WO2024252885A1 - Procédé de production de cellobiose - Google Patents

Procédé de production de cellobiose Download PDF

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WO2024252885A1
WO2024252885A1 PCT/JP2024/018152 JP2024018152W WO2024252885A1 WO 2024252885 A1 WO2024252885 A1 WO 2024252885A1 JP 2024018152 W JP2024018152 W JP 2024018152W WO 2024252885 A1 WO2024252885 A1 WO 2024252885A1
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phosphorylase
cellobiose
sucrose
yeast
seq
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清綱 豊原
智也 ▲濱▼田
淳一 石丸
健太郎 猪熊
寿 安枝
誠久 蓮沼
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Kobe University NUC
Teijin Ltd
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Teijin Ltd
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    • 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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C12N9/10—Transferases (2.)
    • 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
    • C12N9/90—Isomerases (5.)
    • C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides

Definitions

  • the present invention relates to a method for producing cellobiose from sucrose using yeast that expresses an enzyme on its surface.
  • Enzymatic degradation and enzymatic synthesis methods have been known as methods for producing cellobiose.
  • an enzymatic degradation method is known in which a cellulase preparation is reacted with cellulose to obtain cellobiose (Patent Document 1, Patent Document 2).
  • Non-Patent Document 1 an enzymatic synthesis method is known in which starch is used as a starting material and ⁇ -glucan phosphorylase and cellobiose phosphorylase are allowed to act on the starch to obtain cellobiose
  • Patent Document 3 a method is known in which sucrose is used as a starting material and sucrose phosphorylase, glucose isomerase, and cellobiose phosphorylase are allowed to act on the sucrose to obtain cellobiose.
  • cellulose is used as the raw material.
  • Cellulose is the main component of plant cell walls, but it usually exists as a mixture with hemicellulose, lignin, etc. Therefore, when using cellulose in such plant cell walls as a raw material, it is first necessary to remove the hemicellulose, lignin, etc. by methods such as alkali treatment, which poses the problem of high production costs for the raw material.
  • the enzymatic synthesis method has low conversion efficiency from starch to glucose 1-phosphate and from sucrose to cellobiose, and requires reactions to be carried out under mild conditions (around 30°C) using enzymes, resulting in a slow reaction rate and making it unsuitable for low-cost cellobiose production.
  • the object of the present invention is to provide a method for producing cellobiose from sucrose with a high conversion rate and at low cost.
  • the present inventors have discovered that by allowing sucrose to coexist in a reaction solution containing yeast expressing sucrose phosphorylase (hereinafter sometimes referred to as "SP") and cellobiose phosphorylase (hereinafter sometimes referred to as “CBP”) and glucose isomerase (hereinafter sometimes referred to as "XI”), it is possible to produce cellobiose at a high conversion rate and at low cost, and have completed the present invention.
  • SP sucrose phosphorylase
  • CBP cellobiose phosphorylase
  • XI glucose isomerase
  • the present invention includes the following inventions.
  • [Item 1] A method for producing cellobiose, comprising a reaction step of allowing sucrose to coexist in a reaction solution containing a recombinant yeast expressing sucrose phosphorylase and cellobiose phosphorylase and glucose isomerase.
  • [Item 2] The production method according to Item 1, wherein the recombinant yeast is a yeast of the genus Saccharomyces, Pichia, or Yarrowia.
  • [Item 3] The production method according to Item 1 or 2, wherein the sucrose phosphorylase is derived from a bacterium of the genus Bifidobacterium, Leuconostoc, or Streptococcus.
  • sucrose phosphorylase comprises a secreted sucrose phosphorylase secreted and expressed from a recombinant yeast, and an tethered sucrose phosphorylase expressed in a state tethered to the cell wall of the recombinant yeast.
  • the cellobiose phosphorylase is derived from a bacterium of the genus Clostridium, Ruminococcus, Cellulomonas, or Thermotoga.
  • [Item 6] The method according to any one of Items 1 to 5, wherein the recombinant yeast is a recombinant yeast that expresses both sucrose phosphorylase and cellobiose phosphorylase in a single cell.
  • the recombinant yeast is a combination of a recombinant yeast expressing sucrose phosphorylase and a recombinant yeast expressing cellobiose phosphorylase.
  • the recombinant yeast is a yeast transformed with an expression vector carrying a sucrose phosphorylase gene.
  • [Item 9] The production method described in Item 8, wherein the expression vector comprises a combination of a first expression vector carrying both a sucrose phosphorylase gene and a yeast cell wall domain gene, and a second expression vector carrying only the sucrose phosphorylase gene but not the yeast cell wall domain gene.
  • the glucose isomerase is derived from a bacterium of the genus Streptomyces.
  • the method according to any one of Items 1 to 10 further comprising treating the recombinant yeast at 45° C. to 75° C. for 15 to 150 minutes before allowing sucrose to coexist in the reaction solution.
  • the present invention makes it possible to produce cellobiose from sucrose with a high conversion rate and low cost.
  • FIG. 1 is a graph showing the results of measuring the sucrose phosphorylase activity of a recombinant yeast strain expressing sucrose phosphorylase.
  • FIG. 2 is a graph showing the changes over time in the concentrations of sucrose, glucose, fructose, and cellobiose in the reaction supernatant in an experiment to produce cellobiose from sucrose using a recombinant yeast strain expressing sucrose phosphorylase in combination with a recombinant yeast strain expressing cellobiose phosphorylase.
