WO2010095751A1 - High-efficiency lactic acid manufacturing method using candida utilis - Google Patents

Abstract

Disclosed is a strain of Candida utilis yeast produced by a promoter sequence that enables the expression of a gene that codes for a polypeptide that has lactic acid dehydrogenase activity being transformed by at least one copy of the gene that has been functionally bound. The yeast strain is useful for manufacturing lactic acid with high efficiency.

Description

Highly efficient lactic acid production method by Candida utilis Reference to related applications

This patent application is accompanied by a claim of priority based on Japanese Patent Application No. 2009-39820 (filing date: February 23, 2009) which is a previously filed Japanese patent application. The entire disclosure of this earlier patent application is hereby incorporated by reference.

Background of the Invention

TECHNICAL FIELD The present invention relates to a method for producing lactic acid using Candida utilis which is a club tree negative yeast as a host.

Background Art In recent years, interest in biodegradable plastics has increased due to environmental efforts. Biodegradable plastics can circulate resources naturally and have a low impact on the environment because they decompose naturally. Polylactic acid, which is a typical raw material for biodegradable plastics, is produced by polymerizing L-lactic acid. The higher the optical purity of lactic acid, the more stable polylactic acid can be produced. Usually, lactic acid is obtained as a metabolite of a microorganism using a carbohydrate such as glucose as a substrate. In particular, a group of bacteria called lactic acid bacteria has long been known to specifically produce lactic acid, and is involved in the production of yogurt and the like. However, since lactic acid bacteria by-produce several percent of D-lactic acid in addition to L-lactic acid during the fermentation process, the optical purity of the produced lactic acid is lowered.

Yeast is often used for the production of useful substances. In general, yeast can be cultured at a higher cell density than bacteria, and continuous culture is also possible. Yeast secretes proteins into the medium, and the secreted proteins are modified by sugar chains. For this reason, protein production by yeast is advantageous when such modifications are important for biological activity.

Among the yeasts that have been most well studied to date and have accumulated genetic knowledge, there is a yeast of the genus Saccharomyces, which has been studied as a host for the production of various substances.
In recent years, methods for transforming several species such as Pichia yeast, Hansenula yeast, Kluyveromyces yeast, Candida yeast as yeast other than Saccharomyces yeast have been developed and are useful substances. It has been studied as a production host. Among these, Candida yeast has characteristics that are not found in Saccharomyces yeast, such as a wide carbon utilization range.

Among the Candida genus yeasts, Candida utilis exhibits excellent assimilability to pentose including xylose. In addition, unlike Saccharomyces yeast, ethanol is not produced by culturing under aerobic conditions and growth inhibition is not caused thereby, so that efficient microbial cell production by continuous culture at high density is possible. Therefore, Candida utilis has once attracted attention as a protein source, and industrial production of microbial cells using a saccharified solution of broad-leaved trees containing a large amount of pentose and a sulfite pulp waste solution as a sugar source has been performed. In addition, the yeast by the United States FDA (Food and Drug Administration), Saccharomyces cerevisiae (Saccharomyces cerevisiae), along with the Saccharomyces fragilis (Saccharomyces fragilis), has been recognized as safe yeast that can be used as a food additive. In fact, Candida utilis is manufactured in various countries around the world, including Germany, the United States, Taiwan, and Brazil, and is used as a diet. In addition to its use as a microbial protein, Candida utilis has been widely used in industry as a production strain for pentose and xylose fermentation strains, ethyl acetate, L-glutamine, glutathione, invertase and the like. .

As an attempt to produce lactic acid using yeast, a technology for producing lactic acid by introducing a gene encoding a polypeptide having an activity of an exogenous lactate dehydrogenase (LDH) into yeast that does not have the ability to produce lactic acid has been developed. Has been. Yeast that has been subjected to such genetic manipulation can produce lactic acid from glucose via pyruvic acid. Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), which is the most studied in yeast, has a strong ability to perform alcoholic fermentation to produce ethanol from pyruvic acid via acetaldehyde, thus reducing the efficiency of lactic acid production from glucose as a substrate Resulting in. Therefore, in order to suppress alcohol fermentation, attempts have been made to suppress its expression by destroying a gene encoding a polypeptide having pyruvate decarboxylase (PDC) activity in the chromosome of Saccharomyces cerevisiae. (Japanese Patent Publication No. 2001-516584; Japanese Patent Publication No. 2003-500062). However, because Saccharomyces cerevisiae has the property of growing dependent on ethanol fermentation under high glucose (crab tree positive effect), the disruption of the PDC gene contributed to lactic acid production, but it also negatively caused the suppression of cell proliferation. The effect was also brought.

On the other hand, as an example of using the club Tree effect negative yeast, at least a PDC gene using the disrupted Kluyveromyces yeast strain attempt to produce lactic acid it has been made (Kohyo 2001-516584 JP; Laid Table 2005-528106), for example, had a drawback such as a long fermentation time in a stirred tank fermenter.

In addition, as an example of producing lactic acid using Candida recombinant yeast, which is a clubtree-negative yeast, as a host, there is a report using Candida sonorensis (Japanese Patent Laid-Open No. 2007-1111054; Special Table 2005). No. 518197), the lactic acid production efficiency is low, the concentration of lactic acid produced is low, or it takes a long time to produce lactic acid.

Furthermore, no example of producing lactic acid with high efficiency in a medium containing sucrose, which is a main constituent sugar of molasses, as a carbon source using such a lactic acid-producing yeast having a negative club tree effect has been reported.

The present inventors have produced a yeast strain of Candida utilis comprising a gene encoding a polypeptide having lactate dehydrogenase activity so that it can be expressed by transformation, and cultivating the yeast strain. It has been found that lactic acid can be produced efficiently. The present invention is based on this finding.

Therefore, the present invention provides a yeast strain produced using Candida utilis which is a club tree effect negative yeast and producing lactic acid with high efficiency, and a method for producing lactic acid with low cost and high yield. Objective.

The yeast strain according to the present invention is transformed with at least one copy of the gene operably linked to a promoter sequence enabling expression of a gene encoding a polypeptide having lactate dehydrogenase activity. The yeast strain of Candida utilis.

Furthermore, the method for producing lactic acid according to the present invention comprises culturing the yeast strain according to the present invention.

According to the present invention, a novel Candida utilis strain having lactic acid-producing ability is provided, and L-lactic acid can be efficiently produced in a short time by using this yeast strain for fermentation under appropriate conditions. It becomes possible. According to the present invention, in the lactic acid production method using Candida utilis, which is a club tree effect negative yeast, the production of lactic acid can be greatly improved while suppressing the production of by-products such as ethanol and various organic acids. .

Among SEQ ID NO: 36, a nucleotide sequence (codon optimized sequence) from the 13th a to the 1011st a (upstream TGA of the two translation termination codons) and the sequence represented by SEQ ID NO: 38 (bovine It is a figure which shows the alignment of the (wild-type sequence derived). It is a figure which shows the structure of plasmid pCU563. It is a figure which shows the structure of plasmid pCU595. It is a figure which shows the annealing site | part of the primer utilized for destruction of a CuURA3 gene. It is a figure which shows the result of having performed PCR using IM-63 (sequence number 58) and IM-92 (sequence number 59) as a primer. As each template DNA, Hygr and G418s strain (lane 2) in which one copy of CuURA3 gene derived from NBRC0988 strain (lane 1), CuURA3 gene derived from NBRC0988 strain was disrupted, and one copy of CuURA3 gene in Hygs having pCU595 and G418r were disrupted . A strain (lane 3) is a Hygs and G418s strain (lane 4) in which one copy of the CuURA3 gene from which pCU595 has been eliminated is disrupted. M is DNA obtained by digesting Lamda DNA with Sty I. It is a figure which shows the result of having performed PCR using IM-63 (sequence number 58) and IM-223 (sequence number 60) as a primer. As each template DNA, Hygr and G418s strain (lane 2) in which one copy of CuURA3 gene derived from NBRC0988 strain (lane 1), CuURA3 gene derived from NBRC0988 strain was disrupted, and one copy of CuURA3 gene in Hygs having pCU595 and G418r were disrupted . A strain (lane 3) is a Hygs and G418s strain (lane 4) in which one copy of the CuURA3 gene from which pCU595 has been eliminated is disrupted. M is DNA obtained by digesting Lamda DNA with Sty I. It is a figure which shows the growth ability in the non-selective culture medium of the NBRC0988 strain and the CuURA3 gene disruption strain which made the NBRC0988 strain a host, and a selective culture medium. It is a figure which shows the analysis result by the Southern hybridization method for investigating how many types of PDC genes exist in Candida utilis. Lane 1 is a sample obtained by digesting genomic DNA extracted from Saccharomyces cerevisiae S288C with Hind III. For other lanes, genomic DNA extracted from Candida utilis NBRC0988 strain was extracted from Xba I (lane 2), Hind III (lane 3), Bgl II (lane 4), Eco RI (lane 5), Bam HI (lane 6). , Pst I (lane 7) digested sample. Primers IKSM-29 (SEQ ID NO: 1) and IKSM-30 (SEQ ID NO: 2) were prepared, and a DNA fragment of approximately 220 bp (SEQ ID NO: 3) amplified by PCR using the genome of NBRC0988 strain as a probe was probed Used as DNA. It is a figure which shows the annealing part of the primer utilized for destruction of CuPDC1 gene. It is a figure which shows the structure of plasmid pCU681. It is a figure which shows a time-dependent change of the L-lactic acid density | concentration in a culture medium from 4 hours after fermentation start to 13 hours after Pj0404 strain and Pj0957 strain. In a trial using calcium carbonate as a neutralizing agent, sampling was performed several times by 24 hours after the start of fermentation. It is a figure which shows the time-dependent change of the glucose level and L-lactic acid level in a culture medium. In a trial using sodium hydroxide as a neutralizing agent, sampling was performed a plurality of times by 24 hours after the start of fermentation. FIG. 2 is a graph showing changes over time in the amount of glucose and the amount of L-lactic acid in a medium (n = 2). In a trial using sodium hydroxide as a neutralizing agent, sampling was performed a plurality of times by 24 hours after the start of fermentation. FIG. 3 is a graph showing changes over time in the amount of glucose and the amount of L-lactic acid in a medium (n = 3).

DETAILED DESCRIPTION OF THE INVENTION

Since the yeast used in the present invention, Candida utilis, is produced for food and feed, it is known that the yeast is highly safe.

The yeast strain according to the present invention is a strain of Candida utilis comprising at least one of the genes operably linked to a promoter sequence that enables expression of a gene encoding a polypeptide having lactate dehydrogenase activity. It was obtained by transformation with one copy.

Furthermore, it was suggested that Candida utilis has at least one gene encoding a polypeptide having pyruvate decarboxylase activity by analysis using Southern hybridization ( CuPDC1 gene). . When the CuPDC1 gene is disrupted, the reaction in which pyruvic acid is converted to acetaldehyde does not proceed, so alcohol fermentation, which is a subsequent metabolic pathway, is not performed, and ethanol is hardly produced. In the present invention, if a yeast that disrupts a gene encoding a polypeptide having pyruvate decarboxylase activity is used as a host for lactic acid-producing yeast, an excess substance for ethanol-producing lactic acid is not produced, and lactic acid is efficiently produced. Can be manufactured.

Therefore, according to a preferred embodiment of the present invention, there is provided a yeast strain that is capable of expressing a gene encoding a polypeptide having no or reduced activity of pyruvate decarboxylase and having lactate dehydrogenase activity. Is done. In this yeast strain, it is preferable that the endogenous gene encoding the polypeptide having pyruvate decarboxylase activity is disrupted.

Furthermore, the gene encoding a polypeptide having lactate dehydrogenase activity is preferably provided so as to be expressed under the control of a promoter of a gene encoding a polypeptide having pyruvate decarboxylase activity, More preferably, it is provided so that it can be expressed under the control of a promoter of a gene encoding a polypeptide having a pyruvate decarboxylase activity on the yeast chromosome.

According to a particularly preferred embodiment of the present invention, in the lactic acid-producing yeast, a gene encoding a polypeptide having pyruvate decarboxylase activity on the chromosome is disrupted, and the promoter of the disrupted gene is A yeast strain is provided that is capable of expressing a gene encoding a polypeptide having the activity of lactate dehydrogenase under control.

In the yeast strain of any one of these embodiments, the gene encoding the polypeptide having pyruvate decarboxylase activity is preferably pyruvate decarboxylase gene 1 ( CuPDC1 gene), and the lactate dehydrogenase The polypeptide having the enzyme activity is preferably derived from bovine.

The lactic acid produced by the yeast strain according to the present invention may be any of L-lactic acid, D-lactic acid, and DL-lactic acid, but is preferably L-lactic acid.

Hereinafter, the yeast strain according to the present invention will be described, and a method for producing lactic acid using the yeast will be described.

Yeast used for lactic acid production The yeast strain according to the present invention is a transformed yeast having a gene encoding a polypeptide having the activity of an exogenous lactate dehydrogenase. The yeast used for the production of lactic acid is Candida utilis, which is a club tree negative yeast. The strain of Candida utilis may be various strains known in the art, for example, NBRC0626 strain, NBRC0639 strain, NBRC0988 strain, NBRC1086 strain and the like, and preferably NBRC0988 strain.

Pyruvate decarboxylase The yeast strain according to the present invention preferably has no or reduced pyruvate decarboxylase (PDC) activity. This enzyme is an enzyme that converts pyruvate to acetaldehyde in the alcohol fermentation pathway, and yeast that performs alcohol fermentation inherently has a gene encoding a polypeptide having pyruvate decarboxylase activity on its chromosome. Yes. Saccharomyces cerevisiae has three types of genes ( ScPDC1 , ScPDC5, and ScPDC6 ) encoding polypeptides having pyruvate decarboxylase activity, and these function by a so-called autoregulation mechanism. In addition, homology at the nucleotide level of each gene is as high as 70% or more. The proteins encoded by these genes are composed of an N-terminal TPP binding region and a C-terminal PDC active region. The gene encoding PDC is also present in other yeasts. For example, the KlPDC1 gene of Kluyveromyces lactis has high homology with the ScPDC1 gene. On the other hand, Candida utilis has one type of gene ( CuPDC1 ) encoding a polypeptide having pyruvate decarboxylase activity, and there may be another similar gene, but at least CuPDC1 Alcohol fermentation is almost never performed by destroying the gene.

Here, “there is no or reduced PDC activity” means that there is no PDC activity, the enzyme having an activity lower than that of the wild type is produced, or the production amount of the enzyme is wild type. Means less than. The yeast strain having no or reduced PDC activity may be obtained by artificial manipulation or may be found by screening. Artificial manipulations for extinction or reduction of enzyme activity include RNAi, replacement with other genes such as all or part of the selectable marker, and insertion of meaningless sequences inside the gene. It can carry out by a method well-known in this technical field. Among these, it is preferable to destroy (knock out) the gene encoding the polypeptide having the enzyme activity. As such a method, among the above-mentioned methods, all or a part of the sequence of the selectable marker is used. And a method of exchanging the gene of PDC with a gene encoding PDC.

The gene encoding the polypeptide having the activity of pyruvate decarboxylase to be destroyed originally exists in Candida utilis. Among them, the example described in the present invention is CuPDC1 present in NBRC0988 strain. One of the alleles of a gene, the nucleotide sequence of which is represented by SEQ ID NO: 63, and the encoded amino acid sequence is represented by SEQ ID NO: 64. When using other strains of Candida utilis, for example, NBRC0626 strain, NBRC0639 strain, NBRC1086 strain, etc., even if they are different from the sequences, there are those having equivalent functions, that is, activities. It can be targeted for destruction.

According to a preferred embodiment of the present invention, the endogenous gene encoding a polypeptide having pyruvate decarboxylase activity to be destroyed is a gene encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 64 More preferably, the gene comprises the nucleotide sequence represented by SEQ ID NO: 63.

