JP2011160727A - Method for producing ethanol from cellulose at high temperature - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing ethanol efficiently from cellulose. <P>SOLUTION: The method for producing ethanol from biomass includes the process for mixing biomass with a yeast of the transformation Kluyveromyces group which expresses at least two species of cellulose decomposition enzymes and culturing it. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for efficiently producing ethanol from cellulose.

  In recent years, alternative fuels are being developed in the face of fear of exhaustion of fossil fuels. In particular, bioethanol derived from biomass has attracted attention. This is because biomass is a renewable resource, exists in large quantities on the earth, and does not increase carbon dioxide in the atmosphere even if it is used (carbon neutral), thereby contributing to the prevention of global warming.

  Attempts have been made to ferment ethanol directly from non-edible carbon sources by modifying fermentative microorganisms that cannot inherently assimilate the main components of soft biomass, such as cellulose and hemicellulose, using biotechnological techniques. . As such a biotechnological technique, a cell surface display technique is preferably used. For example, yeast that surface-displays a group of enzymes that hydrolyze cellulose (that is, a plurality of types of enzymes) has been produced by cell surface display technology (for example, Patent Documents 1 and 2).

  Since the fermentation temperature of the yeast Saccharomyces cerevisiae is about 30 ° C., ethanol fermentation is usually carried out by culturing the yeast at this temperature. On the other hand, the optimum temperature of the cellulolytic enzyme is about 45-50 ° C. Therefore, in producing ethanol from cellulose, cellulose-degrading enzymes act on cellulose at a temperature much lower than the optimum temperature, so that the degradation from cellulose to glucose is the rate-limiting reaction.

  Kluyveromyces marxianus is known as a thermostable ethanol-producing yeast that can be cultured and grown near the optimum temperature for cellulolytic enzymes (Non-patent Document 1). So far, there has been a report that ethanol was produced from cellobiose using recombinant Kluyveromyces marcyanus into which a cellulolytic enzyme gene was introduced (Non-patent Document 2). However, this recombinant Kluyveromyces marukinus cannot produce ethanol from cellulose.

International Patent Application Publication No. 01/79483 JP 2008-86310 A

I.M.Banat et al., World J. Microbiol. Biotechnol., 1992, 8, 259-263 J. Hong et al., J. Biotechnol., 2007, 130, 114-123 Appl. Microbiol. Biotechnol., 2002, 60, 469-474 Appl. Environmen. Microbiol., 2002, 68, 4517-4522 Appl. Microbiol. Biotechnol., 1997, 48, 339-345 Biotechnology Letters, 2002, 24, 1785-1790 Biotechnol. Prog., 1996, 12, 16-21 Biotechnol. Prog., 1997, 13, 368-373 Appl. Environ. Microbiol., 2004, 70, 1207-1212

  An object of the present invention is to provide a method for efficiently producing ethanol from cellulose.

  The inventors of the present invention have found that, in yeasts of the genus Kluyveromyces that can be cultured and grown near the optimum temperature for cellulose-degrading enzymes, ethanol fermentation can be performed using cellulose as a carbon source by expressing cellulose-degrading enzymes. Completed the invention.

  The present invention provides a method for producing ethanol from biomass, which comprises mixing and culturing transformed Kluyveromyces yeasts expressing at least two cellulolytic enzymes with biomass.

  In one embodiment, the transformed Kluyveromyces yeast that expresses at least two of the cellulolytic enzymes is a transformed Kluyveromyces yeast that presents at least two of the cellulolytic enzymes on the cell surface. .

  In one embodiment, the at least two cellulolytic enzymes are a combination of enzymes with different modes of cellulose hydrolysis.

  In one embodiment, the combination of enzymes with different modes of cellulose hydrolysis is selected from the group consisting of endoglucanase, cellobiohydrolase, and β-glucosidase.

  In one embodiment, the combination of enzymes with different modes of cellulose hydrolysis is a combination of endoglucanase, cellobiohydrolase, and β-glucosidase.

  In one embodiment, in the transformed Kluyveromyces yeast, the genes encoding the at least two cellulolytic enzymes are co-introduced by integration with a yeast δ sequence.

  In one embodiment, the culturing step is performed at 40 ° C. or higher.

  In one embodiment, the transformed Kluyveromyces yeast is a transformed Kluyveromyces marukinus.

  The present invention also provides a transformed Kluyveromyces yeast that expresses at least two cellulolytic enzymes.

  The present invention also provides transformed Kluyveromyces marukinus expressing at least two cellulolytic enzymes.

  According to the method of the present invention, fermentation at a high temperature becomes possible, so that the cooling cost of the fermenter can be significantly reduced. Furthermore, the cellulose decomposition efficiency by the improvement of the cellulolytic enzyme activity can be increased.

