JP5279061B2 - Ethanol production method - Google Patents

Description

  The present invention relates to a method for producing ethanol using microbial fermentation.

  In recent years, it has been required to produce ethanol from soft biomass such as bagasse and corn fiber using fermentation by microorganisms. Soft biomass fibers contain cellulose and hemicellulose. Cellulose is a polysaccharide in which hexose glucose is linked by β-1,4 glucoside. Hemicellulose is a polysaccharide to which various sugars are bonded, and pentose sugar (for example, xylose) is included as a constituent sugar. Compared to cellulose in which glucose is strongly β-1,4-glucoside-bonded, hemicellulose has a weak binding of constituent sugars. Therefore, since it hydrolyzes comparatively easily by heating etc., it is easy to produce xylose in biomass.

  The yeast Saccharomyces cerevisiae, which is considered to have the highest ethanol productivity, can assimilate hexose glucose but not pentose xylose. If Saccharomyces cerevisiae can assimilate not only glucose but also xylose, the yield of ethanol would be further increased.

  Attempts have been made to produce Saccharomyces cerevisiae that assimilate xylose (Non-Patent Document 1). Saccharomyces cerevisiae expressing xylose reductase (XR) gene and xylitol dehydrogenase (XDH) gene (both from Pichia stipitis) and xylulokinase (XK) gene (derived from Saccharomyces cerevisiae) was prepared, It has been confirmed that ethanol can be produced by assimilation (Non-Patent Documents 2 to 7).

  However, any ethanol production method using such yeast still has room for improvement in terms of xylose availability, by-product generation, yeast growth, and the like.

Also, xylanase xylanase II (XYNII) derived from Trichoderma reesei and β-xylosidase (XylA) derived from Aspergillus oryzae, which are xylan-degrading enzymes, are surface-displayed, and XR gene, XDH gene and XK gene are displayed. An expressed Saccharomyces cerevisiae has been produced, and attempts have been made to produce ethanol from xylan as a raw material using this yeast (Non-patent Document 8). However, in Non-Patent Document 8, no consideration is given to the coexistence of hemicellulose and starch or cellulose.
International Patent Application Publication No. 02/42483 International Patent Application Publication No. 03/016525 International Patent Application Publication No. 01/79483 M. Sonderegger et al., Biotechnology and bioengineering, 2004, 87, 90-8 NWY Ho et al., Applied and Environmental Microbiology, 1998, 64, 1852-1859 A. Eliasson et al., Applied and Environmental Microbiology, 2000, 66, 3381-3386 MH Toivari et al., Metabolic Engineering, 2001, 3, 236-249 M. Sedlak et al., Appl. Biochem. Biotechnol., 2004, 113-116, 403-16 B. Johansson et al., Applied and Environmental Microbiology, 2001, 67, 4249-4255 YS Jin et al., Applied and Environmental Microbiology, 2003, 69, 495-503 S. Katahira et al., Applied and Environmental Microbiology, 2004, 70, 5407-5414 Appl. Microbiol. Biotech., 2002, 60, 469-474 Applied and Environmental Microbiology, 2002, 68, 4517-4522 T. Kanai et al., Appl. Microbiol. Biotechnol., 1996, 44, 759-765 H. Sawai-Hatanaka et al., Biosci. Biotechnol. Biochem., 1995, 59, 1221-1228 S. Takahashi et al., Appl. Microbiol. Biotechnol., 2001, 55, 454-462 PN Lipke et al., Mol. Cell. Biol., August 1989, 9 (8), 3155-65 Y. Fujita et al., Applied and Environmental Microbiology, 2002, 68, 5136-41

  An object of the present invention is to provide a method for producing ethanol by efficiently assimilating xylose by a microorganism.

  The present invention provides a method for producing ethanol. This method includes a step of fermenting in a medium containing sugar and xylose composed of two or more glucoses by a microorganism capable of assimilating glucose and xylose in the presence of an enzyme that degrades the sugars.

  In one embodiment, the microorganism is a yeast.

  In a further embodiment, the yeast capable of assimilating glucose and xylose is Saccharomyces cerevisiae having a xylose-assimilating gene.

  In another embodiment, the enzyme is displayed on the cell surface of the microorganism.

  In yet another embodiment, the medium comprises less than 40% glucose relative to the xylose amount (by weight).

