JP5881300B2 - Ethanol-fermenting microorganisms from lignocellulosic biomass - Google Patents

Description

  The present invention relates to an ethanol-fermenting microorganism in which the expression function of a gene for a glycine synthesis protein and / or a gene for a methionine synthesis protein is lost. The present invention also relates to a method for producing a fermentation microorganism having an increased xylose fermentation ability by losing the expression function of a gene for a glycine synthesis protein and / or a gene for a methionine synthesis protein. Furthermore, this invention relates to the manufacturing method of ethanol including the process which the said microorganisms and a xylose containing raw material are made to contact.

In recent years, from the viewpoint of depletion of fossil fuels and the need for CO ₂ gas reduction, research has been conducted on the production of ethanol from biomass, such as corn cob, rice straw, switchgrass, Elianthus, waste materials, etc. It is being advanced. For thousands of years, mankind has had the technology of converting starch into ethanol by fermentation with the yeast Saccharomyces cerevisiae. Starch is a polysaccharide in which glucose is α-1,4-linked and can be easily degraded by hydrolytic enzymes possessed by various organisms. Glucose is the most preferred C source of the yeast Saccharomyces cerevisiae, and two molecules of ethanol are produced from one molecule of glucose by fermentation. On the other hand, biomass contains cellulose or hemicellulose as a polysaccharide.

  Among these, cellulose is a polysaccharide in which glucose is linked by β-1,4 and has a crystal structure. There are problems such as the necessity of pretreatment to break the crystal structure and insufficient activity of cellobiohydrolase I, cellobiohydrolase II, endoglucanase, etc. necessary for decomposing cellulose. Since the sugar produced after decomposition is glucose, there is no problem in fermentation with the yeast Saccharomyces cerevisiae.

  On the other hand, hemicellulose contains pentoses such as xylose and arabinose in addition to glucose. Conventionally, yeast Saccharomyces cerevisiae cannot ferment these pentoses. Therefore, in order to ferment ethanol from xylose, there is a method for highly expressing the xylose reductase gene and xylitol dehydrogenase gene of yeast Pichia stipitis, which has the ability to ferment xylose, in yeast Saccharomyces cerevisiae. Have been used frequently (Non-Patent Documents 1 to 4). Pichia stippitis xylose reductase is an enzyme that mainly uses NADPH as a coenzyme, and a single molecule of NADP is produced after the reaction. On the other hand, xylitol dehydrogenase is an enzyme that mainly uses NAD as a coenzyme, and produces one molecule of NADH after the reaction. Therefore, when fermenting glucose, the balance of NADP / NADPH or NAD / NADH does not change, but when fermenting ethanol from xylose, it tends to increase NADP or NADH. This is considered to be a cause of a decrease in the production amount in ethanol production from xylose. On the other hand, attempts have been made to express enzymes with altered coenzyme specificity and enzymes with different coenzyme specificity, but sufficient effects have not been obtained (Non-patent Documents 2 to 4).

Petschacher B, Nidetzky B. Microb Cell Fact. 2008 Mar 17; 7: 9. Watanabe S, Saleh AA, Pack SP, Annaluru N, Kodaki T, Makino K. J Biotechnol. 2007 Jun 30; 130 (3): 316-9. Epub 2007 Apr 29. Verho R, Londesborough J, Penttile M, Richard P. Appl Environ Microbiol. 2003 Oct; 69 (10): 5892-7. Mikael Anderlund et.al.Appl Environ Microbiol. 1999: June 65 (6): 2333-2340

  Under such circumstances, development of microorganisms with improved xylose fermentability is required.

The present inventors introduced xylose fermentability by introducing xylose reductase gene, xylitol dehydrogenase gene of yeast Pichia stipitis, and xylulose kinase gene of yeast Saccharomyces cerevisiae. Fermentation tests were performed using the yeast of No. 2 and profile analysis of intracellular metabolites in ethanol fermentation from xylose was performed. The obtained data and the numerical values representing the fermentation performance include (1) ethanol production amount / rate / yield, (2) xylose utilization amount / rate / yield, and (3) byproduct production amount / By comparing the rate and yield, etc., and analyzing the correlation between the data and (1) to (3), substances belonging to the metabolic pathway from xylose to ethanol, ie, xylitol, xylulose, xylulose pentaphosphate Then, the relationship between the ethanol fermentation performance and a new substance other than the intermediate metabolite belonging to the glycosylation system and the pentose phosphate cycle was found, the gene as a breeding target was determined, and breeding related to the gene was performed. As a result, it was found that the utilization rate of xylose increased by disrupting the gene GLY1 in the glycine biosynthesis pathway. It was also found that the amount of xylitol produced as a byproduct was reduced by disrupting the methionine biosynthetic pathway gene, MET6 or MET32.
The present invention is based on the above findings.

That is, the present invention relates to the following.
[1] A microorganism in which the expression function of a gene for a glycine synthesis protein and / or a gene for a methionine synthesis system protein is lost and a xylose metabolizing enzyme gene is introduced.
[2] The microorganism according to [1], wherein the gene for the glycine synthesis protein is a GLY1 gene.
[3] The microorganism according to [1] or [2], wherein the gene of the methionine synthesis protein is a MET6 or MET32 gene.
[4] The microorganism according to any one of [1] to [3], which is a yeast.
[5] The microorganism according to any one of [1] to [4], which is Saccharomyces cerevisiae.
[6] The above [1] to [5], wherein the xylose metabolizing enzyme gene is at least one selected from the group consisting of a xylose reductase gene, a xylitol dehydrogenase gene, and a xylulose phosphorylase gene. The microorganism according to any one of the above.
[7] A method for producing a microorganism having an increased ability to ferment xylose by losing the expression function of a gene for a glycine synthesis system protein and / or a gene for a methionine synthesis system protein.
[8] The method according to [7] above, wherein the gene for the glycine synthesis protein is a GLY1 gene.
[9] The method according to [7] or [8] above, wherein the gene of the methionine synthesis protein is a MET6 or MET32 gene.
[10] The method according to any one of [7] to [9], wherein the microorganism is yeast.
[11] The microorganism according to any one of [7] to [10], wherein the microorganism is Saccharomyces cerevisiae.
[12] The method according to any one of [7] to [11], wherein the microorganism is a gene into which a xylose metabolic enzyme gene has been introduced.
[13] The method according to [12], wherein the xylose metabolic enzyme gene is at least one selected from the group consisting of a xylose reductase gene, a xylitol dehydrogenase gene, and a xylulose phosphorylase gene.
[14] A method for producing ethanol, comprising a step of bringing the microorganism according to any one of [1] to [6] into contact with a xylose-containing raw material.

By using the yeast bred in the present invention, the ability to ferment ethanol from xylose or the fermentation rate can be improved. The same breeding can be carried out in any brewing yeast or laboratory yeast. Moreover, as long as it contains xylose, the same effect can be expected with this breeding yeast regardless of the mash obtained from any biomass.
According to the present invention, a microorganism having excellent xylose fermentation ability is provided. Moreover, by using the method for producing a microorganism of the present invention, breeding having a high xylose fermentation ability can be made using a wide range of microorganisms as hosts. By using the microorganism of the present invention, ethanol can be efficiently produced from a xylose-containing raw material. Further, when the microorganism of the present invention is used, ethanol can be efficiently produced even when, for example, biomass having a low amino acid content is used as a raw material.

