JP2009261262A - Polypeptide having d-lactic acid dehydrogenase activity, polynucleotide encoding the polypeptide and method for producing d-lactic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new polypeptide having D-lactic acid dehydrogenase activity for producing D-lactic acid, to provide a polynucleotide encoding the polypeptide, and to provide a host cell to which the polynucleotide is introduced. <P>SOLUTION: The polypeptide having the D-lactic acid dehydrogenase activity comprises any one of the following amino acid sequences (A) to (C): (A) a specific amino acid sequence; (B) an amino acid sequence having one or several amino acids substituted, deleted, inserted and/or added with, from and/or to the amino acid sequence; and (C) an amino acid sequence having at least 80% or more homology with the amino acid sequence. The polynucleotide encodes the polypeptide. The host cell into which the polynucleotide is introduced is also provided. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a novel polypeptide having D-lactic acid dehydrogenase activity for producing D-lactic acid, a polynucleotide encoding the polypeptide, and a host cell into which the polynucleotide has been introduced, and further culturing the host cell. The present invention relates to a method for producing D-lactic acid.

  Polylactic acid, which is a biodegradable polymer, has attracted a great deal of attention from the viewpoint of carbon neutral, and high purity, efficient and inexpensive production methods are required for lactic acid, which is a raw material.

  Most of the polylactic acid currently produced is L-lactic acid polymer, but lactic acid has two types of optical isomers, L-lactic acid and D-lactic acid. In recent years, it has been attracting attention as an intermediate. However, in any application, L-lactic acid and D-lactic acid as raw materials are required to have high substance purity and optical purity, and particularly in production of polylactic acid, very high substance purity and optical purity are required. It is a fact.

In nature, there are bacteria that efficiently produce lactic acid such as lactic acid bacteria, and some lactic acid production methods using these bacteria have already been put into practical use. For example, Lactobacillus delbrueckii ( Lactobacillus delbrueckii) is a bacterium that efficiently produces L-lactic acid, and a Bacillus laevolacticus microorganism is known as a bacterium that efficiently produces D-lactic acid. (Patent Document 1) and a D-lactate dehydrogenase enzyme gene have also been isolated (Patent Document 2) In either case, the amount of accumulated lactic acid has reached a high level in anaerobic culture. In the case of D-lactic acid, D-lactic acid is produced as a by-product in the case of D-lactic acid fermentation, resulting in a decrease in optical purity, and it is very difficult to separate them.

In order to avoid such a decrease in the optical purity of lactic acid, it is effective to produce D-lactic acid by a microorganism that does not originally produce lactic acid. One microorganism that does not produce lactic acid is yeast. Among yeast, baker's yeast is preferable, and Saccharomyces cerevisiae is particularly suitable for this purpose because it has abundant genome information and has a sufficient track record as a genetically modified host.

So far, there have been several attempts to produce D-lactic acid by yeast,
Production of D-lactic acid into which D-lactate dehydrogenase enzyme gene derived from Bacillus laevolacticus has been introduced (Patent Document 2), D-lactate dehydrogenase derived from Leuconostoc mesenteroides , a kind of lactic acid bacteria An example of wine yeast into which an enzyme gene has been introduced (Patent Document 3) is known. However, in the former, the production amount and efficiency of lactic acid, and in the latter, the cultivation time is as long as 4 days and the production rate of lactic acid is slow. In addition, since a natural medium is used for cultivation, a large amount of impurities derived from the medium is contained in the culture medium. Inclusion is a problem, and it has been desired to establish a method for producing D-lactic acid with low impurities and high efficiency and high production rate.
JP2003-088392 JP2007-074939 WO2004 / 104202A1

  An object of the present invention is to provide a polypeptide having D-lactate dehydrogenase activity more suitable for D-lactate fermentation by introduction and expression in a host cell, and a polynucleotide encoding the polypeptide.

  As a result of intensive studies on various D-lactate dehydrogenases, the present inventors have found that a polypeptide having D-lactate dehydrogenase activity that increases the production amount of D-lactate and at the same time generates little by-products, and a polynucleotide encoding the same. The present invention has been completed.

  That is, this invention consists of the following aspects.

(1) A polypeptide having the amino acid sequence of any one of (A) to (C) below and having D-lactate dehydrogenase enzyme activity.
(A) The amino acid sequence set forth in SEQ ID NO: 1.
(B) An amino acid sequence in which one to several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1.
(C) an amino acid sequence having at least 80% homology with the amino acid sequence set forth in SEQ ID NO: 1.

(2) A polynucleotide encoding a polypeptide comprising the base sequence of any one of (a) to (c) below and having D-lactate dehydrogenase enzyme activity.
(A) a base sequence encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(B) A base sequence encoding a polypeptide consisting of an amino acid sequence in which one to several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1.
(C) a base sequence encoding a polypeptide comprising an amino acid sequence having at least 80% homology with the amino acid sequence set forth in SEQ ID NO: 1.

(3) A polynucleotide encoding a polypeptide having the base sequence of any one of (d) to (f) below and having D-lactate dehydrogenase enzyme activity.
(D) The base sequence set forth in SEQ ID NO: 2.
(E) A base sequence in which one to several bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 2.
(F) A polynucleotide comprising a base sequence that hybridizes under stringent conditions with the whole or a part of the base sequence shown in SEQ ID NO: 2 or a part thereof.

  (4) A host cell into which the polynucleotide according to claim 2 or 3 is introduced.

  (5) The host cell according to (4), wherein the cell is a bacterium, yeast, animal cell or insect cell.