It is a graph which shows the time-dependent change of the consumption amount of ethanol and the production amount of ethanol in ethanol fermentation from glucose using yeast Kluyveromyces marukinus wild type | system | group (A) and (DELTA) U strain | stump | stock (B) under high temperature. It is a schematic diagram of the plasmid used in order to display endoglucanase II (A) and (beta) -glucosidase 1 (B) on the cell surface layer of yeast. It is a graph which shows the result of having quantified introduction | transduction of the endoglucanase II gene (A) and (beta) -glucosidase 1 gene (B) in a transformed yeast strain | stump | stock by the real-time PCR method. It is a graph which shows the time-dependent change of the consumption of cellobiose and the production amount of ethanol in ethanol fermentation from cellobiose using a transformed yeast strain | stump | stock at 40 degreeC (A) and 42 degreeC (B). It is a graph which shows the time-dependent change of the consumption of (beta) -glucan and the production amount of ethanol in ethanol fermentation from (beta) -glucan using the transformed yeast strain | stump | stock at high temperature.

  The yeast used in the present invention is a transformed Kluyveromyces yeast that expresses at least two types of cellulolytic enzymes. The species of Kluyveromyces genus yeast (host yeast) used for transformation is not particularly limited, but is preferably Kluyveromyces marxianus. It may be genetically modified so as to enhance the ability to ferment alcohol from a monosaccharide (for example, glucose) which is a fermentation substrate obtained by cellulose degradation. As long as at least two types of cellulolytic enzymes are expressed, they may be presented on the cell surface or secreted.

  Cellulolytic enzyme refers to any enzyme capable of cleaving a β1,4-glucoside bond. It can be derived from any cellulose hydrolase producing bacterium. Cellulose hydrolase producing bacteria typically include the genus Aspergillus (for example, Aspergillus aculeatus, Aspergillus niger, and Aspergillus oryzae), Trichoderma (for example, Trichoderma reesei, genus Clostridium (eg, Clostridium thermocellum, genus Cellulomonas (eg, Cellulomonas fimi and Cellulomonas uda), Examples include microorganisms belonging to the genus Pseudomonas (for example, Pseudomonas fluorescence).

  Hereinafter, endoglucanase, cellobiohydrolase, and β-glucosidase will be described as typical cellulolytic enzymes, but the cellulolytic enzymes are not limited thereto.

  Endoglucanase is an enzyme commonly referred to as cellulase that cleaves cellulose from the interior of the molecule to yield glucose, cellobiose, and cellooligosaccharide (“cellulose intramolecular cleavage”). There are five types of endoglucanases, referred to as endoglucanase I, endoglucanase II, endoglucanase III, endoglucanase IV, and endoglucanase V, respectively. These distinctions are differences in amino acid sequences, but are common in that they have a cellulose intramolecular cleavage action. For example, endoglucanase derived from Trichoderma reesei (particularly, endoglucanase II: EGII) can be used, but is not limited thereto.

  Cellobiohydrolase degrades from either the reducing or non-reducing end of cellulose to release cellobiose (“cellulose molecular end truncation”). There are two types of cellobiohydrolase, called cellobiohydrolase I and cellobiohydrolase II, respectively. These distinctions are differences in amino acid sequences, but are common in that they have a cellulose molecule end cleaving action. For example, cellobiohydrolase derived from Trichoderma reesei (particularly, cellobiohydrolase II: CBHII) can be used, but is not limited thereto.

  β-glucosidase is an exo-type hydrolase that cleaves glucose units from the non-reducing end of cellulose (“glucose unit cleavage”). β-glucosidase can cleave β1,4-glucoside bond between aglycone or sugar chain and β-D-glucose, and can hydrolyze cellobiose or cellooligosaccharide to produce glucose. β-glucosidase is a representative example of an enzyme capable of hydrolyzing cellobiose or cellooligosaccharide. One type of β-glucosidase is currently known and is referred to as β-glucosidase 1. For example, Aspergillus acreatas-derived β-glucosidase (particularly β-glucosidase 1: BGL1) can be used, but is not limited thereto.

  For good hydrolysis of cellulose, it is preferable to combine enzymes with different modes of cellulose hydrolysis. Enzymes that act in a variety of different cellulose hydrolysis modes such as cellulose intramolecular cleavage, cellulose molecular end cleavage, and glucose unit cleavage can be combined as appropriate. Examples of enzymes having respective hydrolysis modes include, but are not limited to, endoglucanase, cellobiohydrolase, and β-glucosidase. The combination of enzymes with different modes of cellulose hydrolysis can be selected, for example, from the group consisting of endoglucanase, cellobiohydrolase, and β-glucosidase. Since it is desirable that glucose, which is a constituent sugar of cellulose, can be finally produced, it is preferable to include at least one enzyme capable of producing glucose. As an enzyme capable of producing glucose, in addition to a glucose unit cleaving enzyme (for example, β-glucosidase), endoglucanase can also produce glucose. Preferably, β-glucosidase, endoglucanase, and cellobiohydrolase can be expressed in yeast.