  The present invention also provides a Saccharomyces cerevisiae yeast having a xylose-assimilating gene and having a amylolytic enzyme and / or a cellulolytic enzyme displayed on the cell surface.

  According to the method of the present invention, ethanol can be produced by efficiently using xylose that has not been effectively used so far (for example, xylose derived from hemicellulose). Yeast suitably used for such a production method is also provided.

  In the present invention, “a medium containing two or more glucose sugars and xylose” is an arbitrary medium in which xylose and two or more glucose sugars coexist in a fermentation medium when a microorganism starts fermentation. The state of. In the present invention, “a sugar comprising two or more glucoses” refers to any sugar obtained by combining two or more glucoses. Oligosaccharides composed of a small number (for example, 2 to 5) of sugars are preferable, and more preferably disaccharides. The linkage can be either an α-1,4 glucoside linkage or a β-1,4 glucoside linkage. Examples of such sugars include starch and glucose degradation products in which glucose is α-1,4-glucoside-bonded, and cellulose and glucose degradation products in which glucose is β-1,4-glucoside-bonded.

  Cellulose is decomposed by endo-β1,4-glucanase (cleaved from the inside of the molecule), cellobiohydrolase (decomposed from either the reducing end or the non-reducing end to release cellobiose), etc. to produce cellooligosaccharides. obtain. Cellooligosaccharides can be broken down to glucose by β-glucosidase, an exo-type hydrolase that cleaves glucose units from the non-reducing end of cellulose.

  Starch can be degraded by α-amylase (cleaved from the inside of the molecule), β-amylase (degraded from the non-reducing end to release maltose), exo-isomaltotriohydrolase, etc. to produce maltooligosaccharides. Furthermore, maltooligosaccharides can be broken down to glucose by glucoamylase, an exo-type hydrolase that cleaves glucose units from the non-reducing end of starch.

  Xylose can be obtained from hemicellulose using xylan degrading enzymes such as β-xylosidase and xylanase. If desired, arabinofuranosidase can further be used to cleave and remove side chains containing arabinose.

  The medium in the method of the present invention may contain cellulose and / or starch and products resulting from degradation of hemicellulose. This decomposition can be any of heating, hydrolysis with acid or alkali, enzymatic decomposition, and the like. The step of degrading cellulose and / or starch or decomposing hemicellulose and the step of producing ethanol from a fermentation substrate by a microorganism may be performed independently or in parallel.

  The above microorganism fermentation substrate can also be derived from, for example, plant materials (corn, rice, etc.) rich in starch, which are conventionally used for alcohol fermentation. Starch and hemicellulose that can be included in such plant material can be utilized for ethanol production. For example, fermentation can be performed after decomposing starch and hemicellulose by cooking.

  In the method of the present invention, the microbial fermentation substrate can be derived from lignocellulose contained in biomass. Lignocellulose includes cellulose and hemicellulose, and lignin. Therefore, cellulose and hemicellulose after removing a non-glycated portion such as lignin from lignocellulose can also be used in the method of the present invention. For example, when the decomposition of cellulose and hemicellulose is compared, hemicellulose having a relatively weak bond can be more easily decomposed than cellulose. Cellulose is a linear molecule in which only glucose is linked by β-1,4 glucoside, and each chain is firmly bound by hydrogen bonds and is strong. By utilizing this fact, xylose can be obtained preferentially in a lignocellulose-derived substrate, and a state of high xylose and low glucose can be created.

  In the method of the present invention, it is important that xylose can be used as a fermentation substrate when a microorganism starts fermentation. As the fermentation progresses, xylose in the medium is consumed as a fermentation substrate, and a sugar composed of two or more glucoses can be used as a fermentation substrate after being decomposed into glucose by an enzyme that degrades the sugar. The “medium containing two or more glucoses containing sugar and xylose” may contain glucose as long as the yeast is not prevented from assimilating xylose. In the fermentation medium, the amount of glucose is preferably less than 40% with respect to the amount of xylose (mass basis). More preferably less than 20%, even more preferably less than 10%, even more preferably less than 4%. For example, as described in the Examples below, when xylose is 50 g / l in the fermentation medium, glucose can be included in the medium at 20-50 g / l. Therefore, in the case of a substrate derived from a material containing a large amount of starch that is usually used for ethanol production, the residue after consumption of starch may be used as the substrate of the present invention. If xylose is present in this residue, ethanol can be produced from this xylose.