Lactate dehydrogenase The yeast strain according to the present invention retains a gene ( LDH gene) encoding a polypeptide having lactate dehydrogenase activity. Since yeast originally has no ability to produce lactic acid, the gene ( LDH ) encoding the polypeptide having the lactate dehydrogenase activity of the yeast strain according to the present invention is foreign. LDH has various congeners depending on the type of organism or in vivo, and L-LDH or D-LDH may be used in the present invention. -LDH. Further, the gene encoding a polypeptide having lactate dehydrogenase activity used in the present invention includes naturally occurring LDH, as well as LDH artificially synthesized by chemical synthesis or genetic engineering techniques. Yes. Examples of organisms having LDH include prokaryotes such as lactic acid bacteria, eukaryotes such as fungi, and higher eukaryotes such as plants, animals and insects. The LDH used in the present invention is preferably derived from higher eukaryotes, particularly those derived from cattle. The nucleotide sequence of a gene encoding a polypeptide having the activity of bovine lactate dehydrogenase (L-LDH) is represented by SEQ ID NO: 38, and the amino acid sequence encoded thereby is represented by SEQ ID NO: 35. Is done.

According to a preferred embodiment of the present invention, the polypeptide having lactate dehydrogenase activity is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 37. The polypeptide having lactate dehydrogenase activity includes an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 37, and lactate dehydration. It may be a polypeptide having an enzyme activity.

Here, amino acid deletion, substitution, addition, or insertion can be performed by modifying a gene encoding the above polypeptide by a technique known in the art. Mutation can be introduced into a gene by a known method such as the Kunkel method or Gapped-duplex method or a method similar thereto, for example, a mutation introduction kit using site-directed mutagenesis, such as Mutant-K (Takara Bio Inc.), Mutant-G (Takara Bio Inc.), etc., or using Takara Bio Inc. LA PCR in vitro Mutageness series kit, KOD-Plus-Mutageness Kit (TOYOBO), etc. be able to. The activity of lactate dehydrogenase can be confirmed by a technique known in the art.

Furthermore, the gene encoding a polypeptide having the activity of lactate dehydrogenase to be introduced into the host is cattle described in SEQ ID NO: 35 (Bos taurus) amino acid sequence derived from the enzyme (DDBJ / EMBL / GenBank Accession number : AAI46211. The nucleotide sequence corresponding to 1) is preferably artificially synthesized in consideration of the Candida utilis codon usage. Such artificial synthesis can be appropriately performed by those skilled in the art, but a particularly preferred nucleotide sequence is the nucleotide sequence from the 13th a to the 1,011st a in SEQ ID NO: 36. . The sequences before and after that are restriction enzyme recognition sites, respectively, a Kpn I recognition site (sequence from the first g to the 6th c in the nucleotide sequence of SEQ ID NO: 36), and an Xba I recognition site (SEQ ID NO: 36). sequences from the seventh t in the nucleotide sequence up to 12 th a), sequences from 1,015 th g in the nucleotide sequence of Bam HI recognition site (SEQ ID NO: 36 to 1,020 th c), and Sac I recognition site (sequence from 1,021st g to 1,025th c in the nucleotide sequence of SEQ ID NO: 36). Among the SEQ ID NO: 36, a nucleotide sequence (codon optimized sequence: SEQ ID NO: 36) from the 13th a to the 1,011st a (upstream tga of two translation termination codons) and SEQ ID NO: 38 The alignment of the nucleotide sequence represented by (wild-type sequence derived from bovine) is shown in FIG. Both sequences had the same 751 bases out of 999 bases, and the homology was 75%. In FIG. 1, the upper sequence is a nucleotide sequence from the 13th a to the 1011st a (upstream tga of two translation termination codons) in SEQ ID NO: 36. The lower sequence of FIG. 1 is the base sequence of L-LDH-A gene derived from Bos taurus represented by SEQ ID NO: 38 (extracted from DDBJ / EMBL / GenBank Accession number: BC146210.1). No. 35). The gene encoding the artificially synthesized polypeptide having the lactate dehydrogenase activity is optimized for codon usage in Candida utilis. L-lactic acid can be produced with high efficiency.

According to a preferred embodiment of the present invention, the gene encoding a polypeptide having lactate dehydrogenase activity has a nucleotide sequence from the 13th a to the 1,011st a in SEQ ID NO: 36. It is considered as a gene containing or equivalent thereof. This equivalent is a gene in which some nucleotide residues are different on the condition that it has a function equivalent to that of a gene containing a nucleotide sequence from the 13th a to the 1011st a in SEQ ID NO: 36. means. Such an equivalent includes a nucleotide sequence from the 13th a to the 1011st a in SEQ ID NO: 36 and 70% or more, preferably 80% or more, more preferably 85% or more, and still more preferably Examples thereof include a gene comprising a nucleotide sequence encoding a polypeptide having 90% or more homology, most preferably 95% or more, and having lactate dehydrogenase activity. As the equivalent, it further hybridizes with the nucleotide sequence from the 13th a to the 1,011st a or its complementary sequence in SEQ ID NO: 36 under stringent conditions, and the activity of lactate dehydrogenase And a gene containing a nucleotide sequence encoding a polypeptide having The equivalent further includes a sequence in which one or several nucleotide residues are deleted, substituted, added, or inserted in the nucleotide sequence from the 13th a to the 1011st a in SEQ ID NO: 36. And a gene comprising a nucleotide sequence encoding a polypeptide having lactate dehydrogenase activity. According to a particularly preferred embodiment of the present invention, the gene encoding a polypeptide having lactate dehydrogenase activity is a nucleotide sequence from the 13th a to the 1,011st a in SEQ ID NO: 36. It is considered as a gene containing

Here, deletion, substitution, addition or insertion of nucleotide residues can be performed by modifying a gene containing the above sequence by a technique known in the art. Mutation can be introduced into a gene by a known method such as the Kunkel method or Gapped-duplex method, or a method equivalent thereto, for example, a mutation introduction kit using site-directed mutagenesis may be used. For example, using Mutant-K (Takara Bio) or Mutant-G (Takara Bio), or using Takara Bio's LA バ イ オ PCR in vitro Mutageness series kit, KOD-Plus-Mutageness Kit (TOYOBO), etc. Mutations can be introduced. The activity of lactate dehydrogenase can be confirmed by a technique known in the art.

The numerical value (%) indicating homology is calculated using default (initial setting) parameters using a base sequence comparison program such as GENETYX-WIN 7.0.0. That is, each gene on the yeast chromosome may be replaced by a gene encoding a polypeptide that is not identical but has an equivalent function, ie, each activity, through homologous recombination or the like. The activity of lactate dehydrogenase can be confirmed by a technique known in the art.

The stringent conditions include, for example, Rapid-Hyb Buffer (manufactured by GE Healthcare Bioscience), the temperature condition is preferably 40 to 70 ° C., more preferably 60 ° C., and others are performed according to the attached protocol. Hybridization conditions. Then, for example using a general method of the person skilled in the art, washing for 5 minutes with a solution consisting of 2 × SSC and 0.1% (w / v) SDS, followed by 1 × SSC and 0.1% (w / v) v) Refers to washing for 10 minutes with a solution consisting of SDS, and further washing for 10 minutes with a solution consisting of 0.1 × SSC and 0.1% (w / v) SDS. However, by setting conditions such as the temperature conditions during hybridization and the salt concentration of the solution used for the subsequent membrane washing, it can be set to a certain level (70%, 80%, 85%, 90%, or 95%). ) DNA containing a base sequence having the above homology can be cloned. The gene thus obtained may be replaced by homologous recombination or the like with a gene that encodes a polypeptide that is not identical in sequence but has an equivalent function, that is, each activity. The activity of lactate dehydrogenase can be confirmed by a technique known in the art.

A gene encoding a polypeptide having the activity of a promoter lactate dehydrogenase used for expression of a structural gene is preferably provided so that it can be expressed under the control of a promoter having a strong promoter activity. For example, in Candida utilis, the promoter of the GAP gene encoding a polypeptide having the activity of glyceraldehyde-3-phosphate dehydrogenase of Candida utilis encodes a polypeptide having the activity of phosphoglycerate kinase. The promoter of PGK gene, the promoter of PMA gene encoding a polypeptide having plasma membrane proton ATPase activity (above, JP-A-2003-144185) and the like are exemplified, but more preferably pyruvate decarboxylase It is a promoter of gene 1 ( CuPDC1 gene) encoding a polypeptide having activity. Among them, what is described in the examples of the present invention is one existing in Candida utilis NBRC0988 strain (SEQ ID NO: 3). When using other strains of Candida utilis, for example, NBRC0626, NBRC0639, NBRC1086, etc., those having equivalent functions (ie, other strains) are present even if they differ from the sequences. You can use it as it is. The other strain sequence can be confirmed by a person skilled in the art by a known method.

The gene encoding a polypeptide having lactate dehydrogenase activity is preferably provided so that it can be expressed under the control of the CuPDC1 gene promoter on the yeast chromosome. Candida utilis used as a host of the yeast strain according to the present invention is presumed to have at least one PDC gene ( CuPDC1 gene). By gene CuPDC1 gene controlled by the CuPDC1 gene promoter encoding a polypeptide having a disrupted by activity of lactate dehydrogenase is expressed in place, lowering the lactic acid effectively pyruvate decarboxylase activity The dehydrogenase activity can be expressed at the same time.

According to a preferred embodiment of the present invention, the promoter sequence is a promoter portion of an endogenous gene encoding pyruvate decarboxylase, and more preferably includes a nucleotide sequence represented by SEQ ID NO: 3. . Alternatively, this promoter sequence may be an equivalent in which some nucleotide residues are different on the condition that it has a function equivalent to that including the nucleotide sequence represented by SEQ ID NO: 3. Such an equivalent includes 70% or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, most preferably 95% or more homology with the nucleotide sequence represented by SEQ ID NO: 3. And DNA having a promoter activity. The equivalent further includes DNA that hybridizes with the nucleotide sequence represented by SEQ ID NO: 3 or its complementary sequence under stringent conditions and has promoter activity. Examples of the equivalent further include DNA having a promoter activity, including a sequence in which one or several nucleotide residues are deleted, substituted, added, or inserted in the nucleotide sequence represented by SEQ ID NO: 3. .

Here, deletion, substitution, addition, or insertion of a nucleotide residue can be performed by modifying the above sequence by a technique known in the art. Mutation can be introduced into a gene by a known method such as the Kunkel method or Gapped-duplex method or a method similar thereto, for example, a mutation introduction kit using site-directed mutagenesis, such as Mutant-K (Takara Bio Inc.), Mutant-G (Takara Bio Inc.), etc., or using Takara Bio Inc. LA PCR in vitro Mutageness series kit, KOD-Plus-Mutageness Kit (TOYOBO), etc. be able to. Promoter activity, ie, transcription activity, can be confirmed by a technique known in the art.

The numerical value (%) indicating homology is calculated using default (initial setting) parameters using a base sequence comparison program such as GENETYX-WIN 7.0.0. That is, each gene on the yeast chromosome may be replaced by a gene that is not identical but has an equivalent function, that is, each activity, through homologous recombination or the like. Promoter activity, ie, transcription activity, can be confirmed by a technique known in the art.

The stringent conditions include, for example, Rapid-Hyb Buffer (manufactured by GE Healthcare Bioscience), the temperature condition is preferably 40 to 70 ° C., more preferably 60 ° C., and others are performed according to the attached protocol. Hybridization conditions. Then, for example using a general method of the person skilled in the art, washing for 5 minutes with a solution consisting of 2 × SSC and 0.1% (w / v) SDS, followed by 1 × SSC and 0.1% (w / v) v) Refers to washing for 10 minutes with a solution consisting of SDS, and further washing for 10 minutes with a solution consisting of 0.1 × SSC and 0.1% (w / v) SDS. However, by setting conditions such as the temperature conditions during hybridization and the salt concentration of the solution used for the subsequent membrane washing, it can be set to a certain level (70%, 80%, 85%, 90%, or 95%). ) DNA containing a base sequence having the above homology can be cloned. The gene thus obtained may be replaced by homologous recombination or the like with a gene that is not identical in sequence but has an equivalent function, that is, each activity. Promoter activity, ie, transcription activity, can be confirmed by a technique known in the art.

Molecular Breeding of Yeast Strain Molecular breeding of a yeast strain according to the present invention can be performed by introducing a gene encoding a polypeptide having lactate dehydrogenase activity into a host yeast in a state where it can be expressed. In that case, it is preferable that the host yeast is accompanied by the destruction of the gene encoding PDC. A DNA construct for PDC disruption has a gene sequence for homologous recombination to be introduced into a specific gene site to destroy the gene. The gene sequence for homologous recombination here is a gene sequence that is homologous to a target site that is a PDC gene to be destroyed or a gene in the vicinity thereof. For example, two types of gene sequences for homologous recombination are made homologous to the upstream and downstream genes of the target gene on the chromosome, and the gene is destroyed between these gene sequences for homologous recombination. The gene at the target site can be destroyed by introducing a DNA fragment comprising the gene for the purpose into the yeast chromosome by homologous recombination. Selection of a gene sequence for homologous recombination to realize such integration on a chromosome is well known to those skilled in the art, and those skilled in the art can select an appropriate gene sequence for homologous recombination as necessary. Thus, a DNA fragment for homologous recombination can be constructed.

According to a preferred embodiment of the present invention, the endogenous gene encoding a polypeptide having pyruvate decarboxylase activity is disrupted by deletion of the gene by insertion of a selectable marker sequence. For example, the PDC gene can be destroyed by incorporating a selectable marker sequence into the nucleotide sequence inserted in place of the PDC gene in the above homologous recombination. The selectable marker is useful for selecting transformed cells. The insertion of the selection marker sequence is not only the introduction of the entire sequence, but also the introduction of a part of the sequence to complete the selection marker sequence by combining this partial sequence with the sequence originally present in the yeast. Is also included. For example, in the case where a yeast strain lacking a part of the originally existing selectable marker sequence is used as a transformation host, any sequence that involves homologous recombination is introduced by introducing the missing partial sequence as a selectable marker sequence. Gene disruption can be performed. Or, when sensitive to drugs such as hygromycin and geneticin (hereinafter also referred to as G418), any gene with homologous recombination is introduced by introducing a gene for imparting resistance to these drugs. Can be destroyed. Therefore, according to one embodiment of the present invention, a yeast strain sensitive to hygromycin B and G418 described above is used as a host, and a gene imparting drug resistance that does not originally exist in the strain is used. It shall destroy the PDC gene. Specific examples of the selection marker include a hygromycin B phosphotransferase gene ( HPT gene, a gene conferring resistance to hygromycin B, which was shown to be usable in the bacterial species in Japanese Patent Application Laid-Open No. 2003-144185. ) And aminoglycoside phosphotransferase ( APT gene, gene conferring resistance to G418).

The position on the chromosome where the gene encoding a polypeptide having lactate dehydrogenase activity is integrated into the yeast genome is not particularly limited, but it encodes a polypeptide having pyruvate decarboxylase activity. It is advantageous to have a locus. Thereby, a gene encoding a polypeptide having lactate dehydrogenase activity can be placed under the control of the full-length promoter of the PDC gene, and thus high expression efficiency can be obtained.

According to one embodiment of the invention, the yeast strain according to the invention comprises an expression comprising a promoter sequence and a DNA sequence encoding a polypeptide having the activity of lactate dehydrogenase under the control of the promoter sequence. It is assumed that it has been transformed with a vector. Moreover, such an expression vector forms one embodiment of the present invention.

Candida utilis has high ploidy and does not form spores. When trying to introduce a mutation into a gene of a highly polyploid strain, it is necessary to add the mutation more severely than in a haploid strain. It is considered that the possibility of mutation is increased. Therefore, when introducing a mutation into the gene of Candida utilis, it is preferable to use a technique that can efficiently add multiple mutations only to the target gene.