  (6) The host cell according to (5), wherein the yeast is Saccharomyces cerevisiae.

  (7) A method for producing D-lactic acid, comprising culturing the host cell according to (4) to (6).

  According to the present invention, a polypeptide having D-lactate dehydrogenase activity suitable for D-lactate fermentation in cells, particularly yeast, and a polynucleotide encoding the same are provided, and a transformant capable of producing D-lactate at high yield is provided. D-lactic acid can be efficiently produced with high purity.

  Hereinafter, embodiments of the present invention will be described in detail.

(Polypeptide, Polynucleotide)
In the present invention, D-lactate dehydrogenase (hereinafter, also referred to as D-LDH) activity means reduced nicotinamide adenine dinucleotide (NADH) and pyruvate to D-lactic acid and oxidized nicotinamide adenine dinucleotide (NAD +). Activity to convert.

Polypeptide having D-LDH activity in the present invention is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 as D-LDH from scallop (Patinopecten yessoensis). Furthermore, the polypeptide in the present invention also includes a homologue of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and these homologues are composed of 1 to several amino acids in the amino acid sequence shown in SEQ ID NO: 1. A polypeptide having a substituted, deleted, inserted and / or added amino acid sequence and having D-LDH activity, or at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1 , Preferably 90%, more preferably 95% or more of a polypeptide having D-LDH activity. In addition, the homology of an amino acid sequence can be easily examined using BLAST which is software widely used in this field. BLAST is available to anyone on the NCBI (National Center for Biotechnology Information) homepage, and can be easily checked for identity using default parameters.

  In addition, the homologue of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 may have improved D-LDH activity and improved thermal stability than the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. May be. Such a polypeptide may be artificially designed based on the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or may be separated from nature.

  The polynucleotide of the present invention is a polynucleotide encoding the polypeptide having the D-LDH activity, preferably DNA or RNA, and derived from cDNA, genomic DNA, synthetic DNA, mRNA, synthetic RNA, replicon RNA, etc. Is not a question. Further, it may be a single strand or a double strand having its complementary strand. In addition, natural or artificial nucleotide derivatives may be included.

  The polynucleotide of the present invention is preferably a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or a homologue thereof. As a homologue of the polynucleotide of the present invention, for example, a polynucleotide comprising a base sequence in which one to several bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 2, or A polynucleotide that hybridizes under stringent conditions with the polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2 or the whole or a part of its complementary strand, and simultaneously encodes a polypeptide having D-LDH activity Things. As the polynucleotide that hybridizes under stringent conditions, for example, one or a plurality of contiguous sequences of at least 20, preferably 25, more preferably at least 30 of the original base sequence were selected. Polynucleotides that can be hybridized using a polynucleotide as a probe and hybridization techniques known to those skilled in the art (Current Protocols I Molecular Biology edit. Ausubel et al., (1987) Publish. John Wily & SonsSectoin 6.3-6.4). It is a nucleotide. Here, as stringent conditions, for example, in the presence of 50% formamide, the hybridization temperature is 37 ° C., more severe conditions are 42 ° C., more severe conditions are 65 ° C., and 0.1 to 2 times the concentration of SSC ( saline-sodium citrate) solution (composition of 1-fold concentration SSC solution: 150 mM sodium chloride, 15 mM sodium citrate). In addition, the homologue of the polynucleotide of the present invention preferably comprises a base sequence having at least 80%, preferably 90% or more, more preferably 95% or more homology with the base sequence of SEQ ID NO: 2. The homology of the nucleotide sequence of the polynucleotide can be determined by a gene analysis program BLAST or the like.

  The polypeptide of the present invention can be extracted from scallops by a known method, or can be prepared by using a known method known as a peptide synthesis method, and a polynucleotide encoding the amino acid sequence of the polypeptide of the present invention is used. It can also be prepared by gene recombination techniques.

  The polynucleotide of the present invention can be prepared by cloning from scallops as described in the Examples. However, the method of Fujimoto et al. Hideya, Synthetic Gene Production Method, Plant Cell Engineering Series 7 Plant PCR Experiment Protocol, 1997, Shujunsha, p95-100).

  In addition, the polypeptide or polynucleotide of the present invention can be converted into a site-specific displacement introduction method (Current Protocols I Molecular Biology edit. Ausubel etal., (1987) Publish. John Wily & Sons Sectoin 8.1-8.5). ) And the like, and can be appropriately modified by introducing substitution, deletion, insertion, and / or addition mutation. Moreover, the modification of the amino acid sequence of such a polypeptide or the base sequence of a polynucleotide is not limited to artificially introduced or synthesized mutations, but based on or not limited to artificial mutation treatment in nature. Those caused by amino acid mutations are also included.

(Polynucleotide construct)
By transforming a host cell with the polynucleotide of the present invention and expressing a polypeptide encoded by this polynucleotide, D-lactic acid can be produced in the host cell by its D-LDH activity. For transformation, a polynucleotide construct that enables expression of a polynucleotide segment comprising the polynucleotide of the present invention in a host cell is used. As a preferable embodiment of the polynucleotide construct for transformation, plasmid (DNA), bacteriophage (DNA), retrotransposon (DNA), artificial chromosome (YAC, PAC, BAC, MAC, etc.), replicon RNA, foreign gene Can be selected and employed according to the type of introduction (extrachromosomal or intrachromosomal) and the type of host cell. Accordingly, the polynucleotide construct of the present invention can comprise the constituent segments of the vector of any of these aspects in addition to the polynucleotide of the present invention. Preferred prokaryotic, eukaryotic, animal and plant cell vectors are well known in the art.

