), and crude pectinase from Aspergillus niger (25 mL, Sigma P2736) was added. The mixture was stirred at 50°C for 6.5 h and subsequently extracted with ethyl acetate (2x100 mL). The organic extract was dried under vacuum and purified with preparative HPLC. Pure mogroside I E1 (11.0 mg) and mogroside I A1 (8.0 mg) were obtained. Their structures were confirmed by N R and mass spectroscopy. See Figure 5.
Example 4: In vitro UGT screening and reactions
[00159] UGT73C3 (SEQ ID NO:21), UGT73C5 (SEQ ID NO:22), UGT73C6 (SEQ ID NO:23), UGT73E1 (SEQ ID NO:24), and UGT85C2 (SEQ ID NO:25) were found to glycosylate mogrol in vitro. The reaction mixtures included 4X Tris buffer, mogrol (250 μΜ), UDP-glucose (750 μΜ), and 1 % alkaline phosphatase. 5 μ1_ of each partially purified UGT enzyme or crude enzyme extract was added to the reaction, and the reaction volume brought to 50 μΙ_ with water. The reactions were incubated overnight at 30°C and performed in sterilized 96 well plates. 25 μΙ_ of DMSO were subsequently added into each reaction, and the reaction plates were centrifuged for 5 min. 40 μΙ_ samples were taken from each well and filtered to be used for LC- MS analysis.
[00160] UGT73C3 (SEQ ID NO:21 ), UGT73C6 (SEQ ID NO:23) and UGT85C2 (SEQ ID NO:25) were found to convert the entire mogrol substrate to mogroside I A1. UGT73C5 (SEQ ID NO:22) produced both mogroside I E1 and mogroside I A1. UGT73E1 (SEQ ID NO:24) converted mogrol to mogroside 1 A1 (major product) and a glycosylated mogrol that was neither mogroside I E1 nor mogroside I A1. The product was caused by a glycosy!ation event on C11- OH; the exact mass was shown as a mogroside I.
Example 5: Monk fruit cucurbitadienol synthase
[00161] The CirCS gene codes for cucurbitadienol synthase in monk fruit, and the partial gene sequence covering 338 of the supposedly 764 amino acid sequence was identified by doing a tBLASTn (translated nucleotide database) analysis of the assembled data with a query cucurbitadienol synthase from Cucurbita pepo (accession number BAD34645.1 , SEQ ID NO:1 ). The partial CirCS is 97.5% identical to the C. pepo gene at the protein level (SEQ ID NO:2; from residues 515 to 764 of SEQ ID NO:1 ).
Example 6: Monk fruit genes encoding P450 enzymes catalyzing formation of mogrol from cucurbitadienol [00162] To identify P450 enzymes catalyzing formation of mogrol from cucurbitadienol, a tBLASTn (translated nucleotide database) analysis was performed using reassembled sequencing reads of an S. grosvenorii transcriptome {see Tang et a/., BMC Genomics 12: 343 (201 1 )). E values of 10E-10 or lower were used to identify sequences homologous to the database query sequences.
[00163] 18 full-length or near full-length genes were identified. The assembled genes were designated CYP533, CYP937, CYP1798, CYP1994, CYP2048, CYP2740, CYP3404, CYP3968, CYP4112, CYP4149, CYP4491 , CYP5491 , CYP6479, CYP7604, CYP8224, CYP8728, CYP10020, and CYP 10285 (see Table 2, SEQ ID NOs: 3-20).
[00164] Fuli-length synthetic S. grosvenorii gene sequences of CYP533 (SEQ ID NO:3), CYP937 (SEQ ID NO:4), CYP 1798 (SEQ ID NO:5), CYP1994 (SEQ ID NO:6), CYP2740 (SEQ ID NO:8), CYP4112 (SEQ ID NO:11), CYP4149 (SEQ ID NO:12), CYP4491 (SEQ ID NO:13), CYP5491 (SEQ ID NO:14, SEQ ID NO:44), CYP7604 (SEQ ID NO:16), CYP8224 (SEQ ID NO:17), and CYP10285 (SEQ ID NO:20) were cloned into yeast expression vectors.
Example 7: Monk fruit genes encoding enzymes catalyzing glycosylation of mogroside II
E
[00165] To identify monk fruit gene sequences encoding UGTs capable of converting mogroside II E into mogroside V, a tBLASTn (translated nucleotide database) analysis was performed using reassembled sequencing reads of an S. grosvenorii transcriptome (see Tang et a/., BMC Genomics 12: 343 (201 1 )). The genes identified were UGT98 (SEQ ID NO:26), UGT1495 (SEQ ID NO:27), UGT1817 (SEQ ID NO:28), UGT3494 (SEQ ID NO:29), UGT5914 (SEQ ID NO:30), UGT8468 (SEQ ID NO:31 ), UGT10391 (SEQ ID NO:32), UGT11789 (SEQ ID NO:33), UGT1 999 (SEQ ID NO:34), UGT13679 (SEQ ID NO:35), and UGT15423 (SEQ ID NO:36).
[00166] Of these, UGT98 (SEQ ID NO:26), UGT1495 (SEQ ID NO:27), UGT1817 (SEQ ID NO:28), UGT5914 (SEQ ID NO:30), UGT8468 (SEQ ID NO:31 ), and UGT10391 (SEQ ID NO:32) were synthesized based on contigs made from the publicaliy-available sequence reads (Tang et a/., BMC Genomics 12: 343 (201 1 )). The genes were inserted into yeast expression vectors. Example 8: Boosting mogrol pathway precursor availability
[00167] To increase the availability of oxidosqualene and dioxidosqualene in yeast, the promoter of the endogenous ERG7 gene (SEQ ID NO:55) was displaced by a PCR fragment comprising the Nurseothricin marker (NatMX) and the CUP1 copper inducible promoter. ERG7 expression was thereby decreased when the yeast strain was grown in normal SC medium. ERG7 encodes lanosterol synthase and lowered expression is known to result in accumulation of oxidosqualene and dioxidosqualene in baker's yeast. Oxidosqualene is generally the precursor of triterpenoids. To further increase oxidosqualene and dioxidosqualene availability, the squalene epoxidase encoded by ERG1 (SEQ ID NO:54) was overexpressed, and a truncated copy of the yeast HMG reductase (tHMG1 , SEQ ID NO:77, SEQ ID NO:78) was expressed.
[00168] Successful boosting of oxidosqualene and dioxidosqualene production in yeast was demonstrated by production of tetrahydroxysqualene when either one of two soluble S. grosvenorii epoxide hydrolases was expressed in this strain. The S. grosvenorii epoxide hydrolase 1 is set forth in SEQ ID NO:38, and the codon-optimized S. grosvenorii epoxide hydrolase 1 is set forth in SEQ ID NO:37. The S. grosvenorii epoxide hydrolase 2 is set forth in SEQ ID NO:40, and the codon-optimized S. grosvenorii epoxide hydrolase 2 is set forth in SEQ ID NO:39. Figure 6 shows the LC-MS mass peak 501 corresponding to the proton plus Na+ adduct of tetrahydroxysqualene in a sample from a yeast strain transformed with a plasmid expressing S. grosvenorii epoxide hydrolase 2. Tetrahydroxysqualene is produced by hydrolysis of 2,3- and 22,23- epoxide bonds of dioxidosqualene. No accumulation of tetrahydroxysqualene was detected in the background yeast strain. Samples were made by boiling culture aliquots in 50% DMSO and then pelleting of cell material by centrifugation. Supernatants were then measured by ESI LC-MS.
Example 9: Production of cucurbitadienoi in yeast strain
[00169] Integration of a codon-optimized gene copy of the S. grosvenorii cucurbitadienoi synthase set forth in SEQ ID NO:42 and SEQ ID NO:43 in S. cerevisiae resulted in production of cucurbitadienoi (see Figure 7B). The yeast strain was grown at 30°C for 5 days in SC medium comprising 2% glucose. Cucurbitadienoi was extracted by boiling a culture sample in 50% ethanol/20% KOH for 5 min followed by extraction with an equal volume of hexane. The samples were then evaporated with hexane, and the dried extract was resuspended in methanol.
[00170] Figures 7A and 7B show LC-MS chromatograms of samples of yeast expressing the cucurbitadienol synthase set forth in SEQ ID NO:42 and SEQ ID NO:43. Figure 7A shows lanosterol peaks, and Figure 7B shows cucurbitadienol and lanosterol peaks. The peak corresponding to lanosterol shows a retention time of -8.05, whereas the peak corresponding to cucurbitadienol has a retention time of 7.85. Both lanosterol and cucurbitadienol show a mass in the LC-MS chromatogram of 409.4 (proton adduct minus mass of one H20 molecule).
Example 10: Modification of cucurbitadienol in S. cerevisiae by CYP5491
[00171] Upon transformation of a cucurbitadienol-producing yeast strain (see Example 9) with a piasmid comprising the S. grosvenorii CYP5491 gene (SEQ ID NO: 14, SEQ ID NO:44) and a piasmid comprising the S. grosvenorii CPR4497 gene (SEQ ID NO:45, SEQ ID NO:46), three peaks were visible with LC-MS (see Figure 8). The upper frame in Figure 8 shows the LC-MS chromatogram with these three peaks, while the three lower frames show the fragmentation spectrum of these three peaks. The masses of the 3 peaks (443.38, 441.37 and
457.36) correspond in weight to proton adducts of hydroxylated cucurbitadienol, oxo cucurbitadienol and hydroxy plus oxo cucurbitadienol respectively. The hydroxylated cucurbitadienol (protonated mass 443.38) and oxidized cucurbitadienol (protonated mass
44 .37) were 1 -hydroxy-cucurbitadieno! and 1-oxo-cucurbitadienol, respectively, as confirmed by NMR (Figure 9).
Example 11 : Glycosylation of mogroi in S. cerevisiae by expression of S. grosvenorii UGT98, UGTSK98, and UGT1576
[00172] UGT98, UGTSK98 and UGT1576 genes were synthesized based on contigs made from publically-available sequence reads (Tang et a/., 2011 , BMC Genomics 12:343). The nucleotide and amino acid sequences of UGT98 are set forth herein as SEQ ID NO:51 and SEQ ID NO:53, respectively, whereas SEQ ID NO:52 corresponds to a codon-optimized version of UGT98. The nucleotide and amino acid sequences of UGTSK98 are set forth herein as SEQ ID NO:49 and SEQ ID NO:50, respectively, and the nucleotide and amino acid sequences of UGT1576 are set forth herein as SEQ ID NO:47 and SEQ ID NO:48, respectively. [00173] When a yeast strain deleted of the exo-1,3-beta g!ucanases EXG1 and EXG2 (to prevent de-giycosylation of produced mogrosides) was fed mogrol (10-100 μΜ) and transformed with a plasmid expressing UGT1576 (SEQ ID NO:47 and SEQ ID NO:48), mogroside I A1 was formed (Fig 11B). Samples were prepared by mixing a culture aliquot 1 :1 with DMSO followed by boiling (80 C) for 5 min and pelleting by centrifugation. The supernatants were then subjected to ESI LC- S. Figure 10A shows the LC-MS chromatogram of reference mogroside I A1 , while Figure 10B shows the peak from a yeast sample expressing UGT1576 in a culture fed with 50 μ mogrol. These data show that the UGT1576 gene encodes a giycosyltransferase with mogrol C24-OH UDP-glycosyltransferase activity.
[00174] When UGT98 (SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53) and UGTSK98 (SEQ ID NO:49, SEQ ID NO:50) were cloned into yeast expression plasmids and subsequently transformed into a yeast strain deleted of the exo-1 ,3-beta glucanases EXG1 and EXG2, no conversion of fed mogrol was detected. In contrast, co-expression of UGT98 (SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53) or UGT SK98 (SEQ ID NO:49, SEQ ID NO:50) with UGT1576 (SEQ ID NO:47 and SEQ ID NO:48) in yeast fed with mogrol resulted in further glycosyiation of mogroside I A1. UGTSK98 co-expressed with UGT1576 resulted in production of di- glycosylated mogrol (mogroside II A, Figure 11 A), while co-expression with UGT98 resulted in di- and tri-glycosylated mogrol (middle and lower frames, Figure 1 1 B). The di-glycosylated mogrol that was formed by both UGT98 and UGTSK98 had a different retention time than mogroside II E and mogroside II A1 during LC-MS.
[00175] Thus, both UGT98 and UGTSK98 were found to be able to catalyze 1 ,2- glycosylation of the glucose of mogroside I A1. UGT98 was found to be multifunctional, catalyzing 1 ,2-glycosylation of mogroside I A1 , resulting in production of mogroside II A, followed by a 1 ,6-glycosylation of mogroside II A to form mogroside III A1 (Figure 1 1 B). UGT98 and UGTSK98 belong to the UGT91 family of UDP-glucose glycosyltransferases, and members of this family are known to be 1 ,2- and 1 ,6-glycosyltransferases. Figure 12 schematically summarizes the glycosyiation reactions from mogrol to mogroside III A1.
Example 12: Glycosyiation of mogrol in S. cerevisiae by expression of S. grosvenorii UGT430
[00176] UGT430 (SEQ ID NO:61 , SEQ ID NO:62) of the 85A UGT family was cloned from synthetic DNA to obtain a sequence identical to that of S. grosvenorii UGT430. The cloned gene was transformed into a yeast strain deleted of EXG1 and EXG2 (to prevent de- g!ycosylation of produced mogrosides). The yeast strain was grown in SC medium minus tryptophan for selection of plasmid maintenance, and comprising 10 μΜ mogrol. Cells were grown for 2 days at 30°C with shaking at 140 rpm. After 2 days, 300 μΙ_ culture samples were mixed with 300 pl_ of 96% ethanol and incubated for 10 min at 80°C. Then, samples were centrifuged, and the supernatant was analyzed by LC-MS.
[00177] LC-MS analyses were performed using a Waters Acquity l-Class UPLC (Waters Corporation, Milford, MA) with Waters Acquity UPLC ®BEH C18 column (2.1 x 50 mm, 1.7 μπι particles, 130 A pore size) coupled to a Waters Xevo TQD triple quadropole mass spectrometer with electrospray ionization (ESI) in negative mode. Compound separation was achieved by a gradient of the two mobile phases A (water with 0.1 % formic acid) and B (MeCN with 0.1 % formic acid) by increasing from 20% to 50% B between 0.3 to 2.0 min, increasing to 100% B at 2.01 min, holding 100% B for 0.6 min and re-equilibrating for another 0.6 min. The flow rate was 0.6 mL/min, and the column temperature 55°C. Mogroside I E1 (m/z 683.5; [M+FA]") was monitored using SIR (Single Ion Recording) and compared with a standard.
[00178] Resulting LC-MS chromatograms are shown in Figure 13. One large peak belonging to a compound of MW = 683.5 was formed by UGT430 (Figure 13B). The mass of this peak corresponds to a formic acid adduct of mono-glycosylated mogrol. This product has the identical retention time of the mogroside I E1 reference compound shown in Figure 13A. UGT430 glycosylated mogrol efficiently and completely since no fed mogrol remained after the 2-day growth period of yeast expressing UGT430. Thus, the S. grosvenorii UGT430 is the UGT responsible for glycosylation of the hydroxy group on C- 3 position of the mogrol molecule in the S. grosvenorii mogroside biosynthetic pathway.
Example 13: Glycosylation of mogrol in S. cerevisiae by expression of S, grosvenorii UGT1697
[00179] UGT 697 (SEQ ID NO:67, SEQ ID NO:68) of the 85A UGT family was cloned from synthetic DNA to obtain a sequence identical to that of S. grosvenorii UGT1697. The cloned gene was transformed into a yeast strain deleted of EXG1 and EXG2 (to prevent de- glycosylation of produced mogrosides. The yeast strain was grown in SC medium minus histidine for selection of plasmid maintenance, and comprising 10 μΜ mogrol. Cells were grown for 2 days at 30°C with shaking at 140 rpm. After 2 days, 300 pL culture samples were mixed with 300 μ[_ of 96% ethanol and incubated for 10 min at 80°C. Then, samples were centrifuged, and the supernatant was analyzed by LC-MS.
[00180] LC-MS analyses were performed using a Waters Acquity !-Class UPLC (Waters Corporation, Milford, MA) with Waters Acquity UPLC ®BEH C18 column (2.1 x 50 mm, 1.7 pm particles, 130 A pore size) coupled to a Waters Xevo TQD triple quadropole mass spectrometer with e!ectrospray ionization (ESI) in negative mode. Compound separation was achieved by a gradient of the two mobile phases A (water with 0.1 % formic acid) and B (MeCN with 0.1 % formic acid) by increasing from 20% to 50% B between 0.3 to 2.0 min, increasing to 100% B at 2.01 min, holding 100% B for 0.6 min and re-equilibrating for another 0.6 min. The flow rate was 0.6 mL/min, and the column temperature 55°C. Mogroside I (m/z 683.5; [M+FA]") was monitored using SIR (Single Ion Recording) and compared with a standard.
[00181] Resulting LC-MS chromatograms are shown in Figure 14. One large peak belonging to a compound of MW = 683.5 was formed by UGT1697 (Figure 14B). The mass of this peak corresponds to a formic acid adduct of mono-glycosylated mogrol. The peak corresponds to mogroside I A1. See Figure 14A. This result shows that the S. grosvenorii UGT1697 glycosylates the hydroxy group at the C-24 position of mogrol. UGT1576 also exhibits C-24 glycosylation of mogrol, as shown in Example 11.
[00182] Moreover, UGT1697 acts on the C-3 position as well, since the presence of mogroside II E (containing one glucose on position C-24 and one on C-3) was detected, as depicted in Figure 14B (retention time of 2.22 min). Thus, UGT1697 glycosylates the C-3 and C-24 position on mogrol and is part of the S. grosvenorii mogroside biosynthetic pathway.
Example 14: Glycosylation of mogrol and mogrosides in S. cerevisiae by expression of S. grosvenor// UGT11789, UGT98, UGT430, and UGT1576
[00183] The full-length sequence for UGT1 789 (SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 , SEQ ID NO:72) was cloned from synthetic DNA to obtain a sequence identical to that of S. grosvenorii UGT11789. A yeast strain deleted of EXG1 and EXG2 was co-transformed with UGT11789 (SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 , SEQ ID NO:72), UGT430 (SEQ ID NO:61 , SEQ ID NO:62), UGT1576 (SEQ ID NO:47, SEQ ID NO:48), and UGT98 (SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53). Separately, a yeast strain deleted of EXG1 and EXG2 was co-transformed with UGT430 (SEQ ID NO:61 , SEQ ID NO:62), UGT1576 (SEQ ID NO:47, SEQ ID NO:48), and UGT98 (SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53). The yeast strains were grown in SC medium minus histidine, uracil, tryptophan, and leucine for selection of plasmid maintenance and comprising 10 μ mogrol. Cells were grown for 2 days at 30°C with shaking at 140 rpm. After 2 days, 300 μ!_ culture samples were mixed with 300 μΙ_ of 96% ethanol and incubated for 10 min at 80°C. Then, samples were centrifuged, and the supernatant was analyzed by LC- S.
[00184] LC-MS analyses were performed using a Waters Acquity !-Class UPLC (Waters Corporation, Milford, MA) with Waters Acquity UPLC ®BEH C18 column (2.1 x 50 mm, 1.7 pm particles, 130 A pore size) coupled to a Waters Xevo TQD triple quadropole mass spectrometer with electrospray ionization (ESI) in negative mode. Compound separation was achieved by gradient I or gradient II. For gradient i, the initial buffer concentration of 80% mobile phase A (water with 0.1 % formic acid) and 20% mobile phase B (MeCN with 0.1 % formic acid) was increased from to 20% to 40% B between 0.3 to 2.0 min, increased to 100% B at 2.01 min, held at 100% B for 0.6 min, and re-equilibrated for another 0.6 min. For gradient II, the initial buffer concentration of 80% mobile phase A (water with 0.1 % formic acid) and 20% mobile phase B (MeCN with 0.1 % formic acid) was increased from to 20% to 50% B between 0.3 to 2.0 min, increased to 100% B at 2.01 min, held at 100% B for 0.6 min, and re-equilibrated for another 0.6 min. For both gradient I and gradient II, the flow rate was 0.6 mL mtn, and the column temperature 55°C. Mogrol and mogrosides were monitored using SIR (Single Ion Recording) and compared with a commercially available mogroside mixture from plant extract (3W botanical extract. Inc.). The SIR traces were as follows: mogrol (m/z 521.4; [M+FA-H]"), mogrol+1 Glucose (m/z 683.5; [M+FA-H]"), mogro!+2Glucose (m/z 799.5; [M-H]"), mogrol+3Glucose (m/z 961.6; [M-H]"), mogrol+4G!ucose (m/z 1123.6; [M-H]") and mogrol+SGIucose (m/z 1285.66; [M-H]'). Resulting LC-MS chromatograms are shown in Figure 15.
[00185] Figure 15A shows mogroside reference standards and indicates peaks corresponding to mogroside V and mogroside II E. Comparison of Figure 15B and Figure 15C demonstrates the effect of expression of the UGT11789 codon-optimized sequence A (SEQ ID NO:70, SEQ ID NO.72). Figure 15B shows that mogroside II E produced upon co-expression of S. grosvenorii UGT1576 (SEQ ID NO:47, SEQ ID NO:48) and UGT430 (SEQ ID NO:61 , SEQ ID NO:62) in an S. cerevisiae strain that was fed mogrol was converted to mogroside V by co-expression of the multifunctional UGT98 (SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53). The intensity of the mogroside V peak in Figure 15B was measured to be 8.65E3 (peak ion intensity in an LC-MS chromatogram). Co-expression of S, grosvenorii UGT1576 (SEQ ID NO:47, SEQ ID NO:48), UGT430 (SEQ ID NO:61 , SEQ ID NO:62), UGT98 (SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53) and UGT11789 (SEQ ID NO:70, SEQ ID NO:72) in an S. cerevisiae strain more efficiently converts fed mogrol to mogroside V, as shown in Figure 15C. The intensity of the mogroside V peak in Figure 15C was measured to be 2.22E5 (peak ion intensity in an LC-MS chromatogram).
[00186] This experiment shows that co-expressed S. grosvenorii UGT98 (SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53) and UGT11789 (SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 , SEQ ID NO:72) catalyze each of the glucose-giucose 1 ,2- and 1 ,6- attachments necessary for efficient mogroside V production in yeast. Mogroside II E can be glycosylated by UGT11789 to form a mogroside with 3 glucoses attached (Figure 15D). Since UGT 1789 is of the UGT91 family and cannot glycosylate the mogrol core, this glycosylation of mogroside HE is by a 1 ,2-bond or 1 ,6-bond, and the product of UGT11789 is therefore mogroside III or mogroside IIIA2.
Example 15: Production of mogrol in S. cerevisiae by expression of S. grosvenorii CYP1798
[00187] CYP1798 was cloned from synthetic DNA to obtain sequence identical to that of S. grosvenorii CYP1798 (SEQ ID NO:5, SEQ ID NO: 74). The nucleotide sequence was codon- optimized for expression in S. cerevisiae (SEQ ID NO:5). To increase the availability of oxidosqualene, the promoter of the endogenous ERG7 gene (SEQ ID NO:55) was disrupted to lower lanosterol synthase expression in an S. cerevisiae strain deleted of the TRP1 gene. To further increase oxidosqualene availability in S. cerevisiae, the squalene epoxidase encoded by ERG1 (SEQ ID NO:54) was overexpressed, and a truncated HMG reductase (tHMG1 , SEQ ID NO:77, SEQ ID NO:78) was expressed. Integration of a codon-optimized optimized gene encoding S. grosvenorii cucurbitadienol synthase (SEQ ID NO:42, SEQ ID NO:43) and of a gene encoding S. grosvenorii CPR4497 (SEQ ID NO:45, SEQ ID NO:46) into the genome of the S. cerevisiae strain resulted in production of cucurbitadienol detectable by ESI LC-MS (Figure 7B).
[00188] Subsequently, the cucurbitadienol-producing S. cerevisiae strain was transformed with plasmids carrying S. grosvenorii CYP5491 (SEQ ID NO:14, SEQ ID NO:44), S. grosvenorii CYP 798 (SEQ ID NO:5, SEQ ID NO:73, SEQ ID NO:74), and S. grosvenorii epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO:40) and grown in SC medium minus uracil, leucin, histidine, and tryptophan for plasmid maintenance. Cells were grown for 4 days at 30°C with shaking at 140 rpm. After 4 days, 300 pL of culture samples were mixed with 300 μ!_ of 96% ethanol and incubated for 10 min at 80°C. Samples were then centrifuged, and the supernatant was analyzed by LC-MS. LC- S analyses were performed using a Waters Acquity l-Class UPLC (Waters Corporation, ilford, MA) with Waters Acquity UPLC ®BEH C18 column (2.1 x 50 mm, 1.7 pm particles, 130 A pore size) coupled to a Waters Xevo TQD triple quadropole mass spectrometer with electrospray ionization (ESI) in negative mode. Compound separation was achieved by a gradient of the two mobile phases A (water with 0.1 % formic acid) and B (MeCN with 0.1 % formic acid) by increasing from 20% to 40% B between 0.3 to 3.5 min, increasing to 100% B within 1 .0 min, holding 100% B for 1 .0 min, and re-equilibrating for another 0.6 min. The flow rate was 0.6 mL/min, and the column temperature 55°C. Mogrol (m/z 521.4; [ +FA- Hj") was monitored using SIR (Single Ion Recording) and compared with a standard.
[00189] Expression of S. grosvenorii cucurbitadienol synthase (SEQ ID NO:42, SEQ ID NO:43), CYP5491 , CYP1798 (SEQ ID NO:5, SEQ ID NO:74), CPR4497 (SEQ ID NO:45, SEQ ID NO:46), and epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO:40) resulted in production of mogrol (Figure 16). Expression of CYP5491 alone in cucurbitadienol producing strain is shown in Figure 8. Peaks of 1 -hydroxy-cucurbitadienol (mass 443) and 11-oxo-cucurbitadienol (mass 441 ) are shown. Mogrol was only efficiently produced upon co-expression of CYP1798 with epoxide hydrolase 2. Thus, CYP1798 catalyzes the epoxidation of the 24-25 carbon double bonds of cucurbitadienol and/or 11 -hydroxy-cucurbitadienol.
Example 16: Production of Mogroside V in S. cerevisiae
[00190] Mogroside V was produced in an EXG1 (SEQ ID NO:63, SEQ ID NO:64) knockout, Mat alpha derivative of S. cerevisiae S288C. S. grosvenorii cucurbitadienol synthase (SEQ ID NO:42, SEQ ID NO:43), CYP5491 (SEQ ID NO:81 , SEQ ID NO:44), CYP1798 (SEQ ID NO:5, SEQ ID NO:74), CYP1798-II (SEQ ID NO:86, SEQ ID NO:74), CPR4497 (SEQ ID NO:82, SEQ ID NO:46), epoxide hydrolase 2 (SEQ ID NO:39, SEQ ID NO;40), UGT1576 (SEQ ID NO:83, SEQ ID NO-.48), UGT430 (SEQ ID NO:84, SEQ ID NO:62), UGT1697 (SEQ ID NO:85, SEQ ID NO:68), UGT98 (SEQ ID NO:52, SEQ ID NO:53), and UGT1 1789 (SEQ ID NO:71 , SEQ ID NO:72) were integrated in expression cassettes flanked by growth selection markers into the S. cerevisiae strain by homologous recombination in actively transcribed chromosomal regions. Codon-optimized S. grosvenorii cucurbitadienol synthase (SEQ ID NO:42, SEQ ID NO:43), CYP1798 (SEQ ID NO:5, SEQ ID NO:74), CPR4497 (SEQ ID NO:81 , SEQ ID NO:46), and UGT98 (SEQ ID NO:52, SEQ ID NO:53) were synthesized by Genscript. Codon-optimized CYP5491 (SEQ ID NO:81 , SEQ ID NO:44), UGT1576 (SEQ ID NO:83, SEQ ID NO:48), UGT430 (SEQ ID NO:84, SEQ ID NO:62), and UGT11789 (SEQ ID NO:71 , SEQ ID NO:72) were synthesized as S. cerevisiae gBlocks® gene fragments (Integrated DNA Technologies). Codon-optimized CYP1798-II (SEQ ID NO:86, SEQ ID N0.74) and UGT1697 (SEQ ID NO:85, SEQ ID NO:68) and native CPR4497 (SEQ ID NO:45, SEQ ID NO:46) were synthesized as GeneArt® Strings™ DNA Fragments (Life Technologies). Codon-optimized epoxide hydrolase 1 (SEQ ID NO:37, SEQ ID NO:38) and epoxide hydroase 2 (SEQ ID NO:39, SEQ ID NO:40) were synthesized by DNA2.0.
[00191] The S. cerevisiae strain was grown for 5 days in SC medium at 30°C. The culture was then frozen with liquid nitrogen, and the residue was concentrated to near dryness. The residue was re-suspended in 50% (v/v) ethanol and heated to 55°C for approximately 30 min. Afterwards, the suspension was centrifuged for 15 min at 4400 rpm and 4°C. The supernatant was filtered using a 0.22 μηι SterilFlip filter (Mil!ipore). Figure 17 shows an LC- S chromatogram of the mogroside V-producing strain after filtration. The crude product was then separated on a semi-preparative Agilent 1200 HPLC system. The system was equipped with a Synergi 4u Hydro RP 80A column (Phenomenex: column dimension 250 x 21.2 mm, 4 micron). Elution was carried out using a mobile phase of eluent B (Acetonitrile with 0.02% trifluoroacetic acid) and eluent A (water with 0.02 % trifluoroacetic acid) by increasing the gradient linearly from 5% to 8% B from min 0.0 to 2.0, increasing linearly from 8% to 25% B from min 2.0 to 12.0, 25% to 50% B from min 12.0 to 20.0, 50% to 100 % B from min 20.0 to 32.0, and finally washing with 100% B and re-equilibrating. A flow rate of 15 mL/min was used for the separation, which was conducted at room temperature. All fractions were analyzed by LC-MS, and fractions comprising a single mogroside compound were pooled and dried under vacuum.
[00192] The combined fractions were utilized for NMR analysis. All R experiments were performed in DMSO-d6 at 25°C using a Bruker Avance III 600MHz NMR spectrometer 15 equipped with a 1.7 mm cryogenic TCI probe. The structures were solved by standard homo- and heteronuclear multipulse NMR experiments, namely 1H,1H-COSY, H, 3C-HSQC, and 1H,13C-HMBC experiments. Purified mogroside peaks from the S. cerevisiae production strain were confirmed to be mogroside I E1 , mogroside II A2, mogroside IV A, and the major product, mogroside V. Figure 18A shows an NMR-elucidated structure, 1H NMR spectrum, and 1H and 13C NMR chemical shifts (in ppm) for mogroside V. Figure 18B shows an NMR-elucidated structure, 1H NMR spectrum, and 1H and 13C NMR chemical shifts (in ppm) for mogroside II A2. Figure 18C shows an NMR-eiucidated structure, 1H NMR spectrum, and 1H and 13C N R chemical shifts (in ppm) for mogroside IV A. Figure 18D shows shows an NMR-eiucidated structure, 1H NMR spectrum, and 1H chemical shifts (in ppm) for mogroside I E1.
[00193] Table 3: Sequences disclosed herein (see also Table 2).
SEQ ID NO: 1
Cucurbi a pepo protein sequence
Met Trp Arg Leu Lys Val Gly Ala Glu Ser Val Gly Glu Glu Asp Glu
1 5 10 15
Lys Trp Val Lys Ser Val Ser Asn His Leu Gly Arg Gin Val Trp Glu
20 25 30
Phe Cys Ala Asp Ala Ala Ala Asp Thr Pro His Gin Leu Leu Gin He
35 40 45
Gin Asn Ala Arg Asn His Phe His His Asn Arg Phe His Arg Lys Gin
50 55 60
Ser Ser Asp Leu Phe Leu Ala lie Gin Tyr Glu Lys Glu He Ala Lys
65 70 75 80
Gly Ala Lys Gly Gly Ala Val Lys Val Lys Glu Gly Glu Glu Val Gly
85 90 95
Lys Glu Ala Val Lys Ser Thr Leu Glu Arg Ala Leu Gly Phe Tyr Ser
100 105 110
Ala Val Gin Thr Arg Asp Gly Asn Trp Ala Ser Asp Leu Gly Gly Pro
115 120 125
Leu Phe Leu Leu Pro Gly Leu Val He Ala Leu His Val Thr Gly Val
130 135 140
Leu Asn Ser Val Leu Ser Lys His His Arg Val Glu Met Cys Arg Tyr
145 150 155 160
Leu Tyr Asn His Gin Asn Glu Asp Gly Gly Trp Gly Leu His He Glu
165 170 175
Gly Thr Ser Thr Met Phe Gly Ser Ala Leu Asn Tyr Val Ala Leu Arg
180 185 190
Leu Leu Gly Glu Asp Ala Asp Gly Gly Asp Gly Gly Ala Met Thr Lys
195 200 205
Ala Arg Ala Trp He Leu Glu Arg Gly Gly Ala Thr Ala He Thr Ser 210 215 220
Trp Gly Lys Leu Trp Leu Ser Val Leu Gly Val Tyr Glu Trp Ser Gly 225 230 235 240
Asn Asn Pro Leu Pro Pro Glu Phe Trp Leu Leu Pro Tyr Ser Leu Pro
245 250 255
Phe His Pro Gly Arg Met Trp Cys His Cys Arg Met Val Tyr Leu Pro
260 265 270
Met Ser Tyr Leu Tyr Gly Lys Arg Phe Val Gly Pro He Thr Pro Lys
275 280 285
Val Leu Ser Leu Arg Gin Glu Leu Tyr Thr He Pro Tyr His Glu He
290 295 300
Asp Trp Asn Lys Ser Arg Asn Thr Cys Ala Lys Glu Asp Leu Tyr Tyr 305 310 315 320
Pro His Pro Lys Met Gin Asp He Leu Trp Gly Ser He Tyr His Val
325 330 335
Tyr Glu Pro Leu Phe Thr Arg Trp Pro Gly Lys Arg Leu Arg Glu Lys
340 345 350
Ala Leu Gin Ala Ala Met Lys His He His Tyr Glu Asp Glu Asn Ser
355 360 365
Arg Tyr lie Cys Leu Gly Pro Val Asn Lys Val Leu Asn Met Leu Cys
370 375 380
Cys Trp Val Glu Asp Pro Tyr Ser Asp Ala Phe Lys Leu His Leu Gin
385 390 395 400
Arg Val His Asp Tyr Leu Trp Val Ala Glu Asp Gly Met Arg Met Gin
405 410 415
Gly Tyr Asn Gly Ser Gin Leu Trp Asp Thr Ala Phe Ser He Gin Ala
420 425 430
He Val Ala Thr Lys Leu Val Asp Ser Tyr Ala Pro Thr Leu Arg Lys
435 440 445
Ala His Asp Phe Val Lys Asp Ser Gin He Gin Glu Asp Cys Pro Gly
450 455 460
Asp Pro Asn Val Trp Phe Arg His He His Lys Gly Ala Trp Pro Leu
465 470 475 480
Ser Thr Arg Asp His Gly Trp Leu He Ser Asp Cys Thr Ala Glu Gly 485 490 495
Leu Lys Ala Ser Leu Met Leu Ser Lys Leu Pro Ser Thr Met Val Gly
500 505 510
Glu Pro Leu Glu Lys Asn Arg Leu Cys Asp Ala Val Asn Val Leu Leu
515 520 525
Ser Leu Gin Asn Asp Asn Gly Gly Phe Ala Ser Tyr Glu Leu Thr Arg 530 535 540
Ser Tyr Pro Trp Leu Glu Leu lie Asn Pro Ala Glu Thr Phe Gly Asp 545 550 555 560 lie Val lie Asp Tyr Pro Tyr Val Glu Cys Thr Ala Ala Thr Met Glu
565 570 575
Ala Leu Thr Leu Phe Lys Lys Leu His Pro Gly His Arg Thr Lys Glu
580 585 590 lie Asp Thr Ala lie Gly Lys Ala Ala Asn Phe Leu Glu Lys Met Gin
595 600 605
Arg Ala Asp Gly Ser Trp Tyr Gly Cys Trp Gly Val Cys Phe Thr Tyr 610 615 620
Ala Gly Trp Phe Gly lie Lys Gly Leu Val Ala Ala Gly Arg Thr Tyr 625 630 635 640
Asn Ser Cys Leu Ala lie Arg Lys Ala Cys Glu Phe Leu Leu Ser Lys
645 650 655
Glu Leu Pro Gly Gly Gly Trp Gly Glu Ser Tyr Leu Ser Cys Gin Asn
660 665 670
Lys Val Tyr Thr Asn Leu Glu Gly Asn Lys Pro His Leu Val Asn Thr
675 680 685
Ala Trp Val Leu Met Ala Leu lie Glu Ala Gly Gin Gly Glu Arg Asp 690 635 700
Pro Ala Pro Leu His Arg Ala Ala Arg Leu Leu Met Asn Ser Gin Leu 705 710 715 720
Glu Asn Gly Asp Phe Val Gin Gin Glu lie Met Gly Val Phe Asn Lys
725 730 735
Asn Cys Met lie Thr Tyr Ala Ala Tyr Arg Asn lie Phe Pro lie Trp
740 745 750
Ala Leu Gly Glu Tyr Cys His Arg Val Leu Thr Glu 755 760
SEQ ID NO: 2
Siraitia grosvenorii protein sequence
Leu Glu Arg Asn Arg Leu Cys Asp Ala Val Asn Val Leu Leu Ser Leu 1 5 10 15
Gin Asn Asp Asn Gly Gly Phe Ala Ser Tyr Glu Leu Thr Arg Ser Tyr
20 25 30
Pro Trp Leu Glu Leu lie Asn Pro Ala Glu Thr Phe Gly Asp lie Val
35 40 45
