JP6343754B2 - Method for imparting acid and salt tolerance and production of useful substances using acid and salt tolerant yeast - Google Patents

Description

  The present invention introduces genes and proteins that are resistant to acids and salts derived from acid- and salt-tolerant yeasts into yeasts that are excellent in fermentation production, or improves their expression levels to provide acid-tolerant and salt-tolerant strong acids. The present invention relates to a method for producing yeast exhibiting various growth inhibition resistance and fermentation inhibition resistance under an environment of (low pH) or high salt concentration, and producing useful substances such as ethanol using the yeast.

  In recent years, bioethanol (ethanol produced from biomass, which is a plant-derived raw material) has attracted attention as an alternative energy for solving the depletion of oil and global warming problems. There are various methods for producing ethanol from biomass. After preparing sugar solution from biomass by processing such as crushing and degrading (saccharification), ethanol from the sugar solution is utilized by reaction (fermentation) by microorganisms such as yeast. Production is the basic flow. In addition to yeast (Saccharomyces cerevisiae), Zymomonas mobilis and corynebacterium glutamicum are also known as ethanol-fermenting microorganisms. However, bacteria have low ethanol resistance, and yeast is around pH 4.5. In most cases, yeast is used in the fermentation process of industrial plants that are currently in operation because, for example, continuous production can be achieved while minimizing contamination by other microorganism species.

  Yeast growth and fermentation capacity is affected by various environmental factors. Most yeasts can grow and ferment under normal environmental conditions of normal temperature and pressure, but cannot grow or ferment in special environments of high temperature, acidity, alkalinity or high salt concentration. However, the existence of yeast (Issatchenkia orientalis) that can grow in such a special environment has recently been clarified (see Patent Document 1, Non-Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3). Issatchenkia orientalis, which excels in heat resistance, acid resistance, and salt resistance, is not only a material suitable for adaptation to special environments or elucidation of resistance mechanisms, but it is also used as a new gene resource and enzyme resource for industrial use It is important from the viewpoint of doing.

  First, the present inventors analyzed the genome sequence of the Issatchenkia orientalis NBRC1279 strain using a next-generation sequencer, and from the obtained genome information, a gene unique to Issatchenkia orientalis related to stress resistance and fermentation inhibition resistance such as acid resistance and salt resistance. Is expected to exist. However, from Issatchenkia orientalis, we identified useful genes related to stress resistance and fermentation inhibition resistance such as acid resistance and salt resistance, and in addition to extracting further resistance, they can grow and ferment only under normal environmental conditions There has been no study to impart resistance to yeast.

JP 2008-278825 A

Kitagawa T et al., Applied Microbiology and Biotechnology, Vol.87, pp.1841-1853 (2010) Isono N et al., Journal of Bioscience and Bioengineering, Vol.113, pp.76-78 (2012) Kwon YJ et al., Bioresource Technology, Vol.102, pp.8099-8104 (2011)

  In the conventional method, during the fermentation of alcohol and useful substances using the yeast Saccharomyces cerevisiae, yeast growth inhibition and fermentation inhibition are prevented under conditions of low pH stress such as pH 2.0 and high salt concentrations such as sodium sulfate. As a result, the target product could not be produced efficiently. Lignocellulosic biomass, in particular, is often hydrolyzed to monosaccharides (glucose and xylose) that become raw materials for fermentation by treatment with acids such as sulfuric acid, so the fermentation solution is neutralized for fermentation with Saccharomyces cerevisiae. A process is required, leading to an increase in cost. For this purpose, it is necessary to proceed with technological development related to imparting acid resistance and salt resistance to yeast.

  Therefore, in the present invention, it is possible to grow in an environment of strong acid or high salt concentration, glucose, a mixed sugar containing glucose, a useful substance such as ethanol can be produced in high yield from a biomass saccharified solution, and the sugar consumption rate is improved. The present invention provides an effective method for producing a useful substance such as ethanol using the produced genetically modified yeast.

The present inventors introduced the Issatchenkia orientalis NBRC1279 strain genomic library into the Saccharomyces cerevisiae BY4742 strain, which can grow on NBRC1279 strain, but cannot grow on BY4742 strain, and can be grown under low pH conditions of pH 2.0. Acquired shares. This strain was resistant to pH 2.5 and 7.5% Na ₂ SO ₄ under strong acid and high salt conditions. From this acid-resistant yeast strain, the LGS1 gene contributing to acid-tolerance and salt tolerance was identified and isolated (see FIG. 1). The function of the LGS1 gene that contributes to acid and salt tolerance has not been reported so far, and this is a surprising finding.

  In the present invention, in order to produce useful substances such as ethanol with high efficiency from glucose, mixed sugar containing glucose, and biomass saccharified solution using yeast with acid resistance, attention is paid to the LGS1 gene derived from Issatchenkia orientalis, and Saccharomyces LGS1 gene was introduced into cerevisiae for constitutive expression. The transformant expressing the LGS1 gene under the conditions of strong acid or strong acid / high salt concentration has significantly improved aerobic growth ability and anaerobic ethanol fermentation ability. Therefore, expression of the LGS1 gene derived from Issatchenkia orientalis in Saccharomyces cerevisiae is important for imparting acid resistance and salt resistance. Yeast that has been imparted acid resistance and salt resistance has a lower pH and higher than conventional yeast strains. The inventors have found that tolerance can be obtained in an environment of salt concentration, and have completed the present invention.

That is, the present invention includes the following.
[1] A genetically modified yeast capable of producing ethanol from glucose with high efficiency, including a plasmid that expresses the LGS1 gene or a homologous gene of LGS1 belonging to the GH72 family.
[2] A genetically modified yeast capable of producing ethanol from glucose with high efficiency, wherein the LGS1 gene or a homologous gene of LGS1 belonging to the GH72 family is introduced by chromosomal integration.

[3] LGS1 homologous genes belonging to the GH72 family are GAS1, GAS2, GAS3, GAS4, GAS5, Gel1, Gel2, Gel3, Gel4, Gel5, Gel6, Gel7, Glt1, PHR1, PHR2, PHR3, EPD1, EPD2, and GCW2 The genetic recombinant yeast according to [1] or [2], which is selected from the group consisting of:

[4] The genetic recombinant yeast according to any one of [1] to [3], wherein the LGS1 gene and the homologous gene of LGS1 belonging to the GH72 family are derived from yeast or bacteria.
[5] The genetic recombinant yeast according to [4], wherein the LGS1 gene and the homologous gene of LGS1 belonging to the GH72 family are derived from Issatchenkia orientalis.
[6] The genetic recombinant yeast according to [4], wherein the LGS1 gene and the homologous gene of LGS1 belonging to the GH72 family are derived from Saccharomyces cerevisiae.
[7] The genetic recombinant yeast according to [4], wherein the LGS1 gene and the homologous gene of LGS1 belonging to the GH72 family are derived from Kluyveromyces marxianus.
[8] The genetic recombinant yeast according to [4], wherein the LGS1 gene and the homologous gene of LGS1 belonging to the GH72 family are derived from Scheffersomyces (Pichia) stipitis.
[9] The genetic recombinant yeast according to any one of [1] to [8], wherein a xylose reductase gene, a xylitol dehydrogenase gene, and a xylulokinase gene are further introduced.
[10] The genetic recombinant yeast according to [9], wherein the xylose reductase gene and the xylitol dehydrogenase gene are derived from Scheffersomyces (Pichia) stipitis, and the xylulokinase gene is derived from Saccharomyces cerevisiae.
[11] The genetic recombinant yeast according to any one of [1] to [10], wherein the genetic recombinant yeast is produced using Saccharomyces cerevisiae as a host.
[12] The genetic recombinant yeast according to any one of [1] to [10], wherein the genetic recombinant yeast is produced using Kluyveromyces marxianus as a host.
[13] The genetic recombinant yeast according to any one of [1] to [10], wherein the genetic recombinant yeast is produced using Scheffersomyces (Pichia) stipitis as a host.
[14] The genetic recombinant yeast according to any one of [1] to [10], wherein the genetic recombinant yeast is produced using Issatchenkia orientalis as a host.

