JP2005102625A - Method for producing d-lactic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing D-lactic acid containing decreased amount of pyruvic acid accumulated as an impurity organic acid and difficult to be removed from a medium containing the produced and accumulated D-lactic acid and provide a method for producing D-lactic acid having high optical purity in high efficiency. <P>SOLUTION: D-lactic acid is produced by the aeration culture of an Escherichia coli prepared by transferring a gene encoding D-lactic acid dehydrogenase (excluding the gene encoding D-lactic acid dehydrogenase derived from Escherichia coli). The production method of D-lactic acid comprises the culture of the Escherichia coli in a medium under aeration to form and accumulate D-lactic acid in the medium. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing D-lactic acid characterized by culturing Escherichia coli and producing and accumulating D-lactic acid in a medium. Specifically, the D-lactic acid has a high purity, particularly a low amount of pyruvic acid produced and accumulated. Involved in efficient manufacturing methods. More specifically, the present invention relates to a gene encoding a D-lactate dehydrogenase (hereinafter sometimes abbreviated as ldh) (hereinafter, referred to as D-lactate dehydrogenase derived from E. coli (hereinafter abbreviated as ldhA)). The present invention relates to Escherichia coli in which D-lactic acid is produced when cultured under aerated conditions, and a method for producing D-lactic acid by culturing the Escherichia coli.

Polylactic acid, which is a biodegradable polymer, has attracted strong attention as a product that supports sustainability and LCA (life cycle assessment) along with the emergence of CO ₂ and energy problems. There is a demand for an efficient and inexpensive production method for lactic acid.

  Incidentally, currently produced polylactic acid is an L-lactic acid polymer, but lactic acid may be abbreviated as L-lactic acid (hereinafter abbreviated as L-form) and D-lactic acid (hereinafter abbreviated as D-form). ), And the D form has recently been attracting attention as an intermediate for polymer raw materials, agricultural chemicals, and pharmaceuticals. However, in any application, high purity is required for the L-form and D-form as raw materials.

  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 and the like are known as bacteria that efficiently produce L-form, and microorganisms belonging to the genus Sporolactobacillus are known as bacteria that produce D-form efficiently. In either case, the amount of lactic acid accumulated in anaerobic culture has reached a high level, but by-products other than lactic acid contained in the culture solution, such as acetic acid, ethanol, acetoin, and pyruvic acid, must be removed during the purification process. The quality of the final product, lactic acid, may be reduced.

  In order to avoid such a decrease in the purity of lactic acid, it is most effective to reduce the amount of by-products produced by bacteria. If a specific gene of a microorganism is destroyed by using a genetic recombination technique that has been developed in recent years, it has become possible to specifically inhibit the production of a target by-product. However, at present, the gene disruption method is not easily adaptable to any bacteria, and it is difficult to adapt to bacteria that can originally produce lactic acid, such as lactic acid bacteria. This is because lactic acid bacteria do not necessarily have sufficient genome information and are not widely used as genetic recombination hosts.

  On the other hand, Escherichia coli can be mentioned as a bacterium with abundant genome information and a sufficient track record as a genetically modified host.

  According to a report by Zou et al. [Zhou, S., Appl Environ Microbiol (2003) 69 399-407], pyruvate formate lyase (pflB), fumarate reductase (frdABCD) (hereinafter abbreviated as frd), Alcohol / aldehyde dehydrogenase (hereinafter sometimes abbreviated as adhE) and acetate kinase (hereinafter sometimes abbreviated as ackA) fourfold disrupted E. coli were cultured in an inorganic salt medium containing 5% glucose for 168 hours under anaerobic conditions. As a result, 48.5 g / L of D-form is produced, and the amount of by-products such as succinic acid, formic acid, acetic acid and ethanol in the medium is significantly reduced. However, there is no mention of pyruvic acid, and the effect of reducing the ability to produce pyruvic acid is unknown. Since pyruvic acid is a precursor of metabolic reaction of D-form, for example, if the enzyme that catalyzes the reaction from phosphoenolpyruvate to pyruvic acid is destroyed, the amount of pyruvic acid accumulated will decrease, but at the same time the amount of D-form accumulated It is expected to cause undesirable results, such as a decline. In general, when pyruvic acid is contained as an impurity in the lactic acid monomer raw material, it is well known to those skilled in the art that undesirable problems such as a decrease in the polymer polymerization rate occur. Is one of the by-products that should be reduced. Nevertheless, no report has been made in the past that the pyruvic acid accumulation amount has been successfully reduced without reducing the D body accumulation amount.