  Examples of plasmid DNA include YCp type E. coli-yeast shuttle vectors such as pRS413, pRS415, pRS416, YCp50, pAUR112 and pAUR123, YEp type E. coli-yeast shuttle vectors such as pYES32 and YEp13, pRS403, pRS404, pRS405, pRS406. YIp type E. coli-yeast shuttle vectors such as pAUR101 or pAUR135, plasmids derived from E. coli (pBR322, pBR325, pUC18, pUC19, pUC119, pTV118N, pTV119N, pBluescript, pHSG298, pHSG396, AC7 such as pCrc P1A plasmid, pM 118, pMW119, pMW218 or pSC101-based plasmids such as pMW219 and the like), plasmids derived from Bacillus subtilis (e.g., and the like pUB110, pTP5, etc.).

  Examples of the phage DNA include λ phage (Charon 4A, Charon 21A, EMBL3, EMBL4, λgt100, gt11, zap), φX174, M13mp18, and M13mp19.

  Examples of the retrotransposon include Ty factor. Examples of YAC include pYACC2.

  Examples of replicon RNA include bromomosaic virus-derived replicons.

  Among the polynucleotide constructs described above, in particular, in order to prepare a DNA construct, a fragment containing the DNA of the present invention is cleaved with an appropriate restriction enzyme and inserted into a restriction enzyme site or a multicloning site of the vector DNA to be used. It depends. The first aspect of the DNA construct of the present invention comprises a promoter segment that is linked so that the DNA segment comprising the DNA of the present invention can be expressed. That is, the DNA segment of the present invention is linked to the downstream side of the promoter so that it can be controlled by the promoter.

  In the expression of the polypeptide of the present invention, since expression in yeast is preferable, it is preferable to use a promoter that is expressed in yeast in the polynucleotide construct. Examples of such promoters include pyruvate decarboxylase gene promoter, glyceraldehyde-3-phosphate dehydrogenase gene promoter, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PH05 promoter, PGK promoter, GAP promoter, It is preferable to use an ADH promoter, an AOX1 promoter, or the like. In particular, a pyruvate decarboxylase 1 (PDC1) promoter or glyceraldehyde-3-phosphate dehydrogenase gene promoter derived from the genus Saccharomyces is preferable, and a pyruvate decarboxylase 1 gene promoter or glyceraldehyde-3 derived from Saccharomyces cerevisiae is preferable. -More preferably, a phosphate dehydrogenase gene promoter is used. This is because these promoters are highly expressed in the ethanol fermentation pathway of Saccharomyces. Such a promoter sequence can be isolated by a PCR amplification method using the PDC1 gene of Saccharomyces yeast or the genomic DNA of glyceraldehyde-3-phosphate dehydrogenase gene as a template.

  The second aspect of the DNA construct which is the polynucleotide construct comprises a DNA segment for homologous recombination of the host chromosome in addition to the DNA of the present invention. The DNA segment for homologous recombination is a DNA sequence that is homologous to the DNA sequence in the vicinity of the target site to which the DNA of the present invention is to be introduced in the host chromosome. At least one, preferably two, DNA segments for homologous recombination are provided. For example, it is preferable that two DNA segments for homologous recombination are DNA sequences that are homologous to DNA upstream and downstream of the target site on the chromosome, and the DNA of the present invention is linked between these DNA segments. .

  When the DNA of the present invention is introduced into a host chromosome by homologous recombination, the DNA of the present invention can be introduced in a controllable manner by a promoter on the host chromosome. In this case, the introduction of the target gene can simultaneously destroy the endogenous gene that should be controlled by the promoter, and the exogenous DNA of the present invention can be expressed in place of the endogenous gene. It is particularly useful when the promoter is a high expression promoter in the host cell.

  In order to create such an expression system on the host chromosome, a highly expressed gene is targeted in the host chromosome, and the DNA of the present invention is introduced downstream of the promoter controlling this gene so as to be controlled by the promoter. Is preferred. When an ethanol-fermenting microorganism such as yeast is used as a host, D-LDH activity is controlled under the control of an endogenous pyruvate decarboxylase gene promoter by targeting a pyruvate decarboxylase gene, particularly a pyruvate decarboxylase 1 gene. DNA encoding the polypeptide can be introduced. In this case, the DNA segment for homologous recombination is the D-LDH structural gene region of pyruvate decarboxylase 1 gene or a sequence in the vicinity thereof (sequence in the vicinity of the start codon, sequence in the upstream region of the start codon, And the like) and the like. Preferably, a Saccharomyces genus, particularly a cerevisiae, is used as a host, and a DNA construct targeting the pyruvate decarboxylase 1 gene of this host is used. According to such a DNA construct, destruction of the pyruvate decarboxylase 1 gene and replacement of this structural gene part with D-LDH can be achieved simultaneously. Pyruvate decarboxylase 1 is an enzyme that mediates an addition reverse reaction from pyruvate to acetaldehyde. By destroying this gene, ethanol production via acetaldehyde is expected to be suppressed, and pyruvate It can be expected that lactic acid production by D-LDH using as a substrate is promoted.