  The gene of the enzyme for the purpose of expression can be obtained from a microorganism producing the enzyme by designing a primer or a probe based on known sequence information and using PCR or a hybridization method. It can also be cut out from an existing vector containing these enzyme genes, preferably in the form of its expression cassette.

  Enzyme genes can be used to construct expression cassettes. The expression cassette may contain so-called regulatory elements such as operators, promoters, terminators, enhancers and the like that regulate the expression of the gene. The promoter or terminator may be that of the gene intended for expression itself, or may be derived from another gene. As the promoter and terminator, promoters and terminators such as GAPDH (glyceraldehyde 3′-phosphate dehydrogenase), PGK (phosphoglycerate kinase), PYK (pyruvate kinase), and TPI (triose phosphate isomerase) can be used. The selection of promoter and terminator depends on the expression of the target enzyme gene and can be appropriately selected by those skilled in the art. If necessary, it may further contain factors (for example, operators and enhancers) that regulate further expression. Expression regulators such as operators and enhancers can also be appropriately selected by those skilled in the art. The expression cassette can further contain necessary functional sequences depending on the purpose of expression of the gene. The expression cassette can also include a linker, if desired.

  Cell surface engineering techniques can be utilized for surface display expression of enzymes to yeast. For example, (a) a method of presenting the cell surface localized protein on the cell surface via the GPI anchor, (b) a method of presenting on the cell surface via the sugar chain binding protein domain of the cell surface localized protein, and (c ) There is a method of presenting on the cell surface via a periplasmic free protein (other receptor molecule or target receptor molecule), but is not limited thereto. The technique of cell surface engineering is also described in Patent Documents 1 and 2, for example.

  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 3: Flo1326 represents a full-length Flo1 protein, Flo protein (having no GPI anchor function and aggregation Floshort or Flolong using non-patent document 4), invertase (not using GPI anchor), which is a periplasm 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 secretory signal sequence generally refers to an amino acid sequence containing many amino acids rich in hydrophobicity, which is bound to the N-terminus of a protein (secretory protein) secreted extracellularly (including periplasm), Normally, secreted proteins are removed when they are secreted from inside the cell through the cell membrane. Any secretory signal sequence can be used as long as it is a secretory signal sequence capable of leading the expression product to the cell membrane, regardless of its origin. For example, as a secretion signal sequence, a glucoamylase secretion signal sequence, a yeast α- or a-agglutinin signal sequence, a secretion signal sequence of the expression product itself, and the like are preferably used. If the activity of other proteins fused to the cell surface binding protein is not affected, a part or all of the secretory signal sequence and the pro sequence may remain at the N-terminus.

  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. For this reason, the “3 ′ half region of the α-agglutinin gene” can be used. 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 sugar chain binding protein domain (aggregation function domain) of the cell surface localized protein 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 sugar chain binding protein domain (aggregation functional domain) of the cell surface localized protein, (2) the enzyme is bound to the C-terminal side, and ( 3) The same or different enzymes can be bound to both the N-terminal side and the C-terminal side. For example, (1) DNA encoding secretory signal sequence-gene encoding target enzyme-structural gene encoding sugar chain binding protein domain (aggregation functional domain) of cell surface localized protein; or (2) secretion signal DNA encoding sequence-Structural gene encoding sugar chain binding protein domain (aggregation functional domain) of cell surface localized protein-Gene encoding target enzyme Recombinant DNA for display can be obtained. 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.

  The elements used in any surface layer display technology of any of (a) to (c) above (also referred to as “cell surface layer display factor” in this specification) are also included in the enzyme gene expression cassette according to the above description. Can be. More specifically, a cell surface display factor can be linked to a gene of an expressed enzyme in a desired arrangement together with a secretory signal sequence depending on the factor, and this linkage can be sandwiched between a promoter and a terminator. . The surface layer display factor can be obtained from a microorganism expressing the factor by designing a primer or a probe based on known sequence information and using a PCR or a hybridization method. Further, from a known plasmid containing a gene for an expression enzyme (for example, endoglucanase, cellobiohydrolase, or β-glucosidase) together with a gene for a cell surface display factor, a secretory signal, and an expression regulatory sequence such as a promoter and a terminator, It can also be used by cutting out in a form suitable for vector preparation and preparing an insert.

  A method for secreting an enzyme out of a cell and expressing it in yeast is well known to those skilled in the art. A recombinant DNA in which the structural gene of the target enzyme is linked to the DNA encoding the secretory signal sequence may be prepared and introduced into yeast.

  Synthesis and binding of DNA containing various sequences can be performed by techniques that can be commonly used by those skilled in the art. For example, the binding between the secretory signal sequence and the structural gene of the target enzyme can be performed using site-directed mutagenesis. By using this method, it is possible to cleave the secretory signal sequence accurately and to express the active enzyme.

  The method for introducing a gene into yeast is not particularly limited. Examples thereof include a lithium acetate method, an electroporation method, a protoplast method, and a δ integration method. The introduced gene may exist in the form of a plasmid, or may exist in a form inserted into a yeast chromosome or in a form integrated into a yeast chromosome by homologous recombination.

