JP2015177760A - Improved methods for ethanol production by yeast of altered metabolism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a recombinant yeast that produces ethanol at a high efficiency from xylose or a mixed sugars of xylose and glucose and methods for producing ethanol at a high efficiency from xylose or a saccharified solution containing xylose and glucose using the same.SOLUTION: Provided is a recombinant yeast of which HAP4 gene has been destroyed (deleted), expressing a gene encoding an enzyme that metabolizes xylose to xylulose 5-phosphate. Also provided is a highly efficient method for producing ethanol from xylose or a saccharified solution containing xylose and glucose by using the recombinant yeast.

Description

  The present invention includes a genetically modified yeast capable of producing ethanol with high efficiency from a mixed sugar containing xylose and xylose, which is imparted with xylose metabolic ability and lacks the HAP4 gene, and xylose and xylose using the same The present invention relates to a method for producing ethanol from a saccharified solution with high efficiency.

  In recent years, in order to combat global warming and replace fossil resources, there is a technology that efficiently produces ethanol from lignocellulosic biomass that does not compete with food and is inexpensive, and uses this bioethanol as a liquid fuel or chemical raw material. Establishment is desired. Yeast (Saccharomyces cerevisiae) has long been used as a fermenting microorganism for industrially producing ethanol because of its strong ethanol fermentability and ethanol tolerance. In addition, S. cerevisiae has the advantage that continuous production is possible with minimal contamination by other microbial species when used near pH 4.5. S. cerevisiae can assimilate and ferment hexoses such as glucose, but not pentoses such as xylose and arabinose. Lignocellulosic biomass, especially hardwoods and herbs, contains a large amount of xylose after glucose, so in ethanol production from lignocellulosic biomass using S. cerevisiae, the low ethanol yield is one of the factors that hinder its practical application. It was. Therefore, the world has developed technology to produce ethanol from xylose by anaerobic culture (fermentation) by introducing xylose metabolic enzyme genes from other organisms such as Schefersomyces (Pichia) stipitis into S. cerevisiae to impart xylose metabolic capacity (See Patent Literature 1, Non-Patent Literature 1, Non-Patent Literature 2, and Non-Patent Literature 3). However, in such a genetically modified yeast, ethanol fermentation efficiency and fermentation rate from xylose are extremely low compared to those from glucose, and many problems still remain for practical use. Therefore, in order to establish a bioethanol high-efficiency conversion technology from lignocellulosic biomass, a yeast strain having a higher ability to produce ethanol from xylose than the yeast is required.

Special Table 2000-509988

Ho NW et al., Applied and Environmental Microbiology, Vol.64, pp.1852-1859 (1998) Eliasson A et al., Applied and Environmental Microbiology, Vol.66, pp.3381-3386 (2000) Matsushika A et al., Applied Microbiology and Biotechnology, Vol.84, pp.37-53 (2009)

  In the conventional method, ethanol could not be efficiently produced from lignocellulosic biomass resources, especially pentose xylose contained in large amounts in saccharified liquids of broad-leaved trees and herbs. There has been a demand for an effective method capable of efficiently producing ethanol at low cost from a mixed sugar containing carbon sugar. This is because the number of microorganisms that can use xylose is limited, the microorganism that converts xylose into ethanol with high efficiency has not been developed, and even the conventional genetically modified microorganisms with xylose fermentability have yielded xylose fermentation yield. The main cause was low xylose fermentation rate.

  Accordingly, an object of the present invention is to provide a microorganism that converts xylose into ethanol with high efficiency and an effective production method of ethanol using the microorganism. The present invention also relates to a genetically modified yeast that produces ethanol from xylose or a mixed sugar containing xylose and glucose with high efficiency and a method for producing ethanol from a saccharified solution containing xylose, xylose and glucose using the same. The issue is to provide.

  The present inventors performed DNA microarray analysis in order to see the difference in gene expression levels when fermented in a glucose medium and fermented in a xylose medium using the xylose-assimilating flocculant practical yeast MA-R4 strain, Genes that were expressed at higher or lower levels in xylose fermentation than in glucose fermentation were extracted (Matsushika A et al., Microbial Cell Factories, vol. 13:16 (2014)). As a result, genes that are originally expressed under aerobic conditions, such as genes for metabolic enzymes in the citrate cycle and genes expressed in mitochondria, are high in anaerobic conditions during xylose fermentation. It was expressed. From this, it was found that the xylose-assimilating yeast in the xylose fermentation was transformed into an oxidative state in the cells. However, in the production of ethanol from xylose, studies have not been conducted on high production of ethanol under aerobic conditions using genetically modified yeast.

  In order to solve the above problems, in the present invention, xylose utilization is imparted by gene manipulation and the HAP4 gene encoding a transcription activator is deleted, and hexoses such as xylose, xylose and glucose are aerobic. Provided is a method for producing a genetically modified yeast that can produce ethanol in a high yield from a mixed sugar containing it and that has improved consumption rates of xylose and glucose, and an effective method for producing ethanol using the same.