  Examples of the “enzyme that degrades sugar” in the present invention include starch degrading enzymes and cellulose degrading enzymes.

  Starch-degrading enzymes include α-amylase, β-amylase, exo-isomaltotriohydrolase, and the like that can degrade to maltooligosaccharide, and glucoamylase that degrades to glucose units. In the present invention, glucoamylase is preferably present in order to produce glucose.

  Cellulose-degrading enzymes include endo-β1,4-glucanase, cellobiohydrolase, etc. that can degrade to cellooligosaccharides, and β-glucosidase that degrades to cellulose units. In the present invention, β-glucosidase is preferably present in order to produce glucose.

  In the method of the present invention, the enzyme itself may be added to the fermentation medium, or a microorganism that produces the enzyme may be used. Such microorganisms may use microorganisms that can naturally produce enzymes, or may be produced by genetic recombination. When utilizing microorganisms, the produced enzyme can be secreted extracellularly or presented to the cell surface. Preferably, a fermenting microorganism in which an enzyme is displayed on the cell surface as described below can be used.

  The microorganism used in the method of the present invention is not particularly limited as long as it is a microorganism that can assimilate both glucose and xylose, and yeast is preferably used. From the viewpoint of ethanol productivity, Saccharomyces cerevisiae is preferred. This yeast can normally assimilate hexose glucose but not pentose xylose. Saccharomyces cerevisiae modified so that xylose can be assimilated by selection screening, mutagenesis, gene recombination, or the like is used. Saccharomyces cerevisiae can be transformed to have a xylose-utilizing gene. Examples of xylose-utilizing genes include xylose metabolic enzyme genes, such as xylose reductase (XR) gene (eg, derived from Pichia stipitis: INSD accession number X59465 or A16164), and xylitol dehydrogenase (XDH) gene (eg, Pichia stipitis derived: INSD accession number X55392 or A16166), and xylulokinase (XK) gene (for example, Saccharomyces cerevisiae derived: INSD accession number X82408). Saccharomyces cerevisiae genetically modified so as to express these three genes can be preferably used.

  As a method for producing a microorganism in which an enzyme is displayed on the cell surface, (a) a method in which the enzyme is displayed on the cell surface via a GPI anchor of the cell surface localized protein, (b) a sugar chain binding protein of the cell surface localized protein There are a method of presenting to the cell surface via a domain and a method of (c) presenting to the cell surface via a periplasm free protein (other receptor molecule or target receptor molecule).

  Examples of cell surface localized proteins that can be used include α- or a-agglutinin (used as a GPI anchor) which is a sex aggregation protein of yeast, Flo1 protein (Flo1 protein has various amino acid lengths on the N-terminal side, Can be used as a GPI anchor: for example, Flo42, Flo102, Flo146, Flo318, Flo428, etc .; Non-Patent Document 9: Flo1326 represents a full-length Flo1 protein, Flo protein (having no GPI anchor function and aggregation Floshort or Flolong using non-patent document 10), invertase (not using GPI anchor), which is a periplasmic localized protein, and the like.

  First, (a) a method using a GPI anchor will be described. A gene encoding a protein localized on the cell surface by a GPI anchor encodes a secretory signal sequence, a cell surface localized protein (sugar chain binding protein domain), and a GPI anchor attachment recognition signal sequence in this order from the N-terminal side. Have a gene. A cell surface localized protein (glycan binding protein) expressed from this gene in the cell is guided to the outside of the cell membrane by a secretion signal, and the GPI anchor attachment recognition signal sequence is selectively cleaved at the C-terminal. It binds to the GPI anchor of the cell membrane via the portion and is fixed to the cell membrane. Then, it is cut by the PI-PLC near the root of the GPI anchor, incorporated into the cell wall, fixed to the cell surface, and presented on the cell surface.

  Here, the GPI anchor refers to a glycolipid having a basic structure of ethanolamine phosphate-6 mannose α1-2 mannose α1-6 mannose α1-4 glucosamine α1-6 inositol phospholipid called glycosylphosphatidylinositol (GPI). , PI-PLC refers to phosphatidylinositol-dependent phospholipase C.