[15] A method for producing ethanol from glucose using the genetically modified yeast according to any one of [1] to [14].
[16] A method for producing ethanol from xylose using the genetically modified yeast according to any one of [9] to [14].
[17] A method for producing ethanol from a mixed sugar of glucose and xylose using the genetically modified yeast according to any one of [9] to [14].
[18] A method for producing ethanol from a saccharified solution prepared from cellulosic biomass, using the genetically modified yeast according to any one of [9] to [14].
[19] The method according to any one of [15] to [18], wherein the culture solution has a pH of 2.0 to 5.0.
[20] The method according to any one of [15] to [19], wherein the culture solution contains 5% or more of Na ₂ SO ₄ .

  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2014-056001, which is the basis for the priority of the present application.

  According to the present invention, the growth inhibition resistance / fermentation inhibition resistance in a strong acid or high salt environment is improved, and glucose, xylose, xylose, and glucose are improved as compared with conventionally known wild-type yeast and genetically modified yeast having excellent fermentation production. Provided is a genetically modified yeast capable of producing ethanol and other useful substances with high efficiency from a mixed sugar contained in a mixed sugar and lignocellulosic biomass saccharified liquid.

FIG. 1 is an explanatory diagram showing a flow until an acid- and salt-resistant gene derived from the acid- and salt-resistant yeast Issatchenkia orientalis in the present invention is identified. First, genomic DNA extracted from Issatchenkia orientalis NBRC1279 strain is cleaved with a restriction enzyme, and a genomic fragment is inserted into a plasmid (pPGK) to prepare a genomic DNA (shotgun) library. This library is transformed into the Saccharomyces cerevisiae BY4742 strain and cultured under conditions of strong acid (pH 2.0) that can grow on the NBRC1279 strain but cannot grow on the BY4742 strain. As a result, one clone was obtained as a viable acid-resistant yeast strain, and it was confirmed that this strain was resistant even under conditions of strong acid / high salt concentration of pH 2.5 and 7.5% Na ₂ SO ₄ . A plasmid was isolated from this acid-tolerant and salt-tolerant yeast strain (approximately 3.8 kbp gene fragment was inserted), and the nucleotide sequence of the inserted DNA was determined by sequence analysis. As a result, genes encoding two types of proteins were included. It was confirmed. Based on the results of homology searches with other yeasts, the functions of the two genes were inferred, and each gene was cloned and transformed again into BY4742 strain. These transformed yeast strains were cultured under the conditions of strong acid (pH 2.0) and strong acid / high salt concentration (pH 2.5, 7.5% Na ₂ SO ₄ ), and strains that could grow under either condition were observed. We clarified that the LGS1 gene held by this acid-salt-resistant yeast strain contributes to acid-salt resistance and identified a gene involved in acid-salt resistance. By constitutively high expression of this gene, the genetically modified yeast in the present invention acquires resistance even under strong acid and high salt concentration environment, exhibits good growth ability, and from sugars such as glucose and xylose to ethanol. It is possible to produce useful substances such as Fig. 2 shows the schematic structure (A) of genomic DNA on a plasmid and four gene sets in SCD agar medium adjusted to various low pH (+ high salt concentration) in acid-resistant yeast strains obtained by screening. It is a characteristic view which shows the growth ability (B) of replacement yeast (B42-Con strain, B42-Clone1 strain, B42-LPR1, and B42-LGS1 strain). FIG. 3 is a characteristic diagram showing alignment of amino acid sequences deduced from four kinds of genes derived from yeast (LGS1, Issatchenkia orientalis, GAS1 of Saccharomyces cerevisiae, PHR1 and PHR2 of Candida albicans). Io Lgs1p represents an LGS1 gene product derived from Issatchenkia orientalis, Sc Gas1p represents a GAS1 gene product derived from Saccharomyces cerevisiae, Ca Phr1p represents a PHR1 gene product derived from Candida albicans, and Ca Phr2p represents a PHR2 gene product derived from Candida albicans. Open circles (◯) indicate positions where amino acids are identical in 3 sequences out of 4 sequences, and solid circles (●) indicate positions where amino acids are identical in 4 sequences out of 4 sequences. FIG. 4 shows four types of genetically modified yeasts (DG1-Con strain, DG1-LGS1 strain, B42-Con strain, and B42-LGS1 strain) in SCD agar medium adjusted to various pH (+ high salt concentration). It is a characteristic view which shows the cell form (B) of two types of genetically modified yeast (DG1-Con strain | stump | stock and DG1-LGS1 strain | stump | stock) cultured with the growth ability (A) and the SCD culture medium adjusted to pH4.0. FIG. 5 is a characteristic diagram showing aerobic growth of two types of genetically modified yeast (B42-Con strain and B42-LGS1 strain) in SCD medium. FIG. 6 shows two types of genetically modified yeasts (B42-Con strain and B42-) in SCD-2 medium adjusted to pH 5.0 (A), 4.0 (B), 3.0 (C), and 2.0 (D). It is a characteristic view which shows the anaerobic ethanol fermentation ability (glucose consumption and ethanol production amount) of LGS1 strain | stump | stock. FIG. 7 shows two types of genetically modified yeasts (B42-Con strain and B42-) in SCD-3 medium adjusted to pH 3.0 (A), 2.5 (B), 2.2 (C), and 2.0 (D). It is a characteristic view which shows the anaerobic ethanol fermentation ability (glucose consumption and ethanol production amount) of LGS1 strain | stump | stock.

  LGS1 protein is a protein having high identity with GAS1 of Saccharomyces cerevisiae, PHR1 and PHR2 of Candida albicans, etc., and GH72 family (Carotti C et al., European Journal of Biochemistry, Vol.271, pp.3635-3645 (2004) ). Members of the GH72 family are also described in Zhang et al., Applied and Environmental Microbiology, 2011, vol. 77, No. 8, pp. 2676-2684, Ragni et al., Yeast 2007, 24, pp. 297-308, etc. It can also be obtained from a known database (CAZY etc.). The Saccharomyces cerevisiae GAS1 gene encodes a GPI-anchored glucanosyltransferase, and its deficiency impairs cell wall synthesis (Nuoffer C et al., Molecular and Cellular Biology, Vol. 11, pp. 27-37 ( 1991) and Popolo L et al., Journal of Bacteriology, Vol. 175, pp. 1879-1885 (1993)). Candida albicans PHR1 and PHR2 are functionally related and their expression is controlled by the pH of the growth environment (Saporito-Irwin SM et al., Molecular and Cellular Biology, Vol. 15, pp. 601-613). (1995) and Muehlschlegel FA et al., Molecular and Cellular Biology, Vol. 17, pp. 5960-5967 (1997)). In the present specification, the homologous gene of LGS1 refers to a gene having the same function as the LGS1 gene with respect to the acid resistance and salt tolerance of yeast.For example, but not limited to, a homologous gene of LGS1 belonging to the GH72 family, for example, Examples include GAS1, GAS2, GAS3, GAS4, GAS5, Gel1, Gel2, Gel3, Gel4, Gel5, Gel6, Gel7, Glt1, PHR1, PHR2, PHR3, EPD1, EPD2, and GCW2. The LGS1 gene and LGS1 homologous genes (genes encoding proteins belonging to the GH72 family such as GAS1, PHR1, and PHR2 genes) are not particularly limited as long as they are genes encoding such proteins. For example, Aspergillus fumigatus, Aspergillus nidulans , Aspergillus oryzae, Candida albicans, Candida dubliniensis, Candida glabrata, Candida maltosa, Coccidioides posadasii, Cryptococcus neoformans, Debaryomyces hansenii, Eromothecium gossypii, Fusarium oxysporum, Issatchenkia orientalis, Kluyveromyces lactis, Kluyveromyces marxianus, Magnaporthe grisea, Neurospora crassa, Paracoccidioides brasiliensis, Pneumocystis Examples include those derived from carinii, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Scheffersomyces (Pichia) stipitis, and Yarrowia lipolytica. In certain embodiments, the LGS1 homologous gene is derived from a yeast such as Saccharomyces cerevisiae, Kluyveromyces marxianus, Schefffersomyces (Pichia) stipitis, and Issatchenkia orientalis. Preferably, the LGS1 gene is derived from Issatchenkia orientalis. The Issatchenkia orientalis LGS1 gene (SEQ ID NO: 1) can be used from the draft genome information of Pichia kudriavzevii (registered as ALNQ01000000 in DDBJ / EMBL / GeneBank), which is synonymous with Issatchenkia orientalis (Chan GF et al., Eukaryotic Cell, Vol. .11, pp.1300-1301 (2012)).

