WO2016001412A1 - Gène et polypeptide impliqués dans la synthèse de valencène et leurs utilisations - Google Patents

Gène et polypeptide impliqués dans la synthèse de valencène et leurs utilisations Download PDF

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WO2016001412A1
WO2016001412A1 PCT/EP2015/065218 EP2015065218W WO2016001412A1 WO 2016001412 A1 WO2016001412 A1 WO 2016001412A1 EP 2015065218 W EP2015065218 W EP 2015065218W WO 2016001412 A1 WO2016001412 A1 WO 2016001412A1
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valencene
synthase
nucleic acid
acid sequence
methyl
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Thorvald Eelco Wallaart
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Eelco Wallaart BV
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
    • C12Y402/03073—Valencene synthase (4.2.3.73)

Definitions

  • the present invention relates to a nucleic acid sequence involved in the synthesis of valencene. Further, the present invention relates to a polypeptide, or enzyme, capable of converting farnesyl pyrophosphate (FPP) into valencene. Furthermore, the present invention relates to the use of the present nucleic acid sequence and polypeptide for the production of valencene, or its derivative nootkatone.
  • FPP farnesyl pyrophosphate
  • nootkatone (4,4a,5,6,7,8-hexahydro-6-isopropenyl-4,3a-dimethyl-2(3H)-naphtalenone) or represented by
  • the flavour and aroma of citrus species is composed of complex combinations of mostly sugars, flavonoids, acids and volatile oils.
  • Mono- and sesquiterpenes are the main components of this volatile, also called essential, oil.
  • essential oil also called essential oil.
  • the monoterpene limonene normally accounts for over 90% of the content of essential oils obtained from citrus species, several unique sesquiterpene compounds, which are present in very low quantities, have an intense effect on the flavor and aroma of the citrus species.
  • valencene nootkatone, alpha- and beta-sinensal
  • Valencene is obtained by the enzymatic activity of valencene synthase and is a major component of citrus essential oils.
  • Nootkatone is a putative -derivative of valencene and can be produced by enzymatic or by synthetic conversion of valencene.
  • Nootkatone occupies a small part of the essential oil but has a dominant role in the flavor and aroma of grapefruit.
  • Valencene and nootkatone are extensively used as an aroma component of citrus fruit and citrus- derived odorants.
  • Valencene is an important compound for the flavour and fragrance (F&F) industry. It is applied in beverage and food. It has also an important place in the cosmetic industry.
  • F&F flavour and fragrance
  • Valencene is present in a number of citrus plant species like grapefruit Citrus paradisi and orange Citrus sinensis but also in non citrus fruit plants like Perilla frutescens (beefsteak-mint) and Vitis vinifera L. (wine grape).
  • Valencene and nootkatone are produced in plants as an secondary metabolite and as most secondary metabolites they are produced only in relatively small quantities.
  • natural valencene is isolated from Valencia oranges Citrus sinensis cv. Valencia. Due to small quantities present in these oranges natural valencene is quite expensive.
  • Biotechnological production of these compounds could be a rational approach to obtain natural valencene and nootkatone in big quantities and at lower costs.
  • a valencene synthase with a high product specificity is a prerequisite for such an approach.
  • not all valencene synthase enzymes have a very high product specificity, which is of paramount importance for biotechnological production of pure compounds.
  • the present invention according to a first aspect, by providing an isolated nucleic acid sequence with a nucleic acid sequence having 70%, preferably 80%, more preferably 85%, most preferably 90% nucleotide sequence identity with SEQ ID NO. 1.
  • the present invention relates to an isolated nucleic acid sequence with a nucleic acid sequence having 95% nucleotide sequence identity with SEQ ID NO.1.
  • Sequence identity is defined as the number of identical nucleic acids, over the full length of the present sequences, divided by the number of nucleic acids, of the full length and multiplied by 100%.
  • the present isolated nucleic acid sequence has a nucleic acid sequence of SEQ ID NO. 1.
  • the present invention is based on the identification and isolation of a gene which is involved in the valencene synthesis.
  • This gene is identified in the plant Coleus forskohlii and encodes valencene synthase.
  • the valencene synthase encoded by the nucleic acid sequence according to the present invention produces nearly exclusive valencene. Due to this very high product specificity, the present polypeptide, or enzyme, provides advantageous properties for biotechnological production of natural valencene and its putative derivative nootkatone.