lie Asp Tyr Pro Tyr Val Glu Cys Thr Ser Ala Thr Met Glu Ala Leu 50 55 60
Thr Leu Phe Lys Lys Leu His Pro Gly His Arg Thr Lys Glu lie Asp 65 70 75 80
Thr Ala lie Val Arg Ala Ala Asn Phe Leu Glu Asn Met Gin Arg Thr
85 30 95
Asp Gly Ser Trp Tyr Gly Cys Trp Gly Val Cys Phe Thr Tyr Ala Gly
100 105 110
Trp Phe Gly lie Lys Gly Leu Val Ala Ala Gly Arg Thr Tyr Asn Asn
115 120 125
Cys Leu Ala lie Arg Lys Ala Cys Asp Phe Leu Leu Ser Lys Glu Leu 130 135 140
Pro Gly Gly Gly Trp Gly Glu Ser Tyr Leu Ser Cys Gin Asn Lys Val 145 150 155 160
Tyr Thr Asn Leu Glu Gly Asn Arg Pro His Leu Val Asn Thr Ala Trp
165 170 175
Val Leu Met Ala Leu lie Glu Ala Gly Gin Ala Glu Arg Asp Pro Thr
180 185 190
Pro Leu His Arg Ala Ala Arg Leu Leu lie Asn Ser Gin Leu Glu Asn
195 200 205
Gly Asp Phe Pro Gin Gin Glu lie Met Gly Val Phe Asn Lys Asn Cys 210 215 220
Met lie Thr Tyr Ala Ala Tyr Arg Asn lie Phe Pro lie Trp Ala Leu 225 230 235 240 Gly Glu Tyr Cys Hxs Arg Val Leu Thr Glu
245 250
SEQ ID NO: 3
Siraitia grosvenorii nucleotide sequence
atggaactct tctctaccaa aactgcagcc gagatcatcg ctgttgtctt gtttttctac 60
gctctcatcc ggctattatc tggaagattc agctctcaac agaagagact gccacctgaa 120
gccggtggcg cctggccact gatcggccat ctccatctcc taggtgggtc ggaacctgca 180
cataaaacct tggcgaacat ggcggacgcc tacggaccag tttttacgtt gaaactgggc 240
atgcatacag ctttggttat gagcagttgg gaaatagcga gagagtgctt tactaaaaac 300
gacagaatct ttgcctcccg ccccatagtc actgcctcaa agcttctcac ctataaccat 360
accatgtttg ggttcagcca atatggtcca ttctggcgcc atatgcgcaa aatagccacg 420
cttcaactcc tctcaaacca ccgcctcgag cagctccaac acatcagaat atcggaggtc 480
cagacttcga ttaagaaact gtacgagttg tgggtcaaca gcagaaataa tggaggcgag 540
aaagtgttgg tggagatgaa gacgtggttc ggaggcataa ccttgaacac catattcagg 600
atggtggtcg gaaagcgatt ctcgactgct ttcgaaggca gtggtggcga acggtatcgg 660
aaggcgttga gggattctct tgaatggttt ggggcattcg ttccgtcaga ttcattcccg
720
tttttaagat ggttggatt gggaggatat gagaaggcga tgaagaagac ggcgagtgtg 780
ctggacgagg tgcttgataa atggctcaaa gagcatcagc agaggagaaa ctccggtgaa 840
ctggagacgg aggagcacga cttcatgcac gtgatgctgt ctattgttaa ggatgatgaa 900
gaactatccg gctacgatgc cgatacagtc acaaaagcta catgtttgaa tttaatagtt 960
ggtggattcg acactacaca agtaactatg acatgggctc tttctttgct tctcaacaat 1020
gaagaggtat taaaaaaggc ccaacttgaa ctagacgaac aagttggaag agagaggttt 1080
gtggaagagt ccgatgttaa aaatctgtta tatctccagg ccatcgtgaa ggaaactttg
1140
cgtttgtacc cttcagcgcc aatctcgaca tttcatgagg ccatggaaga ttgcactgtt 1200
tctggctacc acatcttttc agggacgcgt ttgatggtga atcttcaaaa gcttcaaaga 1260
gatccacttg catgggagga tccatgtgac tttcgaccgg agagatttct gacaactcat 1320 aaggatttcg atcttagagg acatagtcct caattgatac catttgggag tggtcgaaga
1380
atatgccctg gcatctcgtt tgccattcaa gttttgcatc ttacgcttgc aaatctactt 1440
catgggtttg acattggaag gccatctcat gaaccaatcg atatgcagga gagtaaagga 1500
ctaacgagta ttaaaacaac tccacttgag gttgttttag ctccacgcct tgctgctcaa
1560
gtttatgagt
1572
SEQ ID NO: 4
Siraitia grosvenorii nucleotide SEQUENCE
atgccgatcg cagaaggtgc agtctctgat ttgtttggtc gcccactctt ctttgcacta
60
tatgattggt tcttagagca tggatctgtt tataaacttg cctttggacc aaaagccttt 120
gttgttgtat cagatcccat tgtggcaaga tatattcttc gagaaaatgc atttggttat 180
gacaagggag tgcttgctga tattttagaa ccgataatgg gtaaaggact aataccagct
240
gaccttggca cttggaagca gaggagacga gttattgctc caggattcca tgccttgtac 300
ttggaagcta tgaccaaagt atttgccaat tgttcagaac gatcaatatt gaaattggag
360
aagcttctag gagaaggtga actacaggag aataaaacca ttgagttgga tatggaagca
420
gagttttcaa gtttggctct tgatatcatt ggactcggtg ttttcaacta tgattttggt 480
tctgtaacca aagaatctcc ggtgattaag gctgtatatg ggactctttt tgaagcagag
540
catagatcga ctttctatat cccatattgg aaagtacctt tggcaaggtg gatagtccca 600
aggcagcgta aattccatgg tgaccttaag gttattaatg agtgtcttga tggcctaata 660
cgcaacgcaa gagaaacccg agacgaaacg gatgttgaga aattgcagca aagggactac
720
ttaaatctca aggatgccag tcttttgcgt ttcttagttg atatgcgggg agctgatgtt 780
gatgatcgcc agcttaggga cgatctgatg acgatgctta ttgctggcca tgaaacaact 840
gctgctgtgc ttacatgggc tgtttttttg cttgcacaaa atccttcaaa aatgaaaaaa
900
gcgcaagcag agattgattt ggttcttggc atggggaggc caacttttga atcatttaaa 960
gcattgaagt acatcagact tatcgttgca gagactcttc gtttgtttcc tcagcctcca
1020
ttgctgataa gacgagctct caaatcagat atattaccag gaggatacaa tggtgacaaa 1080 actggatatg caattcctgc agggactgac atcttcatct ctgtttacaa tctccacaga 1140
tctccctact tctgggataa tcctcaagaa tttgaaccag gagatttca agtaaagagg 1200
gcaagcgagg gaattgaagg atgggatggt ttcgacccat Ctagaagccc tggagctcta 1260
tacccgaatg agattgtagc agacttttcc ttcttaccat ttggtggagg ccctagaaaa 1320
tgtgtgggag atcaatttgc tctaatggag tcaactatag ca;ttggccat gttactgcag 1380
aagtttgatg tggagctaaa aggaagtcca gaatctgtag actagttac tggagccaca 1440
atacatacca aaagtgggtt gtggtgcaaa ctgagaagaa gatcacaagt aaactga 1497
SEQ ID NO: 5
Codon-optimized DNA sequence encoding CYP1798
atggaaatgt cctcaagtgt cgcagccaca atcagtatct ggatggtcgt cgtatgtatc 60
gtaggtgtag gttggagagt cgtaaattgg gtttggttga gaccaaagaa attggaaaag 120
agattgagag aacaaggttt ggccggtaat tcttacagat tgttgttcgg tgacttgaag 180
gaaagagctg caatggaaga acaagcaaat tcaaagccta taaacttctc ccatgacatc 240
ggtccaagag ttttcccttc aatgtacaag accatccaaa actacggtaa aaactcctac 300
atgtggttag gtccataccc tagagtccac atcatggatc cacaacaatt gaagaccgtt 360
tttactttgg tctacgacat tcaaaagcca aatttgaacc ctttgattaa attcttgtta 420
gatggtatcg ttacacatga aggtgaaaag tgggctaagc acagaaagat tattaaccca 480
gcattccatt tggaaaagtt gaaggatatg atacctgctt tctttcactc atgtaatgaa 540
atcgtcaacg aatgggaaag attgatttca aaagaaggtt cctgcgaatt ggatgtaatg 600
ccttatttgc aaaatttggc cgctgacgcc atttcaagaa ccgcttttgg ttcttcatac 660
gaagaaggta aaatgatctt ccaattgttg aaggaattga ctgatttggt tgtcaaggta 720
gcttttggtg tttatattcc aggttggaga ttcttgccta caaagagtaa caacaaaatg 780
aaggaaatta atagaaaaat caagtctttg ttgttgggta tcattaacaa gagacaaaag 840
gcaatggaag aaggtgaagc cggtcaatct gatttgttgg gtatattaat ggaaagtaat 900
tctaacgaaa tccaaggtga aggtaataac aaggaagatg gcatgtctat tgaagacgtc 960 atcgaagagt gtaaggtatt ttatataggt ggtcaagaaa ctacagcaag attattgatc 1020
tggactatga tattgttgtc cagtcataca gaatggcaag aaagagccag aaccgaagtc
1080
ttgaaggtat ttggtaataa gaaaccagat ttcgacggtt tgtcaagatt gaaggtagtt 1140
actatgatct tgaacgaagt tttaagattg tacccacctg cttccatgtt gacaagaatc 1200
atccaaaagg aaacaagagt tggtaaatta accttgccag caggtgttat cttgataatg
1260
cctatcatct tgatacatag agatcacgac ttgtggggtg aagatgctaa cgagtttaaa 1320
ccagaaagat tcagtaaagg tgtttctaag gcagccaaag tccaaccagc ctttttccct
1380
tttggttggg gtcctagaat ttgcatgggt caaaacttcg ctatgatcga agctaagatg
1440
gcattgagtt tgatcttgca aagattttct ttcgaattgt cttcatccta cgttcatgca 1500
ccaactgtcg tcttcactac acaaccacaa cacggtgccc acatcgtttt gagaaagtta 1560
tga
1563
SEC ID NO: 6
Siraitia grosvenorii nucleotide sequence
atggaaccac aaccaagtgc ggaattcaac tggaatcaca gcctaagcac cgtcgctatc
60
ggtgtcattg ccattatttt cttccgtttt ctcgtcaaaa gagtcaccgg cgccggtgag
120
cgaaagggtc cgaagccgcc aaaagtagcc ggagggtggc ctctaattgg ccacctccct
180
ctcctcggag gacctgaact gccccatgtc aaactgggtg gtttggctga taaatatggt
240
ccaatcttct cgatccggct gggtgtccac tccgccgtcg tgataaacag ttgggaggcg
300
gcgaaacagt tattaaccaa ccatgacgtc gccgtctctt cccgccccca aatgctcggc
360
ggaaaac cc tgggctacaa ctacgccgtg tttggtttcg gaccctacgg ctcttactgg
420
cgcaacatgc gcaagataac cacgcaagag cttctatcca atagcagaat ccagctccta
480
agagacgttc gagcgtcaga agtgaaccaa ggcataaaag agctctacca gcactggaaa
540
gaaagaagag acggtcacga ccaagccttg gtggaactgc agcagtgggt cggggacttg
600
actatgaatc tgattctcgg agtcatcgcc gggaaaaggt tctttggagc tgcagcaacg
660
gtagacgagg aagaggcgcg acggagccat aaagcattga aggagttgtt acattatatg 720 gggctttttc tactgggtga tgctgttcca tatctaggat ggttggacgt cggcggccat 780
gtgaaggcga tgaagaaaac ttcaaaagaa ttggaccgta tgttaacaca gtggttggag 840
gagcacaaga aggaaggacc caagaaagat cataaagact tcatggacgt gatgctttca 900
gttctcaatg aaacatccga tgttctttca gataagaccc atggcttcga tgctgatacc 960
atcatcaaag ctacatgtat gacgatggtt ttaggaggga gtgatacgac ggcggtggtt 1020
gtgatatggg caatctcgct gctgctgaat aatcgccctg cgttgagaaa agtgcaagaa
1080
gaactggaag cccatatcgg ccgagacaga gaactggagg aatcggatct cggtaagcta 1140
gtgtatttgc aggcagtcgt gaaggagaca ttgcggctgt acggagccgg aggccttttc 1200
tttcgtgaaa ccacagagga tgtcaccatc gacggatt.cc atgtcgagaa agggacatgg
1260
ctgttcgtga acgtggggaa gatccacaga gatgggaagg tgtggccgga gccaacggag 1320
ttcaaaccgg agaggt ct gacgacccac aaagattttg atctgaaggg ccagcggttt 1380
gagctcatcc ctttcggggg aggaagaaga tcgtgccctg gaatgtcttt tgggctccaa 1440
atgctacagc ttattttggg taaactgctt caggcttttg atatatcgac gccgggggac 1500
gccgccgttg atatgaccgg atccattgga ctgacgaaca tgaaagccac tccattggaa 1560
gtgctcatca ccccgcgctt gcctctttcg ctttacgatt ga 1602
SEQ ID NO: 7
Siraitia grosvenorii DNA sequence
atggagactc ttcttcttca tcttcaatcg ttatttcatc caatttcctt cactggtttc 60
gttgtcctct ttagcttcct gttcctgctc cagaaatggt tactgacacg tccaaactct 120
tcatcagaag cctcaccccc ttctccacca aagcttccca tcttcggaca ccttctaaac 180
ctgggtctgc atccccacat caccctcgga gcctacgctc gccgctatgg ccctctcttc 240
ctcctccact tcggcagcaa gcccaccatc gtcgtctctt ctgccgaaat cgctcgcgat
300
atcatgaaga cccacgacct cgtcttcgcc aaccgtccta aatcaagcat cagcgaaaag 360
attctttacg gctccaaaga tttagccgca tctccttacg gcgaatactg gaggcagatg 420
aaaagcgttg gcgtgct ca tcttttgagc aacaaaaggg ttcaatcctt tcgctctgtc 480 agagaagaag aagtcgaact gatgatccag aagatccaac agaaccccct atcagttaat 540
ttaagcgaaa tattctctgg actgacgaac gacatagttt g agggtggc tttagggaga
600
aagtatggcg tgggagaaga cggaaagaag ttccggtctc ttctgctgga gtttggggaa
660
gtattgggaa gtttcagtac gagagacttc atcccgtggc tgggttggat tgatcgtatc 720
agtgggctgg acgccaaagc cgagagggta gccaaagagc tcgatgcttt ctttgacaga
780
gtgatcgaag atcacatcca tctaaacaag agagagaata atcccgatga gcagaaggac
840
ttggtggatg tgctgctttg tgtacagaga gaagactcca tcgggtttcc ccttgagatg 900
gatagcataa aagctttaat cttggacatg tttgctgcag gcacagacac gacatacacg
960
gtgttggagt gggcaatgtc ccaactgttg agacacccag aagcgatgaa gaaactgcag 1020
agggaggtca gagaaatagc aggtgagaaa gaacacgtaa gtgaggatga tttagaaaag 1080
atgcattact tgaaggcagt aatcaaagaa acgctgcggc tacacccacc aatcccactc
1140
ctcgtcccca gagaatcaac ccaagacatc aggttgaggg ggtacgatat cagaggcggc
1200
acccgggtta tgatcaatgc atgggccatc ggaaga 1236
SEQ ID NO: 8
Sxraitia grosvenorii sequence
atgtcgatga gtagtgaaat tgaaagcctc tgggttttcg cgctggcttc taaatgctct
60
gctttaacta aagaaaacat cctctggtct ttactcttct ttttcctaat ctgggtttct 120
gtttccattc tccactgggc ccatccgggc ggcccggctt ggggccgcta ctggtggcgc
180
cgccgccgca gcaattccac cgccgctgct attc cggcc cgagaggcct ccccctcgtc
240
ggcagcatgg gcttgatggc cgacttggcc caccaccgga ttgccgccgt ggctgactcc 300
ttaaacgcca cccgcctcat ggccttttcg ctcggcgaca ctcgcgtgat cgtcacatgc 360
aaccccgacg tcgccaaaga gattctcaac agctccctct tcgccgaccg ccccgttaag
420
gagtccgctt actccttgat gttcaaccgc gccattgggt tcgcccccta tggcctttac 480
tggcggaccc tccgccgcat cgcttcccac cacctcttct gccccaagca aatcaagtcc 540
tcccagtccc agcgccgcca aatcgcttcc caaatggtcg caatgttcgc aaaccgcgat
600 gccacacaga gcctctgcgt tcgcgactct ctcaagcggg cttctctcaa caacatgatg 660
ggctctgttt tcggccgagt ttacgacctc tctgactcgg ctaacaatga cgtccaagaa 720
ctccagagcc tcgtcgacga aggctacgac ttgctgggcc tcctcaactg gtccgaccat 780
ctcccatggc tcgccgactt cgactctcag aaaatccggt tcagatgctc ccgactcgtc 840
cccaaggtga accacttcgt cggccggatc atcgccgaac accgcgccaa atccgacaac 900
caagtcctag atttcgtcga cgttttgctc tctctccaag aagccgacaa actctctgac 960
tccgatatga tcgccgttct ttgggaaatg atttttcgtg ggacggacac ggtggcagtt 1020
ttaatcgagt ggatactggc caggatggta cttcacaacg atatccaaag gaaagttcaa 1080
gaggagctag ataacgtggt tgggagtaca cgcgccgtcg cggaatccga cattccgtcg 1140
ctggtgtatc taacggctgt ggttaaggaa gttctgaggt tacatccgcc gggcccactc
1200
ctgtcgtggg cccgcctagc catcactgat acaatcatcg atgggcatca cgtgccccgg 1260
gggaccaccg ctatggttaa catgtggtcg atagcgcggg acccacaggt ctggtcggac 1320
ccactcgaat ttatgcccca gaggtttgtg tccgaccccg gtgacgtgga gttctcggtc 1380
atgggttcgg atctccggct ggctccgttc gggtcgggca gaaggacctg ccccgggaag 1440
gccttcgcct ggacaactgt caccttctgg gtggccacgc ttttacacga cttcaaatgg 1500
tcgccgtccg atcaaaacga cgccgtcgac ttgtcggagg tcctcaagct ctcctgcgag 1560
atggccaatc ccctcaccgt taaagtacac ccaaggcgca gtttaagctt ttaa
1614
SEQ ID NO: 9
Siraitia grosvenorii D A sequence
atggatggt ttcttccaac agtggcggcg agcgtgcctg tgggagtggg tgcaatattg
60
ttcacggcgt tgtgcgtcgt cgtgggaggg gttttggttt atttctatgg accttactgg 120
ggagtgagaa gggtgcctgg tccaccagct attccactgg tcggacatct tcccttgctg 180
gctaagtacg gcccagacgt tttctctgtc cttgccaccc aatatggccc tatcttcagg 240
ttccatatgg gtaggcagcc attgataatt atagcagacc ctgagctttg taaagaagct 300
ggtattaaga aattcaagga catcccaaat agaagtgtcc cttctccaat atcagcttcc 360 cctcttcatc agaagggtct tttcttcaca agggatgcaa gatggtcgac aatgcggaac 420
acgatattat cggtctatca gtcctcccat ctagcgagac taatacctac tatgcaatca 480
atcattgaaa ctgcaactca aaatctccat tcctctgtcc aggaagacat ccctttctcc 540
aatctctccc tcaaattgac caccgatgtg attggaacag cagccttcgg tgtcaacttt 600
gggctctcta atccacaggc aaccaaaact tgtgctacca acggccaaga caacaaaaat 660
gacgaagttt cagacttcat caatcaacac atctactcca caacgcagct caagatggat 720
ttatcaggtt ccttctcaat catacttgga ctgcttgtcc ctatactcca agaaccattt 780
agacaagtcc taaagagaat accattcacc atggactgga aagtggaccg gacaaatcag 840
aaattaagtg gtcggcttaa tgagattgtg gagaagagaa tgaagtgtaa cgatcaaggt 900
tcaaaagact tcttatcgct cattttgaga gcaagagagt cagagacagt atcaaggaat 960
gtcttcactc cagactacat cagtgcagtt acgtatgaac acctacttgc tgggtcggct 1020
accacggcgt ttacgttgtc ttctattgta tatttagttg ctgggcatcc agaagtcgag 1080
aagaagttgc tagaagagat tgacaacttt ggtccatccg atcagatacc aacagctaat 1140
gatcttcatc agaagtttcc atatcttgat caggtgatta aagaggctat gaggttctac 1200
actgtttccc ctctagtagc cagagaaaca gctaaagatg tggagattgg tggatatctt 1260
cttccaaagg ggacatgggt ttggttagca cttggagttc ttgccaagga tccaaagaac 1320
tttccagaac cagataaatt caaaccagag aggtttgatc caaatgaaga agaggagaaa 1380
caaaggcatc cttatgcttt aatccccttt ggaattggtc ctcgagcatg cattggtaaa
1440
aaattcgccc ttcaggagtt gaagctctcg ttgattcatt tgtacaggaa gtttgtattt 1500
cggcat
1506
SEQ ID NO: 10
Siraitia grosvenorii DNA sequence
atggaaatca ttttatcata tctcaacagc tccatagctg gactcttcct cttgcttctc 60
ttctcgtttt ttgttttgaa aaaggctaga acctgtaaac gcagacagcc tcctgaagca 120
gccggcggat ggccgatcat cggccacctg agactgctcg ggggttcgca acttccccat 180 gaaaccttgg gagccatggc cgacaagtat ggaccaatct tcagcatccg agttggtgtc 240
cacccatctc ttgttataag cagttgggaa gtggctaaag agtgctacac caccctcgac 300
tcagttgtct cttctcgtcc caagagtttg ggtggaaagt tgttgggcta caacttcgcc 360
gcttttgggt tcaggcctta tgattccttt taccggagta tccgcaaaac catagcctcc 420
gaggtgctgt cgaaccgccg tctggagttg cagagacaca ttcgagtttc tgaggtgaag 480
agatcggtga aggagcttta caatctgtgg acgcagagag aggaaggctc agaccacata 540
cttattgatg cggatgaatg gattggtaat attaatttga acgtgattct gatgatggtt 600
tgtgggaagc ggtttcttgg cggttctgcc agcgatgaga aggagatgag gcggtgtctc 660
aaagtctcga gagatttctt cgatttgaca gggcagttta cggtgggaga tgccattcct 720
ttcctgcgat ggctggattt gggtggatat gcgaaggcga tgaagaaaac tgcaaaagaa 780
atggactgtc tcgttgagga atggctggaa gaacaccgcc ggaagagaga ctccggcgcc 840
accgacggtg aacgtgactt eatggatgtg atgctttcga ttcttgaaga gatggacctt 900
gctggctacg acgctgacac agtcaacaaa gccacatgcc tgagcattat ttctggggga 960
atcgatacta taacgctaac tctgacatgg gcgatctcgt tattgctgaa caatcgagag 1020
gcactgcgaa gggttcaaga ggagg ggac atccatgtcg gaaacaaaag gcttgtggat 1080
gaatcagact tgagcaagct ggtgtatctc caagccgtcg tgaaagagac attaaggttg 1140
tacccagcag ggccgctgtc gggagctcga gagttcagtc gggactgcac ggtcggaggg 1200
tatgacgtgg ccgccggcac acggctcatc acaaaccttt ggaagataca gacggaccct 1260
cgggtgtggc cggagccact tgagttcagg ccggagaggt. ttctgagcag ccaccagcag
1320
ttggatgtga agggccagaa ctttgaactg gccccatttg gttgtggaag aagagtgtgc 1380
cctggggcgg ggcttggggt tcagatgacg cagttggtgc tggcgagtct gattcattcg
1440
gtggaacttg gaactcgctc cgatgaagcg gtggacatgg ctgctaagtt tggactcaca
1500
atgtacagag ccacccctct tcaggctctc gtcaagccac gcctccaagc cggtgcttat 1560
tcatga
1566
SEQ ID NO: 11 grosvenorii DNA sequence
atgggtgtat tgtccatttt attattcaga tattccgtca agaagaagcc attaagatgc
60
ggtcacgatc aaagaagtac cacagatagt ccacctggtt caagaggttt gccattgata 120
ggtgaaactt tgcaattcat ggctgctatt aattctttga acggtgtata cgatttcgtt
180
agaataagat gtttgagata cggtagatgc tttaagacaa gaatcttcgg tgaaacccat 240
gtttttgtct caactacaga atccgctaag ttgatcttga aggatggtgg tgaaaaattc
300
accaaaaagt acatcagatc aatcgctgaa ttggttggtg acagaagttt gttatgtgca 360
tctcatttgc aacacaagag attgagaggt ttgttgacta atttgttttc tgccacattc
420
ttggcttctt tcgtaactca attcgatgaa caaatcgttg aagcttttag atcatgggaa 480
tccggtagta ccataatcgt tttgaacgaa gcattgaaga tcacttgtaa ggccatgtgc
540
aaaatggtca tgtccttaga aagagaaaac gaattggaag ctttgcaaaa ggaattgggt 600
catgtttgtg aagctatgtt ggcatttcca tgcagattcc ctggtacaag atttcacaat
660
ggtttgaagg caagaagaag aatcattaaa gttgtcgaaa tggccat ag agaaagaaga 720
agatctgaag ctcctagaga agatttcttg caaagattgt tgacagaaga aaaggaagaa
780
gaagacggtg gtggtgtttt aagtgatgcc gaaattggtg acaacatatt gacaatgatg 840
atcgcaggtc aagataccac tgcctctgct attacctgga tggtcaagtt tttggaagaa
900
aaccaagatg tattgcaaaa cttaagagac gaacaattcg aaatcatggg taaacaagaa 960
ggttgtggtt catgcttctt gacattagaa gatttgggta atatgtccta tggtgcaaaa
1020
gtagttaagg aatcattgag attagcctcc gtcgtaccat ggtttcctag attggtttta 1080
caagattctt tgatccaagg ttacaaaatt aaaaagggtt ggaacgtcaa catagacgta
1140
agatctttac attcagatcc atccttgtat aatgacccaa caaagtttaa ccctagtaga 1200
ttcgatgacg aagctaaacc ttactcattt ttggcattcg gtatgggtgg tagacaatgt
1260
ttgggta ga acatggcaaa ggccatgatg ttggttttct tgcacagatt ggtcacctca
1320
ttcagatgga aggttataga ttccgactct tcaatcgaaa aatgggcttt gttctctaag
1380
ttgaagtcag gttgccctat cgtagttacc cacatcggtt cctaa 1425 SEQ ID NO: 12
Siraitia grosvenorii DNA sequence
atggatttct actggatctg tgttcttctg ctttgcttcg catggttttc cattttatcc 60
cttcactcga gaacaaacag cagcggcact tccaaacttc ctcccggacc gaaacccttg 120
ccgatcatcg gaagcctttt ggctctcggc cacgagcccc acaagtcttt ggctaatctc 180
gctaaatctc atggccctct tatgacctta aagctcggcc aaatcaccac cgtcgtagtt
240
tcctccgctg ccatggctaa gcaagttctc caaacgcacg accagtttct gtccagcagg
300
accgttccag acgcaatgac ctctcacaac cacgatgctt tcgcactccc atggattccg 360
gtttcacccc tctggcgaaa ccttcgacga atatgcaaca accagttgtt tgccggcaag 420
attctcgacg ccaacgagaa tctccggcga accaaagtgg ccgagctcgt atccgatatc 480
tcgagaagtg cattgaaagg tgagatggtg gattttggaa acgtggtgtt cgtcacttcg 540
ctcaatctgc tttccaatac gattttctcg gtggatttct tcgacccaaa ttctgaaatt
600
gggaaagagt tcaggcacgc agtacgaggc ctcatggaag aagctgccaa accaaatttg
660
ggggattatt tccctctgct gaagaagata gatcttcaag gaataaagag gagacagacc
720
acttacttcg atcgggtttt taatgttttg gagcacatga tcgaccagcg tcttcagcag 780
cagaagacga cgtctggttc tacctccaac aacaacaacg acttactgca ctaccttctc 840
aacctcagca acgaaaatag cgacatgaaa ttggggaaac ttgagctgaa acacttctta 900
ttggtgctat tcgtcgctgg gactgaaacg agttctgcaa cactgcaatg ggcaatggca
960
gaactactaa gaaacccaga aaagttagca aaagctcaag cggagaccag gcgggtgatt 1020
gggaaaggga acccaattga agaatcagac atttcgaggc tgccttatct gcaagcagtg
1080
gtgaaagaaa ctttcagatt gcacacacca gcgccatttc tactgccgcg caaagcacta 1140
caggacgtgg aaattgcagg tttcacagtc ccaaaggacg ctcaggtact ggtaaattta 1200
tgggctatga gcagagattc aagcatctgg gagaacccag agtggttcga gccagaaagg
1260
tttttggagt cggagctgga cgttagaggg agagattttg agctgatccc gttcggcggt 1320
gggcggagga tttgccccgg tctgccgttg gcgatgagaa tgttgcattt gattttgggt
1380 tctctcatcc acttctttga ttggaagctt gaagatgggt gtcggccgga agacgtgaaa
1440
atggacgaaa agcttggcct cactctggag ttggcttttc ccctcacagc cttgcctgtc 1500
cttgtctaa
1509
SEQ ID NO: 1
Siraitia gr s enorii DN sequence
atgtcctcct gcggtggtcc aactcctttg aatgttatcg gtatcttatt acaatcagaa
60
tcctccagag cctgcaactc agacgaaaac tcaagaattt tgagagattt cgtaacaaga 120
gaagttaacg ctttcttatg gttgtccttg atcactatca cagcagtttt gatcagtaaa
180
gttgtcggtt tgtttagatt gtggtctaag gcaaagcaat tgagaggtcc accttgtcca
240
tcattctacg gtcattctaa gatcatctca agacaaaatt tgactgattt gttatatgac
300
tcccacaaaa agtacggtcc agtagttaaa ttgtggttag gtcctatgca attgttagtc
360
tccgtaaagg aaccaagttt gttgaaggaa atattggtta aagctgagga taagttgcct 420
ttaacaggta gagcctttag attggctttc ggtagatctt cattatttgc atccagtttc
480
gaaaaggttc aaaacagaag acaaagattg gccgaaaagt tgaataagat cgcattccaa 540
agagccaaca tcattccaga aaaggccgta gcttgtttca tgggtagagt tcaagatttg
600
atgatagaag aatctgtcga ctgtaataag gtttctcaac atttggcttt tactttgtta
660
ggttgcacat tgtttggtga cgccttctta ggtt.ggt.cta aggctacaat ctatgaagaa
720
ttgttgatga tgatcgctaa ggacgcatcc ttttgggcta gttatagagt taccccaatc 780
tggaagcaag gtttctggag ataccaaaga ttgtgtatga agttgaagtg cttgactcaa
840
gatatcgttc aacaatacag aaagcattac aagttgtttt ctcactcaca aaaccaaaac
900
ttacacaacg aaaccaagtc aactggtgtt gaagtcgctt ttgatattcc accttgtcct
960
gctgcagacg ttagaaattc ttgctttttc tacggtttga acgatcatgt taacccaaac 1020
gaagaacctt gtggtaatat tatgggtgtc atgtttcacg gttgcttgac tacaacctct 1080
ttgatcgcat caatcttgga aagattggcc actaacccag aaatccaaga aaagattaat
1140 tctgaattga acttagttca aaagggtcca gtcaaggatc atagaaagaa tgttgacaac 1200
atgcctttgt tattggcaac aatctatgaa tcagctagat tattgccagc aggtccttta 1260
ttgcaaagat gtcctttgaa gcaagatttg gttttgaaaa caggtatcac cattccagct 1320
ggtaccttgg tcgtagttcc tattaaattg gttcaaatgg atgactcttc atggggttca 1380
gatgccaatg agtttaatcc atacagattc ttgtccatgg cttgtaatgg tattgacatg 1440
atacaaagaa cccctttagc tggtgaaaac attggtgacc aaggtgaagg ttcatttgtc 1500
ttgaatgacc caattggtaa cgtaggtttc ttaccttttg gtttcggtgc aagagcctgc 15S0
gttggtcaaa agtttataat ccaaggtgtc gctactttgt tcgcaagttt gttggcccat 1620
tacgaaatta aattgcaatc cgagagtaag aatgattcta aaccatccag taacacctct 1680
gccagtcaaa tcgtcccaaa ctcaaaaatc gtattcgtaa gaagaaactc ataa
SEQ ID NO: 14
Siraitia grosvenorii sequence
atgtggactg tcgtgctcgg tttggcgacg ctgtttgtcg cctactacat ccattggatt 60
aacaaatgga gagattccaa gttcaacgga gttctgccgc cgggcaccat gggtttgccg 120
ctcatcggag agacgattca actgagtcga cccagtgact ccctcgacgt tcaccctttc 180
atccagaaaa aagttgaaag atacgggccg atcttcaaaa catgtctggc cggaaggccg 240
gtggtggtgt cggcggacgc agagttcaac aactacataa tgctgcagga aggaagagca
300
gtggaaatgt ggtatttgga tacgctctcc aaatttttcg gcctcgacac cgagtgg tc 360
aaagctctgg gcctcatcca caagtacatc agaagcatta ctctcaatca cttcggcgcc 420
gaggccctgc gggagagatt tcttcctttt attgaagcat cctccatgga agcccttcac 480
tcctggtcta ctcaacctag cgtcgaagtc aaaaatgcct ccgctctcat ggtttttagg 540
acctcggtga ataagatgtt cggtgaggat gcgaagaagc tatcgggaaa tatccctggg 600
aagttcacga agcttctagg aggatttctc agtttaccac tgaattttcc cggcaccacc 660
taccacaaat gcttgaagga tatgaaggaa atccagaaga agctaagaga ggttgtagac 720
gatagattgg ctaatgtggg ccctgatgtg gaagatttct tggggcaagc ccttaaagat 780 aaggaatcag agaagttcat ttcagaggag ttcatcatcc aactgttgtt ttctatcagt 840
tttgctagct ttgagtccat ctccaccact cttactttga ttctcaagct ccttgatgaa
900
cacccagaag tagtgaaaga gttggaagct gaacacgagg cgattcgaaa agctagagca 960
gatccagatg gaccaattac ttgggaagaa tacaaatcca tgacttttac attacaagtc
1020
atcaatgaaa ccctaaggtt ggggag gtc acacctgcct tgttgaggaa aacagttaaa 1080
gatcttcaag taaaaggata cataatcccg gaaggatgga caataatgct tgtcaccgct
1140
tcacgtcaca gagacccaaa agtctataag gaccctcata tcttcaatcc atggcgttgg 1200
aaggacttgg actcaattac catccaaaag aacttcatgc cttttggggg aggcttaagg
1260
cattgtgctg gtgctgagta ctctaaagtc tacttgtgca ccttcttgca catcctctgt 1320
accaaatacc gatggaccaa acttggggga ggaaggattg caagagctca tatattgagt
1380
tttgaagatg ggttac tgt agttcaca cccaaqqi ga 1422
SEQ ID NO: 15
Siraitia grosvenorii DNA sequence
atgaagatga agatggaatc catgcgcacc tccctggata tctccgacca tgacatactt 60
ccaagggttt atcctcatgt tcacctatgg atcaacaaat atgggaaaaa cttcattcag
120
tggaatggca acgtagctca gttgattgtt tcggatcctg acacgatcaa ggagatactc 180
caaaaccgag aacaagctgt tcccaaaata gatctcagcg gagatgcacg gaggatattc
240
gggaatgggc tttcgacttc tgacggtgaa aaatgggcta aggctcgaag aatcgctgat 300
tacgctttcc a ggggatct cctaagaaat atggggccaa ccatggtttc ctgtgctgag
360
gcaatggtgg aaaagtggaa gcatcatcaa ggcaaagagc ttgatttgtt cgaagagttt 420
aaggtgctca cttcagatat cattgcacat acagcctttg gaagcagtta tt ggaaggg
480
aaagttattt ttcagactct aagtaagctg agcatgatat tatttaagaa tcagttcaaa 540
cgaaggattc ctgttatcag caagttcttc agatcaaagg atgcgaggga gggagaggag
600
ctggaaagaa ggttgaaaaa ttccataatt tcaataatgg aaaagagaga agagaaggtg 660 ataagtggtg aagcagataa ctatggtaat gattttcttg gatt ictttt gaaggcaaag 720
aatgagcctg accagaggca gaggatttct gttgatgatg tagt' fgatga atgcaaaaca 780
gtttacttcg ctgggcaaga aactacaagt gttttgcttg cttq. faccgc ctttctttta 840
gcaactcatg agcattggca agaagaagca agaaaggaag faatat gtttggcaac 900
aagaatccaa ctttagaagg catcacaaaa ttaaagatta :atgat catcaaggaa 960
tctctaagat tatatcctcc agccccgccc atgtcaagga aaaaa ggaagtcaga 1020
ttggggaagc tggttctccc ccccaacatt caagtaagca .actat tgcagttcat 1080
catgatactg caatatgggg tgaagatgcc catgtattca aacca .gaaag attttctgaa
1140
ggaacagcta aagatatccc atcagctgca tacatcccat ttggctttgg tcctcgaaac 1200
tgcatcggca atatcttggc catcaacgaa actaagattg cactgtcgat gattctacaa 1260
cgattttctt tcaccatctc cccggcctac gtccacgcac ctttccagtt cctcactatc 1320
tgcccccaac acggggttca ggtaaagctt cagtccctat taagtgaaag gtga 1374
SEQ ID NO: 16
Siraitia grosvenorii DNA sequence
atggaagctg aatttggtgc cggtgctact atggtattat ccgttgtcgc aatcgtcttc
60
tttttcacat ttttacactt gtttgaatct ttctttttga agccagatag attgagatct 120
aagttgagaa agcaaggtat tggtggtcca tctccttcat ttttgttggg taatttgtca 180
gaaattaaat ccatcagagc tttgtcttca caagctaaga acgcagaaga tgcctctgct 240
ggtggtggtg gtggttccgc cagtatagct catggttgga cttcaaattt gtttcctcac 300
ttagaacaat ggagaaacag atatggtcca attttcgtat actccagtgg tacaatccaa 360
atcttgtgta tcacagaaat ggaaaccgtt aaggaaatct ctttgtcaac ctccttgagt 420
ttaggtaaac ctgctcattt gtctaaggat agaggtccat tgttaggttt gggtatctta 480
gcctcttcag gtcctatttg ggttcaccaa agaaagatca tcgctccaca attgtatttg 540
gataaagtaa agggtatgac ctcattgatg gttgaaagtg caaattctat gttaagatcc 600
tgggaaacta aagttgaaaa tcatggtggt caagccgaaa ttaacgtcga tggtgacttg 660 agagcattaa gtgccgatat catttctaag gcttgctttg gttcaaacta ttccgaaggt
720
gaagaaattt tcttgaagtt gagagcattg caagttgtca tgagtaaggg ttctattggt 780
atacctggtt ttagatacat accaactaaa aataacagag aaatgtggaa gttggaaaag
840
gaaatcgaat caatgatctt gaaggttgcc aacgaaagaa cacaacattc cagtcacgaa 900
caagatttgt tgcaaatgat tttggaaggt gcaaagtctt tgggtgaaga caataagagt 960
atgaacatat caagagacaa gtttattgtt gacaattgta agaacatcta tttcgctggt 1020
catgaaacta cagctataac cgcatcttgg tgcttgatgt tgttagctgc acaccctgat 1080
tggcaagcaa gagccagatc tgaagtttta caatgttgcg atgacagacc aatcgatgca 1140
gacacagtca aaaatatgaa gaccttgact atggtaattc aagaaacttt gagattgtac 1200
ccacctgctg tattcgttac aagacaagca ttagaagata cagat caa aaacatcaca 1260
ataccaaagg gtatgaactt tcatatacca atccctatgt tgcaacaaga cttccactta 1320
tggggtcctg atgcttgttc atttgaccca caaagattct ccaatggtgt cttaggtgca 1380
tgcaaaaacc cacaagccta tatgcctttt ggtgttggtc caagagtctg tgccggtcaa
1440
catttcgcta tgatcgaatt gaaagtcatc gtatcattgg ttttgtccag attcgaattt 1500
tctttgtcac cttcctacaa gca tcacca gccttcagat tagt gtcga accagaaaac 1560
ggtgtcatat aaagttgtga 1590
SEQ ID NO: 17
Siraitia grosvenorii DNA sequence
atggaagtgg atatcaatat cttcaccgtc ttttccttcg tattatgcac agtcttcctc
60
ttctttctat ccttcttgat cctcctcctc ctccgaacgc tcgccggaaa atccataacg 120
agctccgagt acacgccagt gtacggcacc gtctacggtc aggctttcta tttcaacaac 180
ctgtacgatc atctaacgga ggtggccaag agacatcgaa ccttccggct gcttgcgccg 240
gcatacagcg agatatacac gaccgatccg agaaacatcg agcatatgtt gaagacgaaa 300
ttcgataagt attcgaaagg aagcaaggat caagaaatcg ttggggatct gtttggagag
360 gggatatttg cag cgatgg agataagtgg aagcagcaga ggaagctggc tagctatgaa 420
ttctcgacga ggattcttag ggattttagc tgctcggttt tcagacgaag tgctgctaaa 480
cttgttggag ttgtttcgga gttttccagc atgggtcggg tttttgatat ccaggatttg 540
ctaatgcggt gcgctttgga ctccattttc aaagtggggt tcggggttga tttgaattgc 600
ttggaggaat caagcaaaga agggagcgat ttcatgaaag ccttcgatga ttctagcgct 660
cagatttttt ggcgctatat cgatcccttc tggaaattga agagattgct taacatcggt 720
tccgaagctt cgtttaggaa caacataaaa accatagatg cttttgtgca ccagttgatc 780
agagacaaga gaaaattgct tcagcaaccg aatcacaaga atgacaaaga ggacatactt 840
tggaggtttc tgatggaaag tgagaaggat ccaacaagaa tgaatgatca atatctaagg
900
gatatagtcc tcaatttcat gttggctggc aaagattcaa gtggaggaac tctgtcctgg 960
ttcttctaca tgctatgcaa gaacccttta atacaggaaa aagttgcaga agaagtgagg 1020
caaattgttg cgtttgaagg ggaagaagtt gacatcaatt tgttcataca aaacttaact 1080
gattcagctc ttgacaaaat gcattatctt catgcagcat tgaccgagac tctgaggcta 1140
tatcctgcag tccctttgga tggaaggact gcagaaatag atgacattct tcctgatggc 1200
tataaactaa gaaaagggga tggagtatac tacatggcct attccatggg caggatgtcc 1260
tccctttggg gagaagatgc tgaagatttt aaacccgaaa gatggcttga aagtggaact 1320
tttcaacccg aatcaccttt caaattcatc gcttttcatg cgggtcctcg aatgtgtttg 1380
ggaaaagagt ttgcttatcg acaaatgaag atagtatctg ctgctttgct tcaatttttt 1440
cgattcaaag tagctgatac aacgaggaat gtgacttata ggatcatgct tacccttcac 1500
attgatggag gtctccctct tcttgcaatt ccgagaatta gaaaatttac ctaa 1554
SEQ ID NO: 18
Siraitia grosvenorii DMA sequence
ttggatagtg gagttaaaag agtgaaacgg ctagttgaag agaaacggcg agcagaattg 60
tctgcccgga ttgcctctgg agaattcaca gtcgaaaaag ctggttttcc atctgtattg 120 aggagtggct tatcaaagat gggtgttccc agtgagattc tggacatatt atttggtttc
180
gttgatgctc aagaagaata tcccaagatt cccgaagcaa aaggatcagt aaatgcaatt 240
cgtagtgagg ccttcttcat acctctctat gagctttatc tcacatatgg tggaatattt 300
aggttgactt ttgggccaaa gtcattcttg atagtttctg atccttccat tgctaaacat 360
atactgaagg ataatccgag gaattattct aagggtatct tagctgaaat tctagagttt 420
gtcatgggga agggacttat accagctgac gagaagatat ggcgtgtacg aaggcgggct 480
atagtcccat ctttgcatct gaagtatgta ggtgctatga ttaatctttt tggagaagct 540
gcagataggc tttgcaagaa gctagatgct gcagcatctg atggggttga tgtggaaatg 600
gagtccctgt tctcccgttt gactttagat atcattggca aggcagtttt taactatgac 660
tttgattcac ttacaaatga cactggcata gttgaggctg tttacactgt gctaagagaa 720
gcagaggatc gcagtgttgc accaattcca gtatgggaaa ttccaatttg gaaggatatt 780
tcaccacggc aaaaaaaggt ctctaaagcc ctcaaattga tcaacgacac cctcgatcaa 840
ctaattgcta tatgcaagag gatggttgat gaggaggagc tgcagtttca tgaggaatac 900
atgaatgagc aagatccaag catccttcat ttccttttgg catcaggaga tgatgtttca 960
agcaagcagc ttcgtgatga cttgatgact atgctta ag ctgggcatga aacatctgct 1020
gcagttttaa catggacctt ttatcttctt tccaaggagc cgaggatcat gtccaagctc 1080
caggaggagg ttgattcagt ccttggggat cggtttccaa ctattgaaga tatgaagaac 1140
ctcaaatatg ccacacgaat aattaacgaa tccttgaggc tttacccaca gccaccagtt
1200
ttaatacgtc gatctcttga caatgatatg ctcgggaagt accccattaa aaagggtgag 1260
gacatattca tttctgtttg gaacttgcat cgcagtccaa aactctggga tgatgcggat 1320
aaatttaatc ctgaaaggtg gcctctggat ggacccaatc caaatgagac aaatcaaaat 1380
ttcagatatt taccttttgg tggcggacca cggaaatgtg tgggagacat gtttgcttcg 1440
tacgagactg ttgtagcact tgcaatgctt gttcggcgat ttgacttcca aatggcactt 1500
ggagcacctc ctgtaaaaat gacaactgga gctacaattc acacaacaga tggattgaaa 1560
atgacagtta cacgaagaat gagacctcca atcataccca cattagagat gcctgcagtg 1620 gtcgttgact cgtctgtcgt ggactcgtcc gtcgccattt tgaaagaaga aacacaaatt
1680
ggttag
1686
SEQ ID NO: 19
Siraitia grosvenorii DNA sequence
cagttcctct cctggtcctc ccagtttggc aagaggttca tcttctggaa tgggatcgag 60
cccagaatgt gcctcaccga gaccgatttg atcaaagagc ttctctctaa gtacagcgcc 120
gtctccggta agtcatggct tcagcaacag ggctccaagc acttcatcgg ccgcggtctc 180
ttaatggcca acggccaaaa ctggtaccac cagcgtcaca tcgtcgcgcc ggccttcatg 240
ggagacagac tcaagagtta cgccgggtac atggtggaat gcacaaagga gatgcttcag 300
tcaattgaaa acgaggtcaa ctcggggcga tccgagttcg aaatcggtga gtatatgacc 360
agactcaccg ccgatataat atcacgaacc gagttcgaaa gcagctacga aaagggaaag 420
caaattttcc atttgctcac cgttttacag catctctgcg ctcaggcgag ccgccacctc 480
tgccttcctg gaagccggtt ttttccgagt aaatacaaca gagagataaa ggcattgaag 540
acgaaggtgg aggggttgtt aatggagata atacagagca gaagagactg tgtggaggtg 600
gggaggagca gttcgtatgg aaatgatctg ttgggaatgt tgctgaa ga gatgcagaag
660
aagaaagatg ggaatgggtt gagcttgaat ttgcagatta taatggatga atgcaagacc 720
ttcttcttcg ccggccatga aaccactgct cttttgctca cttggactgt aatgttattg 780
gccagcaacc cttcttggca acacaaggtt cgagccgaag ttatggccgt ctgcaatgga 840
ggaactctct ctcttgaaca tctctccaag ctctctctgt tgagtatggt gataaatgaa 900
tcgttgaggc tatacccgcc agcaagtatt cttccaagaa tggcatttga agatataaag 960
ctgggagatc ttgagatccc aaaagggctg tcgatatgga tcccagtgct tgcaattcac 1020
cacagtgaag agctatgggg caaagatgca aatgagttca acccagaaag atttgcaaat 1080
tcaaaagcct tcacttcggg gagattcatt ccctttgctt ctggccctcg caactgcgtt 1140
ggccaatcat ttgctctcat ggaaaccaag atcattttgg ctatgctcat ctccaagttt 1200
tccttcacca tctctgacaa ttatcgccat gcacccgtgg tcgtcctcac tataaaaccc 1260 aaatacggag tccaagtttg cttgaagcct ttcaattaa
1299
SEQ ID NO: 20
S raitia grosvenorii DNA sequence
atggaagaca ccttcctact ctatccttcc ctctctcttc tctttcttct ttttgctttc 60
aagctcatcc gtcgatccgg aggagttcgc aggaacttac cgccgagtcc gccctctctt