As a method for transforming Candida utilis, there is a technique described in JP-A-2003-144185. In this document, as a usable vector, a sequence homologous to the chromosomal DNA of Candida utilis and a selection marker gene are included, and a heterologous gene can be incorporated into the chromosomal DNA of Candida utilis by homologous recombination. A DNA sequence that can be transformed into Candida utilis, or a DNA sequence having an autonomous replication function in Candida utilis and a selectable marker gene, has been developed.

A selectable marker gene that can be used in the Candida utilis transformation system is a drug resistance marker that can function in Candida utilis, preferably a cycloheximide-resistant L41 gene, a gene conferring geneticin (G418) resistance, or hygromycin There are genes that confer B resistance. A gene that confers geneticin (G418) resistance or a gene that confers hygromycin B resistance is a sequence that does not exist in wild yeast, and is therefore considered to have a high probability of being incorporated into a target locus. In addition, in other species of yeast, it is considered that the influence on the traits of the host is small (Baganz F et al., 13 (16): 1563-73., 1997) (Cordero Otero R et al., Appl Microbiol Biotechnol, 46 (2): 143-8., 1996). The gene having these characteristics is considered to be useful in breeding Candida utilis.

Another transformation system is the Cre-loxP system derived from bacteriophage P1. This is a site-specific recombination system between the loxP sequences of the two 34 bp, this recombination is catalyzed by Cre recombinase which Cre gene. This system has also been reported to function in yeast cells such as Saccharomyces cerevisiae, and it is known that a selectable marker gene placed between two loxP sequences is removed by recombination between loxP sequences. (Guldener, U., et al., Nucleic Acids Res., 24, 2519-24., 1996). This system is used in several yeast species other than Candida utilis, such as Kluyveromyces lactis (Steensma, HY et al., Yeast, 18, 469-72., 2001). .

Method for Producing Lactic Acid By culturing the yeast strain according to the present invention in the presence of an appropriate carbon source, lactic acid which is a fermentation product of lactic acid dehydrogenase can be produced in the culture. According to the method for producing lactic acid according to the present invention, lactic acid can be obtained by carrying out the step of separating lactic acid from the culture system.
In the present invention, the culture includes cultured cells or microbial cells, cells or disrupted microbial cells, in addition to the culture supernatant.

In culturing the yeast strain according to the present invention, a culture method and culture conditions can be selected according to the type of yeast. For example, a liquid culture method using a test tube, a flask or a jar fermenter can be mentioned, and a culture format such as batch culture or semi-batch culture can be adopted.
Conditions of an amplitude of 35 mm are suitable for culturing in test tubes and flasks, and such culturing can be performed with a table culture apparatus manufactured by TAITEC.

In the method for producing lactic acid according to the present invention, the composition of the medium is not particularly limited as long as it is a composition containing various nutrients that allow yeast to grow and produce lactic acid. As the assimilating carbon source contained in the medium, for example, xylose or sucrose can be used in addition to glucose as long as it can be assimilated. According to a preferred embodiment of the present invention, glucose or sucrose is used as the carbon source, more preferably glucose.

Moreover, as a nutrient source contained in the medium, for example, yeast extract, peptone, whey and the like are used, but a medium obtained by adding the above assimilable carbon source to YP (10 g / L yeast extract, 20 g / L peptone), For example, YPD (20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone), YPX (20 g / L xylose, 10 g / L yeast extract, 20 g / L peptone), YPSuc10 medium (100 g / L sucrose, 10 g / L It is convenient to use a medium such as yeast extract (20 g / L peptone), which has been further adjusted in pH.

It should be noted that inorganic nitrogen such as ammonium salts such as ammonium sulfate and urea are preferred for a medium that is inexpensive and does not impose a burden on the purification process. Moreover, as an inorganic nutrient source, for example, potassium phosphate, magnesium sulfate, Fe (iron), Mn (manganese) compound, or the like is also used. Further, the culture medium may contain a pH adjuster.

Fermentation temperature can be selected within the range where the lactic acid-producing yeast to be used can grow. The fermentation temperature can be, for example, about 15 to 45 ° C., more preferably 25 to 40 ° C., still more preferably 27 to 40 ° C., and most preferably 35 ° C. Further, the pH of the medium during the fermentation process is preferably maintained at 3 to 8, more preferably pH 4 to 7, most preferably pH 6, and neutralization of lactic acid as a fermentation product may be performed as necessary. it can. Examples of the neutralizing agent to be used include calcium carbonate, sodium hydroxide, potassium hydroxide and the like, and calcium carbonate is preferable.

The reaction time required for the production of lactic acid is not particularly limited, and the reaction is carried out at any reaction time as long as the effect of the present invention is recognized. Those skilled in the art can easily optimize these conditions.

In the production of lactic acid, when growing yeast first, it is preferable to carry out pre-culture and pre-culture, and then carry out lactic acid production by fermentation culture.

As pre-culture conditions, cells grown on a YPD agar medium at 30 ° C. for 1 to 3 days are scraped with a sterilized toothpick to obtain. This is preferably cultured using 3 to 5 mL of YPD liquid medium added to a 15 mL tube under shaking conditions of 120 to 150 rpm.

Pre-culture conditions include 50 to 100 mL of YPD liquid medium, YPX10 liquid medium (100 g / L xylose, 10 g / L yeast extract, 20 g / L peptone) or YPSuc10 liquid medium (100 g / L sucrose, 10 g / L). Yeast extract, 20 g / L peptone), inoculate the pre-cultured cells into a new medium so that the OD600 is about 0.1, and then culture at 120 to 150 rpm, 30 ° C. for usually 16 to 30 hours. The culture is preferably performed until the logarithmic growth phase or stationary phase where OD600 is 10 to 25.

As conditions for fermentation culture, the medium contains glucose, xylose or sucrose at a concentration of 95 to 115 g / L (preferably 100 to 115 g / L), and 3 to 5% of calcium carbonate as a neutralizing agent. For example, YPD10 medium (100 g / L glucose, 10 g / L yeast extract, 20 g / L peptone) containing 3-5% calcium carbonate, YPX10 medium (100 g / L xylose, 10 g / L yeast extract, 20 g / L peptone) Alternatively, it is preferable to use YPSuc10 medium (100 g / L sucrose, 10 g / L yeast extract, 20 g / L peptone) and culture under aeration conditions of 70 to 150 rpm, 15 to 45 ° C., and 10 to 40 mL. More preferably, it is 80-100 rpm, 25-40 ° C., 10-20 mL, and more preferably 80 rpm, 27-40 ° C., 10-15 mL. For fermentation, it is preferable to use a 100 mL Erlenmeyer flask with a baffle, and add 10 to 40 mL of medium and cells. In particular, at this stage, it is preferable to adjust OD600 to 1 to 30 (preferably 1 to 20) as an initial amount of bacterial cells from the viewpoint that lactic acid can be efficiently produced in a shorter time, and more preferably, OD600 is adjusted to 5 to 25, more preferably 5-15, and most preferably OD600 of about 10.

In the production of lactic acid at a medium scale of 500 mL or more, when growing yeast, it is preferable to carry out pre-culture, pre-culture, and pre-culture in a liquid medium, and then carry out lactic acid production by fermentation culture. It is preferable to use a jar fermenter in the test on the scale.

As the pre-culture conditions, the cells grown on the YPD agar medium at 30 ° C. for 1 to 3 days are scraped with a sterilized toothpick. This is preferably cultured under shaking conditions using 3 to 5 mL of YPD liquid medium added to a 15 mL tube at 120 to 150 rpm and 30 ° C. for usually 6 to 30 hours.

The pre-culture conditions are as follows: 50 mL to 100 mL of YPD liquid medium is used as the medium, and the pre-culture cells are inoculated into a new medium so that the OD600 is about 0.1, and then 120 to 150 rpm. The culture is preferably carried out at 30 ° C. for usually 10 to 30 hours, and further to the logarithmic growth phase or stationary phase where OD600 is 10 to 25. In the culture, a Sakaguchi flask is preferably used.

As conditions for the pre-culture, it is preferable to use a jar fermenter capable of adjusting temperature, aeration amount, stirring speed and the like. Use 500 mL to 2.5 L YPD liquid medium as the medium, add 20 to 100 mL of the culture medium in advance, inoculate a new medium so that the OD600 is about 0.1, and then stir at a speed of 300 to 400 rpm. It is preferable to culture at a temperature of 30 ° C. and an aeration rate of 1.25 vvm for usually 10 to 30 hours, and to a logarithmic growth phase or a stationary phase where OD600 is 10 to 25.

As conditions for fermentation culture, it is preferable to use a jar fermenter that can adjust temperature, aeration rate, stirring speed, pH control, and the like. YPD10 medium (100 g / L glucose, 10 g / L yeast extract) containing glucose or sucrose as a medium at a concentration of 50 to 220 g / L, preferably 100 to 115 g / L, and 3 to 5% of calcium carbonate medium as a neutralizing agent. 20 g / L peptone) or YPSuc10 medium (100 g / L sucrose, 10 g / L yeast extract, 20 g / L peptone), or the pH is maintained at a pH suitable for fermentation with a neutralizing agent such as sodium hydroxide or potassium hydroxide. It is preferable to cultivate the YPD10 medium or YPSuc10 medium in a damp state under the conditions of a stirring rate of 100 to 300 rpm, 15 to 45 ° C., and 500 mL to 2.5 L of liquid. More preferably, they are 200 to 250 rpm, 27 to 37 ° C., and 1.5 to 2 L. In particular, at this stage, it is preferable to adjust OD600 to 1 to 30 (preferably 1 to 20) as an initial amount of bacterial cells from the viewpoint that lactic acid can be efficiently produced in a shorter time, and more preferably, OD600 is adjusted to 5 to 15 and most preferably an OD600 of about 10.

Here, the aeration conditions during fermentation are preferably aerobic conditions, particularly microaerobic conditions. Usually, the target lactic acid is produced with high efficiency by culturing for 24 to 48 hours.

In the lactic acid production method according to the present invention, the lactic acid component thus produced is separated and collected from the medium, but the separation and collection method is not particularly limited. In the lactic acid production method according to the present invention, for example, a known method used in a conventional production process by lactic acid fermentation can be used as a means for separating and concentrating lactic acid components. As such known methods, for example, 1) calcium lactate recrystallization method comprising adding lime milk to neutralize, 2) organic solvent extraction method using a solvent such as ether, 3) purified lactic acid with alcohol Examples thereof include an esterification separation method for esterification, 4) a chromatographic separation method using an ion exchange resin, and 5) an electrodialysis method using an ion exchange membrane. Accordingly, the lactic acid component obtained by the method for producing lactic acid according to the present invention may be in the form of not only free lactic acid but also salts such as sodium and potassium, and esters such as methyl ester and ethyl ester.

According to the method for producing lactic acid according to the present invention, the ability to produce lactic acid in yeast Candida utilis having the ability to produce lactic acid can be improved, and as a result, lactic acid can be produced in a high yield in a short time. The lactic acid production method according to the present invention can improve the lactic acid production ability of yeast having lactic acid production ability, regardless of the composition of the medium used. Therefore, according to the lactic acid production method of the present invention, it is possible to achieve an improvement in lactic acid production ability even in a poorly nutrient medium such as a relatively inexpensive synthetic medium, and to reduce the cost of lactic acid production. .

In particular, according to the lactic acid production method of the present invention, ethanol production is inhibited when a microorganism having ethanol production ability, such as yeast introduced with a gene encoding a polypeptide having lactate dehydrogenase activity, is used. In addition, lactic acid can be produced with high yield. Further, by suppressing the production of various organic acids such as D-lactic acid, which is a by-product other than ethanol, lactic acid contained in the medium can be more easily recovered. In other words, the steps required for recovery and purification of lactic acid can be simplified, and the cost required for lactic acid production can be suppressed. Such by-products can be analyzed and evaluated according to known techniques. For example, ethanol can be analyzed and evaluated by gas chromatography (GC) or high performance liquid chromatography (HPLC), various aromatic components such as acetaldehyde can be analyzed by GC, and various organic acids such as pyruvic acid can be analyzed and evaluated by HPLC. . Glucose is quantified by HPLC or Biochemistry Analyzer (hereinafter BA) (Wyeth Japan), L-lactic acid is HPLC or BA, D-lactic acid is combined with HPLC or L-lactic acid and F-kit D-lactic acid / Each can be analyzed and evaluated using L-lactic acid (JK International). It is preferable to remove impurities that may adversely affect the analysis, such as clogging the column, by filtering the sample to be subjected to various analyzes, for example, by filtering with a 0.22 μm filter.

Note that pyruvic acid and various organic acids such as citric acid, malic acid, and succinic acid in the culture solution were measured by organic acid analysis by HPLC (detection by electrical conductivity). In addition, methods for measuring other substances such as ethanol are described in the following examples.

The present specification further relates to a yeast strain of Candida utilis in which an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity is disrupted, wherein the lactate dehydrogenase activity is increased. It has been found that pyruvic acid is produced in large quantities by culturing a yeast strain into which a gene encoding the polypeptide it has is not introduced.

Thus, according to another aspect of the present invention, there is provided a Candida utilis yeast strain in which an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity is disrupted, and There is provided a method for producing pyruvic acid comprising culturing the yeast strain. Since pyruvic acid has high reactivity and is used as a synthetic substrate for pharmaceuticals, agricultural chemicals, etc., it is regarded as an important intermediate in the fine chemical field.

The endogenous gene encoding the polypeptide having pyruvate decarboxylase activity in the yeast strain of Candida utilis and the details of the disruption are as described above. As a method for purifying pyruvic acid, any method known as a method for purifying organic compounds may be used. For example, a method by distillation described in JP-A No. 2007-169244 may be used. Distillation can be performed, for example, under the reduced pressure condition or vacuum condition of 70 to 80 ° C. as the first distillation and under the reduced pressure condition or vacuum condition of 90 to 100 ° C. as the second distillation. The pyruvic acid obtained by distillation can be separated from a product such as lactic acid and recovered by appropriately performing treatment with activated carbon, dehydration, deacetic acid and the like. The pyruvic acid thus purified can be used in the fine chemical field as described above.

Specific examples of the present invention will be described below, but this does not impose any restrictions on the technical scope of the present invention.

For gene amplification by PCR, Ex Taq manufactured by TaKaRa or KOD-Plus- manufactured by Toyobo was used unless otherwise specified, and the method followed the attached protocol.
In the PCR amplification reaction, after heat treatment at 94 ° C. for 1 minute, denaturation step: 94 ° C. for 30 seconds, annealing step: X ° C. for 30 seconds (X ° C. is the Tm value of the primer. ), Extension process: Y cycle at 72 ° C. (however, Y second is calculated as about 60 seconds per kilobase pair) from the expected size of the amplified product). The temperature was 4 ° C. As a PCR amplification apparatus, GeneAmp PCR System 9700 (PE Applied Biosystems) was used. Gentoru-kun manufactured by TaKaRa or the potassium acetate method (Methods Enzymol., 65, 404, 1980) was used for extraction of genomic DNA from yeast.
Alkaline Phosphatase ( E. coli C75) manufactured by TaKaRa or Alkaline Phosphatase (Shripmp) manufactured by TaKaRa was used for the dephosphorylation of DNA, and the Ligation Kit ver. 2 and the method followed the attached protocol. Competent cells of DH5α (TOYOBO) were used for transformation of E. coli, and the method followed the attached protocol. For selection of E. coli transformants, an LB plate containing 100 μg / mL of ampicillin (LB + amp plate) or an LB plate containing 50 μg / mL of kanamycin is used according to the drug resistance marker gene contained in the plasmid. Blue-white selection with 20 μg / mLX-gal and 0.1 mM IPTG was performed. For recovering plasmid DNA from E. coli, QIAprep Spin Miniprep Kit manufactured by QIAGEN was used, and the method followed the attached protocol. Transformation of Saccharomyces cerevisiae was performed by the lithium method (Ito et al., J. Bacteriol., 153, 163, 1983). Transformation of Candida utilis was performed by partially modifying the method described in JP-A No. 2003-144185. The base sequence was determined by the following method. PCR was performed using BigDye Terminator v3.1 manufactured by Applied Biosystems, and the method followed the attached protocol. For removal of unreacted BigDye Terminator, CENTRI-SEP COLUMNS (PRINCETON SEPARATIONS) was used, and the method followed the attached protocol. For determination of the base sequence, 3100 Genetic Analyzer manufactured by Applied Biosystems was used, and the method followed the attached protocol. Regarding the notation of degenerate primers described in the sequence listing, “W” is from “A (adenine)” and “T (thymine)”, and “R” is “A (adenine)” and “G ( "Guanine)" and "Y" indicate that "C (cytosine)" and "T (thymine)" and "M" consist of a mixture of "A (adenine)" and "C (cytosine)", respectively. In addition, various numerical values, such as the lactic acid production amount in a table | surface, were shown by the average value +/- standard error.