  Contag et al. (Contag, PR., Appl Environ Microbiol (1990) 56 3760-3765) clones the gene for D-lactate dehydrogenase derived from Clostridium acetobutylicum, and therefore uses E. coli strain FMJ39 that cannot grow under anaerobic conditions as a host. We are conducting expression experiments. Strains obtained by introducing the ldh gene derived from Clostridium acetobutylicum into FMJ39 can be grown under anaerobic conditions, so the ldh gene is cloned using this property. In this experiment, they disclosed lactic acid production concentrations and ldh activity of strains into which two types of expression plasmids pPC37 and pPC58, each incorporating an Ldh gene fragment derived from Clostridium acetobutylicum, were introduced into FMJ39, respectively. As a result, although the ldh activity was higher in FMJ39 (pPC37) than in FMJ39 (pPC58), the amount of lactic acid produced was lower in FMJ39 (pPC37). That is, although Contag et al. Expressed the Ldh gene derived from Clostridium acetobutylicum in Escherichia coli, it did not show that the productivity of lactic acid was improved, and nothing was mentioned about it. Also, there is no mention of pyruvic acid, which is a by-product.

  Moreover, as an example of overexpression of ldh derived from bacteria different from Contag et al. In Escherichia coli, as an example of Ldh derived from Lactobacillus helveticus, Cocker et al. (Kochhar, S., Eur. J. Biochem., (1992) 208, 799-805] and Lactobacillus bulgaricus-derived ldh expression examples include Cocker et al. (Kochhar, S., Biochem. Biophys. Res. Commun., (1992) 185, 705-712). The physicochemical properties of the enzyme were examined, and there is no mention of the accumulated amount of D-form or pyruvic acid.

  In other words, in the past, there was an example of overexpression of lactate dehydrogenase other than E. coli in Escherichia coli, but it was expressed under aeration conditions during the production of lactic acid and succeeded in significantly reducing pyruvate accumulation at the same time as high production of lactic acid. There was nothing I did.

  A mutant having a mutation in pyruvate dehydrogenase (hereinafter abbreviated as pdh) using a transposon and / or a mutation related to pyruvate formate lyase using a mutation agent was introduced in 1983 by Chang et al. [Chang, YY. , J. Bacteriol., (1983) 154,756-762] and is well known. Moreover, the production example of D body using these strains has not been reported.

In the case of lactic acid fermentation using Escherichia coli, it is generally considered that the presence of oxygen is not preferable. This is because in the presence of an electron acceptor such as oxygen, E. coli breathes, not fermented. Only when there is no electron acceptor such as oxygen, E. coli obtains energy (ATP) only by phosphorylation at the substrate level, and reductive organic substances such as lactic acid using the reducing power (NADH) obtained in glycolysis. Produce acid. For this reason, conventional D-form fermentation by Escherichia coli is mostly carried out by anaerobic culture. In rare cases, the first half of the culture is aerated and the second half is anaerobically cultured, but this is intended to ensure a sufficient amount of cells by aeration culture in the first half. Lactic acid fermentation is still anaerobic. However, assuming actual industrial production, corn steep liquor (hereinafter sometimes abbreviated as CSL), which is an inexpensive amino acid source added to the medium, includes not only organic acids that are impurities, but also D-form, L-form. Both bodies contain lactic acid, but in anaerobic culture, the L form is not assimilated and remains in the medium. Since L-lactate dehydrogenase, which is a catalytic enzyme for the reaction of generating pyruvic acid from L-form, is known to be expressed under aeration conditions, if there is a method for efficient D-lactic acid fermentation under aeration conditions, By using this method, it is expected that the L-form contained in the medium will be assimilated into the microbial cells, and it will be possible to produce optically high-purity D-form. There was no technology to do.
Zou et al., Applied and Environmental Microbiology, United States, (2003) 69 399-407 [Zhou, S., Appl Environ Microbiol, United States (2003) 69 399-407] Contag et al., Applied and Environmental Microbiology, United States, (1990) 56 3760-3765 [Contag, PR., Appl Environ Microbiol, United States (1990) 56 3760-3765] Cocker et al., European Journal of Biochemistry, Germany (1992) 208, 799-805 [Kochhar, S., Eur. J. Biochem., Germany (1992) 208, 799-805] Cocker et al., Biochemical and Biophysical Research Communications, USA (1992) 185, 705-712 [Kochhar, S., Biochem. Biophys. Res. Commun., (1992) 185, 705-712] Chang et al., Journal of Biotechnology, United States, (1983) 154 756-762 [Chang, YY., J. Bacteriol. (1983) 154 756-762]