  • the present invention relates to an isolated polypeptide, or enzyme, encoded by the isolated nucleic acid sequence of the present invention.
  • the present invention relates to isolated polypeptides, or enzymes, having 70%, preferably 80%, more preferably 85%, most preferably 90% or 95% amino acid sequence identity with SEQ ID NO. 2.
  • Sequence identity is defined as the number of identical amino acids, over the full length of the present sequence, divided by the number of amino acids, of the full length and multiplied by 100%.
  • the present invention relates to an isolated polypeptide, or enzyme, having an amino acid sequence of SEQ ID NO.2.
  • the present nucleic acids can be used to recombinantly express, or provide, the present polypeptide, or enzyme. Accordingly, according to a third aspect, the present invention relates to expression vectors comprising the present isolated nucleic acid sequence.
  • an expression vector according to the present invention will comprise, besides the present nucleic acid, transcription and translation regulating sequences suitable for expression of the present nucleic acids in a host organism desired.
  • Expression vectors generally also comprise selection and/or marker sequences.
  • a variety of expression vectors for insect, mammal, plant, yeast, bacterium and/or fungus expression of the present nucleic acids are commercially available.
  • the present invention relates to an expression vector which further comprises a gene construct encoding for a polypeptide selected from the group consisting of CoA carboxylase, hydroxyl-methyl-glutaryl CoA reductase, squalene synthase, acetoacetyl-CoA thiolase, 3-hydroxy-3-methyl-glutaryl-CoA synthase, 3-hydroxy-3-methyl-glutaryl-CoA reductase, mevalonate kinase, mevalonate phosphate kinase, mevalonate diphosphate decarboxylase, 1-deoxy-D -xylulose 5-phosphate synthase, 1- deoxy-D -xylulose 5-phosphate reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, 4-
  • valencene precursor preferably farnesyl pyrophosphate
  • This valencene precursor can be converted into valence by the valencene synthase according to the present invention.
  • the present expression vector further comprises a gene construct encoding for the cytochrome P450, or CYP.
  • This embodiment is particularly advantageous if the production of nootkatone is desired since P450 provides the enzymatic conversion of the valence produced to nootkatone. Accordingly, one expression vector provides the genetic information needed for the production of nootkatone.
  • the present invention relates to transgenic organisms comprising the present expression vector or the present nucleic acid sequence.
  • the organisms according to the present invention are preferably selected from the group consisting of plant, bacterium, fungus, such as Mortierella isabellina and yeast.
  • the present plant is selected from the group consisting of the genera Carum, Cichorium, Daucus, Juniperus, Chamomilla, Lactuca, Pogostemon, Vetiveria, Capsicum, Gossyium, Lycopersicon, Nicotiana, Phleum, Solanum and Ulmus or is selected from Glycine max, Helianthus annuus and Brassica napus. It is advantageous to use a plant already producing sesquiterpenes, as these plants already have the basic pathway and storage compartments available.
  • a plant that already is producing nootkatone or is able to catalyse the conversion of valencene into nootkatone is advantageous because the enzyme(s) catalysing the conversion of valencene into nootkatone is already present.
  • the present yeast is selected from the group consisting of Yarrowia lipolytica, Cryptococcus curvatus, Rhodosporidium toruloides, Lipomyces starkeyi, Rhodotorula glutinis, Geotrichum robustum and Saccharomyces cerevisiae and Pichia pastoris.
  • the advantage of using oleaginous yeast host cells since they have the capacity to accumulate up to about 50% (dry weight) of storage carbohydrates in oil bodies, providing the production of large quantities of terpenes.
  • present nucleic acid sequence and the present polypeptide, or enzyme, according to the invention, as outlined above, provide the production of valencene and/or nootkatone.
  • the present invention relates to the use of the present nucleic acid, polypeptide, expression vectors or transgenic organism, as outlined above, for the production of valencene and or nootkatone.
  • the present invention relates also to the use of the plant Coleus forskohlii for the production of valencene and or nootkatone.
  • Figure 1 shows valencene synthase encoding cDNA part between start and stop codon flanked by Ncol and BamHl sites, respectively, subcloned in the NcoUBamHl site of the expression vector pET l id.