Transformation of the Candida utilis strain by electric pulse was performed by partially modifying the method described in Japanese Patent Application Laid-Open No. 2003-144185. The colonies on the YPD plate are cultured with shaking in 5 ml of YPD liquid medium at 30 ° C. for about 8 hours, then inoculated into 200 ml of YPD liquid medium so that OD600 is 0.0024, and cultured at 30 ° C. with shaking. After about 16 hours, the cells grow to the logarithmic growth phase (OD600 = 2.5), and then are collected by centrifugation at 1,400 × g for 5 minutes. The cells are washed once with 100 ml of ice-cooled sterilized water, then once with 40 ml of ice-cold sterilized water, and then once with 40 ml of ice-cooled 1M sorbitol. The cells are suspended in 10 ml of 1M sorbitol, transferred to a sterile polypropylene tube, and collected again by centrifugation at 1,100 × g for 5 minutes. After removing the supernatant, the suspension is suspended in ice-cooled 1 M sorbitol so that the final cell volume is 2.5 ml.

Transformation experiments with electric pulses are performed using a Bio-Rad gene pulser. 50 μl of the bacterial solution, 5 μl of DNA sample containing 100 ng to 10 μg of DNA, and 5 μl of 2.0 mg / ml salmon testis-derived carrier DNA were mixed, and then placed in a 0.2 cm disposable cuvette. Apply electrical pulses. For example, according to a preferred embodiment of the present invention, the electric capacity is 25 μF, the resistance value is 600 to 1000 ohms, and the voltage is 0.75 to 5 KV / cm. After the pulse, 1 ml of ice-cooled YPD medium containing 1 M sorbitol is added, transferred to a sterile polypropylene tube, and cultured with shaking at 30 ° C. for about 6 to 15 hours. After culturing, the bacterial solution was applied to a YPD selection medium containing an appropriate drug according to the selection marker gene, and then the plate was incubated at 28-30 ° C. for 3-4 days to obtain transformant colonies. When the HPT gene was used as a selection marker gene, hygromycin B was added to the YPD medium at a concentration of 600 to 800 μg / ml. When the APT gene was used as a selection marker gene, G418 was added to the YPD medium at a concentration of 200 μg / ml. Hereinafter, each medium is referred to as a HygB medium and a G418 medium. In addition, the resistance to hygromycin B is expressed as HygBr, the sensitivity to hygromycin B is expressed as HygBs, the resistance to G418 is expressed as G418r, and the sensitivity to G418 is expressed as G418s.

Example 1: Development of Candida utilis transformation system using Cre-loxP system
1-1. Construction of Plasmid Required for Multiple Transformation System Using Cre-lox System Plasmid pCU563 for preparing a DNA fragment for gene disruption was constructed by the following procedure. Using the plasmid pGKHPT1 having the PGK gene promoter and hygromycin resistance gene HPT gene described in Shimada et al. (Appl. Environ. Microbiol. 64, 2676-2680) as templates, IM-53 (SEQ ID NO: 16) and IM-57 By performing PCR (elongation reaction 1.5 minutes) with the primer set of (SEQ ID NO: 17), a DNA fragment consisting of loxP (SEQ ID NO: 18), PGK gene promoter, and HPT gene in this order was amplified. In addition, PCR (extension reaction 30 seconds) was performed using primer sets of IM-54 (SEQ ID NO: 19) and IM-55 (SEQ ID NO: 20) using pGAPPT10 (Kondo et al., Nat. Biotechnol. 15, 453-457) as a template. Was performed to amplify a DNA fragment consisting of the GAP gene terminator and loxP.
By mixing these and performing PCR (extension reaction 2 minutes) with IM-1 (SEQ ID NO: 21) and IM-2 (SEQ ID NO: 22), loxP, PGK gene promoter, HPT gene, GAP gene terminator, loxP A DNA fragment consisting of was amplified. The obtained DNA fragment was cloned into a pCR2.1 vector [Invitrogen: TA cloning kit (pCR2.1 vector)]. The plasmid thus obtained was named pCU563 (FIG. 2). Candida utilis cells into which this module has been incorporated by transformation can grow in a medium containing HygB at a concentration of 600 to 800 μg / ml, which cannot grow in a wild strain, for example.

The expression plasmid pCU595 for Cre recombinase was constructed by the following procedure. S. (1) IM-49 (SEQ ID NO: 23) and IM-50 (SEQ ID NO: 23) using as a template the plasmid pSH65 (Gueldener, U., et al., Nucleic Acids Res. 30 (6), E23, 2002) for expressing Cre in cerevisiae 24), (2) PCR was carried out with two types of primer sets, IM-51 (SEQ ID NO: 25) and IM-52 (SEQ ID NO: 26) (both extended for 30 seconds). Each amplified DNA fragment was mixed and then subjected to PCR using IM-49 (SEQ ID NO: 23) and IM-52 (SEQ ID NO: 26) to amplify the gene fragment encoding Cre recombinase. The Cre gene was thus, Bam HI recognition sequence present in the Cre gene pSH65 (GGATCC) is, without changing the amino acid sequence, which is arranged and (GCATAC) that the enzyme does not recognize. Further, a DNA fragment obtained by digesting this with Xba I and Bam HI was inserted into the Xba I- Bam HI gap of pPMAPPT1 (Japanese Patent Laid-Open No. 2003-144185). A Cre expression module obtained by treating this plasmid with Not I, that is, a DNA fragment comprising a PMA gene promoter, a Cre gene, and a PMA gene terminator in this order, pCARS7 (Japanese Patent Laid-Open No. 2003-144185) having an autonomously replicating sequence CuARS2 It was inserted into DNA partially digested with Not I. The plasmid thus obtained was named pCU595 (FIG. 3). This plasmid has an APT gene, and when Candida utilis is transformed with this plasmid, cells into which the plasmid has been introduced grow, for example, in a medium containing G418 at a concentration of 200 μg / ml that cannot be grown in a wild strain. It becomes possible.

1-2. Multiple disruption of CuURA3 gene using Cre-lox system In order to investigate whether the Cre-loxP system functions , multiple disruption of Candida utilis URA3 gene (hereinafter referred to as CuURA3 gene) described in JP-A-2003-144185 Tried. The gene encodes orotidine-5′-phosphate decarboxylase, and a strain in which all the functional gene in the cell is lost becomes uracil-requiring. That is, it is thought that it cannot grow on a medium not containing uracil.

A DNA fragment for disrupting the first and second copies of the CuURA3 gene was prepared as follows. First, two types of PCR shown in the following (1), (2) and (3) were performed: (1) pCU563 was used as a template, and IM-1 (SEQ ID NO: 21) and IM-2 (primers were used as primers. (SEQ ID NO: 22), and the extension reaction time was 2 minutes; (2) NBRC0988 strain genomic DNA was used as a template, and primers IM-59 (SEQ ID NO: 54) and IM-60 (SEQ ID NO: 55) were used. The extension reaction time was 30 seconds; (3) NBRC0988 strain genomic DNA was used as a template, IM-61 (SEQ ID NO: 56) and IM-62 (SEQ ID NO: 57) were used as primers, and the extension reaction time was 30 seconds. It was. In (2) and (3), the upstream part and the downstream part of the CuURA3 gene are amplified. Further, PCR of the following (4) was carried out: (4) Using a mixture of the three kinds of DNA amplified in the above (1), (2) and (3) as a template, IM-59 (sequence) as a primer No. 54) and IM-62 (SEQ ID NO: 57) were used, and the extension reaction time was 3 minutes. Thereby, a DNA fragment consisting of the upstream region of the CuURA3 gene, the loxP, the PGK gene promoter, the HPT gene, the GAP gene terminator, the loxP, and the downstream region of the CuURA3 gene in this order was obtained. Hereinafter, this DNA fragment is referred to as “ CuURA3 disruption first / second fragment”.
If the transformation using this DNA fragment, by occurring double stranded homologous recombination upstream region and downstream region of CuURA3 gene, it is possible to partially deleted allele of CuURA3 gene.

The NBRC0988 strain was transformed with 1 μg of the first and second fragments of CuURA3 disrupted as a DNA fragment. As a result, 119 clones of HygBr transformants were obtained. Genomic DNA was extracted from transformants of 11 clones arbitrarily selected from the NBRC0988 strain and 119 clones, and PCR was performed with IM-63 (SEQ ID NO: 58) and IM-92 (SEQ ID NO: 59) using this as a template ( Elongation reaction 3.5 minutes). As shown in FIG. 4, these primers anneal outside the homologous recombination region. When subjected to 0.8% agarose gel electrophoresis, DNA fragments of 2.3 kb and 2.3 kb were amplified in the NBRC0988 strain with 2.3 kb and in all 11 clones (FIG. 5). From this, it was found that the desired HygBr CuURA3 gene 1-copy disrupted strain was obtained. It was also revealed that there was a high probability that positive clones could be selected from the transformants.

Transformation of one copy disrupted strain of HygBr with CuURA3 gene was performed with Cre expression plasmid pCU595. In the medium containing G418, colonies were not formed in the negative control to which DNA was not added, whereas in the sample to which pCU595 was added, 1,000 or more transformants were obtained. As a result of applying 30 strains of these to G418 medium or HygB medium, all of them were able to grow on G418 medium, but could not grow on HygB medium.

Genomic DNA was extracted from HygBr's CuURA3 gene 1-copy disrupted strain and HygBs' 1st-copy disrupted strain of CuURA3 gene, and this was used as a template for PCR with IM-63 (SEQ ID NO: 58) and IM-92 (SEQ ID NO: 59). (Elongation reaction 3.5 minutes). When subjected to 0.8% agarose gel electrophoresis, DNA fragments of 3.2 kb and 2.3 kb were amplified in the former strain, and 2.3 kb and 1.1 kb in the latter strain. A strain from which the HPT gene was removed as intended was obtained.

HydBs CuURA3 gene 1-copy disrupted strain was cultured overnight in YPD liquid medium, and a portion thereof was applied to YPD medium. After 1 to 3 days, a plurality of single colonies were separated and applied to G418 medium and YPD medium. As a result, most clones grew on the YPD medium but did not grow on the G418 medium.

From the NBRC0988 strain, the Hygr and G418s strain in which one copy of the CuURA3 gene derived from the NBRC0988 strain was disrupted, the Hygs and G418r strain constructed by introducing the Cre expression plasmid, and the Hygs and G418s strain from which the Cre expression plasmid was dropped The results of PCR using the extracted DNA as a template and IM-63 (SEQ ID NO: 58) and IM-92 (SEQ ID NO: 59) as primers are shown in FIG. 5 (extension reaction 3.5 minutes). This corresponds to Lane 1, Lane 2, Lane 3, and Lane 4 in this order.

From the NBRC0988 strain, the Hygr and G418s strain in which one copy of the CuURA3 gene derived from the NBRC0988 strain was disrupted, the Hygs and G418r strain constructed by introducing the Cre expression plasmid, and the Hygs and G418s strain from which the Cre expression plasmid was dropped The results of PCR using the extracted DNA as a template and IM-63 (SEQ ID NO: 58) and IM-223 (SEQ ID NO: 60) as plasmids are shown in FIG. 6 (extension reaction 2 minutes).
This corresponds to Lane 1, Lane 2, Lane 3, and Lane 4 in this order. As shown in FIG. 4, IM-63 (SEQ ID NO: 58) anneals outside the homologous recombination region, and IM-223 (SEQ ID NO: 60) anneals inside the HPT gene. Since the 1.4 kb DNA fragment was amplified only in lane 2 using the Hygr strain as a template, the Cre-loxP system was similar to the results of IM-63 (SEQ ID NO: 58) and IM-92 (SEQ ID NO: 59). Was found to work with Candida utilis.

A primer set for amplifying the Cre gene by extracting genomic DNA from the G418r and G418s strains, which have different growth potentials in a medium containing G418, although one copy of the HyURABs CuURA3 gene has been destroyed. PCR was performed with certain IM-49 (SEQ ID NO: 23) and IM-52 (SEQ ID NO: 26) (extension reaction: 1 minute). As a result, a 1 kb DNA fragment was amplified in the G418r strain, but not amplified in the G418s strain. From this, it was confirmed that pCU595 was dropped and a disrupted strain of HygBs and G418s in the first copy of CuURA3 gene was obtained.

Using the CuURA3 disruption 1st and 2nd fragment as a DNA fragment, a disrupted strain of HygBs and G418s with 1 copy of the CuURA3 gene was transformed. Genomic DNA was extracted from the obtained transformant, and PCR was carried out with IM-63 (SEQ ID NO: 58) and IM-92 (SEQ ID NO: 59) using this as a template (extension reaction 3.5 minutes). When subjected to 0.8% agarose gel electrophoresis, there were several strains in which three types of DNA fragments of 3.2 kb, 2.3 kb, and 1.1 kb were amplified. From this, it was found that the target HygBr disrupted strain of CuURA3 gene 2 copies was obtained.

Transformation of HygBr's CuURA3 gene 2-copy disrupted strain was carried out with pCU595, a Cre expression plasmid. When the obtained transformants were applied to G418 medium and HygB medium, they could all grow on G418 medium, but could not grow on HygB medium. Using genomic DNA extracted from this HygBs and G418r transformant as a template, PCR was performed with IM-63 (SEQ ID NO: 58) and IM-92 (SEQ ID NO: 59) (extension reaction: 3.5 minutes). When subjected to 0.8% agarose gel electrophoresis, two kinds of DNA fragments of 2.3 kb and 1.1 kb were amplified. It was found that the HPT gene could be removed.

HygBs CuURA3 gene 2-copy disrupted strain was cultured overnight in YPD liquid medium, and a portion thereof was applied to YPD medium. Two days later, single colonies were isolated and spread on YPD medium and G418 medium. Then, clones that grew on the YPD medium but did not grow on the G418 medium were isolated. A strain from which pCU595 was eliminated, that is, a disrupted strain of HygBs and G418s in the second copy of the CuURA3 gene could be obtained.

A DNA fragment for disrupting the 3rd and 4th copies of the CuURA3 gene was prepared as follows. First, three types of PCR shown in the following (1), (2) and (3) were performed: (1) pCU563 was used as a template, IM-1 (SEQ ID NO: 21) and IM-2 (primers were used as primers. (SEQ ID NO: 22) and the extension reaction time was 2 minutes; (2) NBRC0988 strain genomic DNA was used as a template, and primers were used with IM-295 (SEQ ID NO: 61) and IM-296 (SEQ ID NO: 62). The extension reaction time was 30 seconds; (3) NBRC0988 strain genomic DNA was used as a template, IM-61 (SEQ ID NO: 56) and IM-62 (SEQ ID NO: 57) were used as primers, and the extension reaction time was 30 seconds. It was. In (2) and (3), the upstream part and the downstream part of the CuURA3 gene are amplified. Furthermore, PCR of the following (4) was carried out: (4) Using a mixture of the three types of DNA amplified in the above (1), (2) and (3) as a template, IM-295 (sequence) as a primer No. 61) and IM-62 (SEQ ID NO: 57) were used, and the extension reaction time was 3 minutes. Thereby, a DNA fragment consisting of the upstream region of the CuURA3 gene, the loxP, the PGK gene promoter, the HPT gene, the GAP gene terminator, the loxP, and the downstream region of the CuURA3 gene in this order was obtained. Hereinafter, this DNA fragment is referred to as “ CuURA3 disrupted third and fourth fragment”. If the transformation using this DNA fragment, by occurring double stranded homologous recombination upstream region and downstream region of CuURA3 gene, it is possible to partially deleted allele of CuURA3 gene. Further, the upstream region of CuURA3 gene amplified by (3), the deleted regions in the transformation for one copy first and two copies th CuURA3 gene disruption was performed using the CuURA3 destruction 1-second fragment Therefore, it is considered that the possibility of being incorporated into two copies of the destroyed allele can be reduced.