  A first object of the present invention is a method for producing D-lactic acid in which the amount of pyruvic acid that has not been easily removed from a medium in which D-lactic acid is conventionally produced and accumulated by microorganisms as an impurity organic acid has been reduced. Is to provide. The second object of the present invention is to provide a method for producing D-lactic acid that efficiently produces D-lactic acid having high optical purity.

  As a result of intensive studies to solve the above-mentioned problems, the inventors have introduced Escherichia coli into which a D-lactate dehydrogenase gene (excluding those derived from Escherichia coli) has been introduced and aerated in a medium containing glucose. E. coli devised to express significantly more D-lactate dehydrogenase gene than wild type even in culture, more preferably pyruvate formate lyase (pfl), and / or pyruvate dehydrogenase (pdh). When E. coli whose activity has been reduced or disappeared is cultured in a medium and D-lactic acid is produced and accumulated, the concentration of pyruvic acid, which is an impurity produced and accumulated in the medium accompanying the production of D-lactic acid, is significantly reduced. Even if D-lactic acid is produced at a high concentration in a shorter time and the optical isomer L-lactic acid is contained in the medium, the optical purity is 99.9% ee or more. - it has reached the onsets present invention viewed to produce lactic acid.

That is, the present invention is as follows.
[1] Escherichia coli introduced with a gene encoding D-lactate dehydrogenase (excluding a gene encoding D-lactate dehydrogenase derived from Escherichia coli), and when cultured under aerated conditions, D-lactate E. coli characterized by producing.
[2] A gene encoding D-lactate dehydrogenase in Escherichia coli having the property of accumulating pyruvate (in culture medium) (when cultured under anaerobic conditions) (except for those derived from Escherichia coli) The Escherichia coli according to [1], which is obtained by introducing
[3] The Escherichia coli according to [1] or [2], which has lipoic acid requirement.
[4] E. coli according to any one of [1] to [3], wherein the function of pyruvate formate lyase is deleted.
[5] The gene encoding D-lactate dehydrogenase is linked to a promoter of a gene responsible for expression of a protein involved in glycolysis, nucleic acid biosynthesis or amino acid biosynthesis [1] to [ [4] The Escherichia coli according to any one of [4].
[6] The E. coli according to any one of [1] to [5], wherein the gene encoding D-lactate dehydrogenase is derived from lactic acid bacteria.
[7] The gene encoding D-lactate dehydrogenase is derived from Lactobacillus plantarum and / or Pediococcus acidilactici [1] ] E. coli according to any one of [5] to [5].
[8] The D-, characterized in that the E. coli according to any one of [1] to [7] is cultured using a medium under aerated conditions, and D-lactic acid is produced and accumulated in the medium. Lactic acid production method.
[9] The method for producing D-lactic acid according to [8], wherein the pH of the medium during culture is 4 to 8.
[10] The method for producing D-lactic acid according to [9], wherein the pH of the medium during the culture is 6 to 7.
[11] The culture was performed using the culture tank, atmospheric air to a temperature 30 ° C. water at operating conditions as aeration conditions at that time is the volumetric oxygen transfer coefficient k _L a a 1h ^-1 or 400h ^-1 or less The method for producing D-lactic acid according to any one of [8] to [10], wherein the conditions are equivalent to an oxygen supply amount that can be achieved when the culture tank is supplied under pressure.