120
ccggttatcg gccacctcca tctcttgaaa aagccactcc accggacttt ccagaaactt 180
tccgccaaat atggtcctgt tatgtccctc cgcctcgggt ctcgcctcgc agtcattgta
240
tcgtcgtcgt cggcggtgga cgagtgtttc actaaaaacg acgtcgtgct cgccaaccgt 300
cctcgtttgc taattggcaa acacctcggc tacaactaca ctaccatggt tggggctccc
360
tacggcgacc actggcgtag cctccgccgc atcggtgccc tcgaaatctt ctcttcatct
420
cgcctcaaca aattcgccga catccgaagg gatgaagtag agggat gct. tcgcaaactc 480
tcacgcaatt cgctccatca attctcgaaa gtggaagttc aatcggcctt gtcggagctg
540
acgttcaaca tctcgatgag aatggcggca gggaaacggt attacggaga tgacgtgacg 600
gacgaggaag aggcgagaaa gttcagagag ttaattaaac agatagtggc gctgggcgga
660
gtatcaaatc caggggattt cgtcccgatt ctgaattgga ttccgaacgg tttcgagagg 720
aagttgatcg agtgtgggaa gaagacggat gcgttcttgc aggggctgat cgaggaccac
780
cggagaaaga aggaagaggg taggaacacg atgatcgatc acctgctctc tctgcaagaa
840
tcggagcctg ctcactacgg agaccaaata a caaaggat ttatactggt gttactgacg
900
gcggggaccg atacatcggc cgtgacaatg gagtgggcgc tatctcatct cctgaacaat
960
cctgaagtgc taaagaaggc aagagatgag gtcgacactg aaattggaca agaacgactt
1020
gtcgaagaat cagacgtagt atctaagtta ccctatcttc aagggatcat ctccgagact 1080
ctccggctga atcccgccgc tccgatgttg ttgccccatt acgcctcgga cgactgcacg
1140
atatgtggat acgacgtgcc acgtgacaca atcgtaatgg tcaatgcatg ggccatacat 1200
agggatccaa acgaatggga ggagcccacg tgtttcagac cagaacgata tgaaaagtcg
1260
tcgtcggaag cggaggtaca caagtcggtg agtttcgggg tgggaaggcg agcttgtcct 1320 gggtctggca tggcgcagag ggtgatgggc ttgactttgg cggcactggt tcagtgcttc 1380
gagtgggaga gagttggaga agaagaagtg gacatgaacg aaggctcagg tgccacaatg 1440
cccaagatgg tgccattgga ggccatgtgc agagctcgtc ccatcgtcca caaccttctt 1500
tactga
1506
SEQ ID NO: 21
Arabidopsis thaliana protein sequence
Met Ala Thr Glu Lys Thr His Gin Phe His Pro Ser Leu His Phe Val
1 5 10 15
Leu Phe Pro Phe Met Ala Gin Gly His Met lie Pro Met He Asp He
20 25 30
Ala Arg Leu Leu Ala Gin Arg Gly Val Thr He Thr He Val Thr Thr
35 40 45
Pro His Asn Ala Ala Arg Phe Lys Asn Val Leu Asn Arg Ala He Glu
50 55 60
Ser Gly Leu Ala lie Asn lie Leu His Val Lys Phe Pro Tyr Gin Glu
65 70 75 80
Phe Gly Leu Pro Glu Gly Lys Glu Asn lie Asp Ser Leu Asp Ser Thr
85 90 35
Glu Leu Met Val Pro Phe Phe Lys Ala Val Asn Leu Leu Glu Asp Pro
100 105 110
Val Met Lys Leu Met Glu Glu Met Lys Pro Arg Pro Ser Cys Leu He
115 120 125
Ser Asp Trp Cys Leu Pro Tyr Thr Ser He He Ala Lys Asn Phe Asn
130 135 140
lie Pro Lys lie Val Phe His Gly Met Gly Cys Phe Asn Leu Leu Cys
145 150 155 160
Met His Val Leu Arg Arg Asn Leu Glu He Leu Glu Asn Val Lys Ser
165 170 175
Asp Glu Glu Tyr Phe Leu Val Pro Ser Phe Pro Asp Arg Val Glu Phe
180 185 190
Thr Lys Leu Gin Leu Pro Val Lys Ala Asn Ala Ser Gly Asp Trp Lys
195 200 205 Glu He Met Asp Glu Met Val Lys Ala Glu Tyr Thr Ser Tyr Gly Val 210 215 220
He Val Asn Thr Phe Gin Glu Leu Glu Pro Pro Tyr Val Lys Asp Tyr 225 230 235 240
Lys Glu Ala Met Asp Gly Lys Val Trp Ser He Gly Pro Val Ser Leu
245 250 255
Cys Asn Lys Ala Gly Ala Asp Lys Ala Glu Arg Gly Ser Lys Ala Ala
260 265 270
He Asp Gin Asp Glu Cys Leu Gin Trp Leu Asp Ser Lys Glu Glu Gly
275 280 285
Ser Val Leu Tyr Val Cys Leu Gly Ser He Cys Asn Leu Pro Leu Ser
290 295 300
Gin Leu Lys Glu Leu Gly Leu Gly Leu Glu Glu Ser Arg Arg Ser Phe
305 310 315 320
He Trp Val He Arg Gly Ser Glu Lys Tyr Lys Glu Leu Phe Glu Trp
325 330 335
Met Leu Glu Ser Gly Phe Glu Glu Arg He Lys Glu Arg Gly Leu Leu
340 345 350
He Lys Gly Trp Ala Pro Gin Val Leu He Leu Ser His Pro Ser Val
355 360 365
Gly Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr Leu Glu Gly He
370 375 380
Thr Ser Gly He Pro Leu He Thr Trp Pro Leu Phe Gly Asp Gin Phe 385 390 395 400
Cys Asn Gin Lys Leu. Val Val Gin Val Leu Lys Ala Gly Val Ser Ala
405 410 415
Gly Val Glu Glu Val Met Lys Trp Gly Glu Glu Asp Lys H Gly Val
420 425 430
Leu Val Asp Lys Glu Gly Val Lys Lys Ala Val Glu Glu Leu Met Gly
435 440 445
Asp Ser Asp Asp Ala Lys Glu Arg Arg Arg Arg Val Lys Glu Leu Gly
450 455 4G0
Glu Leu Ala His Lys Ala Val Glu Lys Gly Gly Ser Ser His Ser Asn
465 470 475 480 Thr Leu Leu Leu Gin Asp lie Met Gin Leu Ala Gin Phe Lys Asn 485 490 495
SEQ ID NO: 22
Arabidopsis thaliana protein sequence
Met Val Ser Glu Thr Thr Lys Ser Ser Pro Leu His Phe Val Leu Phe 1 5 10 15
Pro Phe Met Ala Gin Gly His Met He Pro Met Val Asp lie Ala Arg
20 25 30
Leu Leu Ala Gin Arg Gly Val lie lie Thr lie Val Thr Thr Pro His
35 40 45
Asn Ala Ala Arg Phe Lys Asn Val Leu Asn Arg Ala lie Glu Ser Gly 50 55 60
Leu Pro lie Asn Leu Val Gin Val Lys Phe Pro Tyr Leu Glu Ala Gly 65 70 75 80
Leu Gin Glu Gly Gin Glu Asn lie Asp Ser Leu Asp Thr Met Glu Arg
85 30 95
Met lie Pro Phe Phe Lys Ala Val Asn Phe Leu Glu Glu Pro Val Gin
100 105 110
Lys Leu lie Glu Glu Met Asn Pro Arg Pro Ser Cys Leu lie Ser Asp
115 120 125
Phe Cys Leu Pro Tyr Thr Ser Lys He Ala Lys Lys Phe Asn He Pro 130 135 140
Lys He Leu Phe His Gly Met Gly Cys Phe Cys Leu Leu Cys Met His 145 150 155 160
Val Leu Arg Lys Asn Arg Glu He Leu Asp Asn Leu Lys Ser Asp Lys
165 170 175
Glu Leu Phe Thr Val Pro Asp Phe Pro Asp Arg Val Glu Phe Thr Arg
180 185 190
Thr Gin Val Pro Val Glu Thr Tyr Val Pro Ala Gly Asp Trp Lys Asp
135 200 205
He Phe Asp Gly Met Val Glu Ala Asn Glu Thr Ser Tyr Gly Val He 210 215 220
Val Asn Ser Phe Gin Glu Leu Glu Pro Ala Tyr Ala Lys Asp Tyr Lys 225 230 235 240
Glu Val Arg Ser Gly Lys Ala Trp Thr lie Gly Pro Val Ser Leu Cys
245 250 255
Asn Lys Val Gly Ala Asp Lys Ala Glu Arg Gly Asn Lys Ser Asp lie
260 265 270
Asp Gin Asp Glu Cys Leu Lys Trp Leu Asp Ser Lys Lys His Gly Ser
275 280 285
Val Leu Tyr Val Cys Leu Gly Ser lie Cys Asn Leu Pro Leu Ser Gin 290 295 300
Leu Lys Glu Leu Gly Leu Gly Leu Glu Glu Ser Gin Arg Pro Phe lie
305 310 315 320
Trp Val lie Arg Gly Trp Glu Lys Tyr Lys Glu Leu Val Glu Trp Phe
325 330 335
Ser Glu Ser Gly Phe Glu Asp Arg lie Gin Asp Arg Gly Leu Leu lie
340 345 350
Lys Gly Trp Ser Pro Gin Met Leu lie Leu Ser His Pro Ser Val Gly
355 360 365
Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr Leu Glu Gly lie Thr 370 375 380
Ala Gly Leu Pro Leu Leu Thr Trp Pro Leu Phe Ala Asp Gin Phe Cys 385 390 395 400
Asr. Glu Lys Leu Val Val. Glu Val Leu Lys Ala Gly Val Arg Ser Gly
405 410 415
Val Glu Gin Pro Met Lys Trp Gly Glu Glu Glu Lys lie Gly Val Leu
420 425 430
Val Asp Lys Glu Gly Val Lys Lys Ala Val Glu Glu Leu Met Gly Glu
435 440 445
Ser Asp Asp Ala Lys Glu Arg Arg Arg Arg Ala Lys Glu Leu Gly Asp 450 455 460
Ser Ala His Lys Ala Val Glu Glu Gly Gly Ser Ser His Ser Asn lie
465 470 475 480
Ser Phe Leu Leu Gin Asp lie Met Glu Leu Ala Glu Pro Asn Asn
485 490 495 SEQ ID NO : 23
Arabidopsis thal iana protein sequence
Met Ala Phe Glu Lys Asn Asn Glu Pro Phe Pro Leu His Phe Val Leu 1 5 10 15
Phe Pro Phe Met Ala Gin Gly His Met He Pro Met Val Asp He Ala
20 25 30
Arg Leu Leu Ala Gin Arg Gly Val Leu He Thr He Val Thr Thr Pro
35 40 45
His Asn Ala Ala Arg Phe Lys Asn Val Leu Asn Arg Ala He Glu Ser 50 55 60
Gly Leu Pro He Asn Leu Val Gin Val Lys Phe Pro Tyr Gin Glu Ala 65 70 75 80
Gly Leu Gin Glu Gly Gin Glu Asn Met Asp Leu Leu Thr Thr Met Glu
85 90 95
Gin He Thr Ser Phe Phe Lys Ala Val Asn Leu Leu Lys Glu Pro Val
100 105 110
Gin Asn Leu He Glu Glu Met Ser Pro Arg Pro Ser Cys Leu He Ser
115 120 125
Asp Met Cys Leu Ser Tyr Thr Ser Glu He Ala Lys Lys Phe Lys He 130 135 140
Pro Lys He Leu Phe His Gly Met Gly Cys Phe Cys Leu Leu Cys Val 145 150 155 160
Asn Val Leu Arg Lys Asn Arg Glu He Leu Asp Asn Leu Lys Ser Asp
165 170 175
Lys Glu Tyr Phe He Val Pro Tyr Phe Pro Asp Arg Val Glu Phe Thr
180 185 130
Arg Pro Gin Val Pro Val Glu Thr Tyr Val Pro Ala Gly Trp Lys Glu
195 200 205
He Leu Glu Asp Met Val Glu Ala Asp Lys Thr Ser Tyr Gly Val He 210 215 220
Val Asn Ser Phe Gin Glu Leu Glu Pro Ala Tyr Ala Lys Asp Phe Lys 225 230 235 240
Glu Ala Arg Ser Gly Lys Ala Trp Thr He Gly Pro Val Ser Leu Cys
245 250 255 Asn Lys Val Gly Val Asp Lys Ala Glu Arg Gly Asn Lys Ser Asp lie 260 265 270
Asp Gin Asp Glu Cys Leu Glu Trp Leu Asp Ser Lys Glu Pro Gly Ser
275 280 285
Val Leu Tyr Val Cys Leu Gly Ser lie Cys Asn Leu Pro Leu Ser Gin 290 295 300
Leu Leu Glu Leu Gly Leu Gly Leu Glu Glu Ser Gin Arg Pro Phe lie
305 310 315 320
Trp Val lie Arg Gly Trp Glu Lys Tyr Lys Glu Leu Val Glu Trp Phe
325 330 335
Ser Glu Ser Gly Phe Glu Asp Arg lie Gin Asp Arg Gly Leu Leu lie
340 345 350
Lys Gly Trp Ser Pro Gin Met Leu lie Leu Ser His Pro Ser Val Gly
355 360 365
Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr Leu Glu Gly He Thr
370 375 380
Ala Gly Leu Pro Met Leu Thr Trp Pro Leu Phe Ala Asp Gin Phe Cys 385 390 395 40Q
Asn Glu Lys Leu Val Val Gin He Leu Lys Val Gly Val Ser Ala Glu
405 410 415
Val Lys Glu Val Met Lys Trp Gly Glu Glu Glu Lys He Gly Val Leu
420 425 430
Val Asp Lys Glu Gly Val Lys Lys Ala Val Glu Glu Leu Met Gly Glu
435 440 445
Ser Asp Asp Ala Lys Glu Arg Arg Arg Arg Ala Lys Glu Leu Gly Glu
450 455 460
Ser Ala His Lys Ala Val Glu Glu Gly Gly Ser Ser His Ser Asn He 465 470 475 480
Thr Phe Leu Leu Gin Asp He Met Gin Leu Ala Gin Ser Asn Asn
485 490 495
SEQ ID NO: 24
Stevia rebaudiana protein sequence
Met Ser Pro Lys Met Val Ala Pro Pro Thr Asn Leu His Phe Val Leu 1 5 10 15 Fhe Pro Leu Met Ala Gin Gly His Leu Val Pro Met Val Asp He Ala 20 25 30
Arg He Leu Ala Gin Arg Gly Ala Thr Val Thr He He Thr Thr Pro
35 40 45
Tyr His Ala Asn Arg Val Arg Pro Val He Ser Arg Ala He Ala Thr
50 55 60
Asn Leu Lys He Gin Leu Leu Glu Leu Gin Leu Arg Ser Thr Glu Ala 65 70 75 80
Gly Leu Pro Glu Gly Cys Glu Ser Phe Asp Gin Leu Pro Ser Phe Glu
85 90 95
Tyr Trp Lys Asn He Ser Thr Ala He Asp Leu Leu Gin Gin Pro Ala
100 105 110
Glu Asp Leu Leu Arg Glu Leu Ser Pro Pro Pro Asp Cys He He Ser
115 120 125
Asp Phe Leu Phe Pro Trp Thr Thr Asp Val Ala Arg Arg Leu Asn He 130 135 140
Pro Arg Leu Val Phe Asn Gly Pro Gly Cys Phe Tyr Leu Leu Cys He 145 150 155 160
His Val Ala He Thr Ser Asn He Leu Gly Glu Asn Glu Pro Val Ser
165 170 175
Ser Asn Thr Glu Arg Val Val Leu Pro Gly Leu Pro Asp Arg He Glu
180 185 190
Val Thr Lys Leu Gin He Val Gly Ser Ser Arg Pro Ala Asn Val Asp
195 200 205
Glu Met Gly Ser Trp Leu Arg Ala Val Glu Ala Glu Lys Ala Ser Phe
210 215 220
Gly He Val Val Asn Thr Phe Glu Glu Leu Glu Pro Glu Tyr Val Glu 225 230 235 240
Glu Tyr Lys Thr Val Lys Asp Lys Lys Met Trp Cys He Gly Pro Val
245 250 255
Ser Leu Cys Asn Lys Thr Gly Pro Asp Leu Ala Glu Arg Gly Asn Lys
260 265 270
Ala Ala He Thr Glu His Asn Cys Leu Lys Trp Leu Asp Glu Arg Lys
275 280 285 Leu Gly Ser Val Leu Tyr Val Cys Leu Gly Ser Leu Ala Arg He Ser 290 295 300
Ala Ala Gin Ala He Glu Leu Gly Leu Gly Leu Glu Ser He Asn Arg
3 05 310 315 320
Pro Phe He Trp Cys Val Arg Asn Glu Thr Asp Glu Leu Lys Thr Trp
325 3 3 0 335
Phe Leu Asp Gly Phe Glu Glu Arg Val Arg Asp Arg Gly Leu He Val
34 0 345 350
His Gly Trp Ala Pro Gin Val Leu He Leu Ser His Pro Thr He Gly
355 360 355
Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr He Glu Ser He Thr
370 375 3 8 0
Ala Gly Val Pro Met He Thr Trp Pro Phe Phe Ala Asp Gin Phe Leu 385 3 90 395 4 00
Asn Glu Ala Phe He Val Glu Val Leu Lys He Gly Val Arg He Gly
4 05 410 415
Val Glu Arg Ala Cys Leu Phe Gly Glu Glu Asp Lys Val Gly Val Leu
420 425 43 0
Val Lys Lys Glu Asp Val Lys Lys Ala Val Glu Cys Leu Met Asp Glu
435 440 445
Asp Glu Asp Gly Asp Gin Arg Arg Lys Arg Val He Glu Leu Ala Lys 450 455 460
Met Ala Lys He Ala Met Ala Glu Gly Gly Ser Ser Tyr Glu Asn Val
465 470 475 4 80
Ser Ser Leu He Arg Asp Val Thr Glu Thr Val Arg Ala Pro His
485 4 90 495
SEQ ID NO : 25
Stevia rebaudiana protein sequence
Met Asp Ala Met Ala Thr Thr Glu Lys Lys Pro Hi s Val He Phe He 1 5 10 15
Pro Phe Pro Ala Gin Ser His He Lys Ala Met Leu Lys Leu Ala Gin
20 25 30
Leu Leu His His Lys Gly Leu Gin He Thr Phe Val Asn Thr Asp Phe 35 40 45
lie His Asn Gin Phe Leu Glu Ser Ser Gly Pro His Cys Leu Asp Gly 50 55 60
Ala Pro Gly Phe Arg Phe Glu Thr lie Pro Asp Gly Val Ser His Ser 65 70 75 80
Pro Glu Ala Ser lie Pro lie Arg Glu Ser Leu Leu Arg Ser He Glu
85 90 95
Thr Asn Phe Leu Asp Arg Phe lie Asp Leu Val Thr Lys Leu Pro Asp
100 105 110
Pro Pro Thr Cys lie lie Ser Asp Gly Phe Leu Ser Val Phe Thr He
115 120 125
Asp Ala Ala Lys Lys Leu Gly He Pro Val Met Met Tyr Trp Thr Leu 130 135 140
Ala Ala Cys Gly Phe Met Gly Phe Tyr His He His Ser Leu He Glu 145 150 155 160
Lys Gly Phe Ala Pro Leu Lys Asp Ala Ser Tyr Leu Thr Asn Gly Tyr
165 170 175
Leu Asp Thr Val lie Asp Trp Val Pro Gly Met Glu Gly He Arg Leu
180 185 190
Lys Asp Phe Pro Leu Asp Trp Ser Thr Asp Leu Asn Asp Lys Val Leu
195 200 205
Met Phe Thr Thr Glu Ala Pro Gin Arg Ser His Lys Val Ser His His 210 215 220
lie Phe His Thr Phe Asp Glu Leu Glu Pro Ser He He Lys Thr Leu 225 230 235 240
Ser Leu Arg Tyr Asn His He Tyr Thr He Gly Pro Leu Gin Leu Leu
245 250 255
Leu Asp Gin lie Pro Glu Glu Lys Lys Gin Thr Gly He Thr Ser Leu
260 265 270
His Gly Tyr Ser Leu Val Lys Glu Glu Pro Glu Cys Phe Gin Trp Leu
275 280 285
Gin Ser Lys Glu Pro Asn Ser Val Val Tyr Val Asn Phe Gly Ser Thr 290 295 300
Thr Val Met Ser Leu Glu Asp Met Thr Glu Phe Gly Trp Gly Leu Ala 305 310 315 320
Asn Ser Asn His Tyr Phe Leu Trp He He Arg Ser Asn Leu Val He
325 330 335
Gly Glu Asn Ala Val Leu Pro Pro Glu Leu Glu Glu His He Lys Lys
340 345 350
Arg Gly Phe He Ala Ser Trp Cys Ser Gin Glu Lys Val Leu Lys His
355 360 365
Pro Ser Val Gly Gly Phe Leu Thr His Cys Gly Trp Gly Ser Thr He
370 375 380
Glu Ser Leu Ser Ala Gly Val Pro Met He Cys Trp Pro Tyr Ser Trp
385 390 395 400
Asp Gin Leu Thr Asn Cys Arg Tyr He Cys Lys Glu Trp Glu Val Gly
405 410 415
Leu Glu Met Gly Thr Lys Val Lys Arg Asp Glu Val Lys Arg Leu Val
420 425 430
Gin Glu Leu Met Gly Glu Gly Gly His Lys Met Arg Asn Lys Ala Lys
435 440 445
Asp Trp Lys Glu Lys Ala Arg He Ala lie Ala Pro Asn Gly Ser Ser
450 455 460
Ser Leu Asn He Asp Lys Met Val Lys Glu He Thr Val Leu Ala Arg
465 470 475 480
Asn
SEQ ID NO: 26
Siraitia grosvenorii D A sequence
atggatgccc agcgaggtca caccaccacc attttgatgc ttccatgggt cggctacggc
60
catctcttgc ctttcctcga gctggccaaa agcctctcca ggaggaaatt attccacatc 120
tacttctgtt caacgtctgt tagcctcgac gccattaaac caaagcttcc tccttctatc 180
tcttctgatg attccatcca acttgtggaa cttcgtctcc cttcttctcc tgagttacct
240
cctcatcttc acacaaccaa cggccttccc tctcacctca tgcccgctct ccaccaagcc 300
ttcgtcatgg ccgcccaaca ctttcaggtc attttacaaa cacttgcccc gcatctcctc 360 atttatgaca ttctccaacc ttgggctcct caagtggctt catccctcaa cattccagcc 420
atcaacttca gtactaccgg agcttcaatg ctttctcgaa cgcttcaccc tactcactac 480
ccaagttcta aattcccaat ctcagagttt gttcttcaca atcactggag agccatgtac 540
accaccgccg atggggctct tacagaagaa ggccacaaaa ttgaagaaac acttgcgaat 600
tgcttgcata cttcttgcgg ggtagttttg gtcaatagtt tcagagagct tgagacgaaa 660
tatatcgatt atctctctgt tctcttgaac aagaaagttg ttccggtcgg tcctttggtt 720
tacgaaccga atcaagaagg ggaagatgaa ggttattcaa gcatcaaaaa ttggcttgac 780
aaaaaggaac cgtcctcaac cgtcttcgtt tcatttggaa ccgaatactt cccgtcaaag 840
gaagaaatgg aagagatagc gtatgggtta gagctgagcg aggttaattt catctgggtc 900
cttagatttc ctcaaggaga cagcaccagc accattgaag acgccttgcc gaaggggttt 960
ctggagagag cgggagagag ggcgatggtg gtgaagggtt gggctcctca ggcgaaga a 1020
ctgaagcatt ggagcacagg ggggcttgtg agtcactgtg gatggaactc gatgatggag 1080
ggcatgatgt ttggcgtacc cataatagcg gtcccgatgc atctggacca gccctttaac 1140
gccggactct tggaagaagc tggcgtcggc gtggaagcca agcgaggttc ggacggcaaa 1200
attcaaagag aagaagttgc aaagtcgatc aaagaagtgg tgattgagaa aaccagggaa 1260
gacgtgagga agaaagcaag agaaatgggt gagattttga ggagtaaagg agatgagaaa 1320
attgatgagt tggtggctga aatttctctt ttgcgcaaaa aggctccatg ttcaatttaa 1380
SEQ ID MO: 27
Siraitia grosvenorii DNA sequence
atgcttccat ggctggctca cggccatgtc tcccctttct tcgagctcgc caagttgctc 60
gccgctagaa acttccacat attcttctgc tccaccgccg taaacctccg ctccgtcgaa 120
ccaaaactct ctcagaagct ctcctcccac gtggagctgg tggagctcaa cctaccgccc 180
tcgccggagc tccctccgca ccgccacacc accgccggcc ttccaccgca cctcatgttc 240
tcgctcaagc gagctttcga catggccgct cccgccttcg ccgccatcct ccgcgacctg 300
aacccggact tgctcatcta cgacttcctg cagccgtggg cggcggcgga ggctctgtcg 360 gcggatattc cggccgtgat gttcaaaagc acgggtgcgc tcatggcggc catggtcgcg 420
tacgagctga cgtttccgaa ctctgatttt ttctcgcttt tccctgagat tcgtctctcc
480
gagtgcgaga ttaaacagct gaagaacttg tttcaatgtt ctgtgaatga tgcgaaagac 540
aagcaaagga ttaagggatg ttatgagaga tcttgcggca tgattttggt gaaatctttc eoo
agagaaatcg aaggcaaata tattgatttt ctctctactc tgctgggcaa gaaggttgtt
660
ccagttggtc cacttgttca acaaacagaa gacgacgtcg tatcaggaag ttttgacgaa 720
tggctaaatg gaaaagatag atcgtcttcc atactcgtgt ctttcggaag cgagttctac
780
ctgtccagag aagacatgga agagatcgcg catggcttag agctgagcca ggtgaacttc 840
atatgggtcg tcaggtttcc ggcgggagga gagagaaaca cgacaaaggt ggaagaagaa
900
ctgccaaaag ggtttctaga gagagttaga gagagaggga tggtggtgga gggctgggcg 960
ccgcaggctc agatcttgaa acatccaagc gtcggcggat tcctcagcca ctgcgggtgg 1020
agctccgtcg tggagagcat gaaattcggc gttccgatca tcgccatgcc gatgcacctc
1080
gaccagccgc tgaattcccg gctggtcgag cggctcggcg tcggcgtagt ggtggagaga 1140
gacggccgcc tccggggaga ggtggagaga gttgtcagag aggtggtggt ggagaaaagt
1200
ggagagagag tgaggaagaa ggtggaggag tttgcagaga tcatgaagaa gaaaaaagac
1260
aatgaagaga tggacgtagt cgtggaagag ttggtgacgc tctgcaggaa gaagaagaag 1320
gaggaggatt tacagagtaa ttattggtgc agaaccgcca ttgatgacca ttgttctgaa
1380
gtcgtgaaga ttgaagatgc tgcagcagcc gacgaggagc ctctttgcaa ataa 1434
SEQ ID NO: 28
S raitia grosvenorii DMA sequence
atggctg ca cttacagcct gcacatagca atgtaccctt ggtttgcttt cggccacttg
60
actccatttc tccaagtctc caacaagctt gccaaggaag gccacaaaat ctccttcttc 120
atcccaacga aaacgctaac caaattgcag cctttcaatc tctttccaga tctcattacc
180
tttgtcccca tcactgttcc tcatgttgat ggtctccctc ttggagctga gactactgct 240 gatgtttctc acccttcaca gctcagtctc atcatgactg ctatggattg cacccaaccc 300
gaaatcgagt gtcttcttcg agacataaaa cctgatgcca tcttcttcga tttcgcgcac 360
tgggtgccaa aattggcatg tggattgggc attaagtcga ttgattacag tgtctgttct 420
gcagtatcaa ttggttatgt tttgccccta ttaaggaaag tttgtggaca agatttatta 480
actgaagatg attttatgca gccatctcct ggctacccga gttccaccat caatcttcaa 540
gctcatgagg ctcgatattt tgcatctctg agccgctgga ggtttggcag tgatgtccct 600
ttctttagtc gccatcttac tgcacttaat gaatgcaatg ctttagcatt caggtcatgt 660
agggagattg aagggccttt tatagactat ccagaaagtg aattaaaaaa gcctgtgttg
720
ctttccggag cagtggatct acaaccgcca accacaactg tagaagaaag atgggcaaaa 780
tggctatcag ggttcaacac cgactcggtc gtatattgtg catttggaag tgagtgtacc 840
ttagcaaaag accaattcca agaactgctg ttgggttttg agctttcaaa tatgccattc 900
tttgctgcac ttaaaccacc ttttggtgtt gactcggttg aagcagcctt gcctgaaggt 960
tttgaacaga gagttcaggg aagaggggtg gtctatgggg gatgggtcca acagcagctc 1020
attttggagc acccatcaat tggatgcttt gttacacatt gtggatcagg ctccttatca 1080
gaggcgttag tgaagaagtg tcaattagtg ttgttacctc gtatcggtga ccactttttc 1140
cgagcaagaa tgttgagcaa ttatttgaaa gttggtgtgg aggtagagaa aggagaagga 1200
gatggatctt ttacaaagga aagtgtgtgg aaggcagtga agacagtgat ggatgaagag 1260
aatgaaactg ggaaagagtt cagagcgaac cgtgccaaga taagagagct attgctcgac 1320
gaagatctcg aggagtctta tatcaacaat ttcatccaca gcctgcatac tttgaatgca
1380
tga
1383
SEQ ID NO: 29
Artificial sequence; Partial nucleotide sequence from Siraitia grosvenori atggcggatc ggaaagagag cgttgtgatg ttcccgttca tggggcaggg ccatatcatc 60
ccttttctag ctttggccct ccagattgag cacagaaaca gaaactacgc catatacttg 120
gtaaatactc ctctcaacgt taagaaaatg agatcttctc tccctccaga ttga 174 SEQ ID NO: 30
Siraitia grc svenorii DNA sequence
atggaagcta agaactgcaa aaaggttctg atgttcccat ggctggcgca tggtcacata
60
tcaccatttg tagagctggc caagaagctc acagacaaca acttcgccgt ttttctatgt 120
tcttcccctg caaatcttca aaacgtcaag ccaaaactcc cccatcacta ctctgattcc 180
attgaactcg tggagctcaa ccttccatcg tcgccggagc ttccccctca tatgcacacc
240
accaatggcc tccctttgca tttagttccc accctcgttg acgccttgga catggccgct
300
ccgcacttct ccgccatttt acaggaactg aatccagatt ttctcatatt cgacatcttc 360
caaccctggg cggctgaaat cgcttcctcc ttcggcgttc ctgctatttt gttgcttatc 420
gttggatctg ctataaccgc tttaggggtt cattttgtcc ggagctccgg tacggaattc
480
ccctttcccg agcttactaa atcattcaag aaggaggacg accgaaaacc tccaggagat
540
tccggcaacg atagaggaaa acggctattc aaatgtctgc tggacctgga acattcttca 600
gagactattt tggtgaacag ttttacagag atagagggca aatatatgga ctatctctcg 660
gtcttactga agaagaagat ccttccgatt ggtcctttgg ttcagaaaat tggctccgat
720
gacgatgaat cgggaatcct ccggtggctt gacaagaaga aaccgaattc aactgtgtac 780
gtttcgttcg ggagtgagta ctatttgagc aaagaagaca tagcagagct tgcgcatggt 840
ctggaaatca gcggcgtcaa tttcatctgg attgttcggt ttccaaaggg agagaaaatc 900
gccattgaag aggcattacc agatgaattt cttgaaagag tcggagagag aggcgtcgtc
960
gttgatggat gggcgccgca gatgaaaata ttagggcatt cgagcgtcgg cgggtttctg
1020
tctcactgcg gatggaactc tgtgctggag agtctggtgc tcggcgtgcc gatcatatcc 1080
ctgccgatac acctcgaaca gccgtggaac gccttggtag cggagcacgt cggcgtttgt 1140
gtgagggcga agagagacga cggaggaaat cttcaaagag agttggtggc ggaggccatt
1200
aaagaagtgg tggttgagga aacaggagcg gaactgagaa gcaaagcaag agtaattagt
1260
gaaatcttga aaaataaaga agctgaaaca atacaagatt tggtggctga gcttcaccgg 1320
ctttctgacg caa ttgttga 1347 SEQ ID NO: 31
Siraitia grosvenorii DMA sequence
atggaaaaaa atcttcacat agtgatgctt ccatggtcgg cgttcggcca tctcatacca 60
ttttttcacc tctccatagc cttagccaaa gccaaagttt atatctcctt cgtctccact 120
ccaagaaata ttcagagact yccccaaatc ccgccggact tagcttcttt catagatttg 180
gtggccattc ccttgccgag actcgacgac gatctgttgc tagaatctgc agaggccact 240
tctgatattc cgatcgacaa gattcagtat ttgaagcgag ccgtcgacct cctccgccac 300
cccttcaaga agtttgtcgc cgaacaatcg ccggactggg tcgtcgttga ttttcatgct 360
tattgggccg gcgagatcta ccaggagttt caagttcccg tcgcctactt ctgtattttc 420
tcggccatct gtttgcttta tcttggacct ccagacgtgt attcgaagga tcctcagatc 480
atggcacgaa tatctcccgt taccatgacg gtgccgccgg agtgggtcgg ttttccgtcc 540
gccgtagcct acaacttgca tgaggcgacg gtcatgtact ctgctctcta tgaaacaaat 600
gggtctggaa taagcgactg cgagaggatt cgccggctcg tcctttcctg tcaagccgtg 660
gccattcgaa gctgcgagga gattgaaggc gaatacctta ggttatgtaa gaaactgatt 720
ccaccgcagg ggattgccgt cggcttgctt ccgccggaaa agccaccaaa atcagatcac 780
gagctcatca aatggcttga cgagcaaaag ctccgattcg tcgtgtacgt gacattcggc 840
agcgaatgca acctgacgaa ggaccaagtt cacgagatag cccacgggct ggaactgtcg 900
gagctgccat ttttatgggc actgaggaaa cccagctggg cagctgagga agacgatggg 960
ctgccgtctg ggtttcgtga gagaacgtcc gggagagggg tggtgagcat ggagtgggtg 1020
ccgcagttgg agattctggc gcaccaggcc atcggcgtct ctttagttca cgggggctgg 1080
ggctctatta tcgagtcgct acaagctggg cactgtctgg ttgtgctgcc gtttatcatc 1140
gaccagccgc tgaactcaaa gcttttggtg gagaaaggga tggcgcttga gatcagaagg 1200
aacggttctg atggatggtt tagtagagaa gacatcgccg gaactttgag agaagctatg 1260
cggtcgtctg aggaaggcgg gcagctgagg agccgtgcaa aagaggcggc ggccatcgtt 1320
ggagatgaga agctgcagtg ggaacaatac ttcggcgcgt tcgtacagtt tctgagggac 1380 aagtcttga
1389
SEQ ID NO: 32
Sxraitia grosvenorii sequence,
atgtccgagg agaaaggcag agggcacagc tcgtcgacgg agagacacac tgctgccgcc 60
atgaacgccg agaaacgaag caccaaaatc ttgatgctcc catggctggc tcacggccac 120
atatctccat acttcgagct cgccaagagg ctcaccaaga aaaactgcca cgtttacttg 180
tgttcttcgc ctgtaaatct ccaaggcatc aagccgaaac tctctgaaaa ttactcttcc 240
tccattgaac ttgtggagct tcatcttcca tctctccccg accttcctcc ccatatgcac 300
acgaccaaag gcatccctct acatctacaa tccaccctca tcaaagcctt cgacatggcc
360
gcccctgatt tttccgacct gttgcagaaa ctcgagccgg atctcgtcat ttccgatctc 420
ttccagccat gggcagttca attagcgtcg tctcggaaca ttcccgtcgt caatttcgtt
480
gtcaccggag tcgctgttct tagtcgtttg gctcacgtgt tttgcaactc cgttaaggaa 540
ttccctttcc cggaactcga tctaaccgac cattggatct ccaagagccg ccgcaaaacg 600
tccgacgaat taggtcgcga gtgcgcgatg cgatttttca actgcatgaa acaatcttca 660
aacatcactc tagccaacac tttccccgag ttcgaagaaa aatacatcga ttatctctct 720
tcctcgttta agaaaaagat tcttccggtt gctcctctag ttcctgaaat cgacgcagac
780
gacgagaaat cggaaattat cgagtggctt gacaagaaga aaccgaaatc gactgtttac 840
gtttcgtttg ggagtgagta ttatctgacg aaagaagaca gggaagagct cgcccatggc
900
ttagaaaaga gcggcgtgaa tttcatctgg gttattaggt ttccaaaggg cgagaagatc 960
accattgaag aggctttacc agaaggattt ctcgagagag taggggacag gggagtgatt 1020
atcgacgggt gggcgccgca gttgaaaata ttgaggcatt caagcgtggg cgggttcgtg 1080
tgccactgcg gg ggaactc tgtggtggag agcgtggtgt ttggggtgcc gatcatagcc 1140
ttgccgatgc agctcgatca gccatggcat gcgaaggtgg cggaggacgg cggcgtctgt
1200
gcggaggcga agagagacgt tgaagggagc gttcagagag aagaggtggc: gaaggccatt 1260
aaagaggtgg tgtttgagaa gaaggggggg gttctgagtg gaaaagcaag agagatcagc
1320 gaggccttga gaaagaggga aggggaaatc atagaggaat tggttgctga gtttcaccag 1380
ctctgtgaag cttga 1395
SEQ ID NO: 33
Artificial sequence; Partial nucleotide sequence from Siraitia grosvenorii
ttctgctcca cgcctgtaaa tttggaagcc attaaaccaa agctttccaa aagctactct 60
gattcgatcc aactaatgga ggttcctctc gaatcgacgc cggagcttcc tcctcactat 120
catacagcca aaggccttcc gccgcattta atgcccaaac tcatgaatgc ctttaaaatg 180
gttgctccca atctcgaatc gatcctaaaa accctaaacc cagatctgct catcgtcgac 240
attctccttc catggatgct tccactcgct tcatcgctca aaattccgat ggttttcttc 300
actattttcg gtgccatggc catctccttt atgatttata atcgaaccgt ctcgaacgag 360
cttccatttc cagaatttga acttcacgag tgctggaaat cgaagtgccc ctatttgttc 420
aaggaccaag cggaaagtca atcgttctta gaatacttgg atcaatcttc aggcgtaatt 480
ttgatcaaaa cttccagaga gattgaggct aagtatgtag actttctcac ttcgtcgttt 540
acgaagaagg ttgtgaccac cggtcccctg gttcagcaac cttcttccgg cgaagacgag 600
aagcagtact ccgatatcat cgaatggcta gacaagaagg agccgttatc gacggtgctc 660
gtttcgtttg ggagcgagta ttatctgtca aaggaagaga tggaagaaat cgcctacggg 720
ctggagagcg ccagcgaggt gaatttcatc tggattgtta ggtttccgat gggacaggaa 780
acggaggtcg aggcggcgct gccggagggg ttcatccaga gggcaggaga gagagggaaa
840
gtggtcgagg gctgggctcc gcaggcgaaa atattggcgc atccgagcac cggcggccat 900
gtgagccaca acgggtggag ctcgattgtg gagtgcttga tgtccggtgt accggtgatc
960
ggcgcgccga tgcaacttga cgggccaatc gtcgcaaggc tggtggagga gatcggcgtg 1020
ggtttggaaa tcaagagaga tgaggaaggg agaatcacga ggggcgaagt tgccgatgca 1080
atcaagacgg tggcggtggg caaaaccggg gaagatttta gaaggaaagc aaaaaaaatc 1140
agcagcattt tgaagatgaa agatgaagaa gaggttgaca ctttggcaat ggaattagtg 1200 aggttatgcc aaatgaaaag agggcaggag tctcaggact aa
1242
SEQ ID NO: 34
Artificial sequence; Partial nucleotide sequence from Siraitia grosvenorii
tcccggtcaa cggtagagga cttcacggag cttcgagagt ggatgccttc tggatcgaac 60
atggtctacc ggtaccacga gattaaaaaa tccttagatg gagcaaccgg caacgaatcg
120
gggacgtctg attcggtccg attcggaatt gtgattgagg agagtgttgc tgtggctgta 180
agaagctccc ctgaactgga accggaatgg ttcgatttgc tcgcgaagct ttaccagaag
240
ccagttgttc cggtaggatt tctacctcca gtaattgaag atgcggaaga attgagcagc 3 00
gatatcaagg aatggttaga caaacagagc tcaaactcgg tcctttacgt cgcattcggg
360
accgaggcga ctctgagtca agatgacgtc actgagttag ccatggggct tgagcaatct 420
gggataccat ttttctgggt actgagaacc tcacctcggg acgagtcaga catgttaccg
480
gccgggttca aggagcgagt cgaaggtcga ggaagtgttc acgtgggatg ggtctcgcag 540
gtgaagatac tgagtcacga ctcggttggc ggttgtttga cacactgtgg atggaactcg
600
atcatagagg ggctcggatt cgggcgcg atggtattgt ttccagtcgt gaacgaccag 660
ggattgaacg ctagattgtt gggggagaag aagctcggga tagagataga aagggacgag 720
cgagatggat cgttcacacg cgactcggtg tcggaatcgg tgaggtcggc aatggcggaa 780
agttcaggcg aggccttgag agtgagggcc agggaaatga aggggttgtt tggaaacgga 840
gatgagaacg agcatcaact gaacaagttt gtacaatttc tcgaggcaaa caggaatagg 900
cagtccgagt aa 912
SEQ ID NO: 35
Artificial sequence ; Par ial nucleotide sequence from Siraitia grosvenorii
ctgctgccga ttccgctgcc gaaaccggcc gccgatctct tgccggaagg tgcagaggcg 60
acggtggata ttccgtccga caagattccg tatctgaaat tggccctcga tctcgccgag
120 cagccgtttc ggaagttcgt cgttgatcgt ccgccggatt ggatgatcgt cgattttaat 180
gctacttggg tctgcgatat ttctcgggag cttcaaatcc caatcgtttt ctttcgtgtt 240
ctttcgcctg gatttcttgc tttctttgcg catgttcttg ggagtggtct gccgctgtcg 300
gagatcgaaa gcctgatgac tccgccggtg atcgacgggt cgacggtggc gtaccgccgg 360
catgaagctg ccgttatttg tgctgggttt tttgagaaga acgcttctgg tatgagtgat 420
cgcgatcggg taaccaaaat tctctctgcc agtcaagcaa tcgcagttcg ttcttgctac 480
gaatttgacg ttgagtattt gaaattgtac gagaaatatt gtggaaaaag agtgattcct 540
ctagggtttc tccctccaga aaagccccaa aagtccgagt tcgccgccga ttcgccatgg 600
aaaccgacct tcgagtggct tgacaaacaa aagccccgat cagtggtgtt cgtcggattc 660
ggcagcgaat gcaaactcac gaaagatgat gtttacgaga tagcgcgcgg ggtggagctg 720
tcggagctgc catttttgtg ggctctgaga aaaccgatct ggg ggcggc ggacgattcc 780
gacgctctgc ctgccggatt cctcgagcgg acggcggaga gagggattgt gagcatgggg 840
tgggcgccgc agatggagat tttaacgcac ccgtcgattg gcggctctct gtttcacgcc 900
gggtggggat ccgccattga agctctgcaa ttcgggcatt gccttgttct gttgccattc
960
atcgtggatc agccactgaa tgcaaggctt ctggtggaga agggtgttgc agtcgaagtt 1020
ggaagaaagg aagacgggtc ttttagtgga gaagacatag ctaaagctct gagagaagct 1080
atggtttcag aagaaggtga gcagatgagg aggcaagcga gaaag 1125
SEQ ID NO: 36
Artificial sequence Partial nucleotide sequence from Siraiti grosvenorii
atggaaaacg acggcgtttt gcacgtggtg gtattcccat ggctagcctt gggtcatct 60
attcctttcg ctcgactcgc cacctgctta gcccacaagg gtctcagggt ttcgttcgt 120
tcaaccacaa ggaacctgag cagaattccc aaaatacccc cacatctctc ctcctccgt 180
aacctcgtcg gctttcctct gccccacgtc gacggccttc cggacgccgc cgaggcttc 240
tccgacgtgc cttacaacaa gcaacagtta ctgaagaagg ccttcgactc tctggaatc 300 ccgctcgccg atttgcttcg tgatttgaat cccgattgga ttatctacga ttacgcctct 360
cattggcttc cgcagctcgc ggcggagctc cgtatctcgt ctgttttctt cagcctcttc 420
accgcggcgt ttcttgcttt tcttggccca ccgtcggcgt tgtccggcga cggcagttcc 480
cggtga
486
SEQ ID NO: 37
Artificial Sequence; Codon-optimized nucleotide sequence encoding Epoxide Hydrolase 1
atggacgcga ttgaacatag aaccgtaagt gttaatggta tcaatatgca tgtggcagaa 60
aagggagagg gacctgtcgt gttgttgctt catggtttcc cagaattgtg gtacagttgg 120
agacatcaaa tattggctct ttcctcttta ggttacagag ctgtcgcacc agacttacga 180
ggctacgggg atacagatgc cccagggtca atttcatcat acacatgctt tcacatcgta 240
ggagatctcg tggctctagt tgagtctctg ggtatggaca gggtttttgt tgtagcccac 300
gattggggtg ccatgatcgc ttggtgtttg tgtctgttta gacctgaaat ggttaaagct 360
tttgtttgtc tctccgtccc attcagacag agaaacccta agatgaaacc agttcaaagt
420
atgagagcct ttttcggcga tgattactat atttgcagat ttcaaaatcc tggggaaatc 480
gaagaggaga tggctcaagt gggtgcaagg gaagtcttaa gaggaattct aacatctcgt
540
cgtcctggac caccaatctt accaaaaggg caagctttta gagcaagacc aggagcatcc 600
actgcattgc catcttggct atctgaaaaa gatctgtcat ttttcgcttc taagtatgat
660
caaaagggct ttacaggccc actaaactac tacagagcca tggatcttaa ttgggaattg 720
actgcgtcat ggactggtgt ccaagttaaa gtacctgtca aatacatcgt gggtgacgtt
780
gacatggttt ttacgactcc tggtgtaaag gaatatgtca acggcggtgg tttcaaaaag 840
gacgttccat ttttacagga agtggtaatc atggaaggcg ttggtcattt cattaatcag
900
gaaaaacctg aggagatttc atctcatata cacgatttca taagcaaatt ctaa 954
SEQ ID NO: 38
Siraitia grosvenorii protein sequence Met Asp Ala He Glu His Arg Thr Val Ser Val Asn Gly lie Asn Met 1 5 10 15
His Val Ala Glu Lys Gly Glu Gly Pro Val Val Leu Leu Leu His Gly
20 25 30
Phe Pro Glu Leu Trp Tyr Ser Trp Arg His Gin lie Leu Ala Leu Ser
35 40 45
Ser Leu Gly Tyr Arg Ala Val Ala Pro Asp Leu Arg Gly Tyr Gly Asp
50 55 60
Thr Asp Ala Pro Gly Ser He Ser Ser Tyr Thr Cys Phe His He Val 65 70 75 80
Gly Asp Leu Val Ala Leu Val Glu Ser Leu Gly Met Asp Arg Val Phe
85 90 95
Val Val Ala His Asp Trp Gly Ala Met He Ala Trp Cys Leu Cys Leu
100 105 110
Phe Arg Pro Glu Met Val Lys Ala Phe Val Cys Leu Ser Val Pro Phe
115 120 125
Arg Gin Arg Asn Pro Lys Met Lys Pro Val Gin Ser Met Arg Ala Phe
130 135 140
Phe Gly Asp Asp Tyr Tyr He Cys Arg Phe Gin Asn Pro Gly Glu He 145 150 155 160
Glu Glu Glu Met Ala Gin Val Gly Ala Arg Glu Val Leu Arg Gly He
165 170 175
Leu Thr Ser Arg Arg Pro Gly Pro Pro He Leu Pro Lys Gly Gin Ala
180 185 190
Phe Arg Ala Arg Pro Gly Ala Ser Thr Ala Leu Pro Ser Trp Leu Ser
195 200 205
Glu Lys Asp Leu Ser Phe Phe Ala Ser Lys Tyr Asp Gin Lys Gly Phe
210 215 220
Thr Gly Pro Leu Asn Tyr Tyr Arg Ala Met Asp Leu Asn Trp Glu Leu 225 230 235 240
Thr Ala Ser Trp Thr Gly Val Gin Val Lys Val Pro Val Lys Tyr He
245 250 255
Val Gly Asp Val Asp Met Val Phe Thr Thr Pro Gly Val Lys Glu Tyr
2S0 265 270 Val Asn Gly Gly Gly Phe Lys Lys Asp Val Pro Phe Leu Gin Glu Val
275 280 285
Val He Met Glu Gly Val Gly His Phe He Asn Gin Glu Lys Pro Glu
290 295 300
Glu He Ser Ser His He His Asp Phe He Ser Lys Phe
305 310 315
SEQ ID NO: 39
Artificial Sequence; Codon-optimized nucleotide sequence encoding Epoxide Hydrolase 2
atggatgaaa tcgaacatat taccatcaat acaaatggaa tcaaaatgca tattgcgtca
60
gtcggcacag gaccagttgt tctcttgcta cacggctttc cagaattatg gtactcttgg 120
agacaccaac tactttacct gtcctccgtt gggtacagag caatagctcc agatttgaga 180
ggctatggcg atactgacag tccagctagt cctacctctt atactgctct tcatattgta 240
ggtgacctgg tcggcgcatt agacgaattg ggaatagaaa aggtcttttt agtgggtcat 300
gactggggtg ctattatcgc atggtacttt tgtttgttta gaccagatag aattaaagca
360
cttgtgaatt tgtctgtcca gtttatccca cgtaacccag caataccttt tatagaaggt 420
ttcagaacag cttttggtga tgacttctac atttgtagat ttcaagtacc tggggaagct 480
gaagaggatt tcgcgtctat cgatactgct caattgttta aaacttcatt atgcaataga 540