  Examples of LGS1 homologous genes include, for example, the amino acid sequence of LGS1 protein derived from Issatchenkia orientalis and at least 55%, 60%, 70%, 80%, 90% or 95%, such as 96% or more, 97 A gene consisting of an amino acid sequence having% or more, 98% or more or 99% or more identity and encoding a protein having LGS1 activity; one or several amino acids are deleted in the amino acid sequence of LGS1 derived from Issatchenkia orientalis, A gene consisting of a substituted and / or added amino acid sequence and encoding a protein having LGS1 activity; under stringent conditions with a gene consisting of the base sequence of LGS1 gene derived from Issatchenkia orientalis or a base sequence complementary thereto A gene encoding a protein that hybridizes and has LGS1 activity Murrell. In certain embodiments, LGS1 activity includes an activity that imparts acid and salt tolerance to yeast. The identity and homology of amino acid sequences or base sequences can be determined using known software such as BLAST, ClustalX, and Genetyx.

  In the above amino acid sequence, the number of “several” in one or several amino acids is not particularly limited, but is, for example, 20 or less, preferably 10 or less, more preferably 7 or less, and even more preferably about 5 or less. For example, 5 or less, 4 or less, or 3 or less.

  In addition, the above “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, a complementary strand of DNA consisting of a base sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more identity with DNA having high identity hybridizes, and DNA having lower identity than that The condition that the complementary strands of these do not hybridize is mentioned. More specifically, the sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to 68 ° C, preferably 65 ° C.

  As described above, the GAS1, PHR1, and PHR2 genes belong to the GH72 family. The present inventors have found that the LGS1 gene complements the function of the GAS1 gene and imparts acid and salt tolerance to the yeast. The GAS1 gene is also considered to contribute to acid and salt tolerance, like the LGS1 gene. Therefore, from these findings, it is considered that other members belonging to the GH72 family such as PHR1 and PHR2 also contribute to the acid and salt tolerance of yeast.

  LPR1 is a protein having high identity with PXR1 of Saccharomyces cerevisiae. The PXR1 gene of Saccharomyces cerevisiae encodes a protein essential for ripening ribosomal RNA and small nucleolar RNA (Guglielmi B et al., Journal of Biological Chemistry, Vol.277, pp.35712-35719 (2002)). LPR1 gene and LPR1 homologous gene (such as PXR1 gene) are not particularly limited as long as they encode such proteins, but are derived from yeasts such as Saccharomyces cerevisiae, Kluyveromyces marxianus, Scheffersomyces (Pichia) stipitis, and Issatchenkia orientalis . Preferably, the LPR1 gene is derived from Issatchenkia orientalis. Issatchenkia orientalis LPR1 gene (SEQ ID NO: 2) can be used from the draft genome information of Pichia kudriavzevii (registered as ALNQ01000000 in DDBJ / EMBL / GeneBank) which is synonymous with Issatchenkia orientalis (Chan GF et al., Eukaryotic Cell, Vol. .11, pp.1300-1301 (2012)).

  The above genes can be obtained based on the respective gene sequences by general methods well known to those skilled in the art, for example, hybridization methods, PCR methods, and the like. Furthermore, methods for disrupting (deleting) or inactivating LGS1 gene and LGS1 homologous genes (genes encoding proteins belonging to the GH72 family such as GAS1, PHR1, and PHR2 genes) are well known to those skilled in the art. For example, a deletion plasmid inserted with a deletion DNA fragment prepared by the SOE-PCR method (Gene, 77, 61 (1989)) can be performed using molecular biological techniques. The gene can be deleted or inactivated from the host cell by the homologous recombination used. It can also be obtained by gene disruption using PCR, random mutation, and site-specific mutation. In general, in gene disruption using PCR, an antibiotic resistance gene cassette is amplified by PCR with a primer and replaced with a target gene. In the random mutation method, a mutant pool is constructed using gene shuffling or error-prone PCR, and mutants modified to the desired properties are screened. In site-directed mutagenesis, the cloned LGS1 gene is designed by PCR using LGS1 cloning primers that are designed based on the known LGS1 gene sequence and the homologous gene sequence of LGS1. Mutations can be introduced at predetermined positions.

  In one embodiment of the present invention, these target enzyme genes are expressed in host cells. The method can be performed using general molecular biological techniques known to those skilled in the art (Sambrook J. et al., “Molecular Cloning A LBORATORY MANUAL / second edition”, Cold Spring Harbor Laboratory Press (1989) reference). That is, it can be carried out by incorporating a gene encoding the enzyme into an appropriate vector and transforming an appropriate host organism using the vector.

  As the vector, a general yeast expression vector known to those skilled in the art for gene introduction and expression can be used. As a vector used for introduction into yeast, any of a multicopy type (YEp type), a single copy type (YCp type), and a chromosome integration type (YIp type) can be used.

  The vector can include one or more genes encoding the protein. In addition to the gene encoding the protein of interest, the vector includes an origin of replication that allows replication in the host cell, and a selectable marker that identifies the transformant. Translation control sequences can optionally be included linked to the enzyme gene sequence. Examples of regulatory sequences include transcription promoters, operators or enhancers, mRNA ribosome binding sites, and appropriate sequences that regulate transcription and translation initiation and termination. The transcription promoter that can be used is not particularly limited as long as it can drive gene expression in the host cell. For example, GAL1 promoter, GAL10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH1 A promoter, AOX1 promoter or the like can be used, but it is preferable to use a PGK promoter capable of constitutively expressing the gene. As the selection marker, a commonly used marker can be used by a conventional method. Examples include tetracycline, ampicillin, antibiotic resistance genes such as kanamycin or neomycin, hygromycin or spectinomycin, and auxotrophic genes such as HIS3 and TRP1.

  Examples of host cells that can be used include, but are not limited to, yeasts such as Saccharomyces cerevisiae, Kluyveromyces marxianus, Scheffersomyces (Pichia) stipitis, and Issatchenkia orientalis, with Saccharomyces cerevisiae being particularly preferred.

  Examples of a method for introducing a vector into a host cell include a calcium phosphate method or a calcium chloride / rubidium chloride method, an electroporation method, an electroinjection method, a method using chemical treatment such as PEG, and a method using a gene gun. .