  The present invention provides Escherichia coli that produces D-lactic acid with a small amount of pyruvate. And it is possible to produce D-lactic acid with higher chemical purity and optical purity more economically than existing methods by culturing the cells prepared according to the present invention and producing D-lactic acid. It becomes.

  Hereinafter, the present invention will be described in detail. First, the E. coli of the present invention will be described below.

  The gene (ldh) encoding D-lactate dehydrogenase in the present invention is a gene other than E. coli that encodes an enzyme that produces lactic acid and NAD from pyruvate and NADH, and may be a gene other than those derived from E. coli. There are no particular restrictions on its origin. As such, specifically, Lactobacillus plantarum obtained by Taguchi et al. [Taguchi, H., J Biol Chem 5; 266 (19): 12588-94 (1991)], and / or Illustrate Pediococcus acidilactici obtained by Jamin et al. [Garmyn D., J Bacteriol 177 (12): 3427-37 (1995)] and other D-lactic acid dehydrogenase genes derived from lactic acid bacteria. Can do.

  Moreover, the gene encoding D-lactate dehydrogenase used for such genetic recombination is subjected to mutation after isolation of the gene encoding D-lactate dehydrogenase from a strain other than E. coli, and the gene before mutation is encoded. In addition, a gene encoding a mutant having one or several amino acids deleted, substituted, added or inserted into D-lactate dehydrogenase and having D-lactate dehydrogenase activity is also included. Such mutants can be made, for example, by adding, deleting or substituting one or several bases. Means for addition, deletion or substitution with one or several bases are known per se, such as random mutagenesis, site-directed mutagenesis, gene homologous recombination, or polymerase The chain amplification method (PCR) can be performed alone or in appropriate combination. For example, a method of replacing cytosine base with uracil base by chemical treatment using sodium bisulfite, a method of performing PCR in a reaction solution containing manganese, and reducing the accuracy of nucleotide incorporation during DNA synthesis, site Various commercially available kits for introducing specific mutations can also be used. For example, edited by Sambrook et al. [Molecular Cloning, Laboratory Manual 2nd Edition] Cold Spring Harbor Laboratory, 1989, edited by Masami Muramatsu [Lab Manual Genetic Engineering] Maruzen Co., Ltd., 1988, Ehrlich, HE. Chapter [PCR Technology, Principles and Applications of DNA Amplification] It can be carried out according to the method described in the book of Stockton Press, 1989, etc., or by modifying those methods.

  The Escherichia coli of the present invention is an Escherichia coli that can be obtained by introducing the gene encoding the above-mentioned D-lactic acid dehydrogenase into Escherichia coli that can produce lactic acid by heterolactic fermentation. It is characterized by producing. Here, the introduction of a gene encoding D-lactate dehydrogenase (except for those derived from Escherichia coli) means that D-lactate dehydrogenase is encoded on the chromosome of Escherichia coli and / or on a self-replicating vector. It is shown that a gene encoding D-lactate dehydrogenase other than that derived from E. coli is present in E. coli so that it can be expressed.

  In addition, when cultured under aerated conditions, D-lactic acid is produced when the gene encoding the introduced D-lactate dehydrogenase is expressed under aerated culture conditions and compared with that before introduction of the gene. It shows that it has the capability to improve the accumulated amount of D-lactic acid during production.

  The Escherichia coli of the present invention may accumulate pyruvic acid in the medium along with the production of D-lactic acid. In that case, pyrubin produced in the medium during lactic acid production using microorganisms as conventionally known. Much less than the amount of acid.

  It is characterized by accumulating pyruvic acid in the present invention that, when cultured in a medium containing glucose under aeration conditions, the amount of pyrubin is significantly larger than that of E. coli wild strain MG1655 (ATCC 47076) or W3110 (ATCC 39936). It means Escherichia coli that accumulates acid, and specific examples include Escherichia coli W1485lip2 (ATCC25645) strain, Escherichia coli MT-10934 strain, and strains in which the gene encoding pyruvate dehydrogenase of any Escherichia coli wild strain is disrupted.