agctcagccc ctccttgttt gcctaaagag attggtttta gggctatccc accaccagaa 600
aatctgccat cttggctcac agaggaagat atcaacttct acgcagccaa gtttaaacaa
660
actggtttta ctggtgccct taactattat agagcattcg acttgacatg ggaattaaca 720
gccccatgga caggagccca gatccaagtt cctgtaaagt tcatagttgg tgattcagat 780
ctcacgtacc atttccctgg tgctaaggaa tacatccaca acggagggtt taaaagagat 840
gtgccactat tagaggaagt tgttgtggta aaagatgcct gccacttcat taaccaagag 900
cgaccacaag agattaatgc tcatattcat gacttcatca ataagttcta a
951
SEQ ID NO: 40
Siraitia grosvenorii protein sequence Met Asp Glu He Glu His He Thr He Asn Thr Asn Gly He Lys Met 1 5 10 15
His He Ala Ser Val Gly Thr Gly Pro Val Val Leu Leu Leu His Gly
20 25 30
Phe Pro Glu Leu Trp Tyr Ser Trp Arg His Gin Leu Leu Tyr Leu Ser
35 40 45
Ser Val Gly Tyr Arg Ala He Ala Pro Asp Leu Arg Gly Tyr Gly Asp 50 55 60
Thr Asp Ser Pro Ala Ser Pro Thr Ser Tyr Thr Ala Leu His He Val 65 70 75 80
Gly Asp Leu Val Gly Ala Leu Asp Glu Leu Gly He Glu Lys Val Phe
85 90 95
Leu Val Gly His Asp Trp Gly Ala He He Ala Trp Tyr Phe Cys Leu
100 105 110
Phe Arg Pro Asp Arg He Lys Ala Leu Val Asn Leu Ser Val Gin Phe
115 120 125
He Pro Arg Asn Pro Ala He Pro Phe He Glu Gly Phe Arg Thr Ala 130 135 140
Phe Gly Asp Asp Phe Tyr He Cys Arg Phe Gin Val Pro Gly Glu Ala 145 150 155 160
Glu Glu Asp Phe Ala Ser He Asp Thr Ala Gin Leu Phe Lys Thr Ser
165 170 175
Leu Cys Asn Arg Ser Ser Ala Pro Pro Cys Leu Pro Lys Glu He Gly
180 185 190
Phe Arg Ala He Pro Pro Pro Glu Asn Leu Pro Ser Trp Leu Thr Glu
195 200 205
Glu Asp He Asn Phe Tyr Ala Ala Lys Phe Lys Gin Thr Gly Phe Thr 210 215 220
Gly Ala Leu Asn Tyr Tyr Arg Ala Phe Asp Leu Thr Trp Glu Leu Thr 225 230 235 240
Ala Pro Trp Thr Gly Ala Gin He Gin Val Pro Val Lys Phe He Val
245 250 255
Gly Asp Ser Asp Leu Thr Tyr His Phe Pro Gly Ala Lys Glu Tyr He
260 265 270 His Asn Gly Gly Phe Lys Arg Asp Val Pro Leu Leu Glu Glu Val Val
275 280 285
Val Val Lys Asp Ala Cys His Phe lie Asn Gin Glu Arg Pro Gin Glu
290 295 300
lie Asn Ala His lie His Asp Phe lie Asn Lys Phe
305 310 315
SEQ ID NO: 41
Sxraitia grosvenorii DNA sequence
gtggggccgt cgtctgttga agctcctcag cggacgattt cgaagcctga acagagggag
60
ctaccgttga ggaagattcc cggggactat gggccgccgt tgttgggtcc gattaaggac
120
cgacaagact atttttacaa tcaggggagg gaggagttcc tgagatcacg catgaacagg 180
tacgaatcaa ctgtgtacag aactaatatg ccaccaggtc cctttatctc ctccgattct
240
cgtgtcatcg ttttactcga cggcaagagc ttccctgtac tcttcgacgt ttctaaagtt 300
ctgaaacaag acgtcttcac cggaacttat atgcccttaa cggagctcac tggcggctac
360
cgagttcttt cttatctcga cccctccgag cccgatcacg agaagcttaa acagttcctc 420
ttctacctcc tcaagtaccg tcgcgacaag attctgccgg agtttcactc taccttttcg
480
gagctgtttg agactctgga gaaggaggtg gctgccgccg gtagagcaga ttataatgat 540
cccggtgaac aggcggcgtt taacttcttg gctcggtctc tgttcggcgc caacccgccc
600
gacaccaaac tgggaaacga cgctccgagt ttaatatcca aatgggtgct gttccagctg 660
ggtccggttc tcactcttgg tcttcccaag cctgtcgagg agcttctcct gcgaaccgtc
720
cggctgccac cggcgcttgt gaaatcggat taccagcggc tgtacgattt cttttacgag 780
gcgtcggagg ctgtgtttgc ggaggcggat agattgggca ttgcgagaga ggaagcgtgt
840
cacaacttgg tcttcgccac gtgcttcaat tccttcggag ggatgaagat cctcttcccc 900
aatatgataa aatggatcgg acgtgccgga gtgaatctcc atacggagct cgcacgggag
960
ataagatccg ccgtcaaagc ccacggcggc aagatcacga tggcggctat ggaacagatg 1020
ccgctgatga agtccgtagt gtacgaaacg ctcagaatcg aacccccggt tcctgcgcaa
1080
tacgggcgag cgaaggagga cctggtgatc gagagccacg acgccgcttt cgagatcaaa 1140 gaaggggaaa tgttgtgtgg gtaccagcca ttcgccacta gagatccgaa aatattcgag 1200
agatccgaag aattcgtacc ggatcggttc accggcgacg gcgaggagtt gctgaagcac 1260
gtgctctggt caaacggacc ggagactcaa tccccaaccg ttaaagacaa gcagtgcgct 1320
ggcaaagact tcatagtctt cgtctcccgc ctcctcgtcg tcgaactctt cctccgatac 1380
gactccttcg acattgaagt cgcagcttcg ccgttgggcg ccgccgtcac cataacttcc 1440
ctgaagaagg !aagctttta a 1461
SEQ ID NO: 2
Artificial Sequence Codon-optimized nucleotide sequence encoding cucurbitadienol synthase
atgtggagat tgaaagtagg tgctgaatcc gtaggtgaaa acgacgaaaa gtggttgaaa 60
agtataagta atcatttggg tagacaagtc tgggaatttt gtccagatgc aggtacacaa 120
caacaattgt tgcaagtaca taaggctaga aaggcatttc atgatgacag attccacaga 180
aagcaatctt cagatttgtt catcaccatc caatacggca aggaagtaga aaacggtggc 240
aagactgctg gtgttaaatt gaaggaaggt gaagaagtta gaaaagaagc agttgaatcc 300
agtttggaaa gagccttgtc tttctactct tcaatccaaa cctctgatgg taattgggca 360
tcagacttgg gtggtccaat gttcttgtta cctggtttgg tcattgcctt gtacgtaact 420
ggtgttttga actctgtatt gtcaaagcat cacagacaag aaatgtgtag atacgtttac 480
aaccatcaaa acgaagatgg tggttggggt ttgcacattg aaggtccatc cactatgttt 540
ggtagtgcat tgaattatgt cgccttaaga ttgttaggtg aagatgcaaa cgccggtgct 600
atgcctaagg caagagcctg gatattagac catggtggtg ctactggtat cacatcctgg 660
ggtaaattgt ggttaagtgt cttaggtgta tatgaatggt ctggtaataa cccattgcca 720
cctgaatttt ggttgttccc ttacttttta ccattccatc ctggtagaat gtggtgtcac 780
tgcagaatgg tttacttgcc aatgtcttac ttgtacggca agagattcgt tggtccaata 840
acacctatcg tcttgtcatt gagaaaggaa ttgtacgcag ttccttacca tgaaatcgat 900
tggaacaagt ccagaaacac ctgtgctaag gaagatttgt attacccaca ccctaaaatg 960 caagacattt tgtggggtag tttacatcac gtttacgaac cattatttac tagatggcct
1020
gctaaaagat tgagagaaaa ggcattacaa acagccatgc aacatatcca ctacgaagat 1080
gaaaacacca gatacatctg cttgggtcca gttaacaagg tcttgaactt gttgtgttgc 1140
tgggttgaag atccttattc tgacgctttc aagttgcatt tgcaaagagt acacgattac 1200
ttgtgggttg cagaagacgg tatgaaaatg caaggttaca atggttcaca attgtgggat 1260
acagcttttt ccattcaagc aatagtcagt actaagttgg tagataacta cggtccaaca 1320
ttaagaaaag ctcatgactt cgtaaagtcc agtcaaatac aacaagattg tccaggtgac
1380
cctaatgttt ggtatagaca tatccacaaa ggtgcatggc cattttctac cagagatcat
1440
ggttggttga tttcagactg tactgctgaa ggtttgaagg ctgcattgat gttgtctaag 1500
ttgccatcag aaactgttgg tgaatccttg gaaagaaata gattatgcga tgccgttaac 1560
gtcttgttga gtttgcaaaa cgacaacggt ggtttcgctt cttacgaatt gactagatca 1620
tacccatggt tggaattaat taatcctgct gaaacattcg gtgatatcgt cattgactat 1680
ccatacgtag aatgtacctc cgctactatg gaagcattga ccttgttcaa gaagttgcat
1740
cctggtcaca gaacaaagga aatcgatacc gcaattgtta gagccgctaa tttcttggaa
1800
aacatgcaaa gaacagacgg ttcttggtat ggttgttggg gtgtttgctt tacctacgct
1860
ggttggttcg gtattaaagg tttagtcgca gccggtagaa catacaataa ctgtttggcc 1920
ataagaaaag cttgcgattt cttgttatct aaggaattac caggtggtgg ttggggtgaa 1980
tcctacttga gttgtcaaaa caaggtttac actaatttgg aaggcaacag acctcattta 2040
gttaacacag cctgggtctt gatggcttta atcgaagccg gtcaagctga aagagatcca 2100
actcctttgc atagagctgc aagattgttg atcaactcac aattggaaaa cggtgatttt
2160
ccacaacaag aaatcatggg tgttttcaac aagaactgca tgataacata tgccgcttac
2220
agaaacattt ttcctatatg ggctttgggt gaatactgcc acagagtctt gaccgaataa 2280
SEQ ID NO: 43
Siraitia grosvenori i protein sequence
Met Trp Arg Leu Lys Val Gly Ala Glu Ser Val Gly Glu Asn Asp Glu
1 5 10 15 Lys Trp Leu Lys Ser lie Ser Asn His Leu Gly Arg Gin Val Trp Glu
20 25 30
Phe Cys Pro Asp Ala Gly Thr Gin Gin Gin Leu Leu Gin Val His Lys
35 40 45
Ala Arg Lys Ala Phe His Asp Asp Arg Phe His Arg Lys Gin Ser Ser
50 55 60
Asp Leu Phe lie Thr lie Gin Tyr Gly Lys Glu Val Glu Asn Gly Gly 65 70 75 80
Lys Thr Ala Gly Val Lys Leu Lys Glu Gly Glu Glu Val Arg Lys Glu
85 90 95
Ala Val Glu Ser Ser Leu Glu Arg Ala Leu Ser Phe Tyr Ser Ser lie
100 105 110
Gin Thr Ser Asp Gly Asn Trp Ala Ser Asp Leu Gly Gly Pro Met Phe
115 120 125
Leu Leu Pro Gly Leu Val lie Ala Leu Tyr Val Thr Gly Val Leu Asn 130 135 140
Ser Val Leu Ser Lys His His Arg Gin Glu Met Cys Arg Tyr Val Tyr
145 150 155 160
Asn His Gin Asn Glu Asp Gly Gly Trp Gly Leu His lie Glu Gly Pro
165 170 175
Ser Thr Met Phe Gly Ser Ala Leu Asn Tyr Val Ala Leu Arg Leu Leu
180 185 190
Gly Glu Asp Ala Asn Ala Gly Ala Met Pro Lys Ala Arg Ala Trp lie
195 200 205
Leu Asp His Gly Gly Ala Thr Gly lie Thr Ser Trp Gly Lys Leu rp 210 215 220
Leu Ser Val Leu Gly Val Tyr Glu Trp Ser Gly Asn Asn Pro Leu Pro
225 230 235 240
Pro Glu Phe Trp Leu Phe Pro Tyr Phe Leu Pro Phe His Pro Gly Arg
245 250 255
Met Trp Cys His Cys Arg Met Val Tyr Leu Pro Met Ser Tyr Leu Tyr
260 265 270
Gly Lys Arg Phe Val Gly Pro lie Thr Pro lie Val Leu Ser Leu Arg
275 280 285 Lys Glu Leu Tyr Ala Val Pro Tyr His Glu He Asp Trp Asn Lys Ser 290 295 300
Arg Asn Thr Cys Ala Lys Glu Asp Leu Tyr Tyr Pro His Pro Lys Met 305 310 315 320
Gin Asp lie Leu Trp Gly Ser Leu His His Val Tyr Glu Pro Leu Phe
325 330 335
Thr Arg Trp Pro Ala Lys Arg Leu Arg Glu Lys Ala Leu Gin Thr Ala
340 345 350
Met Gin His lie His Tyr Glu Asp Glu Asn Thr Arg Tyr He Cys Leu
355 360 365
Gly Pro Val Asn Lys Val Leu Asn Leu Leu Cys Cys Trp Val Glu Asp
370 375 380
Pro Tyr Ser Asp Ala Phe Lys Leu His Leu Gin Arg Val His Asp Tyr 385 390 395 400
Leu Trp Val Ala Glu Asp Gly Met Lys Met Gin Gly Tyr Asn Gly Ser
405 410 415
Gin Leu Trp Asp Thr Ala Phe Ser He Gin Ala He Val Ser Thr Lys
420 425 430
Leu Val Asp Asn Tyr Gly Pro Thr Leu Arg Lys Ala His Asp Phe Val
435 440 445
Lys Ser Ser Gin lie Gin Gin Asp Cys Pro Gly Asp Pro Asn Val Trp
450 455 460
Tyr Arg His lie His Lys Gly Ala Trp Pro Phe Ser Thr Arg Asp His
465 470 475 480
Gly Trp Leu lie Ser Asp Cys Thr Ala Glu Gly Leu Lys Ala Ala Leu
485 490 495
Met Leu Ser Lys Leu Pro Ser Glu Thr Val Gly Glu Ser Leu Glu Arg
500 505 510
Asn Arg Leu Cys Asp Ala Val Asn Val Leu Leu Ser Leu Gin Asn Asp
515 520 525
Asn Gly Gly Phe Ala Ser Tyr Glu Leu Thr Arg Ser Tyr Pro Trp Leu
530 535 540
Glu Leu He Asn Pro Ala Glu Thr Phe Gly Asp He Val He Asp Tyr 545 550 555 560 Pro Tyr Val Glu Cys Thr Ser Ala Thr Met Glu Ala Leu Thr Leu Phe 565 570 575
Lys Lys Leu His Pro Gly His Arg Thr Lys Glu He Asp Thr Ala He
580 585 590
Val Arg Ala Ala Asn Phe Leu Glu Asn Met: Gin Arg Thr Asp Gly Ser
595 600 605
Trp Tyr Gly Cys Trp Gly Val Cys Phe Thr Tyr Ala Gly Trp Phe Gly 610 615 620
lie Lys Gly Leu Val Ala Ala Gly Arg Thr Tyr Asn Asn Cys Leu Ala 625 630 635 640
He Arg Lys Ala Cys Asp Phe Leu Leu Ser Lys Glu Leu Pro Gly Gly
645 650 655
Gly Trp Gly Glu Ser Tyr Leu Ser Cys Gin Asn Lys Val Tyr Thr Asn
660 665 670
Leu Glu Gly Asn Arg Pro Hi s Leu Val Asn Thr Ala Trp Val Leu Met
675 680 685
Ala Leu He Glu Ala Gly Gin Ala Glu Arg Asp Pro Thr Pro Leu His
690 695 700
Arg Ala Ala Arg Leu Leu He Asn Ser Gin Leu Glu Asn Gly Asp Phe
705 710 715 720
Pro Gin Gin Glu He Met Gly Val Phe Asn Lys Asn Cys Met He Thr
725 730 735
Tyr Ala Ala Tyr Arg Asn He Phe Pro He Trp Ala Leu Gly Glu Tyr
740 745 750
Cys His Arg Val Leu Thr Glu
755
SEQ ID NO: 44
Siraitia grosvenorii protein sequence
Met Trp Thr Val Val Leu Gly Leu Ala Thr Leu Phe Val Ala Tyr Tyr 1 5 10 15
He His Trp He Asn Lys Trp Arg Asp Ser Lys Phe Asn Gly Val Leu
20 25 30
Pro Pro Gly Thr Met Gly Leu Pro Leu He Gly Glu Thr He Gin Leu 35 40 45
Ser Arg Pro Ser Asp Ser Leu Asp Val His Pro Phe He Gin Lys Lys 50 55 60
Val Glu Arg Tyr Gly Pro lie Phe Lys Thr Cys Leu Ala Gly Arg Pro
65 70 75 80
Val Val Val Ser Ala Asp Ala Glu Phe Asn Asn Tyr He Met Leu Gin
85 90 95
Glu Gly Arg Ala Val Glu Met Trp Tyr Leu Asp Thr Leu Ser Lys Phe
100 105 110
Phe Gly Leu Asp Thr Glu Trp Leu Lys Ala Leu Gly Leu He His Lys
115 120 125
Tyr lie Arg Ser lie Thr Leu Asn His Phe Gly Ala Glu Ala Leu Arg
130 135 140
Glu Arg Phe Leu Pro Phe lie Glu Ala Ser Ser Met Glu Ala Leu His
145 150 155 160
Ser Trp Ser Thr Gin Pro Ser V l Glu Val Lys Asn Ala Ser Ala Leu
165 170 175
Met Val Phe Arg Thr Ser Val Asn Lys Met Phe Gly Glu Asp Ala Lys
180 185 190
Lys Leu Ser Gly Asn lie Pro Gly Lys Phe Thr Lys Leu Leu Gly Gly
195 200 205
Phe Leu Ser Leu Pro Leu Asn Phe Pro Gly Thr Thr Tyr His Lys Cys
210 215 220
Leu Lys Asp Me Lys Glu lie Gin Lys Lys Leu Arg Glu Val Val Asp
225 230 235 240
Asp Arg Leu Ala As Val Gly Pro Asp Val Glu Asp Phe Leu Gly Gin
245 250 255
Ala Leu Lys Asp Lys Glu Ser Glu Lys Phe lie Ser Glu Glu Phe lie
260 265 270
lie Gin Leu Leu Phe Ser lie Ser Phe Ala Ser Phe Glu Ser He Ser
275 280 285
Thr Thr Leu Thr Leu lie Leu Lys Leu Leu Asp Glu His Pro Glu Val
230 295 300
Val Lys Glu Leu Glu Ala Gl His Glu Ala lie Arg Lys Ala Arg Ala 305 310 315 320
Asp Pro Asp Gly Pro lie Thr Trp Glu Glu Tyr Lys Ser Met Thr Phe
325 330 335
Thr Leu Gin Val lie Asn Glu Thr Leu Arg Leu Gly Ser Val Thr Pro
340 345 350
Ala Leu Leu Arg Lys Thr Val Lys Asp Leu Gin Val Lys Gly Tyr He
355 360 365
lie Pro Glu Gly Trp Thr lie Met Leu Val Thr Ala Ser Arg His Arg
370 375 380
Asp Pro Lys Val Tyr Lys Asp o His He Phe Asn Pro Trp Arg Trp
385 3.90 395 400
Lys Asp Leu Asp Ser lie Thr lie Gin Lys Asn Phe Met Pro Phe Gly
405 410 415
Gly Gly Leu Arg His Cys Ala Gly Ala Glu Tyr Ser Lys Val Tyr Leu
420 425 430
Cys Thr Phe Leu His lie Leu Cys Thr Lys Tyr Arg Trp Thr Lys Leu
435 440 445
Gly Gly Gly Arg He Ala Arg Ala His He Leu Ser Phe Glu Asp Gly
450 455 460
Leu His Val Lys Phe Thr Pro Lys Glu
465 470
SEQ ID NO: 45
Siraitia gros enorii DNA sequence
atgaaggtct ctccatttga gttcatgtcg gcaataatta agggcaggat ggacccgtcc 60
aattcttcat ttgagtcgac tggcgaggtt gectcagtta ttttcgagaa ccgtgagctg
120
gttgegatet taaccacctc gatcgccgtc atgattggct gcttcgttgt tctcatgtgg 180
egaagagecg geagteggaa agttaagaac gtggagctac etaagcegtt gattgtgcac
240
gagceggage ccgaagttga agaeggcaag aagaaggttt caatcttctt eggtacacag
300
acaggcaccg ccgaaggatt tgeaaagget ctagctgacg aggegaaage acgatacgag
360
aaggecacat ttagagttgt tgatttggat gattatgeag ctgatgacga tcagtatgaa
420 gagaagttga agaacgagtc tttcgctgtc ttcttattgg caacgtatgg cgatggagag 480
cccactgata atgccgcaag attctataaa tggttcgcgg aggggaaaga gagaggggag
540
tggcttcaga accttcatta tgcggtcttt ggccttggca accgacagta cgagcatttt 600
aataagattg caaaggtggc agatgagctg cttgaggcac agggaggcaa ccgccttgtt 660
aaagtfcggtc ttggagatga cgatcagtgc atagaggatg acttcagtgc ctggagagaa
720
tcattgtggc ctgagttgga tatgttgctt cgagatgagg atgatgcaac aacagtgacc 780
accccttaca cagctgccgt attagaatat cgagttgtat tccatgattc tgcagatgta 840
gctgctgagg acaagagctg gatcaatgca aacggtcatg ctgtacatga tgctcagcat
900
cccttcagat ctaatgtggt tgtgaggaag gagctccata cgtccgcatc tgatcgctcc 960
tgtagtcatc tagaatttaa tatttctggg tctgcactca attatgaaac aggggatcat 1020
gtcggtgttt actgtgaaaa cttaactgag actgtggacg aggcactaaa cttattgggt
1080
ttgtctcctg aaacgtattt ctccatatat actgataacg aggatggcac tccacttggt 1140
ggaagctctt taccacctcc ttttccatcc tgcaccctca gaacagcatt gactcgatat 1200
gcagatctct tgaattcacc caagaagtca gctttgcttg cattagcagc acatgcttca
1260
aatccagtag aggctgaccg attaagatat cttgcatcac ctgccgggaa ggatgaatac 1320
gcccagtctg tgattggtag ccagaaaagc cttcttgagg tcatggctga atttccttct 1380
gccaagcccc cacttggtgt cttcttcgca gctgttgcac cgcgcttgca gcctcgattc
1440
tactccatat catcatctcc aaggatggct ccatctagaa ttcatgttac ttgtgcttta
1500
gtctatgaca aaatgccaac aggacgtatt cataaaggag tgtgctcaac ttggatgaag 1560
aattctgtgc ccatggagaa aagccatgaa tgcagttggg ctccaatttt cgtgagacaa 1620
tcaaacttca agcttcctgc agagagtaaa gtgcccatta tcatggttgg tcctggaact
1680
ggattggctc ctttcagagg tttcttacag gaaagat ag ctttgaagga atctggagta 1740
gaattggggc cttccatatt gttctttgga tgcagaaacc gtaggatgga ttacatatac 1800
gaggatgagc tgaacaactt tgttgagact ggtgctctct ctgagttggt tattgccttc
1860
tcacgcgaag ggccaactaa ggaatatgtg cagcataaaa tggcagagaa ggcttcggat 1920 atctggaatt tgatatcaga aggggcttac ttatatgtat gtggtgatgc aaagggcatg 1980
gctaaggatg tccaccgaac tctccatact atcatgcaag agcagggatc tcttgacagc 2040
tcaaaagctg agagcatggt gaagaatctg caaatgaatg gaaggtatct gcgtgatgtc 2100
tggtga
2106
SEQ ID NO: 46
Siraitia grosvenorii protein sequence
Met Lys Val Ser Pro Phe Glu Phe Met Ser Ala He He Lys Gly Arg
1 5 10 15
Met Asp Pro Ser Asn Ser Ser Phe Glu Ser Thr Gly Glu Val Ala Ser
20 25 30
Val He Phe Glu Asn Arg Glu Leu Val Ala He Leu Thr Thr Ser He
35 40 45
Ala Val Met He Gly Cys Phe Val Val Leu Met Trp Arg Arg Ala Gly
50 55 60
Ser Arg Lys Val Lys Asn Val Glu Leu Pro Lys Pro Leu He Val His
55 70 75 80
Glu Pro Glu Pro Glu Val Glu Asp Gly Lys Lys Lys Val Ser He Phe
85 90 95
Phe Gly Thr Gin Thr Gly Thr Ala Glu Gly Phe Ala Lys Ala Leu Ala
100 105 110
Asp Glu Ala Lys Ala Arg Tyr Glu Lys Ala Thr Phe Arg Val Val Asp
115 120 125
Leu Asp Asp Tyr Ala Ala Asp Asp Asp Gin Tyr Glu Glu Lys Leu Lys
130 135 140
Asn Glu Ser Phe Ala Val Phe Leu Leu Ala Thr Tyr Gly Asp Gly Glu
145 150 155 160
Pro Thr Asp Asn Ala Ala Arg Phe Tyr Lys Trp Phe Ala Glu Gly Lys
165 170 175
Glu Arg Gly Glu Trp Leu Gin Asn Leu His Tyr Ala Val Phe Gly Leu
180 185 190
Gly Asn Arg Gin Tyr Glu His Phe Asn Lys He Ala Lys Val Ala Asp
195 200 205 Glu Leu Leu Glu Ala Gin Gly Gly Asn Arg Leu Val Lys Val Gly Leu
210 215 220
Gly Asp Asp Asp Gin Cys He Glu Asp Asp Phe Ser Ala Trp Arg Glu 225 230 235 240
Ser Leu Trp Pro Glu Leu Asp Met Leu Leu Arg Asp Glu Asp Asp Ala
245 250 255
Thr Thr Val Thr Thr Pro Tyr Thr Ala Ala Val Leu Glu Tyr Arg Val
260 265 270
Val Phe His Asp Ser Ala Asp Val Ala Ala Glu Asp Lys Ser Trp lie
275 280 285
Asn Ala Asn Gly His Ala Val His Asp Ala Gin His Pro Phe Arg Ser
290 295 300
Asn Val Val Val Arg Lys Glu Leu His Thr Ser Ala Ser Asp Arg Ser 305 310 315 320
Cys Ser His Leu Glu Phe Asn lie Ser Gly Ser Ala Leu Asn Tyr Glu
325 330 335
Thr Gly Asp His Val Gly Val Tyr Cys Glu Asn Leu Thr Glu Thr Val
340 345 350
Asp Glu Ala Leu Asn Leu Leu Gly Leu Ser Pro Glu Thr Tyr Phe Ser
355 360 365
lie Tyr Thr Asp Asn Glu Asp Gly Thr Pro Leu Gly Gly Ser Ser Leu
370 375 380
Pro Pro Pro Phe Pro Ser Cys Thr Leu Arg Thr Ala Leu Thr Arg Tyr 385 390 395 400
Ala Asp Leu Le Asn Ser Pro Lys Lys Ser Ala Leu Leu Ala Leu Ala
405 410 415
Ala His Ala Ser Asn Pro Val Glu Ala Asp Arg Leu Arg Tyr Leu Ala
420 425 430
Ser Pro Ala Gly Lys Asp Glu Tyr Ala Gin Ser Val lie Gly Ser Gin
435 440 445
Lys Ser Leu Leu Glu Val Met Ala Glu Phe Pro Ser Ala Lys Pro Pro
450 455 460
Leu Gly Val Phe Phe Ala Ala Val Ala Pro Arg Leu Gin Pro Arg Phe 465 470 475 480 Tyr Ser He Ser Ser Ser Pro Arg Met Ala Pro Ser Arg He His Vai
485 490 495
Thr Cys Ala Leu Val Tyr Asp Lys Met Pro Thr Gly Arg He His Lys
500 505 510
Gly Val Cys Ser Thr Trp Met Lys Asn Ser Val Pro Met Glu Lys Ser
515 520 525
His Glu Cys Ser Trp Ala Pro He Phe Val Arg Gin Ser Asn Phe Lys
530 535 540
Leu Pro Ala Glu Ser Lys Val Pro He He Met Val Gly Pro Gly Thr
545 550 555 560
Gly Leu Ala Pro Phe Arg Gly Phe Leu Gin Glu Arg Leu Ala Leu Lys
565 570 575
Glu Ser Gly Val Glu Leu Gly Pro Ser He Leu Phe Phe Gly Cys Arg
580 585 590
Asn Arg Arg Met Asp Tyr He Tyr Glu Asp Glu Leu Asn Asn Phe Val
595 600 605
Glu Thr Gly Ala Leu Ser Glu Leu Val He Ala Phe Ser Arg Glu Gly
610 615 620
Pro Thr Lys Glu Tyr Val Gin His Lys Met Ala Glu Lys Ala Ser Asp
625 630 635 640
He Trp Asn Leu He Ser Glu Gly Ala Tyr Leu Tyr Val Cys Gly Asp
645 650 655
Ala Lys Gly Met Ala Lys Asp Val His Arg Thr Leu His Thr He Met
660 665 670
Gin Glu Gin Gly Ser Leu Asp Ser Ser Lys Ala Glu Ser Met Val Lys
675 680 685
Asn Leu Gin Met Asn Gly Arg Tyr Leu Arg Asp Val Trp
690 695 700
SEQ ID NO: 47
Siraitia grosvenorii DMA sequence
atggcttctc ctcgccacac tcctcacttt ctgctcttcc ctttcatggc tcaaggccac 60
atgatcccca tgattgacct tgccaggctt ctggctcagc gaggagttat catcactatt 120 atcaccacgc cccacaatgc tgctcgctac cactctgttc ttgctcgcgc catcgattct
180
ggg tacaca tccatgtcct ccaactgcag tttccatgta aggaaggtgg gctgccagaa
240
gggtgcgaga atgtggactt gctaccttca cttgcttcca tacccagatt ctacagagca 300
gcaagtgatc tcctttacga accatctgaa aaactgtttg aggaactcat cccccggccg 360
acctgcataa tctccgatat gtgcctgccc tggaccatgc gaattgctct gaaatatcac 420
gtcccaaggc tcgttttcta cagtttgagc tgcttctttc ttctctgtat gcggagttta 480
aaaaacaatc tagcgcttat aagctccaag tctgattctg agttcgtaac tttctctgac
540
ttgcctgatc cagtcgagtt tctcaagtcg gagctaccta aatccaccga tgaagacttg 600
gtgaagttta gttatgaaat gggggaggcc gatcggcagt catacggcgt tattttaaat 660
ctatttgagg agatggaacc aaagtatctt gcagaatatg aaaaggaaag agaatcgccg 720
gaaagagtct ggtgcgtcgg cccagtttcg ctttgcaacg acaacaaact cgacaaagct 780
gaaagaggca acaaagcctc catcgacgaa tacaaatgca tcaggtggct cgacgggcag
840
cagccatctt cggtggttta cgtctcttta ggaagcttgt gcaatctggt gacggcgcag 900
atcatagagc tgggtttggg tttggaggca tcaaagaaac ccttcatttg ggtcataaga 960
agaggaaaca taacagagga gttacagaaa tggcttgtgg agtacgattt cgaggagaaa 1020
attaaaggga gagggctggt gattcttggc tgggctcccc aagttctgat actgtcacac 1080
cctgcaatcg gatgcttttt gacgcactgc ggttggaact caagcatcga agggatatcg
1140
gccggcgtgc caatggtcac ctggccgctt tttgcggatc aagtcttcaa cgagaagcta
1200
attgtacaaa tactcagaat cggcgtaagt gtaggcacgg aaactactat gaactgggga 1260
gaggaagagg agaaaggggt ggttgtgaag agagagaaag tgagggaagc catagaaata 1320
gtgatggatg gagatgagag agaagagagg agagagagat gcaaagagct tgctgaaacg 1380
gcgaagagag ctatagaaga agggggctcg tctcaccgga acctcacgat gttgattgaa
1440
gatataattc atggaggagg tttgagttat gagaaaggaa gttgtcgctg
1491
SEQ ID NO -.48
Sirait a grosvenorii protein sequence Met Ala Ser Pro Arg His Thr Pro His Phe Leu Leu Phe Pro Phe Met 1 5 10 15
Ala Gin Gly His Met lie Pro Met lie Asp Leu Ala Arg Leu Leu Ala
20 25 30
Gin Arg Gly Val He He Thr He He Thr Thr Pro His Asn Ala Ala
35 40 45
Arg Tyr His Ser Val Leu Ala Arg Ala He Asp Ser Gly Leu His He
50 55 60
His V l Leu Gin Leu Gin Phe Pro Cys Lys Glu Gly Gly Leu Pro Glu
65 70 75 80
Gly Cys Glu Asn Val Asp Leu Leu Pro Ser Leu Ala Ser lie Pro Arg
85 90 95
Phe Tyr Arg Ala Ala Ser Asp Leu Leu Tyr Glu Pro Ser Glu Lys Leu
100 105 110
Phe Glu Glu Leu He Pro Arg Pro Thr Cys He He Ser Asp Met Cys
115 120 125
Leu Pro Trp Thr Met Arg He Ala Leu Lys Tyr His Val Pro Arg Leu
130 135 140
Val Phe Tyr Ser Leu Ser Cys Phe Phe Leu Leu Cys Met Arg Ser Leu 145 150 155 160
Lys Asn Asn Leu Ala Leu lie Ser Ser Lys Ser Asp Ser Glu Phe Val
165 170 175
Thr Phe Ser Asp Leu Pro Asp Pro Val Glu Phe Leu Lys Ser Glu Leu
180 185 190
Pro Lys Ser Thr Asp Glu Asp Leu Val Lys Phe Ser Tyr Glu Me Gly
195 200 205
Glu Ala Asp Arg Gin Ser Tyr Gly Val He Leu Asn Leu Phe Glu Glu
210 215 220
Met Glu Pro Lys Tyr Leu Ala Glu Tyr Glu Lys Glu Arg Glu Ser Pro 225 230 235 240
Glu Arg Val Trp Cys Val Gly Pro Val Ser Leu Cys Asn Asp Asn Lys
245 250 255
Leu Asp Lys Ala Glu Arg Gly Asn Lys Ala Ser He Asp Glu Tyr Lys
260 265 270 Cys lie Arg Trp Leu Asp Gly Gin Gin Pro Ser Ser Val Val Tyr Val 275 280 285
Ser Leu Gly Ser Leu Cys Asn Leu Val Thr Ala Gin He lie Glu Leu
290 295 300
Gly Leu Gly Leu Glu Ala Ser Lys Lys Pro Phe He Trp Val He Arg
305 310 315 320
Arg Gly Asn He Thr Glu Glu Leu Gin Lys Trp Leu Val Glu Tyr Asp
325 330 335
Phe Glu Glu Lys He Lys Gly Arg Gly Leu Val He Leu Gly Trp Ala
340 345 350
Pro Gin Val Leu He Leu Ser His Pro Ala He Gly Cys Phe Leu Thr
355 360 365
His Cys Gly Trp Asn Ser Ser He Glu Gly He Ser Ala Gly Val Pro
370 375 380
Met Val Thr Trp Pro Leu Phe Ala Asp Gin Val Phe Asn Glu Lys Leu
385 390 335 400
He Val Gin He Leu Arg lie Gly Val Ser Val Gly Thr Glu Thr Thr
405 410 415
Met Asn Trp Gly Glu Glu Glu Glu Lys Gly Val Val Val Lys Arg Glu
420 425 430
Lys Val Arg Glu Ala He Glu lie Val Met Asp Gly Asp Glu Arg Glu
435 440 445
Glu Arg Arg Glu Arg Cys Lys Glu Leu Ala Glu Thr Ala Lys Arg Ala
450 455 460
He Glu Glu Gly Gly Ser Ser His Arg Asn Leu Thr Met Leu He Glu
465 470 475 480
Asp He He His Gly Gly Gly Leu Ser Tyr Glu Lys Gly Ser Cys Arg
485 490 495
SEQ ID NO: 9
Sirai ia grosvenorii DNA sequence
atggatgccc agcgaggtca caccaccacc attttgatgc ttccatgggt cggctacggc 60
catctcttgc ctttcctcga gctggccaaa agcctctcca ggaggaaatt attccacatc 120 tacttctgtt caacgtctgt tagcctcgac gccattaaac caaagcttcc tccttctatc 180
tcttctgatg attccatcca acttgtggaa cttcgtctcc cttcttctcc tgagttacct
240
cctcatcttc acacaaccaa cggccttccc tctcacctca tgcccgctct ccaccaagcc 300
ttcgtcatgg ccgcccaaca ctttcaggtc attttacaaa cacttgcccc gcatctcctc 360
atttatgaca ttctccaacc ttgggctcct caagtggctt catccctcaa cattccagcc
420
atcaacttca gtactaccgg agcttcaatg ctttctcgaa cgcttcaccc tactcactac 480
ccaagttcta aattcccaat ctcagagttt gttcttcaca atcactggag agccatgtac
540
accaccgccg atggggctct tacagaagaa ggccacaaaa ttgaagaaac acttgcgaat 600
tgcttgcata cttcttgcgg ggtagttttg gtcaatagtt tcagagagct tgagacgaaa
660
tatatcgatt atctctctgt tctcttgaac aagaaagttg ttccggtcgg tcctttggtt 720
tacgaaccga atcaagaagg ggaagatgaa ggttattcaa gcatcaaaaa ttggcttgac 780
aaaaaggaac cgtcctcaac cgtcttcgtt tcatttggaa ccgaatactt cccgtcaaag
840
gaagaaatgg aagagatagc gtatgggtta gagctgagcg aggttaattt catctgggtc 900
cttagatttc ctcaaggaga cagcaccagc accattgaag acgccttgcc gaaggggttt
960
ctggagagag cgggagagag ggcgatggtg gtgaagggtt gggctcctca ggcgaagata 1020
ctgaagcatt ggagcacagg ggggcttgtg agtcactgtg gatggaactc gatgatggag
1080
ggcatgatgt ttggcgtacc cataatagcg gtcccgatgc atctggacca gccctttaac 1140
gccggactct tggaagaagc tggcgtcggc gtggaagcca agcgaggttc ggacggcaaa 1200
attcaaagag aagaagttgc aaagtcgatc aaagaagtgg tgattgagaa aaccagggaa
1260
gacgtgagga agaaagcaag agaaatgggt gagattttga ggagtaaagg agatgagaaa 1320
attgatgagt tggtggctga aatttctctt ttgcgcaaaa aggctccatg ttcaatttaa
1380
SEQ ID NO: SO
Siraitia grosvenorii protein sequence
Met Asp Ala Gin Arg Gly His Thr Thr Thr lie Leu Met Leu Pro Trp
1 5 10 15
Val Gly Tyr Gly His Leu Leu Pro Phe Leu Glu Leu Ala Lys Ser Leu 20 25 30
Ser Arg Arg Lys Leu Phe His lie Tyr Phe Cys Ser Thr Ser Val Ser
35 40 45
Leu Asp Ala lie Lys Pro Lys Leu Pro Pro Ser He Ser Ser Asp Asp
50 55 60
Ser He Gin Leu Val Glu Leu Arg Leu Pro Ser Ser Pro Glu Leu Pro 65 70 75 80
Pro His Leu His Thr Thr Asn Gly Leu Pro Ser His Leu Met Pro Ala
85 90 95
Leu His Gin Ala Phe Val Met Ala Ala Gin His Phe Gin Val He Leu
100 105 110
Gin Thr Leu Ala Pro His Leu Leu He Tyr Asp He Leu Gin Pro Trp
115 120 125
Ala Pro Gin Val Ala Ser Ser Leu Asn He Pro Ala He Asn Phe Ser
130 135 140
Thr Thr Gly Ala Ser Met Leu Ser Arg Thr Leu His Pro Thr His Tyr
145 150 155 160
Pro Ser Ser Lys Phe Pro He Ser Glu Phe Val Leu His Asn His Trp
165 170 175
Arg Ala Met Tyr Thr Thr Ala Asp Gly Ala Leu Thr Glu Glu Gly His
180 185 1.90
Lys He Glu Glu Thr Leu Ala Asn Cys Leu His Thr Ser Cys Gly Val
195 200 205
Val Leu Val Asn Ser Phe Arg Glu Leu Glu Thr Lys Tyr He Asp Tyr
210 215 220
Leu Ser Val Leu Leu Asn Lys Lys Val Val Pro V l Gly Pro Leu Val
225 230 235 240
Tyr Glu Pro Asn Gin Glu Gly Glu Asp Glu Gly Tyr Ser Ser He Lys
245 250 255
Asn Trp Leu Asp Lys Lys Glu Pro Ser Ser Thr Val Phe Val Ser Phe
260 265 270
Gly Thr Glu Tyr Phe Pro Ser Lys Glu Glu Met Glu Glu He Ala Tyr
275 280 285
Gl Leu Glu Leu Ser Glu Val Asn Phe He Trp Val Leu Arg Phe Pro 295 300
Gin Gly Asp Ser Thr Ser Thr He Glu Asp Ala Leu Pro Lys Gly Phe
305 310 315 320
Leu Glu Arg Ala Gly Glu Arg Ala Met Val Val Lys Gly Trp Ala Pro
325 330 335
Gin Ala Lys He Leu Lys His Trp Ser Thr Gly Gly Leu Val Ser His
340 345 350
Cys Gly Trp Asn Ser Met Met Glu Gly Met Me Phe Gly Val Pro He
355 360 365
He Ala Val Pro Met His Leu Asp Gin Pro Phe Asn Ala Gly Leu Leu
370 375 380
Glu Glu Ala Gly Val Gly Val Glu Ala Lys Arg Gly Ser Asp Gly Lys
385 390 395 400
He Gin Arg Glu Glu Val Ala Lys Ser He Lys Glu Val Val He Glu
405 410 415
Lys Thr Arg Glu Asp Val Arg Lys Lys Ala Arg Glu Met Gly Glu He
420 425 430
Leu Arg Ser Lys Gly Asp Glu Lys He Asp Glu Leu Val Ala Glu He
435 440 445
Ser Leu Leu Arg Lys Lys Ala Pro Cys Ser He
450 455
SEQ ID NO: 51
Si ai ia grosvenorii DNA sequence
atggatgccc agcgaggtca caccacaacc attttgatgt ttccatggct cggctatggc
60
catctttcgg ctttcctaga gttggccaaa agcctctcaa ggaggaactt ccatatctac 120
ttctgttcaa cctctgttaa cctcgacgcc attaaaccaa agcttccttc ttcttcctct 180
tctgattcca tccaacttgt ggaactttgt cttccatctt ctcctgatca gctccctcct
240
catcttcaca caaccaacgc cctcccccct cacctcatgc ccactctcca ccaagccttc 300
tccatggctg cccaacactt tgctgccatt ttacacacac ttgctccgca tctcctcatt
360
tacgactctt tccaaccttg ggctcctcaa ctagcttcat ccctcaacat tccagccatc 420 aacttcaata ctacgggagc ttcagtcctg acccgaatgc ttcacgctac tcactaccca 480
agttctaaat tcccaatttc agagtttgtt ctccacgatt attggaaagc catgtacagc 540
gccgccggtg gggctgttac aaaaaaagac cacaaaattg gagaaacact tgcgaattgc 600
ttgcatgctt cttgtagtgt aattctaatc aatagtttca gagagctcga ggagaaatat 660
atggattatc tctccgttct cttgaacaag aaagttgttc cggttggtcc tttggtttac
720
gaaccgaatc aagacgggga agatgaaggt tattcaagca tcaaaaattg gcttgacaaa 780
aaggaaccgt cctccaccgt cttcgtttca tttggaagcg aatacttccc gtcaaaggaa 840
gaaatggaag agatagccca tgggttagag gcgagcgagg ttcatttcat ctgggtcgtt 900
aggtttcctc aaggagacaa caccagcgcc attgaagatg ccttgccgaa ggggtttctg 960
gagagggtgg gagagagagg gatggtggtg aagggttggg ctcctcaggc gaagatactg 1020
aagcattgga gcacaggggg attcgtgagc cactgtggat ggaactcggt gatggaaagc 1080
atgatgtttg gcgttcccat aataggggtt ccgatgcatc tggaccagcc ctttaacgcc 1140
ggactcgcgg aagaagctgg cgtcggcgtg gaagccaagc gagattcgga cggcaaaatt 1200
caaagagaag aagttgcaaa gtcgatcaaa gaagtggtga ttgagaaaac cagggaagac
1260
gtgaggaaga aagcaagaga aatgggtgag attttgagga gtaaaggaga tgagaaaatt 1320
gatgagttgg tggctgaaat ttctcttttg cgcaaaaagg ctccatgttc aatttaa
1377
SEQ ID NO: 52
Artificial Sequence Codon-o timized nucleotide sequence encoding UGT98 atggatgctc aaagaggtca taccactacc attttgatgt ttccatggtt gggttacggt 60
catttgtctg cttttttgga attggccaag tccttgtcta gaagaaactt ccatatctac 120
ttttgctcca cctccgttaa tttggatgct attaagccaa agttgccatc ctcttcatcc 180
tccgattcta ttcaattggt tgaattgtgc ttgccatctt ccccagatca attgccacca 240
cacttgcata caactaatgc tttaccacca catttgatgc caacattgca tcaagctttt 300
tctatggctg ctcaacattt tgctgctatc ttgcatactt tggctcctca tttgttgatc 360
tacgattctt ttcaaccatg ggctccacaa ttggcttcat ctttgaatat tccagccatc 420 aacttcaaca ctactggtgc ttcagttttg accagaatgt tgcatgctac tcattaccca 480
tcttccaagt tcccaatttc tgaattcgtc ttgcatgatt actggaaggc tatgtattct
540
gctgctggtg gtgctgttac aaaaaaggat cataagattg gtgaaacctt ggccaactgt 600
ttacatgctt cttgctctgt tatcttgatc aactccttca gagaattgga agaaaagtac
660
atggactact tgtccgtctt gttgaacaaa aaggttgttc cagttggtcc attggtctac 720
gaacctaatc aagatggtga agatgaaggt tactcctcca ttaagaattg gttggacaag 780
aaagaaccat cctctaccgt ttttgtttcc ttcggttctg aatacttccc atccaaagaa
840
gaaatggaag aaatcgctca tggtttggaa gcttcagaag ttcatttcat ctgggttgtt 900
agattccctc aaggtgataa cacttccgct attgaagatg ctttgccaaa aggtttcttg
960
gaaagagtcg gtgaaagagg tatggttgtt aagggttggg ctcctcaagc taagattttg 1020
aaacattggt caaccggtgg tttcgtttct cattgtggtt ggaattctgt catggaatct
1080
atgatgttcg gtgttccaat tattggtgtc ccaatgcatt tggatcaacc attcaatgct 1140
ggtttggctg aagaagctgg tgttggtgtt gaagctaaaa gagattctga cggtaagatc
1200
caaagagaag aagttgccaa gtccatcaaa gaagttgtta tcgaaaagac cagagaagat 1260
gtcagaaaga aagctagaga aatgggtgaa atcttgagat ctaaaggtga cgaaaagatc
1320
gatgaattgg tcgccgaaat ttccttgttg agaaaaaaag ctccatgctc tatttga 1377
SEQ ID NO: 53
Siraitia grosvenorii protein sequence
Met Asp Ala Gin Arg Gly His Thr Thr Thr lie Leu Met Phe Pro Trp
1 5 10 15
Leu Gly Tyr Gly His Leu Ser Ala Phe Leu Glu Leu Ala Lys Ser Leu
20 25 30
Ser Arg Arg Asn Phe His lie Tyr Phe Cys Ser Thr Ser Val Asn Leu
35 40 45
Asp Ala lie Lys Pro Lys Leu Pro Ser Ser Ser Ser Ser Asp Ser lie
50 55 60
Gin Leu Val Gl Leu Cys Leu Pro Ser Ser Pro Asp Gin Leu Pro Pro
65 70 75 80 His Leu His Thr Thr Asn Ala Leu Pro Pro His Leu Met Pro Thr Leu
85 90 95
His Gin Ala Phe Ser Met Ala Ala Gin His Phe Ala Ala lie Leu His
100 105 110
Thr Leu Ala Pro His Leu Leu lie Tyr Asp Ser Phe Gin Pro Trp Ala
115 120 125
Pro Gin Leu Ala Ser Ser Leu Asn lie Pro Ala lie Asn Phe Asn Thr
130 135 140
Thr Gly Ala Ser Val Leu Thr Arg Met Leu His Ala Thr His Tyr Pro
145 150 155 160
Ser Ser Lys Phe Pro lie Ser Glu Phe Val Leu His Asp Tyr Trp Lys
165 170 175
Ala Met Tyr Ser Ala Ala Gly Gly Ala Val Thr Lys Lys Asp His Lys
180 185 190 lie Gly Glu Thr Leu Ala Asn Cys Leu His Ala Ser Cys Ser Val lie
195 200 205
Leu lie Asn Ser Phe Arg Glu Leu Glu Glu Lys Tyr Met Asp Tyr Leu
210 215 220
Ser Val Leu Leu Asn Lys Lys Val Val Pro Val Gly Pro Leu Val Tyr 225 230 235 240
Glu Pro Asn Gin Asp Gly Glu Asp Glu Gly Tyr Ser Ser lie Lys Asn
245 250 255
Trp Leu Asp Lys Lys Glu Pro Ser Ser Thr Val Phe Val Ser Phe Gly
260 265 270
Ser Glu Tyr Phe Pro Ser Lys Glu Glu Met Glu Glu lie Ala His Gly
275 280 285
Leu Glu Ala Ser Glu Val His Phe lie Trp Val Val Arg Phe Pro Gin
290 295 300
Gly Asp Asn Thr Ser Ala lie Glu Asp Ala Leu Pro Lys Gly Phe Leu 305 310 315 320
Glu Arg Val Gly Glu Arg Gly Met Val Val Lys Gly Trp Ala Pro Gin
325 330 335
Ala Lys lie Leu Lys His Trp Ser Thr Gly Gly Phe Val Ser His Cys
340 345 350 Gly Trp Asn Ser Val Met Glu Ser Met Met Phe Gly Val Pro He He 355 360 365
Gly Val Pro Met His Leu Asp Gin Pro Phe Asn Ala Gly Leu Ala Glu
370 375 380
Glu Ala Gly Val Gly Val Glu Ala Lys Arg Asp Ser Asp Gly Lys He 385 390 395 400
Gin Arg Glu Glu Val Ala Lys Ser He Lys Glu Val Val He Glu Lys
405 410 415
Thr Arg Glu Asp Val Arg Lys Lys Ala Arg Glu Met Gly Glu He Leu
420 425 430
Arg Ser Lys Gly Asp Glu Lys l ie Asp Glu Leu Val Ala Glu He Ser
435 440 445
Leu Leu Arg Lys Lys Ala Pro Cys Ser He
450 455
SEQ ID NO: 54
Saccharomyces cerevisiae protein sequence
Met Ser Ala Val Asn Val Al Pro Glu Leu He Asn Ala Asp Asn Thr