  The present inventors prepared a gene expression cassette for xylose metabolism (xylose reductase XR, xylitol dehydrogenase XDH and xylulokinase XK) that can be efficiently integrated into yeast chromosomes, and introduced this expression cassette into host yeast cells. Then, a genetically modified yeast imparted with xylose fermentability was produced, and it was reported that the genetically modified yeast can produce xylose to ethanol with high efficiency under anaerobic fermentation conditions (Japanese Patent Laid-Open No. 2009-195220). reference). In this genetically modified yeast, HAP4 gene encoding a transcriptional activator that regulates gene expression in the respiratory system and citrate circuit in addition to the introduction of xylose metabolism enzyme gene expression cassette Improved.

  In the present invention, in order to produce ethanol with high efficiency from xylose or a mixed sugar containing glucose and xylose using xylose-assimilating yeast, it encodes a transcriptional activator that regulates gene expression in the respiratory system and citrate cycle. Focusing on the HAP4 gene, three types of xylose metabolic enzymes (xylose reductase XR, xylitol dehydrogenase XDH, and xylulokinase XK) are introduced into the yeast with disrupted (deleted) HAP4 gene for constant expression. It was. We succeeded in efficiently producing ethanol from xylose or a mixed sugar containing glucose and xylose by culturing a genetically modified yeast strain as a transformant under aerobic conditions. It was found that this genetically modified yeast can produce ethanol from xylose with higher efficiency than conventionally known xylose-fermenting yeast strains, and the present invention has been completed (see FIG. 1). The enzymes that metabolize xylose to xylulose 5-phosphate introduced into yeast in which the HAP4 gene has been disrupted (deleted) can be xylose isomerase XI and xylulokinase XK.

That is, the present invention is as follows.
[1] A gene encoding an enzyme that metabolizes xylose to xylulose 5-phosphate has been introduced, and the HAP4 gene has been disrupted, deleted, or inactivated, and ethanol can be efficiently produced from xylose. Genetically modified yeast.
[2] The genetic recombinant yeast according to [1], wherein the genes encoding an enzyme that metabolizes xylose to xylulose 5-phosphate are a xylose reductase gene, a xylitol dehydrogenase gene, and a xylulokinase gene.
[3] The genetic recombinant yeast according to [1], wherein the genes encoding an enzyme that metabolizes xylose to xylulose 5-phosphate are a xylose isomerase gene and a xylulokinase gene.
[4] The genetic recombinant yeast according to any one of [1] to [3], wherein the HAP4 gene disrupted, deleted, or inactivated is derived from yeast or bacteria.
[5] The genetic recombinant yeast according to [4], wherein the HAP4 gene disrupted, deleted or inactivated is derived from Saccharomyces cerevisiae.
[6] The genetic recombinant yeast according to [2], [4] or [5], 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 .
[7] The genetic recombinant yeast according to any one of [3] to [5], wherein the xylose isomerase gene is derived from Reticulitermes speratus and the xylulokinase gene is derived from Saccharomyces cerevisiae.
[8] The genetic recombinant yeast according to any one of [1] to [7], wherein the genetic recombinant yeast is produced using Saccharomyces cerevisiae as a host.
[9] A method for producing ethanol from xylose using the genetically modified yeast according to any one of [1] to [8].
[10] A method for producing ethanol from a mixed sugar of glucose and xylose using the genetically modified yeast according to any one of [1] to [8].
[11] A method for producing ethanol from a saccharified solution prepared from lignocellulosic biomass using the genetically modified yeast according to any one of [1] to [8].
[12] The production method according to any one of [9] to [11], which is performed under anaerobic or aerobic conditions.

  According to the present invention, ethanol is more efficiently produced from xylose, mixed sugar containing xylose and glucose, and mixed sugar contained in lignocellulosic biomass saccharified liquid under aerobic conditions than conventionally known genetically modified yeast capable of xylose fermentation. Provided is a genetically modified yeast that can be produced.

1-1 is explanatory drawing which shows the xylose metabolic pathway in the genetically modified yeast in this invention. In the figure, XR means xylose reductase, XDH means xylitol dehydrogenase, and XK means xylulokinase. By introducing genes encoding XR and XDH derived from Scheffersomyces (Pichia) stipitis and XK derived from Saccharomyces cerevisiae into a yeast, a genetically modified yeast imparted with xylose fermentability can be produced (JP 2009- No. 195220). Furthermore, in genetically modified yeast introduced with genes encoding each of XR, XDH and XK, the gene for the transcriptional activator HAP4 is disrupted, deleted, or inactivated under aerobic conditions. It is possible to produce a genetically modified yeast having excellent pentose utilization efficiency that produces ethanol from a mixed sugar containing xylose. Thereby, the genetically modified yeast in the present invention has an excellent conversion efficiency from xylose to ethanol, and can produce ethanol with high efficiency. 1-2 is explanatory drawing which shows the xylose metabolic pathway in the genetically modified yeast in this invention. In the figure, XI means xylose isomerase, and XK means xylulokinase. By introducing a gene encoding each of XI and XK into the yeast, a genetically modified yeast imparted with xylose fermentability can be produced (see, for example, JP-A-2013-055948). Furthermore, in transgenic yeast introduced with genes encoding XI and XK, the gene for the transcriptional activator HAP4 is disrupted, deleted, or inactivated, so that xylose or xylose can be obtained under aerobic conditions. It is considered that a genetically modified yeast having an excellent pentose utilization ability that produces ethanol at a high yield from a mixed sugar containing sucrose can be produced. FIG. 2 is a characteristic diagram showing the anaerobic ethanol fermentation ability (ethanol production amount in the SX medium) of the two types of genetically modified yeast (MA-B42 strain and B42-DHAP4 strain). FIG. 3 shows the aerobic ethanol production ability (xylose consumption (A) and ethanol production (B)) in SX medium of two types of genetically modified yeasts (MA-B42 strain and B42-DHAP4 strain). FIG. FIG. 4 shows the aerobic ethanol production capacity (xylose and glucose consumption (A) and ethanol production (B)) in SDX medium of two types of genetically modified yeast (MA-B42 strain and B42-DHAP4 strain). FIG. FIG. 5 shows the aerobic ethanol production capacity (xylose and glucose consumption (A) and ethanol production (B) of cedar saccharified solution of two types of genetically modified yeasts (MA-B42 strain and B42-DHAP4 strain). FIG.