  Pyruvate dehydrogenase (pdh) refers to an enzyme that produces acetyl CoA from pyruvate. Pyruvate dehydrogenase requires lipoic acid to express its activity. Therefore, the lipoic acid-requiring strain that has lost the ability to biosynthesize lipoic acid can be controlled in its activity by lipoic acid supplied from the medium. The lipoic acid requirement in the present invention is that growth is not possible in a medium not containing lipoic acid, but growth is possible by adding lipoic acid to the medium. Such Escherichia coli is specifically W1485lip2 strain ( ATCC 25645). For the reasons described above, the Escherichia coli of the present invention may have lipoic acid requirement.

  The Escherichia coli of the present invention may further lack the pyruvate formate lyase function. Pyruvate formate lyase refers to an enzyme that produces formic acid from pyruvate. Note that pyruvate formate lyase is composed of two gene products, the pflA gene and the pflB gene. When it is not necessary to distinguish the two, it is simply written as pfl, and when only the pflB gene is pointed out, it is written as pflB.

  In the present invention, the lack of the function of pyruvate formate lyase is a strain in which the activity of pyruvate formate lyase is significantly reduced compared to the E. coli wild strain MG1655 strain or W3110 strain. Including. Specifically, strains in which the gene encoding pyruvate formate lyase (pflB) of Escherichia coli MT-10934 strain and any wild strain of Escherichia coli is disrupted can be exemplified.

  MT-10934 has already been completely destroyed by pyruvate dehydrogenase mutation using a transposon, and pyruvate formate lyase is reduced in its activity by a mutagen. Therefore, the present invention can be easily used to carry out the present invention. Is possible. This strain is the deposit number of FERM P-19092 and has been deposited on November 8, 2002 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi 1-1-1, Tsukuba, Ibaraki Has been.

  In the Escherichia coli of the present invention, a gene encoding D-lactate dehydrogenase can be linked to a promoter of a gene that controls expression of a protein involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis.

  In the present invention, the promoter of a gene that controls the expression of a protein involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis is a strong promoter that functions constantly in E. coli and suppresses expression even in the presence of glucose. Specific examples of promoters that are not easily received include glyceraldehyde 3-phosphate dehydrogenase promoter (GPA) and serine hydroxymethyltransferase (glyA) promoter.

  The lactic acid bacterium in the present invention is a microorganism in which 50% or more of an organic acid produced by fermentatively degrading sugar is converted to lactic acid. Specifically, Lactobacillus plantarum (Lactobacillus plantarum), Pediococcus reed An example is Pediococcus acidilactici.

  The Escherichia coli of the present invention is designed to be expressed in the presence of 50 g / L or more of glucose, and is 10% or more of D-lactic acid as compared to before introduction when producing 50 g / L or more of D-lactic acid. It is preferable that the amount of pyruvic acid is improved and the pyruvic acid produced at the same time is designed and introduced so that it has the ability to significantly decrease. Specific examples include MT-10934 / pGLPldh strain into which lactobacillus plantarum lactate dehydrogenase has been introduced, and MT-10934 / pGPAldh strain into which lactate dehydrogenase from Pediococcus acidilacticis has been introduced.

  Next, the method for culturing Escherichia coli and the method for producing D-lactic acid according to the present invention will be described.

  The culture for lactic acid production in the present invention means culturing Escherichia coli according to the present invention using a medium. At that time, the medium to be used is not particularly limited as long as the prepared microbial cells contain a nutrient source necessary for producing D-lactic acid. Examples of such nutrient sources include carbon sources, nitrogen sources, inorganic ions, organic trace elements required by cells, nucleic acids, vitamins, and the like. Can do.

  In order to obtain more preferable results, a medium containing two or more amino acids is preferable. As the form of addition of such amino acids, a combination of two or more kinds of natural amino acids, a natural extract containing a plurality of amino acids such as yeast extract, casamino acid, peptone, whey, molasses, corn steep liquor, etc. as a mixture And hydrolysates of natural product extracts. A medium containing at least one kind selected from yeast extract, peptone, whey, molasses, corn steep liquor, etc., or a mixture thereof is more preferably 0.5% by mass to 20% by mass, more preferably 1% by mass to 10% by mass. Then, it is more preferable. In particular, the addition of corn steep liquor or yeast extract is more effective than the addition of peptone or casamino acid. At this time, it is better to add no salt such as ammonium sulfate. The medium is usually a liquid medium.