  Even the first DNA construct can be made into a DNA construct for homologous recombination by providing a DNA segment for homologous recombination with the host chromosome. In the first DNA construct, the promoter segment in the DNA construct can also be used as a DNA segment for homologous recombination with the host chromosome. For example, for a host Saccharomyces cerevisiae, a DNA construct having a promoter segment in the Saccharomyces cerevisiae host chromosome, for example, a pyruvate decarboxylase 1 gene promoter, as a promoter segment constitutes a targeting vector having the gene as a target site. . In this case, it is preferable to provide a homologous sequence to the structural gene region of the pyruvate decarboxylase 1 gene.

  In addition, a terminator, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, and a ribosome binding sequence (SD sequence) can be linked to the DNA construct as necessary. The selectable marker is not particularly limited, and various known selectable marker genes such as drug resistance genes and auxotrophic genes can be used. For example, a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, etc. can be used.

(Transformation with polynucleotide construct)
Once the polynucleotide construct is constructed, transformation, transfection, conjugation, protoplast fusion, electroporation, lipofection, lithium acetate, particle gun, calcium phosphate precipitation can be performed on appropriate host cells. It can be introduced by any suitable means such as Agrobacterium method, PEG method, direct microinjection method. After introduction of the polynucleotide construct, the recipient cells are cultured in a selective medium.

Host cells are not particularly limited, but bacteria such as Escherichia coli and Bacillus subtilis , insect cells such as yeast, sf9 and sf21, COS cells, and Chinese hamster ovary cells (CHO cells) It can be an animal cell or the like. Preferably, yeasts such as Saccharomyces genus, Schizosaccharomyces genus, Pichia genus, particularly baker's yeast used for alcoholic fermentation and bread production, more preferably Saccharomyces cerevisiae , as a specific example Is Saccharomyces cerevisiae NBRC10505 strain. In the present invention, the baker's yeast is a yeast used first for the production of bread, and secondly a yeast obtained by mating with the baker's yeast, and is used as an experimental yeast. It may be a thing.

  In the transformant transformed with the polynucleotide construct, the components of the polynucleotide construct are present on the chromosome or on extrachromosomal factors (including artificial chromosomes). When the polynucleotide construct is maintained extrachromosomally or is integrated into the chromosome by random integration, other enzymes using pyruvate, which is a substrate of D-LDH, for example, pyruvin The gene for acid decarboxylase (pyruvate decarboxylase 1 gene in Saccharomyces yeasts) is preferably knocked out by a targeting vector. When a polynucleotide construct capable of achieving homologous recombination is introduced, it can be controlled by the promoter downstream of a desired promoter on the host chromosome or a homologue of the promoter replaced with the promoter. There will be a polynucleotide segment which is a polynucleotide of the present invention linked to. In the transformant of Saccharomyces yeast, on the host chromosome, the polynucleotide of the present invention is controllable by the promoter downstream of the pyruvate decarboxylase 1 gene promoter or a homologue of the promoter replaced with the promoter. It is preferable to provide. Further, usually, a selection marker gene and a part of a disrupted structural gene (site corresponding to the homologous sequence on the polynucleotide construct) are present downstream of the polynucleotide of the present invention in the homologous recombinant.

  Whether or not the polynucleotide of the present invention has been introduced under a desired promoter can be confirmed by PCR or Southern hybridization. For example, it can be confirmed by preparing DNA from a transformant, performing PCR with an introduction site-specific primer, and detecting the expected band in electrophoresis for the PCR product. Alternatively, it can be confirmed by performing PCR with a primer labeled with a fluorescent dye or the like. These methods are well known to those skilled in the art.

(Production of D-lactic acid)
By culturing the transformant obtained by introducing the polynucleotide construct, D-lactic acid is produced in the culture by the action of D-lactate dehydrogenase, which is an expression product of a foreign gene. D-lactic acid can be obtained by carrying out the step of separating D-lactic acid from the culture. In the present invention, the culture includes cultured cells or microbial cells, or crushed cells or cells in addition to the culture supernatant. In culturing the transformant of the present invention, culture conditions can be selected according to the type of transformant. Such culture conditions are well known to those skilled in the art.

  As a medium for cultivating transformants obtained using microorganisms such as bacteria and yeast as a host, it contains carbon sources, nitrogen sources, inorganic salts, etc. that can be assimilated by the microorganisms, so that the transformants can be cultured efficiently. As long as the medium can be used, either a natural medium or a synthetic medium can be used. As the carbon source, carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used. As the nitrogen source, ammonium salt of inorganic acid or organic acid such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, etc. may be used. it can. Examples of inorganic substances that can be used include potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

As a medium for culturing a transformant obtained by using animal cells as a host, generally used RPMI 1640 medium, DMEM medium, or a medium obtained by adding fetal calf serum or the like to these mediums can be used. The culture is usually performed at 37 ° C. for 1 to 30 days in the presence of 5% CO ₂ . During the culture, antibiotics such as kanamycin and penicillin may be added to the medium as necessary.

  As a medium for culturing a transformant obtained using insect cells as a host, generally used Grace medium, TC-100 medium, or a medium obtained by adding fetal calf serum or the like to these mediums can be used. . The culture is usually performed at 27 ° C. for 1 to 30 days. During the culture, antibiotics such as kanamycin and penicillin may be added to the medium as necessary.

  The culture of the transformant should usually be performed under conditions where productivity of lactic acid is good, in a range from aerobic conditions such as shaking culture or aeration and agitation culture to anaerobic conditions in which aeration is not performed. However, conditions of slight aerobic to anaerobic are preferably used. The culture temperature can be 25 ° C to 37 ° C, preferably 30 ° C to 33 ° C. Since the pH of the culture solution decreases as D-lactic acid accumulates in the medium, it can be neutralized with an alkali, preferably a hydroxide of divalent ions. Further, in order to facilitate the purification of lactic acid in the subsequent stage, it can be cultured without neutralization.