1 5 10 15
He Thr Tyr Asp Ala He Val He Gly Ala Gly Val He Gly Pro Cys
20 25 30
Val Ala Thr Gly Leu Ala Arg Lys Gly Lys Lys Val Leu He Val Glu
35 40 45
Arg Asp Trp Ala Met Pro Asp Arg He Val Gly Glu Leu Met Gin Pro
50 55 60
Gly Gly Val Arg Ala Leu Arg Ser Leu Gly Met He Gin Ser He Asn 65 70 75 80
Asn He Glu Ala Tyr Pro Val Thr Gly Tyr Thr Val Phe Phe Asn Gly
85 90 95
Glu Gin Val Asp He Pro Tyr Pro Tyr Lys Ala Asp He Pro Lys Val
100 105 110
Glu Lys Leu Lys Asp Leu Val Lys Asp Gly Asn Asp Lys Val Leu Glu
115 120 125
Asp Ser Thr He His He Lys Asp Tyr Glu Asp Asp Glu Arg Glu Arg 130 135 140
Gly Val Ala Phe Val His Gly Arg Phe Leu Asn Asn Leu Arg Asn lie 145 150 155 160
Thr Ala Gin Glu Pro Asn Val Thr Arg Val Gin Gly Asn Cys lie Glu
165 170 175 lie Leu Lys Asp Glu Lys Asn Glu Val Val Gly Ala Lys Val Asp lie
180 185 190
Asp Gly Arg Gly Lys Val Glu Phe Lys Ala His Leu Thr Phe lie Cys
195 200 205
Asp Gly lie Phe Ser Arg Phe Arg Lys Glu Leu His Pro Asp His Val 210 215 220
Pro Thr Val Gly Ser Ser Phe Val Gly Met Ser Leu Phe Asn Ala Lys 225 230 235 240
Asn Pro Ala Pro Met His Gly His Val lie Leu Gly Ser Asp His Met
245 250 255
Pro lie Leu Val Tyr Gin lie Ser Pro Glu Glu Thr Arg lie Leu Cys
260 265 270
Ala Tyr Asn Ser Pro Lys Val Pro Ala Asp lie Lys Ser Trp Met lie
275 280 285
Lys Asp Val Gin Pro Phe lie Pro Lys Ser Leu Arg Pro Ser Phe Asp
290 295 300
Glu Ala Val Ser Gin Gly Lys Phe Arg Ala Met Pro Asn Ser Tyr Leu
305 310 315 320
Pro Ala Arg Gin Asn Asp Val Thr Gly Met Cys Val lie Gly Asp Ala
325 330 335
Leu Asn Met Arg His Pro Leu Thr Gly Gly Gly Met Thr Val Gly Leu
340 345 350
His Asp Val Val Leu Leu lie Lys Lys lie Gly Asp Leu Asp Phe Ser
355 360 365
Asp Arg Glu Lys Val Leu Asp Glu Leu Leu Asp Tyr His Phe Glu Arg 370 375 380
Lys Ser Tyr Asp Ser Val lie Asn Val Leu Ser Val Ala Leu Tyr Ser 385 390 395 400
Leu Phe Ala Ala Asp Ser Asp Asn Leu Lys Ala Leu Gin Lys Gly Cys 405 410 415
Phe Lys Tyr Phe Gin Arg Gly Gly Asp Cys Val Asn Lys Pro Val Glu
420 425 430
Phe Leu Ser Gly Val Leu Pro Lys Pro Leu Gin Leu Thr Arg Val Phe
435 440 445
Phe Ala Val Ala Phe Tyr Thr lie Tyr Leu Asn Met Glu Glu Arg Gly 450 455 460
Phe Leu Gly Leu Pro Met Ala Leu Leu Glu Gly lie Met lie Leu lie
465 470 475 480
Thr Ala lie Arg Val Phe Thr Pro Phe Leu Phe Gly Glu Leu lie Gly
485 490 495
SEQ ID NO: 55
Saccharomyces cerevisiae protein sequence
Met Thr Glu Phe Tyr Ser Asp Thr lie Gly Leu Pro Lys Thr Asp Pro
1 5 10 15
Arg Leu Trp Arg Leu Arg Thr Asp Glu Leu Gly Arg Glu Ser Trp Glu
20 25 30
Tyr Leu Thr Pro Gin Gin Ala Ala Asn Asp Pro Pro Ser Thr Phe Thr
35 40 45
Gin Trp Leu Leu Gin Asp Pro Lys Phe Pro Gin Pro His Pro Glu Arg
50 55 60
Asn Lys His Ser Pro Asp Phe Ser Ala Phe Asp Ala Cys His Asn Gly 65 70 75 80
Ala Ser Phe Phe Lys Leu Leu Gin Glu Pro Asp Ser Gly lie Phe Pro
85 90 95
Cys Gin Tyr Lys Gly Pro Met Phe Met Thr lie Gly Tyr Val Ala Val
100 105 110
Asn Tyr lie Ala Gly lie Glu lie Pro Glu His Glu Arg lie Glu Leu
115 120 125
He Arg Tyr He Val Asn Thr Ala His Pro Val Asp Gly Gly Trp Gly 130 135 140
Leu His Ser Val Asp Lys Ser Thr Val Phe Gly Thr Val Leu Asn Tyr
145 150 155 160 Val He Leu Arg Leu Leu Gly Leu Pro Lys Asp His Pro Val Cys Ala 165 170 175
Lys Ala Arg Ser Thr Leu Leu Arg Leu Gly Gly Ala He Gly Ser Pro
180 185 190
His Trp Gly Lys He Trp Leu Ser Ala Leu Asn Leu Tyr Lys Trp Glu
195 200 205
Gly Val Asn Pro Ala Pro Pro Glu Thr Trp Leu Leu Pro Tyr Ser Leu 210 215 220
Pro Met His Pro Gly Arg Trp Trp Val His Thr Arg Gly Val Tyr He 225 230 235 240
Pro Val Ser Tyr Leu Ser Leu Val Lys Phe Ser Cys Pro Met Thr Pro
245 250 255
Leu Leu Glu Glu Leu Arg Asn Glu He Tyr Thr Lys Pro Phe Asp Lys
260 265 270
He Asn Phe Ser Lys Asn Arg Asn Thr Val Cys Gly Val Asp Leu Tyr
275 280 285
Tyr Pro His Ser Thr Thr Leu Asn He Ala Asn Ser Leu Val Val Phe
290 295 300
Tyr Glu Lys Tyr Leu Arg Asn Arg Phe He Tyr Ser Leu Ser Lys Lys 305 310 315 320
Lys Val Tyr Asp Leu He Lys Thr Glu Leu Gin Asn Thr Asp Ser Leu
325 330 335
Cys He Ala Pro Val Asn Gin Ala Phe Cys Ala Leu Val Thr Leu He
340 345 350
Glu Glu Gly Val Asp Ser Glu Ala Phe Gin Arg Leu Gin Tyr Arg Phe
355 360 365
Lys Asp Ala Leu Phe His Gly Pro Gin Gly Met Thr He Met Gly Thr 370 375 380
Asn Gly Val Gin Thr Trp Asp Cys Ala Phe Ala He Gin Tyr Phe Phe 385 390 395 400
Val Ala Gly Leu Ala Glu Arg Pro Glu Phe Tyr Asn Thr He Val Ser
405 410 415
Ala Tyr Lys Phe Leu Cys His Ala Gin Phe Asp Thr Glu Cys Val Pro
420 425 430 Gly Ser Tyr Arg Asp Lys Arg Lys Gly Ala Trp Gly Phe Ser Thr Lys
435 440 445
Thr Gin Gly Tyr Thr Val Ala Asp Cys Thr Ala Glu Ala lie Lys Ala
450 455 460
lie lie Met Val Lys Asn Ser Pro Val Phe Ser Glu Val His His Met
465 470 475 480 lie Ser Ser Glu Arg Leu Phe Glu Gly lie Asp Val Leu Leu Asn Leu
485 490 495
Gin Asn lie Gly Ser Phe Glu Tyr Gly Ser Phe Ala Thr Tyr Glu Lys
500 505 510
He Lys Ala Pro Leu Ala Met Glu Thr Leu Asn Pro Ala Glu Val Phe
515 520 525
Gly Asn lie Met Val Glu Tyr Pro Tyr Val Glu Cys Thr Asp Ser Ser 530 535 540
Val Leu Gly Leu Thr Tyr Phe His Lys Tyr Phe Asp Tyr Arg Lys Glu 545 550 555 560
Glu lie Arg Thr Arg lie Arg lie Ala lie Glu Phe lie Lys Lys Ser
565 570 575
Gin Leu Pro Asp Gly Ser Trp Tyr Gly Ser Trp Gly lie Cys Phe Thr
580 585 590
Tyr Ala Gly Met Phe Ala Leu Glu Ala Leu His Thr Val Gly Glu Thr
595 600 605
Tyr Glu Asn Ser Ser Thr Val Arg Lys Gly Cys Asp Phe Leu Val Ser
610 615 620
Lys Gin Met Lys Asp Gly Gly Trp Gly Glu Ser Met Lys Ser Ser Glu 625 630 635 640
Leu His Ser Tyr Val Asp Ser Glu Lys Ser Leu Val Val Gin Thr Ala
645 650 655
Trp Ala Leu lie Ala Leu Leu Phe Ala Glu Tyr Pro Asn Lys Glu Val
660 665 670 lie Asp Arg Gly He Asp Leu Leu Lys Asn Arg Gin Glu Glu Ser Gly
675 680 685
Glu Trp Lys Phe Glu Ser Val Glu Gly Val Phe Asn His Ser Cys Ala 690 695 700 He Glu Tyr Pro Ser Tyr Arg Phe Leu Phe Pro He Lys Ala Leu Gly 705 710 715 720
Met Tyr Ser Arg Ala Tyr Glu Thr His Thr Leu
725 730
SEQ ID NO: 56
Arabidopsis thaliana protein sequence
Met Ala Thr Glu Lys Thr His Gin Phe His Pro Ser Leu His Phe Val
1 5 10 15
Leu Phe Pro Phe Met Ala Gin Gly His Met He Pro Met He Asp He
20 25 30
Ala Arg Leu Leu Ala Gin Arg Gly Val Thr He Thr He Val Thr Thr
35 40 45
Pro His Asn Ala Ala Arg Phe Lys Asn Val Leu As Arg Ala He Glu
50 55 60
Ser Gly Leu Ala He Asn lie Leu His Val Lys Phe Pro Tyr Gin Glu 65 70 75 80
Phe Gly Leu Pro Glu Gly Lys Glu Asn He Asp Ser Leu Asp Ser Thr
85 90 35
Glu Leu Met Val Pro Phe Phe Lys Ala Val Asn Leu Leu Glu Asp Pro
100 105 110
Val Met Lys Leu Met Glu Glu Met Lys Pro Arg Pro Ser Cys Leu He
115 120 125
Ser Asp Trp Cys Leu Pro Tyr Thr Ser He He Ala Lys Asn Phe Asn
130 135 140
He Pro Lys He Val Phe His Gly Met Gly Cys Phe Asn Leu Leu Cys 145 150 155 160
Met His Val Leu Arg Arg Asn Leu Glu He Leu Glu Asn Val Lys Ser
165 170 175
Asp Glu Glu Tyr Phe Leu Val Pro Ser Phe Pro Asp Arg Val Glu Phe
180 185 190
Thr Lys Leu Gin Leu Pro Val Lys Ala Asn Ala Ser Gly Asp Trp Lys
195 200 205 Glu He Met Asp Glu Met Val Lys Ala Glu Tyr Thr Ser Tyr Gly Val 210 215 220
He Val Asn Thr Phe Gin Glu Leu Glu Pro Pro Tyr Val Lys Asp Tyr
225 230 235 240
Lys Glu Ala Met Asp Gly Lys Val Trp Ser He Gly Pro Val Ser Leu
245 250 255
Cys Asn Lys Ala Gly Ala Asp Lys Ala Glu Arg Gly Ser Lys Ala Ala
260 265 270
He Asp Gin Asp Glu Cys Leu Gin Trp Leu Asp Ser Lys Glu Glu Gly
275 280 285
Ser Val Leu Tyr Val Cys Leu Gly Ser He Cys Asn Leu Pro Leu Ser
290 295 300
Gin Leu Lys Glu Leu Gly Leu Gly Leu Glu Glu Ser Arg Arg Ser Phe
305 310 315 320
He Trp Val He Arg Gly Ser Glu Lys Tyr Lys Glu Leu Phe Glu Trp
325 330 335
Met Leu Glu Ser Gly Phe Glu Glu Arg He Lys Glu Arg Gly Leu Leu
340 345 350
He Lys Gly Trp Ala Pro Gin Val Leu He Leu Ser His Pro Ser Val
355 360 365
Gly Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr Leu Glu Gly He
370 375 380
Thr Ser Gly He Pro Leu He Thr Trp Pro Leu Phe Gly Asp Gin Phe
385 390 395 400
Cys Asn Gin Lys Leu Val V l Gin Val Leu Lys Ala Gly Val Ser Ala
405 410 415
Gly Val Glu Glu Val Me Lys Trp Gly Glu Glu Asp Lys He Gly Val
420 425 430
Leu Val Asp Lys Glu Gly Val Lys Lys Ala Val Glu Glu Leu Met Gly
435 440 445
Asp Ser Asp Asp Ala Lys Glu Arg Arg Arg Arg Val Lys Glu Leu Gly
450 455 460
Glu Leu Ala His Lys Ala Val Glu Lys Gly Gly Ser Ser His Ser Asn
465 470 475 480 lie Thr Leu Leu Leu Gin Asp lie Met Gin Leu Ala Gin Phe Lys Asn
485 490 495
SEQ ID NO: 57
Arabidopsis thaiiana protein sequence
Met Val Ser Glu Thr Thr Lys Ser Ser Pro Leu His Phe Val Leu Phe 1 5 10 15
Pro Phe Met Ala Gin Gly His Met He Pro Met Val Asp He Ala Arg
20 25 30
Leu Leu Ala Gin Arg Gly Val lie He Thr He Val Thr Thr Pro His
35 40 45
Asn Ala Ala Arg Phe Lys Asn Val Leu Asn Arg Ala He Glu Ser Gly
50 55 60
Leu Pro lie Asn Leu Val Gin Val Lys Phe Pro Tyr Leu Glu Ala Gly
65 70 75 80
Leu Gin Glu Gly Gin Glu Asn lie Asp Ser Leu As Thr Met Glu Arg
85 90 95
Met lie Pro Phe Phe Lys Ala Val Asn Phe Leu Glu Glu Pro Val Gin
100 105 110
Lys Leu lie Glu Glu Met Asn Pro Arg Pro Ser Cys Leu He Ser Asp
115 120 125
Phe Cys Leu Pro Tyr Thr Ser Lys He Ala Lys Lys Phe Asn He Pro
130 135 140
Lys lie Leu Phe His Gly Met Gly Cys Phe Cys Leu Leu Cys Met His
145 150 155 160
Val Leu Arg Lys Asn Arg Glu lie Leu Asp Asn Leu Lys Ser Asp Lys
165 170 175
Glu Leu Phe Thr Val Pro Asp Phe Pro Asp Arg Val Glu Phe Thr Arg
180 185 190
Thr Gin Val Pro Val Glu Thr Tyr Val Pro Ala Gly Asp Trp Lys Asp
195 200 205
He Phe Asp Gly Met Val Glu Ala Asn Glu Thr Ser Tyr Gly Val He 210 215 220
Val Asn Ser Phe Gin Glu Leu Glu Pro Ala Tyr Ala Lys Asp Tyr Lys 225 230 235 240 Glu Val Arg Ser Gly Lys Ala Trp Thr lie Gly Pro Val Ser Leu Cys
245 250 255
Asn Lys Val Gly Ala Asp Lys Ala Glu Arg Gly Asn Lys Ser Asp lie
260 265 270
Asp Gin Asp Glu Cys Leu Lys Trp Leu Asp Ser Lys Lys His Gly Ser
275 280 285
Val Leu Tyr Val Cys Leu Gly Ser lie Cys Asn Leu Pro Leu Ser Gin 290 295 300
Leu Lys Glu Leu Gly Leu Gly Leu Glu Glu Ser Gin Arg Pro Phe lie 305 310 315 320
Trp Val lie Arg Gly Trp Glu Lys Tyr Lys Glu Leu Val Glu Trp Phe
325 330 335
Ser Glu Ser Gly Phe Glu Asp Arg lie Gin Asp Arg Gly Leu Leu lie
340 345 350
Lys Gly Trp Ser Pro Gin Met Leu lie Leu Ser His Pro Ser Val Gly
355 360 365
Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr Leu Glu Gly He Thr
370 375 380
Ala Gly Leu Pro Leu Leu Thr Trp Pro Leu Phe Ala Asp Gin Phe Cys
385 390 395 400
Asn Glu Lys Leu Val Val Glu Val Leu Lys Ala Gly Val Arg Ser Gly
405 410 415
Val Glu Gin Pro Met Lys Trp Gly Glu Glu Glu Lys He Gly Val Leu
420 425 430
Val Asp Lys Glu Gly Val Lys Lys Ala Val Glu Glu Leu Met Gly Glu
435 440 445
Ser Asp Asp Ala Lys Glu Arg Arg Arg Arg Ala Lys Glu Leu Gly Asp
450 455 460
Ser Ala His Lys Ala Val Glu Glu Gly Gly Ser Ser His Ser Asn He
465 470 475 480
Ser Phe Leu Leu Gin Asp He Met Glu Leu Ala Glu Pro Asn Asn
485 490 495
SEQ ID NO: 58 Arabidopsis thaliana protein sequence
Met Ala Phe Glu Lys Asn Asn Glu Pro Phe Pro Leu His Phe Val Leu 1 5 10 15
Phe Pro Phe Met Ala Gin Gly His Met lie Pro Met Val Asp lie Ala
20 25 3 0
Arg Leu Leu Ala Gin Arg Gly Val Leu lie Thr lie Val Thr Thr Pro
35 40 45
His Asn Ala Ala Arg Phe Lys Asn Val Leu Asn Arg Ala lie Glu Ser
50 55 60
Gly Leu Pro He Asn Leu Val Gin Val Lys Phe Pro Tyr Gin Glu Ala
65 70 75 80
Gly Leu Gin Glu Gly Gin Glu Asn Met Asp Leu Leu Thr Thr Met Glu
85 90 95
Gin lie Thr Ser Phe Phe Lys Ala Val Asn Leu Leu Lys Glu Pro Val
100 105 110
Gin Asn Leu He Glu Glu Met Ser Pro Arg Pro Ser Cys Leu He Ser
115 120 125
Asp Met Cys Leu Ser Tyr Thr Ser Glu He Ala Lys Lys Phe Lys He
13 0 135 14 0
Pro Lys lie Leu Phe His Gly Met Gly Cys Phe Cys Leu Leu Cys Val
145 150 155 160
Asn Val Leu Arg Lys Asn Arg Glu lie Leu Asp Asn Leu Lys Ser Asp
165 170 175
Lys Glu Tyr Phe He Val Pro Tyr Phe Pro Asp Arg Val Glu Phe Thr
180 185 190
Arg Pro Gin Val Pro Val Glu Thr Tyr Val Pro Ala Gly Trp Lys Glu
195 200 205
He Leu Glu Asp Met Val Glu Ala Asp Lys Thr Ser Tyr Gly Val He 210 215 220
Val Asn Ser Phe Gin Glu Leu Glu Pro Ala Tyr Ala Lys Asp Phe Lys 225 230 235 240
Glu Ala Arg Ser Gly Lys Ala Trp Thr He Gly Pro Val Ser Leu Cys
245 250 255
Asn Lys Val Gly Val Asp Lys Ala Glu Arg Gly Asn Lys Ser Asp He 260 265 270
Asp Gin Asp Glu Cys Leu Glu Trp Leu Asp Ser Lys Glu Pro Gly
275 280 285
Val Leu Tyr Val Cys Leu Gly Ser lie Cys Asn Leu Pro Leu Ser Gin 290 295 300
Leu Leu Glu Leu Gly Leu Gly Leu Glu Glu Ser Gin Arg Pro Phe lie 305 310 315 320
Trp Val lie Arg Gly Trp Glu Lys Tyr Lys Glu Leu Val Glu Trp Phe
325 330 335
Ser Glu Ser Gly Phe Glu Asp Arg lie Gin Asp Arg Gly Leu Leu lie
340 345 350
Lys Gly Trp Ser Pro Gin Met Leu lie Leu Ser His Pro Ser Val Gly
355 360 365
Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr Leu Glu Gly lie Thr 370 375 380
Ala Gly Leu Pro Met Leu Thr Trp Pro Leu Phe Ala Asp Gin Phe Cys 385 390 395 400
Asn Glu Lys Leu Val Val Gin lie Leu Lys Val Gly Val Ser Ala Glu
405 410 415
Val Lys Glu Val Met Lys Trp Gly Glu Glu Glu Lys lie Gly Val Leu
420 425 430
Val Asp Lys Glu Gly Val Lys Lys Ala Val Glu Glu Leu Met Gly Glu
435 440 445
Ser Asp Asp Ala Lys Glu Arg Arg Arg Arg Ala Lys Glu Leu Gly Glu 450 455 460
Ser Ala His Lys Ala Val Glu Glu Gly Gly Ser Ser His Ser Asn lie 465 470 475 480
Thr Phe Leu Leu Gin Asp lie Met Gin Leu Ala Gin Ser Asn Asn
485 490 495
SEQ ID MO: 59
Stevia rebaudian protein sequence
Met Ser Pro Lys Met Val Ala Pro Pro Thr Asn Leu His Phe Val Leu 1 5 10 15
Phe Pro Leu Met Ala Gin Gly His Leu Val Pro Met Val Asp lie Ala
20 25 30
Arg He Leu Ala Gin Arg Gly Ala Thr Val Thr He He Thr Thr Pro
35 40 45
Tyr His Ala Asn Arg Val Axg Pro Val lie Ser Arg Ala He Ala Thr 50 55 60
Asn Leu Lys He Gin Leu Leu Glu Leu Gin Leu Arg Ser Thr Glu Ala
65 70 75 80
Gly Leu Pro Glu Gly Cys Glu Ser Phe Asp Gin Leu Pro Ser Phe Glu
85 90 95
Tyr Trp Lys Asn He Ser Thr Ala He Asp Leu Leu Gin Gin Pro Ala
100 105 110
Glu Asp Leu Leu Arg Glu Leu Ser Pro Pro Pro Asp Cys He He Ser
115 120 125
Asp Phe Leu Phe Pro Trp Thr Thr Asp Val Ala Arg Arg Leu Asn He 130 135 140
Pro Arg Leu Val Phe Asn Gly Pro Gly Cys Phe Tyr Leu Leu Cys He
145 150 155 160
His Val Ala He Thr Ser Asn He Leu Gly Glu Asn Glu Pro Val Ser
165 170 175
Ser Asn Thr Glu Arg Val Val Leu Pro Gly Leu Pro Asp Arg He Glu
180 185 190
Val Thr Lys Leu Gin He Val Gly Ser Ser Arg Pro Ala Asn Val Asp
195 200 205
Glu Met Gly Ser Trp Leu Arg Ala Val Glu Ala Glu Lys Ala Ser Phe
210 215 220
Gly He Val Val Asn Thr Phe Glu Glu Leu Glu Pro Glu Tyr Val Glu 225 230 235 240
Glu Tyr Lys Thr Val Lys Asp Lys Lys Met Trp Cys lie Gly Pro Val
245 250 255
Ser Leu Cys Asn Lys Thr Gly Pro Asp Leu Ala Glu Arg Gly Asn Lys
260 265 270
Ala Ala He Thr Glu His Asn Cys Leu Lys Trp Leu Asp Glu Arg Lys 275 280 285
Leu Gly Ser Val Leu Tyr Val Cys Leu Gly Ser Leu Ala Arg lie Ser 290 295 300
Ala Ala Gin Ala lie Glu Leu Gly Leu Gly Leu Glu Ser lie Asn Arg 305 310 315 320
Pro Phe lie Trp Cys Val Arg Asn Glu Thr Asp Glu Leu Lys Thr Trp
325 330 335
Phe Leu Asp Gly Phe Glu Glu Arg Val Arg Asp Arg Gly Leu lie Val
340 345 350
His Gly Trp Ala Pro Gin Val Leu lie Leu Ser His Pro Thr lie Gly
355 360 365
Gly Phe Leu Thr His Cys Gly Trp Asn Ser Thr He Glu Ser He Thr 370 375 380
Ala Gly Val Pro Met He Thr Trp Pro Phe Phe Ala Asp Gin Phe Leu 385 390 395 400
Asn Glu Ala Phe He Val Glu Val Leu Lys lie Gly Val Arg He Gly
405 410 415
Val Glu Arg Ala Cys Leu Phe Gly Glu Glu Asp Lys Val Gly Val Leu
420 425 430
Val Lys Lys Glu Asp Val Lys Lys Ala Val Glu Cys Leu Met Asp Glu
435 440 445
Asp Glu Asp Gly Asp Gin Arg Arg Lys Arg Val He Glu Leu Ala Lys 450 455 460
Met Ala Lys He Ala Met Ala Glu Gly Gly Ser Ser Tyr Glu Asn Val 465 470 475 480
Ser Ser Leu He Arg Asp Val Thr Glu Thr Val Arg Ala Pro His
485 490 495
SEQ ID NO: 60
Stevia rebaudian protein sequence
Met Asp Ala Met Ala Thr Thr Glu Lys Lys Pro His Val He Phe He 1 5 10 15
Pro Phe Pro Ala Gin Ser His He Lys Ala Met Leu Lys Leu Ala Gin
20 25 30 Leu Leu His His Lys Gly Leu Gin lie Thr Phe Val Asn Thr Asp Phe 35 40 45
lie His Asn Gin Phe Leu Glu Ser Ser Gly Pro His Cys Leu Asp Gly
50 55 60
Ala Pro Gly Phe Arg Phe Glu Thr He Pro Asp Gly Val Ser His Ser
65 70 75 80
Pro Glu Ala Ser He Pro He Arg Glu Ser Leu Leu Arg Ser He Glu
85 90 95
Thr Asn Phe Leu Asp Arg Phe He Asp Leu Val Thr Lys Leu Pro Asp
100 105 110
Pro Pro Thr Cys He He Ser Asp Gly Phe Leu Ser Val Phe Thr He
115 120 125
Asp Ala Ala Lys Lys Leu Gly He Pro Val Met Met Tyr Trp Thr Leu 130 135 140
Ala Ala Cys Gly Phe Met Gly Phe Tyr His He His Ser Leu He Glu
145 150 155 160
Lys Gly Phe Ala Pro Leu Lys Asp Ala Ser Tyr Leu Thr Asn Gly Tyr
165 170 175
Leu Asp Thr Val He Asp Trp Val Pro Gly Met Glu Gly He Arg Leu
180 185 190
Lys Asp Phe Pro Leu Asp Trp Ser Thr Asp Leu Asn Asp Lys Val Leu
195 200 205
Met Phe Thr Thr Glu Ala Pro Gin Arg Ser His Lys Val Ser His His 210 215 220
He Phe His Thr Phe Asp Glu Leu Glu Pro Ser He He Lys Thr Leu
225 230 235 240
Ser Leu Arg Tyr Asn His He Tyr Thr He Gly Pro Leu Gin Leu Leu
245 250 255
Leu Asp Gin He Pro Glu Glu Lys Lys Gin Thr Gly He Thr Ser Leu
260 265 270
His Gly Tyr Ser Leu Val Lys Glu Glu Pro Glu Cys Phe Gin Trp Leu
275 280 285
Gin Ser Lys Glu Pro Asn Ser Val Val Tyr Val Asn Phe Gly Ser Thr
290 295 300 Thr Val Met Ser Leu Glu Asp Met Thr Glu Phe Gly Trp Gly Leu Ala
305 310 315 320
Asn Ser Asn His Tyr Phe Leu Trp lie lie Arg Ser Asn Leu Val lie
325 330 335
Gly Glu Asn Ala Val Leu Pro Pro Glu Leu Glu Glu His lie Lys Lys
340 345 350
Arg Gly Phe lie Ala Ser Trp Cys Ser Gin Glu Lys Val Leu Lys His
355 3S0 365
Pro Ser Val Gly Gly Phe Leu Thr His Cys Gly Trp Gly Ser Thr He
370 375 380
Glu Ser Leu Ser Ala Gly Val Pro Met lie Cys Trp Pro Tyr Ser Trp
385 390 395 400
Asp Gin Leu Thr Asn Cys Arg Tyr lie Cys Lys Glu Trp Glu Val Gly
405 410 415
Leu Glu Met Gly Thr Lys Val Lys Arg Asp Glu Val Lys Arg Leu Val
420 425 430
Gin Glu Leu Met Gly Glu Gly Gly His Lys Met Arg Asn Lys Ala Lys
435 440 445
Asp Trp Lys Lys Ala Arg lie Ala lie Ala Pro Asn Gly Ser Ser
450 455 460
Ser Leu Asn lie Asp Lys Met Val Lys Glu He Thr Val Leu Ala Arg
465 470 475 480
Asn
SEQ ID NO: 61
Siraitia grosvenorii DNA sequence
atggagcaag ctcatgatct tcttcacgtc ctcctttttc cgtatccggc gaagggccac 60
atcaagccct tcctctgcct cgccgagctc ctctgcaacg ccggtctcaa cgtcaccttc 120
ctcaacaccg actacaacca ccgccgcctc cacaatctcc atctcctcgc cgcctgcttt
180
ccctctcttc atttcgagtc catttccgac ggcctccagc ccgatcagcc tcgagatata 240
ctggacccca agttttatat atccatctgt caagtcacta aacccctttt ccgggagctc 300
ctcctttcct acaaacgaac ttccagtgtc cagaccggcc gcccgccaat aacttgcgtt
360 attacagatg tgatttttcg ttttccgatc gacgtagctg aagaactgga tattcctgtg 420
tttagtttct gtactttcag tgcccgtttc atgtttcttt acttctggat tcccaagctc
480
attgaagatg gccagcttcc atacccaaac ggcaatatca accagaaact ctacggtgtt 540
gctcctgagg cggaaggcct tttaagatgt aaagatttgc cgggaca tg ggctttcgca 600
gacgaactaa aagatgatca acttaacttt gtggaccaga caacggcgtc acttcgatcc
660
tccggtctca ttctcaacac attcgacgac ctcgaagctc catttctggg gcgtctctcc 720
accatcttta agaaaatcta cgccgttgga cccatccacg ctctgttgaa ctcccaccac
780
tgtggtcttt ggaaagaaga tcacagttgc ctggcgtggc tcgactcccg ggcggcgaga
840
tccgtcgtgt tcgtcagctt cgggagcttg gtgaagataa caagtaggca gctgatggag
900
ttttggcatg gcttgctcaa cagtggaacg tcgttcctct tcgtgttgag atctgacgta 960
gttgagggcg atggtgaaaa acaagtcgtc aaagaaattt acgagacgaa ggcagagggg
1020
aaatggttgg ttgtggggtg ggctccgcaa gagaaggtgt tagcccatga agctgttggt 1080
ggatttctga cccattcggg ctggaactcc attttagaga gcattgctgc tggggttcct
1140
atgatctcct gccccaaaat tggagaccag tccagtaact gtacgtggat cagtaaagta 1200
tggaaaattg ggctcgaaat ggaggaccaa tacgaccggg ccacggtcga ggcaatggtt
1260
aggtctataa tgaaacatga aggagaaaaa attcaaaaga caattgcaga gttagcaaaa 1320
cgagccaagt ataaagttag taaagatggg acatcgtatc gaaatttaga aattttaatt
1380
gaggatatta aaaaaattaa accaaattaa 1410
SEQ ID NO: 62
Siraitia grosvenorii protein sequence
Met Glu Gin Ala His Asp Leu Leu His Val Leu Leu Phe Pro Tyr Pro
1 5 10 15
Ala Lys Gly His lie Lys Pro Phe Leu Cys Leu Ala Glu Leu Leu Cys
20 25 30
Asn Ala Gly Leu Asn Val Thr Phe Leu Asn Thr Asp Tyr Asn His Arg
35 40 45
Arg Leu His Asn Leu His Leu Leu Ala Ala Cys Phe Pro Ser Leu His 50 55 60
Phe Glu Ser lie Ser Asp Gly Leu Gin Pro Asp Gin Pro Arg Asp lie 65 70 75 80
Leu Asp Pro Lys Phe Tyr lie Ser lie Cys Gin Val Thr Lys Pro Leu
85 90 95
Phe Arg Glu Leu Leu Leu Ser Tyr Lys Arg Thr Ser Ser Val Gin Thr
100 105 110
Gly Arg Pro Pro lie Thr Cys Val lie Thr Asp Val lie Phe Arg Phe
115 120 125
Pro lie Asp Val Ala Glu Glu Leu Asp lie Pro Val Phe Ser Phe Cys 130 135 140
Thr Phe Ser Ala Arg Phe Met Phe Leu Tyr Phe Trp lie Pro Lys Leu 145 150 155 160 lie Glu Asp Gly Gin Leu Pro Tyr Pro Asn Gly Asn lie Asn Gin Lys
165 170 175
Leu Tyr Gly Val Ala Pro Glu Ala Glu Gly Leu Leu Arg Cys Lys Asp
180 185 190
Leu Pro Gly His Trp Ala Phe Ala Asp Glu Leu Lys Asp Asp Gin Leu
195 200 205
Asn Phe Val Asp Gin Thr Thr Ala Ser Leu Arg Ser Ser Gly Leu lie 210 215 220
Leu Asn Thr Phe Asp Asp Leu Glu Ala Pro Phe Leu Gly Arg Leu Ser 225 230 235 240
Thr lie Phe Lys Lys lie Tyr Ala Val Gly Pro lie His Ala Leu Leu
245 250 255
Asn Ser His His Cys Gly Leu Trp Lys Glu Asp His Ser Cys Leu Ala
260 265 270
Trp Leu Asp Ser Arg Ala Ala Arg Ser Val Val Phe Val Ser Phe Gly
275 280 285
Ser Leu Val Lys lie Thr Ser Arg Gin Leu Met Glu Phe Trp His Gly 290 295 300
Leu Leu Asn Ser Gly Thr Ser Phe Leu Phe Val Leu Arg Ser Asp Val 305 310 315 320
Val Glu Gly Asp Gly Glu Lys Gin Val Val Lys Glu lie Tyr Glu Thr 325 330 335
Lys Ala Glu Gly Lys Trp Leu Val Val Gly Trp Ala Pro Gin Glu Lys
340 345 350
Val Leu Ala His Glu Ala Val Gly Gly Phe Leu Thr His Ser Gly Trp
355 360 365
Asn Ser lie Leu Glu Ser lie Ala Ala Gly Val Pro Met lie Ser Cys
370 375 380
Pro Lys lie Gly Asp Gin Ser Ser Asn Cys Thr Trp lie Ser Lys Val
385 390 395 400
Trp Lys lie Gly Leu Glu Met Glu Asp Gin Tyr Asp Arg Ala Thr Val
405 410 415
Glu Ala Met Val Arg Ser lie Met Lys His Glu Gly Glu Lys lie Gin
420 425 430
Lys Thr lie Ala Glu Leu Ala Lys Arg Ala Lys Tyr Lys Val Ser Lys
435 440 445
Asp Gly Thr Tyr Arg Asn Leu Glu lie Leu lie Glu Asp lie Lys
450 455 460
Lys lie Lys Pro Asn
465
SEQ ID NO: 63
Saccharomyces cerevisiae DNA sequence
atgctttcgc ttaaaacgtt actgtgtacg ttgttgactg tgtcatcagt actcgctacc 60
ccagtccctg caagagaccc ttcttccatt caatttgttc atgaggagaa caagaaaaga 120
tactacgatt atgaccacgg ttccctcgga gaaccaatcc gtggtgtcaa cattggtggt 180
tggttacttc ttgaaccata cattactcca tctttgttcg aggctttccg tacaaatgat 240
gacaacgacg aaggaattcc tgtcgacgaa tatcacttct gtcaatattt aggtaaggat 300
ttggctaaaa gccgtttaca gagccattgg tctactttct accaagaaca agatttcgct 360
aatattgctt cccaaggttt caaccttgtc agaattccta tcggttactg ggctttccaa 420
actttggacg atgatcctta tgttagcggc ctacaggaat cttacctaga ccaagccatc 480
ggttgggcta gaaacaacag cttgaaagtt tgggttgatt tgcatggtgc cgctggttcg 540 cagaacgggt ttgataactc tggtttgaga gattcataca agtttttgga agacagcaat 600
ttggccgtta ctacaaatgt cttgaactac atattgaaaa aatactctgc ggaggaatac 660
ttggacactg ttattggtat cgaattgatt aatgagccat tgggtcctgt tctagacatg
720
gataaaatga agaatgacta cttggcacct gcttacgaat acttgagaaa caacatcaag
780
agtgaccaag ttatcatcat ccatgacgct ttccaaccat acaattattg ggatgacttc
840
atgactgaaa acgatggcta ctggggtgtc actatcgacc atcatcacta ccaagtcttt
900
gcttctgatc aattggaaag atccattgat gaacatatta aagtagcttg tgaatggggt
960
accggagttt tgaatgaatc ccactggact gtttgtggtg agtttgctgc cgctttgact 1020
gattgtacaa aatggttgaa tagtgttggc ttcggcgcta gatacgacgg ttcttgggtc
1080
aatggtgacc aaacatcttc ttacattggc tcttgtgcta acaacgatga tatagcttac
1140
tggtctgacg aaagaaagga aaacacaaga cgttatgtgg aggcacaact agatgccttt
1200
gaaatgagag ggggttggat tatctggtgt tacaagacag aatctagttt ggaatgggat 1260
gctcaaagat tgatgttcaa tggtttattc cctcaaccat tgactgacag aaagtatcca
1320
aaccaatgtg gcacaatttc taactaa
1347
SEQ ID NO: 64
Saccharomyces cerevisiae protein sequence
Met Leu Ser Leu Lys Thr Leu Leu Cys Thr Leu Leu Thr Val Ser Ser
1 5 10 15
Val Leu Ala Thr Pro Val Pro Ala Arg Asp Pro Ser Ser He Gin Phe
20 25 30
Val His Glu Glu Asn Lys Lys Arg Tyr Tyr Asp Tyr Asp His Gly Ser
35 40 45
Leu Gly Glu Pro He Arg Gly Val Asn He Gly Gly Trp Leu Leu Leu
50 55 60
Glu Pro Tyr lie Thr Pro Ser Leu Phe Glu Ala Phe Arg Thr Asn Asp
65 70 75 80
Asp Asn As Glu Gly He Pro Val Asp Glu Tyr His Phe Cys Gin Tyr
85 90 95 Leu Gly Lys Asp Leu Ala Lys Ser Arg Leu Gin Ser His Trp Ser Thr 100 105 110
Phe Tyr Gin Glu Gin Asp Phe Ala Asn He Ala Ser Gin Gly Phe Asn
115 120 125
Leu Val Arg lie Pro He Gly Tyr Trp Ala Phe Gin Thr Leu Asp Asp 130 135 140
Asp Pro Tyr Val Ser Gly Leu Gin Glu Ser Tyr Leu Asp Gin Ala He 145 150 155 160
Gly Trp Ala Arg Asn Asn Ser Leu Lys Val rp Val Asp Leu His Gly
165 170 175
Ala Ala Gly Ser Gin Asn Gly Phe Asp Asn Ser Gly Leu Arg Asp Ser
180 185 190
Tyr Lys Phe Leu Glu Asp Ser Asn Leu Ala Val Thr Thr Asn Val Leu
195 200 205
Asn Tyr lie Leu Lys Lys Tyr Ser Ala Glu Glu Tyr Leu Asp Thr Val 210 215 220
He Gly He Glu Leu He Asn Glu Pro Leu Gly Pro Val Leu Asp Met 225 230 235 240
Asp Lys Met Lys Asn Asp Tyr Leu Ala Pro Ala Tyr Glu Tyr Leu Arg
245 250 255
Asn Asn He Lys Ser Asp Gin Val He lie He His Asp Ala Phe Gin
260 265 270
Pro Tyr Asn Tyr Trp Asp Asp Phe Met Thr Glu Asn Asp Gly Tyr Trp
275 280 285
Gly Val Thr He Asp His His His Tyr Gin Val Phe Ala Ser Asp Gin
290 295 300
Leu Glu Arg Ser He Asp Glu His He Lys Val Ala Cys Glu Trp Gly 305 310 315 320
Thr Gly Val Leu Asn Glu Ser His Trp Thr Val Cys Gly Glu Phe Ala
325 330 335
Ala Ala Leu Thr Asp Cys Thr Lys Trp Leu Asn Ser Val Gly Phe Gly
340 345 350
Ala Arg Tyr Asp Gly Ser Trp Val Asn Gly Asp Gin Thr Ser Ser Tyr
355 360 365 He Gly Ser Cys Ala Asn Asn Asp Asp lie Ala Tyr Trp Ser Asp Glu 370 375 380
Arg Lys Glu Asn Thr Arg Arg Tyr Val Glu Ala Gin Leu Asp Ala Phe
385 390 395 400
Glu Met Arg Gly Gly Trp lie lie Trp Cys Tyr Lys Thr Glu Ser Ser
405 410 415
Leu Glu Trp Asp Ala Gin Arg Leu Met Phe Asn Gly Leu Phe Pro Gin
420 425 430
Pro Leu Thr Asp Arg Lys Tyr Pro Asn Gin Cys Gly Thr lie Ser Asn
435 440 445
SEQ ID NO: 65
Saccharomyces cerevisiae DNA sequence
atgcctttga agtcgttttt tttttcagca tttctagttt tatgcctgtc taaattcacg 60
caaggcgttg gcaccacaga gaaggaagaa tcgttatcgc ctttggaact aaatatttta 120
caaaacaaat tcgcctccta ctatgcaaac gacactatca ccgtgaaagg tattactatt
180
ggcggctggc tagtaacaga accttatatc acgccatcat tatatcgtaa tgctacgtca 240
ctggcaaaac agcaaaac c ttccagcaat atctccattg tcgacgaa tactctttgt
300
aaaaccttag gatataacac ctctctaact ttattggata atcacttcaa aacttggatt 360
acagaggatg attttgaaca aatcaaaacc aacggtttca atttagttag gatccccatc 420
ggatattggg cgtggaaaca aaatactgat aaaaacttgt acatcgataa cataactttc
480
aatgatccat acgtaagtga tggattacaa ctgaaatatt taaataatgc tctcgaatgg 540
gcgcaaaagt acgaactaaa tgtatggtta gatctacatg gtgctcctgg atcccagaat
600
ggattcgata attccggtga aagaatactc tatggcgatt taggctggtt aaggttgaat 660
aataetaaag aactgactct ggctatttgg agagatatgt tccagacatt tttaaataaa 720
ggtgacaaaa gtcctgtggt gggtattcaa atcgtcaacg aaccgcttgg tggcaaaatc
780
gatgtttcag acataacgga gatgtattac gaagcatttg acttgctcaa gaaaaatcag 840
aattcgagtg acaacactac gtttgttatt catgacggtt ttcaaggaat cggtcactgg
900 aacttggagc taaacccaac ctaccagaat gtatcgcatc attatttcaa tttgactggt
960
gcaaattaca gctctcaaga tatattggtc gaccatcatc attatgaagt gtttactgat
1020
gcgcaattgg ccgaaactca gtttgcacgt attgaaaaca ttatcaatta tggggactct
1080
atccacaaag aactttcttt tcacccagca gtagtcggag aatggtcagg cgctattact
1140
gattgtgcaa cctggctaaa tggtgttggg gtgggtgcac gttacgatgg atcatactac
1200
aatacaacgt tgtttaccac caacgacaag ccagttggaa catgtatatc ccaaaatagc
1260
ttagctgatt ggacgcaaga ttaccgtgac cgtgtgagac aattcattga ggcacagcta
1320
gccacttatt cgtcaaaaac aacgggatgg attttttgga attggaagac cgaagacgcc
1380
gtagaatggg attatttgaa gctaaaagaa gctaaccttt tcccttcccc tttcgacaac
1440
tacacgtact tcaaagcaga tggatctatc gaagaaaaat tctcatcctc tttatcagca
1500
caggcatttc caagaacaac gtcatcggtt ttgtcctcca ctacgacttc caggaagagt
1560
aagaatgctg caatttctaa taaactaaca acttcgcagc tattaccaat caaaaatatg
1620
agtttgacct ggaaagcgag cgtatgcgca ctcgctatca ccattgccgc tctttgcgct
1680
tctctttaa
1689
SEQ ID NO: 66
Saccharomyces cerevisiae protein sequence
Met Pro Leu Lys Ser Phe Phe Phe Ser Ala Phe Leu Val Leu Cys Leu
1 5 10 15
Ser Lys Phe Thr Gin Gly Val Gly Thr Thr Glu Lys Glu Glu Ser Leu
20 25 30
Ser Pro Leu Glu Leu Asn lie Leu Gin Asn Lys Phe Ala Ser Tyr Tyr
35 40 45
Ala Asn Asp Thr lie Thr Val Lys Gly lie Thr lie Gly Gly Trp Leu
50 55 60
Val Thr Glu Pro Tyr lie Thr Pro Ser Leu Tyr Arg Asn Ala Thr Ser
65 70 75 80
Leu Ala Lys Gin Gin Asn Ser Ser Ser Asn lie Ser lie Val Asp Glu
85 90 95 Phe Thr Leu Cys Lys Thr Leu Gly Tyr Asn Thr Ser Leu Thr Leu Leu 100 105 110
Asp Asn His Phe Lys Thr Trp lie Thr Glu Asp Asp Phe Glu Gin lie
115 120 125
Lys Thr Asn Gly Phe Asn Leu Val Arg lie Pro lie Gly Tyr Trp Ala 130 135 140
Trp Lys Gin Asn Thr Asp Lys Asn Leu Tyr He Asp Asn He Thr Phe 145 150 155 160
Asn Asp Pro Tyr Val Ser Asp Gly Leu Gin Leu Lys Tyr Leu Asn Asn
165 170 175
Ala Leu Glu Trp Ala Gin Lys Tyr Glu Leu Asn Val Trp Leu Asp Leu
180 185 190
His Gly Ala Pro Gly Ser Gin Asn Gly Phe Asp Asn Ser Gly Glu Arg
195 200 205
He Leu Tyr Gly Asp Leu Gly Trp Leu Arg Leu Asn Asn Thr Lys Glu 210 215 220
Leu Thr Leu Ala He Trp Arg Asp Met Phe Gin Thr Phe Leu Asn Lys 225 230 235 240
Gly Asp Lys Ser Pro Val Val Gly He Gin He Val Asn Glu Pro Leu
245 250 255
Gly Gly Lys He Asp Val Ser Asp He Thr Glu Met Tyr Tyr Glu Ala
260 265 270
Phe Asp Leu Leu Lys Lys Asn Gin Asn Ser Ser Asp Asn Thr Thr Phe
275 280 285
Val He His Asp Gly Phe Gin Gly He Gly His Trp Asn Leu Glu Leu 290 295 300
Asn Pro Thr Tyr Gin Asn Val Ser His His Tyr Phe Asn Leu Thr Gly 305 310 315 320
Ala Asn Tyr Ser Ser Gin Asp He Leu Val Asp His His His Tyr Glu
325 330 335
Val Phe Thr Asp Ala Gin Leu Ala Glu Thr Gin Phe Ala Arg He Glu
340 345 350
Asn He He Asn Tyr Gly Asp Ser He His Lys Glu Leu Ser Phe His
355 360 365 Pro Ala Val Val Gly Glu Trp Ser Gly Ala lie Thr Asp Cys Ala Thr
370 375 380
Trp Leu Asn Gly Val Gly Val Gly Ala Arg Tyr Asp Gly Ser Tyr Tyr
385 390 395 400
Asn Thr Thr Leu Phe Thr Thr Asn Asp Lys Pro Val Gly Thr Cys He
405 10 415
Ser Gin Asn Ser Leu Ala Asp Trp Thr Gin Asp Tyr Arg Asp Arg Val
420 425 430
Arg Gin Phe lie Glu Ala Gin Leu Ala Thr Tyr Ser Ser Lys Thr Thr
435 440 445
Gly Trp lie Phe Trp Asn Trp Lys Thr Glu Asp Ala Val Glu Trp Asp
450 455 460
Tyr Leu Lys Leu Lys Glu Ala Asn Leu Phe Pro Ser Pro Phe Asp Asn
465 470 475 480
Tyr Thr Tyr Phe Lys Ala Asp Gly Ser He Glu Glu Lys Phe Ser Ser
485 490 495
Ser Leu Ser Ala Gin Ala Phe Pro Arg Thr Thr Ser Ser Val Leu Ser
500 505 510
Ser Thr Thr Thr Ser Arg Lys Ser Lys Asn Ala Ala He Ser Asn Lys
515 520 525
Leu Thr Thr Ser Gin Leu Leu Pro lie Lys Asn Met Ser Leu Thr Trp
530 535 540
Lys Ala Ser Val Cys Ala Leu Ala lie Thr He Ala Ala Leu Cys Ala
545 550 555 560
Ser Leu
SEQ ID NO: 67
Si.r it.ia grosvenorii DMA sequence
atggtgcaac ctcgggtact gctgtttcct ttcccggcac tgggccacgt gaagcccttc
60
ttatcactgg cggagctgct ttccgacgcc ggcatagacg tcgtcttcct cagcaccgag 120
tataaccacc gtcggatctc caacactgaa gccctagcct cccgcttccc gacgcttcat
180 ttcgaaacta taccggatgg cctgccgcct aatgagtcgc gcgctcttgc cgacggccca
240
ctgtatttct ccatgcgtga gggaactaaa ccgagattcc ggcaactgat tcaatctctt
300
aacgacggtc gttggcccat cacctgcatt atcactgaca tcatgttatc ttctccgatt 360
gaagtagcgg aagaatttgg gattccagta attgccttct gcccctgcag tgctcgctac
420
ttatcgattc acttttttat accgaagctc gttgaggaag gtcaaattcc atacgcagat
480
gacgatccga ttggagagat ccagggggtg cccttgttcg aaggtctttt gcgacggaat
540
catttgcctg gttcttggtc tgataaatct gcagatatat ctttctcgca tggcttgatt
600
aatcagaccc ttgcagctgg tcgagcctcg gctcttatac tcaacacctt cgacgagctc
660
gaagctccat ttctgaccca tctctcttcc attttcaaca aaatctacac cattggaccc
720
ctccatgctc tgtccaaatc aaggctcggc gactcctcct cctccgcttc tgccctctcc 780
ggattctgga aagaggatag agcctgcatg tcctggctcg actgtcagcc gccgagatct
840
gtggttttcg tcagtttcgg gagtacgatg aagatgaaag ccgatgaatt gagagagttc
900
tggtatgggt tggtgagcag cgggaaaccg ttcctctgcg tgttgagatc cgacgttgtt
960
tccggcggag aagcggcgga attgatcgaa cagatggcgg aggaggaggg agctggaggg 1020
aagctgggaa tggtagtgga gtgggcagcg caagagaagg tcctgagcca ccctgccgtc 1080
ggtgggtttt tgacgcactg cgggtggaac tcaacggtgg aaagcattgc cgcgggagtt
1140
ccgatgatgt gctggccgat tctcggcgac caacccagca acgccacttg gatcgacaga 1200
gtgtggaaaa ttggggttga aaggaacaat cgtgaatggg acaggttgac ggtggagaag
1260
atggtgagag cattgatgga aggccaaaag agagtggaga ttcagagatc aatggagaag
1320
ctttcaaagt tggcaaatga gaaggttgtc aggggtgggt tgtcttttga taacttggaa
1380
gttctcgttg aagacat ittgaaa ccatataaat tttaa 1425
SEQ ID NO: 68
Siraitia grosvenorii protein sequence
Met Val Gin Pro Arg Val Leu Leu Phe Pro Phe Pro Ala Leu Gly His
1 5 10 15 Val Lys Pro Phe Leu Ser Leu Ala Glu Leu Leu Ser Asp Ala Gly He 20 25 30