Using the CuURA3 disrupted 3rd and 4th fragment as a DNA fragment, the CuURA3 gene 2 copy disrupted strain of HygBs and G418s was transformed for the 3rd copy of the CuURA3 gene disruption. Genomic DNA was extracted from the obtained transformant, and PCR was carried out with IM-63 (SEQ ID NO: 58) and IM-92 (SEQ ID NO: 59) using this as a template (extension reaction 3.5 minutes). When subjected to 0.8% agarose gel electrophoresis, there were strains in which three types of DNA fragments of 3.6 kb, 2.3 kb, and 1.1 kb were amplified. From this, it was found that the disrupted strain of the target HygBr in the third copy of the CuURA3 gene was obtained.

Transformation of the HyURA Br CuURA3 gene 3-copy disruption strain was performed with Cre expression plasmid pCU595. As a result of applying the obtained transformant to G418 medium and HygB medium, all were able to grow on G418 medium, but were not able to grow on HygB medium. Using genomic DNA extracted from this HygBs and G418r transformant as a template, PCR was performed with IM-63 (SEQ ID NO: 58) and IM-92 (SEQ ID NO: 59) (extension reaction: 3.5 minutes). When subjected to 0.8% agarose gel electrophoresis, three types of DNA fragments of 2.3 kb, 1.5 kb, and 1.1 kb were amplified. It was found that the HPT gene could be removed.

HygBs CuURA3 gene 3-copy disrupted strain was cultured overnight in YPD liquid medium, and a portion thereof was applied to YPD medium. Two days later, single colonies were isolated and spread on YPD medium and G418 medium. Then, clones that grew on the YPD medium but did not grow on the G418 medium were isolated. A strain in which pCU595 was eliminated, that is, a strain in which the third copy of the CuURA3 gene of HygBs and G418s was disrupted was obtained.

Using CuURA3 fracture 3-fourth fragment as DNA fragment, was transformed HygBs and G418s of CuURA3 gene 3 copies disrupted strain. Genomic DNA was extracted from the obtained transformant, and PCR was performed using this as a template with IM-63 (SEQ ID NO: 58) and IM-92 (SEQ ID NO: 59) (extension reaction: 3.5 minutes). When subjected to 0.8% agarose gel electrophoresis, three types of DNA fragments of 3.6 kb, 1.5 kb and 1.1 kb were amplified in a plurality of transformants. From this, it was found that the desired HygBr CuURA3 gene 4 copy disruption strain was obtained. Also, DNA fragments of 2.3kb was observed in a wild strain NBRC0988 strain, that is, from the DNA fragment allele not destroyed is amplified is not detected, this strain when is CuURA3 gene completely disrupted strain it was thought.

Transformation of the HygBr CuURA3 gene 4 copy disruption strain was carried out with pCU595, a plasmid for Cre expression. As a result of applying the obtained transformant to G418 medium and HygB medium, all were able to grow on G418 medium, but were not able to grow on HygB medium. Using genomic DNA extracted from this HygBs and G418r transformant as a template, PCR was performed with IM-63 (SEQ ID NO: 58) and IM-92 (SEQ ID NO: 59) (extension reaction: 3.5 minutes). When subjected to 0.8% agarose gel electrophoresis, there were several strains in which two kinds of DNA fragments of 1.5 kb and 1.1 kb were amplified. It was found that the HPT gene could be removed.

HygBs CuURA3 gene 4 copy-disrupted strain was cultured overnight in YPD liquid medium, and a part thereof was applied to YPD medium. Two days later, single colonies were isolated and spread on YPD medium and G418 medium. Then, clones that grew on the YPD medium but did not grow on the G418 medium were isolated. A strain in which pCU595 was eliminated, that is, a disrupted strain of HygBs and G418s at the fourth copy of CuURA3 gene could be obtained.

SC medium, SC-Ura medium (medium not containing uracil), which is a non-selective medium of a strain in which the CuURA3 gene is disrupted (HygBs and G418s from which the HPT gene and the APT gene have been removed) using the NBRC0988 strain and the NBRC0988 strain as hosts And the growth ability in 5-FOA medium was examined. In addition, these culture medium compositions followed what was described in Methods In Yeast Genetics 1997 Edition (Cold Spring Harbor Laboratory Press). As shown in FIG. 7, only 4 copies of the CuURA3 gene, that is, all the disrupted strains, were different from the other 4 strains including the NBRC0988 strain, and could not grow on the SC-Ura medium, but could grow on the 5-FOA medium.

Using the recombinant DNA technology, the entire allele of the CuURA3 gene was disrupted and a uracil-requiring strain was successfully obtained. This is the first report of Candida utilis showing that there are four alleles in the cell. The Candida utilis transformation system using the Cre-loxP system, which enabled efficient destruction of the target gene while repeatedly using the selectable marker gene, was considered significant.

Example 2: Construction of a gene-disrupted strain encoding PDC
2-1. Cloning of a gene encoding PDC Primers IKSM-29 (SEQ ID NO: 1) and IKSM-30 (SEQ ID NO: 2) for amplifying a C-terminal base sequence having a lot of common sequences in ScPDC1 gene and KlPDC1 gene were prepared, and NBRC0988 PCR was performed using the strain genome as a template (elongation time 30 seconds). When the sequence of the amplified DNA fragment of about 220 bp (base pair) (hereinafter referred to as CuP-Fg) was decoded (SEQ ID NO: 3), it was found to be highly homologous to the ScPDC1 gene. From this, this DNA fragment was considered to be a part of the gene encoding PDC.

Southern analysis was performed using this DNA fragment as a probe. First, genomic DNA extracted from Saccharomyces cerevisiae S288C strain (NBRC1136 strain) was digested with Hind III, and genomic DNA extracted from Candida utilis NBRC0988 was digested with Xba I, Hind III, Bgl II, Eco RI, Bam HI, Digested with Pst I and subjected to 0.8% agarose gel electrophoresis. The separated genomic DNA was transferred to Hybond N + nylon membrane manufactured by Amersham Biosciences according to a conventional method. Radiolabeling of the probe was performed using Random Primer DNA Labeling Kit Ver. The method followed the attached protocol. As the labeled dCTP, 1.85 MBq of [α-32P] dCTP manufactured by Amersham Biosciences was used. Hybridization was performed using Rapid-Hyb buffer according to the attached protocol. However, the hybridization temperature was 60 ° C. The result is shown in FIG.

In Saccharomyces cerevisiae, three bands thought to be derived from three types of PDC genes ( ScPDC1 gene, ScPDC5 gene, ScPDC6 gene) were detected (lane 1). In contrast, in the Candida utilis NBRC0988 strain, Bgl II (lane 4) there is a restriction enzyme recognition site in the probe, and the sample except Eco RI (lane 5) was detected only one band. This suggested that there was one kind of gene having PDC activity in NBRC0988. Although there is no Eco RI recognition sequence in the probe, there is a locus where the Eco RI recognition sequence exists and a heterogeneous region where there is no Eco RI recognition sequence between alleles of homologous chromosomes in the vicinity of the region where the probe hybridizes. The possibility that it existed was considered.

As genomic library to be used for colony hybridization, a pBR322 was dephosphorylated after digestion with Bam HI (Nippon Gene), the reactions to link fragments of 5 ~ 10 kb of the partial digested DNA with Sau 3AI went. With this solution, 50,000 clones grown on LB + Amp agar were obtained. In addition, less than 5% of the clones were self-closed.

Further, a plurality of clones containing a site homologous to the probe sequence were obtained by the colony hybridization method using the aforementioned DNA fragment CuP-Fg as a probe. When the sequences of these clones were decoded by the primer walking method, one type of contig was completed (SEQ ID NO: 63). When this was subjected to BLAST search of SGD (Saccharomyces Genome Database), it was highly homologous to the ScPDC1 gene and the ScPDC5 gene, so this gene was named CuPDC1 gene. When this DNA fragment was subjected to homology search by NCBI (National Center for Biotechnology Information) BLAST, high homology was recognized with genes encoding polypeptides having pyruvate decarboxylase activity of various yeasts. Further by utilizing the database of NCBI, by performing a homology search of the PDC gene of the present sequence and other species, we try to control certain ORF (Open Reading Frame) region, ORF of CuPDC1 gene 1,692nt (Nucleotides) (SEQ ID NO: 63). The sequence of the gene was 76% homologous to the ScPDC1 gene at the amino acid level, and was considered to have a high possibility of having PDC activity. In addition, it was considered that the sequence 2,246 bases in the upstream region of the gene ORF region described in SEQ ID NO: 63 correspond to the promoter region of the CuPDC1 gene, and the 1076 bases in the downstream region correspond to the terminator region of the CuPDC1 gene. The sequences reported here were all contained in the plasmid pCU530 obtained by the colony hybridization method.

The presence or absence of a cis element involved in transcriptional control was examined in the 1.2 kb promoter region upstream of the ORF. ORF transcription start ATG was positioned with the base immediately before ATG as “−1”. As a result, there was a sequence considered to be a TATA box composed of TATATAA in the vicinity of -149. UAS-PDC sequence (GCACCACTACCTT) (Butler G. et al., Curr Genet., 14 (5): 405), which is considered necessary for the transcriptional activation of the PDC genes of Saccharomyces cerevisiae and Kluyveromyces lactis 12., 1988) at least three sequences having high homology were found (near -1,084, -998, and -812). Furthermore, there are at least 4 Gcr1 binding domains that do not overlap with them (near -1,538, -556, -518, -430), and at least 5 CAAT sequences (near -1,224, -1,116). Near, -979, -658, -556)).

Southern hybridization was performed on samples digested with Hind III of genomic DNA extracted from Candida utilis strains such as NBRC0626, NBRC0639, and NBRC1086 using the DNA fragment CuP-Fg. A band was detected. Moreover, in the nucleotide sequence of the CuPDC1 gene, a single nucleotide polymorphism was found among the NBRC0626 strain, the NBRC0988 strain, and the NBRC1086 strain.

In order to analyze the function of the CuPDC1 gene, it was examined whether the lethality of the double disruption of the ScPDC1 gene and the ScPDC5 gene in Saccharomyces cerevisiae can be suppressed by expressing the CuPDC1 gene with the ScPDC1 gene promoter. Therefore, first, BY4741 origin of the yeast-all gene-disrupted strain series (ura3 gene and his3 gene mutant strain) of ScPDC1 gene disrupted strain and (Invitrogen), ScPDC5 gene disrupted strain (Open from BY4742 (ura3 gene and his3 gene mutant strain) BioSystems) hybrid strain SGY107 was constructed.

Plasmid pCU546 for expressing the ScPDC1 gene in Saccharomyces cerevisiae was constructed as follows. A centromeric plasmid pRS316 (Sikorski, R et al., Genetics. 122, 19-27.1989) (having the URA3 gene) that functions in Saccharomyces cerevisiae was cleaved with Cla I and Bam HI. Using BY4741 (Invitrogen) as a template, PCR was performed with a primer set of IM-135 (SEQ ID NO: 4) and IM-136 (SEQ ID NO: 5) (extension time: 3 minutes). This amplified fragment was digested with Cla I and Bam HI. Plasmid pCU546 was constructed by ligating this DNA fragment with the plasmid fragment previously treated with the restriction enzyme.

Next, the SGY107 strain was transformed with pCU546. This strain was transferred to a spore-forming agar medium (0.5 g / L glucose, 1 g / L Yeast Extract, 10 g / L potassium acetate, 20 g / L agarose) and allowed to stand at 25 ° C. for 3 days. The genomic DNA extracted from the obtained spore was used as a template and subjected to the following two types of PCR (extension time: 2 minutes): (1) IM-19 (SEQ ID NO: 6) and IM-331 (SEQ ID NO: 7) (About 1.5 kb DNA fragment is amplified only in the strain in which the ScPDC1 gene is disrupted in this combination); (2) IM-20 (SEQ ID NO: 8) and IM-334 (SEQ ID NO: 9) (In this combination, a DNA fragment of about 1.5 kb is amplified only in a strain in which the ScPDC5 gene is disrupted). The SGY116 strain was obtained in which DNA fragments were amplified with both primer sets and pCU546 was retained.

A hybrid strain of SGY116 strain and BY4742-derived ScPDC6 gene disruption strain (Open BioSystems) was constructed. It was transferred to a sporulation medium and allowed to stand at 25 ° C. for 3 days. The genomic DNA extracted from the obtained spore was used as a template for the following three types of PCR (extension time 2 minutes): (1) of IM-19 (SEQ ID NO: 6) and IM-331 (SEQ ID NO: 7) Primer set; (2) Primer set of IM-20 (SEQ ID NO: 8) and IM-334 (SEQ ID NO: 9); (3) Primer set of IM-339 (SEQ ID NO: 10) and IM-340 (SEQ ID NO: 11) (DNA fragment amplified from strain ScPDC6 gene is destroyed by this combination, larger DNA fragment ScPDC6 gene is amplified by the strain that has not been destroyed (approximately 3.4 kb)). As a result, in the PCRs (1) and (2), a DNA fragment of about 1.5 kb was amplified, and in the PCR of (3), a DNA fragment larger than 3.4 kb was amplified. That is, the SGY389 strain in which all of the ScPDC1 gene, ScPDC5 gene, and ScPDC6 gene were destroyed and pCU546 was retained was obtained.

Plasmid pCU655 for expressing the CuPDC1 gene in Saccharomyces cerevisiae was constructed as follows. First, the following PCRs (1), (2) and (3) were performed: (1) IM-135 (SEQ ID NO: 4) and IM-147 (SEQ ID NO: 12) using BY4741 genomic DNA as a template (2) Using the BY4741 strain genomic DNA as a template, IM-150 (SEQ ID NO: 13) and IM-136 (SEQ ID NO: 5) as primers (extension reaction 1 minute) (3) IM-148 (SEQ ID NO: 14) and IM-149 (SEQ ID NO: 15) were used as primers with pCU530 having a putative ORF region of the CuPDC1 gene (extension reaction 2 minutes); Next, PCR was performed using the DNA fragments amplified in (1), (2) and (3) as templates and IM-135 (SEQ ID NO: 4) and IM-136 (SEQ ID NO: 5) as primers. Consequently, in order ScPDC1 gene, to obtain DNA fragments consisting predicted ORF region of CuPDC1 gene, and ScPDC1 gene terminator region. All PCRs were performed using KOD-Plus-. A DNA fragment obtained by digesting this fragment with Sma I from a centromeric type plasmid pRS313 (Sikorski, R. et al., Genetics. 122, 19-27.1989 ) (having the HIS3 gene) that functions in Saccharomyces cerevisiae. And linked. The resulting plasmid was named pCU655.

The SGY389 strain in which all of the ScPDC1 gene, ScPDC5 gene, and ScPDC6 gene were disrupted was transformed with pRS313 or pCU655 to obtain SGY393 strain and SGY392 strain, respectively.