  In the genetically modified yeast capable of producing ethanol from xylose of the present invention with high efficiency, a gene encoding an enzyme that metabolizes xylose to xylulose 5-phosphate is introduced, and the HAP4 gene is disrupted, deleted, or Inactive. There are mainly two methods for metabolizing xylose to xylulose 5-phosphate. One is a method of introducing XR, XDH, and XK genes (see FIG. 1-1). Another method is to introduce XI and XK genes (see FIG. 1-2). That is, in one embodiment, the genes encoding enzymes that metabolize xylose to xylulose 5-phosphate are the XR, XDH, and XK genes. In another example, the genes encoding enzymes that metabolize xylose to xylulose 5-phosphate are the XI and XK genes. These will be described below.

  XR is an enzyme that catalyzes the reaction of converting xylose to xylitol. The XR gene is not particularly limited as long as it encodes such an enzyme, but is derived from yeasts such as Candida Shehatae, Scheffersomyces (Pichia) stipitis, Pachysolen tannophilus, and Kluyveromyces marxianus. Preferably, the XR gene is derived from Scheffersomyces (Pichia) stipitis. The XR gene of Scheffersomyces (Pichia) stipitis is registered in GeneBank as registration number XM_001385144 (SEQ ID NO: 1), and these gene information can be used.

  XDH is an enzyme that catalyzes a reaction for converting xylitol into xylulose. The XDH gene is not particularly limited as long as it encodes such an enzyme, but is derived from yeasts such as Candida Shehatae, Scheffersomyces (Pichia) stipitis, Pachysolen tannophilus, and Kluyveromyces marxianus. Preferably, the XDH gene is derived from Scheffersomyces (Pichia) stipitis. The XDH gene of Scheffersomyces (Pichia) stipitis is registered in GeneBank as registration number AF127801 or X55392 (SEQ ID NO: 2), and information on these genes can be used.

  XK is an enzyme that catalyzes the reaction of converting xylulose and ATP into xylulose pentaphosphate and ADP. The XK gene is not particularly limited as long as it encodes such an enzyme. . Preferably, the XK gene is derived from Saccharomyces cerevisiae. The XK gene of Saccharomyces cerevisiae is registered as NC_001139.7 (SEQ ID NO: 3) in GeneBank, and such gene information can be used.

  XI is an enzyme that catalyzes the reaction of converting xylose to xylulose. The XI gene is not particularly limited as long as it encodes such an enzyme, but is derived from, for example, Reticulitermes speratus. The gene information of SEQ ID NO: 5 can be used for the XI gene derived from Reticulitermes speratus.

  HAP4 is an activator subunit of the transcription factor HAP2 / 3/4/5 complex that regulates the expression of genes in the oxidative phosphorylation, respiratory system, electron transport system, and citrate cycle. is there. In addition, its expression is suppressed by glucose (Brons JF et al., Yeast, Vol. 19, pp. 923-932 (2002)). The HAP4 gene is not particularly limited as long as it encodes a factor that activates transcription of a gene involved in respiration and the like, but is derived from yeasts or filamentous fungi such as Kluyveromyces lactis, Saccharomyces cerevisiae, Schizosaccaromyces pombe, and Aspergillus. Preferably, the HAP4 gene is derived from Saccharomyces cerevisiae. The HAP4 gene of Saccharomyces cerevisiae is registered in GeneBank as registration number X16727 or X71133 (SEQ ID NO: 4), and information on these genes can be used.

  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.

  In this specification, to disrupt, delete, or inactivate the HAP4 gene means to disrupt, delete, or suppress the expression of the HAP4 gene so that the protein encoded by the HAP4 gene cannot perform its function. To do or to inactivate. Methods for disrupting, deleting, or inactivating the HAP4 gene can be performed using general molecular biology techniques well known to those skilled in the art, and are not particularly limited, for example, homologous recombination, such as SOE- Deletion or inactivation of the gene from the host cell by homologous recombination using a deletion plasmid inserted with the deletion DNA fragment prepared by PCR (Gene, 77, 61 (1989)) Can do.

  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, PCR is performed using a primer for cloning HAP4 that has been designed based on a known HAP4 gene sequence and mutations are introduced at a predetermined position, thereby mutating at a predetermined position of the cloned HAP4 gene. Can be introduced. In some embodiments, the HAP4 gene disruption strain or deletion strain may be referred to as a HAP4 disruption strain, ΔHAP4 strain, or DHAP4 strain.