  In order to purify D-lactic acid from the D-lactic acid culture solution obtained as described above, it can be purified by a conventionally known method. For example, after separating cells from the obtained D-lactic acid culture solution with a filter, the pH is adjusted to 1 or less and then extracted with diethyl ether, ethyl acetate or the like, or the method is eluted after adsorbing and washing with an ion exchange resin. There are a method of distilling as an ester by reacting with alcohol in the presence of an acid catalyst, a method of crystallizing as a calcium salt or a lithium salt, a method of purifying with a nanofilter, or a combination thereof. Examples thereof include a method of purification or a method of distilling a concentrated D-lactic acid solution obtained by evaporating moisture after separating cells from the obtained D-lactic acid culture solution using a filter. Here, when distilling, it is preferable to distill while supplying moisture so that the concentration of water in the distillation stock solution is constant. After distilling off the D-lactic acid aqueous solution, the water can be concentrated by heating to evaporate to obtain purified D-lactic acid having a target concentration. When a D-lactic acid aqueous solution containing a low-boiling component (ethanol, acetic acid, etc.) is obtained as a distillate, it is preferable to remove the low-boiling component during the D-lactic acid concentration process. In addition, impurities can be removed from the distillate after the distillation operation by ion exchange resin, activated carbon, chromatographic separation, or the like, if necessary, to obtain higher purity D-lactic acid.

  Hereinafter, embodiments of the present invention will be described by way of examples, but these are merely examples and do not limit the present invention in any way.

(Example 1) were crushed scallop from D-LDH gene cloning scallop (Patinopecten yessoensis) scallops, using the obtained crushed material from Quick prep mRNA purification kit (GE Healthcare Bio-Sciences KK) poly A (+) RNA was isolated, and cDNA was synthesized using Oligo d (T) as a primer using SuperScript II Reverse Transcriptase (Invitrogen). Next, using this reaction solution as a template, PCR was performed using oligo DNAs of SEQ ID NO: 3 and SEQ ID NO: 4 designed by paying attention to highly conserved DNA sequences in D-LDH, and scallop-derived D- A partial sequence of LDH was amplified. PCR was performed under the following conditions. 95 ° C. (3 minutes): {95 ° C. (30 seconds) −45 ° C. (30 seconds) −72 ° C. (30 seconds)} × 35 cycles: 72 ° C. (5 minutes). Subsequently, the amplified DNA fragment was confirmed by electrophoresis using a 1.0% agarose gel, the 600 bp amplified fragment was purified using QIA quick Gel extraction kit (manufactured by QIAGEN), and then the plasmid vector pGEMt-easy ( And the DNA sequence was determined. The base sequence was determined according to the method of Sanger using Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biochemical).

  Next, the entire ORF sequence of scallop-derived D-LDH was revealed using 5'RACE and 3'RACE methods. 5 'RACE was performed by the following method. First, cDNA was synthesized by using SuperScript II Reverse Transcriptase (manufactured by Invitrogen) as a template and scallop poly A (+) RNA as a template and using the oligo DNA described in SEQ ID NO: 5 as a primer. Next, using terminal deoxynucleotidyl transferase (manufactured by New England Biolabs), poly (C) tail was added to the 3 ′ end of the cDNA by adding dCTP to the reaction solution. This reaction solution was purified using a QIA quick PCR purification kit (Qiagen), and the PCR was performed for the first time using the oligo DNAs described in SEQ ID NO: 5 and SEQ ID NO: 6 as primers as a template. PCR was performed a second time using the oligo DNAs described in SEQ ID NO: 7 and SEQ ID NO: 8 as primers. Subsequently, the amplified DNA fragment was confirmed by electrophoresis using a 1.0% agarose gel, the amplified fragment was purified using a QIA quick Gel extraction kit (manufactured by QIAGEN), and then plasmid vector pGEMt-easy (Promega stock) The DNA sequence was determined. The base sequence was determined according to the method of Sanger using Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biochemical).

  3'RACE was performed by the following method. First, cDNA was synthesized using scallop poly A (+) RNA as a template and SuperScript II Reverse Transcriptase (manufactured by Invitrogen) using Oligo d (T) as a primer. Next, the first PCR is performed using this reaction solution as a template and Oligo d (T) and the oligo DNA described in SEQ ID NO: 9 as primers. Further, Oligo d (T) and SEQ ID NO: 10 are described using the reaction solution as a template. PCR was performed a second time using the oligo DNA as a primer. Subsequently, the amplified DNA fragment was confirmed by electrophoresis using a 1.0% agarose gel, the amplified fragment was purified using QIA quick Gel extraction kit (manufactured by Qiagen), and then plasmid vector pGEMt-easy (Promega stock) The DNA sequence was determined. The base sequence was determined according to the method of Sanger using Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biochemical).

  The amino acid sequence (SEQ ID NO: 1) and ORF sequence (SEQ ID NO: 2) of scallop-derived D-LDH of the present invention were determined by joining the sequences obtained by the above method.