  • the graph in Figure 2(a) shows the results when a strain expressing cellobiose phosphorylase (CsCBP) derived from Clostridium stercorarium was used
  • the graph in Figure 2(b) shows the results when a strain expressing cellobiose phosphorylase (CuCBP) derived from Cellulomonas uda was used
  • the graph in Figure 2(c) shows the results when a strain expressing cellobiose phosphorylase (TaCBP) derived from Thermosipho africanus was used
  • the graph in Figure 2(d) shows the results when a strain expressing cellobiose phosphorylase (TnCBP) derived from Thermotoga neapolitana was used.
  • Figure 3 is a graph showing the time course of the concentrations of sucrose, glucose, fructose, and cellobiose in the reaction supernatant in an experiment to produce cellobiose from sucrose using a single recombinant yeast strain co-expressing sucrose phosphorylase and cellobiose phosphorylase.
  • the graph in Figure 3(a) shows the results when a recombinant yeast with Saccharomyces cerevisiae as the host was used
  • the graph in Figure 3(b) shows the results when a recombinant yeast with Pichia pastoris as the host was used.
  • Figure 4 is a graph showing the time course of the concentrations of sucrose, glucose, fructose, and cellobiose in the reaction supernatant of an experiment to produce cellobiose from sucrose using a single recombinant yeast strain co-expressing sucrose phosphorylase and cellobiose phosphorylase with Pichia pastoris as the host.
  • the graph in Figure 4(a) shows the results when the Pp-SP2CBP strain was cultured using glucose as the carbon source
  • the graph in Figure 4(b) shows the results when the Pp-SP2CBP strain was cultured using glycerol as the carbon source.
  • Figure 5 is a graph showing the time course of sucrose, glucose, fructose, and cellobiose concentrations in the reaction supernatant of a repeated experiment of cellobiose production from sucrose using a single recombinant yeast strain co-expressing sucrose phosphorylase and cellobiose phosphorylase.
  • the graph in Figure 5(a) shows the results when recombinant yeast with Saccharomyces cerevisiae as the host was cultured using glucose as the carbon source
  • the graph in Figure 5(b) shows the results when recombinant yeast with Pichia pastoris as the host was cultured using glycerol as the carbon source.
  • identity of amino acid sequences means the proportion of identical amino acid residues
  • similarity means the proportion of identical or similar amino acid residues.
  • homology and identity of amino acid sequences can be determined, for example, by the BLAST method (NCBI PBLAST default conditions). Furthermore, for example, when it is expressed as “80% or more homology,” it clearly includes the case of "80% or more identity.”
  • similar amino acid residues refers to amino acid residues having side chains with similar chemical properties (e.g., charge or hydrophobicity).
  • similar amino acid residues include the following combinations: (1) Amino acid residues having an aliphatic side chain: glycine (Gly or G), alanine (Ala or A), valine (Val or V), leucine (Leu or L), and isoleucine (Ile or I) residues. (2) Amino acid residues having aliphatic hydroxyl side chains: serine (Ser or S) and threonine (Thr or T) residues.
  • Amino acid residues having amide-containing side chains asparagine (Asn or N) and glutamine (Gln or Q) residues.
  • Amino acid residues having aromatic side chains phenylalanine (Phe or F), tyrosine (Tyr or Y), and tryptophan (Trp or W) residues.
  • Amino acid residues having basic side chains lysine (Lys or K), arginine (Arg or R), and histidine (His or H) residues.
  • Amino acid residues having acidic side chains aspartic acid (Asp or D) and glutamic acid (Glu or E) residues.
  • Amino acid groups having sulfur-containing side chains cysteine (Cys or C) and methionine (Met or M) residues. Furthermore, the combination of (1) and a methionine (Met or M) residue, and the combination of (4) and a histidine (His or H) residue are also treated as similar amino acid residues.
  • One aspect of the present invention relates to a method for producing cellobiose.
  • the method includes a step of allowing sucrose to coexist in a reaction solution containing a recombinant yeast expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP) and glucose isomerase (XI).
  • SP sucrose phosphorylase
  • CBP cellobiose phosphorylase
  • XI glucose isomerase
  • the yeast species is not particularly limited as long as it is capable of expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP).
  • examples include yeasts of the genus Saccharomyces, Pichia, or Yarrowia.
  • yeasts that do not have invertase activity on the cell surface such as Pichia pastoris.
  • yeasts that have strong invertase activity on the cell surface such as Saccharomyces cerevisiae, it is preferable to use a strain in which invertase activity has been lost by gene knockout or the like.
  • Pichia pastoris since Pichia pastoris has been classified and renamed as Komagataella pastoris and Komagataella phaffii, Pichia pastoris is a synonym for both Komagataella pastoris and Komagataella phaffii.
  • sucrose phosphorylase The origin (species) of sucrose phosphorylase (SP) is not particularly limited. Examples include those derived from bacteria of the genus Bifidobacterium, Leuconostoc, or Streptococcus. Of these, those derived from bacteria of the genus Bifidobacterium are preferred, and those derived from Bifidobacterium longum are particularly preferred.