  The GPI anchor attachment recognition signal sequence is a sequence recognized when the GPI anchor binds to the cell surface localized protein, and is usually located at or near the C-terminal of the cell surface localized protein. As the GPI anchor attachment signal sequence, for example, the sequence of the C-terminal part of yeast α-agglutinin is preferably used. Since the GPI anchor adhesion recognition signal sequence is included in the C-terminal side of the sequence of 320 amino acids from the C-terminal of α-agglutinin, the gene used in the above method encodes a sequence of 320 amino acids from the C-terminal. DNA sequences are particularly useful.

  Therefore, for example, a DNA encoding a secretory signal sequence-a structural gene encoding a cell surface localized protein-a sequence having a DNA sequence encoding a GPI anchor adhesion recognition signal, and a structural gene encoding this cell surface localized protein By substituting all or part of the sequence with a DNA sequence encoding the target enzyme, recombinant DNA for presenting the target enzyme on the cell surface via the GPI anchor can be obtained. When the cell surface localized protein is α-agglutinin, it is preferable to introduce DNA encoding the target enzyme so as to leave a sequence encoding a 320 amino acid sequence from the C-terminus of α-agglutinin. The enzyme presented on the cell surface by introducing such DNA into yeast and expressing it has its C-terminal side immobilized on the surface.

  Next, (b) a method using a sugar chain binding protein domain will be described. When the cell surface localized protein is a sugar chain binding protein, the sugar chain binding protein domain has a plurality of sugar chains, and this sugar chain interacts with or entangles with sugar chains in the cell wall. It is possible to stay. Examples thereof include sugar chain binding sites such as lectins and lectin-like proteins. Typically, an aggregation functional domain of GPI anchor protein and an aggregation functional domain of FLO protein can be mentioned. The aggregation functional domain of the GPI anchor protein is a domain that is located on the N-terminal side of the GPI anchoring domain, has a plurality of sugar chains, and is considered to be involved in aggregation.

  By binding the cell surface localized protein (aggregation functional domain) and the target enzyme, the enzyme is presented on the cell surface. Depending on the type of the target enzyme, (1) the enzyme is bound to the N-terminal side of the cell surface localized protein (aggregation functional domain), (2) the enzyme is bound to the C-terminal side, and (3) the N-terminal side and The same or different enzymes can be bound to both the C-terminal side. In the present invention, (1) DNA encoding secretory signal sequence-gene encoding target enzyme-structural gene encoding cell surface localized protein (aggregation functional domain); or (2) encoding secretory signal sequence DNA-a structural gene encoding a cell surface localized protein (aggregation functional domain)-a gene encoding a target enzyme, thereby producing a recombinant DNA for presenting the target enzyme on the cell surface It is done. When the aggregation functional domain is used, since the GPI anchor is not involved in the cell surface presentation, only a part of the DNA sequence encoding the GPI anchor attachment recognition signal sequence may be present in the recombinant DNA. It does not have to exist. In addition, when the aggregation functional domain is used, since the length of the domain can be easily adjusted (for example, either Floshort or Flolong can be selected), the enzyme can be displayed on the cell surface with a more appropriate length, and This is very useful in that it can be bound on either the N-terminus or C-terminus of the enzyme.

  Next, (c) a method using a periplasm free protein (another receptor molecule or a target receptor molecule) will be described. In this case, the target enzyme can be expressed on the cell surface as a fusion protein with a periplasm free protein. Examples of the periplasmic free protein include invertase (Suc2 protein). The target enzyme can be appropriately fused to the N-terminal or C-terminal depending on these periplasmic free proteins.

  A gene construct is introduced so that the xylose-utilizing gene as described above is also expressed in cells.

  The gene construct is incorporated into a cloning vector and expression vector appropriate for the microorganism. Such appropriate vectors are known in the art and appropriately contain elements necessary for expression in microorganisms. Introduction of a gene construct into a microorganism means that the gene construct is introduced into a cell and expressed. Methods for introducing a gene construct include various methods known to those skilled in the art, such as transformation, transduction, transfection, co-transfection, and electroporation. Specifically, methods using lithium acetate, protoplasts, etc. There are laws. The introduced gene construct may be incorporated into the chromosome in the form of a plasmid, inserted into a host gene, or subjected to homologous recombination with the host gene. Cells into which the gene construct has been introduced are selected by a selectable marker (eg, TRP) and selected by measuring the activity of the expressed protein. Whether the protein is immobilized on the cell surface layer can be confirmed, for example, by an immunoantibody method using an anti-protein antibody and a FITC-labeled anti-IgG antibody.