  Preferably, the LGS1 gene or a homologous gene of LGS1 (a gene encoding a protein belonging to the GH72 family such as GAS1, PHR1, and PHR2 genes) is constitutively expressed in the host cell. For example, the LGS1 gene or the homologous gene of LGS1 may be expressed on a multi-copy plasmid, or after introduction into a chromosome integration type vector or the like, integration into a yeast chromosome and expression in a single or several copies. Preferably, the LGS1 gene or a homologous gene of LGS1 is expressed on a multicopy plasmid. Further, in the present invention, it is possible to use a genetically modified yeast in which xylose reductase XR, xylitol dehydrogenase XDH, and xylulokinase XK genes described in JP-A-2009-195220 are incorporated into chromosomal DNA. it can. The XR gene can be derived from Scheffersomyces stipitis, GeneBank accession number XM — 001385144, SEQ ID NO: 12). The XDH gene can be derived from Scheffersomyces stipitis (GeneBank accession number AF127801 or X55392, SEQ ID NO: 13). The XK gene can be derived from Saccharomyces cerevisiae (GeneBank accession number NC — 001139.7, SEQ ID NO: 14).

  By constitutively overexpressing the above LGS1 gene or LGS1 homologous genes (genes encoding proteins belonging to the GH72 family such as GAS1, PHR1, and PHR2 genes), acid resistance and salt tolerance can be imparted and strengthened. Using the acid-salt-tolerant yeast that is produced, a genetically modified yeast that can efficiently produce useful substances by avoiding growth inhibition and fermentation inhibition is obtained, compared with the case of using conventionally known yeast with excellent fermentation production. be able to.

In the present specification, acid resistance means that microorganisms (such as yeast) can survive or grow even under acidic conditions, specifically pH 2.0 to 5.0, 2.0 to 4.0, 2.0 to 3.0, 2.0 to 2.5, 2.0 to 2.4, 2.0-2.3, 2.0-2.2 or 2.0-2.1, preferably 2.0 means that it can survive or proliferate. In this specification, salt tolerance means that microorganisms can survive or grow even under conditions of high salt concentration. Specifically, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6 % Or 7% or 7.5% or more in the presence of Na ₂ SO ₄ . In this specification, both acid resistance and salt resistance may be collectively referred to as acid resistance and salt resistance. The genetically modified yeast of the present invention preferably has acid and salt resistance. For example, in certain embodiments, the genetically modified yeast of the present invention having acid and salt tolerance has a pH of 2.0 to 5.0, 2.0 to 4.0, 2.0 to 3.0, 2.0 to 2.5, 2.0 to 2.4, 2.0 to 2.3, 2.0 to 2.2, or 2.0 to 2.1. 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, or 7.5% or more in the presence of Na ₂ SO ₄ , for example, pH 2.0 to 5.0 and 5% In the presence of Na ₂ SO ₄ , pH 2.0-4.0 and 6% Na ₂ SO ₄ , pH 2.0-3.0 and 7% Na ₂ SO ₄ , or pH 2.0-2.5 and 7.5% Na ₂ SO ₄ It can grow in the presence. In the present specification, when a concentration, for example, a salt concentration or the like is described as%, this means that the final concentration is expressed in weight% unless otherwise specified. In this specification, to give acid resistance to salt tolerance in yeast, the introduction of a gene (for example, LGS1 gene or LGS1 homologous gene) in yeast that cannot survive or proliferate under acidic and high salt conditions. It means that the resulting genetically modified yeast can survive or grow under acidic and high salt conditions. For example, GAS1-deficient yeast cannot survive or proliferate under acidic and high salt conditions, but can be proliferated by introducing the LGS1 gene thereto, and is imparted with acid and salt tolerance (FIG. 4A).

  A host cell and a method for transformation using a gene in which the host cell has been deleted or inactivated are as defined above.

  The genetic recombinant yeast according to the present invention may be immobilized on a solid phase. Examples of the solid phase include, but are not limited to, polyacrylamide gel, polystyrene resin, porous glass, and metal oxide. By immobilizing the genetically modified yeast according to the present invention to a solid phase, it is advantageous in that continuous repeated use is possible.

  The genetically modified yeast according to the present invention can produce useful substances such as ethanol from hexoses such as glucose and xylose of pentoses under aerobic and anaerobic conditions. At that time, the concentration of sugar contained in the medium is 0.1 to 20%, preferably 0.5% to 10%, and more preferably 2% to 4%.

  Moreover, the genetically modified yeast according to the present invention can produce useful substances such as ethanol from a saccharified solution prepared from lignocellulosic biomass by fermentation reaction. The saccharified solution is not particularly limited, but can be prepared from one derived from lignocellulosic biomass such as woody materials (hardwood and coniferous trees) and herbs such as agricultural waste. As a saccharification technique for preparing a saccharified solution, a general method in the field can be used, and either an acid decomposition method or an enzymatic saccharification method may be used, but it is preferable that high efficiency and low environmental impact can be expected. This is a sulfuric acid pretreatment / enzymatic saccharification method. In fact, when fermenting using yeast, a saccharified solution that has not been detoxified may be used directly, or a saccharified solution that has been detoxified may be used. The pH of the saccharified solution may be an untreated acidic saccharified solution or may be used after being adjusted to near neutral, but preferably an untreated acidic saccharified solution is used.

The ethanol production reaction by the culture can be performed by a general method known to those skilled in the art. The culture temperature is controlled to 25 ° C to 38 ° C, preferably 27 ° C to 33 ° C, more preferably 30 ° C. The pH of the medium is controlled to 1.5 to 6.0, preferably 2.0 to 5.0, 2.0 to 4.0, 2.0 to 3.0, 2.0 to 2.5, 2.0 to 2.4, 2.0 to 2.3, 2.0 to 2.2 or 2.0 to 2.1, more preferably 2.0. To do. Since fermentation proceeds under anaerobic conditions, it is necessary that oxygen be absent. Therefore, the operation to remove the oxygen in the system and the dissolved oxygen in the medium before fermentation, that is, the operation to blow nitrogen gas into the medium. It is preferable to carry out. The reaction may be performed continuously or batchwise. Furthermore, the salt concentration of the medium may be high, which can suppress the growth of contaminating microorganisms. For example, the medium may contain 1%, 2%, 3%, 4%, 5%, 6%, 7%, or 7.5% Na ₂ SO ₄ . The salt can be other salts such as NaCl, KCl, etc. as long as they do not affect ethanol production.

  The culture medium after 0 to 192 hours, preferably 0 to 96 hours, more preferably 72 hours after the start of the culture is collected to separate ethanol. As a method for separating ethanol from the medium, known methods such as distillation and pervaporation membrane are used, but a method by distillation is preferred. Subsequently, ethanol can be obtained by further purifying the separated ethanol (a known method such as distillation can be used as an ethanol purification method).

  The present inventors made a yeast GAS1 gene deletion strain and confirmed that the LGS1 gene complements the GAS1 function. Like the LGS1 gene, the GAS1 gene is considered to have a function that contributes to acid and salt tolerance. From these findings, other members belonging to the GH72 family such as PHR1 and PHR2 are considered to contribute to the acid and salt tolerance of yeast as well. A person skilled in the art can easily confirm whether other genes belonging to the GH72 family have the activity of imparting acid resistance and salt tolerance to yeast using conventional techniques. For this, for example, the GAS1 gene-deficient yeast strain of the present invention can be used. This GAS1-deficient strain may be transformed with a recombinant vector having a gene belonging to the GH72 family to confirm the acid resistance and salt resistance of the genetically modified yeast.