1 is a schematic representation of the plasmid pIUX1X2XKO used in the examples. 1 is a schematic representation of the plasmid pBIITGRFRT used in the examples. 1 is a schematic representation of the plasmid pUGRFRT used in the examples. 1 is a schematic representation of the plasmid YRNFLP used in the examples. The result of the ethanol fermentation test from xylose using the microorganisms of this invention is shown (xylose utilization amount). The result of the ethanol fermentation test from xylose using the microorganisms of this invention is shown (ethanol production amount). The result of the ethanol fermentation test from the xylose using the microorganism of this invention is shown (xylitol production amount). The results of an ethanol fermentation test from xylose using the microorganism of the present invention are shown (XCR: xylose utilization rate, EPR ethanol production rate, XPR: xylitol production rate, EY: ethanol yield, XY: xylitol yield, Met6: MET6 gene disruption strain, Met32: MET32 gene disruption strain, Gly1: GLY1 gene disruption strain, MT: parent strain). 2 is a diagram showing the results of a xylose fermentation test of Example 1 and a comprehensive analysis of intracellular components of yeast cells. FIG.

  The present inventors have found that when a microbial strain in which the expression of a gene for a glycine synthesis protein is suppressed is used, the utilization rate of xylose increases as compared to a wild-type homologous strain. Further, the methionine synthesis protein It was found that the production of xylitol, which is a byproduct, in the production of ethanol using xylose as a starting material was reduced in the microbial strain in which the expression of the above gene was suppressed, and the present invention was completed. Each embodiment of the present invention will be described below.

1. Glycine synthesizing protein gene and / or methionine synthesizing protein gene functional loss microorganism In one embodiment, the present invention relates to a microorganism that has lost the expression function of glycine synthesizing protein gene and / or methionine synthesizing protein gene. provide.

In the present invention, the term “microorganism” means an organism having a size that is less than the extent that its presence cannot be discerned with the naked eye and can be observed with a microscope or the like. Examples of microorganisms include prokaryotes such as bacteria, cyanobacteria and archaea, and eukaryotes such as filamentous fungi, yeast, deformed fungi, basidiomycetes, unicellular algae and protozoa.
Preferably, the microorganism is a species capable of fermenting sugar, and more preferably yeast. The yeast may be budding yeast or fission yeast. In one embodiment, the yeast is Saccharomyces cerevisiae NBRC1951, NBRC1952, NBRC1953, NBRC1954, X2180-1A (ATCC26786), CB11 (Berkley Stock Center), W303-1A (BY4848), MT8-1 (BY2685), NBRC1440, NBRC1445. Saccharomyces cerevisiae such as In another embodiment, the yeast is Schizosaccharomyces japonicus (Hasegawaea japonicus), Schizosaccharomyces octosporus (Octosporomyces octosporus) and Schizoscharcharomyces It may be.

  In the present invention, the term “glycine synthesis protein gene” or “glycine synthesis protein gene” means a gene encoding an enzyme protein or transcriptional regulatory factor involved in glycine synthesis, and may be either DNA or RNA. . For example, for yeast-derived ones, superpathway of serine and glycine biosynthesis (http://www.yeastgenome.org/cgi-bin/search/textSearch) described in the Saccharomyces genome database (http://www.yeastgenome.org/) .pl? query = glycine & type = pathway) genes, SHM2, SHM1, GLY1, SER3, AGX1, SER1, SER2, SER33, and the like. A glycine synthesis protein is an enzyme protein involved in glycine synthesis and a transcriptional regulator thereof.For example, those derived from yeast are encoded by the above superpathway SHM2, SHM1, GLY1, SER3, AGX1, SER1, SER2, SER33. There is a protein. Preferably, it is a protein (hereinafter referred to as “L-threonine aldolase activity”) having an activity of hydrolyzing L-threonine to produce glycine (hereinafter referred to as “L-threonine aldolase activity”). In the case of yeast, GLY1 Coded to

As used herein, the “glycine synthesis protein gene” is a gene (SEQ ID NO: 1, Saccharomyces Genome Database Accession) encoding L-threonine aldolase “GLY1” (SEQ ID NO: 2) derived from Saccharomyces cerevisiae. No: YEL046C), and may be a gene encoding a homologous protein belonging to the same family as L-threonine aldolase and exhibiting L-threonine aldolase activity. Proteins belonging to the same family as L-threonine aldolase have been found in many plants and are highly conserved throughout the family. Since L-threonine aldolase is an essential protein for glycine synthesis, its gene is highly conserved throughout the family, and the genes for L-threonine aldolase or its homologous protein of several microorganisms are already bases. The sequence is analyzed and the sequence information is registered in the database. Table 1 shows genes encoding L-threonine aldolase derived from microorganisms and homologs thereof, but is not limited thereto.
When producing the microorganism of the present invention using Saccharomyces cerevisiae, the glycine synthesis protein gene is preferably the GLY1 gene.

  In one embodiment, the glycine synthesis protein gene is a mutation in which one or more bases are deleted, inserted, substituted, or added in the base sequence of a polynucleotide encoding a polypeptide having L-threonine aldolase activity. It may be a body. Variants can be mutated in coding or non-coding regions, or both. Mutations in the coding region can generate conservative or non-conservative amino acid deletions, insertions, substitutions and / or additions. As used herein, the gene encoding the amino acid sequence of SEQ ID NO: 2, or one or more of the amino acid sequence (for example, 1 to 40, 1 to 20, 1 to 15, 1 to 10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1 etc.) or 1 or A gene encoding an amino acid sequence in which several amino acids are deleted, substituted or added is also included in the “gene of glycine synthesis protein”.

  In the present invention, the term “methionine synthesis protein gene” or “methionine synthesis protein gene” means a gene encoding an enzyme protein or transcriptional regulatory factor involved in methionine synthesis, and may be either DNA or RNA. . For example, for yeast-derived ones, see superpathway of threonine and methionine biosynthesis (http://pathway.yeastgenome.org/YEAST/new-image?type=PATHWAY&object=P4-PWY&detail-level=2) described in the Saccharomyces genome database. The genes involved, MET2, HOM3, FOL3, HOM2, MET7, MET17, THR1, THR4, HOM6, MET6 and superpathway of methionine salvage pathway (http: // pathway. Yeastgenome.org/YEAST/new-image?type=PATHWAY&object = PWY3O-351 & detail-level = 2), MRI1, BAT1, ARO9, MDE1, SAM2, ARO8, SPE2, SPE3, SPE4, UTR4, MEU1, ADI1, BAT2, SAM1, and transcriptional regulation of these genes The genes involved are MET4, MET28, MET31, MET32. The methionine synthesis system protein is an enzyme protein involved in methionine synthesis and its transcriptional regulator, for example, those derived from yeast, MET2, HOM3, FOL3, HOM2, MET7, MET17, THR1, THR4, HOM6 of the above superpathway, There are proteins encoded by MET6, MRI1, BAT1, ARO9, MDE1, SAM2, ARO8, SPE2, SPE3, SPE4, UTR4, MEU1, ADI1, BAT2, SAM1, MET4, MET28, MET31, and MET32. Preferably, a protein (hereinafter referred to as “methionine synthase”) having an activity of methylating sulfur of homocysteine to generate methionine to produce methionine (hereinafter referred to as “methionine synthase activity”) and a transcription regulator of methionine synthase. In the case of yeast, an example of a gene for methionine synthase is MET6, and a gene encoding the transcriptional regulatory factor is MET32.