  • the amino acid sequence of sucrose phosphorylase (SP) from Bifidobacterium longum is shown in SEQ ID NO: 34, and an example of a base sequence encoding this is shown in SEQ ID NO: 5.
  • sucrose phosphorylase (SP) derived from Bifidobacterium longum examples include proteins consisting of an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more homology (preferably identity) with SEQ ID NO:34.
  • sucrose phosphorylase When sucrose phosphorylase (SP) is expressed in recombinant yeast, it can be in the form of a secreted enzyme secreted and expressed from the recombinant yeast, or in the form of an tethered enzyme expressed in a state tethered to the cell wall of the recombinant yeast, but either form is acceptable. However, in the present invention, it is preferable to allow the secreted sucrose phosphorylase (SP) secreted and expressed from the recombinant yeast and the tethered sucrose phosphorylase (SP) expressed in a state tethered to the cell wall of the recombinant yeast to coexist in the reaction solution. This makes it possible to improve the conversion efficiency by sucrose phosphorylase (SP). The reason for this is unclear, but it is speculated to be due to the formation of a dimer between the secreted sucrose phosphorylase (SP) and the tethered sucrose phosphorylase (SP).
  • CBP cellobiose phosphorylase
  • SEQ ID NO: 35 amino acid sequence of cellobiose phosphorylase (CBP) from Clostridium stercorarium is shown in SEQ ID NO: 35, and an example of the base sequence encoding this is shown in SEQ ID NO: 6.
  • CBP cellobiose phosphorylase
  • CBP cellobiose phosphorylase
  • it may be in the form of a secreted enzyme secreted and expressed from the recombinant yeast, or in the form of an tethered enzyme expressed in a state tethered to the cell wall of the recombinant yeast, either of which may be used.
  • it is preferable to express it in the form of tethered cellobiose phosphorylase (CBP) expressed in a state tethered to the cell wall of the recombinant yeast.
  • recombinant yeast As the recombinant yeast, a recombinant yeast that co-expresses sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP) in a single cell may be used, or a recombinant yeast that expresses sucrose phosphorylase (SP) may be used in combination with a recombinant yeast that expresses cellobiose phosphorylase (CBP).
  • SP sucrose phosphorylase
  • CBP cellobiose phosphorylase
  • sucrose phosphorylase SP
  • CBP cellobiose phosphorylase
  • a recombinant yeast that co-expresses the secreted enzyme and the tethered enzyme in a single cell may be used, or a recombinant yeast that expresses the secreted enzyme may be used in combination with a recombinant yeast that expresses the tethered enzyme.
  • the method for preparing recombinant yeast expressing sucrose phosphorylase (SP) and/or cellobiose phosphorylase (CBP) is not particularly limited.
  • one or more types of yeast are transformed with one or more expression vectors carrying the sucrose phosphorylase (SP) gene and/or the cellobiose phosphorylase (CBP) gene.
  • expression vectors include plasmids, cosmids, lambda phages, etc., with plasmids being preferred.
  • sucrose phosphorylase (SP) gene and/or cellobiose phosphorylase (CBP) gene are operably linked to regulatory sequences such as a promoter and a terminator, and carried in the expression vector in the form of an expression cassette that can be expressed autonomously in yeast cells.
  • regulatory sequences such as a promoter and a terminator
  • Such expression vectors can be constructed using standard genetic engineering techniques known to those skilled in the art, by referring appropriately to literature such as Michael R. Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, (2012), Cold Spring Harbor Laboratory.
  • the recombinant yeast is preferably pre-cultured in the presence of a carbon source prior to the reaction.
  • a carbon source there are no particular limitations on the type of carbon source used when culturing the recombinant yeast cells, and it may be selected appropriately depending on the type of yeast. Examples include glucose and glycerol. Of these, in the present invention, it is preferable to use at least glycerol as a carbon source. This can improve the conversion rate of sucrose to cellobiose.
  • a pretreatment prior to the reaction to stop the uptake of glucose.
  • An example of such a pretreatment is a heat treatment in which the yeast is exposed to a certain temperature for a certain period of time.
  • the treatment conditions are sufficient as long as they can stop the uptake of glucose by the yeast used.
  • the heating temperature can be, for example, 45°C or higher, or 50°C or higher, or 55°C or higher, and can be, for example, 75°C or lower, or 70°C or lower, or 65°C or lower.
  • the heating time can be, for example, 15 minutes or more, or 45 minutes or more, or 70 minutes or more, and can be, for example, 150 minutes or less, 120 minutes or less, or 100 minutes or less.
  • the temperature can be about 60°C, and the time can be about 90 minutes.
  • One example of such a pretreatment method is shaking a suspension of the transformed yeast under the above conditions.
  • glucose isomerase (XI) is allowed to coexist in a reaction solution containing recombinant yeast expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP).
  • the amount of recombinant yeast cells in the reaction solution is not particularly limited, but can be, for example, 25 g wet cells/L or more, 50 g wet cells/L or more, or 75 g wet cells/L or more, and can be, for example, 200 g wet cells/L or less, 150 g wet cells/L or less, or 100 g wet cells/L or less.
  • Glucose isomerase also known as xylose isomerase, XI
  • XI xylose isomerase
  • Glucose isomerase may be expressed in yeast or other microorganisms in the same manner as other enzymes, but it is preferable to use the isolated glucose isomerase (XI) enzyme itself.