The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
Example 1: Search for acid and salt tolerant yeast strains
From the genome information obtained from the results of genome analysis of Issatchenkia orientalis NBRC1279 strain, it was predicted that there are genes unique to Issatchenkia orientalis related to stress resistance and fermentation inhibition resistance such as acid resistance and salt resistance. Therefore, an attempt was made to identify these useful genes. The flow up to the identification of an acid / salt-tolerant gene derived from the acid-salt-tolerant yeast Issatchenkia orientalis in the present invention is shown in FIG. First, genomic DNA extracted from Issatchenkia orientalis NBRC1279 is randomly cleaved with a restriction enzyme, then 3 to 5 kp or more of DNA fragments are fractionated, the fractionated DNA ends are smoothed, and inserted into a plasmid (pPGK). A DNA (shotgun) library was prepared. The prepared genomic DNA library was transformed into Saccharomyces cerevisiae BY4742 so that the genome coverage was 10 times or more, and an auxotrophic agar medium adjusted to pH 2.0 (6.7 g / l yeast nitrogen base w / o amino) acids, 20 g / l glucose, 2 g / l drop out mix excluding the amino acid to be assayed, 17 g / l agar (SCD agar medium), and cultured at 30 ° C. It has been confirmed that NBRC1279 strain can grow on this agar medium, but BY4742 strain cannot grow. As a result, 1 colony (1 clone) was obtained as a viable acid-resistant yeast strain and named B42-Clone1 strain. The B42-Clone1 strain was resistant to pH 2.5 and 7.5% Na ₂ SO ₄ under strong acid and high salt conditions. From these results, it was predicted that the acid-salt-tolerant gene derived from the Issatchenkia orientalis NBRC1279 strain functions effectively in the obtained acid-salt-tolerant yeast strain.
Example 2: Identification of acid-tolerant salt-tolerant gene A plasmid (named pPGK-Clone1) was isolated from the obtained acid-tolerant yeast-tolerant yeast strain (B42-Clone1 strain) and cleaved with a restriction enzyme. As a result, 3819 bp on pPGK It was confirmed that a gene fragment (named Clone 1) (SEQ ID NO: 3) was inserted. As a result of determining the base sequence of the genomic DNA inserted by sequence analysis, it was confirmed that genes encoding two kinds of proteins were contained (see A in FIG. 2). A homology search with other yeasts based on the deduced amino acid sequences of these genes revealed that they had high identity with Saccharomyces cerevisiae PXR1 and GAS1, respectively. LPR1 (Like PXR1) and LGS1 (Like GAS1) Named. It was also found that Issatchenkia orientalis LGS1 is identical to PHR1 and PHR2 of pathogenic Candida species such as Candida albicans and Candida glabrata, and belongs to the GH72 family (see Fig. 3). The alignment of the deduced amino acid sequence of LGS1 showed 60% identity with Saccharomyces cerevisiae GAS1 and 61% and 58% identity with Candida albicans PHR1 and PHR2, respectively, indicating high identity. The Saccharomyces cerevisiae PXR1 gene encodes a protein essential for ripening ribosomal RNA and small nucleolar RNA, while the GAS1 gene encodes a GPI-anchored glucanosyltransferase and is required for cell wall synthesis Therefore, the gene that contributes to the function of acid tolerance in the acid-tolerance-resistant yeast strain obtained this time was predicted to be LGS1, a homologous gene of GAS1. Therefore, in order to investigate whether the LGS1 gene contributes to acid-tolerance and salt tolerance among the two types of genes (LPR1 and LGS1 genes) contained on pPGK-Clone1, the LPR1 gene was cloned in addition to the LGS1 gene. .
Example 3: Production of pPGK-LPR1
In order to prepare the wild type LPR1 gene, the following two primers were designed with reference to the information of the base sequence obtained as a result of sequence analysis of Clone1 derived from Issatchenkia orientalis. A sequence recognizing an EcoR I cleavage site at the 5 ′ end of the LPR1 gene and a BamH I cleavage site at the 3 ′ end were added to the primer.
5'-ATgaattcATGGGTCTCGCAGGAACAAG-3 '(SEQ ID NO: 4)
5'-GAggatccTCATTTGATCATGAAGATTTC-3 '(SEQ ID NO: 5)
PCR was performed using PrimeSTAR Max DNA polymerase (Takara Bio Inc.). Using 20 pmol of each primer and 100 ng of Issatchenkia orientalis genomic DNA, the LPR1 gene was amplified under the conditions of denaturation reaction at 98 ° C for 10 seconds, annealing reaction at 55 ° C for 5 seconds, and extension reaction at 72 ° C for 5 seconds. . The obtained DNA fragment was introduced into the EcoR I and BamH I restriction enzyme cleavage sites of the plasmid pPGK and named pPGK-LPR1.
Example 4: Production of pPGK-LGS1
In order to produce the wild type LGS1 gene, the following two primers were designed with reference to the information of the base sequence obtained as a result of sequence analysis of Clone1 derived from Issatchenkia orientalis. A sequence recognizing an EcoR I cleavage site at the 5 ′ end and a BamH I cleavage site at the 3 ′ end of the LGS1 gene was added to the primer.
5'-ATgaattcATGAAGTTCTCAAAGTCTCTCGC-3 '(SEQ ID NO: 6)
5'-GAggatccTCAGATCAAAATCATAGAGATGGAGC-3 '(SEQ ID NO: 7)
PCR was performed using PrimeSTAR Max DNA polymerase (Takara Bio Inc.). Using 20 pmol of each primer and 100 ng of Issatchenkia orientalis genomic DNA, the LGS1 gene was amplified under the conditions of denaturation reaction at 98 ° C for 10 seconds, annealing reaction at 55 ° C for 5 seconds, and extension reaction at 72 ° C for 5 seconds. . The obtained DNA fragment was introduced into the EcoR I and BamH I restriction enzyme cleavage sites of the plasmid pPGK and named pPGK-LGS1.
Example 5: Production of genetically modified yeast strain (1)
The plasmids pPGK-LPR1 and pPGK-LGS1 were transformed into Saccharomyces cerevisiae BY4742 strain by the lithium acetic acid method using YEASTMAKER yeast transformation system 2 (Clontech). BY4742 strain was transformed with pPGK-LPR1 to produce genetically modified yeast B42-LPR1 strain. Also, pPGK-LGS1 was transformed into BY4742 strain to produce genetically modified yeast B42-LGS1 strain. On the other hand, a B42-Con strain was prepared by transformation into BY4742 strain using pPGK not containing any gene, and used as a control strain.
Example 6: Culture of genetically modified yeast (1)
In order to examine the acid and salt resistance, in addition to the B42-Clone1 strain obtained by screening, the B42-LPR1 and B42-LGS1 strains and their control yeast (B42-Con) strains were added to a normal auxotrophic agar medium ( SCD agar medium grown at 30 ° C on SCD agar medium and then adjusted to various low pH (pH 2.5, 2.3, 2.2, 2.1, and 2.0) and low pH / high salt concentration (pH 2.5, 7.5% Na ₂₎ It was transferred to a SCD agar medium of SO ₄ ) and cultured at 30 ° C.

B in FIG. 2 shows the results (pH 2.1) of culturing in the above SCD agar medium using four types of genetically modified yeasts (B42-Con strain, B42-Clone1 strain, B42-LPR1 strain, and B42-LGS1 strain). And 13 days for 2.0 and 2.0 SCD media, and 6 days for other SCD media). First, at pH 2.5, all four types of genetically modified yeasts grew well. However, at pH 2.3, the B42-Clone1 and B42-LGS1 strains grew well, but the control strains B42-Con and B42-LPR1 had poor growth ability. Below pH 2.2 (pH 2.2, 2.1, 2.0), the B42-Clone1 and B42-LGS1 strains grew, but the B42-Con and B42-LPR1 strains hardly grew. Further, even under the conditions of pH 2.5 and 7.5% Na ₂ SO ₄ , the B42-Clone1 strain and the B42-LGS1 strain could grow, but the B42-Con strain and the B42-LPR1 strain could not grow. Under the conditions of pH 2.2 or lower, pH 2.5, and 7.5% Na ₂ SO ₄ , the growth ability of the B42-LGS1 strain was better than that of the B42-Clone1 strain. From these results, it was clarified that the gene contributing to acid and salt tolerance is LGS1.
Example 7: Production of genetically modified yeast strain (2)
In order to investigate whether the LGS1 gene derived from Issatchenkia orientalis complements the function of the GAS1 gene derived from Saccharomyces cerevisiae, a transformed yeast was prepared by introducing the LGS1 gene into a knockout yeast lacking the GAS1 gene. The acid resistance and salt resistance performance of the yeast strain was examined.