As used herein, a “methionine synthesis protein gene” is a gene encoding a methionine synthase “MET6” (SEQ ID NO: 4) derived from Saccharomyces cerevisiae (SEQ ID NO: 3, Saccharomyces Genome Database Accession No: YER091C) and the gene encoding its transcription factor “MET32” (SEQ ID NO: 6) (SEQ ID NO: 5, Saccharomyces Genome Database Accession No: YDR253C) are not limited to the same family as MET6, which exhibits methionine synthase activity It may be a gene encoding a homologous protein to which it belongs (namely, a homologous gene of MET6) or a gene encoding a transcription factor of the MET6 homologous gene (that is, a homologous gene of MET32). Proteins belonging to the same family as MET6 and MET32 have been found in many plants and are highly conserved throughout the family. Table 2 shows genes encoding MET6 derived from microorganisms and homologues thereof, and Table 3 shows genes encoding MET32 derived from microorganisms and homologues thereof, but is not limited thereto.
When the microorganism of the present invention is produced using Saccharomyces cerevisiae, the methionine synthesis protein gene is preferably a MET6 or MET32 gene.

  In one embodiment, the methionine synthesis protein gene has one or more bases deleted, inserted, substituted, or added in the base sequence of a polynucleotide encoding a polypeptide having methionine synthase activity or a transcription factor thereof. It may be a mutant. Variants can be mutated in coding or non-coding regions, or both. Mutations in the coding region can generate conservative or non-conservative amino acid deletions, insertions, substitutions and / or additions. As used herein, a gene encoding the amino acid sequence of SEQ ID NO: 4 or 6, or one or more of the amino acid sequences (for example, 1 to 40, 1 to 20, 1 to 15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1 etc.) or A gene encoding an amino acid sequence in which one or several amino acids are deleted, substituted or added is also included in the “gene of methionine synthesis system protein”.

  In the present invention, “the expression function of the gene for the glycine synthesis protein and / or the gene for the methionine synthesis system protein has been lost” means that the glycine synthesis system protein gene and / or the methionine synthesis system protein gene (hereinafter “target protein”). It means that the target protein having the original biological function is not expressed from the gene generically). Examples of such a state include not only the state where no expression product of the target protein gene is generated, but also the expression product of the gene (for example, hnRNA, mRNA or protein) is expressed, The state which does not have a normal function is mentioned. Such loss of function of the target protein gene may be caused by deletion, substitution, and / or insertion of one or more nucleotides on the target protein gene or an expression control region including the transcriptional regulatory region or promoter region thereof. it can. The site for the deletion, substitution, and / or insertion and the sequence to be deleted, substituted, and / or inserted are not particularly limited as long as the normal function of the target protein gene can be lost, but is preferably Preferably, at least one of the gene sequences encoding the active site of the target protein is deleted.

  The microorganism of the present invention may be effective if the expression function of the target protein is lost in at least one allele on the chromosome (heterozygous type), particularly in the case of a eukaryote, Loss in both alleles (homozygous) is preferred.

2． BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a fermentation microorganism having an increased xylose fermentation ability by losing the expression function of a gene of a glycine synthesis protein and / or a gene of a methionine synthesis protein of a host microorganism. A method of producing a microorganism is provided.
Examples of the method for losing the expression function of a glycine synthesis protein gene and / or a methionine synthesis protein gene include gene targeting or knockout or knockdown techniques such as RNAi.

(1) Gene targeting Gene targeting is a technique for introducing a mutation into a specific gene on a chromosome using homologous recombination (Capeccchi, MR Science, 244, 1288-1292, 1989).

  First, a targeting vector for losing the target protein gene function is constructed. In constructing the targeting vector, a genomic DNA library of the target microorganism is prepared. This genomic DNA library is prepared from microorganisms of the same strain as the microbial species targeted for gene targeting, preferably from genomic DNA derived from the same type of microorganisms, so that the frequency of recombination does not decrease due to polymorphisms, etc. It is preferable to use it. A commercially available library may be used as such a library. Alternatively, screening may be performed using the cDNA of the target protein or a partial sequence thereof as a probe, and the DNA sequence of the genomic protein of the target protein may be cloned, or a primer is created based on the cDNA of the target protein or a partial sequence thereof, It can be prepared by PCR amplification of the target protein gene of the genome. For details of the method for preparing a genomic DNA library, the following can be referred to. "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor Laboratory Press 2001", "Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997"

  Next, the cloned genomic DNA is analyzed by sequencing, Southern blotting, restriction enzyme digestion and the like to identify the position of exons (in the case of eukaryotes) and restriction enzyme sites. Based on the sequence information obtained by such analysis, a mutation introduction site and the like are determined.

  In the present invention, the mutation (deletion, substitution, and / or insertion) to be introduced onto the chromosome is not particularly limited as long as the normal function of the target protein gene is impaired, and these mutations are the intron region of the target protein gene, It may exist in the exon region or the expression control region of the target protein gene. The mutation is preferably present in an exon region of the target protein gene, more preferably a mutation in which at least one exon of the target protein gene is deleted, and most preferably a mutation in which all exons are deleted. This is because such a mutation can surely delete the function of the target protein gene.

The targeting vector may contain a selection marker for selection of recombinants in addition to the 3 ′ and 5 ′ homologous regions (3 ′ arm and 5 ′ arm, respectively) of the mutation introduction site. Examples of selectable markers include Nourseothricin dihydrogen sulfate (NAT) resistance gene, neomycin resistance gene, hygromycin B phosphotransferase gene, kanamycin resistance gene and other positive selection markers, LacZ, GFP (Green Fluorescence Protein) and luciferase genes Examples include an expression reporter of a target gene, a herpes simplex virus thymidine kinase gene (HSV-TK), and a negative selection marker such as diphtheria toxin A fragment (DTA). In particular, since the present invention uses a microorganism as a host, if a gene that complements auxotrophy, such as URA3, is used, selection can be performed on an agar medium that does not contain uracil, and depending on the auxotrophy of the host, it can be used. Is possible. In addition, if a drug resistance gene (YAP1) for cycloheximide is used, selection can be performed in a medium containing cycloheximide. The target gene can also be destroyed by using a sequence in which these selection markers are inserted between the 3 ′ arm and the 5 ′ arm. The vector may contain an appropriate restriction enzyme cleavage site for linearizing the vector outside the homologous region.
Such a targeting vector can be constructed based on a commercially available plasmid vector (for example, pENTR / D-TOPO (registered trademark: Invitrogen), pBluescriptII (Stratagene), etc.).