  • the isolated glucose isomerase (XI) enzyme may be either a commercially available product or one obtained by culturing a microorganism that produces these enzymes. It may also be either a purified product or an unpurified product. In this case, it is preferable to add the isolated glucose isomerase (XI) enzyme to a reaction solution containing recombinant yeast that expresses sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP) and allow them to coexist.
  • SP sucrose phosphorylase
  • CBP cellobiose phosphorylase
  • the amount of glucose isomerase (XI) is not particularly limited, but is usually preferably 0.01 units or more, 0.1 units or more, or 1.0 units or more per mole of sucrose.
  • 1 unit (1 U) means the amount of enzyme that breaks down 1 ⁇ mol of fructose in 1 wt/vol % sucrose at 50°C and pH 7.0 per minute.
  • the reaction is carried out in the presence of sucrose in a reaction solution containing recombinant yeast expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP) and glucose isomerase (XI).
  • sucrose may be naturally occurring or chemically synthesized.
  • concentration as high as possible is preferable.
  • solubility of the cellobiose produced is about 15 to 20%, if it is desired to keep it dissolved, it is preferable to use a sucrose concentration of about 10 to 20%.
  • the conditions for the cellobiose synthesis reaction are not limited, but may be, for example, as follows. That is, the reaction temperature may be, for example, 35°C or higher, 45°C or higher, or 50°C or higher, and, for example, 60°C or lower, or 70°C or lower.
  • the reaction time may be, for example, 60 minutes or more, 12 hours or more, or 24 hours or more, and may be, for example, 36 hours or less, or 48 hours or less.
  • the pressure during the reaction is not particularly limited, and may be reduced pressure, normal pressure, or pressurized, but is usually carried out under normal pressure.
  • the atmosphere during the reaction is also not particularly limited, and any atmosphere may be used as long as the enzyme activity is maintained even in an atmosphere in which the recombinant yeast used cannot grow.
  • cellobiose can be obtained by separating and purifying it from the reaction solution as necessary.
  • any method can be used, including, for example, filtration separation, centrifugation, membrane separation, etc. These methods allow efficient separation of the used bacteria and cellobiose.
  • it may be purified to a higher degree by treatment with an ion exchange resin or recrystallization.
  • the recombinant yeast may be separated and recovered from the reaction solution as necessary and reused in the cellobiose synthesis reaction. Even when the recombinant yeast of the present invention is separated and recovered from the reaction solution and reused in this way, the enzyme activity may be maintained, and high cellobiose synthesis efficiency may be repeatedly obtained. That is, according to a preferred embodiment, the cellobiose production method of the present invention further includes recovering the recombinant yeast from the reaction solution after the reaction step and subjecting it to another reaction step.
  • the method for separating and recovering the recombinant yeast from the reaction solution is not limited, but examples include centrifugation.
  • the number of times the recombinant yeast is separated and recovered and reused is not particularly limited, but according to one embodiment, it can be subjected to a reaction, for example, two or more times, three or more times, four or more times, or five or more times.
  • the activity of the recombinant yeast when reused is not particularly limited, but according to one embodiment, it is preferable that 60% or more, 70% or more, 80% or more, or 90% or more of the activity in the previous reaction is maintained in the next reaction.
  • Example Group I Cellobiose production using transformed yeast (yeast displaying sucrose phosphorylase on the surface, yeast displaying cellobiose phosphorylase on the surface, and yeast displaying sucrose phosphorylase + cellobiose phosphorylase on the surface)]
  • yeast Saccharomyces cerevisiae BY4741 strain MAT ⁇ his3 leu2 met15 ura3 strain
  • yeast Saccharomyces cerevisiae BY4741 ⁇ SUC2 strain MAT ⁇ his3 leu2 met15 ura3 suc2 strain
  • the yeast Saccharomyces cerevisiae BY4741 strain was obtained from ATCC (American Type Culture Collection), and the yeast Saccharomyces cerevisiae BY4741 ⁇ SUC2 strain was obtained from Yeast deletion MAT-A complete set (Thermo Fisher Scientific).
  • Plasmids containing the following expression cassettes X1 to X9 were prepared by the following procedure.
  • vector plasmid pIEG-SSSD (a surface expression vector having an expression cassette in which the SED1 promoter derived from Saccharomyces cerevisiae, the secretory signal peptide sequence of SED1 derived from Saccharomyces cerevisiae, the coding region of the endoglucanase II (TrEGII) gene derived from Trichoderma reesei, the coding region (SED1 anchoring region) of the SED1 gene derived from Saccharomyces cerevisiae, and the terminator region of the DIT1 gene derived from Saccharomyces cerevisiae are arranged in this order: Applied Microbiology and Biotechnology, Vol.105, 2021,
  • the resulting vector plasmid was amplified by PCR using primer pair SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) with pIBISP-SSSD (SEQ ID NO: 5895-590
  • the resulting plasmid containing expression cassette X1 (SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + BlSP (SEQ ID NO: 5) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)) was named pIBlSP-SSSD.
  • the primer pair BlSP-F (SEQ ID NO: 18) and DIT1t-R (SEQ ID NO: 19) were used to amplify by PCR to prepare a DNA fragment containing the coding region of the sucrose phosphorylase (BlSP) gene derived from Bifidobacterium longum and the terminator region of the DIT1 gene derived from Saccharomyces cerevisiae.