For this purpose, a yeast ΔGAS1 strain lacking the GAS1 gene was acquired from the collection of knockout yeasts from Open Biosystems (parent strain BY4742), and the above plasmid pPGK-LGS1 was used using YEASTMAKER yeast transformation system 2 (Clontech). Then, transformation was performed by the lithium acetic acid method to produce a genetically modified yeast strain DG1-LGS1. In addition, DG1-Con strain was prepared by transforming into ΔGAS1 strain using pPGK not containing LGS1 gene, and used as a control strain.
Example 8: Culture of genetically modified yeast (2)
In order to investigate acid and salt tolerance, in addition to the DG1-Con and DG1-LGS1 strains, the previously prepared B42-Con and B42-LGS1 strains were incubated at 30 ° C in a normal auxotrophic agar medium (SCD agar medium). , And then transferred to SCD agar medium adjusted to low pH (pH 2.5 and 2.2) and low pH / high salt concentration (pH 2.5, 7.5% Na ₂ SO ₄ ) SCD agar medium at 30 ° C. Cultured.

FIG. 4A shows the results (pH 2.5) of culturing in the above SCD agar medium using four types of genetically modified yeasts (DG1-Con strain, DG1-LGS1 strain, B42-Con strain, and B42-LGS1 strain). 3 days for SCD agar medium, 6 days for pH 2.2 SCD agar medium, 9 days for pH 2.5 and 7.5% Na ₂ SO ₄ SCD agar medium). FIG. First, on the SCD agar medium at pH 2.5, only the DG1-Con strain could not grow, but the remaining three types of genetically modified yeast strains all grew well. The DG1-Con strain did not grow even when cultured on an SCD agar medium at pH 2.5 for 16 days or more, but grew on the third day on an SCD agar medium at pH 2.8. In the SCD agar medium at pH 2.2, the B42-Con strain could not grow in addition to the DG1-Con strain, but the DG1-LGS1 and B42-LGS1 strains could grow well. Further, even under the conditions of pH 2.5 and 7.5% Na ₂ SO ₄ , the DG1-LGS1 strain and the B42-LGS1 strain could grow, but the DG1-Con strain and the B42-Con strain could not grow. From these results, it was found that, when the GAS1 gene is deleted, the acid and salt tolerance performance decreases, but the expression of the LGS1 gene increases the acid and salt tolerance performance compared to wild strains. Furthermore, it was suggested that the GAS1 gene also has a function that contributes to acid and salt tolerance, like the LGS1 gene.

  Next, DG1-LGS1 strain expressing LGS1 gene in ΔGAS1 strain and its control yeast DG1-Con strain were adjusted to pH 4.0 auxotrophic medium (6.7 g / l yeast nitrogen base w / o amino acids, 20 g / l glucose, 2 g / l drop out mix excluding amino acids to be assayed: SCD medium) was cultured aerobically at 30 ° C. for 12 hours, and the morphology of each yeast cell was observed with a microscope (B in FIG. 4). See). The DG1-Con strain has a large cell size, a large percentage of vacuoles, and one or more daughter cells that budding, and the GAS1 gene-deficient strains reported so far (Popolo L et al., Journal of Bacteriology, Vol. 175, pp. 1879-1885 (1993)). On the other hand, the DG1-LGS1 strain did not show the characteristic morphology observed in the DG1-Con strain, and showed the same morphology as the wild strain. Thus, the LGS1 gene was able to complement the function of the GAS1 gene.

The GAS1, PHR1, and PHR2 genes belong to the GH72 family. As described above, the LGS1 gene complements the function of the GAS1 gene. In addition, the GAS1 gene is considered to have a function contributing to acid and salt tolerance, similar to the LGS1 gene. Therefore, from these, it is considered that other LGS1 homologous genes belonging to the GH72 family such as PHR1 and PHR2 also contribute to the acid and salt tolerance of yeast.
Example 9: Culture of genetically modified yeast (3)
For aerobic growth experiments, the B42-LGS1 strain expressing the LGS1 gene in the BY4742 strain and its control yeast B42-Con strain were mixed with an auxotrophic medium containing 20 g / l glucose (6.7 g / l yeast nitrogen base). w / o amino acids, 20 g / l glucose, 2 g / l drop out mix (amino acid to be assayed: SCD medium) was aerobically cultured at 30 ° C. for 12 hours. Bacteria were collected by centrifugation, washed with sterilized water, and an appropriate amount was inoculated into 300 μl of SCD medium adjusted to pH 2.2 (unified cell mass). The culture was aerobically cultured at 30 ° C. for 216 hours with gentle shaking in a microplate reader HiTS (Scinics). As a result, the B42-Con strain hardly grew, while the B42-LGS1 strain grew well. Similarly, the B42-Con strain did not grow in the SCD medium with a strong acid / high salt concentration (pH 2.2, 5% Na ₂ SO ₄ ), but the B42-LGS1 strain could grow well. . Please refer to FIG.
Example 10: Culture of genetically modified yeast (4)
For ethanol fermentation experiments, the B42-LGS1 strain that expresses the LGS1 gene in the BY4742 strain and its control yeast B42-Con strain were cultured in an auxotrophic medium containing 20 g / l glucose (6.7 g / l yeast nitrogen base w / o Amino acids, 20 g / l glucose, 2 g / l Dropout mix (amino acid to be assayed: SCD medium) was aerobically cultured at 30 ° C. for 24 hours. The cells were collected by centrifugation, washed with sterilized water, and inoculated with 20 ml of 4 types of fermentation medium (SCD-2 medium) containing 40 g / l glucose (unified cell mass). The pH of the fermentation medium (SCD-2 medium) was adjusted to 5.0, 4.0, 3.0, and 2.0, and each fermentation broth was stirred in a 50 ml sealed vial with a stir bar at 30 ° C with gentle stirring. Cultured anaerobically for hours.
Example 11: Measurement of ethanol concentration (1)
The concentrations of ethanol, glucose, glycerol, and acetic acid were measured using high performance liquid chromatography (HPLC; JASCO Corporation). The separation column was an HPX-87H column (Bio-Rad), and the HPLC apparatus was run at 65 ° C. with 5 mM H ₂ SO ₄ at a flow rate of 0.6 ml / min. Yeast growth was measured at a wavelength of 600 nm using a spectrophotometer Biowave II (WPA). As a result of the analysis, in the SCD-2 medium adjusted to pH 5.0, 4.0, and 3.0, the growth rate was not so much different between B42-Con strain and B42-LGS1 strain, but the pH was adjusted to 2.0. In the SCD-2 medium, the growth rate was significantly different between these genetically modified yeast strains. That is, the B42-Con strain hardly grew, whereas the B42-LGS1 strain grew well.

  FIG. 6 shows an anaerobic culture using a fermentation medium (SCD-2 medium) containing 40 g / L of glucose using two types of genetically modified yeasts (B42-Con strain and B42-LGS1 strain). It is the figure which showed the glucose consumption of the genetically modified yeast strain | stump | stock, and the ethanol production amount with time. The results of fermentation experiments in SCD-2 media with pH adjusted to 5.0, 4.0, 3.0 and 2.0 are shown in A, B, C and D of FIG.

  First, in SCD-2 medium adjusted to pH 5.0 and 4.0, ethanol production was slightly higher in B42-LGS1 strain expressing LGS1 gene after 72 hours compared to B42-Con strain of control strain. Both glucose consumption and ethanol production were almost unchanged between the two strains (see A and B in FIG. 6). Under either condition, 75-78% glucose was consumed after 72 hours, producing 13.8-14.8 g / L ethanol.