  In the microorganism of the present invention, it is preferable that the selection marker used for homologous recombination is dropped from the cell after the target protein gene is destroyed. For example, a recombination sequence is inserted into both ends of the selectable marker gene encoded by the targeting vector, recombination occurs between the recombination sequences, and the selectable marker gene is looped out, whereby the selectable marker is transferred to the host cell. Can be dropped from. Since the microorganism of the present invention produced in this manner does not have a selection marker, it has an excellent feature that the selection marker is not restricted when a plurality of target protein genes are destroyed.

Recombinant sequences that can be used to drop selectable markers include Flippase Recognition Target sequence (FRT sequence), loxP sequence derived from bacteriophage P1, RS sequence derived from soy yeast (Zygosaccharomyces rouxii), gix sequence derived from bacteriophage Mu, etc. Is mentioned.
In order to cause recombination between these recombination sequences, it is necessary to express an appropriate recombinase. The recombinases that cause recombination between the above recombination sequences are Flippase (FLP: FLP-FRT recombination), Cre (Cre-loxP recombination), R Recombinase (R: RS recombination), GIX recombinase ( R: RS recombination).
Furthermore, it is preferable that the plasmid encoding recombinase is also removed from the host cell. This is because the selection marker is not limited in disrupting a plurality of target protein genes in the host cell.
Accordingly, the plasmid encoding recombinase is preferably unstable in the host cell, and in particular, a plasmid that is removed from the host cell in a state in which the selection pressure for the selection marker of the plasmid is not applied.
Examples of such plasmids include, but are not limited to, YRp type plasmids.

  Selectable markers include any drug resistance (NAT resistance, neomycin resistance, hygromycin B phosphotransferase resistance, kanamycin resistance, hygromycin resistance, zeocin resistance, geneticin resistance), auxotrophic markers (ura3, ura5, niaD), copper resistance Marker (CUP1) (Marin et al., Proc. Natl. Acad. Sci. USA, 81, 337 1984), cerulenin resistance marker (fas2m, PDR4), etc. may be used, but it is not limited to these. .

2 and 3 show examples of vectors for obtaining a fragment used for knockout of the target protein gene of Saccharomyces cerevisiae used in the examples. This construct contains a G418 resistance gene, FRT-G418R-FRT fragment having FRT sequences upstream and downstream. G418R is a geneticin resistance gene and is used as a positive selection marker for strains in which the target protein gene has been disrupted. Using these plasmids as templates, a primer in which 20 bases immediately upstream of the FRT-G418R-FRT fragment are aligned with the sequence of 80 bases downstream from the start codon of each ORF of the target protein gene, and FRT- PCR is performed using a primer with 20 bases immediately downstream of the G418R-FRT fragment. The obtained PCR amplified fragment contains an FRT-G418R-FRT fragment in which a sequence of 80 bases downstream from the start codon of each ORF of the target protein gene or a sequence of 80 bases upstream from the start codon is added to both ends. Using this fragment, a host cell is transformed to obtain a strain that has acquired geneticin resistance by homologous recombination.
Next, a plasmid encoding FLP (pYRNFLP shown in FIG. 4) is introduced into the obtained strain to cause recombination between the FRT sequences, thereby dropping out the geneticin resistance gene.
Furthermore, since pYRNFLP is also unstable, it is shed from the host cell under non-selective pressure. Since the microorganism of the present invention thus prepared does not have a selection marker, the same selection marker as that used for the previous gene disruption is repeatedly used when disrupting multiple genes. Is possible.

(2) RNA interference In addition to the above, RNA interference (RNAi) method using siRNA (small interfering RNA) or the like can be used as a method for deleting the expression function of the target protein gene. RNAi is a multi-step process that takes place in multiple stages. First, double-stranded RNA (Double Stranded RNA: dsRNA) or hairpin-shaped shRNA (Small Hairpin RNA) expressed from an RNAi expression vector is recognized by Dicer and degraded into 21-23 nucleotide siRNAs. The siRNAs are then incorporated into an RNAi target complex called the RNA-Induced Silencing Complex (RISC), where the RISC and siRNAs complex contains a sequence that is complementary to the siRNA sequence. Binds to mRNA and degrades mRNA. The target mRNA is cleaved at the center of the region complementary to the siRNA. Finally, the target mRNA is rapidly degraded and the protein expression level decreases. The most potent siRNA duplex is known to be a 21 nucleotide long sequence with two uridine residue overhangs at each 3 'end of the 19 bp duplex (Elbashir SM et al. Genes and Dev, 15, 188-200 (2001)).

  Therefore, in order to obtain a target protein gene knockdown microorganism, first, nucleotides including a sequence complementary to a part of (but not limited to) the target protein gene sequence as shown in Tables 1 to 3 are appropriately RNAi. An RNAi vector is prepared by inserting into an expression vector in a state where it can be expressed as dsRNA or shRNA. Next, the target protein gene knockdown microorganism can be obtained by introducing the vector into a host cell and selecting transformants.

  The design and synthesis of dsRNA or shRNA can be performed with a commercially available DNA / RNA synthesizer, eg, Applied Biosystems 394, or can be commissioned to a third party (eg, TAKARA Bio).

  Loss of the expression function of the target protein gene can be confirmed as a decrease in the expression level of the normal target protein gene. The expression level of the target protein gene can be measured by RT-PCR using the microorganism extract of the present invention, agarose gel electrophoresis, Real-Time PCR, Northern blotting, microarray analysis, mass spectrometry, etc. it can. Primers or probes used in these measurement methods can be designed and synthesized based on the sequence of the target protein gene. Whether or not the expression product of the target protein gene is normal can be confirmed by performing sequence analysis of the expression product.

The microorganism of the present invention is characterized in that the expression function of the target protein gene is lost, but it may further be introduced in a state where the xylose metabolizing enzyme gene can be expressed. The xylose metabolizing enzyme gene means a gene encoding a protein having an activity of catalyzing any one of a series of chemical reactions for producing ethanol from xylose (hereinafter, “xylose metabolizing enzyme”). Examples of the xylose metabolic enzyme gene include a xylose reductase gene, a xylitol dehydrogenase gene, and a xylulose phosphorylase gene.
Such xylose metabolizing enzyme genes are, for example, in Pichia stippitis, Saccharomyces cerevisiae, E. coli, Lactobacillus casei, Lactobacillus acidophilus, Aspergillus fumigatus, Staphylococcus aureus, Pichia pastoris, Schizosaccharomyces pombe, etc. It may be derived.
The microorganism of the present invention is preferably introduced with at least one selected from the group consisting of a xylose reductase gene, a xylitol dehydrogenase gene or a xylulose phosphorylase gene among the xylose metabolizing enzymes, Two or more genes or all three genes may be introduced.

Examples of the xylose reductase gene, xylitol dehydrogenase gene or xylulose phosphorylase gene include, but are not limited to, the following.

Xylose reductase is an enzyme that catalyzes the following reaction in xylose metabolism.

Xylose + NADPH → Xylitol + NADP

Xylitol dehydrogenase is an enzyme that catalyzes the following reaction in xylose metabolism.

Xylitol + NAD → Xylulose + NADH

Xylulose kinase is an enzyme that catalyzes the following reaction in xylose metabolism.