In addition, XDH may be used in a modified form in which coenzyme requirement is changed as necessary. It is preferable to substitute at least one of the amino acids corresponding to the 207th to 211st amino acids of XDH with another amino acid, for example, alanine, arginine, serine or threonine. XDH amino acid sequence with 207th aspartic acid substituted with alanine, 208th isoleucine with arginine, amino acid sequence with 209th phenylalanine substituted with serine or threonine, and amino acid sequence Of these, those obtained by substituting arginine for the 211st asparagine are particularly preferred. More preferably, 207th aspartic acid in the amino acid sequence of XDH is substituted with alanine, 208th isoleucine with arginine, 209th phenylalanine with serine, and 211st asparagine with arginine (Japanese Patent Laid-Open No. 2009-195220). No. Publication; see Watanabe S. et al., The Journal of Biological Chemistry Vol. 280, No. 11, pp. 10340-10349 (2005)). Modified XDH can be converted to NADPH using NADP ⁺ as a coenzyme.

  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 contain one or more of the enzyme genes and / or transporter genes. In addition to the target enzyme gene and / or transporter gene, the vector includes a replication origin that enables replication in a host cell, a selection marker for identifying a transformant, and preferably a yeast-derived appropriate gene. Transcriptional or translational control sequences may be included, optionally 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 Candida Shehatae, Scheffersomyces (Pichia) stipitis, Pachysolen tannophilus, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Schizosaccaromyces pombe. preferable.

  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, XR, XDH, and XK, or XI and XK are constitutively expressed in the host cell. For example, it is preferable to introduce the XR, XDH, and XK genes, or the XI and XK genes into a chromosome integration type vector or the like, and then integrate the yeast chromosome into a single chromosome or several copies. These genes may be integrated together into chromosomal DNA by homologous recombination on one allele, or each gene may be integrated into chromosomal DNA by homologous recombination on separate alleles. . Preferably, the XR, XDH, and XK genes, or the XI and XK genes are combined together on one allele on the host DNA. In the present invention, XR, XDH, and XK gene chromosomal integration vectors described in JP-A-2009-195220 can be used. In the present invention, a genetically modified yeast in which the XR, XDH, and XK genes described in JP-A-2009-195220 are incorporated into chromosomal DNA can be used. In the present invention, vectors encoding the XI and XK genes and genetically modified yeasts into which the XI and XK genes are incorporated include, for example, van Maris AJ et al., Advances in Biochemical Engineering / Biotechnology Vol. 108, pp.179-204 (2007 ) Can be used.

  By using the genetically modified yeast introduced with the XR, XDH, and XK genes or the genetically modified yeast introduced with the XI and XK genes as a host strain, the HAP4 gene is disrupted, deleted, or inactivated, The target genetically modified yeast can be obtained. Alternatively, a genetically modified yeast in which the HAP4 gene is disrupted, deleted, or inactivated is used as a host strain, and the XR, XDH, and XK genes are constitutively overexpressed, or the XI and XK genes are constitutively The target genetically modified yeast can be obtained by overexpression.

  The method of constitutively expressing XR, XDH, and XK, or XI and XK can be performed by the method described above.

  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 be obtained from xylose under aerobic conditions as compared with a conventionally known genetically modified yeast capable of xylose fermentation (for example, a genetically modified yeast described in JP-A-2009-195220). Ethanol production is possible, xylose consumption rate and glucose consumption rate are fast, and these mixed sugars can be converted into ethanol with high efficiency.

  The genetic recombinant yeast according to the present invention can produce ethanol from xylose under aerobic and anaerobic conditions. At that time, the concentration of xylose contained in the medium is 0.1 to 20%, preferably 0.5% to 5%, more preferably 0.7% to 2%, and the concentration of glucose contained in the medium is 0.1 to 20%. %, Preferably 0.2 to 10%, and more preferably 4% to 8%.

  Moreover, the genetically modified yeast according to the present invention can produce ethanol from a saccharified solution prepared from lignocellulosic biomass by fermentation reaction. The saccharified solution is not particularly limited, but can be prepared from lignocellulosic biomass such as woody materials (particularly hardwoods rich in xylan) 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. 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 neutrality, but is preferably used after being adjusted to near neutrality. Nutrient sources such as yeast extract and peptone may or may not be added to the saccharified solution, but preferably 1% yeast extract is added.

  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 3.0 to 7.6, preferably 5.0 to 6.0, and more preferably 5.5. The culture conditions may be anaerobic or aerobic. For example, although the genetically modified yeast according to the present invention can produce ethanol from a mixed sugar with xylose and glucose even in anaerobic fermentation reaction, ethanol productivity superior to conventionally known genetically modified yeast capable of xylose fermentation can be obtained. Requires an oxygen-existing state, so the oxygen concentration in the system and the dissolved oxygen concentration in the medium are increased before culturing (for example, when performed in an Erlenmeyer flask, etc.) It is preferable to take a large upper space, plug it with a silicon tube or the like, and perform aeration culture or shaking culture). The reaction may be performed continuously or batchwise.