  In the SCD-2 medium adjusted to pH 3.0, there was a difference between yeast strains in terms of glucose consumption and ethanol production (see C in FIG. 6). That is, the control strain B42-Con consumed 72% glucose after 72 hours, whereas the B42-LGS1 strain consumed 80% glucose after 72 hours, and the glucose consumption rate was higher than that of the B42-Con strain. B42-LGS1 strain was faster. In addition, in proportion to the glucose consumption rate, the ethanol production rate was faster in the B42-LGS1 strain than in the B42-Con strain. The B42-Con strain produced 13.0 g / L ethanol after 72 hours, whereas the B42-LGS1 strain produced 15.0 g / L ethanol after 72 hours, and the ethanol yield from glucose consumption was B42 -Con and B42-LGS1 were 86% and 94%, respectively (relative to 100% of the theoretical yield). Thus, the final ethanol production and ethanol yield were higher in the B42-LGS1 strain than in the B42-Con strain. Both yeast strains produced small amounts of by-products glycerol and acetic acid in SCD-2 medium adjusted to pH 3.0-5.0 (0.9 g / l and 0.6 g / l or less, respectively).

Furthermore, in the SCD-2 medium adjusted to pH 2.0, there were further differences between yeast strains in terms of glucose consumption and ethanol production (see D in FIG. 6). That is, the control strain B42-Con strain consumed only 42% glucose after 72 hours, whereas the B42-LGS1 strain consumed 77% glucose after 72 hours, and the glucose consumption rate was B42-Con strain. The B42-LGS1 strain was significantly faster than that. In addition, in proportion to the glucose consumption rate, the ethanol production rate was significantly faster in the B42-LGS1 strain than in the B42-Con strain. The B42-Con strain produced only 6.7 g / L ethanol after 72 hours, whereas the B42-LGS1 strain was able to produce 13.3 g / L ethanol after 72 hours. Yields were 81% and 87% for B42-Con and B42-LGS1 strains (relative to 100% of the theoretical yield), respectively. Thus, the final ethanol production and ethanol yield were higher in the B42-LGS1 strain than in the B42-Con strain not only under the pH 3.0 conditions but also under the low pH conditions of pH 2.0. Both strains produced small amounts of glycerol and acetic acid on SCD-2 medium adjusted to pH 2.0 (1.3 g / l and 0.8 g / l or less, respectively), but the amount of these by-products under pH 3.0-5.0 conditions. Slightly increased. In this way, the control strain B42-Con strain markedly decreased ethanol productivity from glucose in the fermentation medium at pH 2.0, but the B42-LGS1 strain expressing the LGS1 gene had a pH of 2.0, even in the fermentation medium. However, the ethanol fermentability was hardly impaired, as was the case with 5.0, 4.0, and 3.0 fermentation media. These results show that the ethanol fermentation ability of the yeast strain that expresses the LGS1 gene is hardly inhibited even under low pH conditions, and that the LGS1 gene plays an important role in obtaining acid resistance. It was suggested.
Example 12: Culture of genetically modified yeast (5)
For further ethanol fermentation experiments, B42-LGS1 and B42-Con strains were aerobically cultured in SCD medium at 30 ° C for 24 hours, collected by centrifugation, washed with sterile water, and washed with 40 g Appropriate amounts were inoculated into 20 ml of 4 types of fermentation media (SCD-3 media) containing 1 / l glucose and 7.5% Na ₂ SO ₄ (unified bacterial mass). The pH of the fermentation medium (SCD-3 medium) was adjusted to 3.0, 2.5, 2.2, and 2.0, respectively. As in Example 10, each fermentation broth was gently added in a 50 ml sealed vial containing a stir bar. Cultured anaerobically at 30 ° C. for 72 hours with stirring.
Example 13: Measurement of ethanol concentration (2)
As in Example 11, the concentrations of ethanol, glucose, glycerol and acetic acid were measured using high performance liquid chromatography (HPLC; JASCO Corporation), and the separation column was HPX-87H column (Bio-Rad). The HPLC apparatus was flowed with 5 mM H ₂ SO ₄ at a flow rate of 0.6 ml / min and operated at 65 ° C. Yeast growth was measured at a wavelength of 600 nm using a spectrophotometer Biowave II (WPA). As a result of analysis, in SCD-3 medium adjusted to pH 3.0, growth rate was not so different between B42-Con strain and B42-LGS1 strain, but pH was adjusted to 2.5, 2.2, and 2.0. In the SCD-3 medium, the growth rate was significantly different between these genetically modified yeast strains. That is, the B42-LGS1 strain grew better than the B42-Con strain.

  FIG. 7 shows a fermentation medium (SCD-3 medium) containing 40 g / L glucose and 7.5% Na2SO4 using two types of genetically modified yeasts (B42-Con strain and B42-LGS1 strain). It is the figure which showed the glucose consumption and ethanol production amount of these genetically modified yeast strains in anaerobic culture over time. The results of fermentation experiments in SCD-3 medium with pH adjusted to 3.0, 2.5, 2.2, and 2.0 are shown in A, B, C, and D of FIG.

  First, in the SCD-3 medium adjusted to pH 3.0, there was a difference between yeast strains in terms of glucose consumption and ethanol production (see A in FIG. 7). That is, the B42-Con strain of the control strain consumed all glucose after 72 hours, whereas the B42-LGS1 strain consumed all glucose after 55 hours, and the glucose consumption rate was higher than that of the B42-Con strain. The LGS1 stock was slightly faster. Also, in proportion to the glucose consumption rate, the ethanol production rate was slightly faster in the B42-LGS1 strain than in the B42-Con strain. The B42-Con strain produced 18.2 g / L ethanol after 72 hours, whereas the B42-LGS1 strain produced 19.4 g / L ethanol after 72 hours, and the ethanol yield from glucose consumption was B42 -Con and B42-LGS1 were 86% and 92%, respectively (relative to 100% of the theoretical yield). Thus, the final ethanol production and ethanol yield were slightly higher in the B42-LGS1 strain than in the B42-Con strain. Both yeast strains produced small amounts of by-products glycerol and acetic acid in SCD-3 medium adjusted to pH 3.0 (less than 2.2 g / l and 0.8 g / l, respectively).

  In SCD-3 medium with pH adjusted to 2.5 and 2.2, there were further differences between yeast strains in terms of glucose consumption and ethanol production (see B and C in FIG. 7). That is, in the SCD-3 medium at pH 2.5, the control B42-Con strain consumed only 57% glucose after 72 hours, whereas the B42-LGS1 strain consumed all glucose after 72 hours. The glucose consumption rate was significantly faster in the B42-LGS1 strain than in the B42-Con strain. In addition, in proportion to the glucose consumption rate, the ethanol production rate was significantly faster in the B42-LGS1 strain than in the B42-Con strain. The B42-Con strain produced only 9.8 g / L ethanol after 72 hours, whereas the B42-LGS1 strain was able to produce 19.6 g / L ethanol after 72 hours. Yields were 80% and 95% for B42-Con and B42-LGS1 strains (relative to 100% of theoretical yield), respectively. In addition, in the SCD-3 medium at pH 2.2, the control B42-Con strain consumed only 36% glucose after 72 hours, whereas the B42-LGS1 strain consumed 86% glucose after 72 hours. As in the case of pH 2.5, the glucose consumption rate was significantly faster in the B42-LGS1 strain than in the B42-Con strain. In addition, in proportion to the glucose consumption rate, the ethanol production rate was significantly faster in the B42-LGS1 strain than in the B42-Con strain. The B42-Con strain was able to produce only 6.2 g / L ethanol after 72 hours, whereas the B42-LGS1 strain was able to produce 16.0 g / L ethanol after 72 hours. Thus, the final ethanol production was higher in the B42-LGS1 strain than in the B42-Con strain not only under the pH 2.5 conditions but also under the low pH / high salt concentration conditions of pH 2.2. The ethanol yield from glucose consumption was 82% and 83% for B42-Con and B42-LGS1 strains (relative to 100% of the theoretical yield), respectively. For both yeast strains, the by-products glycerol (less than 2.2 g / l and 3.0 g / l respectively) and acetic acid (less than 0.6 g / l and 0.5 g / l respectively) in SCD-3 medium adjusted to pH 2.5 and 2.2 Was produced in small quantities.