  A vector can be constructed for integration of the cellulolytic enzyme with the yeast δ sequence. Vectors used for delta integration may include pairs of delta sequences (which allow homologous recombination with a large number of delta sequences present on the yeast chromosome) and enzyme genes.

  It has been reported that the yeast δ sequence is a long terminal repeat of retrotransposons Ty1 and Ty2 for Saccharomyces cerevisie (for example, Non-Patent Documents 5 to 8), and is also referred to as a Ty sequence. The δ sequence is known and readily available to those skilled in the art (Genebank accession number M18706). Pairs of 5 'and 3' sequences of the δ sequence can be prepared so as to allow homologous recombination with a large number of δ sequences present on the yeast chromosome. For example, such a pair of δ sequences can be prepared by designing a primer pair based on the sequence information and performing PCR amplification using yeast genomic DNA as a template. In the present invention, commercially available vectors for δ integration can also be used.

  The enzyme gene to be expressed can be usually designed in a vector so as to be in the form of an expression cassette as described above. That is, it can be included in a vector together with expression control elements such as a promoter and a terminator. The expression cassette can also be designed such that the expressed enzyme is expressed on the cell surface or secreted as described above.

  In the vector used in the present invention, an enzyme expression cassette may be sandwiched and linked between a pair of δ sequences so as to allow homologous recombination with a large number of δ sequences present on the yeast chromosome. For convenience, this vector is also referred to as a δ integration vector. Preferably, it may be in the form of a plasmid. From the viewpoint of simplification of DNA acquisition, a shuttle vector of yeast and E. coli is preferable. Optionally, the vector can include regulatory sequences as described above. Such vectors have, for example, a yeast 2 μm plasmid replication origin (Ori) and a ColE1 replication origin, and yeast selection markers (described below) and E. coli selection markers (such as drug resistance genes). ).

  Any known marker can be used as the yeast selection marker. For example, a drug resistance gene, an auxotrophic marker gene (for example, a gene encoding imidazoleglycerol phosphate dehydrogenase (HIS3), a gene encoding malate beta-isopropyl dehydrogenase (LEU2), a gene encoding tryptophan synthase (TRP5) A gene encoding argininosuccinate lyase (ARG4), a gene encoding N- (5′-phosphoribosyl) anthranilate isomerase (TRP1), a gene encoding histidinol dehydrogenase (HIS4), orotidine-5-phosphate deoxygenase A gene encoding carboxylase (URA3), a gene encoding dihydroorotate dehydrogenase (URA1), a gene encoding galactokinase (GAL1), and al And a gene encoding pha-aminoadipate reductase (LYS2)). For example, an auxotrophic marker gene (for example, HIS3, LEU2, URA1, TRP1-deficient marker, etc.) can be preferably used. A yeast selectable marker can be sandwiched between a pair of δ sequences along with an enzyme expression cassette. The position (upstream or downstream) or orientation (forward or reverse) of the yeast selection marker with respect to the enzyme expression cassette is not particularly limited.

  In order to express a plurality of types of cellulolytic enzyme genes, respective δ integration vectors containing the respective enzyme genes can be constructed as the respective enzyme expression vectors. For example, for the expression of three cellulolytic enzyme genes, endoglucanase, cellobiohydrolase, and β-glucosidase, three δ integration vectors containing the genes of the respective enzymes can be constructed. Preferably, each δ integration vector containing the gene for each of these enzymes is designed to have the same yeast selectable marker.

  The final prepared vector contains the desired elements (eg, δ sequence, enzyme gene and its expression regulatory sequence, and yeast selectable marker; preferably, depending on the expression pattern of the enzyme gene, a secretory signal sequence, And an additional element such as a cell surface display factor (which may be located 3 ′ or 5 ′ relative to the expressed enzyme gene depending on the factor used). The procedure may depend on the material used (eg, a backbone vector, an enzyme gene that can be excised from a known vector or an insert of an element such as a cell surface display factor).

  A plurality of δ integration vectors designed for the expression of multiple types of cellulolytic enzymes can be co-introduced into the host yeast. “Introduction” of a vector into a host yeast means not only the introduction of a gene or DNA in the vector into a cell, but also expression. Examples of gene or DNA introduction include transformation, transduction, transfection, co-transfection, and electroporation. Specific examples of the introduction into yeast cells include a method using lithium acetate and a protoplast method. The introduced DNA can be incorporated into the chromosome by undergoing homologous recombination with the δ sequence of the host yeast. In “co-introduction”, these plural vectors may be introduced simultaneously or sequentially. In the case of sequential introduction, the order of introduction is not limited.

  Co-introduction into a host yeast of a vector for δ integration for the expression of genes for a plurality of cellulolytic enzymes can be performed repeatedly. Preferably, during the initial co-introduction as well as during this co-introduction, multiple integration vectors can be designed to have the same yeast selectable marker. More preferably, repeated co-introduction may use a yeast selection marker that is different from the initial or previous co-introduction yeast selection marker.