  • FIG. 2 SDS-PAGE gel: lanes 1 - 5 are all showing the pellets fractions: lane 1 and 2 valencene synthase-pETl Id construct in BL21(DE3), lane 3 and 4 amorpha-4, l l-diene synthase-pETl ld construct in BL21(DE3) (positive control). Lane 5 pETl ld without insert in BL21(DE3) (negative control). M: PageRulerTM 10-200 KDa ladder (Fermentas).
  • Lanes 6 - 10 are all showing the supernatant fractions: lane 6 and 7 valencene synthase- pETl Id construct in BL21(DE3), lane 8 and 9 amorpha-4, 11-diene synthase-pETl ld construct in BL21(DE3) (positive control). Lane 10 pETl ld without insert in BL21(DE3) (negative control). Amorpha-4, 11- diene synthase-pETl ld construct in BL21(DE3) (positive control) and valencene synthase-pETl ld construct in BL21(DE3) showed a intens spot which was not present in the negative control pET l id.
  • FIG. 3 Upper part: Flame Ionization Detector (FID) signals of the FPP assay with terpene synthases in expression vector pETl Id in BL21(DE3).
  • FID Flame Ionization Detector
  • Mass spectrum of the FPP assay with terpene synthases in expression vector pETl Id in BL21(DE3) lower part: Mass spectrum of the FPP assay with terpene synthases in expression vector pETl Id in BL21(DE3).
  • Figure 4 Mass spectrum of reference valencene compared with mass spectrum of the FPP assay with the terpene synthases of the invention (SEQ ID NO.2) in expression vector pETl Id in BL21(DE3). This comparison yielded a quality score of 99%, corresponding with a maximum score of identicalness.
  • Figure 5 Schematic representation of pSYL092. Valencene synthase flanked by the
  • TEF promoter and XPR2 terminator of Y lipolytica and with the URA3 auxotrophic selection marker are included in the TEF promoter and XPR2 terminator of Y lipolytica and with the URA3 auxotrophic selection marker.
  • FIG. 6 Schematic representation of pSYL094. tHMG-CoA encoding gene flanked by the TEF promoter and XPR2 terminator of Y lipolytica and with the Leu2 auxotrophic selection marker.
  • Total RNA was isolated from the roots of Coleus forskohlii by using the
  • the reverse transcription reaction was carried out in a 20 ⁇ reaction containing 2,5 ⁇ g total RNA, 0.2 ig Oligo dT primer (SEQ ID NO.10); 5' -AAT CGA TAG GCC GAG GCG GCC GAT CAG-(T)28-(A/C/G)-(A/T/C/G)-3', 0.5 niM each dATP, dTTP, dCTP and dGTP, 10 niM DTT, 2 U "RiboLockTM" ribonuclease inhibitor (Fermentas), first strand synthesis buffer
  • terpene synthases of the biosynthetic pathways leading to production of mono-, sesqui-, and di-terpenes are ubiquitous to all plant species. Although sequence conservation is not very high among terpene synthases of different plant species, discrete conserved domains are present. These conserved domains have been the basis for isolation of a number of terpene synthases encoding genes from a variety of plant species using degenerate-primer based RT-PCR.
  • degenerated primers were designed for two conserved regions.
  • the degenerated primers were designed in such a way that they have high homology with sesquiterpene synthase encoding genes.
  • the sequence of the degenerated sense primer (primer A) was 5'-GA(C/T) GA(G/A) AA(C/T) GGI AA(G/A) TT(C/T) AA(G/A)
  • PCR was performed in a total volume of 100 ⁇ containing 0.5 ⁇ of each of these two primers, 0.2 mM each dNTP, 1,5 U Pfu DNA polymerase/lx Pfu PCR buffer (Fermentas) and 2 ⁇ cDNA.
  • the reaction was incubated in a thermocycler (Biometra T-gradient) with 45 seconds denaturation at 94°C, 45 seconds annealing at 40°C and 1 minute and 15 seconds elongation at
  • the by PCR obtained sequence was used to design a specific sense (primer C) and anti sense (primer D) primer.
  • the sequence of the sense primer C was 5' -CCA GGG GTT TGC TGA GTC TGT A-3 ' (SEQ ID NO.13) and the sequence of the anti sense primer D was 5 ' -GCG ATG GTC TTG GTG AGC ATG AT-3' (SEQ ID NO.14).