(Example 2) Construction of D-LDH expression plasmid derived from scallop D-LDH Using scallop poly A (SEQ ID NO: 11 and SEQ ID NO: 12 corresponding to the ORF 5 'side and ORF 3' side) as primers, +) PCR was performed using cDNA synthesized from RNA as a template. The obtained DNA fragment was cleaved with restriction enzymes XhoI and NotI, and introduced into the cleavage site of yeast expression vector pTRS11 (FIG. 1) cleaved with restriction enzymes XhoI and NotI. Hereinafter, this plasmid vector is referred to as pTRS11PyDLDH. Here, the pTRS11 vector was prepared by cleaving the pNV11 vector (Nature, vol357, 25 JUNE 1992, p700) with the restriction enzyme XhoI and self-ligating.

(Example 3) Introduction of scallop-derived D-LDH expression plasmid into yeast pTRS11PyDLDH obtained as in Example 2 was transformed into the yeast Saccharomyces cerevisiae NBRC10505 strain. Transformation was performed by the lithium acetate method using YEASTMAKER Yeast Transformation System (Clontech). For details, the attached protocol was followed. The Saccharomyces cerevisiae NBRC10505 strain used as a host is a strain lacking the ability to synthesize uracil, and by the action of the URA3 gene possessed by pTRS11PyDLDH, a transformant having pTRS11PyDLDH introduced therein can be selected on a uracil-free medium.

  Confirmation of D-LDH gene expression vector introduction into the transformant thus obtained was performed using a genomic DNA extraction kit Gen Toru-kun (manufactured by Takara Bio Inc.) from a transformant cultured in a liquid medium not added with uracil. The genomic DNA containing plasmid DNA was extracted by PCR and PCR was performed using this as a template. The primer used when cloning the scallop-derived D-LDH gene was used. As a result, it was confirmed that the scallop-derived D-LDH gene was introduced into all the transformants.

(Example 4) D-lactic acid productivity test A D-lactic acid productivity test was carried out using the Saccharomyces cerevisiae NBRC10505 strain (hereinafter referred to as NBRC10505 / pTRS11PyDLDH strain) into which pTRS11PyDLDH obtained as in Example 3 was introduced. went.

  10 mL of a medium having the composition shown in Table 1 (hereinafter abbreviated as SC-Ura medium) was placed in a test tube, a small amount of each strain was inoculated therein, and cultured overnight at 30 ° C. (preculture). Next, 10 mL of SC-Ura medium was put in a test tube, 100 μL of each preculture was inoculated, and cultured with shaking at 30 ° C. (main culture). The culture solution for 40 hours after the start of the main culture was centrifuged, and lactic acid contained in the supernatant was measured by HPLC under the following conditions.

Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
Detection method: electrical conductivity Temperature: 45 ° C.

  Table 2 shows the accumulated concentration of D-lactic acid calculated from the measurement results.

(Example 5) Introduction of scallop-derived D-LDH gene into yeast chromosome PCR using pTRS11PyDLDH containing scallop-derived D-LDH gene obtained in Example 2 as a template and oligo DNAs described in SEQ ID NOs: 13 and 14 as primers Thus, a DNA fragment of about 1.1 kb containing each D-LDH gene was amplified (corresponding to step 1 in FIG. 2). Here, the oligo DNA described in SEQ ID NO: 13 was designed so that a sequence having homology was added to the upstream 65 bp of the PDC1 gene.

Next, a DNA fragment of about 1.4 kb containing the TRP1 gene as a yeast selection marker was amplified by PCR using plasmid pRS424 (GenBank Accession Number: U03453) as a template and the oligo DNAs described in SEQ ID NOs: 15 and 16 as primers (see FIG. Equivalent to step 2 in FIG. 2). Here, the oligo DNA described in SEQ ID NO: 16 was designed so that a sequence having homology was added to 65 bp downstream of the PDC1 gene.
The DNA fragments were separated by 1.5% agarose gel electrophoresis, and then purified using QIA quick Gel extraction kit (Qiagen). D-LDH was obtained by PCR using a mixture of the obtained 1.1 kb fragment containing the D-LDH gene and the 1.4 kb fragment containing the TRP1 gene as a template and using the oligo DNAs described in SEQ ID NOs: 13 and 16 as primers. A DNA fragment of about 2.5 kb to which the gene, GAPDH terminator and TRP1 gene were linked was amplified (corresponding to step 3 in FIG. 2).

  The above DNA fragments are separated by 1.5% agarose gel electrophoresis, purified using QIA quick Gel extraction kit (manufactured by Qiagen), transformed into Saccharomyces cerevisiae NBRC10505 strain, and cultured in a tryptophan-free medium. Thus, a transformant in which the D-LDH gene was introduced downstream of the PDC1 gene promoter on the chromosome was selected.

  Confirmation that the transformant obtained as described above was a yeast in which the D-LDH gene was introduced downstream of the PDC1 gene promoter on the chromosome was performed as follows. First, the genomic DNA of the transformant was prepared with a genomic DNA extraction kit Gen Toru-kun (manufactured by Takara Bio Inc.), and this was used as an amplification template, and PCR was performed using oligonucleotides (SEQ ID NOs: 17 and 18) as primer sets. This was confirmed by obtaining a 3.2 kb amplified DNA fragment. In the non-transformed strain, an amplified DNA fragment of about 2.4 kb can be obtained by the PCR. Hereinafter, a transformant in which the D-LDH gene derived from scallop is introduced downstream of the PDC1 gene promoter on the chromosome is referred to as a PyDLDH strain.

(Example 6) D-lactic acid productivity test Example 4 was performed using the PyDLDH strain obtained as in Example 5 and a medium having the composition shown in Table 3 (hereinafter abbreviated as SC-Trp medium). A D-lactic acid productivity test was conducted in the same manner. As a result of the measurement, L-lactic acid was not detected in any experimental group. Table 4 shows the optical purity derived from the accumulation of lactic acid and the measurement limit.