  After the co-introduction described above, a yeast having a desired cellulose resolution can be selected by screening using the cellulose resolution. For this purpose, the activity of decomposing phosphoric acid-swollen cellulose (PASC) can be used. For example, after screening using a yeast selection marker, screening by measuring PASC degradation activity can be used. Since cellulolytic enzymes are displayed on the surface, colony formation in media using PASC as a single carbon source can also be utilized.

  Kluyveromyces yeasts obtained by co-introduction of vectors for δ integration for the expression of a plurality of types of cellulolytic enzymes as described above are also within the scope of the present invention. Such yeast suitably expresses a plurality of co-introduced cellulolytic enzymes and can hydrolyze cellulose. Such yeast can also be used for hydrolysis of cellulose-containing materials (eg, bagasse, rice straw, etc.).

  In the method of the present invention, transformed Kluyveromyces spp. Yeast is mixed with biomass and the transformed yeast is cultured. Ethanol is produced in the medium.

  Culture of the transformed yeast can be appropriately performed by methods well known to those skilled in the art. The culture temperature is about 40 to about 50 ° C, preferably about 45 to about 50 ° C, and most preferably about 48 ° C. The pH of the medium is preferably about 4 to about 6, most preferably about 5. 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 ppm. Culturing is preferably carried out until the amount of yeast cells is 10 g (wet amount) / L or more, preferably 25 g (wet amount) / L, more preferably 37.5 g (wet amount) / L or more. About 20 to about 50 hours. The transformed yeast can increase the amount of cells by culturing under aerobic conditions before being subjected to fermentation.

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

  The yeast Kluyveromyces marxianus NBRC1777 strain used in this example was obtained from the NITE Biological Resource Center.

  For all the PCR methods shown in this example, KOD-Plus-DNA polymerase (manufactured by Toyobo Co., Ltd.) was used.

  The gene introduction into all yeasts shown in this example was performed by the lithium acetate method using a YAST Maker yeast transformation system (Clontech).

(Reference example: ethanol fermentation from glucose using the yeast Kluyveromyces marukinus)
Whether yeast Kluyveromyces marukinus ΔU strain used for transformation by introducing a cellulase expression cassette can be fermented with ethanol at high temperature using glucose as a sugar source was examined together with the wild strain. In addition to 10 g / L yeast extract, 20 g / L polypeptone and 0.5 g / L potassium disulfate, a medium containing 100 g / L D-glucose as a sugar source was used for fermentation. Fermentation was performed at an initial microbial cell concentration of OD ₆₀₀ = 20 at each temperature of 40 ° C., 42 ° C., and 45 ° C. under anaerobic conditions. For the fermentation, yeast that had undergone seed culture and main culture was used. For seed culture, seed culture was performed at 30 ° C. for 24 hours using an SD medium containing 6.7 g / L YNB, 20 g / L D-glucose and an appropriate amino acid. For the main culture, a YPD medium containing 10 g / L yeast extract, 20 g / L polypeptone and D-glucose was used, and main culture was performed at 30 ° C. for 72 hours under anaerobic conditions. The concentration of glucose in the medium during fermentation was measured by HPLC, and the concentration of ethanol was measured by HPLC. The results are shown in FIG. Squares represent results at 40 ° C, circles at 42 ° C, and diamonds at 45 ° C. White represents the glucose concentration, and black represents the ethanol concentration. The bar attached to each value represents the standard deviation.

  As is apparent from FIG. 1, the yeast Kluyveromyces marukinus ΔU strain (requiring uracil) and the wild strain all grow using D-glucose as a sugar source even at high temperatures of 40 ° C., 42 ° C. and 45 ° C. It was confirmed that ethanol was produced. Further, at 42 ° C., the ethanol production rate in the initial stage of fermentation and the final yield of ethanol were the highest, and it was found that there was an optimum temperature for fermentation from glucose between 40 ° C. and 45 ° C. In this way, it was found that the yeast Kluyveromyces marukinus can be fermented from glucose at high temperatures. It was suggested that fermentation is possible.

(Example 1: Construction of pδUAGBGL and pδUAGEGII)
A plasmid for presenting one of cellulases, Trichoderma reesei-derived endoglucanase II (EGII) and Aspergillus aculeatus-derived β-glucosidase 1 (BGL1) on the cell surface of yeast Constructed as follows.

(Construction of pIHPGBGL and pIWPGAGEGII)
PGK1 promoter sequence (pPGK), one of the phosphoglycerate kinases present on the yeast genome, using primers pPGKF (XhoI) (SEQ ID NO: 1) and pPGKR (SmaI) (SEQ ID NO: 2), and The terminator sequence (tPGK) was amplified by PCR using the genomic DNA of the yeast Saccharomyces cerevisiae BY4741 strain as a template, using tPGKF (SmaI) (SEQ ID NO: 3) and tPGKR (NotI) (SEQ ID NO: 4).