In the 5-FOA medium, the BY4741 strain, the BY4742 strain, the ScPDC1 gene disruption strain derived from the BY4741 strain, the ScPDC5 gene disruption strain derived from the BY4742 strain, and the ScPDC6 gene disruption strain derived from the BY4742 strain were able to grow. That is, these strains are uracil-requiring strains. Next, the SGY389 strain in which all of the ScPDC1 gene, the ScPDC5 gene, and the ScPDC6 gene were disrupted, and pRS313 was introduced into the SGY389 strain, which retained pCU546, and the SGY393 strain in which pRS313 was introduced could not grow in a 5-FOA medium. This indicates that pCU546 expressing the ScPDC1 gene cannot be removed. That is, it is shown that expression of a gene encoding a polypeptide having PDC activity is essential for growth in a 5-FOA medium. On the other hand, the SGY392 strain (a strain that expresses the CuPDC1 gene) obtained by introducing pCU655 into the SGY389 strain was able to grow on a 5-FOA medium. This indicates that the CuPDC1 gene contained in pCU655 retains the PDC function of the ScPDC1 gene contained in pCU547 . Therefore, it was suggested that the polypeptide encoded by the CuPDC1 gene is PDC.

2-2. Preparation of a DNA fragment for disrupting the first and second copies of the CuPDC1 gene using the Cre-lox system was performed as follows. First, three types of PCR shown in the following (1), (2) and (3) were performed: (1) pCU563 was used as a template, and IM-1 (SEQ ID NO: 21) and IM-2 (primers were used as primers. (SEQ ID NO: 22) and the extension reaction time was 2 minutes; (2) NBRC0988 strain genomic DNA was used as a template, and primers IM-277 (SEQ ID NO: 27) and IM-278 (SEQ ID NO: 28) were used. The extension reaction time was 30 seconds; (3) NBRC0988 strain genomic DNA was used as a template, IM-279 (SEQ ID NO: 29) and IM-280 (SEQ ID NO: 30) were used as primers, and the extension reaction time was 30 seconds. It was. In (2) and (3), the upstream part and the downstream part of the CuPDC1 gene are amplified. Furthermore, PCR of the following (4) was performed: (4) Using a mixture of the three types of DNA amplified in the above (1), (2) and (3) as a template, and using IM-277 ( SEQ ID NO: 27) and IM-280 (SEQ ID NO: 30) were used, and the extension reaction time was 3 minutes. Thereby, a DNA fragment consisting of the upstream region of the CuPDC1 gene, the loxP, the PGK gene promoter, the HPT gene, the GAP gene terminator, the loxP, and the downstream region of the CuPDC1 gene was obtained in this order. Hereinafter, this DNA fragment is referred to as “ CuPDC1 disruption first and second fragment”. If the transformation using this DNA fragment, by duplex homologous recombination upstream region and downstream region of CuPDC1 gene occurs, it is possible to an allele of CuPDC1 gene partially deleted.

The NBRC0988 strain was transformed using the first and second fragments of CuPDC1 disrupted as DNA fragments. Genomic DNA was extracted from the NBRC0988 strain and the resulting transformant, and PCR was carried out with IM-281 (SEQ ID NO: 31) and IM-282 (SEQ ID NO: 32) using this as a template (extension reaction: 4 minutes). As shown in FIG. 9, these primers anneal outside the homologous recombination region. When subjected to 0.8% agarose gel electrophoresis, a 3.7 kb DNA fragment was amplified in the NBRC0988 strain, and a 3.9 kb and 3.7 kb DNA fragment was amplified in the plurality of transformants. From this, it was found that the desired HygBr CuPDC1 gene 1-copy disruption strain was obtained.

Transformation of a HyPd CuPDC1 gene 1-copy disrupted strain was performed with Cre expression plasmid pCU595. In the medium containing G418, colonies were not formed in the negative control to which DNA was not added, whereas in the sample to which pCU595 was added, 1,000 or more transformants were obtained. As a result of applying 30 of these strains to the G418 medium or HygB medium, all of them were able to grow on the G418 medium, but could not grow on the HygB medium.

Genomic DNA was extracted from the HygBr CuPDC1 gene 1-copy disrupted strain and the HygBs CuPDC1 gene 1-copy disrupted strain. (Elongation reaction 4 minutes). When subjected to 0.8% agarose gel electrophoresis, DNA fragments of 3.9 kb and 3.7 kb were amplified in the former strain, and DNA fragments of 3.7 kb and 1.9 kb were amplified in the latter strain. A strain from which the HPT gene was removed as intended was obtained.

A disrupted strain of 1 copy of HyPBs CuPDC1 gene was cultured overnight in YPD liquid medium, and a portion thereof was applied to YPD medium. Two days later, single colonies were separated and applied to G418 medium and YPD medium. As a result, most clones grew on the YPD medium but did not grow on the G418 medium.

A primer set for amplifying the Cre gene by extracting genomic DNA from G418r and G418s strains, which have different growth potentials in a medium containing G418, although one copy of the HyPB1 CuPDC1 gene is destroyed. PCR was performed with certain IM-49 (SEQ ID NO: 23) and IM-52 (SEQ ID NO: 26) (extension reaction: 1 minute). As a result, a 1 kb DNA fragment was amplified in the G418r strain, but not amplified in the G418s strain. From this, pCU595 was lost, and a disrupted strain of HygBs and G418s CuPDC1 gene in the first copy was obtained.

Using the CuPDC1 disruption 1st and 2nd fragment as a DNA fragment, transformation of a HydBs and G418s CuPDC1 gene disrupted one copy was performed. Genomic DNA was extracted from the obtained transformant, and PCR was carried out using IM-281 (SEQ ID NO: 31) and IM-282 (SEQ ID NO: 32) as a template (extension reaction: 4 minutes). When subjected to 0.8% agarose gel electrophoresis, there were several strains in which three types of DNA fragments of 3.9 kb, 3.7 kb, and 1.9 kb were amplified. From this, it was found that the target HygBr disrupted strain of CuPDC1 gene 2 copies was obtained.

Transformation of the HyPd CuPDC1 gene 2-copy disruption strain was carried out with pCU595, a Cre expression plasmid. When the obtained transformants were applied to G418 medium and HygB medium, they could all grow on G418 medium, but could not grow on HygB medium. Using genomic DNA extracted from this HygBs and G418r transformant as a template, PCR was performed with IM-281 (SEQ ID NO: 31) and IM-282 (SEQ ID NO: 32) (extension reaction: 4 minutes). When subjected to 0.8% agarose gel electrophoresis, two kinds of DNA fragments of 3.7 kb and 1.9 kb were amplified. It was found that the HPT gene could be removed.

HydBs CuPDC1 gene 2-copy disrupted strain was cultured overnight in YPD liquid medium, and a portion thereof was applied to YPD medium. Two days later, single colonies were isolated and spread on YPD medium and G418 medium. Then, clones that grew on the YPD medium but did not grow on the G418 medium were isolated. A strain in which pCU595 was eliminated, that is, a disrupted strain of HyGBs and G418s in the second copy of the CuPDC1 gene could be obtained.

A DNA fragment for disrupting the third and fourth copies of the CuPDC1 gene was prepared as follows. First, three types of PCR shown in the following (1), (2) and (3) were performed: (1) pCU563 was used as a template, IM-1 (SEQ ID NO: 21) and IM-2 (primers were used as primers. (SEQ ID NO: 22), and the extension reaction time was 2 minutes; (2) genomic DNA of NBRC0988 strain was used as a template, and IM-277 (SEQ ID NO: 27) and IM-278 (SEQ ID NO: 28) were used as primers. The extension reaction time was 30 seconds; (3) NBRC0988 strain genomic DNA was used as a template, IM-185 (SEQ ID NO: 33) and IM-168 (SEQ ID NO: 34) were used as primers, and the extension reaction time was 30 seconds. It was. In (2) and (3), the upstream part and the downstream part of the CuPDC1 gene are amplified. Furthermore, PCR of the following (4) was performed: (4) Using a mixture of the three types of DNA amplified in the above (1), (2) and (3) as a template, IM-277 ( SEQ ID NO: 27) and IM-168 (SEQ ID NO: 34) were used, and the extension reaction time was 3 minutes. Thereby, a DNA fragment consisting of the upstream region of the CuPDC1 gene, the loxP, the PGK gene promoter, the HPT gene, the GAP gene terminator, the loxP, and the downstream region of the CuPDC1 gene was obtained in this order. Hereinafter, this DNA fragment is referred to as “ CuPDC1 disruption 3rd and 4th fragment”. If the transformation using this DNA fragment, by duplex homologous recombination upstream region and downstream region of CuPDC1 gene occurs, it is possible to an allele of CuPDC1 gene partially deleted. The downstream region of CuPDC1 gene amplified by (3), the deleted regions in the transformation for one copy first and two copies th CuPDC1 gene disruption was performed using the CuPDC1 destruction 1-second fragment Is likely to greatly reduce the possibility of incorporation into two copies of a destroyed allele.

Using the CuPDC1 disruption 3rd and 4th fragment as a DNA fragment, the CuPDC1 gene 2-copy disruption strain of HygBs and G418s was transformed for the third copy of the CuPDC1 gene. Genomic DNA was extracted from the obtained transformant, and PCR was carried out using IM-281 (SEQ ID NO: 31) and IM-282 (SEQ ID NO: 32) as a template (extension reaction: 4 minutes). When subjected to 0.8% agarose gel electrophoresis, there were strains in which three types of DNA fragments of 4.4 kb, 3.7 kb, and 1.9 kb were amplified. From this, it was found that the target HygBr disrupted strain of the third copy of the CuPDC1 gene was obtained.

Transformation of a HyPd CuPDC1 gene 3 copy disruption strain was carried out with pCU595, a plasmid for Cre expression. As a result of applying the obtained transformant to G418 medium and HygB medium, all were able to grow on G418 medium, but were not able to grow on HygB medium. Using genomic DNA extracted from this HygBs and G418r transformant as a template, PCR was performed with IM-281 (SEQ ID NO: 31) and IM-282 (SEQ ID NO: 32) (extension reaction: 4 minutes). When subjected to 0.8% agarose gel electrophoresis, three types of DNA fragments of 3.7 kb, 2.4 kb, and 1.9 kb were amplified. It was found that the HPT gene could be removed.

HygBs CuPDC1 gene 3-copy disrupted strain was cultured overnight in YPD liquid medium, and a part thereof was applied to YPD medium. Two days later, single colonies were isolated and spread on YPD medium and G418 medium. Then, clones that grew on the YPD medium but did not grow on the G418 medium were isolated. A strain in which pCU595 was eliminated, that is, a strain in which the third copy of the CuPDC1 gene of HygBs and G418s was disrupted was obtained.

Using the CuPDC1 disrupted 3rd and 4th fragment as a DNA fragment, a CuPDC1 gene 3 copy disrupted strain of HygBs and G418s was transformed. Genomic DNA was extracted from the obtained transformant, and PCR was carried out using IM-281 (SEQ ID NO: 31) and IM-282 (SEQ ID NO: 32) as a template (extension reaction: 4 minutes). When subjected to 0.8% agarose gel electrophoresis, three types of DNA fragments of 4.4 kb, 2.4 kb, and 1.9 kb were amplified in a plurality of transformants. From this, it was found that the desired HygBr CuPDC1 gene 4 copy disruption strain was obtained. Also, DNA fragments of 3.7kb was observed in a wild strain NBRC0988 strain, that is, from the DNA fragment allele not destroyed is amplified is not detected, this strain is a CuPDC1 gene completely disrupted strain it was thought.

Transformation of HygBr CuPDC1 gene 4 copy disruption strain was performed with Cre expression plasmid pCU595. As a result of applying the obtained transformant to G418 medium and HygB medium, all were able to grow on G418 medium, but were not able to grow on HygB medium. Using genomic DNA extracted from this HygBs and G418r transformant as a template, PCR was performed with IM-281 (SEQ ID NO: 31) and IM-282 (SEQ ID NO: 32) (extension reaction: 4 minutes). When subjected to 0.8% agarose gel electrophoresis, there were several strains in which two types of DNA fragments of 2.4 kb and 1.9 kb were amplified. It was found that the HPT gene could be removed.

A 4 copy disrupted strain of HyPBs CuPDC1 gene was cultured overnight in a YPD liquid medium, and a portion thereof was applied to the YPD medium. Two days later, single colonies were isolated and spread on YPD medium and G418 medium. Then, clones that grew on the YPD medium but did not grow on the G418 medium were isolated. A strain from which pCU595 was eliminated, that is, a disrupted strain of HyGBs and G418s at the fourth copy of the CuPDC1 gene could be obtained. This strain was named Cu8402g strain.

2-3. Characterization of CuPDC1 gene disrupted strain The CuPDC1 gene is thought to encode a polypeptide having pyruvate decarboxylase activity that catalyzes the conversion of pyruvate to acetaldehyde. In the fermentation pathway, acetaldehyde is further metabolized to ethanol by alcohol dehydrogenase. That is, it is expected that by destroying the CuPDC1 gene, the metabolic pathway to ethanol is shut down and the ethanol production ability is reduced. Therefore, the CuPDC1 gene 1-copy disrupted strain, the CuPDC1 gene 2-copy-disrupted strain, the CuPDC1 gene 3-copy-disrupted strain, and the CuPDC1 gene completely disrupted strain Cu8402g strain were subjected to fermentation tests (all strains of HygBs and G418s), ethanol production ability and organic acid Was analyzed.

For the purpose of investigating the ethanol production ability, aroma component production ability and organic acid production ability of the Cu8402g strain, the wild strain NBRC0988 strain and the Cu8402g strain were used in a YPD agar medium for 1 to 3 days using a 50 mL to 100 mL YPD liquid medium as a medium. The grown cells were inoculated into a new medium so that the OD600 was about 0.1, and cultured for 16 to 30 hours at 30 ° C. with an amplitude of 35 mm, 120 to 150 rpm, using a table culture apparatus manufactured by TAITEC. Then, the cells were collected by centrifugation under conditions of 4 ° C. and 3,000 rpm for 5 minutes, and after removing the supernatant, the cells were washed with a medium used for fermentation (not including a neutralizing agent). The yeast cells thus obtained were inoculated into 50 mL of YPD10 (100 g / L glucose, 10 g / L yeast extract, 20 g / L peptone) medium in a 100 mL Erlenmeyer flask with baffle so that the initial OD600 was 0.5. Then, the cells were cultured at 30 ° C. for 48 hours with an amplitude of 35 mm and a shaking speed of 80 rpm using a table culture apparatus manufactured by TAITEC. The culture solution was filtered through a 0.22 μm filter, and the ethanol concentration, aroma component concentration, and various organic acid concentrations in the medium were measured. The results are shown in Table 1. Various data are values calculated from the results of three independent trials.

48 hours after the start of fermentation, NBRC0988 strain produced 3.96 g / L ethanol, but Cu8402g strain could not detect ethanol.

48 hours after the start of fermentation, as for acetaldehyde, 26.2 mg / L was produced in the NBRC0988 strain, but 1.14 mg / L in the Cu8402g strain.

48 hours after the start of fermentation, 660.3 mg / L of acetic acid was produced for NBRC0988 strain, but 273.1 mg / L for Cu8402g strain.

48 hours after the start of fermentation, the concentrations of ethanol and acetic acid in which acetaldehyde was a precursor were both lower in the Cu8402g strain than in the NBRC0988 strain.

The pyruvate concentration of NBRC0988 strain 48 hours after the start of fermentation was 462.4 mg / L, whereas it was observed that 3659.9 mg / L was present in the Cu8402g strain. The L-lactic acid concentration was lower in both the NBRC 0988 strain and the Cu8402g strain than in the medium without yeast added. The concentration of D-lactic acid decreased in the NBRC0988 strain, but increased in the Cu8402g strain, compared to the medium without yeast added.

As a characteristic of the Cu8402g strain, by disrupting the CuPDC1 gene, the pathway for converting pyruvate to acetaldehyde is shut down, thereby accumulating pyruvate without being metabolized, which further affects the methylglyoxal pathway. It is considered that D-lactic acid, which is a precursor of pyruvic acid, is easy to accumulate.