  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 invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1: Production of genetically modified yeast strain
In order to examine the importance of HAP4 transcriptional activator in ethanol production, a transformed yeast is prepared by introducing a xylose metabolic gene expression cassette into knockout yeast lacking the HAP4 gene, and a wild type yeast in which the HAP4 gene is not destroyed. The ability of ethanol production from xylose of these genetically modified yeast strains was compared with control strains into which xylose metabolic enzyme genes were introduced.

  For this purpose, a yeast lacking the HAP4 gene from the knockout yeast collection was obtained from Open Biosystems (the parent strain is BY4742), and an xylose metabolic enzyme (XR, XDH and XK) gene is integrated into the yeast chromosome. pAURXKXDH (WT) XR (see Japanese Patent Application No. 2008-211274 (JP 2009-195220)) was transformed by the lithium acetic acid method using YEASTMAKER yeast transformation system 2 (Clontech) to impart xylose utilization A recombinant yeast strain B42-DHAP4 was prepared. On the other hand, pAURXKXDH (WT) XR was introduced into BY4742 strain to produce genetically modified yeast MA-B42 strain (control strain). When XR, XDH, and XK activities in these recombinant yeast strains were measured (see Japanese Patent Application No. 2008-211274 (Japanese Patent Application Laid-Open No. 2009-195220)), it was found that all retained high enzyme activity.

Example 2: Culture of genetically modified yeast (1)
First, for a normal ethanol fermentation experiment, knockout yeast lacking the HAP4 gene (B42-DHAP4 strain) and its control yeast (MA-B42 strain) were synthesized in a synthetic medium containing 20 g / l glucose (20 g / l polypeptone). And 10 g / l yeast extract (YPD medium) at 30 ° C. for 36 hours. Bacteria are collected by centrifugation, washed with sterile water, and 20 ml of fermentation medium (minimum medium containing 20 g / l xylose: SX medium (20 g / l xylose, 7 g / l NH ₄ Cl, 5 g / l l KH ₂ PO ₄ , 0.8 g / l MgSO ₄ · 7H ₂ O, pH 5.0)) or SDX medium (20 g / l xylose, 40 g / l glucose, 7 g / l NH ₄ Cl, 5 g / l KH ₂ PO ₄ , 0.8 g / l MgSO ₄ .7H ₂ O, pH 5.0)) was inoculated with an appropriate amount (unified cell mass). The fermentation broth was anaerobically cultured at 30 ° C. with gentle stirring in a 50 ml sealed vial containing a stir bar.

Example 3: Measurement of ethanol concentration (1)
The concentrations of ethanol, glucose, xylose, xylitol and other by-products 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, there was no significant difference in the anaerobic growth rate between these genetically modified yeasts. Moreover, the xylose consumption rate of control yeast (MA-B42 strain) and knockout yeast (B42-DHAP4 strain) was hardly changed in the SX medium. In SDX medium, the glucose consumption rate was almost the same in both recombinant yeast strains, but the xylose consumption rate was slightly slower in the knockout yeast (B42-DHAP4 strain) than in the control yeast (MA-B42 strain). It was.

  Fig. 2 shows these gene sets in anaerobic culture using 20 g / L xylose-containing fermentation medium (SX medium) using two types of genetically modified yeasts (MA-B42 and B42-DHAP4). It is the figure which showed ethanol production amount (refer FIG. 2) of a change yeast strain with time.

  In the SX medium, the B42-DHAP4 strain had a slightly faster ethanol production start time than the control strain, but there was no significant difference in the ethanol production rate between the two recombinant yeast strains after the ethanol production started. The control strain MA-B42 produced 3.4 g / L ethanol after 72 hours, while the knockout yeast strain B42-DHAP4 produced 3.8 g / L ethanol after 72 hours. In SDX medium, the ethanol production rate of B42-DHAP4 was slightly higher than that of the control strain until 24 hours. Thus, under anaerobic fermentation conditions, when xylose is the only carbon source, the B42-DHAP4 strain has a slightly faster start time for ethanol production than the control strain, but a mixed sugar of glucose and xylose is used as the carbon source. In this case, there is no change in the ethanol production start time. When mixed sugar is used as a carbon source, the amount of ethanol produced in the B42-DHAP4 strain is slightly increased in the initial stage of fermentation compared to the control strain.

Example 4: Culture of genetically modified yeast (2)
Next, in order to investigate ethanol production ability under aerobic conditions, knockout yeast lacking the HAP4 gene (B42-DHAP4 strain) and its control yeast (MA-B42 strain) were synthesized in a synthetic medium containing 20 g / l glucose. The cells were aerobically cultured at 30 ° C. for 36 hours in (20 g / l polypeptone, 10 g / l yeast extract: YPD medium). Bacteria are collected by centrifugation, washed with sterile water, and cultured in 50 ml culture medium (minimum medium containing 18 g / l xylose: SX medium (18 g / l xylose, 7 g / l NH ₄ Cl, 5 g / l l KH ₂ PO ₄ , 0.8 g / l MgSO ₄ · 7H ₂ O, pH 5.0)) or SDX medium (18 g / l xylose, 40 g / l glucose, 7 g / l NH ₄ Cl, 5 g / l KH ₂ PO ₄ , 0.8 g / l MgSO ₄ .7H ₂ O, pH 5.0)) was inoculated with an appropriate amount (unified cell mass). The culture was aerobically cultured at 30 ° C. in a 300 ml Erlenmeyer flask fitted with a silicon stopper while stirring with a shaking incubator.