  • BlSP sucrose phosphorylase
  • vector plasmid pIL2-BG-SSS (a surface expression vector having an expression cassette in which the SED1 promoter derived from Saccharomyces cerevisiae, the secretory signal peptide sequence of SED1 derived from Saccharomyces cerevisiae, the coding region of the ⁇ -glucosidase 1 (AaBGL1) gene derived from Aspergillus aculeatus, the coding region (SED1 anchoring region) of the SED1 gene derived from Saccharomyces cerevisiae, and the terminator region of the ⁇ -agglutinin gene derived from Saccharomyces cerevisiae are arranged in this order: Metabolic Engineering, Vol.
  • pRS403-F SEQ ID NO: 20
  • pRS403-S SEQ ID NO: 15
  • pRS403-F SEQ ID NO: 20
  • pRS403-S SEQ ID NO: 15
  • pRS403-F SEQ ID NO: 20
  • a DNA fragment was prepared that contained the entire length of the vector plasmid excluding the coding region of the ⁇ -glucosidase 1 (AaBGL1) gene derived from Aspergillus aculeatus, the coding region (SED1 anchoring region) of the SED1 gene derived from Saccharomyces cerevisiae, and the terminator region of the ⁇ -agglutinin gene derived from Saccharomyces cerevisiae. These fragments were ligated by the In-Fusion method.
  • the resulting plasmid containing the expression cassette X2 (SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + BlSP (SEQ ID NO: 5) + DIT1 terminator (SEQ ID NO: 4)) was named pIL2-BlSP-SSD.
  • Preparation Example 1-3 Preparation of vector plasmid containing Clostridium stercorarium-derived cellobiose phosphorylase (CsCBP) surface expression cassette X3) A DNA fragment containing the coding region of the cellobiose phosphorylase (CsCBP) gene derived from Clostridium stercorarium was prepared by gene synthesis.
  • CsCBP Clostridium stercorarium-derived cellobiose phosphorylase
  • vector plasmid pIEG-SSSD (a surface expression vector having a cassette in which the SED1 promoter derived from Saccharomyces cerevisiae, the secretory signal peptide sequence of SED1 derived from Saccharomyces cerevisiae, the coding region of the endoglucanase II (TrEGII) gene derived from Trichoderma reesei, the coding region (SED1 anchoring region) of the SED1 gene derived from Saccharomyces cerevisiae, and the terminator region of the DIT1 gene derived from Saccharomyces cerevisiae are arranged in this order: Applied Microbiology and Biotechnology, Vol.105, 2021, Using the primer pair SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) and PCR amplification was performed using the 5'-terminal end of the primer pair SED1a-F (SEQ ID NO: 14)
  • SED1p-F SEQ ID NO: 21
  • DIT1t-R SEQ ID NO: 19
  • vector plasmid pIU5-CBH D (a surface expression vector having a cassette in which the Saccharomyces cerevisiae-derived SED1 promoter, the coding region of the Talaromyces emersonii-derived cellobiohydrolase (TsCBH1) gene, the coding region of the Saccharomyces cerevisiae-derived SED1 gene (SED1 anchoring region), and the terminator region of the Saccharomyces cerevisiae-derived ⁇ -agglutinin gene are arranged in this order: Biotechnology and Bioengineering, Vol.
  • TsCBH1 Talaromyces emersonii-derived cellobiohydrolase
  • the vector plasmid was amplified by PCR using primer pair pRS403-F (SEQ ID NO: 20) and pRS403-R (SEQ ID NO: 22) to prepare a DNA fragment containing the entire length of the vector plasmid excluding the expression cassette region. These fragments were ligated by the In-Fusion method.
  • the resulting plasmid containing expression cassette X3 (SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + CsCBP (SEQ ID NO: 6) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)) was named pIU5-CsCBP-SSSD.
  • the vector plasmid pIU5-CsCBP-SSSD was used as a template, and the primer pair SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) were used to amplify by PCR to prepare a DNA fragment containing the full length of the vector plasmid excluding the coding region of the CsCBP gene. These fragments were ligated by the In-Fusion method.
  • the resulting plasmid containing the expression cassette X4 (SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + CuCBP (SEQ ID NO: 7) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)) was named pIU5-CuCBP-SSSD.
  • the vector plasmid pIU5-CsCBP-SSSD was used as a template, and the primer pair SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) were used to amplify the vector plasmid by PCR, to prepare a DNA fragment containing the full length of the vector plasmid excluding the coding region of the CsCBP gene. These fragments were ligated by the In-Fusion method.
  • the resulting plasmid containing the expression cassette X5 (SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + TaCBP (SEQ ID NO: 8) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)) was named pIU5-TaCBP-SSSD.
  • Preparation Example 1-6 Preparation of vector plasmid containing Thermotoga neapolitana-derived cellobiose phosphorylase (TnCBP) surface expression cassette X6) A DNA fragment containing the coding region of the Thermotoga neapolitana-derived cellobiose phosphorylase (TnCBP) gene was prepared by gene synthesis.