Xylulose + ATP → Xylulose-5P + ADP

  In one embodiment, the xylose metabolizing enzyme gene (such as xylose reductase, xylitol dehydrogenase or xylulose phosphorylase) is a mutant of the xylose metabolizing enzyme gene as long as it encodes a protein having xylose metabolic activity. It may be. Examples of the mutant may be a mutant in which one or more bases are deleted, inserted, substituted, or added in the base sequence of the polynucleotide encoding the xylose metabolizing enzyme protein. Variants can be mutated in coding or non-coding regions, or both. Mutations in the coding region can generate conservative or non-conservative amino acid deletions, insertions, substitutions and / or additions. As used herein, a gene (SEQ ID NO: 7, 9, or 11) encoding the amino acid sequence of SEQ ID NO: 8, 10, or 12, or one or more (eg, 1 to 40) in the amino acid sequence 1-20 pieces, 1-15 pieces, 1-10 pieces, 1-9 pieces, 1-8 pieces, 1-7 pieces, 1-6 pieces, 1-5 pieces, 1-4 pieces, 1-3 pieces , 1-2, 1, etc.) or a gene encoding an amino acid sequence in which one or several amino acids have been deleted, substituted or added are also included in the “xylose metabolizing enzyme gene”.

  It is well known in the art that some amino acids in the amino acid sequence of a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide. Furthermore, it is also well known that there are variants in natural proteins that do not significantly alter the structure or function of the protein, not only artificially modified.

  One skilled in the art can readily modify one or more amino acids in the amino acid sequence of a polypeptide using well-known techniques. For example, according to a known point mutation introduction method, any base of a polynucleotide encoding a polypeptide can be modified. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide. Furthermore, if the method described in this specification is used, it can be easily determined whether or not the produced variant has a desired activity.

  Preferred variants have conservative or non-conservative amino acid substitutions, deletions, or additions. Silent substitution, addition, and deletion are preferred, and conservative substitution is particularly preferred. These do not alter the polypeptide activity according to the invention.

  Representative conservative substitutions are substitutions of one amino acid for another in the aliphatic amino acids Ala, Val, Leu, and Ile; exchange of hydroxyl residues Ser and Thr, acidic residues Exchange of Asp and Glu, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe, Tyr.

  As detailed above, further guidance on which amino acid changes are likely to be phenotypically silent (ie likely not to have a significantly detrimental effect on function) can be found in Bowie, JU “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions”, Science 247: 1306-1310 (1990) (incorporated herein by reference).

  When the polynucleotide according to the present invention mentioned above is used, a polypeptide having xylose metabolizing enzyme activity can be synthesized in a transformant or a cell.

The xylose metabolizing enzyme gene is preferably introduced in a state of being incorporated into an expression vector that can be expressed in a host cell. The expression vector may include the following elements (i) to (iii).
(I) a promoter capable of being transcribed in a host cell;
(Ii) a xylose metabolizing enzyme gene linked to the promoter; and (iii) an expression cassette containing a signal that functions in the host cell as a component for transcription termination and polyadenylation of RNA molecules.

  Examples of such vectors include pYE22m (for yeast: Biosci. Biotech. Biochem., 59, 1221-1228, 1995), YCp50 (for yeast: X70276), and YIp1 (for yeast: X70480).

The promoter / terminator for regulating gene expression in the host cell may be any combination as long as it functions in the host cell. For example, when used in yeast, promoters such as TDH3, PYK1, PGK, GAP, TPI, GAL1, GAL10, ADH2, PHO5, CUP1, and MFα1 can be used, but are not limited thereto. As the constitutive high expression promoter, promoters such as TDH3, TPI1, PGK1, PGI1, PYK1, and ADH1 can be used.
Selectable markers used for transformation include auxotrophic markers (ura3, ura5, niaD), drug resistance markers (hygromycin resistance, zeocin resistance, geneticin resistance), copper resistance gene (CUP1) (Marin et al., Proc. Natl. Acad. Sci. USA, 81, 337 1984), cerulenin resistance gene (fas2m, PDR4) (Rei, Koshiwa, et al., Biochemistry, 64, 660, 1992; Hussain et al., Gene, 101, 149, respectively) , 1991) are available.

  As a method for transforming host cells, generally known methods can be used. For example, in the case of prokaryotes, the electroporation method (Mackenxie D.A. et al. Appl. Environ. Microbiol., 66, 4655-4661, 2000) and the heat shock method can be used. In the case of yeast, electroporation method, spheroplast method (Proc. Natl. Acad. Sci. USA, 75 p1929 (1978)), lithium acetate method (J. Bacteriology, 153, p163 (1983)), Proc. Natl. Acad. Sci. USA, 75 p1929 (1978), Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual, etc., but is not limited thereto.

  For other general cloning techniques, see "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor Laboratory Press 2001", "Methods in Yeast Genetics, A laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) ".

3． Method for Producing Ethanol The microorganism of the present invention produced as described above has an increased xylose fermentation ability compared to the same type of wild-type microorganism. Here, xylose fermentation means producing ethanol by assimilating xylose. Therefore, when the microorganism of the present invention is cultured in the presence of a xylose-containing raw material, xylose can be assimilated faster than when the same type of wild-type microorganism is cultured under the same conditions, and xylitol as a by-product Production amount of ethanol is small, and ethanol fermentation ability from xylose increases.
From this, this invention provides the manufacturing method of ethanol including the process which the microorganisms of this invention and a xylose containing raw material are made to contact in another embodiment.

The step of bringing the microorganism of the present invention into contact with the xylose-containing material can be achieved by mixing the microorganism of the present invention and the xylose-containing material. Preferably, the microorganism of the present invention is contacted with the xylose-containing material in an active state.
As an example of a method for bringing the microorganism of the present invention into contact with the xylose-containing material in an active state, the microorganism of the present invention is cultured under culture conditions in which the microorganism of the present invention can grow and in the presence of a xylose-containing raw material. A method is mentioned. Such culture conditions vary depending on the microorganism species.For example, in the case of yeast, a YPD medium (yeast extract 10 g / L, polypeptone 20 g / L, glucose 20 g / L), SX medium (xylose 50 g / l, 6.7 g / l yeast nitrogen base without amino acids, adenine 20 mg / l, histidine 20 mg / l, leucine 100 mg / l, tryptophan 20 mg / l), etc. It can be cultured at 30 ° C. in a suitable medium.
Alternatively, before culturing in the presence of a xylose-containing raw material, the microorganism of the present invention is cultured under culture conditions that allow the microorganism of the present invention to grow, and then the cells are grown, and then in the presence of the xylose-containing raw material. Fermentation may be performed using the microorganism of the present invention.
However, the culture conditions are not limited to the above. A person skilled in the art can appropriately perform culture by selecting appropriate culture conditions according to the microorganism species.

  The ability to ferment ethanol from xylose is obtained by contacting the microorganism of the present invention with a xylose-containing raw material, and for a certain period of time (for example, 72 hours, 48 hours, 24 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, It can be confirmed by measuring the amount of ethanol produced in the system after 2 hours and 1 hour).

  The amount of xylose assimilated and the amount of ethanol produced can be easily measured by analyzing the components of the fermentation system containing the microorganism of the present invention and the xylose-containing raw material by a known analytical method such as high performance liquid chromatography. .