Example 5: Measurement of ethanol concentration (2)
The concentration of ethanol, glucose, xylose, xylitol, and other by-products was operated in the same manner as in Example 3 using high performance liquid chromatography (HPLC; JASCO Corporation). Yeast growth was measured at a wavelength of 600 nm using a spectrophotometer Biowave II (WPA). As a result of the analysis, there was no significant difference in the aerobic growth rate between these genetically modified yeasts.

  Fig. 3 shows these genes in an aerobic culture using a fermentation medium (SX medium) containing 18 g / L xylose using two types of genetically modified yeasts (MA-B42 and B42-DHAP4). It is the figure which showed the xylose consumption (refer A of FIG. 3) and ethanol production (refer B of FIG. 3) with time of the recombinant yeast strain.

  First, regarding the xylose consumption, compared to the control yeast strain MA-B42, the knockout yeast strain B42-DHAP4 had a faster xylose consumption rate. After 72 hours, the MA-B42 strain consumed 43% xylose, whereas the B42-DHAP4 strain consumed 50% xylose. Along with xylose consumption, there was a difference between yeast strains in ethanol production. The control yeast strain MA-B42 produced no ethanol during the fermentation period, whereas the knockout yeast strain B42-DHAP4 produced 2.2 g / L ethanol after 72 hours. In both yeast strains, glycerol, an intermediate metabolite, was produced in a small amount (2.0 g / l or less), but no xylitol was produced. On the other hand, the MA-B42 strain did not produce any acetic acid, whereas the B42-DHAP4 strain produced a small amount of acetic acid (1.8 g / l or less). From these results, even though recombinant yeast originally imparted with xylose assimilation ability cannot produce ethanol from xylose under aerobic conditions, it is aerobic by destroying (deleting) the HAP4 gene. It was revealed that ethanol can be produced from xylose under conditions.

  On the other hand, FIG. 4 shows a fermentation medium (SDX medium) containing 18 g / L xylose and 40 g / L glucose using two types of genetically modified yeasts (MA-B42 strain and B42-DHAP4 strain). FIG. 4 is a graph showing sugar (xylose and glucose) consumption (see A in FIG. 4) and ethanol production (see B in FIG. 4) over time in these aerobic cultures used. is there.

  The xylose consumption rate of the knockout yeast strain B42-DHAP4 was slightly faster than that of the control yeast strain MA-B42, but not so markedly. On the other hand, the glucose consumption rate of the B42-DHAP4 strain was significantly faster than that of the control yeast strain MA-B42. The MA-B42 strain required 72 hours to completely consume glucose, whereas the B42-DHAP4 strain completely consumed glucose in 55 hours. Furthermore, the amount of ethanol produced by the B42-DHAP4 strain greatly exceeded the amount produced by the MA-B42 strain. The MA-B42 strain produced 17.9 g / L ethanol after 72 hours, and the ethanol yield from the total sugar consumption was 60%, whereas the B42-DHAP4 strain was 20.2 g / L after 72 hours. Ethanol was produced, and the ethanol yield based on the total sugar consumption was 68%. These results suggest that the B42-DHAP4 strain, compared to the MA-B42 strain, increases the ethanol production from the mixed sugar of xylose and glucose and improves the conversion efficiency from sugar to ethanol. It was. Neither yeast strain produced any intermediate metabolite xylitol, but small amounts of glycerol and acetic acid (less than 2.9 g / l and 4.0 g / l, respectively). Interestingly, glycerol production was greater in the B42-DHAP4 strain than in the MA-B42 strain, and conversely, acetic acid production was greater in the MA-B42 strain than in the B42-DHAP4 strain.

Example 6: Culture of genetically modified yeast (3)
Next, in order to examine the efficiency of the genetically modified yeast of the present invention to produce ethanol from glucose or xylose contained in a saccharified solution prepared from lignocellulosic biomass, a knockout yeast lacking the HAP4 gene (B42-DHAP4 strain) ) And its control yeast (MA-B42 strain) in a synthetic medium containing 20 g / l glucose (20 g / l polypeptone, 10 g / l yeast extract: YPD medium) aerobically at 30 ° C. for 36 hours. Cultured. Collected by centrifugation, washed with sterile water, and saccharified solution prepared from softwood cedar (contains 72.0 g / l glucose, 1.1 g / l mannose, 1.1 g / l galactose, 6.5 g / l xylose) ) 20 ml was inoculated with an appropriate amount (unified bacterial mass). The fermentation broth was anaerobically cultured at 30 ° C. with gentle stirring in a 50 ml sealed vial containing a stir bar. The method for preparing the saccharified solution was in accordance with Japanese Patent Application No. 2008-211274 (Japanese Patent Laid-Open No. 2009-195220).

Example 7: Measurement of ethanol concentration (3)
The concentrations of ethanol, glucose, xylose, mannose, galactose, xylitol, and other by-products were measured using high performance liquid chromatography (HPLC; JASCO Corporation). The separation column was an HPX-87P column (Bio-Rad), and the HPLC apparatus was run at 70 ° C. with H ₂ O 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, the aerobic growth rate of the knockout yeast (B42-DHAP4 strain) was slightly slower than that of the control yeast (MA-B42 strain), but this was not a significant difference.