  • TnCBP Thermotoga neapolitana-derived cellobiose phosphorylase
  • the vector plasmid pIU5-CsCBP-SSSD was used as a template, and the primer pair SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) were used to amplify the vector plasmid by PCR, to prepare a DNA fragment containing the full length of the vector plasmid excluding the coding region of the CsCBP gene. These fragments were ligated by the In-Fusion method.
  • the resulting plasmid containing the expression cassette X6 (SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + TnCBP (SEQ ID NO: 9) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)) was named pIU5-TnCBP-SSSD.
  • Preparation Example 1-7 Preparation of vector plasmid containing Bifidobacterium longum-derived sucrose phosphorylase (BISP) surface expression cassette X7) Using the plasmid pIBlSP-SSSD as a template, amplified by PCR using the primer pair BlSP-NheI-F (SEQ ID NO: 23) and BlSP-XhoI-R (SEQ ID NO: 24), and further treated with NheI and XhoI, to prepare a DNA fragment containing the coding region of the BlSP gene.
  • BlSP-NheI-F SEQ ID NO: 23
  • BlSP-XhoI-R SEQ ID NO: 24
  • vector plasmid pIBG-PpSSG61 (a surface expression vector having an expression cassette in which the SPI1 promoter derived from Pichia pastoris, the secretory signal peptide sequence of SPI1 derived from Pichia pastoris, the coding region of the ⁇ -glucosidase 1 (AaBGL1) gene derived from Aspergillus aculeatus, the coding region of the GCW61 gene derived from Pichia pastoris (GCW61 anchoring region), and the terminator region of the AOX1 gene derived from Pichia pastoris are arranged in this order: Biotechnology and Bioengineering, Vol.120, 2023, 1097-1107) was treated with NheI and XhoI to prepare a DNA fragment containing the entire length of the vector plasmid excluding the coding region of the AaBGL1 gene.
  • plasmid pIBG-PpGMG30 (a surface expression vector having an expression cassette in which a GAPDH promoter derived from Pichia pastoris, a secretory signal peptide sequence of ⁇ -factor derived from Saccharomyces cerevisiae, a coding region of ⁇ -glucosidase 1 (AaBGL1) gene derived from Aspergillus aculeatus, a coding region of GCW30 gene derived from Pichia pastoris (GCW30 anchoring region), and a terminator region of AOX1 gene derived from Pichia pastoris are arranged in this order: Biotechnology and Bioengineering, Vol.120, 2023, 1097-1107) was treated with XhoI and Ml
  • the vector plasmid was amplified by PCR using pIH-PpSS (SEQ ID NO: 28) and a DNA fragment containing the entire length of the vector plasmid excluding the coding region of the TrEGII gene and the GCW34 anchoring region was prepared. These fragments were ligated by the In-Fusion method.
  • the resulting plasmid containing the expression cassette X8 (SPI1 promoter (SEQ ID NO: 10) + SPI1 secretion signal (SEQ ID NO: 11) + BlSP (SEQ ID NO: 5) + AOX1 terminator (SEQ ID NO: 13)) was named pIH-BlSP-PpSS.
  • a DNA fragment containing the full length of the vector plasmid excluding the coding region of the BlSP gene was prepared by treating the vector plasmid pIBlSP-PpSSG30 with NheI and XhoI. These fragments were ligated.
  • this plasmid was used as a template and amplified by PCR using the primer pair CsCBP-F (SEQ ID NO: 31) and AOX1t-R (SEQ ID NO: 32) to prepare a DNA fragment containing the coding region of the CsCBP gene, the GCW30 anchoring region, and the AOX1 terminator region.
  • a vector plasmid pIZ-CBH1-PpSSG34 (a surface expression vector having an expression cassette in which the coding region of SPI1 derived from Pichia pastoris, the coding region of the cellobiohydrolase (TsCBH1) gene derived from Talaromyces emersonii, the coding region of the GCW34 gene derived from Pichia pastoris (GCW34 anchoring region), and the terminator region of the AOX1 gene derived from Pichia pastoris are arranged in this order: Biotechnology and Bioengineering, Vol.
  • RNA fragment was prepared containing the entire length of the vector plasmid excluding the coding region of the TsCBH1 gene, the GCW34 anchoring region, and the AOX1 terminator region. These fragments were ligated by the In-Fusion method.
  • the resulting plasmid containing the expression cassette X9 (SPI1 promoter (SEQ ID NO: 10) + SPI1 secretion signal (SEQ ID NO: 11) + CsCBP (SEQ ID NO: 6) + GCW30 anchoring region (SEQ ID NO: 12) + AOX1 terminator (SEQ ID NO: 13)) was named pIZ-CsCBP-PpSSG30.
  • Preparation Example 2-1 Production of sucrose phosphorylase surface-displaying yeast
  • the plasmid pIBlSP-SSSD described in Preparation Example 1-1 was treated with NdeI, and then used in yeast Saccharomyces cerevisiae BY4741 ⁇ SUC2 strain, which was transformed by the lithium acetate method.
  • This transformed strain is referred to as the ⁇ SUC2-BlSP strain.
  • the plasmid pIBlSP-PpSSG30 described in Preparation Example 1-7 was treated with EcoRV, and then used in yeast Pichia pastoris CBS7435 strain, which was transformed by the lithium acetate method. This transformed strain is referred to as the Pp-BlSP strain.