  • the 3' part of the mature cDNA was amplified by PCR with the sense primer C and the oligo dT primer (5'-AAT CGA TAG GCC GAG GCG GCC GAT CAG-(T)28-(A/C/G)-(A/T/C/G)-3') as the anti sense primer.
  • PCR was performed in a total volume of 50 ⁇ containing 0.5 ⁇ of each of these two primers, 0.2 mM each dNTP, 1.5 U Pfu DNA polymerase/lx Pfu PCR buffer (Fermentas) and 2 ⁇ cDNA.
  • the reaction was incubated in a thermocycler (Biometra T-gradient) with 45 seconds denaturation at 92°C, 45 seconds annealing at 58°C and 1 minute and 30 seconds elongation at 68°C during 40 cycles.
  • new cDNA was synthesised with primer E (5 ' -GCC AACTCTTGTTCAAATTGAT-3 ' ) (SEQ ID NO.15) and under the conditions as described under B "cDNA Synthesis".
  • the obtained cDNA was tailed by Terminal deoxynucleotidyl Transferase (TdT) yielding a poly A tail at the 3 'part of the cDNA.
  • TdT Terminal deoxynucleotidyl Transferase
  • the poly A tailing was performed in a total volume of 30 ⁇ containing 20 ⁇ of approximately 1 pmol cDNA, 6 ⁇ 5x TdT buffer (Fermentas), and 3 ⁇ 2mM dATP.
  • the PCR was performed in a total volume of 50 ⁇ containing 0.5 ⁇ of each of these two primers, 0.2 mM each dNTP, 1.5 U Pfu DNA polymerase/lx Pfu PCR buffer
  • the reaction was incubated in a thermocycler (Biometra T-gradient) with 45 seconds denaturation at 92°C, 45 seconds annealing at 58°C and 1 minute and 30 seconds elongation at 68°C during 40 cycles.
  • Agarose gel electrophoresis revealed a major PCR product of
  • primer G (SEQ ID NO.17), with an Ncol site (underlined) introduced at the start codon ATG (Italic), and primer H (5'-
  • the cDNA clone was subcloned, in frame, into the expression vector pET l id (Stratagene). After digestion with Bamffl and Ncol of the PCR product of SEQ ID NO.1 and the expression vector pET l id both were gel purified and ligated together to yield a construct as revealed in figure 1.
  • this gene construct (figure 1), pET l id without an insert as negative control, and pET l id with the Artemisia annua L. amorpha-4,11-diene synthase (ADS) gene (Wallaart et al., Planta 212(3); 460-465 (2001)); as positive control were transformed to E.coli BL21 (DE3) (Stratagene), and grown overnight on LB agar plates supplemented with ampicillin at 37°C.
  • ADS Artemisia annua L. amorpha-4,11-diene synthase
  • the obtained supernatant was used for terpene synthase enzyme activity determinations.
  • Supernatant and pellet of the valencene synthase -pET 1 Id construct in E.coli BL21 (DE3) yielded on SDS-PAGE gel a protein band of approximately 50 to 60 kDa. This was about the same size as seen for, the positive control, amorpha-4,l l-diene synthase.
  • the pET l id vector without an insert, the negative control did not reveal such a protein band on the SDS-PAGE gel, as is shown in figure 2.
  • FPP Farnesyl pyrophosphate
  • pentane/diethyl-ether mixture was slowly concentrated under a stream of N 2 gas.
  • GC-MS analyses of the concentrated pentane/diethyl-ether mixtures were performed using a HP 5890 series II GC and HP 5972A Mass Selective Detector (Hewlett- Packard) equipped with an HP-5 MS or HP-Innowax column (both 30 m x 0.25 mm i.d., 0.25 ⁇ df).
  • the oven was programmed at an initial temperature of 70°C. for 1 min, with a ramp of 5°C. min "1 , to 210°C. and final time of 5 min.
  • the injection port (splitless mode), interface and MS source temperatures were 175, 290 and 180°C., respectively, and the He inlet pressure was controlled by electronic pressure control to achieve a constant column flow of 1.0 ml min "1 .
  • Ionization potential was set at 70 eV, and scanning was performed from 30-250 amu.
  • GC-MS analysis on the two different GC-columns of FPP assay products by the sonicated preparation showed that the main product had a mass spectrum and retention time equal to that of the reference valencene (see figure 3 and figure 4).