The “xylose-containing raw material” is not particularly limited as long as it is a raw material containing xylose, but is preferably a biological raw material containing xylose, that is, “xylose-containing biomass”. Biomass is defined as “renewable, bio-derived organic resources excluding fossil resources” in the “Biomass Nippon Integrated Strategy” issued by the Ministry of Agriculture, Forestry and Fisheries of Japan.
Therefore, based on this definition, “xylose-containing biomass” can be defined as “a renewable, organic-derived organic resource excluding fossil resources and containing xylose”.
Specific examples of xylose-containing biomass include sugarcane, corn, sugar beet, potato, sweet potato, wheat, sorghum (sorghum), sorghum and other non-edible parts, waste wood, pulp waste liquid, bagasse, rice husk, corn cob, Examples include, but are not limited to, rice straw, switchgrass, Eliansus, and Napiergrass.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to the aspect described in the Example.

Test method:
Test items and test methods used in this example are shown below. Unless otherwise noted, the test methods in this example were based on this.

<Yeast strain>
The strains used in the present invention are as shown in the following (a) to (e). In both strains, the P. stipitis xylose reductase gene (XYL1: SEQ ID NO: 7), the xylitol dehydrogenase gene (XYL2: SEQ ID NO: 9) and the S. cerevisiae xylulose kinase gene (XKS1: SEQ ID NO: 11) ) And a plasmid pIUX1X2XK (FIG. 1) having an expression unit linked under the promoter control of the S. cerevisiae TDH3 gene has been introduced into the URA3 gene region on the chromosome by homologous recombination. About a strain | stump | stock, it will not be limited to this, What is necessary is just a yeast which has a xylose fermentation ability.

(a) MT8-1 / pIUX1X2XK (Mata ade his3 leu2 trp1 / pIUX1X2XK)
(b) MT8-1 / pIUX1X2XK / Ad (strain adapted to Mata ade his3 leu2 trp1, xylose medium)
(c) MT8-1 / pIUX1X2XK / pGK404-ScTal (Mata ade his3 leu2 / pIUX1X2XK / pGK404-ScTal)
(d) NBRC1440 / pIUX1X2XK (Matα / his3 trp1 / pIUX1X2XK)
(e) NBRC1445 / pIUX1X2XK (Mata his3 trp1 / pIUX1X2XK)

MT8-1 is registered with the National BioResource Project as NBRP No. BY2685.

<Gene disruption method>
Using each of the following primers (1) to (16), plasmid pBIITGRFRT (FIG. 2) ((1) + (2)), or pUGRFRT (FIG. 3) ((3) + (4), (5) PCR was performed using + (6)) as a template. By these, the sequence of 80 bases downstream from the start codon of each ORF of MET6 ((1) + (2)), MET32 ((3) + (4)), GLY1 ((5) + (6)) or The FRT-G418R-FRT fragment in which a sequence of 80 bases upstream from the codon is added to both ends is amplified. FRT is a recombination site on a yeast 2μ plasmid where recombination occurs by recombinase, and its sequence is 5′-GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC-3 ′ (SEQ ID NO: 13). G418R is a G418 resistance marker gene (PGK1p :: KanMX), and both pBIITGRFRT and pUGRFRT are plasmids having an FRT-G418R-FRT fragment. MT8-1 / pIUX1X2XK was transformed with the amplified FRT-G418R-FRT fragment having both ends of each ORF, and the transformed cells were transformed into YPD (yeast extract 10 g / L) containing geneticin 300 μg / ml. Polypeptone 20 g / L, glucose ^* 20 g / L (filter sterilization addition ^* )). In order to confirm that the FRT-G418R-FRT fragment replaced the ORFs of MET6, MET32, and GLY1 in the grown strain, genomic DNA was prepared from several colonies. Using this as a template, PCR was performed using (7) and (10) for MET6, (8) and (10) for MET32, and (9) and (10) for GLY1. Primers (7), (8) and (9) are sequences 5 ′ upstream of each ORF, and (10) is a sequence complementary to the G418 resistance gene. For a strain in which DNA of an appropriate size was amplified by PCR, plasmid pYRNFLP (FIG. 4) was further transformed and applied to YPD containing 50 μg / ml of Nourseothricin dihydrogen sulfate.

(1) MET6DF1: 5'-ATGGTTCAATCTGCTGTCTTAGGGTTCCCAAGAATCGGTCCAAACAGAGAATTAAAGAAGGCCACTGAAGGTTACTGGAAACTCAAGCTATGCATCAAGC-3 '(SEQ ID NO: 14)
(2) MET6DR1: 5'-TTAATTCTTGTATTGTTCACGGAAGTACTTGGCGGCTTCGACCATATGAGTCAAAGACAATCT
AGTTTCTTCCCAGCCTCAATACGACTCACTATAGGGC-3 '(SEQ ID NO: 15)
(3) MET32DF1: 5'-ATGGAGGATCAGGATGCTGCATTTATCAAACAGGCTACAGAAGCAATAGTGGATGTATCATTA
AATATAGATAACATAGAGTAAAACGACGGCCAGTGCC-3 '(SEQ ID NO: 16)
(4) MET32DR2: 5'-TCAGCCATTACTGCTACCATTGTGGTTGTTATCTTGACGATGAACGATGTTGGATTGCTTG
ACTTTTTTCAGTAATTCATCCGGCTCGTATGTTGTGTGG-3 '(SEQ ID NO: 17)
(5) GLY1DF1: 5'-ATGACTGAATTCGAATTGCCTCCAAAATATATCACCGCTGCTAACGACTTGCGGTCAGACACA
TTCACCACTCCAACTGCGTAAAACGACGGCCAGTGCC-3 '(SEQ ID NO: 18)
(6) GLY1DR1: 5'-TCAGTATTTGTAGGTTTTTATTTCGCGGATAGCGTTGCCATCAACGTCGACCTCGGTGGATTC
ACTACGGTAAATCTGGGCCGGCTCGTATGTTGTGTGG-3 '(SEQ ID NO: 19)
(7) MET6-5UF1: 5'-AAACTGTGGTAGTCATAGCTC-3 '(SEQ ID NO: 20)
(8) MET32-5UF1: 5'-TATACTAGTCAAAGATAGTATCCACC-3 '(SEQ ID NO: 21)
(9) GLY1-UF1: 5'-ATGTCACGCGAAACGGACC-3 '(SEQ ID NO: 22)
(10) G418MER: 5'-AAATGCTTGATGGTCGGAAG-3 '(SEQ ID NO: 23)
(11) MET6NF1: 5'-ATGGTTCAATCTGCTGTCTTAGGGT-3 '(SEQ ID NO: 24)
(12) MET6CR: 5'-TTAATTCTTGTATTGTTCACGGAAGTAC-3 '(SEQ ID NO: 25)
(13) MET32NF1: 5'-ATGGAGGATCAGGATGCTGCATTT-3 '(SEQ ID NO: 26)
(14) MET32CR2: 5'-TCAGCCATTACTGCTACCATTGTG-3 '(SEQ ID NO: 27)
(15) GLY1NF1: 5'-TGACTGAATTCGAATTGCCTC-3 '(SEQ ID NO: 28)
(16) GLY1CR1: 5'-ATAGCGTTGCCATCAACGTC-3 '(SEQ ID NO: 29)

  pYRNFLP is a YRp type plasmid that expresses the FLP recombinase on the yeast 2μ plasmid under the control of TDH3p. Using the genomic DNA of the grown strain as a template, PCR was performed using primers (11) and (12) for MET6, (13) and (14) for MET32, and (15) and (16) for GLY1. went. The primers (11) to (16) are primers that can amplify each full length ORF.When PCR is performed using these ORFs, in the strain in which recombination occurs in the FRT region and the G418 resistance gene is looped out, the ORF length Regardless, a short fragment of about 350 bp is amplified. Each acquired gene-disrupted strain has no G418 resistance gene. In addition, YRp-type plasmids are poorly stable and will drop out of cells immediately without application of selective pressure. Therefore, growth in a medium that does not contain nursesericin dihydrogen sulfate (NAT) will also remove pYRNFLP. be able to. Therefore, since the strain in which the gene is disrupted by this method does not have a selection marker (drug resistance), a plurality of genes can be further disrupted by the same method.