  The culture may be carried out using a flask or the like and a liquid medium in the flask for culturing. Usually, the liquid medium is placed in a culture tank and the temperature is adjusted and stirred.

  The culture conditions can be appropriately changed depending on the type of E. coli cells produced and the culture apparatus, but are suitable conditions for culturing E. coli. , Preferably 6-7.

For example, when MT-10934 having a reduced pyruvate formate lyase activity is used, the culture temperature is preferably 20 ° C. to 40 ° C., more preferably 25 ° C. to 35 ° C., and the pH is NaOH, NH _3. It is preferable to adjust and culture at 4.0 to 8.0, more preferably 6 to 7, etc.

  The aeration condition of the present invention does not necessarily require air to pass through the culture medium, and depending on the shape of the culture tank, the top surface ventilation may be used to ventilate the air layer above the culture liquid while stirring the culture liquid appropriately. It means a state in which a gas containing oxygen is introduced into the inside of the culture tank.

  The aeration conditions during culturing of the present invention can produce D-lactic acid without aeration at all. However, aeration is performed to obtain more favorable results such as reduction of impurities and improvement of optical purity. Is good.

  When aerated in the culture solution in the culture tank, the dissolved oxygen concentration changes depending on the combination of the internal pressure of the culture tank, the position of the stirring blade, the shape of the stirring blade, and the stirring speed. The optimum conditions can be obtained as follows using the amount of organic acid as an index. For example, when culturing the above E. coli in a relatively small culture vessel such as the culturing apparatus BMJ-01 manufactured by ABLE, when using 500 g of culture solution, the air is 0.01 vvm to 1 vvm at normal pressure, and the stirring speed is 50 rpm. Favorable results can be obtained under aeration conditions that can be achieved at 500 rpm, more preferably 0.1 vvm to 0.5 vvm at normal pressure and a stirring speed of 100 rpm to 400 rpm.

The above aeration conditions are such that when the aeration and stirring conditions are for water at a temperature of 30 ° C., the oxygen transfer rate coefficient k _L a is 1 h ^{−1 to} 400 h ^{−1 and} more preferably 1 h ^{−1 to} 200 h at normal pressure. It is a condition that enables oxygen supply that can be achieved under conditions of ⁻¹ or less. In addition, as another index of optimum aeration conditions, formic acid, acetic acid, succinic acid, glycerol, ethanol, etc. that MT-10934 normally produces in anaerobic culture is 5.0 g / L or less, more preferably 1.0 g / L or less. And the aeration condition achieved by the aeration rate and the stirring speed at which D-lactic acid is produced. Further, another index of optimum aeration conditions is that within 10 to 100 hours when cultured in a culture solution containing 0.3% optical isomer L-lactic acid during the production of D-lactic acid by MT-10934. The aeration amount and the stirring speed are such that the concentration of L-lactic acid is reduced to 0.02% by mass or less.

  The aeration conditions described above do not need to be consistently performed from the beginning of the culture to the end, and preferable results can be obtained by performing it in part of the culture process.

  The culture in the present invention refers to the microbial cells produced by the above-described method, a culture solution containing the microbial cells, a culture solution from which the microbial cells have been removed, and a processed product thereof.

  As a method for recovering D-lactic acid from a culture such as the culture solution obtained as described above, a conventionally known method can be used. For example, a method of acidifying the culture and then directly distilling the lactate lactate A method in which lactic acid is formed and distilled, a method in which lactic acid is esterified by adding an alcohol and a catalyst, a method in which distillation is performed, a method in which lactic acid is extracted into an organic solvent, a method in which lactic acid is separated with an ion exchange column, and lactic acid is concentrated and separated by electrodialysis A method of combining them or a method of combining them can be adopted.