  • DNA there are many ways to introduce DNA into a plant cell. Suitable methods by which DNA can be introduced into the plant cell include Agrobacterium infection or direct delivery of DNA such as, for example, by PEG-mediated transformation of protoplasts (Omirulleh et al., Plant Molecular Biology 21, 415-428 (1993)) or electroporation, by acceleration of DNA coated microprojectiles (for example, microprojectile bombardment) microinjection, etc.
  • a suitable promoter for example, the cauliflower mosaic virus (CaMV) 35S promoter
  • a suitable terminator for example, the nopaline synthase transcription terminator (nos-tail)
  • suitable restriction sites for subcloning were introduced by using PCR with a sense primer I 5'-GCGGATCCArGGCTCAAGTGCAATCGGAAATTG -3' (SEQ ID NO.19) introducing a BamHl site (underlined) preceding the start codon (Italic) and an anti-sense primer J 5'-GCGGATCCTCTAAATTAGAATGGGGTCGACGAAC-3' (SEQ ID NO.20) (introducing a BamHl site (underlined) directly behind the stop codon (Italic).
  • the plant-expression casette pLV399 is a pUC 19 vector (Yanisch-Perron, C. et al., Gene 33, 103-119 (1985)) in which the multiple cloning site (polylinker) is replaced by a CaMV 35 S promoter BamHl fused to a nos-tail
  • terminal flanked by the 'unique' sites; EcoRl, Kpnl, Xhol, and a HmdIII site downstream from the promoter and EcoRl, Xhol, Pstl, Sphl, Kpnl, Hindlll upstream from the terminator.
  • the orientation of the valencene synthase encoding gene in pLV399 was checked by restriction analysis with Sphl. After digestion of this construct with Kpnl the valencene synthase encoding gene flanked by the 35S promotor and nos terminator was ligated into the Kpnl digested binary vector pCGN1548.
  • This transformed Agrobacterium strain was used for transformation of explants from the target plant species. Only the transformed tissue carrying a resistance marker (kanamycin- resistance, present between the binary plasmid T-DNA borders) regenerated on a selectable (kanamycin containing) regeneration medium. (According to Rogers S G, Horsch R B, Fraley R T Methods Enzymol (1986)118: 627-640).
  • the plants regenerated out of the transformed tissue expressed the valencene synthase gene as followed from the presence therein of valencene as confirmed by GC-MS analyses.
  • valencene synthase encoding cDNA in Yarrowia lipolytica expression signals like a promoter and terminator sequence were cloned.
  • the XPR2 terminator was amplified by PCR using primer K (5 ' -CAC AAACTAGTTTGCCACCTAC AAGCC AGAT-3 ' ) (SEQ ID N0.21) and primer L (5'-
  • Genomic DNA from wild type strain Yarrowia lipolytica SYL291 was isolated using the G-nome DNA kit (MP Biomedicals, Solon, USA) according to the manufactures protocol. The PCR was performed in a total volume of 50 ⁇ containing 0.5 ⁇ of each of these two primers, 0.2 mM of each dNTP, 1.5 U Pfu DNA polymerase/ lx Pfu PCR buffer (Fermentas) and 2 ⁇ genomic DNA of wild type strain Yarrowia lipolytica SYL291. The reaction was incubated in a
  • thermocycler Biometra T-gradient
  • initial denaturation step 2 minutes at 92°C followed by 40 cycles of 45 seconds denaturation at 92°C, 45 seconds annealing at 58°C and 1 minute and 30 seconds elongation at 68°C.
  • Agarose gel electrophoresis revealed a specific PCR product of approximately 200 bp. A 3 ⁇ overhang was introduced by using Taq DNA polymerase (Fermentas). The obtained sticky DNA was "TA” subcloned in pGEM-Teasy (Promega Corporation, Madison, USA) yielding pSYL076.
  • the XPR2 terminator flanked by Notl and Spel and subcloned into pGEM-Teasy pSYL076 was cut with Notl and Spel yielding a 200 bp fragment containing the XPR2 terminator.
  • the TEF promoter flanked by and Kpnl and Notl and subcloned into pGEM-Teasy pSYL077 was cut with Kpnl and Notl yielding a 400 bp fragment containing the TEF promotor. Both fragments were ligated in a 3 points ligation in Kpnl and Spel cut pBluescriptll KS (-) yielding vector pSYL083.