  Examples of the amylolytic enzyme displayed on the surface include a single glucoamylase and a combination of glucoamylase and α-amylase. When only one glucoamylase is presented on the cell surface, the host microorganism can preferably be further transformed to secrete α-amylase. The cell surface display of the amylolytic enzyme can be performed according to the description of Patent Document 1 or 2, for example.

  Cellulose-degrading enzymes displayed on the surface include, for example, only one β-glucosidase, a combination of endo β1,4-glucanase and β-glucosidase, a combination of cellobiohydrolase and β-glucosidase, and endo β1,4- A combination of glucanase, cellobiohydrolase and β-glucosidase can be mentioned. The cell surface display of the cellulolytic enzyme can be performed, for example, according to the description in Patent Document 3.

  Furthermore, xylan-degrading enzymes such as β-xylosidase and xylanase can also be displayed on the cell surface. Such enzymes include Trichoderma reesei xylanase II (XYNII) and Aspergillus oryzae β-xylosidase (XylA). The cell surface display of the xylan-degrading enzyme can be performed according to the description of Non-Patent Document 8, for example.

  When there are multiple enzymes that are intended for cell surface presentation, use a combination of microorganisms that present each enzyme on the cell surface, or a microorganism that has been recombined to present the combination of those enzymes on the cell surface. Also good.

  A Saccharomyces cerevisiae yeast having a xylose-assimilating gene and having a amylolytic enzyme and / or a cellulolytic enzyme displayed on the cell surface can be obtained as described above.

  The microorganism used in the method of the invention and, if necessary, the enzyme are preferably immobilized on a carrier. As a result, reuse becomes possible.

  As the carrier and method for immobilizing the enzyme, those commonly used by those skilled in the art are used, and examples thereof include a carrier binding method, a comprehensive method, and a crosslinking method.

  A porous body is preferably used as a carrier for immobilizing microorganisms. For example, foams or resins such as polyvinyl alcohol, polyurethane foam, polystyrene foam, polyacrylamide, polyvinyl formal resin porous body, and silicon foam are preferable. The size of the opening of the porous body may be determined in consideration of the microorganism to be used and its size. In the case of yeast, 50-1000 micrometers is preferable.

  Moreover, the shape of a support | carrier is not ask | required. Considering the strength of the carrier, the culture efficiency, etc., a spherical shape or a cubic shape is preferable. The size may be determined depending on the microorganism to be used. In general, the diameter is preferably 2 to 50 mm in the case of a sphere, and 2 to 50 mm in the case of a cube.

  Microorganisms capable of assimilating glucose and xylose used in the method of the present invention can be increased in number by culturing under aerobic conditions before being subjected to fermentation. The medium may be a selective medium or a non-selective medium. The pH of the medium during culture is preferably about 4.0 to about 6.0, and most preferably about 5.0. The dissolved oxygen concentration in the medium during aerobic culture is preferably about 0.5 to about 6 ppm, more preferably about 1 to about 4 ppm, and most preferably about 2.0 ppm. Moreover, the temperature at the time of culture is about 20 to about 45 ° C, preferably about 25 to about 35 ° C, and most preferably about 30 ° C. The culture time is preferably cultured until the bacterial cell concentration is 10 g / l or more, and is about 20 to about 50 hours.

  In the method of the present invention, a microorganism capable of assimilating glucose and xylose is fermented under anaerobic conditions to produce ethanol. Examples of the fermentation process include a batch process, a fed-batch process, a repeated batch process, and a continuous process, and any of these may be used.

  The dissolved oxygen concentration in the medium during anaerobic fermentation is preferably about 1.0 ppm or less, more preferably about 0.1 ppm or less, and most preferably about 0.05 ppm or less. The temperature during fermentation is about 20 to about 45 ° C, preferably about 25 to about 35 ° C, and most preferably about 30 ° C.

  As the fermentation conditions change as the fermentation progresses, it is preferable to adjust these to a certain range. What is necessary is just to monitor the time-dependent change of fermentation with the means normally used by those skilled in the art, such as a gas chromatograph and HPLC.