<Method for preparing template DNA for PCR>
Suspend yeast cells in 50 μl of Lysis buffer (0.125 mg / ml zymolyase 100T, 1 M sorbitol, 40 mM potassium phosphate buffer pH 6.8, 1 mM dithiothreitol). After incubation at 30 ° C for 1 hour, 5 µl of protease E (1 mg / ml) is added, and further incubated at 55 ° C for 20 minutes and 99 ° C for 10 minutes. Centrifugation was performed at 15,000 rpm for 10 minutes, and the supernatant was used as template DNA.

<Ethanol fermentation test from xylose>
Yeast pre-cultured in 5 ml of SD medium (20 g / l glucose, 6.7 g / l yeast nitrogen base without amino acids, adenine 20 mg / l, histidine 20 mg / l, leucine 100 mg / l, tryptophan 20 mg / l) Was transferred to 500 ml of SD medium (containing 20 g / l of casamino acid) so that the initial OD ₆₀₀ was 0.03, and aerobically cultured at 30 ° C. for 48 hours at 30 ° C. Cells are collected in a centrifuge, washed with sterile water, and then 100 ml of SX medium (xylose 50 g / l, 6.7 g / l yeast nitrogen base without amino acids, adenine 20 mg so that the initial OD ₆₀₀ is 20 / l, histidine 20 mg / l, leucine 100 mg / l, tryptophan 20 mg / l), and fermentation was started. Fermentation was performed in a 100 ml medium bottle with an outlet for releasing generated carbon dioxide gas, and the culture solution was stirred with a stirrer at 100-150 rpm. In order to monitor the progress of the fermentation, 1 ml of the medium was collected and centrifuged at 1000 g, and then the supernatant was collected and analyzed by HPLC.

<Preparation method of sample for metabolome analysis>
Yeast cells sampled over time from the fermentation broth were harvested by centrifugation (1000 g, 3 min). Cell pellets were washed twice with MilliQ water (Millipore, Tokyo, Japan) and then frozen in liquid nitrogen. Cells were lyophilized and stored at -80 ° C until use.

<HPLC analysis of fermentation moromi>
The xylose, xylitol, and ethanol concentrations in the fermentation medium were quantified by an HPLC method (GL-7400 system, GL Sciences) using a ShimPack SPR-Pb column (Shimadzu, Kyoto, Japan). The column was kept at 80 ° C., MilliQ water was used as the mobile phase, and the flow rate was set to 0.6 ml / min. Each compound was detected by a differential refractive index detector (Shimadzu) of GL-7454.

Intracellular metabolite analysis method <GC-MS analysis>
- 10 mg of dry cells and mixed solvent 1000 ml Add a 2 ml tube of yeast _{(MeOH: CHCl 3: H 2} O = 2.5: 1: 1) as the internal standard solution 60 ml (2-isopropylmalic acid 1 mg / ml )-Mix at 1200 rpm, 37 ° C, 30 minutes with a shaker-Centrifuge at 15000 xg, 4 ° C, 3 minutes with a centrifuge-Transfer 900 ml of the supernatant to a 1.5 ml tube, add 400 ml of MilliQ water, Mix with vortex ・ Centrifuge at 15000 xg, 4 ℃, 3 minutes ・ Transfer 200 ml of supernatant to 1.5 ml tube and dry with centrifugal concentrator and freeze dryer ・ Methoxyamine (dissolved with pyrimidine) Add 100 ml of 20 mg / ml ・ Mix at 1200 rpm, 30 ℃, 90 minutes with shaker ・ Add 50 ml of MSTFA 1 mg / ml, mix at 1200 rpm, 37 ℃, 30 minutes with shaker ・ Transfer to vial 1 Inject ml into GC-TOF / MS and analyze

Analysis conditions
GC system: 6890N (Agilent)
Column: CP-Sil 8 CB-Low Bleed 30 mx 0.25 mm ID (DF = 0.25 mm)
Oven temperature: 80 ° C (hold for 2 minutes), rise to 330 ° C at 15 ° C / minute, then hold for 6 minutes Injector temperature: 230 ° C
Ion source temperature: 200 ° C
MS system: PegasusIII TOF-MS (Leco)
Mass range: 85-500 m / z
Scan speed: 20 scans / second

<CE-MS analysis>
- Add 10 mg of dry cell of yeast in a 2 ml tube _{· MeOH 500ml, CHCl 3 500ml,} MilliQ water 180 ml, and the internal standard solution 20ml (100mM ribitol, 100mM PIPES) was added, at-centrifuge mixed in vortex Centrifuge at 15000 xg, 4 ° C for 3 minutes ・ Transfer 350 ml of the supernatant to a 1.5 ml tube, add 100 ml of MilliQ water and mix with vortex ・ Add 100 ml of MilliQ water to the precipitate and mix with vortex ・ Both samples Centrifuge at 15000 xg at 4 ° C for 3 minutes in a centrifugeTransfer the supernatant to two ultrafiltration tubes (Millpore Ultrafree-MC 5kDa MWCO) ・ Dry it overnight in a centrifugal concentrator ・ MilliQ water 50ml Dissolve in ・ Transfer to vial and analyze

Analysis conditions
CE system: P / ACE MDQ (Beckman Coulter)
Capillary: Fused silica capillary 50μm id x 80 cm
Seeds solution: 5 mM HCOONH ₃ in 50% MeOH
Electrolyte: 50 mM CH ₃ COONH ₃ (pH 9.0)
Voltage: 30.0 kV
MS system: 4000 QTRAP (Applied Biosystems)
Ionization method: ESI
Liquid feed pump: MP-711 (GL Sciences)

Example 1: Ethanol fermentation test 1 with a plurality of xylose-fermenting yeasts
Seven types of xylose-fermenting yeast ((1) MT8-1 / pIUX1X2XK (2) MT8-1 / pIUX1X2XK / Ad (3) MT8-1 / pIUX1X2XK / pGK404-ScTal (4) NBRC1440 / pIUX1X2XK (5) NBRC1445 / pIUX1X2X ) Was precultured in SD medium by the method shown in the experimental method, and the cells were collected and washed with sterilized water. Yeast cells were inoculated into 100 ml of SX medium so that OD ₆₀₀ = 20, and an ethanol fermentation test from xylose was performed. 1 ml of the fermentation mash was sampled in a timely manner, and the xylose, xylitol and ethanol concentrations were measured by HPLC. At the same time, yeast cells were sampled and subjected to GC-MS and CE-MS analysis by the method shown in the experimental method, and a comprehensive analysis of intracellular components was performed. As a result of constructing an OPLS regression model with the obtained metabolite profile as an explanatory variable and various performance evaluation values as a response variable, and analyzing the explanatory variables important for model construction, the following compounds were found to have the ability to ferment ethanol from xylose. I found that I was deeply involved.