  • the Coleus forskohlii valencene synthase encoding cDNA from example 1 (SEQ ID NO.l) was amplified by PCR as described above using the sequence of SEQ ID NO.l as template with primers O (5'-
  • This vector (pSYL085) was cut with Xbal and Mlul yielding a fragment of approximately 1500 bp containing the valencene synthase encoding cDNA.
  • XPR2 terminator containing vector pSYL083 was cut open with Mlul and Nhel.
  • the valencene synthase encoding cDNA flanked by Xbal and Mlul was ligated into Mlul and Nhel opened pSYL083. Yielding a valencene synthase encoding cDNA flanked by the TEF promoter and XPR2 terminator in pBluescriptll KS (-) pSYL086.
  • the Leu2 selection marker containing vector pSYL090 and the valencene synthase encoding cDNA flanked by the TEF promoter and XPR2 terminator pSYL086 were cut with Xbal.
  • the 2.5 kb fragment from pSYL090 was ligated in pSYL086 yielding pSYL092 see figure 5.
  • tHMG-CoA red SEQ ID NO.l 1
  • primers were designed to amplify a part of the HMG CoA reductase gene to obtain truncated HMG CoA reductase by PCR.
  • the PCR was performed as described above with genomic DNA isolated from wild type strain Yarrowia lipolytica SYL291 as template.
  • Primers used in this reaction were primer S (5'- CACACTCTAGAC ACAAAAATGACCCAGTCTGTGAAGGTGGTTGAGAAG-3 ' ) (SEQ ID N0.29) and primer T (5 ' -C ACAC ACGCGT ACACCTATGACCGTATGCAAAT-3 ' ) (SEQ ID NO.30) yielding a PCR product of about 1400 bp.
  • the PCR product was ligated in pGem-T easy (Promega Corporation, Madison, USA) according to the manufactures protocol yielding pSYL075.
  • the tHMG-CoA red gene was cut out pSYL075 with Xbal and Mlul and ligated into Mlul, Nhel cut pSYL083 yielding pSYL087.
  • auxotrophic selection marker "orotidine 5- phosphate decarboxylase” (URA3) was together with its own promoter and terminator amplified by PCR as described above with the primers U (5'-
  • Ura3 selection marker containing vector pSYL091 and the tHMG-CoA encoding gene flanked by the TEF promoter and XPR2 terminator pSYL087 were cut with Xbal.
  • the 1.9 kb fragment from pSYL091 was ligated in pSYL087 yielding pSYL094, see figure 6.
  • Yarrowia lipolytica SYL312 (Aura3, Aleu2) was transformed with linearized pSYL092 according to Chen et al., 1997 yielding Yarrowia lipolytica SYL320 (valencene, Aleu2).
  • Possible transformants were checked by PCR with the primers O and P based on the valencene synthase encoding cDNA. Stability of the transformants was checked via culturing without selection pressure (YPD plates containing uracil and leucine) followed by culturing on YNB plates without uracil. Yarrowia lipolytica SYL320 (valencene, Aleu2) was transformed with linearized pSYL094 according to Chen et al., 1997 yielding Yarrowia lipolytica SYL321 (valencene, tHMG- CoA). These double transformants were checked by PCR with the primers T and S for the presence of the tHMG-CoA gene.

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Abstract

La présente invention concerne une séquence d'acide nucléique isolée dont la séquence d'acide nucléique correspond à 70 %, de préférence à 80 %, de préférence encore à 85 %, de préférence encore à 90 % à la séquence nucléotidique SEQ ID NO. 1. La présente invention concerne spécifiquement un gène, et le polypeptide qu'il code, impliqués dans la synthèse de valencène.
PCT/EP2015/065218 2014-07-04 2015-07-03 Gène et polypeptide impliqués dans la synthèse de valencène et leurs utilisations Ceased WO2016001412A1 (fr)

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CN107723252A (zh) * 2017-09-22 2018-02-23 天津大学 生产巴伦西亚橘烯和诺卡酮的重组解脂耶氏酵母菌及构建方法
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CN114107078A (zh) * 2021-10-29 2022-03-01 湖北冠众通科技有限公司 一种高产瓦伦烯基因工程菌及其构建方法与应用

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