The above results indicate that the CuPDC1 gene encodes pyruvate decarboxylase, which is involved in the conversion of pyruvate to acetaldehyde, and the activity of the enzyme in the cell is lost by completely deleting this gene. Or it is thought that it originates in having fallen.

In the process of purifying the target product L-lactic acid, a technique of adding calcium carbonate to the culture supernatant and recovering it as an L-calcium lactate salt is widely used. The fact that the concentration of various organic acids in ethanol and TCA cycle is greatly reduced compared to the wild type strain indicates that it is possible to reduce the production of by-products other than L-lactate in this process. It is considered to be a trait that is a useful index for evaluating performance.

After 48 hours from the start of fermentation, the CuPDC1 gene 1-copy disrupted strain, the CuPDC1 gene 2-copy-disrupted strain, and the CuPDC1 gene 3-copy disrupted strain had the same ability to produce ethanol as the Candida utilis wild strain NBRC0988.

Example 3: Construction of Candida utilis strain into which L-LDH gene was introduced
3-1. Design of DNA sequence of L-LDH gene encoding polypeptide having L-lactate dehydrogenase activity Polypeptide having L- lactate dehydrogenase activity derived from bovine, which is a higher eukaryote, is transformed into yeast Candida utilis. In order to efficiently express the protein, the activity of lactate dehydrogenase described in JP-A No. 2003-259878 and described in the amino acid sequence of a bovine-derived enzyme (DDBJ / EMBL / GenBank Accession number: AAI46211.1) is used. With respect to the gene encoding the polypeptide possessed, Takara Bio Inc. was requested to design and synthesize a new gene sequence that does not exist in nature, using the following items as design guidelines.

(A) Codons frequently used in Candida utilis were used.
(B) The unstable sequences and repetitive sequences of mRNA were eliminated as much as possible.
(C) There was no difference in the GC content bias over the entire region.
(D) Restricted restriction enzyme sites that are inappropriate for gene cloning were made in the designed sequence.
(E) A useful restriction enzyme site was added to both ends for incorporation into an L-LDH gene expression vector (upstream of L-LDH coding region: Kpn I, Xba I; downstream of L-LDH coding region: Bam HI, Sac I). Here, the Kpn I recognition site indicates the sequence GGTACC from the first g to the sixth c in the nucleotide sequence of SEQ ID NO: 36, and the Xba I recognition site is from the seventh t in the nucleotide sequence of SEQ ID NO: 36. The sequence TCTAGA up to the 12th a, the Bam HI recognition site represents the sequence GGATCC from the 1,015th g to the 1,020th c in the nucleotide sequence of SEQ ID NO: 36, and the Sac I recognition site is The sequence GAGCTC from the 1,021st g to the 1,026th c in the nucleotide sequence of SEQ ID NO: 36 is shown.

The synthesized DNA sequence is shown in SEQ ID NO: 36. Of these, the amino acid sequence corresponding to the nucleotide sequence from the 13th a to the 1,011st a encoding the above-mentioned polypeptide having the activity of L-lactate dehydrogenase is derived from bovine itself. (DDBJ / EMBL / GenBank Accession number: AAI46211.1). Note that the 1,009 to 1,011st TGAs of SEQ ID NO: 36 and the subsequent 1,012 to 1,014th TGAs are translation end codons. The plasmid having this DNA fragment was named pCU669 (also known as GA07033).

Of this SEQ ID NO: 36, the nucleotide sequence (codon optimized sequence) from the 13th a to the 1,011st a (upstream TGA of the two translation termination codons) is represented by SEQ ID NO: 38 The alignment of the nucleotide sequence (wild-type sequence derived from bovine) is shown in FIG. Both sequences had the same 751 bases out of 999 bases, and the homology was 75%. In FIG. 1, the upper sequence is a nucleotide sequence from the 13th a to the 1011st a (upstream TGA of two translation termination codons) in SEQ ID NO: 36. The lower sequence of FIG. 1 is the base sequence of L-LDH-A gene derived from Bos taurus represented by SEQ ID NO: 38 (extracted from DDBJ / EMBL / GenBank Accession number: BC146210.1). No. 35).

3-2. Construction of manufacturing L-LDH gene expression plasmid for L-LDH gene expression plasmid, unless otherwise stated, was performed as follows using the KOD-Plus-.

PCR was performed with IM-345 (SEQ ID NO: 39) and IM-346 (SEQ ID NO: 40) to amplify the downstream region of the CuPDC1 gene (extension reaction 1 minute). After digesting the amplified fragment with Bss HII, and ligated with pBluescriptIISK it was completely digested with Bss HII (+) (TOYOBO Co.). The resulting plasmid was named pCU670 (alias: pPt).

A plasmid pCU621 in which the PGK gene promoter of the plasmid pCU563 for preparing a DNA fragment for gene disruption was elongated was constructed by the following procedure. Using the plasmid pGKHPT1 having the PGK gene promoter and hygromycin resistance gene HPT gene described in Shimada et al. (Appl. Environ. Microbiol. 64, 2676-2680) as a template, By performing PCR (extension reaction 2 minutes) with the primer set of (SEQ ID NO: 17), DNA fragments consisting of loxP, PGK gene promoter, and HPT gene were sequentially amplified.
In addition, PCR (extension reaction 30 seconds) was carried out using the primer set of IM-54 (SEQ ID NO: 19) and IM-55 (SEQ ID NO: 20) using pGAPPT10 (Kondo et al., Nat. Biotechnol. 15, 453-457) as a template. By performing, a DNA fragment consisting of the GAP gene terminator and loxP was amplified. By mixing these and performing PCR with IM-1 (SEQ ID NO: 21) and IM-2 (SEQ ID NO: 22) (elongation reaction 2.5 minutes, LA Taq manufactured by Takara Bio Inc. was used as the enzyme), A DNA fragment consisting of loxP, PGK gene promoter, HPT gene, GAP gene terminator, and loxP was amplified in this order. The resulting DNA fragment was cloned into the pCR2.1 vector. The plasmid thus obtained was named pCU621 (alias: pNNLHL).

First, the following three types of PCR were performed: (1) PCR was performed using pCU621 as a template and IM-349 (SEQ ID NO: 42) and IM-350 (SEQ ID NO: 43) as primers (extension reaction 2.5). Min); (2) PCR was carried out using pPGKPT2 (Japanese Patent Laid-Open No. 2003-144185) as a template and IM-347 (SEQ ID NO: 44) and IM-348 (SEQ ID NO: 45) as primers (extension reaction 30 seconds), PGK The terminator region of the gene was amplified with a single base mutation so that the Bgl II recognition sequence was eliminated; (3) using the DNA fragment amplified in (1) and (2) as a template and IM-347 (sequence) as a primer No. 44) and IM-350 (SEQ ID NO: 43) were used for PCR (extension reaction: 3 minutes). (3) The resulting DNA fragment with and ligated into pBluescriptIISK (+) digested with Sma I. The resulting plasmid was named pCU672 (alias: pPGtH).

PCU672 (aka: pPGtH) PGK gene terminator was obtained by digesting with Bam HI and Cla I, loxP, PGK gene promoter, HPT gene, GAP gene terminator, a DNA fragment of about 3kbp consisting loxP, Bam HI and Cla A new plasmid pCU675 (also known as pPGtHPt) was constructed by ligation to pCU670 digested with I.

First, the following three types of PCR were performed: (1) PCR was performed using pCU530 as a template and IM-341 (SEQ ID NO: 46) and IM-342 (SEQ ID NO: 47) as primers (extension reaction 2 minutes), CuPDC1 gene promoter region was amplified; (2) PCR was performed using pCU669 (also known as GA07033) as a template and IM-343 (SEQ ID NO: 48) and IM-379 (SEQ ID NO: 49) as primers (extension reaction 1 min) L-LDH structural gene was amplified; (3) The DNA fragment amplified in (1) and (2) was used as a template, and IM-341 (SEQ ID NO: 46) and IM-379 (SEQ ID NO: 49) were used as primers. PCR was performed using (extension reaction 3 minutes). The DNA fragment amplified in (3) was digested with Not I and Bgl II, and the obtained DNA fragment was ligated to pCU675 (also called pPGtHPt) cleaved with Not I and BamH I. The resulting plasmid PCU681 (aka: pPLPGtHPt) (FIG. 10) is the Bss HII site of pBluescriptIISK (+), in order CuPDC1 gene promoter region, L-LDH structural gene, PGK gene terminator, loxP, PGK gene promoter, HPT gene A DNA fragment consisting of the GAP gene terminator, loxP, and the downstream region of the CuPDC1 gene has been inserted. The inserted DNA fragment side two Bss HII recognition sequence, since it is equipped with Bgl II recognition sequence immediately following the recognition sequence, PCU681 (aka: pPLPGtHPt) a by digestion with Bgl II, above CuPDC1 gene It is possible to obtain a DNA fragment consisting of the downstream region of the CuPDC1 gene from the promoter. Transformants are selected on a YPD medium containing 600 μg / mL hygromycin B.

3-3. Was transformed NBRC0988 shares at Candida utilis pCU681 was digested with the introduction Bgl II of the L-LDH gene into the wild strain NBRC0988 Ltd. (pPLPGtHPt) 3μg. PCR was performed using the DNA extracted from the obtained transformant as a template and IM-362 (SEQ ID NO: 50) and IM-174 (SEQ ID NO: 51) as a primer set (extension reaction 4 minutes). Among them, a transformant Pj0202 strain in which a 3.6 kb DNA fragment that is not amplified in the NBRC0988 strain was amplified was obtained. Further, when PCR was carried out using IM-163 (SEQ ID NO: 52) and IM-164 (SEQ ID NO: 53) as a primer set (elongation reaction 30 seconds), a DNA fragment of about 500 bp was amplified. This indicates that the Pj0202 strain has at least one copy of the undisrupted CuPDC1 gene.

3-4. In NBRC0988 strain and CuPDC1 genes total destruction was digested with L-LDH gene introduction Bgl II to strain Cu8402g strain pCU681 (pPLPGtHPt) 3μg was transformed Cu8402g strain. PCR was carried out using the DNA extracted from the obtained HygBr transformant as a template and IM-362 (SEQ ID NO: 50) and IM-174 (SEQ ID NO: 51) as a primer set (extension reaction: 4 minutes). Among them, a transformant Pj0404 strain was obtained in which a 3.6 kb DNA fragment that was not amplified in the Cu8402g strain was amplified. This strain is a strain of L-LDH gene is integrated into CuPDC1 locus, the expression of the L-LDH gene is controlled by the native CuPDC1 gene promoter.

The Pj0404 strain is a strain into which at least one copy of the L-LDH gene has been introduced.
In particular, since the HPT gene expressed in this study can select a transformant exhibiting the HygBr phenotype by introducing only one copy, the Pj0404 strain is a strain into which one copy of the L-LDH gene is incorporated. Conceivable. As a result of PCR using IM-281 (SEQ ID NO: 31) and IM-282 (SEQ ID NO: 32) as a primer set (extension reaction 4 minutes), at least two kinds of DNA fragments of 2.4 kb and 1.9 kb were amplified. It was. As a result, it was revealed that the CuPDC1 locus in a disrupted state exists in the Pj0404 strain without incorporating the L-LDH gene.

The Pj0404 strain was transformed with the Cre recombinase expression plasmid pCU595 to obtain a HygBs and G418r clone. The clone was cultured overnight in a YPD liquid medium, and a part thereof was applied to the YPD medium. Two days later, single colonies were isolated and spread on YPD medium and G418 medium. Then, clones that grew on the YPD medium but did not grow on the G418 medium were isolated. As a result of PCR using DNA extracted from this clone Pj0707a strain as a template and IM-362 (SEQ ID NO: 50) and IM-174 (SEQ ID NO: 51) as a primer set (extension reaction 4 minutes), a DNA of 1.2 kb Was amplified.
Pj0707a strain has a HygBs and G418s phenotype, destroyed all CuPDC1 gene and a strain CuPDC1 gene promoter inducible L-LDH gene integrated was introduced into CuPDC1 locus.

The Pj0707a strain was transformed with 3 μg of pCU681 (pPLPGtHPt) digested with Bgl II. PCR was carried out using the DNA extracted from the obtained HygBr transformant as a template and IM-362 (SEQ ID NO: 50) and IM-174 (SEQ ID NO: 51) as a primer set (extension reaction: 4 minutes). As a result, a transformant Pj0957 strain was obtained in which two types of DNA fragments of 3.6 kb and 1.2 kb were amplified. This strain L-LDH gene is a strain incorporated in CuPDC1 locus different alleles and CuPDC1 locus L-LDH gene is integrated at Pj0457 strain. Expression of the L-LDH gene introduced by this transformation is also controlled by the original CuPDC1 gene promoter.

The Pj0957 strain is a strain into which at least two copies of the L-LDH gene have been introduced.
In particular, since the HPT gene expressed in this study can select a transformant exhibiting a HygBr phenotype by introducing only one copy, the Pj0957 strain is a strain into which two copies of the L-LDH gene are incorporated. Conceivable.

It was confirmed that the Cre-loxP system in Candida utilis can be used not only for gene destruction but also for introduction of arbitrary genes.

Example 4 Fermentation Test in Flask As shown below, NBRC0988 strain and the newly constructed recombinant yeast strain were evaluated for lactic acid production ability. The ethanol concentration in the medium was measured using GC or HPLC, and the glucose concentration and L-lactic acid concentration in the medium were measured using Biochemistry Analyzer (BA) manufactured by Wyeth Japan. For the discrimination of optical isomers, J. et al. K. Using F-kit D-lactic acid / L-lactic acid manufactured by International Co., Ltd., the method followed the attached protocol. Other various organic acid production amounts were performed using HPLC. As a sample subjected to analysis, a culture solution previously filtered through a 0.22 μm filter was used. Each type of data is an average of the results of independent trials at least three times.

Inoculate a 3 mL YPD liquid medium in a 15 mL tube from a yeast cell cultured on a YPD agar medium for 2 to 3 days at 30 ° C. with a platinum loop. Pre-culture was performed for 20 to 30 hours at an amplitude of 35 mm, 130 rpm, and 30 ° C. using a culture apparatus. This is inoculated into 100 mL of YPD medium in a Sakaguchi flask so that the OD600 is about 0.1, and precultured at a 35 mm amplitude, 130 rpm, 30 ° C. for 15 to 22 hours with a table culture apparatus manufactured by TAITEC. went. Then, the cells were collected by centrifugation under conditions of 4 ° C., 3,000 rpm, and 5 minutes, and the supernatant was removed, followed by washing with a medium used for fermentation (without a neutralizing agent). The bacterial cells thus obtained were inoculated into a 15 mL medium containing 100 to 115 g / L glucose in a baffled 100 mL Erlenmeyer flask and fermented by a table culture apparatus manufactured by TAITEC at an amplitude of 35 mm and 80 rpm. . The amount of cells to be inoculated for fermentation by the preculture was planted so that OD600 would be 10 unless otherwise specified. Unless otherwise specified, calcium carbonate was added to the medium to a concentration of 4.5% (w / v) as a neutralizing agent. The temperature at the time of fermentation was 25 degreeC, 30 degreeC, or 35 degreeC. In addition to glucose at the above concentration (100 to 115 g / L), 10 g / L yeast extract and 20 g / L peptone are added to the medium, and the medium having this composition is hereinafter referred to as YPD10 medium.

The total sugar conversion rate (%) is a value obtained by dividing the weight of L-lactic acid in the medium by the initial glucose weight in the medium and multiplying by 100. The optical purity (%) of L-lactic acid is a value obtained by dividing the value of L-lactic acid concentration by the value obtained by adding D-lactic acid concentration to L-lactic acid concentration and multiplying by 100.