  After completion of the fermentation process, the ethanol-containing medium is withdrawn from the fermenter, and ethanol is isolated by a separation process commonly used by those skilled in the art, such as a separation operation using a centrifuge and a distillation operation.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by this Example.

(Example 1: Production of Saccharomyces cerevisiae having a xylose-utilizing gene and β-glucosidase displayed on the cell surface)
Both XYL1 (INSD accession number X59465) and XYL2 (INSD accession number X55392), and xylulokinase (XK) from Saccharomyces cerevisiae, which are xylose reductase (XR) gene and xylitol dehydrogenase (XDH) gene derived from Pichia stipitis A plasmid pIUX1X2XK (FIG. 1a) for intracellular expression of the gene XKS1 (INSD accession number X82408) gene was constructed based on the procedure described in Non-Patent Document 8.

  The multicopy plasmid pRS405 + 2 was constructed by introducing a 2.24 kb EcoRI-EcoRI DNA fragment blunted at the end of plasmid pWI3 (Non-patent Document 11) containing a 2 μm DNA portion at the AatII site of plasmid pRS405 (Stratagene). . A 1.3 kb HindIII-HindIII DNA fragment blunted with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter and GAPDH terminator derived from plasmid pYE22m (Non-patent Document 12) was introduced into the PvuII site of plasmid pRS405 + 2. The resulting plasmid was named pLGP3.

  A 0.95 kb SacI-BamHI DNA fragment containing the XYL1 gene derived from Pichia stipitis, a 1.09 kb SacI-BamHI DNA fragment containing the XYL2 gene derived from Pichia stipitis, and a 1.8 kb BamHI-SalI DNA containing the XKS1 gene derived from Saccharomyces cerevisiae Using the genomic DNA of Pichia stipitis and Saccharomyces cerevisiae as templates, XYL1F (SacI) (SEQ ID NO: 1) and XYL1R (BamHI) (SEQ ID NO: 2); XYL2F (SacI) (SEQ ID NO: 3) and XYL2R ( BamHI) (SEQ ID NO: 4); and XKS1F (BamHI) (SEQ ID NO: 5) and XKS1R (SalI) (SEQ ID NO: 6) primer pairs were used to prepare by polymerase chain reaction (PCR). In addition, PCR of the present Example was performed using KOD-Plus-DNA polymerase (Toyobo). These DNA fragments were introduced into the SacI-BamHI section of plasmids pWGP3 and pUGP3 (both non-patent document 13) and the BamHI-SalI section of plasmid pLGP3, respectively, and were designated as pWPXYL1, pUPXYL2, and pLXKS1.

  A 2.2 kbp KpnI-XhoI DNA fragment composed of the GAPDH promoter, XYL1 gene, and GAPDH terminator was used as a template for pWPXYL1 as a template, and a primer pair of XYL1cF (KpnI) (SEQ ID NO: 7) and XYL1cR (XhoI) (SEQ ID NO: 8). And prepared by PCR. A 2.3 kbp XhoI-NotI DNA fragment composed of the GAPDH promoter, XYL2 gene, and GAPDH terminator was used as a template pair of XYL2cF (XhoI) (SEQ ID NO: 9) and XYL2cR (NotI) (SEQ ID NO: 10) using pUPXYL2 as a template. And prepared by PCR. A 3.04 kbp NotI-SacII DNA fragment composed of the GAPDH promoter, XKS1 gene, and GAPDH terminator was used as a template pair of pKSX1 (SEQ ID NO: 11) and XKS1cR (SacII) (SEQ ID NO: 12) using pLXKS1 as a template. And prepared by PCR. These DNA fragments were inserted into the KpnI-XhoI section, XhoI-NotI section, and NotI-SacII section of plasmid pRS406 (Stratagene), respectively, and the resulting plasmid was named pIUX1X2XK.

  A plasmid pIBG13 for cell surface display of β-glucosidase BGL1 derived from Aspergillus aculeatus No. F-50 was constructed as follows. A 2.5 kbp NcoI-XhoI DNA fragment encoding the bgl1 gene was used using plasmid pBG211 (gift from Kyoto University) as a template and a primer pair of bgl1 primer 1 (SEQ ID NO: 13) and bgl1 primer 2 (SEQ ID NO: 14). And prepared by PCR. This DNA fragment is digested with NcoI and XhoI and contains the gene encoding the secretory signal sequence of the glucoamylase gene derived from Rhizopus oryzae and the 3 ′ half region of the α-agglutinin gene (Non-patent Document 14) The cell surface expression plasmid pIHCS (Non-patent Document 15) was inserted into the NcoI-XhoI site. The resulting plasmid was named pIBG13 (FIG. 1b).