Asp Val Val Phe Leu Ser Thr Glu Tyr Asn His Arg Arg He Ser Asn
35 40 45
Thr Glu Ala Leu Ala Ser Arg Phe Pro Thr Leu His Phe Glu Thr He
50 55 60
Pro Asp Gly Leu Pro Pro Asn Glu Ser Arg Ala Leu Ala Asp Gly Pro 65 70 75 80
Leu Tyr Phe Ser Met Arg Glu Gly Thr Lys Pro Arg Phe Arg Gin Leu
85 90 95
He Gin Ser Leu Asn Asp Gly Arg Trp Pro He Thr Cys He He Thr
100 105 110
Asp He Met Leu Ser Ser Pro He Glu Val Ala Glu Glu Phe Gly He
115 120 125
Pro Val He Ala Phe Cys Pro Cys Ser Ala Arg Tyr Leu Ser He His 130 135 140
Phe Phe He Pro Lys Leu Val Glu Glu Gly Gin He Pro Tyr Ala Asp
145 150 155 160
Asp Asp Pro lie Gly Glu He Gin Gly Val Pro Leu Phe Glu Gly Leu
165 170 175
Leu Arg Arg Asn His Leu Pro Gly Ser Trp Ser Asp Lys Ser Ala Asp
180 185 190
He Ser Phe Ser His Gly Leu He Asn Gin Thr Leu Ala Ala Gly Arg
195 200 205
Ala Ser Ala Leu He Leu Asn Thr Phe Asp Glu Leu Glu Ala Pro Phe 210 215 220
Leu Thr His Leu Ser Ser He Phe Asn Lys He Tyr Thr He Gly Pro 225 230 235 240
Leu His Ala Leu Ser Lys Ser Arg Leu Gly Asp Ser Ser Ser Ser Ala
245 250 255
Ser Ala Leu Ser Gly Phe Trp Lys Glu Asp Arg Ala Cys Met Ser Trp
260 265 270
Leu Asp Cys Gin Pro Pro Arg Ser Val Val Phe Val Ser Phe Gly Ser
275 280 285 Thr Met Lys Met Lys Ala Asp Glu Leu Arg Glu Phe Trp Tyr Gly Leu 290 295 300
Val Ser Ser Gly Lys Pro Phe Leu Cys Val Leu Arg Ser Asp Val Val
305 310 315 320
Ser Gly Gly Glu Ala Ala Glu Leu He Glu Gin Met Ala Glu Glu Glu
325 330 335
Gly Ala Gly Gly Lys Leu Gly Met Val Val Glu Trp Ala Ala Gin Glu
340 345 350
Lys Val Leu Ser His Pro Ala Val Gly Gly Phe Leu Thr His Cys Gly
355 360 365
Trp Asn Ser Thr Val Glu Ser lie Ala Ala Gly Val Pro Met Met Cys
370 375 380
Trp Pro lie Leu Gly Asp Gin Pro Ser Asn Ala Thr Trp lie Asp Arg
385 390 335 400
Val Trp Lys lie Gly Val Glu Arg Asn Asn Arg Glu Trp Asp Arg Leu
405 410 415
Thr Val Glu Lys Met Val. Arg Ala Leu Met Glu Gly Gin Lys Arg Val
420 425 430
Glu lie Gin Arg Ser Met Glu Lys Leu Ser Lys Leu Ala Asn Glu Lys
435 440 445
Val Arg Gly Gly Leu Ser Phe Asp Asn Leu Glu Val Leu Val Glu
450 455 460
Asp He Lys Lys Leu Lys Pro Tyr Lys Phe
465 470
SEQ ID NO: 69
Siraitia grosvenorii DNA sequence
atggatgcaa aagaagaaag cttgaaagtt tttatgcttc catggttggc ccatggtcat 60
atatcgccct acctagagct agccaagagg cttgcaaaga gaaaatttct tgtttatttc 120
tgctccacgc ctgtaaattt ggaagccatt aaaccaaagc tttccaaaag ctactctgat
180
tcgatccaac taatggaggt tcctctcgaa tcgacgccgg agcttcctcc tcactatcat 240
acagccaaag gccttccgcc gcatttaatg cccaaactca tgaatgcctt taaaatggtt 300 gctcccaatc tcgaatcgat cctaaaaacc ctaaacccag atctgctcat cgtcgacatt
360
ctccttccat ggatgcttcc actcgcttca tcgctcaaaa ttccgatggt tttcttcact 420
attttcggtg ccatggccat ctcctttatg atttataatc gaaccgtctc gaacgagctt 480
ccatttccag aatttgaact tcacgagtgc tggaaatcga agtgccccta tttgttcaag
540
gaccaagcgg aaagtcaatc gttcttagaa tacttggatc aatcttcagg cgtaattttg 600
atcaaaactt ccagagagat tgaggctaag tatgtagact ttctcacttc gtcgtttacg 66 0
aagaaggttg tgaccaccgg tcccctggtt cagcaacctt cttccggcga agacgagaag
720
cagtactccg atatcatcga atggctagac aagaaggagc cgttatcgac ggtgctcgtt 780
tcgtttggga gcgagtatta tctgtcaaag gaagagatgg aagaaatcgc ctacgggctg 840
gagagcgcca gcgaggtgaa tttcatctgg attgttaggt ttccgatggg acaggaaacg
900
gaggtcgagg cggcgctgcc ggaggggttc atccagaggg caggagagag agggaaagtg 960
gtcgagggct gggctccgca ggcgaaaata ttggcgcatc cgagcaccgg cggccatgtg
1020
agccacaacg ggtggagctc gattgtggag tgcttgatgt ccggtgtacc ggtgatcggc
1080
gcgccgatgc aacttgacgg gccaatcgtc gcaaggctgg tggaggagat cggcgtgggt 1140
ttggaaatca agagagatga ggaagggaga atcacgaggg gcgaagttgc cgatgcaatc
1200
aagacggtgg cggtgggcaa aaccggggaa gattt agaa ggaaagcaaa aaaaatcagc
1260
agcattttga agatgaaaga tgaagaagag gttgacactt tggcaatgga attagtgagg
1320
ttatgccaaa :ct caggactaa
1359
SEQ ID NO: 70
Artificial Sequence; Codon-optimized nucleotide sequence A encoding UGT11789
atggacgcca aagaagaatc cttgaaggtt tttatgttgc catggttggc tcatggtcat 60
atttctccat atttggaatt ggctaagaga ttggccaaga gaaagttctt ggtttacttc 120
tgttctaccc cagttaactt ggaagctatt aagccaaagt tgtccaagtc ctactccgat
180
tctattcaat tgatggaagt cccattggaa tccactccag aattgccacc acattatcat 240 actgctaaag gtttgccacc tcatttgatg ccaaaattga tgaacgcttt caagatggtt 300
gctccaaact tggaatcaat cttgaaaacc ttgaacccag acttgttgat cgttgatatt 360
ttgttgcctt ggatgttgcc tttggcctcc tctttgaaaa ttcctatggt tttcttcacc 420
atcttcggtg ctatggctat ttctttcatg atctacaaca gaaccgtttc caacgaattg 480
ccatttccag aatttgaatt gcacgaatgc tggaagtcta agtgtccata cttgtttaag 540
gatcaagceg aatcccaatc cttcttggaa tatttggatc aatcctccgg tgtcattttg 600
atcaagacct ctagagaaat tgaagccaag tacgttgatt tcttgacctc ttcattcacc 660
aagaaggttg ttactactgg tccattggtt caacaaccat catctggtga agatgaaaag 720
caatactccg atatcattga atggttggac aagaaagaac cattgtccac tgttttggtt 780
tctttcggtt ccgaatatta cttgtctaaa gaagaaatgg aagaaatcgc ctacggtttg 840
gaatctgctt ctgaagttaa tttcatctgg atcgtcagat tcccaatggg tcaagaaact 900
gaagttgaag ctgctttgcc agaaggtttt attcaaagag ctggtgaaag aggtaaagtt 960
gttgaaggtt gggctccaca agctaagatt ttggctcatc catctactgg tggtcacgtt 1020
tctcataatg gttggtcatc tatcgttgaa tgcttgatgt ctggtgttcc agttattggt 1080
gctccaatgc aattggatgg tccaatagtt gctagattgg tcgaagaaat tggfcgttggt 1140
ttggaaatca agagagatga agaaggtaga atcaccagag gtgaagttgc tgatgctatt 1200
aagactgttg ctgttggtaa aaccggtgaa gattttagaa gaaaggccaa gaagatctcc 1260
tccattttaa agatgaagga cgaagaagaa gttgacacct tggctatgga attggttaga 1320
ttgtgtcaaa tcaagaatcc caagactga 1359
SEQ ID NO: 71
Artificial Sequence Codon-o timized nucleotide sequence B encoding UGT11789
atggatgcta aggaagaatc tttgaaagtc tttatgctgc cttggttggc tcacggtcat 60
atttccccgt atttggaatt ggcaaaaaga ctggccaaga gaaaattctt agtgtatttc 120
tgttcaactc cagtgaattt ggaagccatc aaaccaaaat tgtctaagtc atattctgac 180 tctatacaac tgatggaagt tcctttggaa agtacaccgg aactgccacc ccattatcat 240
acagctaaag ggttaccccc acacttgatg cccaagctaa tgaatgcatt taagatggtc 300
gcaccaaatc tggaaagtat acttaagacg ctaaaccctg atttattaat tgtagatatc
360
cttctaccat ggatgttgcc cttagcttca tctttaaaaa ttccgatggt ttttttcact 420
atctttggag ccatggcaat ttcctttatg atttacaata gaacagtctc aaatgagtta
480
cctttcccag agtttgaatt acatgaatgc tggaaatcta aatgtccata tttgttcaaa 540
gaccaagcag aatcccaatc tttcttagaa tacttagatc agagttccgg agttatcttg 600
atcaagacat ctagggaaat tgaagcaaag tatgtggact ttttgacctc cagttttact
660
aagaaagtcg taacaacggg tcctctagtc caacaaccta gttcaggaga ggatgagaaa 720
caatatagcg atataatcga atggttagat aaaaaagagc cattgagtac cgttctagtg
780
tcctttggtt cagaatatta tttgtctaaa gaagagatgg aagagattgc ctacggctta 840
gaatcagctt ccgaagtaaa ctttatatgg attgtcagat ttcccatggg acaagaaacc 900
gaggtcgaag cagctttgcc cgaaggtttt attcaacgtg ccggcgaaag aggaaaagta
960
gtggaaggtt gggctccaca agccaaaatt ctagctcacc cgtccactgg tggtcatgtc 1020
tctcataacg gatggagttc aattgttgaa tgtt ga ga gtggtgttcc agtgatagga
1080
gctcctatgc agctggacgg tccaatagtc gccaggttag tcgaagaaat tggtgttggt 1140
ttagaaataa agagagacga agaaggtaga attactagag gtgaagtagc agatgcaatt
1200
aaaactgttg ctgtcggcaa gactggagag gattttcgta gaaaagccaa aaaaatatca
1260
tctatactaa aaatgaaaga cgaagaggag gttgatacgc tggcgatgga actagttaga 1320
ttgtgtcaga caagactaa
1359
SEQ ID NO: 72
Siraitia grosvenori i protein sequence
Met Asp Ala Lys Glu Glu Ser Leu Lys Val Phe Met Leu Pro Trp Leu
1 5 10 15
Ala His Gly His lie Ser Pro Tyr Leu Glu Leu Ala Lys Arg Leu Ala
20 25 30 Lys Arg Lys Phe Leu Val Tyr Phe Cys Ser Thr Pro Val Asn Leu Glu 35 40 45
Ala lie Lys Pro Lys Leu Ser Lys Ser Tyr Ser Asp Ser lie Gin Leu 50 55 60
Met Glu Val Pro Leu Glu Ser Thr Pro Glu Leu Pro Pro His Tyr His 65 70 75 80
Thr Ala Lys Gly Leu Pro Pro His Leu Met Pro Lys Leu Met Asn Ala
85 90 95
Phe Lys Met Val Ala Pro Asn Leu Glu Ser He Leu Lys Thr Leu Asn
100 105 110
Pro Asp Leu Leu He Val Asp He Leu Leu Pro Trp Met Leu Pro Leu
115 120 125
Ala Ser Ser Leu Lys He Pro Met Val Phe Phe Thr He Phe Gly Ala 130 135 140
Met Ala He Ser Phe Met He Tyr Asn Arg Thr Val Ser Asn Glu Leu 145 150 155 160
Pro Phe Pro Glu Phe Glu Leu His Glu Cys Trp Lys Ser Lys Cys Pro
165 170 175
Tyr Leu Phe Lys Asp Gin Ala Glu Ser Gin Ser Phe Leu Glu Tyr Leu
180 185 190
Asp Gin Ser Ser Gly Val He Leu He Lys Thr Ser Arg Glu He Glu
135 200 205
Ala Lys Tyr Val Asp Phe Leu Thr Ser Ser Phe Thr Lys Lys Val Val 210 215 220
Thr Thr Gly Pro Leu Val Gin Gin Pro Ser Ser Gly Glu Asp Glu Lys 225 230 235 240
Gin Tyr Ser Asp He He Glu Trp Leu Asp Lys Lys Glu Pro Leu Ser
245 250 255
Thr Val Leu Val Ser Phe Gly Ser Glu Tyr Tyr Leu Ser Lys Glu Glu
260 265 270
Met Glu Glu He Ala Tyr Gly Leu Glu Ser Ala Ser Glu Val Asn Phe
275 280 285
He Trp He Val Arg Phe Pro Met Gly Gin Glu Thr Glu Val Glu Ala 290 295 300 Ala Leu Pro Glu Gly Phe He Gin Arg Ala Gly Glu Arg Gly Lys Val 305 310 315 320
Val Glu Gly Trp Ala Pro Gin Ala Lys He Leu Ala His Pro Ser Thr
325 330 335
Gly Gly His Val Ser His Asn Gly Trp Ser Ser He Val Glu Cys Leu
340 345 350
Met Ser Gly Val Pro Val He Gly Ala Pro Met Gin Leu Asp Gly Pro
355 360 365
He Val Ala Arg Leu Val Glu Glu He Gly Val Gly Leu Glu He Lys
370 375 380
Arg Asp Glu Glu Gly Arg He Thr Arg Gly Glu Val Ala Asp Ala He
385 390 395 400
Lys Thr Val Ala Val Gly Lys Thr Gly Glu Asp Phe Arg Arg Lys Ala
405 410 415
Lys Lys He Ser Ser He Leu Lys Met Lys Asp Glu Glu Glu Val Asp
420 425 430
Thr Leu Ala Met Glu Leu Val Arg Leu Cys Gin Met Lys Arg Gly Gin
435 440 445
Glu Ser Gin Asp
450
SEQ ID NO: 73
Siraitia grosvenorii DNA sequence
atggaaatgt cgtcgtctgt tgcagctacg atttcaatat ggatggttgt ggtgtgcata 60
gtgggagtgg gatggagagt tgtgaactgg gtttggttga ggccgaagaa gcttgagaag 120
cggctgagag agcaaggcct cgccggaaac tcttaccggc ttctgttcgg agacttgaag 180
gagagggcgg cgatggagga gcaggccaac tccaagccca tcaacttctc ccatgatatc 240
ggaccacgtg tcttcccctc catgtacaaa accatccaga attatggtaa gaattcgtac 300
atgtggcttg gcccatatcc aagagtgcac atcatggacc ctcagcaact taaaactgtt 360
tttactctag tctatgatat ccaaaagcca aatttgaacc cccttatcaa gtttcttttg 420
gatggaatag taactcatga aggagaaaaa tgggctaaac acagaaagat aatcaaccct 480 gcatttcatt tggaaaagtt gaaggatatg ataccagcat tctttcatag ttgtaatgag
540
atagttaacg aatgggaaag attaatctcg aaagagggtt cgtgtgagtt ggatgttatg
600
ccatatctgc aaaatttggc agctgatgcc atttctcgaa ctgcatttgg gagtagctat 660
gaagaaggaa aaatgatctt ccaactttta aaagaactaa ctgatttggt ggttaaagtt 720
gcatttggag tttatattcc cggatggagg tttctaccaa ctaagtcaaa caataaaatg 780
aaagaaataa atagaaaaat taaaagtttg cttttgggta ttataaacaa aaggcaaaag 840
gctatggaag aaggtgaagc tggacaaagt gatttattag gcattctcat ggaatccaat
900
tcaaacgaaa ttcaaggaga aggaaacaat aaagaagatg gaatgagcat agaagatgtt 960
attgaagaat gcaaggtttt ctatattggt ggccaagaaa ccacagccag attactgatt 1020
tggaccatga ttttgttgag ttcacacacg gaatggcaag agcgagcaag aactgaggta 1080
ttaaaagtat ttggtaacaa gaagccagat tttgatggtt tgagtcgact aaaagttgta 1140
actatgattt tgaacgaggt tctcaggtta tacccaccag caagtatgct tactcgtatt
1200
attcaaaagg aaacaagagt tggaaaattg actctaccag ctggtgtgat attgatcatg
1260
ccaattattc ttatccatcg tgatcatgac ctatggggtg aagatgcaaa cgaatttaaa 1320
ccagaaagat tttctaaggg agtctctaaa gcagcaaaag ttcaacccgc tttcttccca 1380
tttggatggg gtcctcgaat atgcatgggg cagaactttg cgatgattga agcaaaaatg 1440
gcattatcat taattctaca acgcttctca tttgagcttt cttcgtcgta tgttcatgct 1500
cctaccgtcg ttttcactac tcaacctcaa catggagctc atatcgtcct gcgcaaactg
1560
tag
1563
SEQ ID NO: 74
Siraitia grosvenorii protein sequence
Met Glu Met Ser Ser Ser Val Ala Ala Thr lie Ser lie Trp Met Val
1 5 10 15
Val Val Cys He Val Gly Val Gly Trp Arg Val Val Asn Trp Val Trp
20 25 30
Leu Arg Pro Lys Lys Leu Glu Lys Arg Leu Arg Glu Gin Gly Leu Ala
35 40 45 Gly Asn Ser Tyr Arg Leu Leu Phe Gly Asp Leu Lys Glu Arg Ala Ala
50 55 60
Met Glu Glu Gin Ala Asn Ser Lys Pro He Asn Phe Ser His Asp He 65 70 75 80
Gly Pro Arg Val Phe Pro Ser Met Tyr Lys Thr He Gin Asn Tyr Gly
85 90 95
Lys Asn Ser Tyr Met Trp Leu Gly Pro Tyr Pro Arg Val His He Met
100 105 110
Asp Pro Gin Gin Leu Lys Thr Val Phe Thr Leu Val Tyr Asp He Gin
115 120 125
Lys Pro Asn Leu Asn Pro Leu He Lys Phe Leu Leu Asp Gly He Val
130 135 140
Thr His Glu Gly Glu Lys Trp Ala Lys His Arg Lys He He Asn Pro
145 150 155 160
Ala Phe His Leu Glu Lys Leu Lys Asp Met He Pro Ala Phe Phe His
165 170 175
Ser Cys Asn Glu lie Val Asn Glu Trp Glu Arg Leu He Ser Lys Glu
180 185 190
Gly Ser Cys Glu Leu Asp Val Met Pro Tyr Leu Gin Asn Leu Ala Ala
195 200 205
Asp Ala lie Ser Arg Thr Ala Phe Gly Ser Ser Tyr Glu Glu Gly Lys 210 215 220
Met lie Phe Gin Leu Leu Lys Glu Leu Thr Asp Leu Val Val Lys Val 225 230 235 240
Ala Phe Gly Val Tyr lie Pro Gly Trp Arg Phe Leu Pro Thr Lys Ser
245 250 255
Asn Asn Lys Met Lys Glu He Asn Arg Lys He Lys Ser Leu Leu Leu
260 265 270
Gly lie lie Asn Lys Arg Gin Lys Ala Met Glu Glu Gly Glu Ala Gly
275 280 285
Gin Ser Asp Leu Leu Gly He Leu Met Glu Ser Asn Ser Asn Glu He 290 295 300
Gin Gly Glu Gly Asn Asn Lys Glu Asp Gly Met Ser He Glu Asp Val
305 310 315 320 He Glu Glu Cys Lys Val Phe Tyr He Gly Gly Gin Glu Thr Thr Ala
325 330 335
Arg Leu Leu He Trp Thr Met He Leu Leu Ser Ser His Thr Glu Trp
340 345 350
Gin Glu Arg Ala Arg Thr Glu Val Leu Lys Val Phe Gly Asn Lys Lys
355 360 365
Pro Asp Phe Asp Gly Leu Ser Arg Leu Lys Val Val Thr Met He Leu
370 375 380
Asn Glu Val Leu Arg Leu Tyr Pro Pro Ala Ser Met Leu Thr Arg He
385 330 335 400
He Gin Lys Glu Thr Arg Val Gly Lys Leu Thr Leu Pro Ala Gly Val
405 410 415
He Leu He Met Pro He He Leu He His Arg Asp His Asp Leu Trp
420 425 430
Gly Glu Asp Ala Asn Glu Phe Lys Pro Glu Arg Phe Ser Lys Gly Val
435 440 445
Ser Lys Ala Ala Lys Val Gin Pro Ala Phe Phe Pro Phe Gly Trp Gly
450 455 460
Pro Arg He Cys Met Gly Gin Asn Phe Ala Met He Glu Ala Lys Met
465 470 475 480
Ala Leu Ser Leu He Leu Gin Arg Phe Ser Phe Glu Leu Ser Ser Ser
485 490 435
Tyr Val His Ala Pro Thr Val Val Phe Thr Thr Gin Pro Gin His Gly
500 505 510
Ala His He Val Leu Arg Lys Leu
515 520
SEQ ID HO: 7
Saccharoinyc s cerevisiae DNA sequence
atgtctgtta ttaatttcac aggtagttct ggtccattgg tgaaagtttg cggcttgcag 60
agcacagagg ccgcagaatg tgctctagat tccgatgctg acttgctggg tattatatgt 120
gtgcccaata gaaagagaac aattgacccg gttattgcaa ggaaaatttc aagtcttgta 180 aaagcatata aaaatagttc aggcactccg aaatacttgg ttggcgtgtt tcgtaatcaa 240
cctaaggagg atgttttggc tctggtcaat gattacggca ttgatatcgt ccaactgcat
300
ggagatgagt cgtggcaaga ataccaagag ttcctcggtt tgccagttat taaaagactc 360
gtatttccaa aagactgcaa catactactc agtgcagctt cacagaaacc tcattcgttt 420
attcccttgt ttgattcaga agcaggtggg acaggtgaac ttttggattg gaactcgatt 480
tctgactggg ttggaaggca agagagcccc gaaagcttac attttatgtt agctggtgga 540
ctgacgccag aaaatgttgg tgatgcgctt agattaaatg gcgttattgg tgttgatgta
600
agcggaggtg tggagacaaa tggtgtaaaa gactctaaca aaatagcaaa tttcgtcaaa
660
aatgctaaga aatag
675
SEQ ID NO: 76
Saccharomyces cerevisiae protein sequence
Met Ser Vai He Asn Phe Thr Gly Ser Ser Gly Pro Leu Val Lys Val.
1 5 10 15
Cys Gly Leu Gin Ser Thr Glu Ala Ala Glu Cys Ala Leu Asp Ser Asp
20 25 30
Ala Asp Leu Leu Gly He He Cys Val Pro Asn Arg Lys Arg Thr He
35 40 45
Asp Pro Val He Ala Arg Lys He Ser Ser Leu Val Lys Ala Tyr Lys
50 55 60
Asn Ser Ser Gly Thr Pro Lys Tyr Leu Val Gly Val Phe Arg Asn Gin
65 70 75 80
Pro Lys Glu Asp Val Leu Ala Leu Val Asn Asp Tyr Gly He Asp He
85 90 95
Val Gin Leu His Gly Asp Glu Ser Trp Gin Glu Tyr Gin Glu Phe Leu
100 105 110
Gly Leu Pro Val He Lys Arg Leu Val Phe Pro Lys Asp Cys Asn He
115 120 125
Leu Leu Ser Ala Ala Ser Gin Lys Pro His Ser Phe He Pro Leu Phe
130 135 140 Asp Ser Glu Ala Gly Gly Thr Gly Glu Leu Leu Asp Trp Asn Ser lie
145 150 155 160
Ser Asp Trp Val Gly Arg Gin Glu Ser Pro Glu Ser Leu His Phe Met
165 170 175
Leu Ala Gly Gly Leu Thr Pro Glu Asn Val Gly Asp Ala Leu Arg Leu
180 185 190
Asn Gly Val He Gly Val Asp Val Ser Gly Gly Val Glu Thr Asn Gly
195 200 205
Val Lys Asp Ser Asn Lys He Ala Asn Phe Val Lys Asn Ala Lys Lys
210 215 220
SEQ ID NO: 77
Saccharomyces cerevisiae DNA sequence
atggcagctg accaattggt gaaaactgaa gtcaccaaga agtcttttac tgctcctgta 60
caaaaggctt ctacaccagt tttaaccaat aaaacagtca tttctggatc gaaagtcaaa 120
agtttatcat ctgcgcaatc gagctcatca ggaccttcat catctagtga ggaagatgat
180
tcccgcgata ttgaaagctt ggataagaaa atacgtcctt tagaagaatt agaagcatta 240
ttaagtagtg gaaatacaaa acaattgaag aacaaagagg tcgctgcctt ggttattcac 300
ggtaagttac ctttgtacgc tttggagaaa aaattaggtg atactacgag agcggttgcg
360
gtacgtagga aggctctttc aattttggca gaagctcctg tattagcatc tgatcgttta 420
ccatataaaa attatgacta cgaccgcgta tttggcgctt gttgtgaaaa tgttataggt 480
tacatgcctt tgcccgttgg tgttataggc cccttggtta tcgatggtac atcttatcat
540
ataccaatgg caactacaga gggttgtttg gtagcttctg ccatgcgtgg ctgtaaggca 600
atcaatgctg gcggtggtgc aacaactgtt ttaactaagg atggtatgac aagaggccca 660
gtagtccgtt tcccaacttt gaaaagatct ggtgcctgta agatatggtt agactcagaa
720
gagggacaaa acgcaattaa aaaagctttt aactctacat caagatttgc acgtctgcaa
780
catattcaaa cttgtctagc aggagattta ctcttcatga gatttagaac aactactggt 840
gacgcaatgg gtatgaatat gatttctaaa ggtgtcgaat actcattaaa gcaaatggta 900
gaagagtatg gctgggaaga tatggaggtt gtctccgttt ctggtaacta ctgtaccgac 960 aaaaaaccag ctgccatcaa ctggatcgaa ggtcgtgg a agagtgtcgt cgcagaagct 1020
actattcctg gtgatgttgt cagaaaagtg ttaaaaagtg atgtttccgc attggttgag 1080
ttgaacattg ctaagaattt ggttggatct gcaatggctg ggtctgttgg tggatttaac
1140
gcacatgcag ctaatttagt gacagctgtt ttcttggcat taggacaaga tcctgcacaa 1200
aatgttgaaa gttccaactg tataacattg atgaaagaag tggacggtga tttgagaatt 1260
tccgtatcca tgccatccat cgaagtaggt accatcggtg gtggtactgt tctagaacca
1320
caaggtgcca tgttggactt attaggtgta agaggcccgc atgctaccgc tcctggtacc 1380
aacgcacgtc aattagcaag aatagttgcc tgtgccgtct tggcaggtga attatcctta 1440
tgtgctgccc tagcagccgg ccatttggtt caaagtcata tgacccacaa caggaaacct
1500
gctgaaccaa caaaacctaa caatttggac gccactgata taaatcgttt gaaagatggg 1560
tccgtcacct gcattaaatc ctaa 1584
SEQ ID NO: 78
Saccharomyces cerevisiae protein sequence
Met Ala Ala Asp Gin Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe
1 5 10 15
Thr Ala Pro Val Gin Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr
20 25 30
Val lie Ser Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gin Ser Ser
35 40 45
Ser Ser Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp lie
50 55 60
Glu Ser Leu Asp Lys Lys lie Arg Pro Leu Glu Glu Leu Glu Ala Leu
65 70 75 80
Leu Ser Ser Gly Asn Thr Lys Gin Leu Lys Asn Lys Glu Val Ala Ala
85 90 95
Leu Val lie His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu
100 105 110
Gly Asp Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser lie
115 120 125 Leu Ala Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro Tyr Lys Asn 130 135 140
Tyr Asp Tyr Asp Arg Val Phe Gly Ala Cys Cys Glu Asn Val He Gly
145 150 155 160
Tyr Met Pro Leu Pro Val Gly Val He Gly Pro Leu Val He Asp Gly
165 170 175
Thr Ser Tyr His lie Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala
180 185 190
Ser Ala Met Arg Gly Cys Lys Ala He Asn Ala Gly Gly Gly Ala Thr
195 200 205
Thr Val Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe 210 215 220
Pro Thr Leu Lys Arg Ser Gly Ala Cys Lys He Trp Leu Asp Ser Glu
225 230 235 240
Glu Gly Gin Asn Ala lie Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe
245 250 255
Ala Arg Leu Gin His He Gin Thr Cys Leu Ala Gly Asp Leu Leu Phe
260 265 270
Met Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met He
275 280 285
Ser Lys Gly Val Glu Tyr Ser Leu Lys Gin Met Val Glu Glu Tyr Gly 290 295 300
Trp Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp 305 310 315 320
Lys Lys Pro Ala Ala He Asn Trp He Glu Gly Arg Gly Lys Ser Val
325 330 335
Val Ala Glu Ala Thr lie Pro Gly Asp Val Val Arg Lys Val Leu Lys
340 345 350
Ser Asp Val Ser Ala Leu Val Glu Leu Asn He Ala Lys Asn Leu Val
355 360 365
Gly Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala 370 375 380
Asn Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gin Asp Pro Ala Gin 385 390 395 400 Asn Val Glu Ser Ser Asn Cys lie Thr Leu Met Lys Glu Val Asp Gly
405 410 415
Asp Leu Arg lie Ser Val Ser Met Pro Ser lie Glu Val Gly Thr lie
420 425 430
Gly Gly Gly Thr Val Leu Glu Pro Gin Gly Ala Met Leu Asp Leu Leu
435 440 445
Gly Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gin
450 455 4S0
Leu Ala Arg lie Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu
465 470 475 480
Cys Ala Ala Leu Ala Ala Gly His Leu Val Gin Ser His Met Thr His
485 490 495
Asn Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala Thr
500 505 510
Asp lie Asn Arg Leu Lys Asp Gly Ser Val Thr Cys lie Lys Ser
515 520 525
SEQ ID NO: 79
Siraitia grosvenorii DNA sequence
atggacgaga ttgagcatat caccatcaac accaatggca tcaaaatgca cattgcctct 60
gtagggacgg gcccagtagt tcttcttctc catggcttcc cggagctctg gtactcatgg
120
cgccaccagc ttctgtatct ttcttccgta ggatatcgag ctattgcgcc ggacctccgc
180
ggctatggcg acacggactc gccggcgtct cctacctcct acaccgcgct ccacatcgtc
240
ggcgatttgg ttggggctct ggacgagctt gggatcgaga aggtgttcct ggtcggacat
300
gactgggggg cgatcatcgc ctggtacttt tgcttgttca ggcccgatag aatcaaggcg 360
ctggtgaatc tgagcgtcca gttcataccc agaaacccag cgattccttt catcgagggt
420
ttcagaactg cgttcggtga tgacttctat atttgcaggt ttcaggttcc aggagaggca
480
gaagaagatt ttgcctccat cgacacagct cagctgttca agacatcatt atgtaataga
540
agttctgcac ctccatgctt gcctaaagaa attggatttc gtgcgatccc acctccagag
600
aaccttcctt cttggctgac agaagaagat atcaactttt atgctgccaa atttaagcag 660 acaggcttca ccggagcgtt gaactactat cgagcttttg acctaacttg ggagctcacg 720
gcgccatgga cgggagcaca gattcaggta ccggtgaagt tcatcgtcgg ggattcggat 780
ctaacttacc attttccggg agccaaggaa tatatccata atggcggatt caaaagggac 840
gtgccgttgc tggaggaagt agttgtagta aaagatgctt gtcacttcat caaccaagaa 900
aggccacaag aaatcaatgc tcacatccat gacttcatca ataaattctg a 951
SEQ ID NO: 80
Siraitia grosvenorii sequence
atgtggaggt taaaggtcgg agcagaaagc gttggggaga atgatgagaa atggttgaag 60
agcataagca atcacttggg acgccaggtg tgggagttct gtccggatgc cggcacccaa 120
caacagctct tgcaagtcca caaagctcgt aaagctttcc acgatgaccg tttccaccga 180
aagcaatctt ccgatctctt tatcactatt cagtatggaa aggaagtaga aaatggtgga 240
aagacagcgg gagtgaaatt gaaagaaggg gaagaggtga ggaaagaggc agtagagagt 300
agcttagaga gggcattaag tttctactca agcatccaga caagcgatgg gaactgggct 360
tcggatcttg gggggcccat gtttttactt ccgggtctgg tgattgccct ctacgttaca 420
ggcgtcttga attctgtttt atccaagcac caccggcaag agatgtgcag atatgtttac 480
aatcaccaga atgaagatgg ggggtggggt ctccacatcg agggcccaag caccatgttt 540
ggttccgcac tgaattatgt tgcactcagg ctgcttggag aagacgccaa cgccggggca 600
atgccaaaag cacgtgcttg gatcttggac cacggtggcg ccaccggaat cacttcctgg 660
ggcaaattgt ggctttctgt acttggagtc tacgaatgga gtggcaataa tcctcttcca 720
cccgaatttt ggttatttcc ttacttccta ccatttcatc caggaagaat gtggtgccat 780
tgtcgaatgg tttatctacc aatgtcatac ttatatggaa agagatttgt tgggccaatc 840
acacccatag ttctgtctct cagaaaagaa ctctacgcag ttccatatca tgaaatagac 900
tggaataaat ctcgcaatac atgtgcaaag gaggatctgt actatccaca tcccaagatg 960
caagatattc tgtggggatc tctccaccac gtgtatgagc ccttgtttac tcgttggcct 1020
gccaaacgcc tgagagaaaa ggctttgcag actgcaatgc aacatattca ctatgaagat 1080 gagaataccc gatatatatg ccttggccct gtcaacaagg tactcaatct gctttgttgt
1140
tgggttgaag atccctactc cgacgccttc aaacttcatc ttcaacgagt ccatgactat 12 CO
ctctgggttg ctgaagatgg catgaaaatg cagggttata atgggagcca gttgtgggac
1260
actgctttct ccatccaagc aatcgtatcc accaaacttg tagacaacta tggcccaacc
1320
ttaagaaagg cacacgactt cgttaaaagt tctcagattc agcaggactg tcctggggat 1380
cctaatgttt ggtaccgtca cattcataaa ggtgcatggc cattttcaac tcgagatcat
1440
ggatggctca tctctgactg tacagcagag ggattaaagg ctgctttgat gttatccaaa 1500
cttccatccg aaacagttgg ggaatcatta gaacggaatc gcctttgcga tgctgtaaac
1560
gttctccttt ctttgcaaaa cgataatggt ggctttgcat catatgagtt gacaagatca 1620
tacccttggt tggagttgat caaccccgca gaaacgtttg gagatattgt cattgattat 1680
ccgtatgtgg agtgcacctc agccacaatg gaagcactga cgttgtttaa gaaattacat
1740
cccggccata ggaccaaaga aattgatact gctattgtca gggcggccaa cttccttgaa 1800
aatatgcaaa ggacggatgg ctcttggtat ggatgttggg gggtttgctt cacgtatgcg
1860
gggtggtttg gcataaaggg attggtggct gcaggaagga catataataa ttgccttgcc 1920
attcgcaagg cttgcgattt tttactatct aaagagctgc ccggcggtgg atggggag g 1980
agttaccttt catgtcagaa taaggtatac acaaatcttg aaggaaacag accgcacctg
2040
gttaacacgg cctgggtttt aatggccctc atagaagctg gccaggctga gagagaccca 2100
acaccattgc atcgtgcagc aaggttgtta atcaattccc agttggagaa tggtgatttc
2160
ccccaacagg agatcatggg agtctttaat aaaaattgca tgatcacata tgctgcatac 2220
cgaaacattt ttcccatttg ggctcttgga gagtattgcc atcgggtttt gactgaataa
2280
SEQ ID NO -.81
Artificial Sequence; Codon-optimized nucleotide sequence encoding CYP5491 atgtggactg ttgttttggg tttggctact ttgtttgttg cctactacat tcactggatc 60
aacaagtgga gagactctaa gtttaatggt gttttgccac caggtactat gggtttgcca
120 ttgattggtg aaaccatcca attgtcaaga ccatccgatt ctttggatgt tcatccattc
180
atccaaaaaa aggtcgaaag atacggtcca atcttcaaga cttgtttggc tggtagacca 240
gttgttgttt ctgctgatgc tgaatttaac aactacatca tgttgcaaga aggtagagct 300
gttgaaatgt ggtacttgga tactttgtct aagttcttcg gtttggatac cgaatggttg
360
aaggctttgg gtttaatcca taagtacatc agatccatca ccttgaatca ttttggtgct 420
gaagccttga gagaaagatt cttgcctttt attgaagcct cttctatgga agccttgcat 480
tcttggtcta ctcaaccatc tgttgaagtt aagaatgctt ccgctttgat ggttttcaga
540
acctctgtta acaagatgtt tggtgaagat gccaagaagt tgtctggtaa tattccaggt 600
aagttcacca agttgttggg tggttttttg tctttgcctt tgaatttccc aggtacaacc
660
taccataagt gcttgaaaga tatgaaggaa atccaaaaga agttgagaga agtcgttgat
720
gatagattgg ctaatgttgg tccagatgtc gaagattttt tgggtcaagc cttgaaggac
780
aaagaatccg aaaagttcat ctccgaagaa tttatcattc aattgttgtt ctctatctcc
840
ttcgcctcct tcgaatctat ttctactact ttgaccttga tcttgaagtt gttagacgaa
900
catccagaag tcgtcaaaga attggaagct gaacatgaag ctattagaaa ggctagagct 960
gatccagatg gtccaattac ttgggaagaa tacaagtcta tgaccttcac cttgcaagtt
1020
atcaacgaaa ctttgagatt gggttctgtt actccagctt tgttgagaaa aactgtcaag
1080
gacttacaag tcaagggtta cattattcct gaaggttgga ccattatgtt ggttactgct 1140
tcaagacata gagatccaaa ggtttacaaa gacccacata ttttcaatcc ttggagatgg
1200
aaggatttgg actccattac tattcaaaag aacttcatgc cattcggtgg tggtttgaga 1260
cattgtgctg gtgcagaata ctctaaggtt tacttgtgta ctttcttgca catcttgtgc 1320
actaagtaca gatggacaaa attgggtggt ggtagaattg ctagagccca tattttgtca
1380
ttcgaagatg gtttacatgt caagttcacc ccaaaagaat ga 1422
SEQ ID NO: 82
Artificial Sequence Codon-o timized nucleotide sequence encoding CYP4497 atgaaggtca gtccattcga attcatgtcc gctattatca agggtagaat ggacccatct
60
aactcctcat ttgaatctac tggtgaagtt gcctccgtta tctttgaaaa cagagaattg 120
gttgccatct tgaccacttc tattgctgtt atgattggtt gcttcgttgt cttgatgtgg
180
agaagagctg gttctagaaa ggttaagaat gtcgaattgc caaagccatt gattgtccat 240
gaaccagaac ctgaagttga agatggtaag aagaaggttt ccatcttctt cggtactcaa 300
actggtactg ctgaaggttt tgctaaggct ttggctgatg aagctaaagc tagatacgaa
360
aaggctacct tcagagttgt tgatttggat gattatgctg ccgatgatga ccaatacgaa 420
gaaaaattga agaacgaatc cttcgccgtt ttcttgttgg ctacttatgg tgatggtgaa
480
cctactgata atgctgctag attttacaag tggttcgccg aaggtaaaga aagaggtgaa
540
tggttgcaaa acttgcacta tgctgttttt ggtttgggta acagacaata cgaacacttc 600
aacaagattg c taaggttgc cgacgaatta ttggaagctc aaggtggtaa tagattggtt
660
aaggttggtt taggtgatga cgatcaatgc atcgaagatg atttttctgc ttggagagaa 720
tctttgtggc cagaattgga tatgttgttg agagatgaag atgatgctac tactgttact 780
actccatata ctgctgctgt cttggaatac agagttgtct ttcatgattc tgctgatgtt
840
gctgctgaag ataagtcttg gattaacgct aatggtcatg ctgttcatga tgctcaacat 900
ccattcagat ctaacgttgt cgtcagaaaa gaattgcata cttctgcctc tgatagatcc
960
tgttctcatt tggaattcaa catttccggt tccgctttga attacgaaac tggtgatcat
1020
gttggtgtct actgtgaaaa cttgactgaa actgttgatg aagccttgaa cttgttgggt 1080
ttgtctccag aaacttact t ctctatctac accgataacg aagatggtac tccattgggt
1140
ggttcttcat tgccaccacc atttccatca tgtactttga gaactgcttt gaccagatac 1200
gctgatttgt tgaactctcc aaaaaagtct gctttgttgg ctttagctgc tcatgcttct 1260
aatccagttg aagctgatag attgagatac ttggcttctc cagctggtaa agatgaatat
1320
gcccaatctg ttatcggttc ccaaaagtct ttgttggaag ttatggctga attcccatct 1380
gctaaaccac cattaggtgt tttttttgct gctgttgctc caagattgca acctagattc 1440
tactccattt catcctctcc aagaatggct ccatctagaa tccatgttac ttgtgctttg
1500 gtttacgata agatgccaac tggtagaatt cataagggtg tttgttctac ctggatgaag
1560
aattctgttc caatggaaaa gtcccatgaa tgttcttggg ctccaatttt cgttagacaa
1620
tccaatttta agttgccagc cgaatccaag gttccaatta tcatggttgg tccaggtact 1680
ggtttggctc cttttagagg ttttttacaa gaaaga gg ccttgaaaga atccggtgtt
1740
gaattgggtc catccatttt gtttttcggt tgcagaaaca gaagaatgga ttacatctac
1800
gaagatgaat tgaacaactt cgttgaaacc ggtgctttgt ccgaattggt tattgctttt 1860
tctagagaag gtcctaccaa agaatacgtc caacataaga tggctgaaaa ggcttctgat 1920
atctggaact tgatttctga aggtgcttac ttgtacgttt gtggtgatgc taaaggtatg
1980
gctaaggatg ttcatagaac cttgcatacc atcatgcaag aacaaggttc tttggattct 2040
tccaaagctg aatccatggt caagaacttg caaatgaatg gtagatactt aagagatgtt
2100
tggtaa
2106
SEQ ID NO: 83
Artificial Sequence ; Codon-optimized nucleotide sequence encoding UGT1576 atggcgtcac ctagacatac tcctcatttc ttgttatttc catttatggc tcaaggacat
60
atgataccta tgattgatct ggctaggcta ctagcacaaa gaggtgttat tatcactatt 120
attactactc cacataatgc agctcgttat catagtgttt tagctcgtgc cattgactct 180
ggtttacata tccacgtttt acaactacaa ttcccttgca aagaaggcgg actaccggaa
240
ggttgtgaga acgtagactt acttccatcc ttagcgagca ttccaagatt ttacagagct 300
gcctctgatc tactatatga acctagcgaa aaacttttcg aagagttgat accgagacca 360
acttgtatca tttctgatat gtgtttacca tggactatga gaattgcctt aaagtatcat
420
gtgcccagac ttgttttcta ctctttgtct tgcttttttc tgctgtgcat gagaagctta 480
aagaacaatt tagcattaat ttctagcaag tcagattccg agttcgtaac tttctctgat 540
ttacccgatc cagttgaatt tttgaagtct gagcttccta agtccacaga cgaagacttg
600
gttaaatttt catatgaaat gggtgaggcei gacagacaat catatggcgt tatactaaac
660
ttgtttgaag aaatggagcc caaatatttg gcagagtatg aaaaagaaag agaaagtccc
720 gaaagagttt ggtgtgttgg tccagtatct ttgtgcaacg ataacaaatt agataaagca 780
gagaggggta acaaagcatc aattgacgaa tataagtgta ttagatggtt agatgggcaa
840
caacctagca gtgttgttta tgttagtctt ggatcattat gcaacttggt tactgctcaa 900
attattgaat tggggttggg gttggaagct tctaaaaagc cattcatttg ggttattagg
960
aggggcaaca taacagaaga actacaaaaa tggctggttg aatatgactt tgaggagaag 1020
attaagggac gtggattagt catattaggg tgggcgcccc aagtacttat tctatctcat
1080
ccagctattg gttgcttctt aactcattgc ggttggaatt cctctatcga aggtatttcc 1140
gccggtgttc ctatggttac ctggcctcta tttgcagatc aggttttcaa cgaaaaatta
1200
atagttcaaa tcttgagaat cggagttagc gttggtacag aaacaaccat gaactggggt 1260
gaggaagaag aaaaaggtgt ggtggtcaaa agggagaaag tgagagaggc gatagagatc
1320
gtaatggatg gcgacgaaag agaagaaaga agagaaaggt gtaaagaact agcagaaact 1380
gccaaacgtg ctatcgagga aggtggtagc agtcatagaa atttgaccat gctaattgaa
1440
gatattatcc acggtggtgg cttatcttac gagaaagggt cctgcaggta
1491
SEQ ID NO: 84
Artificial Sequence; Codon-o timized nucleotide sequence encoding UGT430 atggaacaag cccacgattt gctgcatgtt ttactttttc catatccagc taaagggcat 60
attaagccct ttttgtgtct tgcggaactt ttatgcaacg caggtcttaa tgttacgttt
120
ttgaataccg attataatca cagaagatta cacaatctgc acctattagc ggcttgtttt 180
cctagtttgc attttgaaag tatcagtgat ggtttgcagc cagatcaacc tagagatatc
240
ttggacccaa agttttacat ctctatttgc caagttacca agccattatt cagagaattg 300
ttattatcct ataaaaggac atcctcagta caaaccggca ggccgccaat aacttgtgtt
360
ataacagatg ttatatttcg ttttccaatc gatgtagccg aggaattaga tatccctgtt 420
ttttctttct gtacttttag cgcgcgtttt atgtttcttt acttctggat cccaaagctt
480
atcgaggatg ggcaattgcc ttacccaaac ggtaacataa atcagaaact gtatggtgtt 540 gcacctgaag cagaaggatt attaaggtgt aaggatttac cgggacactg ggctttcgct 600
gatgagttaa aagacgatca gttgaacttt gttgatcaaa ctaccgccag tttgagatca 660
tctggtttga tcttaaacac tttcgacgat ttggaagctc cattcctggg acgtttgtca 720
acaatattta agaagatcta cgctgttggg ccaatacatg cgttgctaaa cagtcaccat 780
tgcggtttat ggaaagaaga ccacagctgt ttggcctggt tagatagtag agcggcacgt 840
tctgtcgtgt tcgtcagttt cggttctttg gttaagatca cttctaggca attgatggaa 300
ttctggcatg gattgttgaa tagcgggaca agctttttgt ttgtcttgag aagtgatgtt 960
gtagaaggtg atggggaaaa gcaagttgtc aaagaaatct acgaaacgaa agcagagggt 1020
aaatggttag ttgttggttg ggctccacaa gaaaaagtat tggcacatga agccgttgga 1080
ggtttcttaa ctcattccgg ttggaactca atcttagagt ctatagccgc aggtgtacct 1140
atgataagtt gcccaaaaat aggagaccaa tcttctaatt gtacctggat tagtaaagtt 1200
tggaagattg gtttagaaat ggaagaccag tatgacagag caactgtgga agctatggtg 1260
agatcaatta tgaaacacga agg gagaag atacaaaaga ctattgcgga acttgcaaaa 1320
agagcaaaat ataaagtttc caaggacggc acttcatata gaaatctgga aattttgatc 1380
gaagatatca gccgaattag 1410
SEQ ID NO: 85
Artificial Sequence ; Codon-optimized nucleotide sequence encoding UGT1637 atggttcaac ctagggtctt attgtttccc ttccctgctt tgggacatgt caaacccttt 60
ctgtcactgg cagaattact ttccgatgct gggatagacg ttgtatttct tagtacagaa 120