  FIG. 5 shows the results of aerobic culture in a cedar saccharified solution containing 72.0 g / l glucose and 6.5 g / l xylose using two types of genetically modified yeasts (MA-B42 strain and B42-DHAP4 strain). FIG. 6 is a graph showing sugar (xylose and glucose) consumption (see A in FIG. 5) and ethanol production (see B in FIG. 5) over time of these genetically modified yeast strains.

  Although all yeast strains consumed glucose completely within 48 hours, the glucose consumption rate of the B42-DHAP4 strain was similar to that of the MA-B42 strain of the control yeast, as in the mixed sugar culture results of Example 5. Than noticeably faster. However, xylose was not consumed within 72 hours in any yeast strain. On the other hand, all yeast strains consumed mannose within 31 hours but did not consume galactose within 72 hours. As a result, after 48 hours, the MA-B42 and B42-DHAP4 strains produced 30.2 g / l and 33.3 g / l ethanol, respectively, from the mixed sugar contained in the cedar saccharified solution, and the B42-DHAP4 strain from the control yeast. Even ethanol was able to produce high. The ethanol yields from the total sugar consumption of MA-B42 and B42-DHAP4 strains were 68.3% and 79.9%, respectively. The ethanol yield of B42-DHAP4 strain was significantly higher than that of control yeast. In addition, glycerol, an intermediate metabolite, was produced in a small amount mainly during glucose fermentation (1.0 g / l or less), but no xylitol was produced. From these results, the yeast strain of the present invention can produce ethanol highly from a saccharified solution prepared from lignocellulosic biomass, imparts xylose utilization, and disrupts (deletes) the HAP4 gene. By aerobically cultivating the yeast thus produced, it was shown that it is useful for high production of ethanol from lignocellulosic biomass-derived mixed sugars.

  From these examples, the genetically modified yeast of the present invention (B42-DHAP4 strain) is not limited to xylose and glucose / xylose mixed sugar in a synthetic medium, but also glucose contained in a saccharified solution prepared from lignocellulosic biomass. It has become clear that sugars such as xylose can be efficiently converted to ethanol.

Alternative method As described above, for the recombinant yeast strain (B42-DHAP4 strain) in which the HAP4 gene is deleted and XR, XDH and XK are introduced, not only the mixed sugar of xylose and glucose and xylose in the synthetic medium, but also glucose and It was demonstrated that sugars such as xylose can be efficiently converted to ethanol. As a method for imparting xylose fermentability to yeast, a method of introducing xylose isomerase (XI) and XK instead of introducing XR, XDH and XK is also known (van Maris AJ et al., Advances in Biochemical Engineering / Biotechnology Vol.108, pp.179-204 (2007)). Therefore, for genetically modified yeast strains lacking the HAP4 gene and introduced with XI and XK, xylose, mixed sugars of glucose and xylose, and sugars such as glucose and xylose in the synthetic medium can also be efficiently converted to ethanol. Conceivable. This alternative method is described below.

  First, in the same manner as described in Example 1, yeast lacking the HAP4 gene was acquired from the collection of knockout yeasts from Open Biosystems (parent strain BY4742), and xylose metabolic enzymes (XI and XK) An expression cassette capable of integrating the gene into the yeast chromosome is transformed by the lithium acetic acid method using YEASTMAKER yeast transformation system 2 (Clontech) to produce a genetically modified yeast strain having xylose utilization. As the XI gene, the one described in SEQ ID NO: 5 can be used. If necessary, a genetically modified yeast strain in which XI and XK are introduced into BY4742 strain is prepared as a control strain. XI and XK activities in these recombinant yeast strains are measured to confirm that they retain enzyme activity.

Next, the produced genetically modified yeast is cultured in the same manner as described in Example 2. Specifically, for a normal ethanol fermentation experiment, a knockout yeast strain lacking the HAP4 gene was synthesized in a synthetic medium containing 20 g / l glucose (20 g / l polypeptone, 10 g / l yeast extract: YPD medium). Incubate aerobically at 30 ° C for 36 hours. Bacteria are collected by centrifugation, washed with sterile water, and 20 ml of fermentation medium (minimum medium containing 20 g / l xylose: SX medium (20 g / l xylose, 7 g / l NH ₄ Cl, 5 g / l l KH ₂ PO ₄ , 0.8 g / l MgSO ₄ · 7H ₂ O, pH 5.0)) or SDX medium (20 g / l xylose, 40 g / l glucose, 7 g / l NH ₄ Cl, 5 g / l KH ₂ PO ₄ , 0.8 g / l MgSO ₄ · 7H ₂ O, pH 5.0))). The fermentation broth is anaerobically cultured at 30 ° C. with gentle agitation in a 50 ml sealed vial containing a stir bar.

Next, the ethanol concentration is measured in the same manner as described in Example 3. Specifically, the concentrations of ethanol, glucose, xylose, xylitol, and other by-products are measured using high performance liquid chromatography (HPLC; JASCO Corporation). The separation column is an HPX-87H column (Bio-Rad), and the HPLC apparatus is flowed with 5 mM H ₂ SO ₄ at a flow rate of 0.6 ml / min and operated at 65 ° C. Yeast growth is measured at a wavelength of 600 nm using a spectrophotometer Biowave II (WPA). Compare with control yeast if necessary.