  • the plasmid pIL2-BlSP-SSD described in Preparation Example 1-2 was treated with NdeI, and then used in the ⁇ SUC2-BlSP strain to transform it by the lithium acetate method. This transformed strain is called the ⁇ SUC2-BlSP2 strain.
  • the plasmid pIH-BlSP-PpSS described in Preparation Example 1-8 was treated with BsrGI, and then used in the Pp-BlSP strain to transform it by the lithium acetate method. This transformed strain is called the Pp-BlSP2 strain.
  • Preparation Example 2-2 Production of cellobiose phosphorylase surface-displaying yeast
  • the plasmids pIU5-CsCBP-SSSD, pIU5-CuCBP-SSSD, pIU5-TaCBP-SSSD, and pIU5-TnCBP-SSSD described in Preparation Examples 1-3 to 1-6 were treated with SpeI, and then used in yeast Saccharomyces cerevisiae BY4741 ⁇ SUC2 strain to transform the strains by the lithium acetate method.
  • These transformed strains are referred to as ⁇ SUC2-CsCBP strain, ⁇ SUC2-CuCBP strain, ⁇ SUC2-TaCBP strain, and ⁇ SUC2-TnCBP strain, respectively.
  • Preparation Example 2-3 Production of yeast displaying sucrose phosphorylase + cellobiose phosphorylase on the surface
  • the plasmid pIU5-CsCBP-SSSD described in Preparation Example 1-3 was treated with SpeI, and then used in the ⁇ SUC2-B1SP2 strain to transform the strain by the lithium acetate method. This transformed strain is referred to as the ⁇ SUC2-SP2CBP strain.
  • the plasmid pIZ-CsCBP-PpSSG30 described in Preparation Example 1-9 was treated with NsiI, and then used in the Pp-B1SP2 strain to transform the strain by the electroporation method. This transformed strain is referred to as the Pp-SP2CBP strain.
  • yeasts transformed in A the ⁇ SUC2-BlSP strain, ⁇ SUC2-BlSP2 strain, ⁇ SUC2-CsCBP strain, ⁇ SUC2-CuCBP strain, ⁇ SUC2-TaCBP strain, ⁇ SUC2-TnCBP strain, and ⁇ SUC2-SP2CBP strains using Saccharomyces cerevisiae as a host were pretreated with 5 mL of YPD medium (1% dried yeast extract (Nacalai Tesque), Bacto TM Peptone (Life Technologies, Inc.)).
  • YPD medium 1% dried yeast extract (Nacalai Tesque), Bacto TM Peptone (Life Technologies, Inc.)
  • the bacteria were transferred to 2% Nacalai Tesque (Nippon Biotechnology) and 2% D-glucose (Nacalai Tesque) and pre-cultured at 30°C and 200 rpm for 18 hours, and then inoculated into 50 mL of YPD medium to an OD600 of 0.05 and cultured with shaking at 30°C and 150 rpm for 48 hours.
  • Pp-BlSP strain, Pp-BlSP2 strain, and Pp-SP2CBP strain which use Pichia pastoris as a host, were transplanted into 5 mL of YPG medium (1% dry yeast extract (Nacalai Tesque), 2% Bacto TM Peptone (Life Technologies), 2% glycerol (Nacalai Tesque)), pre-cultured for 18 hours at 30 ° C. and 200 rpm, and then inoculated into 50 mL of YPG medium so that the OD600 was 0.05, and cultured with shaking at 30 ° C. and 150 rpm for 48 hours.
  • YPG medium 1% dry yeast extract (Nacalai Tesque), 2% Bacto TM Peptone (Life Technologies), 2% glycerol (Nacalai Tesque)
  • reaction solution composition: 10 g/L sucrose (Nacalai Tesque); 80 mM sodium phosphate buffer (pH 7.0); and pretreated cells of the Pp-BlSP strain or Pp-BlSP2 strain prepared in Preparation Example 2-1 (final cell concentration 100 g wet cells/L)) was prepared and reacted at 35 rpm and 60°C for 60 minutes.
  • the reaction solution was kept at 100°C for 10 minutes, then cooled on ice for 10 minutes, centrifuged to precipitate the cells, etc., and the supernatant was collected and used for analysis of fructose concentration.
  • the amount of enzyme that liberates 1 ⁇ mol of fructose in 1 minute is defined as 1 IU.
  • Sampling was performed twice, for 4 hours and 24 hours. To stop the reaction, the sampling liquid was placed in a sampling container and kept at 100°C for 10 minutes, then cooled on ice for 10 minutes, and centrifuged to precipitate the cells, etc., and the supernatant was collected and used for analysis.
  • sampled liquid was placed in a sampling container, kept at 100° C. for 10 minutes, then cooled on ice for 10 minutes and centrifuged to precipitate the bacteria and the like, and the supernatant was collected and used for analysis.
  • Figure 2 shows the time course of the sucrose, glucose, fructose, and cellobiose concentrations in the reaction supernatant of an experiment to produce cellobiose from sucrose using a combination of yeast D that displays sucrose phosphorylase and yeast D that displays cellobiose phosphorylase.
  • yeast D displays sucrose phosphorylase
  • yeast D displays cellobiose phosphorylase
  • Figure 3 shows the time course of the concentrations of sucrose, glucose, fructose, and cellobiose in the reaction supernatant of an experiment to produce cellobiose from sucrose using a sucrose phosphorylase + cellobiose phosphorylase surface-displaying yeast ( ⁇ SUC2-SP2CBP strain) with Saccharomyces cerevisiae as the host.