tacaatcata ggaggattag taacacggag gctctggcct caagatttcc aaccttgcat 180
tttgaaacaa taccagatgg tcttccacct aacgagagca gggctttggc agacggccct 240
ttgtacttta gcatgcgtga ggggacaaaa cccagattca gacagctgat acagagcctg 300
aacgatggca gatggcctat cacgtgtatc attaccgata tcatgttgag tagccccatc 360
gaagtagctg aggagtttgg aattccagta attgcctttt gtccctgctc cgctagatac 420 ttgtctattc attttttcat acccaagttg gttgaagagg gtcagatccc ttatgcagat
480
gatgatccaa tcggtgaaat tcaaggtgtg ccacttttcg aagggcttct gaggagaaat 540
catttgccag gcagctggag tgataagtct gcagacatct cattttccca tggtttgatc 600
aaccaaacat tagcagccgg tagagcttct gcattaatct tgaatacgtt tgatgagttg
660
gaagctccat ttctgactca tctttctagt atttttaata agatttatac aattggtcct 720
ttgcatgcct tatctaagtc aaggt agga gactcctcat ctagtgctag tgcacttagt
780
ggattc gga aggaagatag ggcttgtatg tcttggttgg attgtcaacc tcctagatct 840
gttgttttcg tctcttttgg cagtactatg aaaatgaagg cggacgaact aagagaattt 900
tggtatggat tagtatcttc aggaaaacca tttttatgcg ttttaagatc cgatgtagtc
960
tcaggcggag aagctgcgga gttaattgaa caaatggcag aagaggaagg tgccgggggt 1020
aagttgggca tggttgttga atgggcagct caggagaagg tacttagcca tccagcggtt
1080
ggtggatttt tgacgcattg cgggtggaat agcactgtgg aaagtatagc agcaggggtc
1140
ccgatgatgt gttggccaat cttgggagat caaccatcca acgcgacctg gatcgataga 1200
gtttggaaaa tcggtgtaga aagaaataat agagaatggg atagattaac tgttgaaaaa
1260
atggttagag ccttgatgga aggacagaaa agagttgaaa ttcagcgttc aatggaaaag 1320
ctatcaaagt tggccaatga aaaagtagtt agggggggtc tttcatttga taatcttgaa 1380
gttcttgtcg .gttaaag ccgtacaagt tttaa
1425
SEQ ID NO: 86
Artificial Sequence,- Codon-optimized nucleotide sequence encoding CYP1798 atggaaatgt cctcttctgt tgctgccacc atttctattt ggatggttgt tgtatgtatc 60
gttggtgttg gttggagagt tgttaattgg gtttggttaa gaccaaagaa gttggaaaag
120
agattgagag aacaaggttt ggctggtaac tcttacagat tgttgttcgg tgacttgaaa 180
gaaagagctg ctatggaaga acaagctaac tctaagccaa tcaacttctc ccatgatatt
240
ggtccaagag ttttcccatc tatgtacaag accattcaaa actacggtaa gaactcctat 300 atgtggttgg gtccataccc aagagttcat attatggatc cacaacaatt gaaaaccgtc
350
tttaccttgg tttacgacat ccaaaagcca aacttgaacc cattgatcaa gttcttgttg 420
gatggtattg tcacccatga aggtgaaaaa tgggctaaac atagaaagat tatcaaccca 480
gccttccact tggaaaag t gaaagatatg attccagcct tcttccactc ttgcaacgaa 540
atagttaatg aatgggaaag attgatctcc aaagaaggtt cttgcgaatt ggatgttatg 600
ccatacttgc aaaatttggc tgctgatgct atttctagaa ctgcttttgg ttcctcttac 660
gaagaaggta agatgatctt ccaattattg aaagaattga ccgacttggt tgttaaggtt 720
gctttcggtg tttacattcc aggttggaga tttttgccaa ctaagtccaa caacaagatg 780
aaggaaatca acagaaagat caagtctttg ttgttaggta tcatcaacaa gagacaaaag 840
gccatggaag aaggtgaagc tggtcaatct gatttgttgg gtattttgat ggaatccaac 900
tccaacgaaa ttcaaggtga aggtaacaac aaagaagatg gtatgtccat cgaagatgtt 960
atcgaagaat gcaaggtttt ctacatcggt ggtcaagaaa ctaccgccag attattgatt 1020
tggaccatga tcttgttgag ttcccatact gaatggcaag aaagagcaag aactgaagtc 1080
ttgaaggttt tcggtaacaa aaagccagat ttcgacggtt tgtctagatt gaaggttgtc 1140
accatgattt tgaacgaagt tttgagatta tacccaccag cttctatgtt gaccagaatc 1200
attcaaaaag aaaccagagt cggtaagttg actttgccag ctggtgttat tttgatcatg 1260
ccaatcatct tgatccacag agatcatgat ttgtggggtg aagatgctaa tgaattcaag 1320
ccagaaagat tctccaaggg tgtttctaaa gctgctaaag ttcaaccagc tttctttcca 1380
tttggttggg gtccaagaat atgtatgggt caaaatttcg ctatgatcga agctaagatg 1440
gccttgtctt tgatcttgca aagattttcc ttcgaattgt cctcctcata tgttcatgct 1500
ccaactgttg ttttcaccac tcaaccacaa catggtgctc atatcgtttt gagaaagttg
1560
taa
1563
SEQ ID NO: 87
Saccharomyces cerevisiae protein sequence
Met Gly Lys Leu Leu Gin Leu Ala Leu His Pro Val Glu Met Lys Ala 1 5 10 15
Ala Leu Lys Leu Lys Phe Cys Arg Thr Pro Leu Phe Ser lie Tyr Asp
20 25 30
Gin Ser Thr Ser Pro Tyr Leu Leu His Cys Phe Glu Leu Leu Asn Leu
35 40 45
Thr Ser Arg Ser Phe Ala Ala Val lie Arg Glu Leu His Pro Glu Leu 50 55 60
Arg Asn Cys Val Thr Leu Phe Tyr Leu lie Leu Arg Ala Leu Asp Thr 65 70 75 80 lie Glu Asp Asp Met Ser lie Glu His Asp Leu Lys lie Asp Leu Leu
85 90 95
Arg His Phe His Glu Lys Leu Leu Leu Thr Lys Trp Ser Phe Asp Gly
100 105 110
Asn Ala Pro Asp Val Lys Asp Arg Ala Val Leu Thr Asp Phe Glu Ser
115 120 125
lie Leu lie Glu Phe His Lys Leu Lys Pro Glu Tyr Gin Glu Val lie
130 135 140
Lys Glu lie Thr Glu Lys Met Gly Asn Gly Met Ala Asp Tyr lie Leu 145 150 155 160
Asp Glu Asn Tyr Asn Leu Asn Gly Leu Gin Thr Val His Asp Tyr Asp
165 170 175
Val Tyr Cys His Tyr Val Ala Gly Leu Val Gly Asp Gly Leu Thr Arg
180 185 190
Leu lie Val lie Ala Lys Phe Ala Asn Glu Ser Leu Tyr Ser Asn Glu
195 200 205
Gin Leu Tyr Glu Ser Met Gly Leu Phe Leu Gin Lys Thr Asn lie lie
210 215 220
Arg Asp Tyr Asn Glu Asp Leu Val Asp Gly Arg Ser Phe Trp Pro Lys
225 230 235 240
Glu lie Trp Ser Gin Tyr Ala Pro Gin Leu Lys Asp Phe Met Lys Pro
245 250 255
Glu Asn Glu Gin Leu Gly Leu Asp Cys lie Asn His Leu Val Leu Asn
260 265 270
Ala Leu Ser His Val lie Asp Val Leu Thr Tyr Leu Ala Gly lie His 275 280 285
Glu Gin Ser Thr Phe Gin Phe Cys Ala He Pro Gin Val Met Ala He
290 295 300
Ala Thr Leu Ala Leu Val Phe Asn Asn Arg Glu Val Leu His Gly Asn
305 310 315 320
Val Lys lie Arg Lys Gly Thr Thr Cys Tyr Leu He Leu Lys Ser Arg
325 330 335
Thr Leu Arg Gly Cys Val Glu He Phe Asp Tyr Tyr Leu Arg As He
340 345 350
Lys Ser Lys Leu Ala Val Gin Asp Pro Asn Phe Leu Lys Leu Asn He
355 360 365
Gin lie Ser Lys He Glu Gin Phe Met Glu Glu Met Tyr Gin Asp Lys 370 375 380
Leu Pro Pro Asn Val Lys Pro Asn Glu Thr Pro He Phe Leu Lys Val 385 390 395 400
Lys Glu Arg Ser Arg Tyr Asp Asp Glu Leu Val Pro Thr Gin Gin Glu
405 410 415
Glu Glu Tyr Lys Phe Asn Met Val Leu Ser He He Leu Ser Val Leu
420 425 430
Leu Gly Phe Tyr Tyr He Tyr Thr Leu His Arg Ala
435 440
SEQ ID NO: 88
Gynostemma pentaphyllum Squalene epoxidase protein sequence
Met Val Asp Gin Phe Ser Leu Ala Phe He Phe Ala Ser Val Leu Gly 1 5 10 15
Ala Val Ala Phe Tyr Tyr Leu Phe Leu Arg Asn Arg He Phe Arg Val
20 25 30
Ser Arg Glu Pro Arg Arg Glu Ser Leu Lys Asn He Ala Thr Thr Asn
35 40 45
Gly Glu Cys Lys Ser Ser Tyr Ser Asp Gly Asp He He He Val Gly 50 55 SO
Ala Gly V l Ala Gly Ser Ala Leu Ala Tyr Thr Leu Gly Lys As Gly 65 70 75 80 Arg Arg Val His Val lie Glu Arg Asp Leu Thr Glu Pro Asp Arg Thr 85 90 95
Val Gly Glu Leu Leu Gin Pro Gly Gly Tyr Leu Lys Leu Thr Glu Leu
100 105 110
Gly Leu Glu Asp Cys Val Asn Glu lie Asp Ala Gin Arg Val Tyr Gly
115 120 125
Tyr Ala Leu Phe Lys Asp Gly Lys Asp Thr Lys Leu Ser Tyr Pro Leu 130 135 140
Glu Lys Phe His Ser Asp Val Ser Gly Arg Ser Phe His Asn Gly Arg 145 150 155 160
Phe lie Gin Arg Met Arg Glu Lys Ala Ala Thr Leu Pro Asn Val Arg
165 170 175
Leu Glu Gin Gly Thr Val Thr Ser Leu Leu Glu Glu Asn Gly lie lie
180 185 190
Lys Gly Val Gin Tyr Lys Ser Lys Thr Gly Gin Glu Met Thr Ala Tyr
195 200 205
Ala Pro Leu Thr lie Val Cys Asp Gly Cys Phe Ser Asn Leu Arg Arg
210 215 220
Ser Leu Cys Asn Pro Lys Val Asp Val Pro Ser Cys Phe Val Ala Leu
225 230 235 240
Val Leu Glu Asn Cys Glu Leu Pro His Ala Asn Tyr Gly His Val Tie
245 250 255
Leu Ala Asp Pro Ser Pro lie Leu Phe Tyr Pro lie Ser Ser Thr Glu
260 265 270
Val Arg Cys Leu Val Asp Val Pro Gly Gin Lys Val Pro Ser lie Ser
275 280 285
Asn Gly Glu Met Ala Asn Tyr Leu Lys Ser Val Val Ala Pro Gin lie
290 295 300
Pro Pro Gin lie Tyr Asp Ala Leu Arg Ser Cys Tyr Asp Lys Gly Asn
305 310 315 320 lie Arg Thr Met Pro Asn Arg Ser Met Pro Ala Asp Pro Tyr Pro Thr
325 330 335
Pro Gly Ala Leu Leu Met Gly Asp Ala P e Asn Met Arg His Pro Leu
340 345 350 Thr Gly Gly Gly Met Thr Val Ala Leu Ser Asp lie Val Val Leu Arg 355 360 365
Asp Leu Leu Lys Pro Leu Arg Asp Leu His Asp Ala Pro He Leu Ser 370 375 380
Asn Tyr Leu Glu Ala Phe Tyr Thr Leu Arg Lys Pro Val Ala Ser Thr
385 390 395 400 lie Asn Thr Leu Ala Gly Ala Leu Tyr Lys Val Phe Cys Ala Ser Pro
405 410 415
Asp Gin Ala Arg Arg Glu Met Arg Gin Ala Cys Phe Asp Tyr Leu Ser
420 425 430
Leu Gly Gly Val Phe Ser Asn Gly Pro Val Ser Leu Leu Ser Gly Leu
435 440 445
Asn Pro Arg Pro Leu Ser Leu Val Leu His Phe Phe Ala Val Ala He 450 455 460
Tyr Gly Val Gly Arg Leu Leu lie Pro Phe Pro Ser Pro Arg Arg Val 465 470 475 480
Trp lie Gly Ala Arg Leu lie Ser Gly Ala Ser Gly He He Phe Pro
485 430 495 lie lie Lys Ala Glu Gly Val Arg Gin He Phe Phe Pro Ala Thr Leu
500 505 510
Pro Ala Tyr Tyr Arg Ala Pro Pro Leu Val Arg Gly Arg
515 520 525
SEQ ID NO : 89
Arabidopsis thaliana Squalene epoxidase 1 protein sequence
Met Glu Ser Gin Leu Trp Asn Trp He Leu Pro Leu Leu He Ser Ser 1 5 10 15
Leu Leu He Ser Phe Val Ala Phe Tyr Gly Phe Phe Val Lys Pro Lys
20 25 30
Arg Asn Gly Leu Arg Hi s Asp Arg Lys Thr Val Ser Thr Val Thr Ser
35 40 45
Asp Val Gly Ser Val Asn He Thr Gly Asp Thr Val Ala Asp Val He 50 55 60 Val Val Gly Ala Gly Val Ala Gly Ser Ala Leu Ala Tyr Thr Leu Gly 65 70 75 80
Lys Asp Lys Arg Arg Val His Val lie Glu Arg Asp Leu Ser Glu Pro
85 90 95
Asp Arg lie Val Gly Glu Leu Leu Gin Pro Gly Gly Tyr Leu Lys Leu
100 105 110
Leu Glu Leu Gly He Glu Asp Cys Val Glu Glu lie Asp Ala Gin Arg
115 120 125
Val Tyr Gly Tyr Ala Leu Phe Lys Asn Gly Lys Arg lie Arg Leu Ala
130 135 140
Tyr Pro Leu Glu Lys Phe His Glu Asp Val Ser Gly Arg Ser Phe His 145 150 155 160
Asn Gly Arg Phe He Gin Arg Met Arg Glu Lys Ala Ala Ser Leu Pro
1S5 170 175
Asn Val Gin Leu Glu Gin Gly Thr Val Leu Ser Leu Leu Glu Glu Asn
180 185 190
Gly Thr He Lys Gly Val Arg Tyr Lys Asn Lys Ala Gly Glu Glu Gin
195 200 205
Thr Ala Phe Ala Ala Leu Thr He Val Cys Asp Gly Cys Phe Ser Asn
210 215 220
Leu Arg Arg Ser Leu Cys Asn Pro Gin Val Glu Val Pro Ser Cys Phe 225 230 235 240
Val Gly Leu Val Leu Glu Asn Cys Asn Leu Pro Tyr Ala Asn His Gly
245 250 255
His Val Val Leu Ala Asp Pro Ser Pro He Leu Met Tyr Pro He Ser
260 265 270
Ser Thr Glu Val Arg Cys Leu Val Asp Val Pro Gly Gin Lys Val Pro
275 280 285
Ser He Ala Asn Gly Glu Met Lys Asn Tyr Leu Lys Thr Val Val Ala
290 295 300
Pro Gin Met Pro His Glu Val Tyr Asp Ser Phe He Ala Ala Val Asp 305 310 315 320
Lys Gly Asn lie Lys Ser Met Pro Asn Arg Ser Met Pro Ala Ser Pro
325 330 335 Tyr Pro Thr Pro Gly Ala Leu Leu Met Gly Asp Ala Phe Asn Met Arg 340 345 350
His Pro Leu Thr Gly Gly Gly Met Thr Val Ala Leu Ala Asp He Val
355 360 365
Val Leu Arg Asn Leu Leu Arg Pro Leu Arg Asp Leu Ser Asp Gly Ala 370 375 380
Ser Leu Cys Lys Tyr Leu Glu Ser Phe Tyr Thr Leu Arg Lys Pro Val
385 390 395 400
Ala Ala Thr He Asn Thr Leu Ala Asn Ala Leu Tyr Gin Val Phe Cys
405 410 415
Ser Ser Glu Asn Glu Ala Arg Asn Glu Met Arg Glu Ala Cys Phe Asp
420 425 430
Tyr Leu Gly Leu Gly Gly Met Cys Thr Ser Gly Pro Val Ser Leu Leu
435 440 445
Ser Gly Leu Asn Pro Arg Pro Leu Thr Leu Val Cys His Phe Phe Ala
450 455 460
Val Ala Val Tyr Gly Val He Arg Leu Leu He Pro Phe Pro Ser Pro
465 470 475 480
Lys Arg He Trp Leu Gly Ala Lys Leu He Ser Gly Ala Ser Gly He
485 490 495
He Phe Pro He He Lys Ala Glu Gly Val Arg Gin Met Phe Phe Pro
500 505 510
Ala Thr Val Pro Ala Tyr Tyr Tyr Lys Ala Pro Thr Val Gly Glu Thr
515 520 525
Lys Cys Ser
530
SEQ ID NO: 90
Arabidopsis thai iana Squalene epoxidase 4 protein sequence
Met Thr Tyr Ala Trp Leu Trp Thr Leu Leu Ala Phe Val Leu Thr Trp 1 5 10 15
Met Val Phe His Leu He Lys Met Lys Lys Ala Ala Thr Gly Asp Leu
20 25 30
Glu Ala Glu Ala Glu Ala Arg Arg Asp Gly Ala Thr Asp Val He He 35 40 45
Val Gly Ala Gly Val Ala Gly Ala Ser Leu Ala Tyr Ala Leu Ala Lys 50 55 60
Asp Gly Arg Arg Val His Val lie Glu Arg Asp Leu Lys Glu Pro Gin
65 70 75 80
Arg Phe Met Gly Glu Leu Met Gin Ala Gly Gly Arg Phe Met Leu Ala
85 90 95
Gin Leu Gly Leu Glu Asp Cys Leu Glu Asp lie Asp Ala Gin Glu Ala
100 105 110
Lys Ser Leu Ala lie Tyr Lys Asp Gly Lys His Ala Thr Leu Pro Phe
115 120 125
Pro Asp Asp Lys Ser Phe Pro His Glu Pro Val Gly Arg Leu Leu Arg 130 135 140
Asn Gly Arg Leu Val Gin Arg Leu Arg Gin Lys Ala Ala Ser Leu Ser 145 150 155 160
Asn Val Gin Leu Glu Glu Gly Thr Val Lys Ser Leu lie Glu Glu Glu
165 170 175
Gly Val Val Lys Gly Val Thr Tyr Lys Asn Ser Ala Gly Glu Glu lie
180 185 190
Thr Ala Phe Ala Pro Leu Thr Val Val Cys Asp Gly Cys Tyr Ser Asn
195 200 205
Leu Arg Arg Ser Leu Val. Asp Asn Thr Glu Glu Val Leu Ser Tyr Met 210 215 220
Val Gly Tyr Val Thr Lys Asn Ser Arg Leu Glu Asp Pro His Ser Leu 225 230 235 240
His Leu lie Phe Ser Lys Pro Leu Val Cys Val lie Tyr Gin lie Thr
245 250 255
Ser Asp Glu Val Arg Cys Val Ala Glu Val Pro Ala Asp Ser lie Pro
260 265 270
Ser lie Ser Asn Gly Glu Met Ser Thr Phe Leu Lys Lys Ser Met Ala
275 280 285
Pro Gin lie Pro Glu Thr Gly Asn Leu Arg Glu lie Phe Leu Lys Gly
290 295 300
lie Glu Glu Gly Leu Pro Glu lie Lys Ser Thr Ala Thr Lys Ser Met 305 310 315 320
Ser Ser Arg Leu Cys Asp Lys Arg Gly Val lie Val Leu Gly Asp Ala
325 330 335
Phe Asn Met Arg His Pro lie lie Ala Ser Gly Me Met Val Ala Leu
340 345 350
Ser Asp lie Cys lie Leu Arg Asn Leu Leu Lys Pro Leu Pro Asn Leu
355 360 365
Ser Asn Thr Lys Lys Val Ser Asp Leu Val Lys Ser Phe Tyr lie lie
370 375 380
Arg Lys Pro Met Ser Ala Thr Val Asn Thr Leu Ala Ser lie Phe Ser 385 390 395 400
Gin Val Leu Val Ala Thr Thr Asp Glu Ala Arg Glu Gly Met Arg Gin
405 410 415
Gly Cys Phe Asn Tyr Leu Ala Arg Gly Asp Phe Lys Thr Arg Gly Leu
420 425 430
Met Thr lie Leu Gly Gly Met Asn Pro His Pro Leu Thr Leu Val Leu
435 440 445
His Leu Val Ala lie Thr Leu Thr Ser Met Gly His Leu Leu Ser Pro 450 455 460
Phe Pro Ser Pro Arg Arg Phe Trp His Ser Leu Arg lie Leu Ala Trp
465 470 475 480
Ala Leu Gin Met Leu Gly Ala His Leu Val Asp Glu Gly Phe Lys Glu
485 490 495
Met Leu lie Pro Thr Asn Ala Ala Ala Tyr Arg Arg Asn Tyr lie Ala
500 505 510
Thr Thr Thr Val
515
SEQ ID NO: 91
Arabidopsis thaliana Squalene epoxidase 6 protein sequence
Met Ala Phe Thr His Val Cys Leu Trp Thr Leu Val Ala Phe Val Leu
1 5 10 15
Thr Trp Thr Val Phe Tyr Leu Thr Asn Met Lys Lys Lys Ala Thr Asp 20 25 30
Leu Ala Asp Thr Val Ala Glu Asp Gin Lys Asp Gly Ala Ala Asp Val
35 40 45
lie lie Val Gly Ala Gly Val Gly Gly Ser Ala Leu Ala Tyr Ala Leu 50 55 60
Ala Lys Asp Gly Arg Arg Val His Val lie Glu Arg Asp Met Arg Glu 65 70 75 80
Pro Glu Arg Met Met Gly Glu Phe Met Gin Pro Gly Gly Arg Leu Met
85 90 95
Leu Ser Lys Leu Gly Leu Gin Asp Cys Leu Glu Asp lie Asp Ala Gin
100 105 110
Lys Ala Thr Gly Leu Ala Val Tyr Lys Asp Gly Lys Glu Ala Asp Ala
115 120 125
Pro Phe Pro Val Asp Asn Asn Asn Phe Ser Tyr Glu Pro Ser Ala Arg
130 135 140
Ser Phe His Asn Gly Arg Phe Val Gin Gin Leu Arg Arg Lys Ala Phe 145 150 155 160
Ser Leu Ser Asn Val Arg Leu Glu Glu Gly Thr Val Lys Ser Leu Leu
165 170 175
Glu Glu Lys Gly Val Val Lys Gly Val Thr Tyr Lys Asn Lys Glu Gly
180 185 190
Glu Glu Thr Thr Ala Leu Ala Pro Leu Thr Val Val Cys Asp Gly Cys
195 200 205
Tyr Ser Asn Leu Arg Arg Ser Leu Asn Asp Asp Asn Asn Ala Glu lie 210 215 220
Met Ser Tyr lie Val Gly Tyr lie Ser Lys Asn Cys Arg Leu Glu Glu
225 230 235 240
Pro Glu Lys Leu His Leu lie Leu Ser Lys Pro Ser Phe Thr Met Val
245 250 255
Tyr Gin lie Ser Ser Thr Asp Val Arg Cys Gly Phe Glu Val Leu Pro
260 265 270
Glu Asn Phe Pro Ser lie Ala Asn Gly Glu Met Ser Thr Phe Met Lys
275 280 285
Asn Thr lie Val Pro Gin Val Pro Pro Lys Leu Arg Lys lie Phe Leu 290 295 300
Lys Gly lie Asp Glu Gly Ala His lie Lys Val Val Pro Ala Lys Arg 305 310 315 320
Met Thr Ser Thr Leu Ser Lys Lys Lys Gly Val lie Val Leu Gly Asp
325 330 335
Ala Phe Asn Met Arg His Pro Val Val Ala Ser Gly Met Met Val Leu
340 345 350
Leu Ser Asp lie Leu lie Leu Arg Arg Leu Leu Gin Pro Leu Ser Asn
355 360 365
Leu Gly Asp Ala Asn Lys Val Ser Glu Val lie Asn Ser Phe Tyr Asp 370 375 380
lie Arg Lys Pro Met Ser Al Thr Val Asn Thr Leu Gly Asn Ala Phe 385 390 395 400
Ser Gin Val Leu lie Gly Ser Thr Asp Glu Ala Lys Glu Ala Met Arg
405 410 415
Gin Gly Val Tyr Asp Tyr Leu Cys Ser Gly Gly Phe Arg Thr Ser Gly
420 425 430
Met Me Ala Leu Leu Gly Gly Met Asn Pro Arg Pro Leu Ser Leu Val
435 440 445
Tyr His Leu Cys Ala lie Thr Leu Ser Ser lie Gly Gin Leu Leu Ser
450 455 460
Pro Phe Pro Ser Pro Leu Arg lie Trp His Ser Leu Lys Leu Phe Gly 465 470 475 480
Leu Ala Met Lys Met Leu Val Pro Asn Leu Lys Ala Glu Gly Val Ser
485 490 495
Gin Met Leu Phe Pro Ala Asn Ala Ala Ala Tyr His Lys Ser Tyr Met
500 505 510
Ala Ala Thr Thr Leu
515
SEQ ID NO: 92
Arabidopsis thaliana Squalene epoxidase 5 protein sequence
Met Ala Phe Thr Asn Val Cys Leu Trp Thr Leu Leu Ala Phe Met Leu
1 5 10 15 Thr Trp Thr Val Phe Tyr Val Thr Asn Arg Gly Lys Lys Ala Thr Gin 20 25 30
Leu Ala Asp Ala Val Val Glu Glu Arg Glu Asp Gly Ala Thr Asp Val
35 40 45
He He Val Gly Ala Gly Val Gly Gly Ser Ala Leu Ala Tyr Ala Leu
50 55 60
Ala Lys Asp Gly Arg Arg Val His Val He Glu Arg Asp Leu Arg Glu 65 70 75 80
Pro Glu Arg He Met Gly Glu Phe Met Gin Pro Gly Gly Arg Leu Met
85 90 95
Leu Ser Lys Leu Gly Leu Glu Asp Cys Leu Glu Gly He Asp Ala Gin
100 105 110
Lys Ala Thr Gly Met Thr Val Tyr Lys Asp Gly Lys Glu Ala Val Ala
115 120 125
Ser Phe Pro Val Asp Asn Asn Asn Phe Pro Phe Asp Pro Ser Ala Arg 130 135 140
Ser Phe His Asn Gly Arg Phe Val Gin Arg Leu Arg Gin Lys Ala Ser 145 150 155 160
Ser Leu Pro Asn Val Arg Leu Glu Glu Gly Thr Val Lys Ser Leu He
165 170 175
Glu Glu Lys Gly Val He Lys Gly Val Thr Tyr Lys Asn Ser Ala Gly
180 185 190
Glu Glu Thr Thr Ala Leu Ala Pro Leu Thr Val Val Cys Asp Gly Cys
195 200 205
Tyr Ser Asn Leu Arg Arg Ser Leu Asn Asp Asn Asn Ala Glu Val Leu
210 215 220
Ser Tyr Gin Val Gly Phe He Ser Lys Asn Cys Gin Leu Glu Glu Pro
225 230 235 240
Glu Lys Leu Lys Leu He Met Ser Lys Pro Ser Phe Thr Met Leu Tyr
245 250 255
Gin He Ser Ser Thr Asp Val Arg Cys Val Phe Glu Val Leu Pro Asn
260 265 270
Asn He Pro Ser He Ser Asn Gly Glu Met Ala Thr Phe Val Lys Asn
275 280 285 Thr lie Ala Pro Gin Val Pro Leu Lys Leu Arg Lys He Phe Leu Lys
290 295 300
Gly lie Asp Glu Gly Glu His lie Lys Ala Met Pro Thr Lys Lys Met
305 310 315 320
Thr Ala Thr Leu Ser Glu Lys Lys Gly Val He Leu Leu Gly Asp Ala
325 330 335
Phe Asn Met Arg His Pro Ala He Ala Ser Gly Met Met Val Leu Leu
340 345 350
Ser Asp lie Leu lie Leu Arg Arg Leu Leu Gin Pro Leu Ser Asn Leu
355 360 365
Gly Asn Ala Gin Lys He Ser Gin Val He Lys Ser Phe Tyr Asp He 370 375 380
Arg Lys Pro Met Ser Ala Thr Val Asn Thr Leu Gly Asn Ala Phe Ser 385 390 395 400
Gin Val Leu Val Ala Ser Thr Asp Glu Ala Lys Glu Ala Met Arg Gin
405 410 415
Gly Cys Tyr Asp Tyr Leu Ser Ser Gly Gly Phe Arg Thr Ser Gly Met
420 425 430
Met Ala Leu Leu Gly Gly Me Asn Pro Arg Pro He Ser Leu He Tyr
435 440 445
His Leu Cys Ala He Thr Leu Ser Ser He Gly His Leu Leu Ser Pro 450 455 460
Phe Pro Ser Pro Leu Arg He Trp His Ser Leu Arg Leu Phe Gly Leu 465 470 475 480
Ala Met Lys Me Leu Val Pro His Leu Lys Ala Glu Gly Val Ser Gin
485 490 495
Met Leu Phe Pro Val Asn Ala Ala Ala Tyr Ser Lys Ser Tyr Met Ala
500 505 510
Ala Thr Ala Leu
515
SEQ ID NO: 93
Arabidopsis thaliana Squalene epoxidase 2 protein sequence Met Lys Pro Phe Val lie Arg Asn Leu Pro Arg Phe Gin Ser Thr Leu 1 5 10 15
Arg Ser Ser Leu Leu Tyr Thr Asn His Arg Pro Ser Ser Arg Phe Ser
20 25 30
Leu Ser Thr Arg Arg Phe Thr Thr Gly Ala Thr Tyr lie Arg Arg Trp
35 40 45
Lys Ala Thr Ala Ala Gin Thr Leu Lys Leu Ser Ala V l Asn Ser Thr
50 55 60
Val Met Met Lys Pro Ala Lys lie Ala Leu Asp Gin Phe lie Ala Ser 65 70 75 80
Leu Phe Thr Phe Leu Leu Leu Tyr lie Leu Arg Arg Ser Ser Asn Lys
85 90 95
Asn Lys Lys Asn Arg Gly Leu Val Val Ser Gin Asn Asp Thr Val Ser
100 105 110
Lys Asn Leu Glu Thr Glu Val Asp Ser Gly Thr Asp Val lie lie Val
115 120 125
Gly Ala Gly Val Ala Gly Ser Ala Leu Ala His Thr Leu Gly Lys Glu 130 135 140
Gly Arg Arg Val His Val Tie Glu Arg Asp Phe Ser Glu Gin Asp Arg
145 150 155 160 lie Val Gly Glu Leu Leu Gin Pro Gly Gly Tyr Leu Lys Leu lie Glu
165 170 175
Leu Gly Leu Glu Asp Cys Val Lys Lys lie Asp Ala Gin Arg Val Leu
180 185 190
Gly Tyr Val Leu Phe Lys Asp Gly Lys His Thr Lys Leu Ala Tyr Pro
135 200 205
Leu Glu Thr Phe Asp Ser Asp Val Ala Gly Arg Ser Phe His Asn Gly
210 215 220
Arg Phe Val Gin Arg Met Arg Glu Lys Ala Leu Thr Leu Ser Asn Val 225 230 235 240
Arg Leu Glu Gin Gly Thr Val Thr Ser Leu Leu Glu Glu His Gly Thr
245 250 255 lie Lys Gly Val Arg Tyr Arg Thr Lys Glu Gly Asn Glu Phe Arg Ser
260 265 270 Phe Ala Pro Leu Thr He Val Cys Asp Gly Cys Phe Ser Asn Leu Arg
275 280 285
Arg Ser Leu Cys Lys Pro Lys Val Asp Val Pro Ser Thr Phe Val Gly 290 295 300
Leu Val Leu Glu Asn Cys Glu Leu Pro Phe Ala Asn His Gly His Val 305 310 315 320
Val Leu Gly Asp Pro Ser Pro He Leu Met Tyr Pro He Ser Ser Ser
325 330 335
Glu Val Arg Cys Leu Val Asp Val Pro Gly Gin Lys Leu Pro Pro He
340 345 350
Ala Asn Gly Glu Met Ala Lys Tyr Leu Lys Thr Arg Val Ala Pro Gin
355 360 365
Val Pro Thr Lys Val Arg Glu Ala Phe He Thr Ala Val Glu Lys Gly
370 375 380
Asn He Arg Thr Met Pro Asn Arg Ser Met Pro Ala Asp Pro He Pro
385 390 395 400
Thr Pro Gly Ala Leu Leu Leu Gly Asp Ala Phe Asn Met Arg His Pro
405 410 415
Leu Thr Gly Gly Gly Met Thr Val Ala Leu Ala Asp He Val Val Leu
420 425 430
A g Asp Leu Leu Arg Pro He Arg Asn Leu Asn Asp Lys Glu Ala Leu
435 440 445
Ser Lys Tyr He Glu Ser Phe Tyr Thr Leu Arg Lys Pro Val Ala Ser
450 455 460
Thr He Asn Thr Leu Ala Asp Ala Leu Tyr Lys Val Phe Leu Ala Ser
465 470 475 480
Ser Asp Glu Ala Arg Thr Glu Met Arg Glu Ala Cys Phe Asp Tyr Leu
485 490 495
Ser Leu Gly Gly Val Phe Ser Ser Gly Pro Val Ala Leu Leu Ser Gly
500 505 510
Leu Asn Pro Arg Pro Leu Ser Leu Val Leu His Phe Phe Ala Val Ala
515 520 525
He Tyr Ala Val Cys Arg Leu Met Leu Pro Phe Pro Ser He Glu Ser
530 535 540 Phe Trp Leu Gly Ala Arg lie lie Ser Ser Ala Ser Ser l ie He Phe
545 550 555 560
Pro l ie lie Lys Ala Glu Gly Val Arg Gin Met Phe Phe Pro Arg Thr
565 570 575
Pro Ala lie Tyr Arg Ala Pro Pro
580 585
SEQ ID NO : 94
Arabidopsis th i iana Squalene epoxidase 3 protein sequence
Met Ala Pro Thr He Phe Val Asp His Cys He Leu Thr Thr Thr Phe
1 5 10 15
Val Ala Ser Leu Phe Ala Phe Leu Leu Leu Tyr Val Leu Arg Arg Arg
20 25 30
Ser Lys Thr He His Gly Ser Val Asn Val Arg Asn Gly Thr Leu Thr
35 40 45
Val Lys Ser Gly Thr Asp Val Asp He He He Val Gly Ala Gly Val 50 55 60
Ala Gly Ala Ala Leu Ala His Thr Leu Gly Lys Glu Gly Arg Arg Val 65 70 75 80
His Val He Glu Arg Asp Leu Thr Glu Pro Asp Arg He Val Gly Glu
85 90 95
Leu Leu Gin Pro Gly Gly Tyr Leu Lys Leu He Glu Leu Gly Leu Glu
100 105 110
Asp Cys Val Lys Asp He Asp Ala Gin Arg Val Leu Gly Tyr Ala Leu
115 120 125
Phe Lys Asp Gly Lys His Thr Lys Leu Ser Tyr Pro Leu Asp Gin Phe 130 135 140
Asp Ser Asp Val Ala Gly Arg Ser Phe His Asn Gly Arg Phe Val Gin 145 150 155 160
Arg Met Arg Glu Lys Ala Ser Leu Leu Pro Asn Val Arg Met Glu Gin
1S5 170 175
Gly Thr Val Thr Ser Leu Val Glu Glu Asn Gly He He Lys Gly Val
180 185 190 Gin Tyr Lys Thr Lys Asp Gly Gin Glu Leu Lys Ser Phe Ala Pro Leu
195 200 205
Thr lie Val Cys Asp Gly Cys Phe Ser Asn Leu Arg Arg Ser Leu Cys
210 215 220
Lys Pro Lys Val Glu Val Pro Ser Asn Phe Val Gly Leu Val Leu Glu 225 230 235 240
Asn Cys Glu Leu Pro Phe Pro Asn His Gly His Val Val Leu Gly Asp
245 250 255
Pro Ser Pro lie Leu Phe Tyr Pro He Ser Ser Ser Glu Val Arg Cys
260 265 270
Leu Val Asp Val Pro Gly Ser Lys Leu Pro Ser Val Ala Ser Gly Glu
275 280 285
Met Ala His His Leu Lys Thr Met Val Ala Pro Gin Val Pro Pro Gin
290 295 300
He Arg Asp Ala Phe He Ser Ala Val Glu Lys Gly Asn He Arg Thr 305 310 315 320
Met Pro Asn Arg Ser Met Pro Ala Asp Pro He His Thr Pro Gly Ala
325 330 335
Leu Leu Leu Gly Asp Ala Phe Asn Met Arg His Pro Leu Thr Gly Gly
340 345 350
Gly Met Thr Val Ala Leu Ser Asp He Val He Leu Arg Asp Leu Leu
355 360 365
Asn Pro Leu Val Asp Leu Thr Asn Lys Glu Ser Leu Ser Lys Tyr He
370 375 380
Glu Ser Phe Tyr Thr Leu Arg Lys Pro Val Ala Ser Thr He Asn Thr 385 390 395 400
Leu Ala Gly Ala Leu Tyr Lys Val Phe Leu Ala Ser Pro Asp Asp Ala
405 410 415
Arg Ser Glu Met Arg Arg Ala Cys Phe Asp Tyr Leu Ser Leu Gly Gly
420 425 430
Val Cys Ser Ser Gly Pro Val Ala Leu Leu Ser Gly Leu Asn Pro Arg
435 440 445
Pro Met Ser Leu Val Leu His Phe Phe Ala Val Ala He Phe Gly Val
450 455 460 Gly Arg Leu Leu Val Pro Leu Pro Ser Val Lys Arg Leu Trp Leu Gly
465 470 475 480
Ala Arg Leu lie Ser Ser Ala Ser Gly lie lie Phe Pro lie lie Lys
485 490 495
Ala Glu Gly Val Arg Gin Met Phe Phe Pro Arg Thr lie Pro Ala He
500 505 510
Tyr Arg Ala Pro Pro Thr Pro Ser Ser Ser Ser Pro Gin
515 520 525
SEQ ID NO: 95
Brassica napus Squalene raonooxygenase 1,1 protein sequence
Met Asp Leu Ala Phe Pro His Val Cys Leu Trp Thr Leu Leu Ala Phe 1 5 10 15
Val Leu Thr Trp Thr Val Phe Tyr Val Asn Asn Arg Arg Lys Lys Val
20 25 30
Ala Lys Leu Pro Asp Ala Ala Thr Glu Val Arg Arg Asp Gly Asp Ala
35 40 45
Asp Val He He Val Gly Ala Gly Val Gly Gly Ser Ala Leu Ala Tyr
50 55 60
Ala Leu Ala Lys Asp Gly Arg Arg Val His Val He Glu Arg Asp Met 65 70 75 80
Arg Glu Pro Val Arg Met Met Gly Glu Phe Met Gin Pro Gly Gly Arg
85 90 95
Leu Leu Leu Ser Lys Leu Gly Leu Glu Asp Cys Leu Glu Gly He Asp
100 105 110
Glu Gin He Ala Thr G y Leu Ala Val Tyr Lys Asp Gly Gin Lys Ala
115 120 125
Leu Val Ser Phe Pro Glu Asp Asn Asp Phe Pro Tyr Glu Pro Thr Gly 130 135 140
Arg Ala Phe Tyr Asn Gly Arg Phe Val Gin Arg Leu Arg Gin Lys Ala 145 150 155 160
Ser Ser Leu Pro Thr Val Gin Leu Glu Glu Gly Thr Val Lys Ser Leu
165 170 175 lie Glu Glu Lys Gly Val lie Lys Gly Val Thr Tyr Lys Asn Ser Ala
180 185 190
Gly Glu Glu Thr Thr Ala Phe Ala Pro Leu Thr Val Val Cys Asp Gly
195 200 205
Cys Tyr Ser Asn Leu Arg Arg Ser Val Asn Asp Asn Asn Ala Glu Val
210 215 220
He Ser Tyr Gin Val Gly Tyr Val Ser Lys Asn Cys Gin Leu Glu Asp 225 230 235 240
Pro Glu Lys Leu Lys Leu He Met Ser Lys Pro Ser Phe Thr Met Leu
245 250 255
Tyr Gin He Ser Ser Thr Asp Val Arg Cys Val Met Glu He Phe Pro
260 265 270
Gly Asn He Pro Ser He Ser Asn Gly Glu Met Ala Val Tyr Leu Lys
275 280 285
Asn Thr Met Ala Pro Gin Val Pro Pro Glu Leu Arg Lys He Phe Leu
290 295 300
Lys Gly He Asp Glu Gly Ala Gin He Lys Ala Met Pro Thr Lys Arg 305 310 315 320
Met Glu Ala Thr Leu Ser Glu Lys Gin Gly Val He Val Leu Gly Asp
325 330 335
Ala Phe Asn Met Arg His Pro Ala He Ala Ser Gly Met Met Val Val
340 345 350
Leu Ser Asp He Leu He Leu Arg Arg Leu Leu Gin Pro Leu Arg Asn
355 360 365
Leu Ser Asp Ala Asn Lys Val Ser Glu Val He Lys Ser Phe Tyr Val
370 375 380
He Arg Lys Pro Met Ser Ala Thr Val Asn Thr Leu Gly Asn Ala Phe
385 330 395 400
Ser Gin Val Leu He Ala Ser Thr Asp Glu Ala Lys Glu Ala Met Arg
405 410 415
Gin Gly Cys Phe Asp Tyr Leu Ser Ser Gly Gly Phe Arg Thr Ser Gly
420 425 430
Met Met Ala Leu Leu Gly Gly Met Asn Pro Arg Pro Leu Ser Leu He
435 440 445 Phe His Leu Cys Gly He Thr Leu Ser Ser He Gly Gin Leu Leu Ser 450 455 460
Pro Phe Pro Ser Pro Leu Gly He Trp His Ser Leu Arg Leu Phe Gly
465 470 475 480
Ala Glu Gly Val Ser Gin Met Leu Ser Pro Ala Tyr Ala Ala Ala Tyr
485 490 495
Arg Lys Ser Tyr Met Thr Ala Thr Ala Leu
500 505
SEQ ID NO: 96
Brassica napus Squal ne monooxygenase 1,2 protein sequence
Met Asp Met Ala Phe Val Glu Val Cys Leu Arg Met Leu Leu Val Phe
1 5 10 15
Val Leu Ser Trp Thr He Phe His Val Asn Asn Arg Lys Lys Lys Lys
20 25 30
Ala Thr Lys Leu Ala Asp Leu Ala Thr Glu Glu Arg Lys Glu Gly Gly
35 40 45
Pro Asp Val He He Val Gly Ala Gly Val Gly Gly Ser Ala Leu Ala 50 55 60
Tyr Ala Leu Ala Lys Asp Gly Arg Arg Val His Val He Glu Arg Asp 65 70 75 80
Met Arg Glu Pro Val Arg Met Met Gly Glu Phe Met Gin Pro Gly Gly
85 90 95
Arg Leu Met Leu Ser Lys Leu Gly Leu Gin Asp Cys Leu Glu Glu He
100 105 110
Asp Ala Gin Lys Ser Thr Gly He Arg Leu Phe Lys Asp Gly Lys Glu
115 120 125
Thr Val Ala Cys Phe Pro Val Asp Thr Asn Phe Pro Tyr Glu Pro Ser 130 135 140
Gly Arg Phe Phe His Asn Gly Arg Phe Val Gin Arg Leu Arg Gin Lys 145 150 155 160
Ala Ser Ser Leu Pro Asn Val Arg Leu Glu Glu Gly Thr Val Arg Ser
165 170 175
Leu He Glu Glu Lys Gly Val Val Lys Gly Val Thr Tyr Lys Asn Ser 180 185 190
Ser Gly Glu Glu Thr Thr Ser Phe Ala Pro Leu Thr Val Val Cys Asp
195 200 205
Gly Cys His Ser Asn Leu Arg Arg Ser Leu Asn Asp Asn Asn Ala Glu 210 215 220
Val Thr Ala Tyr Glu lie Gly Tyr lie Ser Arg Asn Cys Arg Leu Glu
225 230 235 240
Gin Pro Asp Lys Leu His Leu lie Met Ala Lys Pro Ser Phe Ala Met
245 250 255
Leu Tyr Gin Val Ser Ser Thr Asp Val Arg Cys Asn Phe Glu Leu Leu
260 265 270
Ser Lys Asn Leu Pro Ser Val Ser Asn Gly Glu Met Thr Ser Phe Val
275 280 285
Arg Asn Ser lie Ala Pro Gin Val Pro Leu Lys Leu Arg Lys Thr Phe 290 295 300
Leu Lys Gly Leu Asp Glu Gly Ser His lie Lys lie Thr Gin Ala Lys 305 310 315 320
Arg lie Pro Ala Thr Leu Ser Arg Lys Lys Gly Val lie Val Leu Gly
325 330 335
Asp Ala Phe Asn Met Arg His Pro Val lie Ala Ser Gly Met Met Val
340 345 350
Leu Leu Ser Asp lie Leu lie Leu Ser Arg Leu Leu Lys Pro Leu Gly
355 360 365
Asn Leu Gly Asp Glu Asn Lys Val Ser Glu Val Met Lys Ser Phe Tyr 370 375 380
Ala Leu Arg Lys Pro Met Ser Ala Thr Val Asn Thr Leu Gly Asn Ser
385 390 395 400
Phe Trp Gin Val Leu lie Ala Ser Thr Asp Glu Ala Lys Glu Ala Met
405 410 415
Arg Gin Gly Cys Phe Asp Tyr Leu Ser Ser Gly Gly Phe Arg Thr Ser
420 425 430
Gly Leu Met Ala Leu lie Gly Gly Met Asn Pro Arg Pro Leu Ser Leu
435 440 445
Phe Tyr His Leu Phe Val lie Ser Leu Ser Ser lie Gly Gin Leu Leu 450 455 460
Ser Pro Phe Pro Thr Pro Leu Arg Val Trp His Ser Leu Arg Leu Leu 465 470 475 480
Asp Leu Ser Leu Lys Met Leu Val Pro His Leu Lys Ala Glu Gly He
485 490 495
Gly Gin Met Leu Ser Pro Thr Asn Ala Ala Ala Tyr Arg Lys Ser Tyr
500 505 510
Met Ala Ala Thr Val Val
515
SEQ ID NO: 97
Euphorbia tirucalli Squalene epoxidase protein sequence
Met Glu Val lie Phe Asp Thr Tyr He Phe Gly Thr Phe Phe Ala Ser
1 5 10 15
Leu Cys Ala Phe Leu Leu Leu Phe He Leu Arg Pro Lys Val Lys Lys
20 25 30
Me Gly Lys lie Arg Glu lie Ser Ser He Asn Thr Gin Asn Asp Thr
35 40 45
Ala He Thr Pro Pro Lys Gly Ser Gly Thr Asp Val He He Val Gly
50 55 60
Ala Gly Val Ala Gly Ala Ala Leu Ala Cys Thr Leu Gly Lys Asp Gly 65 70 75 80
Arg Arg Val His Val He Glu Arg Asp Leu Lys Glu Pro Asp Arg He
85 90 95
Val Gly Glu Leu Leu Gin Pro Gly Gly Tyr Leu Lys Leu Val Glu Leu
100 105 110
Gly Leu Gin Asp Cys Val Glu Glu He Asp Ala Gin Arg He Val Gly
115 120 125
Tyr Ala Leu Phe Met Asp Gly Asn Asn Thr Lys Leu Ser Tyr Pro Leu 130 135 140
Glu Lys Phe Asp Ala Glu Val Ser Gly Lys Ser Phe His Asn Gly Arg 145 150 155 160
Phe He Gin Arg Met Arg Glu Lys Ala Ala Ser Leu Pro Asn Val Gin
165 170 175 Leu Glu Gin Gly Thr Val Thr Ser Leu Leu Glu Glu Asn Gly Thr lie 180 185 190
Lys Gly Val Gin Tyr Lys Thr Lys Asp Gly Gin Glu His Lys Ala Tyr
195 200 205
Ala Pro Leu Thr Val Val Cys Asp Gly Cys Phe Ser Asn Leu Arg Arg
210 215 220
Ser Leu Cys Lys Pro Lys Val Asp Val Pro Ser His Phe Val Gly Leu 225 230 235 240
Val Leu Glu Asn Cys Asp Leu Pro Phe Ala Asn His Gly His Val lie
245 250 255
Leu Ala Asp Pro Ser Pro lie Leu Phe Tyr Pro lie Ser Ser Thr Glu
260 265 270
Val Arg Cys Leu Val Asp Val Pro Gly Gin Lys Leu Pro Ser lie Ala
275 280 285
Ser Gly Glu Met Ala Lys Tyr Leu Lys Thr Met Val Ala Lys Gin lie
290 295 300
Pro Pro Val Leu His Asp Ala Phe Val Ser Ala lie Asp Lys Gly Asn 305 310 315 320 lie Arg Thr Met Pro Asn Arg Ser Met Pro Ala Asp Pro Leu Pro Thr
325 330 335
Pro Gly Ala Leu Leu Met Gly Asp Ala Phe Asn Met Arg His Pro Leu
340 345 350
Thr Gly Gly Gly Met Thr Val Ala Leu Ala Asp He Val Leu Leu Arg
355 360 365
Asp Leu Leu Lys Pro Leu Arg Asp Leu Asn Asp Ala Pro Ala Leu Ala
370 375 380
Lys Tyr Leu Glu Ser Phe Tyr Thr Leu Arg Lys Pro Val Ala Ser Thr
385 390 395 400
He Asn Thr Leu Ala Gly Ala Leu Tyr Lys Val Phe Ser Ala Ser Pro
405 410 415
Asp Glu Ala Arg Lys Glu Met Arg Gin Ala Cys Phe Asp Tyr Leu Ser
420 425 430
Leu Gly Gly Glu Cys Ala Met Gly Pro Val Ser Leu Leu Ser Gly Leu
435 440 445 Asn Pro Ser Pro Leu Thr Leu Val Leu His Phe Phe Gly Val Ala He 450 455 460
Tyr Gly Val Gly Arg Leu Leu He Pro Phe Pro Thr Pro Lys Gly Met 465 470 475 480
Trp He Gly Ala Arg He He Ser Ser Ala Ser Gly He He Phe Pro
485 490 495 lie He Lys Ala Glu Gly Val Arg Gin Val Phe Phe Pro Ala Thr Val
500 505 510
Pro Ala He Tyr Arg Asn Pro Pro Val Asn Gly Lys Ser Val Glu Val
515 520 525
Pro Lys Ser
530
SEQ ID NO: 98
Medicago truncatula Squalene epoxidase protein sequence
Met He Asp Pro Tyr Gly Phe Gly Trp He Thr Cys Thr Leu He Thr
1 5 10 15
Leu Ala Ala Leu Tyr Asn Phe Leu Phe Ser Arg Lys Asn His Ser Asp
20 25 30
Ser Thr Thr Thr Glu Asn He Thr Thr Ala Thr Gly Glu Cys Arg Ser
35 40 45
Phe Asn Pro Asn Gly Asp Val Asp He He He Val Gly Ala Gly Val 50 55 60
Ala Gly Ser Ala Leu Ala Tyr Thr Leu Gly Lys Asp Gly Arg Arg Val
65 70 75 80
Leu He He Glu Arg Asp Leu Asn Glu Pro Asp Arg He Val Gly Glu
85 90 95
Leu Leu Gin Pro Gly Gly Tyr Leu Lys Leu He Glu Leu Gly Leu Asp
100 105 110
Asp Cys Val Glu Lys He Asp Ala Gin Lys Val Phe Gly Tyr Ala Leu
115 120 125
Phe Lys Asp Gly Lys His Thr Arg Leu Ser Tyr Pro Leu Glu Lys Phe 130 135 140 His Ser Asp He Ala Gly Arg Ser Phe His Asn Gly Arg Phe He Leu
145 150 155 160
Arg Met Arg Glu Lys Ala Ala Ser Leu Pro Asn Val Arg Leu Glu Gin
165 170 175