(Example 7) D-lactic acid productivity test A D-lactic acid productivity test was carried out using the PyDLDH strain obtained as in Example 5. 10 mL of SC-Trp medium was placed in a test tube, a small amount of PyDLDH strain and the like were inoculated therein, and cultured overnight at 30 ° C. (pre-culture). Next, 100 mL of SC-Trp medium was placed in a 500 ml Erlenmeyer flask, and each pre-culture solution was inoculated in its entirety and cultured with shaking at 30 ° C. for 24 hours (pre-culture). Subsequently, the pre-culture solution 24 hours after the start of pre-culture was inoculated in a mini jar fermenter (manufactured by Maruhishi Bio-Engineering Co., Ltd., volume 5 L) charged with 1 L of SC-Trp medium, and the stirring speed (120 rpm ), Aeration volume (0.1 L / min), temperature (30 ° C.), and 1N calcium hydroxide to maintain the pH (pH 5) constant (main culture). After centrifuging the culture solution for 40 hours after the start of the main culture, lactic acid and acetic acid were measured under the conditions described in Example 4, and the yield to sugar was calculated by the following equation.
Sugar yield (%) = total amount of lactic acid / total amount of glucose added to the culture system.

  As a result of the measurement, L-lactic acid was not detected. Table 5 shows the optical purity derived from the accumulation of lactic acid and the measurement limit.

(Example 8) Purification of lactic acid Concentrated sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise to the culture solution obtained in Example 7 until the pH was 2.0, and then stirred at 25 ° C for 1 hour. Calcium lactate was converted into lactic acid and calcium sulfate. Next, the precipitated calcium sulfate was filtered off by suction filtration using qualitative filter paper No. 2 (manufactured by Advantech). Further, in order to separate impurities, the filtrate was passed through a nanofilter (piperazine polyamide semipermeable membrane UTC60, manufactured by Toray Industries, Inc.) to collect permeated water. As a result of analyzing the concentration of lactic acid and acetic acid contained in the permeated water by the method described in Example 4, the permeated water contained 18.6 g / L lactic acid and 0.18 g / L acetic acid. Purified lactic acid was purified (Table 6).

Comparative Example 1 D-Lactic Acid Productivity Test of Yeast Introduced with Bacillus laevolacticus-derived D-LDH Expression Plasmid NBRC10505 / pTM63 strain into which a D-LDH expression plasmid derived from Bacillus laevolacticus was introduced (See JP 2007-74939), and the D-lactic acid productivity test was performed under the same conditions as in Example 4. Table 2 shows the accumulated concentration of D-lactic acid calculated from the measurement results.
(Comparative Example 2) Leuconostoc Looking At Troy Death (Leuconostoc mesenteroides) derived from, Fusobacterium Nukureatomu (Fusobacterium nucleatum) derived from Clostridium acetobutylicum (Clostridium acetobutylicum) derived from, cyclo-off Lexus Torukuizu (Psychroflexus torquis) from each D- Cloning of LDH gene Leuconostoc mescentroides (NBRC3426) from the National Institute of Biotechnology Resources (NBRC), Fusobacterium nucleatom (ATCC25586), Clostridium acetobutylicum (ATCC824), Cycloflexus Torque (A TCC70075) was obtained from American Type Culture Collection (ATCC), and genomic DNA was purified using Gen Toru-kun (manufactured by Takara Bio Inc.). PCR was performed using this genomic DNA as a template, and DNA fragments encoding each D-LDH described in SEQ ID NOs: 19 to 22 were obtained.

  As a primer, for amplification of the D-LDH gene derived from Leuconostoc mescentroides (Accession No. AB233384, SEQ ID NO: 19), the oligo DNA described in SEQ ID NOs: 23 and 24 was used, and the D-LDH gene derived from Fusobacterium nucleatom For the amplification of (Accession No. AE009951, SEQ ID NO: 20), the oligo DNA described in SEQ ID NOs: 25 and 26 was used. For the amplification of the D-LDH gene derived from Clostridium acetobutylicum (Accession No. AE001437, SEQ ID NO: 21), SEQ ID NO: The oligo DNAs described in 27 and 28 were used to amplify the cycloflexus torques-derived D-LDH gene (refer to the D-LDH gene region from the whole genome sequence registered under Accession No. NZ_AAPR01000005, SEQ ID NO: 22). Oligo DNAs described in SEQ ID NOs: 29 and 30 were used.

  The amplified fragment obtained as described above was cloned into the plasmid vector pTRS11. Hereinafter, these plasmid vectors are referred to as pTRS11LmDLDH, pTRS11FnDLDH, pTRS11CaDLDH, and pTRS11PtDLDH, respectively.

(Comparative Example 3) Introduction of each D-LDH gene into yeast chromosome pTM63 containing Bacillus laevolacticus-derived D-LDH gene disclosed in JP-A-2007-74939, Leucono obtained in Comparative Example 2 SEQ ID NOs: 14 and 31, SEQ ID NOs: 14 and 31, respectively, using pTRS11LmDLDH, pTRS11FnDLDH, pTRS11CaDLDH, pTRS11PtDLDH including D-LDH genes derived from Stock Mecentroides, Fusobacterium nucleatum, Clostridium acetobutylicum, and Cycloflexus torqueis, respectively. 32, a DNA fragment of about 1.1 kb containing each D-LDH gene was amplified by PCR using oligo DNAs described in SEQ ID NOs: 14 and 33, SEQ ID NOs: 14 and 34, and SEQ ID NOs: 14 and 35 as primers. (Corresponding to step 1 in FIG. 2). Here, the oligo DNAs described in SEQ ID NOs: 31 to 35 were designed so that a sequence having homology was added to the upstream 65 bp of the PDC1 gene.