  • the graph in Figure 3(a) shows the results when the ⁇ SUC2-SP2CBP strain was used with Saccharomyces cerevisiae as the host, and the graph in Figure 3(b) shows the results when the Pp-SP2CBP strain was used with Pichia pastoris as the host.
  • Figure 3(a) which used a single recombinant yeast strain co-expressing sucrose phosphorylase and cellobiose phosphorylase with the same Saccharomyces cerevisiae as a host, shows that sucrose was converted to cellobiose at a high conversion rate (about 70%) despite the same total amount of yeast cells used in the reaction.
  • Figure 4 shows the time course of the concentrations of sucrose, glucose, fructose, and cellobiose in the reaction supernatant of an experiment to produce cellobiose from sucrose using a sucrose phosphorylase + cellobiose phosphorylase surface-displaying yeast (Pp-SP2CBP strain) with E. Pichia pastoris as the host.
  • the graph in Figure 4(a) shows the results when the Pp-SP2CBP strain was cultured using glucose as the carbon source
  • the graph in Figure 4(b) shows the results when the Pp-SP2CBP strain was cultured using glycerol as the carbon source.
  • the conversion rate of sucrose to cellobiose in the Pp-SP2CBP strain was approximately 40% when it was cultured using glucose as the carbon source, but increased to approximately 80% when it was cultured using glycerol as the carbon source. This indicates that it is preferable to use glycerol as a carbon source when culturing a yeast that displays sucrose phosphorylase and cellobiose phosphorylase on the surface using Pichia pastoris as a host.
  • Example Group II Repeated cellobiose production by reusing transformed yeast (sucrose phosphorylase + cellobiose surface display yeast)]
  • ⁇ SUC2-SP2CBP strain prepared in A was transferred to 5 mL of YPD medium (1% dried yeast extract (Nacalai Tesque), 2% Bacto TM Peptone (Life Technologies), 2% D-glucose (Nacalai Tesque)) and pre-cultured at 30°C and 200 rpm for 18 hours, and then inoculated into 50 mL of YPD medium to an OD600 of 0.05, and cultured with shaking at 30°C and 150 rpm for 48 hours.
  • YPD medium 1% dried yeast extract (Nacalai Tesque), 2% Bacto TM Peptone (Life Technologies), 2% D-glucose (Nacalai Tesque)
  • the Pp-SP2CBP strain was transferred to 5 mL of YPG medium (1% dried yeast extract (Nacalai Tesque), 2% Bacto TM Peptone (Life Technologies), 2% glycerol (Nacalai Tesque)), pre-cultured at 30 ° C. and 200 rpm for 24 hours, and then inoculated into 50 mL of YPG medium so that the OD600 was 0.05, and cultured with shaking at 30 ° C. and 150 rpm for 48 hours.
  • YPG medium 1% dried yeast extract (Nacalai Tesque), 2% Bacto TM Peptone (Life Technologies), 2% glycerol (Nacalai Tesque)
  • cellobiose production reaction solution 100 g/L sucrose (Nacalai Tesque); 80 mM sodium phosphate buffer (pH 7.0); 1 wt% magnesium sulfate; 1.5 IU/mL glucose isomerase (xylose isomerase, Godo Shusei Co., Ltd., derived from Streptomyces griseofuscus); and pretreated cells of ⁇ SUC2-SP2CBP strain or Pp-SP2CBP strain prepared in G-1 (final cell concentration 100 g wet cells/L)) were prepared and reacted at 35 rpm and 60 ° C. for 24 hours (first time).
  • the yeast cells were collected by centrifugation of the reacted liquid, resuspended in a new reaction liquid, and reacted at 35 rpm and 60 ° C. for 24 hours (second time).
  • the third reaction was carried out in the same manner. Sampling was performed three times for each reaction, at 4 hours, 8 hours, and 24 hours. To stop the reaction, each sample was placed in a sampling container, kept at 100°C for 10 minutes, and then cooled on ice for 10 minutes and centrifuged to precipitate the bacteria and the like. The supernatant was collected and used for analysis.
  • sucrose phosphorylase + cellobiose phosphorylase surface-displaying yeast with Pichia pastoris cultured using glycerol as a carbon source was used as a transformed yeast, it was confirmed that the yeast of the Pp-SP2CBP strain maintained a high cellobiose conversion rate of about 80% even when the reaction was repeated three times at 60 ° C. for 24 hours.
  • the manufacturing method of the present invention can be used as a method for manufacturing cellobiose for use in food or pharmaceutical preparations.

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Abstract

La présente invention concerne un procédé de production de cellobiose peu coûteux et efficace. Le procédé comprend une étape de réaction du saccharose dans un liquide de réaction contenant de la glucose isomérase et de la levure qui a été conçue pour exprimer la sucrose phosphorylase et la cellobiose phosphorylase à sa surface.
PCT/JP2024/018152 2023-06-07 2024-05-16 Procédé de production de cellobiose Ceased WO2024252885A1 (fr)

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Citations (4)

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JPH03130086A (ja) * 1989-10-17 1991-06-03 Natl Food Res Inst セロビオースの製造方法
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