  The amplified pPGK and tPGK were respectively inserted into the XhoI / SmaI site and SmaI / NotI site of the vector plasmid pBluescript II KS + (Stratagen).

  A sequence containing pPGK and tPGK was obtained from the constructed plasmid by restriction enzyme treatment with BSSHII, and yeast expression vectors pRS403 (HIS3 yeast expression vector: Stratagen) and pRS404 (TRP1 yeast expression vector: Stratagen) The resulting plasmids were named pIHPG and pIWPG.

  The BGL1 / AG-anchor and EGII / AG-anchor genes were converted to primers BGLF (XbaI) (SEQ ID NO: 5) and BGLR (XbaI) (SEQ ID NO: 6), and EGIIF (NheI) (SEQ ID NO: 7) and EGIIR (SmaI), respectively. ) (SEQ ID NO: 8), pBG211 (a 3 ′ half region of the α-agglutinin gene (a region consisting of nucleotides from position 991 to position 1953 of the coding region of the α-agglutinin gene) and 445 bp downstream of the coding region Β-glucosidase 1 surface expression vector having non-patent document 9) and pEG23u31H6 (endoglucanase II surface expression vector having the 3 ′ half region of α-agglutinin gene: non-patent document) Amplification was performed by PCR using 9) as a template.

  The amplified BGL1 / AG-anchor was inserted into pIHPG, the resulting plasmid was named pIHPGBGL, the amplified EGII / AG-anchor gene was inserted into pIWPG, and the resulting plasmid was named pIWPGAGEGII.

(Construction of pδU)
A vector plasmid pδU for δ integration was constructed as follows.

  First, the 5′-side 167 bp (5′δ sequence) of the δ sequence present on the yeast genome was used using the primers 5′DSF (SacI) (SEQ ID NO: 9) and 5′DSR (SacI) (SEQ ID NO: 10). The yeast Saccharomyces cerevisiae BY4741 genomic DNA was used as a template for amplification by the PCR method.

  Subsequently, the amplified insert DNA (5′δ sequence) was inserted into the SacI site of the vector plasmid pBluescript II KS +.

  Similarly, 167 bp (3′δ sequence) on the 3 ′ side of the δ sequence was amplified by PCR using primers 3′DSF (KpnI) (SEQ ID NO: 11) and 3′DSR (KpnI) (SEQ ID NO: 12), It was introduced into the kpnI site of pBluescript II KS + into which the 5′δ sequence had been introduced.

  Using the URA3 deficient marker (URA3d) from the URA3 gene present on the plasmid pRS406 (URA3 yeast expression vector: Stratagen), using the primers URA3dF (XhoI) (SEQ ID NO: 13) and URA3dR (XhoI) (SEQ ID NO: 14) The 5′δ sequence and the 3′δ sequence described above were inserted into the XhoI site of the introduced vector plasmid pBluescript II KS +, and the resulting plasmid was named pδU. Therefore, pδU is a vector plasmid for δ inlation containing a URA3 deletion marker as a selectable marker and a yeast δ sequence.

  Various cellulase expression cassettes were inserted into the δ integration vector plasmid pδU to construct two cellulase expression δ integration vector plasmids pδU-PGAGBGL and pδU-PGAGEGII. These construction procedures will be described below.

(Construction of pδUAGBGL and pδUAGEGII)
Using the plasmids pIHPGBGL and pIWPGAGEGII as templates, primers pPGKF (NotI) (SEQ ID NO: 15) and tAGR (NotI) (SEQ ID NO: 16), respectively, by PCR, the promoter of each cellulase expression cassette, secretion signal, A region containing an expressed enzyme gene and a cell surface display factor in this order (ie, a PGK promoter, a secretion signal, a β-glucosidase 1 gene, a region on the 3 ′ half of the α-agglutinin gene; PGK promoter, secretion signal, cello (The region of the 3 ′ half of the biohydrolase II gene and the α-agglutinin gene; and the region of the 3 ′ half of the PGK promoter, secretion signal, endoglucanase II gene, and α-agglutinin gene are arranged in the order of description) Was amplified.

  The cellulase expression cassettes described above were inserted into the NotI site of the pδU, and the resulting plasmids were named pδUAGBGL and pδUAGEGII, respectively. A schematic diagram of each plasmid is shown in FIG. 2 (A: pδUAGBGL, B: pδUAGEGII). Β-glucosidase 1 and endoglucanase II expressed from these cellulase expression cassettes are displayed on the cell surface of the host yeast into which the gene has been introduced.

(Example 2: Production of transformed yeast)
Two plasmids pδUAGBGL and pδUAGEGII having the URA3 deletion marker prepared in Example 1 were co-introduced into the yeast Kluyveromyces marukinus NRBC1777 strain. For screening of transformed yeast, screening was performed by visual observation of colony formation on a selective medium plate containing no uracil and glucose as a single carbon source, followed by measurement of endoglucanase activity and β-glucosidase activity To obtain 3 strains of transformed yeast strains KdUEB1, KdUEB2 and KdUEB3 by δ integration.