Further, even in the SCD-3 medium adjusted to pH 2.0, there was a difference between yeast strains in terms of glucose consumption and ethanol production (see D in FIG. 7), but in the SCD-3 medium at pH 3.0 to 2.2 Compared with the case, the fermentation ability of B42-LGS1 strain was slightly weakened. That is, the control strain B42-Con consumed 31% glucose after 72 hours, and the B42-LGS1 strain consumed 46% glucose after 72 hours. Even in this case, the glucose consumption rate was faster in the B42-LGS1 strain than in the B42-Con strain. In addition, in proportion to the glucose consumption rate, the ethanol production rate was also faster in the B42-LGS1 strain than in the B42-Con strain. The B42-Con strain produced 4.0 g / L ethanol after 72 hours, while the B42-LGS1 strain produced 7.7 g / L ethanol after 72 hours, and the ethanol yield from glucose consumption was B42 -Con and B42-LGS1 strains (relative to 100% of theoretical yield) were 59% and 77%, respectively. Thus, the final ethanol production and ethanol yield were higher in the B42-LGS1 strain than in the B42-Con strain even under the low pH / high salt concentration conditions of pH 2.0. Both strains produced a small amount of glycerol (1.9 g / l or less) on SCD-3 medium adjusted to pH 2.0, but no acetic acid. In this way, the B42-LGS1 strain expressing the LGS1 gene is less isolated from glucose than the control strain B42-Con, even in fermentation media with pH 2.0 and 7.5% Na ₂ SO ₄ at low pH and high salt concentration. Ethanol productivity was high. These results show that the ethanol fermentation ability of yeast strains expressing the LGS1 gene is less susceptible to inhibition even under conditions of low pH and high salt concentration, and the LGS1 gene is important in obtaining acid and salt tolerance. It was suggested that it is working.

  From these examples, the genetically modified yeast of the present invention (B42-LGS1 strain) produced by constitutively high expression of the LGS1 gene involved in acid and salt tolerance is capable of inhibiting growth under low pH and high salt environments. It became clear that fermentation inhibition was avoided, sugars such as glucose were successfully grown using carbon as a carbon source, and useful substances such as ethanol could be produced with high efficiency.

  Since LGS1 was able to complement GAS1 (Example 8, FIG. 4), it is considered that other LGS1 homologous genes belonging to the GH72 family also impart acid and salt tolerance to yeast, as with LGS1 and GAS1. Yeast to which such acid and salt resistance is imparted can also be used for ethanol production.

  In the present invention, the method for imparting acid and salt tolerance, and the acid and salt tolerant yeast produced thereby, avoid ethanol growth and fermentation inhibition as compared with the case of using a conventionally known yeast with excellent fermentation production. Can be produced efficiently. This enables high-efficiency conversion of biomass-derived sugars to ethanol and other useful substances that are expected as next-generation liquid energy even under environmental stress conditions such as strong acids and high salt environments. It can greatly contribute to the practical use of biomass resources and the economic improvement of the fermentation process.

All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
Brief Description of Sequences SEQ ID NO: 1 LGS1 gene base sequence SEQ ID NO: 2 LPR1 gene base sequence SEQ ID NO: 3 Clone 1 gene base sequence SEQ ID NO: 4-7 Primer SEQ ID NO: 8 LGS1 protein amino acid sequence SEQ ID NO: 9 GAS1 protein Amino acid sequence SEQ ID NO: 10 PHR1 deduced amino acid sequence SEQ ID NO: 11 PHR2 deduced amino acid sequence SEQ ID NO: 12 XR gene SEQ ID NO: 13 XDH gene SEQ ID NO: 14 XK gene

Claims (12)

Containing a plasmid expressing a homologous gene LGS1 belonging to LGS1 genes or GH72 family, ethanol from glucose a production can recombinant yeast further xylose reductase gene, xylitol dehydrogenase gene and xylulokinase gene is introduced The genetically modified yeast,
Here, the homologous gene of LGS1 is
(i) a gene having a nucleotide sequence identity of 90% or more with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a protein having LGS1 activity;
(ii) a protein having 90% or more amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 8, and encoding a protein having LGS1 activity, or
(iii) GAS1, GAS2, GAS3, GAS4, GAS5, Gel1, Gel2, Gel3, Gel4, Gel5, Gel6, Gel7, Glt1, PHR1, PHR2, PHR3, EPD1, EPD2, and GCW2 is there,
The genetically modified yeast . Homologous genes LGS1 belonging to LGS1 genes or GH72 family have been introduced by chromosomal integration, ethanol from glucose a production can recombinant yeast further xylose reductase gene, xylitol dehydrogenase gene and xylulokinase gene The genetically modified yeast introduced,
Here, the homologous gene of LGS1 is
(i) a gene having a nucleotide sequence identity of 90% or more with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a protein having LGS1 activity;
(ii) a protein having 90% or more amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 8, and encoding a protein having LGS1 activity, or
(iii) GAS1, GAS2, GAS3, GAS4, GAS5, Gel1, Gel2, Gel3, Gel4, Gel5, Gel6, Gel7, Glt1, PHR1, PHR2, PHR3, EPD1, EPD2, and GCW2 is there,
The genetically modified yeast . The genetic recombinant yeast according to claim 1 or 2 , wherein the xylose reductase gene and the xylitol dehydrogenase gene are derived from Scheffersomyces (Pichia) stipitis, and the xylulokinase gene is derived from Saccharomyces cerevisiae. The genetic recombinant yeast according to any one of claims 1 to 3 , wherein the genetic recombinant yeast is produced using Saccharomyces cerevisiae as a host. The genetic recombinant yeast according to any one of claims 1 to 3 , wherein the genetic recombinant yeast is produced using Kluyveromyces marxianus as a host. The genetic recombinant yeast according to any one of claims 1 to 3 , wherein the genetic recombinant yeast is produced using Scheffersomyces (Pichia) stipitis as a host. The genetic recombinant yeast according to any one of claims 1 to 3 , wherein the genetic recombinant yeast is produced using Issatchenkia orientalis as a host. With either recombinant yeast of claim 1 to 7, a method to produce ethanol from glucose. A method for producing ethanol from xylose using the genetically modified yeast according to any one of claims 1 to 7 . With either recombinant yeast of claim 1 to 7, a method for producing ethanol from a mixed sugar of glucose and xylose. A method for producing ethanol from a saccharified solution prepared from cellulosic biomass, using the genetically modified yeast according to any one of claims 1 to 7 . A genetically modified yeast capable of producing ethanol from glucose, comprising a plasmid expressing a LGS1 gene or a homologous gene of LGS1 belonging to the GH72 family,
Here, the homologous gene of LGS1 is
(i) a gene having a nucleotide sequence identity of 90% or more with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a protein having LGS1 activity;
(ii) a protein having 90% or more amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 8, and encoding a protein having LGS1 activity, or
(iii) GAS1, GAS2, GAS3, GAS4, GAS5, Gel1, Gel2, Gel3, Gel4, Gel5, Gel6, Gel7, Glt1, PHR1, PHR2, PHR3, EPD1, EPD2, and GCW2 is there,
A method for producing ethanol from glucose using the genetically modified yeast.

Images