Next, the genetically modified yeast is cultured in the same manner as described in Example 4. Specifically, the yeast lacking the HAP4 gene and having the XI and XK genes introduced into it was synthesized at 30 ° C in a synthetic medium containing 20 g / l glucose (20 g / l polypeptone, 10 g / l yeast extract: YPD medium). Incubate aerobically for 36 hours. Bacteria are collected by centrifugation, washed with sterile water, and cultured in 50 ml culture medium (minimum medium containing 18 g / l xylose: SX medium (18 g / l xylose, 7 g / l NH ₄ Cl, 5 g / l l KH ₂ PO ₄ , 0.8 g / l MgSO ₄ · 7H ₂ O, pH 5.0)) or SDX medium (18 g / l xylose, 40 g / l glucose, 7 g / l NH ₄ Cl, 5 g / l KH ₂ PO ₄ , 0.8 g / l MgSO ₄ · 7H ₂ O, pH 5.0))). The culture is aerobically cultured at 30 ° C. in a 300 ml Erlenmeyer flask fitted with a silicon stopper while stirring in a shaking incubator.

  Next, the ethanol concentration is measured in the same manner as described in Example 5. Specifically, the concentration of ethanol, glucose, xylose, xylitol, and other by-products is operated in the same manner as in Example 3 using high performance liquid chromatography (HPLC; JASCO Corporation). Yeast growth is measured at a wavelength of 600 nm using a spectrophotometer Biowave II (WPA).

  Next, the genetically modified yeast is cultured in the same manner as described in Example 6. Specifically, the yeast lacking the HAP4 gene and having the XI and XK genes introduced into it was synthesized at 30 ° C in a synthetic medium containing 20 g / l glucose (20 g / l polypeptone, 10 g / l yeast extract: YPD medium). Incubate aerobically for 36 hours. Collected by centrifugation, washed with sterile water, and saccharified solution prepared from softwood cedar (contains 72.0 g / l glucose, 1.1 g / l mannose, 1.1 g / l galactose, 6.5 g / l xylose) ) Inoculate an appropriate amount in 20 ml (unify the amount of cells). The fermentation broth is anaerobically cultured at 30 ° C. with gentle agitation in a 50 ml sealed vial containing a stir bar. The method for adjusting the saccharified solution is in accordance with Japanese Patent Application No. 2008-211274 (Japanese Patent Laid-Open No. 2009-195220).

Next, the ethanol concentration is measured in the same manner as described in Example 7. Specifically, the concentrations of ethanol, glucose, xylose, mannose, galactose, xylitol, and other by-products are measured using high performance liquid chromatography (HPLC; JASCO Corporation). The separation column is an HPX-87P column (Bio-Rad), and the HPLC apparatus is run at 70 ° C. with H ₂ O at a flow rate of 0.6 ml / min. Yeast growth is measured at a wavelength of 600 nm using a spectrophotometer Biowave II (WPA).

  Mixing xylose or glucose and xylose as compared to the case of using a genetically modified yeast expressing only XR, XDH, and XK, which is conventionally known, by aerobic culture using the genetically modified yeast of the present invention. It is possible to efficiently produce ethanol from sugar. Thereby, xylose contained in lignocellulosic biomass can be converted to ethanol, which is expected as the next generation liquid energy, with high efficiency.

SEQ ID NO: 1 XR gene SEQ ID NO: 2 XDH gene SEQ ID NO: 3 XK gene SEQ ID NO: 4 HAP4 gene SEQ ID NO: 5 XI gene

Claims (12)

  A gene that encodes an enzyme that metabolizes xylose to xylulose 5-phosphate, has been introduced, and the HAP4 gene has been disrupted, deleted, or inactivated. yeast.   The genetic recombinant yeast according to claim 1, wherein the genes encoding an enzyme that metabolizes xylose to xylulose 5-phosphate are a xylose reductase gene, a xylitol dehydrogenase gene, and a xylulokinase gene.   The genetic recombinant yeast according to claim 1, wherein the genes encoding an enzyme that metabolizes xylose to xylulose 5-phosphate are a xylose isomerase gene and a xylulokinase gene.   The genetic recombinant yeast according to any one of claims 1 to 3, wherein the HAP4 gene disrupted, deleted or inactivated is derived from yeast or bacteria.   The genetically modified yeast according to claim 4, wherein the HAP4 gene disrupted, deleted or inactivated is derived from Saccharomyces cerevisiae.   The genetic recombinant yeast according to claim 2, 4 or 5, 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 3 to 5, wherein the xylose isomerase gene is derived from Reticulitermes speratus and the xylulokinase gene is derived from Saccharomyces cerevisiae.   The genetic recombinant yeast according to any one of claims 1 to 7, wherein the genetic recombinant yeast is produced using Saccharomyces cerevisiae as a host.   The method to produce ethanol from xylose using the genetically modified yeast of any one of Claims 1-8.   The method to produce ethanol from the mixed sugar of glucose and xylose using the genetically modified yeast of any one of Claims 1-8.   A method for producing ethanol from a saccharified solution prepared from lignocellulosic biomass using the genetically modified yeast according to any one of claims 1 to 8.   The production method according to any one of claims 9 to 11, which is carried out under anaerobic or aerobic conditions.

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