  The plasmids pIUX1X2XK and pIBG13 were introduced into Saccharomyces cerevisiae MT8-1 (MATa ura3 trp1 ade leu2 his3) by the lithium acetate method using Yeast Maker (manufactured by CLONTEC). Transformants are in SD medium (6.7 g / l yeast nitrogen base w / o amino acids, 20 g / l glucose, 0.03 g / l leucine, 0.02 g / l tryptophan, 0.02 g / l adenine, 0.02 g / l uracil). Selected. The obtained transformant was named MT8-1 / pIUX1X2XK / pIBG13. This transformant presents BGL1 on the cell surface.

(Example 2: Verification of xylose utilization of transformed Saccharomyces cerevisiae and ethanol production by transformed Saccharomyces cerevisiae in a fermentation medium containing glucose and xylose)
In this example, the transformed Saccharomyces cerevisiae prepared in Example 1 is xylose-assimilating and, in practice, it can be used in the presence of glucose and xylose. The production of ethanol in was investigated.

  The yeast transformant MT8-1 / pIUX1X2XK / pIBG13 prepared in Example 1 was added to SDC medium (6.7 g / l yeast nitrogen base w / o amino acids, 20 g / l glucose, 20 g / l) at 30 ° C. for 48 hours. The cells were aerobically cultured in casamino acid, 0.02 g / l uracil, 0.02 g / l histidine). The cells were harvested by centrifugation at 3,000 × g for 10 minutes at 4 ° C. and washed 3 times with distilled water.

The cells were then inoculated into a fermentation medium containing 6.7 g / l yeast nitrogen base w / o amino acids, 20 g / l casamino acids, and sugar as the carbon source. The sugars used were: xylose 50 g / l only (FIG. 2A); glucose 2 g / l and xylose 50 g / l (FIG. 2B); glucose 5 g / l and xylose 50 g / l (FIG. 2C); glucose 10 g / l and xylose 50 g / l (FIG. 2D); glucose 20 g / l and xylose 50 g / l (FIG. 2E); and glucose 50 g / l and xylose 50 g / l (FIG. 2F). All fermentations were performed in a 100 ml sealed bottle with a bubbled CO ₂ outlet at 30 ° C. with gentle stirring at 100 rpm.

The initial cell density in the fermentation medium was adjusted so that the OD ₆₀₀ was 20. During fermentation, cell growth was observed by monitoring absorbance at 600 nm. The dry cell weight (DW) was determined as follows: a culture broth (10 ml) adjusted to various OD ₆₀₀ values, pre-weighed by centrifuging at 3,000 × g for 10 minutes. Precipitated in. After resuspension with 10 ml of distilled water and further centrifugation, the precipitate was lyophilized with FreeZone FZ-1 (Labconco, Kansas, MO, USA). DW was calculated by reweighing the test tube. The cell concentration was estimated from the correlation between OD ₆₀₀ and DW (0.31 g DW / OD ₆₀₀ value).

  Sugars and sugar alcohols in the fermentation medium were analyzed by high performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan). A Shim-pack SPR-Pb column (Shimadzu) was used with a refractive index detector (model RID-10A, Shimadzu). The HPLC system was operated at a flow rate of 0.4 ml / min with water as the mobile phase.

  The ethanol concentration was measured by gas chromatography. A gas chromatograph (model GC-8A; Shimadzu) equipped with a flame ionization detector was operated under the following conditions: glass column (3.2 mm x 2.0 m, packed with Thermon-3000 (Shimadzu)); column, injector And a detector temperature of 180 ° C .; and a nitrogen carrier gas flow rate of 25 ml / min.

  FIG. 2A to FIG. 2F are graphs showing the time course of fermentation by transformed Saccharomyces cerevisiae in each of the above fermentation media. Black circles represent glucose, black diamonds represent xylose, black squares represent ethanol, black triangles represent glycerol, and x represents xylitol. The left vertical axis represents the xylose concentration (g / l) in the fermentation medium, the right vertical axis represents the product concentration (g / l), and the horizontal axis represents time (hours).