In addition, the endoglucanase activity of the bacterial cells was measured as follows:
(1) Inoculate yeast cells in 5 mL of YPD medium and culture for 72 hours;
(2) Washing the cells twice with distilled water;
(3) Prepare a reaction solution 250 μL (composition: 1% CMC (carboxymethylcellulose; Sigma-Aldrich) 200 μL; yeast 50 μL) and react at 50 ° C. for 6 hours;
(4) The sample after the reaction is centrifuged, 100 μL of the supernatant is mixed with 100 μL of Somogyi copper reagent (Sigma-Aldrich), incubated at 100 ° C. for 20 minutes, and then immediately cooled on ice;
(5) After cooling, 250 μL of Nelson reagent (manufactured by Sigma-Aldrich) is mixed to dissolve and develop the reduced copper precipitate;
(6) After standing for 30 minutes, the mixture was centrifuged at 14,000 rpm for 10 minutes at 20 ° C., 625 μL of distilled water was mixed with 250 μL of the supernatant, and the absorbance at 520 nm was measured. The amount of enzyme that liberates 1 μmol of glucose converted reducing sugar in 1 minute is defined as 1 U.

The measurement of the β-glucosidase activity of the bacterial cells was performed as follows:
(1) Inoculate yeast cells in 5 mL of YPD medium and culture for 72 hours;
(2) Washing the cells twice with distilled water;
(3) Prepare 1000 μL of reaction solution (composition: 10 mM pNPG (p-nitrophenyl-β-D-glucoside; Sigma-Aldrich) 100 μL; 50 μL of 1M NaAc (pH 5.0); 333 μL of yeast; 517 μL of distilled water) Reaction at 30 ° C. for 30 minutes;
(4) The sample after the reaction is centrifuged, 500 μL of 3M Na ₂ CO ₃ is mixed with 500 μL of the supernatant, and the absorbance at 400 nm is measured. The amount of enzyme that releases 1 μmol of pNP in 1 minute is defined as 1 U.

(Example 3: Confirmation of gene introduction into transformed yeast)
About the transformed yeast strain obtained in Example 2, introduction of the co-introduced β-glucosidase 1 gene and endoglucanase II gene was confirmed by a real-time PCR method.

Real-time PCR was performed as follows:
(1) Inoculate yeast cells in 5 mL of SD medium and culture for 24 hours;
(2) Washing the cells twice with distilled water;
(3) Buffer solution (100 mM NaCl; 1 mM EDTA; 2% Triton-X; 1% SDS; 10 mM Tris-HCl) 200 μL, PCI (phenol: chloroform: isoamyl alcohol = 25: 24: 1) solution in cells 200 μL and 400 μL of glass beads (φ0.5 mm; manufactured by Yasui Kikai Co., Ltd.) are added, mixed and treated with a multi-bead shocker (manufactured by Yasui Kikai Co., Ltd.);
(4) Add 200 μL of PCI solution and 200 μL of distilled water to the treatment solution, mix and centrifuge;
(5) The supernatant is collected and treated with RNase;
(6) After PCI treatment and ethanol precipitation, the precipitate is dissolved in 20 μL of TE buffer (genomic DNA solution);
(7) After measuring the DNA concentration of the yeast genomic DNA solution, the genomic DNA is treated with restriction enzyme NotI, and after PCI treatment and ethanol precipitation, the precipitate is dissolved in 20 μL of TE buffer (restriction enzyme-treated genomic DNA solution);
(8) Real-time PCR using a restriction enzyme-treated genomic DNA solution as a template (BGL1 gene amplification PCR primer: consisting of the nucleotide sequences shown in SEQ ID NOs: 17 and 18; EGII gene amplification PCR primer: the nucleotide sequences shown in SEQ ID NOs: 19 and 20) PCR primer for amplification of endogenous phosphoglycerate kinase (PGK) gene: consisting of the nucleotide sequences shown in SEQ ID NOs: 21 and 22).

  The results are shown in FIG. The copy number of the introduced cellulase gene is expressed as a relative amount with respect to the copy number of the endogenous phosphoglycerate kinase (PGK) gene.

  From FIG. 3, among KdUEB1, KdUEB2 and KdUEB3, KdUEB1 had the highest number of copies of the EGII gene, but the copy number of the BGL1 gene was the smallest. KdUEB3 had the highest copy number of the BGL1 gene, but the lowest copy number of the EGII gene. It was confirmed that all strains were introduced with β-glucosidase 1 gene and endoglucanase II gene.