Gly Thr Val Thr Ser Leu Leu Glu Glu Asn Gly Thr He Lys Gly Val
180 185 190
Gin Tyr Lys Thr Lys Asp Ala Gin Glu Phe Ser Ala Cys Ala Pro Leu
195 200 205
Thr He Val Cys Asp Gly Cys Phe Ser Asn Leu Arg Arg Ser Leu Cys
210 215 220
Asn Pro Lys Val Glu Val Pro Ser Cys Phe Val Gly Leu Val Leu Glu 225 230 235 240
Asn Cys Glu Leu Pro Cys Ala Asp His Gly His Val He Leu Gly Asp
245 250 255
Pro Ser Pro Val Leu Phe Tyr Pro He Ser Ser Thr Glu He Arg Cys
260 265 270
Leu Val Asp Val Pro Gly Gin Lys Val Pro Ser He Ser Asn Gly Glu
275 280 285
Met Ala Lys Tyr Leu Lys Thr Val Val Ala Pro Gin Val Pro Pro Glu
290 295 300
Leu His Ala Ala Phe He Ala Ala Val Asp Lys Gly His He Arg Thr 305 310 315 320
Met Pro Asn Arg Ser Met Pro Ala Asp Pro Tyr Pro Thr Pro Gly Ala
325 330 335
Leu Leu Met Gly Asp Ala Phe Asn Met Arg His Pro Leu Thr Gly Gly
340 345 350
Gly Met Thr Val Ala Leu Ser Asp He Val Val Leu Arg Asn Leu Leu
355 360 365
Lys Pro Leu Arg Asp Leu Asn Asp Ala Ser Ser Leu Cys Lys Tyr Leu 370 375 380
Glu Ser Phe Tyr Thr Leu Arg Lys Pro Val Ala Ser Thr He Asn Thr
385 390 395 400
Leu Ala Gly Ala Leu Tyr Lys Val Phe Cys Ala Ser Pro Asp Pro Ala
405 410 415 Arg Lys Glu Met Arg Gin Ala Cys Phe Asp Tyr Leu Ser Leu Gly Gly
420 425 430
Leu Phe Ser Glu Gly Pro Val Ser Leu Leu Ser Gly Leu Asn Pro Cys
435 440 445
Pro Leu Ser Leu Val Leu His Phe Phe Ala Val Ala He Tyr Gly Val
450 455 460
Gly Arg Leu Leu Leu Pro Phe Pro Ser Pro Lys Arg Leu Trp He Gly
465 470 475 480 lie Arg Leu lie Ala Ser Ala Ser Gly lie lie Leu Pro He He Lys
485 490 495
Ala Glu Gly lie Arg Gin Met Phe Phe Pro Ala Thr Val Pro Ala Tyr
500 505 510
Tyr Arg Ala Pro Pro Asp Ala
515
SEQ ID NO: 99
Medicago t ruricatul a Squa lene monooxygenase protein sequence
Met Asp Leu Tyr Asn He Gly Trp H e Leu Ser Ser Val Leu Ser Leu 1 5 10 15
Phe Ala Leu Tyr Asn Leu He Phe Ala Gly Lys Lys Asn Tyr Asp Val
20 25 30
Asn Glu Lys Val Asn Gin Arg Glu Asp Ser Val Thr Ser Thr Asp Ala
35 40 45
Gly Glu He Lys Ser Asp Lys Leu Asn Gly Asp Ala Asp Val He He
50 55 60
Val Gly Ala Gly He Ala Gly Ala Ala Leu Ala His Thr Leu Gly Lys 65 70 75 80
Asp Gly Arg Arg Val His lie He Glu Arg Asp Leu Ser Glu Pro Asp
85 90 95
Arg He Val Gly Glu Leu Leu Gin Pro Gly Gly Tyr Leu Lys Leu Val
100 105 110
Glu Leu Gly Leu Gin Asp Cys Val Asp Asn He Asp Ala Gin Arg Val
115 120 125
Phe Gly Tyr Ala Leu Phe Lys Asp Gly Lys His Thr Arg Leu Ser Tyr 130 135 140
Pro Leu Glu Lys Phe His Ser Asp Val Ser Gly Arg Ser Phe His Asn 145 150 155 160
Gly Arg Phe lie Gin Arg Me Arg Glu Lys Ala Ala Ser Leu Pro Asn
165 170 175
Val Asn Met Glu Gin Gly Thr Val lie Ser Leu Leu Glu Glu Lys Gly
180 185 190
Thr lie Lys Gly Val Gin Tyr Lys Asn Lys Asp Gly Gin Ala Leu Thr
195 200 205
Ala Tyr Ala Pro Leu Thr lie Val Cys Asp Gly Cys Phe Ser Asn Leu 210 215 220
Arg Arg Ser Leu Cys Asn Pro Lys Val Asp Asn Pro Ser Cys Phe Val 225 230 235 240
Gly Leu lie Leu Glu Asn Cys Glu Leu Pro Cys Ala Asn His Gly His
245 250 255
Val lie Leu Gly Asp Pro Ser Pro lie Leu Phe Tyr Pro lie Ser Ser
260 265 270
Thr Glu lie Arg Cys Leu Val Asp Val Pro Gly Thr Lys Val Pro Ser
275 280 285
lie Ser Asn Gly Asp Met Thr Lys Tyr Leu Lys Thr Thr Val Ala Pro
290 295 300
Gin Val Pro Pro Glu Leu Tyr Asp Ala Phe lie Ala Ala Val Asp Lys
305 310 315 320
Gly Asn lie Arg Thr Met Pro Asn Arg Ser Met Pro Ala Asp Pro Arg
325 330 335
Pro Thr Pro Gly Ala Val Leu Met Gly Asp Ala Phe Asn Met Arg His
340 345 350
Pro Leu Thr Gly Gly Gly Met Thr Val Ala Leu Ser Asp lie Val Val
355 360 365
Leu Arg Asn Leu Leu Lys Pro Met Arg Asp Leu Asn Asp Ala Pro Thr 370 375 380
Leu Cys Lys Tyr Leu Glu Ser Phe Tyr Thr Leu Arg Lys Pro Val Ala
385 390 395 400
Ser Thr lie Asn Thr Leu Ala Gly Ala Leu Tyr Lys Val Phe Ser Ala 410 415
Ser Pro Asp Glu Arg Lys Glu Met Arg Gin Ala Cys Phe Asp Tyr
420 425 430
Leu Ser Leu Gly Gly Leu Phe Ser Glu Gly Pro lie Ser Leu Leu Ser
435 440 445
Gly Leu Asn Pro Arg Pro Leu Ser Leu Val Leu His Phe Phe Ala Val 450 455 460
Ala Val Phe Gly Val Gly Arg Leu Leu Leu Pro Phe Pro Ser Pro Lys
465 470 475 480
Arg Val Trp lie Gly Ala Arg Leu Leu Ser Gly Ala Ser Gly lie lie
485 490 495
Leu Pro lie lie Lys Ala Glu Gly lie Arg Gin Met Phe Phe Pro Ala
500 505 510
Thr Val Pro Ala Tyr Tyr Arg Pro Pro Val Asn Ala Phe
515 525
SEQ ID NO: 100
Ricinus communis Squalene monooxygenase protein sequence
Met Ala Asp Asn Tyr Leu Leu Gly Trp lie Leu Cys Ser lie lie Gly
1 5 10 15
Leu Phe Gly Leu Tyr Tyr Met Val Tyr Leu Val Val Lys Arg Glu Glu
20 25 30
Glu Asp Asn Asn Arg Lys Ala Leu Leu Gin Ala Arg Ser Asp Ser Ala
35 40 45
Lys Thr Met Ser Ala Val Ser Gin Asn Gly Glu Cys Arg Ser Asp Asn
50 55 60
Pro Ala Asp Ala Asp lie lie lie Val Gly Ala Gly Val Ala Gly Ser 65 70 75 80
Ala Leu Ala His Thr Leu Gly Lys Asp Gly Arg Arg Val His Val lie
85 90 95
Glu Arg Asp Leu Thr Glu Pro Asp Arg lie Val Gly Glu Leu Leu Gin
100 105 110
Pro Gly Gly Tyr Leu Lys Leu lie Glu Leu Gly Leu Glu Asp Cys Val
115 120 125 Glu Glu lie Asp Ala Gin Arg Val Phe Gly Tyr Ala Leu Phe Met Asp
130 135 140
Gly Lys His Thr Gin Leu Ser Tyr Pro Leu Glu Lys Phe His Ser Asp 145 150 155 160
Val Ala Gly Arg Ser Phe His Asn Gly Arg Phe He Gin Arg Met Arg
165 170 175
Glu Lys Ala Ser Ser lie Pro Asn Val Arg Leu Glu Gin Gly Thr Val
180 185 190
Thr Ser Leu lie Glu Glu Lys Gly He He Arg Gly Val Val Tyr Lys
195 200 205
Thr Lys Thr Gly Glu Glu Leu Thr Ala Phe Ala Pro Leu Thr He Val
210 215 220
Cys Asp Gly Cys Phe Ser Asn Leu Arg Arg Ser Leu Cys Asn Pro Lys
225 230 235 240
Val Asp Val Pro Ser Cys Phe Val Gly Leu Val Leu Glu Asp Cys Lys
245 250 255
Leu Pro Tyr Gin Tyr His Gly His Val Val Leu Ala Asp Pro Ser P o
260 265 270 lie Leu Phe Tyr Gin lie Ser Ser Thr Glu Val Arg Cys Leu Val Asp
275 280 285
Val Pro Gly Gin Lys Val Pro Ser He Ser Asn Gly Glu Met Ala Lys 290 295 300
Tyr Leu Lys Asn Val Val Ala Pro Gin Val Pro Pro Glu He Tyr Asp 305 310 315 320
Ser Phe Val Ala Ala Val Asp Lys Gly Asn He Arg Thr Met Pro Asn
325 330 335
Arg Ser Met Pro Ala Ser Pro Tyr Pro Thr Pro Gly Ala Leu Leu Met
340 345 350
Gly Asp Ala Phe Asn Met Arg His Pro Leu Thr Gly Gly Gly Met Thr
355 360 365
Val Ala Leu Ser Asp He Val Val Leu Arg Glu Leu Leu Lys Pro Leu
370 375 380
Arg Asp Leu His Asp Ala Pro Thr Leu Cys Arg Tyr Leu Glu Ser Phe
385 390 395 400 Tyr Thr Leu Arg Lys Pro Val Ala Ser Thr He Asn Thr Leu Ala Gly
405 410 415
Ala Leu Tyr Lys Val Phe Cys Ala Ser Ser Asp Glu Ala Arg Asn Glu
420 425 430
Met Arg Gin Ala Cys Phe Asp Tyr Leu Ser Leu Gly Gly Val Phe Ser
435 440 445
Thr Gly Pro lie Ser Leu Leu Ser Gly Leu Asn Pro Arg Pro Leu Ser
450 455 460
Leu Val Val His Phe Phe Ala Val Ala He Tyr Gly Val Gly Arg Leu 465 470 475 480
Leu Leu Pro Phe Pro Ser Pro Lys Arg Val Trp Val Gly Ala Arg Leu
485 490 495
He Ser Gly Ala Ser Gly He He Phe Pro He He Lys Ala Glu Gly
500 505 510
Val Arg Gin Met Phe Phe Pro Ala Thr Val Pro Ala Tyr Tyr Arg Ala
515 520 525
Pro Pro Val Glu Cys Asn
530
SEQ ID NO : 101
Ricinus communis Squalene monooxygenase protein sequence
Met Glu Tyr Lys Leu Ala Val Ala Gly He He Ala Ser Leu Trp Ala 1 5 10 15
Leu Phe Met Leu Cys Ser Leu Lys Arg Lys Lys Asn He Thr Arg Ala
20 25 30
Ser Phe Asn Asn Tyr Thr Asp Glu Thr Leu Lys Ser Ser Ser Lys Glu
35 4 0 45
He Cys Gin Pro Glu He Val Ala Ser Pro Asp He He He Val Gly
50 55 60
Ala Gly Val Ala Gly Ala Ala Leu Ala Tyr Ala Leu Gly Glu Asp Gly 65 70 75 80
Arg Gin Val His Val He Glu Arg Asp Leu Ser Glu Pro Asp Arg He
85 90 95 Val Gly Glu Leu Leu Gin Pro Gly Gly Tyr Leu Lys Leu He Glu Leu 100 105 110
Gly Leu Glu Asp Cys Val Glu Lys He Asp Ala Gin Gin Val Phe Gly
115 120 125
Tyr Ala He Phe Lys Asp Gly Lys Ser Thr Lys Leu Ser Tyr Pro Leu
130 135 140
Asp Gly Phe Gin Thr Asn Val Ser Gly Arg Ser Phe His Asn Gly Arg 145 150 155 160
Phe He Gin Arg Met Arg Glu Lys Ala Thr Ser Leu Pro Asn Leu He
165 170 175
Leu Gin Gin Gly Thr Val Thr Ser Leu Val Glu Lys Lys Gly Thr Val
180 185 190
Lys Gly Val Asn Tyr Arg Thr Arg Asn Gly Gin Glu Met Thr Ala Tyr
195 200 205
Ala Pro Leu Thr He Val Cys Asp Gly Cys Phe Ser Asn Leu Arg Arg
210 215 220
Ser Leu Cys Asn Pro Lys Val Glu He Pro Ser Cys Phe Val Ala Leu 225 230 235 240
Val Leu Glu Asn Cys Asp Leu Pro Tyr Ala Asn His Gly His Val He
245 250 255
Leu Ala Asp Pro Ser Pro He Leu Phe Tyr Pro He Ser Ser Thr Glu
260 265 270
Val Arg Cys Leu Val Asp He Pro Gly Gin Lys Val Pro Ser He Ser
275 280 285
Asn Gly Glu Leu Ala Gin Tyr Leu Lys Ser Thr Val Ala Lys Gin He
290 295 300
Pro Ser Glu Leu His Asp Ala Phe He Ser Ala He Glu Lys Gly Asn
305 310 315 320
He Arg Thr Met Pro Asn Arg Ser Met Pro Ala Ser Pro His Pro Thr
325 330 335
Pro Gly Ala Leu Leu Val Gly Asp Ala Phe Asn Met Arg His Pro Leu
340 345 350
Thr Gly Gly Gly Met Thr Val Ala Leu Ser Asp He Val Leu Leu Arg
355 360 365 Asn Leu Leu Arg Pro Leu Glu Asn Leu Asn Asp Ala Ser Val Leu Cys
370 375 380
Lys Tyr Leu Glu Ser Phe Tyr lie Leu Arg Lys Pro Met Ala Ser Thr 385 390 395 400 lie Asn Thr Leu Ala Gly Ala Leu Tyr Lys Val Phe Ser Ala Ser Thr
405 410 415
Asp Arg Ala Arg Ser Glu Met Arg Gin Ala Cys Phe Asp Tyr Leu Ser
420 425 430
Leu Gly Gly Val Phe Ser Asn Gly Pro He Ala Leu Leu Ser Gly Leu
435 440 445
Asn Pro Arg Pro Leu Asn Leu Val Leu His Phe Phe Ala Val Ala Val
450 455 460
Tyr Gly Val Gly Arg Leu lie Leu Pro Phe Pro Ser Pro Lys Ser He 465 470 475 480
Trp Asp Gly Val Lys Leu l ie Ser Gly Ala Ser Ser Val He Phe Pro
485 490 495 lie Met Lys Ala Glu Gly He Gly Gin He Phe Phe Pro He Thr Lys
500 505 510
Pro Pro Asn His Lys Ser Gin Thr Trp
515 520
SEQ ID NO: 102
Ricinus communis Squalene monoo ygena se protein sequence
Met Gly Val Ser Arg Glu Glu Asn Ala Arg Asp Glu Lys Cys His Tyr 1 5 10 15
Tyr Glu Asn Gly He Ser Leu Ser Glu Lys Ser Met Ser Thr Asp He
20 25 30
He He Val Gly Ala Gly Val Ala Gly Ser Ala Leu Ala Tyr Thr Leu
35 40 45
Gly Lys Asp Gly Arg Arg Val His Val He Glu Arg Asp Leu Ser Leu
50 55 60
Gin Asp Arg He Val Gly Glu Leu Leu Gin Pro Gly Gly Tyr Leu Lys 65 70 5 80 Leu lie Glu Leu Gly Leu Glu Asp Cys Val Glu Glu lie Asp Ala Gin 85 90 95
Gin Val Phe Gly Tyr Ala Leu Tyr Lys Asn Gly Arg Ser Thr Lys Leu
100 105 110
Ser Tyr Pro Leu Glu Ser Phe Asp Ser Asp Val Ser Gly Arg Ser Phe
115 120 125
His Asn Gly Arg Phe lie Gin Arg Met Arg Glu Lys Ala Ala Ser Leu 130 135 140
Pro Asn Val Arg Leu Glu Glu Gly Thr Val Thr Ser Leu Leu Glu Val 145 150 155 1G0
Lys Gly Thr lie Lys Gly Val Gin Tyr Lys Thr Lys Asn Gly Glu Glu
165 170 175
Leu Thr Ala Ser Ala Pro Leu Thr lie Val Cys Asp Gly Cys Phe Ser
180 185 190
Asn Leu Arg Arg Ser Leu Cys Asn Pro Lys Val Asp lie Pro Ser Cys
195 200 205
Phe Val Ala Leu lie Leu Glu Asn Ser Gly Gin Lys Leu Pro Ser lie 210 215 220
Ser Asn Gly Asp Met Ala Asn Tyr Leu Lys Ser Val Val Ala Pro Gin
225 230 235 240 lie Pro Pro Val Leu Ser Glu Ala Phe lie Ser Ala lie Glu Lys Gly
245 250 255
Lys lie Arg Thr Met Pro Asn Arg Ser Met Pro Ala Ala Pro His Pro
260 265 270
Thr Pro Gly Ala Leu Leu Leu Gly Asp Ala Phe Asn Met Arg His Pro
275 280 285
Leu Thr Gly Gly Gly Met Thr Val Ala Leu Ser Asp He Val Val Leu 290 295 300
Arg Asn Leu Leu Lys Pro Leu His Asp Leu Thr Asp Ala Ser Ala Leu 305 310 315 320
Cys Glu Tyr Leu Lys Ser Phe Tyr Ser Leu Arg Lys Pro Val Ala Ser
325 330 335
Thr lie Asn Thr Leu Ala Gly Ala Leu Tyr Lys Val Phe Ser Ala Ser
340 345 350 His Asp Pro Ala Arg Asn Glu Met Arg Gin Ala Cys Phe Asp Tyr Leu
355 360 365
Ser Leu Gly Gly Val Phe Ser Asn Gly Pro He Ala Le Leu Ser Gly
370 375 380
Leu Asn Pro Arg Pro Leu Ser Leu Val Ala His Phe Phe Ala Val Ala 385 390 395 400
He Tyr Gly Val Gly Arg Leu He Phe Pro Leu Pro Ser Ala Lys Gly
405 410 415
Met Trp Met Gly Ala Arg Met He Lys Val Ala Ser Gly He He Phe
420 425 430
Pro He He Arg Ala Glu Gly Val Gin His Met Phe Phe Ser Lys Thr
435 440 445
Leu Ser Ala Phe Ser Arg Ser Gin Thr Ser
450 455
SEQ ID NO: 103
Ricinus communis Squalene monooxygenase protein sequence
Met Glu Tyr Gin Tyr Phe Val Gly Gly He He Ala Ser Ala. Leu Leu 1 5 10 15
Phe Val Leu Val Cys Arg Leu Ala Gly Lys Arg Gin Arg Arg Ala Leu
20 25 30
Arg Asp Thr Val Asp Arg Asp Glu He Ser Gin Asn Ser Glu Asn Gly
35 40 45
He Ser Gin Ser Glu Lys Asn Met Asn Thr Asp He He He Val Gly
50 55 60
Ala Gly Val Ala Gly Ser Thr Leu Ala Tyr Thr Leu Gly Lys Asp Gly
65 70 75 80
Arg Arg Val Arg Val He Glu Arg As Leu Ser Leu Gin Asp Arg He
85 90 95
Val Gly Glu Leu Leu Gin Pro Gly Gly Tyr Leu Lys Leu He Glu Leu
100 105 110
Gly Leu Glu Asp Cys Val Glu Glu He Asp Ala Leu Gin Val Phe Gly
115 120 125 Tyr Ala Leu Tyr Lys Asn Gly Arg Ser Thr Lys Leu Ser Tyr Pro Leu 130 135 140
Asp Ser Phe Asp Ser Asp Val Ser Gly Arg Ser Phe His Asn Gly Arg
145 150 155 160
Phe lie Gin Arg Met Arg Glu Lys Ala Ala Ser Leu Pro Asn Val Arg
165 170 175
Met Glu Gly Gly Thr Val Thr Ser Leu Leu Glu Val Lys Gly Thr lie
180 185 190
Lys Gly Val Gin Tyr Lys Asn Lys Asn Gly Glu Glu Leu He Ala Cys
195 200 205
Ala Pro Leu Thr lie Val Cys Asp Gly Cys Phe Ser Asn Leu Arg Arg
210 215 220
Ser Leu Cys Asn Ser Lys Val Asp He Pro Phe Cys Phe Val Ala Leu 225 230 235 240 lie Leu Glu Asn Cys Glu Leu Pro Tyr Pro Asn His Gly His Val He
245 250 255
Leu Ala Asp Pro Ser Pro He Leu Phe Tyr Arg He Ser He Ser Glu
260 265 270 lie Arg Cys Leu Val Asp He Pro Ala Gly Gin Lys Leu Pro Ser He
275 280 285
Ser Asn Gly Glu Met Ala Asn Tyr Leu Lys Ser Val Val Ala Pro Gin
290 295 300
lie Pro Pro Glu Leu Ser Asn Ala Phe Leu Ser Ala He Glu Lys Gly 305 310 315 320
Lys lie Arg Thr Met Pro Lys Arg Ser Met Pro Ala Ala Pro His Pro
325 330 335
Thr Pro Gly Ala Leu Leu Leu Gly Asp Ala Phe Asn Met Arg His Pro
340 345 350
Leu Thr Gly Gly Val Met Thr Val Ala Leu Ser Asp He Val Val Leu
355 360 365
Arg Ser Leu Leu Arg Pro Leu His Asp Leu Thr Asp Ala Ser Ala Leu
370 375 380
Cys Glu Tyr Leu Lys Ser Phe Tyr Ser Leu Arg Lys Pro Met Val Ser 385 390 395 400 Thr lie Asn Thr Leu Ala Gly Ala Leu Tyr Arg Val Phe Ser Ala Ser
405 410 415
Gin Asp Pro Ala Arg Asp Glu Met Arg Gin Ala Cys Phe Asp Tyr Leu
420 425 430
Ser Leu Gly Gly Val Phe Ser Asn Gly Pro lie Ala Leu Leu Ser Gly
435 440 445
Leu Asn Pro Arg Pro Leu Ser Leu He Val His Phe Phe Ala Val Ala 450 455 460
Val Tyr Gly Val Gly Arg Leu lie Phe Pro Leu Pro Ser Ala Lys Arg 465 470 475 480
Met Trp Met Gin Glu
485
SEQ ID NO: 104
Ricinus communis Squalene monooxygenase protein sequence
Met Glu Tyr Gin Tyr Leu Met Gly Gly Gly He Met Thr Leu Leu Phe 1 5 10 15
Val Leu Ser Tyr Arg Leu Lys Arg Glu Thr Arg Ala Ser Val Glu Asn
20 25 30
Ala Arg Asp Glu Val Leu Gin Asn Ser Glu Asn Gly He Ser Gin Ser
35 40 45
Glu Lys Ala Met Asn Thr As He Lys Leu Leu Leu Glu Gin He Val
50 55 60
Gin Lys lie Ala Met Leu Asn Ser He Arg Leu Glu Glu Gly Thr Val 65 70 75 80
Thr Ser Leu Leu Glu Val Lys Arg Asp He Lys Gly Val Gin Tyr Lys
85 90 95
Thr Lys Asn Gly Glu Glu Leu Thr Ala Cys Ala Pro Leu Thr He Val
100 105 110
Ser His Gly Cys Phe Ser As Leu Arg Leu His Val Thr Pro Ser Thr
115 120 125
Ser Lys Phe Lys Ser Phe He Gly Leu Glu Val Asp He Pro Ser Ser
130 135 140
Phe Ala Ala Leu He Leu Gly Asn Cys Glu Leu Pro Phe Pro Asn His 145 150 155 160
Gly His Val He Leu Ala Asp Pro Ser Ser He Leu Phe Tyr Arg He
165 170 175
Ser Ser Ser Glu He Cys Cys Leu Val Asp Val Pro Ala Gly Gin Lys
180 185 190
Leu Pro Ser lie Asn Gly Glu Met Ala Asn Tyr Leu Lys Ser Val
195 200 205
Val Ala His Gin Phe Lys Val Gly Leu Ala Tyr
210 215 220
SEQ ID NO: 105
Ricinus communis Squalene monooxygenase protein sequence
Met Ser Pro He Ser He Gin Leu Pro Pro Arg Pro Gin Leu Tyr Arg 1 5 10 15
Ser Leu He Ser Ser Leu Ser Leu Ser Thr Tyr Lys Gin Pro Pro Ser
20 25 30
Pro Pro Ser Phe Ser Leu Thr He Ala Asn Ser Pro Pro Gin Pro Gin
35 40 45
Pro Gin Ala Thr Val Ser Ser Lys Thr Arg Thr He Thr Arg Leu Ser
50 55 60
Asn Ser Ser Asn Arg Val Asn Leu Leu Gin Ala Glu Gin His Pro Gin 65 70 75 80
Glu Pro Ser Ser Asp Leu Ser Tyr Ser Ser Ser Pro Pro His Cys Val
85 90 95
Ser Gly Gly Tyr Asn He Lys Leu Met Glu Val Gly Thr Asp Asn Tyr
100 105 110
Ala Val He He He Leu Gly Thr Phe Phe Ala Ser Leu Phe Ala Phe
115 120 125
Val Phe Leu Ser He Leu Arg Tyr Asn Phe Lys Asn Lys Asn Lys Ala 130 135 140
Lys He His Asp Glu Thr Thr Leu Lys Thr Gin Asn Asp Asn Val Arg 145 150 155 160
Leu Pro Asp Asn Gly Ser Gly Asn Asp Val He He Val Gly Ala Gly
165 170 175 Val Ala Gly Ala Ala Leu Ala Tyr Thr Leu Gly Lys Asp Gly Arg Arg
180 185 130
Val His Val lie Glu Arg Asp Leu Thr Glu Pro Asp Arg He Val Gly
195 200 205
Glu Leu Leu Gin Pro Gly Gly Tyr Leu Lys Leu lie Glu Leu Gly Leu 210 215 220
Glu Asp Cys Val Gin Glu lie Asp Ala Gin Arg Val Leu Gly Tyr Ala 225 230 235 240
Leu Phe Lys Asp Gly Lys Asn Thr Arg Leu Ser Tyr Pro Leu Glu Lys
245 250 255
Phe His Ala Asp Val Ala Gly Arg Ser Phe His Asn Gly Arg Phe He
260 265 270
Gin Arg Met Arg Glu Lys Ala Ala Ser Leu Pro As Val Lys Leu Glu
275 280 285
Gly Thr Val Thr Ser Leu Leu Glu Glu Asn Gly Thr He Lys Gly
290 295 300
Val Gin Tyr Lys Thr Lys Asp Gly Gin Glu He Arg Ala Tyr Ala Pro
305 310 315 320
Leu Thr He Val Cys Asp Gly Cys Phe Ser Asn Leu Arg Arg Ser Leu
325 330 335
Cys Asn Pro Lys Val Asp Val Pro Ser Cys Phe Val Gly Leu Val Leu
340 345 350
Glu Asn Cys Gin Leu Pro Phe Ala Asn His Gly His Val Val Leu Ala
355 360 365
Asp Pro Ser Pro He Leu Phe Tyr Pro He Ser Ser Thr Glu Val Arg 370 375 380
Cys Leu Val Asp Val Pro Gly Gin Lys Val Pro Ser He Ala Asn Gly 385 390 395 400
Glu Met Ala Lys Tyr Leu Lys Asn Val Val Ala Pro Gin He Pro Pro
405 410 415
Val Leu His Asp Ala Phe He Ser Ala lie Asp Lys Gly Asn He Arg
420 425 430
Thr Met Pro Asn Arg Ser Met Pro Ala Asp Pro His Pro Thr Pro Gly 435 440 445
Ala Leu Leu Met Gly Asp Ala Phe Asn Met Arg His Pro Leu Thr Gly
450 455 460
Gly Gly Met Thr Val Ala Leu Ser Asp lie Val Val Leu Arg Asp Leu
465 470 475 480
Leu Lys Pro Leu Arg Asp Leu Asn Asp Ala Thr Ser Leu Thr Lys Tyr
485 490 495
Leu Glu Ser Phe Tyr Thr Leu Arg Lys Pro Val Ala Ser Thr lie Asn
500 505 510
Thr Leu Ala Gly Ala Leu Tyr Lys Val Phe Ser Ala Ser Pro Asp Gin
515 520 525
Ala Arg Lys Glu Met Arg Gin Ala Cys Phe Asp Tyr Leu Ser Leu Gly
530 535 540
Gly lie Phe Ser Ser Gly Pro Val Ala Leu Leu Ser Gly Leu Asn Pro
545 550 555 560
Arg Pro Leu Ser Leu Val Met His Phe Phe Ala Val Ala lie Tyr Gly
565 570 575
Val Gly Arg Leu Leu Leu Pro Phe Pro Ser Pro Lys Ser Val Trp lie
580 585 590
Gly Ala Arg Leu lie Ser Ser Ala Ser Gly lie lie Phe Pro lie lie
595 600 605
Lys Ala Glu Gly Val Arg Gin Met Phe Phe Pro Ala Thr lie Pro Ala
610 615 620
lie Tyr Arg Pro Pro Pro Val Lys Asp Thr Ser Asp Asp Glu Gin Lys
625 630 635 640
Ser Arg
[00194] Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.

Claims

WHAT IS CLAIMED IS:
1. A recombinant host comprising one or more of:
(a) a gene encoding a squalene epoxidase polypeptide;
(b) a gene encoding a cucurbitadienol synthase polypeptide;
(c) a gene encoding a cytochrome P450 polypeptide;
(d) a gene encoding a cytochrome P450 reductase polypeptide;
(e) a gene encoding an epoxide hydrolase polypeptide;
(f) a gene encoding a UGT1576 polypeptide having 60% or greater identity to an amino acid sequence set forth in SEQ ID NO:48;
(g) a gene encoding a UGT430 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:62;
(h) a gene encoding a UGT1697 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:68;
(i) a gene encoding a UGT11789 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:72;
(j) a gene encoding a UGT98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:53;
(k) a gene encoding a UGTSK98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:50;
wherein at least one of the genes is a recombinant gene;
wherein the host is capable of producing a mogrol precursor, a mogroside precursor, and/or a mogroside compound.
2. The recombinant host of claim 1 , wherein:
(a) the squalene epoxidase polypeptide comprises a polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:54;
(b) the cucurbitadienol synthase polypeptide comprises a polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:43;
(c) the cytochrome P450 polypeptide comprises a CYP5491 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:44 and/or a CY 1798 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:74; (d) the cytochrome P450 reductase polypeptide comprises a CPR4497 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:46; and/or
(e) the epoxide hydrolase polypeptide comprises an epoxide hydrolase 1 polypeptide having 75% or greater identity to an amino acid sequence set forth in SEQ ID NO:38 or an epoxide hydrolase 2 polypeptide having 65% or greater identity to an amino acid sequence set forth in SEQ ID NO:40.
A recombinant host comprising one or more of:
(a) one or more genes encoding one or more enzymes capable of catalyzing conversion of dioxidosqualene to produce 24,25 epoxy cucurbitadienol;
(b) one or more genes encoding one or more enzymes capable of catalyzing conversion of oxidosqualene to produce cucurbitadienol;
(c) one or more genes encoding one or more enzymes capable of catalyzing hydroxylation of 24,25 epoxy cucurbitadienol to produce 11-hydroxy- 24,25 epoxy cucurbitadienol;
(d) one or more genes encoding one or more enzymes capable of catalyzing hydroxylation of cucurbitadienol to produce 11 -hydroxy-cucurbitadienol;
(e) one or more genes encoding one or more enzymes capable of catalyzing epoxidation of cucurbitadienol to produce 24,25 epoxy cucurbitadienol; or
(f) one or more genes encoding one or more enzymes capable of catalyzing epoxidation of 1 1 -hydroxy-cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol;
(g) one or more genes encoding one or more enzymes capable of catalyzing conversion of 11-hydroxy-24,25 epoxy cucurbitadienol to produce mogro!; or
(h) one or more genes encoding one or more enzymes capable of catalyzing glycosylation of a mogroside precursor to produce a mogroside compound;
wherein at least one of the genes is a recombinant gene.
The recombinant host of claim 3, further comprising a gene encoding squaiene epoxidase polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:54.
5. The recombinant host of any one of claims 1-4, wherein the recombinant host has been modified to reduce expression of a lanosterol synthase (ERG7) polypeptide.
6. The recombinant host of claim 5, wherein the ERG7 polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:55.
7. A method of producing a mogroside precursor and/or a mogroside compound, comprising:
(a) growing the recombinant host of any one of claims 1-6 in a culture medium, under conditions in which the genes disclosed in any one of claims 1-6 are expressed;
wherein the mogroside precursor and/or the mogroside compound is synthesized by the recombinant host; and
(b) optionally isolating the mogroside precursor and/or the mogroside compound.
8. The method of claim 7, wherein the mogroside precursor is mogrol synthesized by epoxidation of 11-hydroxy-cucurbitadienol to synthesize 11 -hydroxy-24,25 epoxy cucurbitadienol and hydrolysis of 11-hydroxy-24,25 epoxy cucurbitadienol to synthesize mogrol.
9. The method of claim 8, wherein epoxidation of 11-hydroxy-cucurbitadienol to synthesize 11-hydroxy-24,25 epoxy cucurbitadienol is catalyzed by the CYP1798 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:74.
10. A method of producing a mogroi precursor in vitro, comprising:
(a) contacting dioxidosqualene with one or more enzymes capable of catalyzing conversion of dioxidosqualene to produce 24,25 epoxy cucurbitadienol; or
(b) contacting oxidosqualene with one or more enzymes capable of catalyzing conversion of oxidosqualene to produce cucurbitadienol; or (c) contacting 24,25 epoxy cucurbitadienol with one or more enzymes capable of catalyzing hydroxy!ation of 24,25 epoxy cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol; or
(d) contacting cucurbitadienol with one or more enzymes capable of catalyzing hydroxylation of cucurbitadienol to produce 11-hydroxy- cucurbitadienol; or
(e) contacting cucurbitadienol with one or more enzymes capable of catalyzing epoxidation of cucurbitadienol to produce 24,25 epoxy cucurbitadienol; or
(f) contacting 11-hydroxy-cucurbitadienol with one or more enzymes capable of catalyzing epoxidation of 11-hydroxy-cucurbitadienol to produce 1 1 - hydroxy-24,25 epoxy cucurbitadienol.
11. A method of producing a mogrol in vitro, comprising contacting 11-hydroxy-24,25 epoxy cucurbitadienol with one or more enzymes capable of catalyzing conversion of 1 1 - hydroxy-24,25 epoxy cucurbitadienol to produce mogrol.
12. A method of producing a mogroside compound in vitro, comprising contacting a mogroside precursor with one or more enzymes capable of catalyzing glycosylation of the mogroside precursor to produce a mogroside compound.
13. The method of any one of claims 10-12, further comprising isolating the mogrol precursor, mogrol or the mogroside compound.
14. The recombinant host of claim 3 or the method of any one of claims 10-13, wherein:
(a) the one or more enzymes capable of catalyzing conversion of dioxidosqualene to produce 24,25 epoxy cucurbitadienol comprise a cucurbitadienol synthase having 70% or greater identity to an amino acid sequence set forth in SEQ ID N0.43;
(b) the one or more enzymes capable of catalyzing conversion of oxidosqualene to produce cucurbitadienol comprise a cucurbitadienol synthase having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:43;
(c) the one or more enzymes capable of catalyzing conversion of 24,25 epoxy cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol comprise CYP5491 having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:44;
(d) the one or more enzymes capable of catalyzing conversion of cucurbitadienol to produce 11 -hydroxy-cucurbitadienoi comprise CYP5491 having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:44;
(e) the one or more enzymes capable of catalyzing epoxidation of cucurbitadienol to produce 24,25 epoxy cucurbitadienol comprise CYP1798 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:74;
(f) the one or more enzymes capable of catalyzing epoxidation of 1 1 -hydroxy- cucurbitadienoi to produce 1 1-hydroxy-24,25 epoxy cucurbitadienol comprise CYP1798 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:74;
(g) the one or more enzymes capable of catalyzing conversion of 11 -hydroxy-24,25 epoxy cucurbitadienol to produce mogrol comprise a polypeptide comprising epoxide hydrolase 1 having 75% or greater identity to an amino acid sequence set forth in SEQ ID NO:38 or epoxide hydrolase 2 having 65% or greater identity to an amino acid sequence set forth in SEQ ID NO:40; and/or
(h) the one or more enzymes capable of catalyzing conversion of the mogroside precursor to a mogroside compound comprise UGT1576 having 60% or greater identity to an amino acid sequence set forth in SEQ ID NO:48; UGT98 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:53; UGTSK98 having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:50; UGT430 having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:62; UGT1697 having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:68; or UGT1 1789 having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:72.
15. A method of producing a mogroside compound, comprising contacting a recombinant host expressing one or more of:
(a) a UGT1576 polypeptide having 60% or greater identity to an amino acid sequence set forth in SEQ ID NO:48;
(b) a UGT430 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:62; (c) a UGT1697 polypeptide having 45% or greater identity to an amino acid sequence set forth in SEQ ID NO:68;
(d) a UGT1 1789 polypeptide having 50% or greater identity to an amino acid sequence set forth in SEQ ID NO:72;
(e) a UGT98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:53; or
(f) a UGTSK98 polypeptide having 70% or greater identity to an amino acid sequence set forth in SEQ ID NO:50
with a mogroside precursor.
16. The method of claim 15, wherein the mogroside precursor is plant-derived or synthetic.
17. The method of claim 15, further comprising isolating the mogroside compound.
18. The recombinant host of any one of claims 1-6 or the method of claims 7 or 12-14,
wherein the mogroside compound is:
(a) mogroi glycosylated at C3 position; or
(b) mogroi glycosylated at C24 position; or
(c) mogroi glycosylated at C3 position and C24 position.
19. The recombinant host of any one of claims 1-6 or the method of claims 7 or 2-17, wherein the mogroside compound is one or more of mogroside I A1 , mogroside I E1 , mogroside II A, mogroside II A1 , mogroside If A2, mogroside II E, mogroside III A1 , mogroside III A2, mogroside III, mogroside III E, mogroside IV, mogroside IV A, mogroside V or siamenoside.
20. The recombinant host of any one of claims 1 -6 or the method of claims 10 or 13, wherein the mogroi precursor is one or more of squalene, dioxidosqualene, oxidosqualene, 24,25 epoxy cucurbitadienol, cucurbitadienol, 11-hydroxy-cucurbitadieno!, 11 -hydroxy 24, 25 epoxy cucurbitadienol or 1 1-oxo-mogrol.
21. The recombinant host of any one of claims 1 -6 or the method of claims 7-9, 12 or 14-17, wherein the mogroside precursor is one or more of mogroi, glycosylated mogroi, di- glycosylated mogroi or tri-glycosylated mogroi.
22. The method of any one of claims 7, 8-13 or 15-17 or the recombinant host of any one of claims 1-6, 14 or 18-21 , wherein the recombinant host comprises a microorganism that is a yeast cell, a plant cell, a mammalian cell, an insect cell, a fungal cell, or a bacterial cell.
23. The bacterial cell according to 22, wherein the bacterial cell comprises Escherichia bacteria cells, Lactobacillus bacteria cells, Lactococcus bacteria cells, Cornebacterium bacteria cells, Acetobacter bacteria cells, Acinetobacter bacteria cells, or Pseudomonas bacterial cells.
24. The yeast cell according to claim 22, wherein the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ash by a gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species.
25. The yeast cell of claim 24, wherein the yeast cell is a Saccharomycete.
26. The yeast cell of claim 25, wherein the yeast ceil is a cell from the Saccharomyces cerevisiae species.
27. The recombinant host of any one of claims 1-6 or 14, wherein one or more of the genes further comprise a nucleotide sequence coding a fusion tag.
28. The recombinant host of claim 27, wherein the fusion tag is a protein or polypeptide.
29. The recombinant host of claim 28, wherein the fusion tag is green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), a polyhistidine-tag (HIS tag), and a FLAG-tag, a chlorop!ast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, a signal peptide, or a secretion tag.
30. The recombinant host of any one of claims 1-6, 12, or 25-27, wherein one or more of the genes are expressed as fusion proteins.
31. A mogroside composition produced by the recombinant host of any one of claims 1-6, 16, or 27-29 or the method of any one of claims 7, 8-13 or 13-17, wherein the composition comprises one or more of mogroside I A1, mogroside I E1, mogroside II A, mogroside II E, mogroside III A1 , mogroside III A2, mogroside III, mogroside III E, mogroside IV, mogroside V, and siamenoside.
32. A food or drink product comprising the composition of claim 31.
PCT/EP2015/072645 2012-12-04 2015-09-30 Methods and materials for biosynthesis of mogroside compounds Ceased WO2016050890A2 (en)

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CN201580062037.XA CN107466320B (en) 2014-10-01 2015-09-30 Methods and materials for biosynthesis of mogroside compounds
JP2017517783A JP2017529860A (en) 2014-10-01 2015-09-30 Methods and materials for biosynthesis of mogroside compounds
SG11201702123SA SG11201702123SA (en) 2014-10-01 2015-09-30 Methods and materials for biosynthesis of mogroside compounds
US15/511,565 US10633685B2 (en) 2012-12-04 2015-09-30 Methods and materials for biosynthesis of mogroside compounds
EP15797018.7A EP3201315A2 (en) 2014-10-01 2015-09-30 Methods and materials for biosynthesis of mogroside compounds
IL251380A IL251380B (en) 2014-10-01 2017-03-26 Methods and materials for biosynthesis of mogroside compounds
US16/806,812 US11091787B2 (en) 2012-12-04 2020-03-02 Methods and materials for biosynthesis of mogroside compounds
IL276781A IL276781A (en) 2014-10-01 2020-08-18 Methods and materials for biosynthesis of mogroside compounds
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US62/059,136 2014-10-02
US201462087726P 2014-12-04 2014-12-04
US62/087,726 2014-12-04
US201462090836P 2014-12-11 2014-12-11
US62/090,836 2014-12-11
US201462091895P 2014-12-15 2014-12-15
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