Homoserine
Homocysteine
Methionine
Threonine
Glycine
Serine

  Homoserine, Threonine, Glycine are XCR (xylose utilization rate g-comsumed xylose / h), EPR (ethanol production rate g-ethanol / h), EY (ethanol yield g-ethanol / g-comsumed xylose), SEPR (ratio It was thought that the metabolite had an important meaning in the prediction model of each fermentation performance of ethanol production rate g-ethanol / g-cell-dried weight / h). Since these metabolites are intermediates in the glycine biosynthetic pathway, the above fermentation performance is expected to be related to the glycine biosynthetic pathway. When the coefficient values of the explanatory variables were analyzed from the prediction model, it was predicted that the fermentation performance was improved by reducing the intracellular accumulation of these metabolites. Therefore, in order to reduce the intracellular accumulation of these substances, we selected one of the genes in the glycine biosynthesis pathway as a target gene that disrupts GLY1.

  On the other hand, Homocysteine, Methionine and Serine are XPR (xylitol production rate g-xylitol / h), XY (xylitol yield g-xylitol / g-comsumed xylose), SXPR (specific xylitol production rate g-xylitol / g-cell- It was considered to be an important metabolite in the prediction model of each fermentation performance of dried weight / h). These metabolites are intermediates in the methionine biosynthetic pathway and the above fermentation performance is expected to be associated with this pathway. Analyzing the coefficient values of the explanatory variables from the prediction model, it was predicted that the xylitol-related performance (XPR, XY, SXPR) would decrease by decreasing the intracellular accumulation of Homocysteine. Therefore, it was predicted that xylitol production could be suppressed if the accumulated amount of Homocysteine could be reduced by disrupting genes related to the methionine synthesis pathway. MET6 and MET32 were selected as target genes to be destroyed. Fig. 9 summarizes metabolites that are closely related to fermentation performance, their maps, coefficients, and so on.

Example 2: Preparation of disrupted strains of MET6, MET32 and GLY1
The MET6, MET32, and GLY1 genes of MT8-1 / pIUX1X2XK strain were disrupted as shown in the experimental method. That is, a DNA fragment having a sequence of 80 bp downstream from the start codon of each gene and 80 bp upstream from the stop codon at both ends and having FRT-G418R-FRT between them is prepared by PCR, and MT8-1 / pIUX1X2XK strain A strain having geneticin resistance was selected. From the multiple strains, those in which FRT-G418R-FRT was inserted into each gene of MET6, MET32, and GLY1 were selected by PCR, and then pYRNFLP was transformed. A strain having resistance to Nourseothricin dihydrogen sulfate was selected, and a strain in which recombinase expressed from pYRNFLP caused recombination between FRT sequences and the G418 resistance gene was looped out was selected by PCR.

Example 3: Ethanol fermentation from xylose in xylose medium of disrupted strains of MET6, MET32, GLY1
Using MT8-1 / pIUX1X2XK strain (parent strain) and disrupted strains of MET6, MET32, GLY1, pre-cultured in SD medium by the method shown in the experimental method, collected the cells, and washed with sterile water . Yeast cells were inoculated into 100 ml of SX medium so that OD = 20, and an ethanol fermentation test was performed (n = 3). 1 ml of the fermentation mash was sampled in a timely manner, and the xylose, xylitol and ethanol concentrations were measured by HPLC. FIG. 5 shows the xylose utilization process, FIG. 6 shows the ethanol production process, and FIG. 7 shows the xylitol production process. In addition, some indexes representing fermentation performance are shown in FIG.

  From this result, it was possible to accelerate the utilization rate of xylose by disrupting the gene GLY1 in the glycine biosynthesis pathway. In addition, by disrupting the methionine biosynthetic pathway gene, MET6 or MET32, it was possible to reduce the amount of xylitol produced as one of the by-products. It is considered that the ethanol yield can be improved by combining these breeds or by combining with other breeds.

<Summary>
As described above, it is not directly related to glycolysis, pentose phosphate pathway, or enzymes that are metabolic pathways from xylose to ethanol, such as xylose reductase and xylitol dehydrogenase. By destroying genes involved in the glycine biosynthesis pathway and methionine biosynthesis pathway, we were able to improve the ethanol production performance from xylose.

The ethanol production yield can be increased in performing ethanol fermentation using pentose produced from biomass as a carbon source. This is particularly effective when the hemicellulose content is high.
By using the yeast bred in the present invention, the ability to ferment ethanol from xylose or the fermentation rate can be improved. The same breeding can be carried out in any brewing yeast or laboratory yeast. Moreover, as long as it contains xylose, the same effect can be expected with this breeding yeast regardless of the mash obtained from any biomass.

Sequence number 13: Synthetic DNA
Sequence number 14: Synthetic DNA
SEQ ID NO: 15: synthetic DNA
Sequence number 16: Synthetic DNA
Sequence number 17: Synthetic DNA
Sequence number 18: Synthetic DNA
Sequence number 19: Synthetic DNA
Sequence number 20: Synthetic DNA
SEQ ID NO: 21: synthetic DNA
Sequence number 22: Synthetic DNA
Sequence number 23: Synthetic DNA
Sequence number 24: Synthetic DNA
Sequence number 25: Synthetic DNA
SEQ ID NO: 26: synthetic DNA
SEQ ID NO: 27: synthetic DNA
Sequence number 28: Synthetic DNA
Sequence number 29: Synthetic DNA

Claims (10)

  A microorganism in which the expression function of the GLY1, MET6 or MET32 gene is lost and a xylose metabolic enzyme gene is introduced.   2. The microorganism according to claim 1, which is a yeast.   The microorganism according to claim 1 or 2, which is Saccharomyces cerevisiae.   The xylose metabolic enzyme gene is at least one selected from the group consisting of a xylose reductase gene, a xylitol dehydrogenase gene and a xylulose phosphorylase gene. Microorganisms.   A method for producing a microorganism having an increased ability to ferment xylose by losing the expression function of GLY1 gene, MET6 gene or MET32 gene.   6. The method according to claim 5, wherein the microorganism is yeast. The method according to claim 5 or 6, wherein the microorganism is Saccharomyces cerevisiae.   The method according to any one of claims 5 to 7, wherein the microorganism is a gene into which a xylose-metabolizing enzyme gene has been introduced.   9. The method according to claim 8, wherein the xylose metabolic enzyme gene is at least one selected from the group consisting of a xylose reductase gene, a xylitol dehydrogenase gene, and a xylulose phosphorylase gene.   The manufacturing method of ethanol including the process which the microorganisms of any one of Claims 1-4, and a xylose containing raw material are made to contact.