(Example 4: Ethanol fermentation from cellobiose using transformed yeast)
It was examined whether the transformed yeast strains KdUEB1, KdUEB2, and KdUEB3 of Kluyveromyces marukinus obtained in Example 2 can be fermented with ethanol at high temperatures using cellobiose as a sugar source. In addition to 10 g / L yeast extract, 20 g / L polypeptone and 0.5 g / L potassium disulfate, a medium containing 50 g / L cellobiose as a sugar source was used for the fermentation. Fermentation was carried out at an initial cell concentration of OD ₆₀₀ = 20 under anaerobic conditions at temperatures of 40 ° C. and 42 ° C. For the fermentation, as in the reference example, yeast that had undergone seed culture and main culture was used. The concentration of cellobiose in the medium during fermentation was measured by HPLC, and the concentration of ethanol was measured by HPLC or GC. The results are shown in FIG. Squares represent the results of the KdUEB1 strain, circles represent the results of the KdUEB2 strain, and diamonds represent the results of the KdUEB3 strain. White indicates the cellobiose concentration, and black indicates the ethanol concentration. The bar attached to each value represents the standard deviation.

  As is clear from FIG. 4, even at high temperatures of 40 ° C. and 42 ° C., the transformed yeast strains of Kluyveromyces marukinus with endoglucanase II gene and β-glucosidase 1 gene grow using cellobiose as a sugar source. , Confirmed to produce ethanol. Moreover, it became clear that any transformant yeast strain | stump | stock improves fermentability by the temperature rise from 40 degreeC to 42 degreeC. Furthermore, KdUEB3, which had the highest expression of β-glucosidase 1 at any temperature, had the highest ethanol production rate at the beginning of fermentation, but the lowest ethanol yield. This is thought to be because glucose produced by saccharification of cellobiose by β-glucosidase 1 inhibited the activity of β-glucosidase 1.

(Example 5: Ethanol fermentation from β-glucan using transformed yeast)
It was examined whether the transformed yeast strain KdUEB1 of Kluyveromyces marukinus obtained in Example 2 can be ethanol-fermented at high temperatures using β-glucan as a sugar source. In addition to 10 g / L yeast extract, 20 g / L polypeptone and 0.5 g / L potassium disulfate, a medium containing 10 g / L β-glucan as a sugar source was used for fermentation. Fermentation was performed at an initial microbial cell concentration of OD ₆₀₀ = 20 at each temperature of 40 ° C., 42 ° C., and 45 ° C. under anaerobic conditions. The concentration of cellobiose in the medium during fermentation was measured by HPLC, and the concentration of ethanol was measured by HPLC or GC. The results are shown in FIG. Squares represent results at 40 ° C, circles at 42 ° C, and diamonds at 45 ° C. White indicates the β-glucan concentration, and black indicates the ethanol concentration. The bar attached to each value represents the standard deviation.

  As is apparent from FIG. 5, even at high temperatures of 40 ° C., 42 ° C. and 45 ° C., the transformed yeast strains of Kluyveromyces marukinus endoglucanase II gene and β-glucosidase 1 gene As a source, it was confirmed to grow and produce ethanol. In addition, when β-glucan was used as a sugar source, it became clear that the fermentation ability was improved when the temperature increased from 40 ° C. to 42 ° C. and from 42 ° C. to 45 ° C. At 45 ° C., the ethanol production rate at the beginning of fermentation and the final yield of ethanol were the highest.

It can be applied to the production of biofuels (ethanol, butanol, isoprenoids, etc.) and chemical polymer raw materials (isopropanol, amino acids, organic acids, quinones, etc.) from cellulosic biomass. Effective use of biomass can be expected not only to reduce the environmental burden, but also to suppress CO ₂ emissions due to the use of fossil resources.

Claims (10)

A method for producing ethanol from biomass,
A method comprising the step of mixing and culturing transformed Kluyveromyces yeast expressing at least two types of cellulolytic enzymes with biomass.   The transformed Kluyveromyces yeast that expresses at least two of the cellulolytic enzymes is a transformed Kluyveromyces yeast that presents at least two of the cellulolytic enzymes on the cell surface. the method of.   The method according to claim 1 or 2, wherein the at least two cellulolytic enzymes are a combination of enzymes having different modes of cellulose hydrolysis.   4. The method of claim 3, wherein the combination of enzymes with different modes of cellulose hydrolysis is selected from the group consisting of endoglucanase, cellobiohydrolase, and β-glucosidase.   The method according to claim 4, wherein the combination of enzymes having different cellulose hydrolysis modes is a combination of endoglucanase, cellobiohydrolase, and β-glucosidase.   6. The transformed Kluyveromyces genus yeast according to claim 1, wherein the genes encoding the at least two cellulolytic enzymes are co-introduced by integration with a yeast δ sequence. Method.   The method according to any one of claims 1 to 6, wherein the culturing step is performed at 40 ° C or higher.   The method according to any one of claims 1 to 7, wherein the transformed Kluyveromyces genus yeast is transformed Kluyveromyces marcianus.   A transformed Kluyveromyces yeast that expresses at least two cellulolytic enzymes.   Transformed Kluyveromyces marukinus expressing at least two cellulolytic enzymes.

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