  As shown in FIG. 2A, with the passage of time, xylose was consumed and ethanol was produced. As described above, when xylose metabolic enzymes (XR, XDH, and XK) were added to Saccharomyces cerevisiae, xylose that Saccharomyces cerevisiae cannot normally assimilate could be converted into ethanol.

  When glucose is included in the medium, as shown in FIG. 2E, when glucose is 20 g / l, the rate of xylose utilization is affected, and as shown in FIG. 2F, glucose is 50 g / l. In some cases, the xylose utilization rate was significantly reduced. When glucose was 20 g / l or more, xylose still remained after 100 hours (FIGS. 2E and F). This is presumably because the Saccharomyces cerevisiae preferentially takes in glucose that can be assimilated and converts it into ethanol, thereby inhibiting the route to assimilation of xylose. On the other hand, as shown in FIG. 2B to FIG. 2D, it seemed that the fermentation medium with glucose up to 10 g / l had little influence on the utilization of xylose.

(Example 3: Ethanol production by transformed Saccharomyces cerevisiae in a fermentation medium containing cellobiose and xylose)
Except that the transformed Saccharomyces cerevisiae MT8-1 / pIUX1X2XK / pIBG13 prepared in Example 1 contains 50 g / l cellobiose and 50 g / l xylose as carbon sources in the fermentation medium, The ethanol production was investigated.

  FIG. 3 is a graph showing the time course of fermentation by transformed Saccharomyces cerevisiae in a fermentation medium containing cellobiose and xylose. White triangles represent cellobiose, black diamonds represent xylose, black squares represent ethanol, black triangles represent glycerol, and x represents xylitol. The left vertical axis represents the sugar concentration (g / l) in the fermentation medium, the right vertical axis represents the product concentration (g / l), and the horizontal axis represents time (hour). Over time, xylose and cellobiose were consumed and ethanol was produced. Unlike the results observed with glucose and xylose co-fermentation in Example 2, xylose could be assimilated in a fermentation medium containing cellobiose and xylose with no significant difference from that of xylose alone (FIG. 3).

  According to the method of the present invention, the pentose xylose can be efficiently converted to ethanol in Saccharomyces cerevisiae, which is said to be most excellent in ethanol production by microbial fermentation. The method of the present invention may be useful for the production of ethanol utilizing microbial fermentation from soft biomass such as bagasse and corn fiber.

It is a schematic diagram of plasmid pIUX1X2XK (a) and pIBG13 (b). It is a graph which shows the time-dependent change of fermentation by the transformed Saccharomyces cerevisiae in a fermentation medium containing xylose 50 g / l. It is a graph which shows the time-dependent change of fermentation by transformation Saccharomyces cerevisiae in the fermentation medium containing 2 g / l of glucose and 50 g / l of xylose. It is a graph which shows the time-dependent change of fermentation by transformation Saccharomyces cerevisiae in the fermentation medium containing glucose 5g / l and xylose 50g / l. It is a graph which shows the time-dependent change of fermentation by transformed Saccharomyces cerevisiae in a fermentation medium containing glucose 10 g / l and xylose 50 g / l. It is a graph which shows the time-dependent change of fermentation by transformed Saccharomyces cerevisiae in a fermentation medium containing glucose 20 g / l and xylose 50 g / l. It is a graph which shows the time-dependent change of fermentation by transformed Saccharomyces cerevisiae in a fermentation medium containing glucose 50 g / l and xylose 50 g / l. It is a graph which shows the time-dependent change of fermentation by the transformed Saccharomyces cerevisiae in a fermentation medium containing cellobiose 50 g / l and xylose 50 g / l.

Claims (1)

A method of producing ethanol, the method comprising:
In a medium containing xylose and sugars composed of two or more glucoses, and in the presence of an enzyme that decomposes the sugars composed of two or more glucoses, fermenting with a microorganism capable of assimilating glucose and xyloses,
The microorganism is a Saccharomyces cerevisiae having a xylose-assimilating gene and presenting an enzyme that degrades a sugar composed of two or more glucoses on a cell surface,
The enzyme that degrades a sugar composed of two or more glucoses includes an enzyme that is presented on the cell surface of the Saccharomyces cerevisiae;
The medium comprises an amount of glucose less than 40% xylose amount (by weight),
Method.

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