  Next, a DNA fragment of about 1.4 kb containing the TRP1 gene, which is a yeast selection marker, was amplified by the method described in Example 5 (corresponding to step 2 in FIG. 2). Each DNA fragment was purified by the method described in Example 5. The obtained mixture of 1.1 kb fragment containing each D-LDH gene and 1.4 kb fragment containing TRP1 gene was used as a template to obtain SEQ ID NO: 16 and 31, SEQ ID NO: 16 and 32, SEQ ID NO: 16 and 33, respectively. A DNA fragment of about 2.5 kb to which each D-LDH gene, GAPDH terminator and TRP1 gene were linked was amplified by PCR using oligo DNAs described in SEQ ID NOs: 16 and 34 and SEQ ID NOs: 16 and 35 as primers (FIG. 2). Equivalent to step 3).

  After purifying the above DNA fragment by the method described in Example 5, it is transformed into Saccharomyces cerevisiae NBRC10505 strain and cultured in a medium without tryptophan, so that each D-LDH gene is located downstream of the PDC1 gene promoter on the chromosome. An introduced transformant was selected. It was confirmed by the method described in Example 5 that the transformant thus obtained was a yeast in which each D-LDH gene was introduced downstream of the PDC1 gene promoter on the chromosome. In the following, D-LDH genes derived from Bacillus laevolyticus, Leuconostoc mescentroides, Fusobacterium nucleatum, Clostridium acetobutylicum and Cycloflexus torques are introduced downstream of the PDC1 gene promoter on the chromosome. These transformed strains are designated as BLDLDH strain, LmDLDH strain, FnDLDH strain, CaDLDH strain, and PtDLDH strain, respectively.

Comparative Example 4 D-Lactic Acid Productivity Test Using the BlDLDH strain, FnDLDH strain, CaDLDH strain, and PtDLDH strain obtained as in Comparative Example 3, the D-lactic acid productivity test was performed according to the method described in Example 6. Went. Table 4 shows the accumulated concentration of D-lactic acid calculated from the measurement results.

(Comparative Example 5) D-lactic acid productivity test A D-lactic acid productivity test was carried out by the method described in Example 7 using the LmDLDH strain obtained as in Comparative Example 3. Table 5 shows the sugar yield of D-lactic acid calculated from the measurement results, and the accumulated concentrations of lactic acid and acetic acid. The optical purity of D-lactic acid was measured under the conditions described in Example 6. As a result of the measurement, L-lactic acid was not detected. Table 5 shows the optical purity derived from the accumulation of lactic acid and the measurement limit. As a result, the fermentation results of D-lactic acid using the synthetic medium of yeast introduced with D-LDH gene derived from Leuconostoc mescentroides and yeast introduced with D-LDH gene derived from scallop are the latter, It was excellent in both yield and by-product formation.

(Comparative Example 6) Purification of lactic acid The culture solution obtained in Comparative Example 5 was purified by the same method as in Example 8. As a result, 16.5 g / L lactic acid and 0.62 g / L acetic acid were used as permeated water. (Table 6).

  As described above, as shown in Examples and Comparative Examples, various D-LDHs were introduced into yeast, and as a result of examining the lactic acid fermentation titer, when yeast introduced with a scallop-derived D-LDH gene was used, impurities were mixed. It has been clarified that high-purity lactic acid with a small amount can be produced in a high yield.

FIG. 1 is a diagram showing a physical map of the expression vector pTRS11 for yeast used in the present invention. FIG. 2 is a schematic diagram illustrating a method for introducing the D-LDH gene downstream of the PDC1 gene promoter.

Claims (7)

A polypeptide having the amino acid sequence of any one of (A) to (C) below and having D-lactate dehydrogenase enzyme activity.
(A) The amino acid sequence set forth in SEQ ID NO: 1.
(B) An amino acid sequence in which one to several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1.
(C) an amino acid sequence having at least 80% homology with the amino acid sequence set forth in SEQ ID NO: 1. A polynucleotide comprising a nucleotide sequence of any one of (a) to (c) below and encoding a polypeptide having D-lactate dehydrogenase enzyme activity.
(A) a base sequence encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(B) A base sequence encoding a polypeptide consisting of an amino acid sequence in which one to several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1.
(C) a base sequence encoding a polypeptide comprising an amino acid sequence having at least 80% homology with the amino acid sequence set forth in SEQ ID NO: 1. A polynucleotide encoding a polypeptide having the nucleotide sequence of any one of (d) to (f) below and having D-lactate dehydrogenase enzyme activity.
(D) The base sequence set forth in SEQ ID NO: 2.
(E) A base sequence in which one to several bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 2.
(F) A polynucleotide comprising a base sequence that hybridizes under stringent conditions with the whole or a part of the base sequence shown in SEQ ID NO: 2 or a part thereof.   A host cell into which the polynucleotide according to claim 2 or 3 has been introduced.   The host cell according to claim 4, wherein the cell is a bacterium, yeast, animal cell or insect cell.   The host cell according to claim 5, wherein the yeast is Saccharomyces cerevisiae.   A method for producing D-lactic acid, comprising culturing the host cell according to claim 4.

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