NBRC0988 strain, Cu8402g strain, and Pj0202 strain were examined for glucose concentration, ethanol concentration, L-lactic acid concentration, D-lactic acid concentration, and other organic acid concentrations in the medium 24 hours after the start of fermentation. Table 2 shows the results when the fermentation temperature was 30 ° C.

From the above results, it was found that the Cu8402g strain in which the CuPDC1 gene was completely disrupted produced almost no L-lactic acid and ethanol, and accumulated a large amount of pyruvic acid and D-lactic acid, as compared with the NBRC0988 strain. .

From the results shown in Table 2, first, the wild strain NBRC0988 strain consumed almost all glucose and produced ethanol. In the NBRC0988 strain, the concentration of L-lactic acid was decreased. The Pj0202 strain is a strain having both the undisrupted CuPDC1 gene and the L-LDH gene, and the strain produced both ethanol and L-lactic acid. In order to improve the production efficiency of L-lactic acid from glucose, it was considered effective to reduce the production amount of ethanol, for example, to delete all of the CuPDC1 gene.

For the Pj0404 strain and the Pj0957 strain, the L-lactic acid concentration in the medium from 4 hours to 13 hours after the start of fermentation was measured every hour. Regarding the fermentation temperature, the Pj0404 strain was set to one condition of 30 ° C, and the Pj0957 strain was set to two conditions of 30 ° C and 35 ° C. A first-order approximation formula was also obtained for each data. The results are shown in FIG.

Regarding the production rate of L-lactic acid per unit time, 3.41 g / L / hour (r2 value = 0.998) for Pj0404 strain (30 ° C.) and 4.13 g / L / hour for Pj0957 strain (30 ° C.). It was 4.80 g / L / hour (r-square value = 0.998) for the Pj0957 strain (35 ° C.). From this, it was found that the Pj0957 strain having a higher L-LDH gene copy number than the Pj0404 strain has the ability to produce lactic acid faster. In addition, in order to increase the fermentation rate of the Pj0957 strain, it was considered that 35 ° C. was better selected than 30 ° C.

For the Pj0404 and Pj0957 strains, the glucose concentration, ethanol concentration, L-lactic acid concentration, D-lactic acid concentration, and other organic acid concentrations in the medium 24 hours after the start of fermentation were examined. Regarding the fermentation temperature, the Pj0404 strain was set to one condition of 30 ° C, and the Pj0957 strain was set to two conditions of 30 ° C and 35 ° C. The results are listed in Table 3.

In 24 hours after the start of fermentation, 4.5% (w / v) calcium carbonate added as a neutralizing agent remained in a powder state in all samples. In addition, the amount of lactic acid produced by the Pj0404 strain was lower than that of the Pj0957 strain. This indicates that the higher the L-LDH gene copy number, the faster the lactic acid production.

24 hours after the start of fermentation, the amount of lactic acid produced was higher when the Pj0957 strain was cultured at 35 ° C than when the Pj0957 strain was cultured at 30 ° C. From this, it was considered that the fermentation temperature is preferably 35 ° C rather than 30 ° C for the production of L-lactic acid.

The Pj0957 strain was fermented under the conditions with and without the neutralizer. The fermentation temperature was 35 ° C. Table 4 shows the results of examining the glucose concentration, ethanol concentration, L-lactic acid concentration, D-lactic acid concentration, and other organic acid concentrations in the medium after 33 hours.

When the neutralizer was not added, the amount of lactic acid produced was smaller than when the neutralizer was added. This is considered to be caused by the high acidity of the medium. From this, it was thought that adding a neutralizing agent was effective for efficient lactic acid production.

The Pj0957 strain in which the CuPDC1 gene has been completely disrupted and the L-LDH gene has been introduced is capable of rapidly removing L-lactic acid from a medium containing 108.7 g / L glucose at a high efficiency of 95.10% in terms of total sugar. Manufactured out of.

For the Pj0957 strain, the initial OD was 10, the fermentation temperature was 25 ° C., and the concentration of L-lactic acid in YPD10 medium (containing 100 g / L glucose) supplemented with 4.5% (w / v) calcium carbonate was determined. Measurements were made every 2 hours from 4 hours to 12 hours after the start. The first-order approximate expression was obtained, and the L-lactic acid production rate per unit time was calculated to be 3.0 g / L / h. Further, when the L-lactic acid concentration at 33 hours after the start of fermentation was examined, the L-lactic acid concentration in the medium was 95 g / L. Although the L-lactic acid production rate was inferior to the conditions of 25 ° C. and 30 ° C. and 35 ° C., a considerable amount of L-lactic acid was produced. Therefore, it has been clarified that the Pj0957 strain can produce L-lactic acid with high efficiency at a wide temperature range of 25 ° C to 35 ° C.

The amount of cells used for fermentation was examined. The Pj0404 strain and the Pj0957 strain were inoculated at an OD600 of 2, 5, or 10 at the start of fermentation, and the glucose concentration and L-lactic acid concentration in the medium 42.5 hours after the start of fermentation were examined. A medium with a sugar concentration of 100 g / L was used. The liquid volume was 15 mL.

For the Pj0404 strain, the OD2 condition is 88.2 g / L, the OD5 condition is 92.0 g / L, the OD10 condition is 93.0 g / L, the Pj0957 strain is the OD2 condition, 93.8 g / L, and the OD5 condition is It became 92.8 g / L on condition of 92.2 g / L and OD10. This indicates that even when the initial OD is lower than 10, L-lactic acid can be produced with the efficiency almost the same as the OD10 condition by increasing the fermentation time.

Example 5: Fermentation test using a jar fermenter As shown below, the lactic acid-producing ability of the Pj0957 strain was evaluated. The ethanol concentration in the medium was measured using GC or HPLC, and the glucose concentration and L-lactic acid concentration in the medium were measured using Biochemistry Analyzer (BA) manufactured by Wyeth Japan.
For the discrimination of optical isomers, J. et al. K. Using F-kit D-lactic acid / L-lactic acid manufactured by International Co., Ltd., the method followed the attached protocol. Other various organic acid production amounts were performed using HPLC. As a sample subjected to analysis, a culture solution previously filtered through a 0.22 μm filter was used.

Inoculate a 3 mL YPD liquid medium in a 15 mL tube from a yeast cell cultured on a YPD agar medium for 2 to 3 days at 30 ° C. with a platinum loop. Pre-culture was performed for 6 to 15 hours at an amplitude of 35 mm, 130 rpm, and 30 ° C. using a culture apparatus. Next, as a pre-culture, 50 mL of YPD liquid medium is used as a medium, and the cells of the pre-pre-culture are inoculated into a new medium so that the OD600 is about 0.1, and usually at 130 rpm and 30 ° C. The cells were cultured for 12 to 18 hours and cultured until the logarithmic growth phase or the stationary phase where OD600 was 10 to 25. In this culture, a Sakaguchi flask was used. The pre-culture conditions for preparing the cells to be used for fermentation were as follows: YPD medium 2 in a 5 L jar fermenter (desktop culture device, Bioneer-C 5 L (S), manufactured by Maruhishi Bioengineering). 5 L was inoculated with the entire amount of the cells previously cultured and cultured at 400 rpm, 30 ° C., 1 vvm for 21 to 27 hours. At this time, the OD600 was usually 10-25. Then, the cells were collected by centrifugation under conditions of 4 ° C. and 3,000 rpm for 5 minutes, and after removing the supernatant, the cells were washed with a medium used for fermentation (not including a neutralizing agent). The bacterial cells thus obtained were inoculated into a 2 L medium containing 100 to 120 g / L glucose, and a 5 L capacity jar fermenter (desktop culture device, Bioneer-C 5 L (S), Maruhishi Bioengineering Co., Ltd. Made). In addition, it planted so that the quantity which inoculates the microbial cell obtained by preculture for fermentation may be set to OD600.10. Pj0957 strain was used for this fermentation test. The stirring speed was 250 rpm, the temperature was 35 ° C., and the air flow rate was 1 vvm. As neutralization conditions, when calcium carbonate is added to the medium so that the concentration becomes 5% (w / v) at the start of fermentation, the pH during fermentation becomes 5.5 with 2.5N sodium hydroxide. The case where the program was built to control feedback was examined. The results when using calcium carbonate are the values obtained from only one trial, and the results when using sodium hydroxide are the average of the values obtained from two independent trials. In addition, when sodium hydroxide was used, the volume of the fermentation broth changed greatly, so the amount of medium was measured from the amount of sodium hydroxide added. In this study, since the value obtained by analysis such as HPLC or BA is the concentration, a value obtained by multiplying the value by the amount of medium, that is, the total weight of the substance was obtained.

The total sugar conversion rate (%) is a value obtained by dividing the weight of L-lactic acid in the medium by the initial glucose weight in the medium and multiplying by 100. The optical purity (%) of L-lactic acid is a value obtained by dividing the value of L-lactic acid concentration by the value obtained by adding D-lactic acid concentration to L-lactic acid concentration and multiplying by 100.

In a trial using calcium carbonate as a neutralizing agent, sampling was performed a plurality of times by 24 hours after the start of fermentation. FIG. 12 shows the change over time in the amount of glucose and the amount of L-lactic acid in the medium.

In a trial using sodium hydroxide as a neutralizing agent, sampling was performed a plurality of times by 24 hours after the start of fermentation. FIG. 13A shows the change over time in the amount of glucose and the amount of L-lactic acid in the medium (n = 2).

The amount of glucose, the amount of ethanol, the amount of L-lactic acid, the amount of D-lactic acid, the amount of other organic acids, and the pH in the medium 24 hours after the start of fermentation were examined. The results are listed in Table 5 (n = 2). After 24 hours, the added calcium carbonate powder (solid matter) was not observed at all.

There was no significant difference in the amount of L-lactic acid produced after 24 hours between the conditions using calcium carbonate and the conditions using sodium hydroxide.

Under the condition where calcium carbonate was added at 5% (w / v), when the concentration of lactic acid increased to about 80 to 100 g / L, calcium lactate precipitated at 30 ° C. or 35 ° C., and the medium gelled. Since the gelation occurs in a state where the bacterial cells and lactate are mixed, it is expected that the process of purifying L-lactic acid will be complicated due to this event. However, when sodium hydroxide is used, it is considered that this phenomenon can be avoided. Therefore, depending on the purification method of L-lactic acid, the availability of sodium hydroxide can be a favorable property in the production process of L-lactic acid. .

At 24 hours after the start of fermentation, the optical purity of L-lactic acid was higher with sodium hydroxide than with calcium carbonate.

At 24 hours after the start of fermentation, the amount of acetic acid as a by-product was lower when sodium hydroxide was used than when calcium carbonate was used.

Furthermore, in addition to FIG. 13A showing the results of two independent experiments, the results of three independent experiments are shown in FIG. 13B. According to the experimental data shown in FIG. 13B, the total sugar conversion rate after 24 hours of culture is 90.4 ± 8.1%.

Example 6: Evaluation of L-lactic acid-producing ability of Pj0957 strain in a medium using sucrose as a single sugar source The lactic acid-producing ability of Pj0957 strain was evaluated as described below. The L-lactic acid concentration in the medium was measured using a biochemistry analyzer (BA) manufactured by Wyeth Japan. For the discrimination of optical isomers, J. et al. K. Using F-kit D-lactic acid / L-lactic acid manufactured by International Co., Ltd., the method followed the attached protocol. Other various organic acid production amounts were performed using HPLC. As a sample subjected to analysis, a culture solution previously filtered through a 0.22 μm filter was used. Each type of data is an average of the results of independent trials at least three times.

Inoculate a 3 mL YPD liquid medium in a 15 mL tube from a yeast cell cultured on a YPD agar medium for 2 to 3 days at 30 ° C. with a platinum loop. Pre-culture was performed for 20 to 30 hours at an amplitude of 35 mm, 130 rpm, and 30 ° C. using a culture apparatus. This is inoculated into 100 mL of YPD medium in a Sakaguchi flask so that the OD600 is about 0.1, and precultured at a 35 mm amplitude, 130 rpm, 30 ° C. for 15 to 22 hours with a table culture apparatus manufactured by TAITEC. went. Then, the cells were collected by centrifugation under conditions of 4 ° C., 3,000 rpm, and 5 minutes, and the supernatant was removed, followed by washing with a medium used for fermentation (without a neutralizing agent).

The bacterial cells thus obtained were inoculated into a 15 mL medium containing 100 g / L sucrose in a 100 mL Erlenmeyer flask with baffles, and fermented at 35 mm, 80 rpm, and 35 ° C. using a table-top culture apparatus manufactured by TAITEC. It was. The amount of cells to be inoculated for fermentation by the preculture was planted so that OD600 would be 10 unless otherwise specified. Unless otherwise specified, calcium carbonate was added to the medium to a concentration of 4.5% (w / v) as a neutralizing agent. The temperature during fermentation was 30 ° C. or 35 ° C. In addition to sucrose at the above concentration, 10 g / L yeast extract and 20 g / L peptone are added to the medium, and the medium having this composition is hereinafter referred to as YPsuc10 medium.

The total sugar conversion rate (%) is a value obtained by dividing the L-lactic acid weight in the medium by the initial sucrose weight in the medium, and further multiplying by (342/360) and 100. The optical purity (%) of L-lactic acid is a value obtained by dividing the value of L-lactic acid concentration by the value obtained by adding D-lactic acid concentration to L-lactic acid concentration and multiplying by 100.

As a result, regarding the amount of L-lactic acid produced after 33 hours, the total sugar conversion rate was 94.8 ± 3.5%, and the optical purity of L-lactic acid exceeded 99.9%. When the ethanol concentration was quantified by HPLC, the ethanol concentration was less than the detection limit (less than 0.01 g / L).

Claims (10)

1. Candida utilis yeast strain transformed with at least one copy of the gene operably linked to a promoter sequence enabling expression of a gene encoding a polypeptide having lactate dehydrogenase activity .
2. The yeast strain according to claim 1, wherein an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity is disrupted.
3. The yeast strain according to claim 1, wherein an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity is destroyed by deletion of the gene by insertion of a selection marker sequence.
4. The transformant according to any one of claims 1 to 3, which is transformed with an expression vector comprising a promoter sequence and a DNA sequence encoding a polypeptide having lactate dehydrogenase activity under the control of the promoter sequence. A yeast strain according to 1.
5. A polypeptide having lactate dehydrogenase activity is
   (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 37, or (b) an amino acid in which one or several amino acids are deleted, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 37 The yeast strain according to any one of claims 1 to 4, which is a polypeptide comprising a sequence and having a lactate dehydrogenase activity.
6. A gene encoding a polypeptide having lactate dehydrogenase activity is
   (A) Nucleotide sequence from 13th a to 1,011st a in SEQ ID NO: 36, or (b) Nucleotide sequence from 13th a to 1,011st a in SEQ ID NO: 36 A nucleotide sequence encoding a polypeptide having a homology of 85% or more and having lactate dehydrogenase activity, or (c) from SEQ ID NO: 36 from the 13th a to the 1,011st a The nucleotide sequence according to any one of claims 1 to 4, which comprises a nucleotide sequence that hybridizes with a nucleotide sequence or a complementary sequence thereof under stringent conditions and encodes a polypeptide having lactate dehydrogenase activity. Yeast strain.
7. An endogenous gene encoding a polypeptide having pyruvate decarboxylase activity comprises a nucleotide sequence encoding an amino acid sequence represented by SEQ ID NO: 64 or a nucleotide sequence represented by SEQ ID NO: 63; The yeast strain according to any one of claims 2 to 4.
8. The yeast according to any one of claims 1 to 4, wherein the promoter sequence is a promoter portion of an endogenous gene encoding pyruvate decarboxylase or comprises a nucleotide sequence represented by SEQ ID NO: 3. Strain.
9. A method for producing lactic acid, comprising culturing the yeast strain according to any one of claims 1 to 8.
10. 10. The method according to claim 9, wherein the OD600 of the microbial cell at the initial stage of fermentation culture is 1 to 30 in culturing a yeast strain.

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