Although an example of the present invention is shown below by an example, these do not limit the present invention at all. Unless otherwise specified, “%” is based on mass.
Example 1 (Construction of D-lactate dehydrogenase expression vector and D-lactate producing bacterium)
In order to obtain the serine hydroxymethyltransferase (glyA) promoter, the Escherichia coli genomic DNA was used as a template, amplified by PCR with SEQ ID NO: 1 and SEQ ID NO: 2, and the resulting DNA fragment was digested with the restriction enzyme EcoRI. As a result, a fragment encoding the glyA promoter of about 850 bp was obtained. Furthermore, in order to obtain the D-lactate dehydrogenase structural gene (ldhA), Escherichia coli genomic DNA (obtained by extraction from the ATCC 47076 strain by a conventional method) was used as a template and amplified by the PCR method with SEQ ID NO: 3 and SEQ ID NO: 4. The obtained DNA fragment was digested with restriction enzymes EcoRI and HindIII to obtain an about 1.0 kbp D-lactate dehydrogenase structural gene (ldhA) fragment.

  The above two DNA fragments and the fragment obtained by digesting plasmid pUC18 with restriction enzymes EcoRI and HindIII are mixed, ligated with ligase, and transformed into E. coli (DH5α strain) to obtain plasmid pGlydhA. It was.

  Similarly, a genomic DNA obtained from Lactobacillus plantarum (ATCC8041) was used as a template, and a DNA fragment encoding a Lactobacillus plantarum-derived D-lactate dehydrogenase gene was amplified using SEQ ID NO: 5 and SEQ ID NO: 6 to obtain an expression plasmid pGLPldh. Furthermore, Escherichia coli genomic DNA was used as a template, EcoRI and SacI cleaved fragments of DNA fragments obtained by PCR using SEQ ID NO: 1 and SEQ ID NO: 9 and genomic DNA obtained from Pediococcus acidilactici (ATCC) were used as templates, and SEQ ID NO: 7 SacI, HindIII digestion fragment of DNA fragment obtained by PCR method using SEQ ID NO: 8 and fragment obtained by digesting plasmid pUC18 with restriction enzymes EcoRI and HindIII were ligated to express P-diococcus acidilactici-derived D-lactate dehydrogenase Plasmid pGPAldh was obtained.

  The three plasmids pGlyldA, pGLPldh, and pGPAldh obtained as described above were transformed into E. coli W1485lip2 (ATCC25645) and E. coli MT-10934 as D-lactic acid-producing bacteria. Six transformants of pGPAldh, MT-10934 / pGlyldA, MT-10934 / pGLPldh, MT-10934 / pGPAldh were obtained.

  W1485lip2 strain is available as ATCC25645 from American Type Culture Collection. The vector plasmid pUC18 is obtained by extraction from ATCC 37253 available from the American Type Culture Collection by a conventional method. In addition, MT-10934 strain is deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, as described above.

  Example 2 (D-lactic acid production by D-lactic acid-producing bacteria W1485lip2 / pGlydhA strain, W1485lip2 / pGLPldh strain, W1485lip2 / pGPAldh strain)

  Prepare a plurality of LB Broth, Miller culture medium (Difco244620) 25 ml in a 100 ml Erlenmeyer flask with baffle. After inoculation, stirring culture was performed overnight at 120 rpm to prepare a preculture solution.

  The total amount of the preculture was separately inoculated into 4 culture mediums having 475 g of the composition described in (Table 1) in the culture tank of the culture device BMJ-01 manufactured by ABLE. Culturing was carried out under atmospheric pressure for 24 hours at an aeration rate of 0.5 vvm, a stirring speed of 600 rpm, a culture temperature of 37 ° C., and a pH of 7.3 (adjusted with NaOH). After completion of the culture, quantification of D-lactic acid in the culture and measurement of optical purity were performed by HPLC according to a conventional method. The results are shown in Table 2. The concentrations of formic acid, acetic acid, and succinic acid in each culture solution were all 0.3 g / L or less.

Example 3 (D-lactic acid production by D-lactic acid-producing bacteria MT-10934 / pGlyldA strain, MT-10934 / pGLPldh strain, MT-10934 / pGPAldh strain (addition effect of casamino acid))
A plurality of Erlenmeyer flasks containing 25 g of LB Broth, Miller culture solution (Difco244620) were prepared as precultures. Three strains of D-lactic acid producing strain MT-10934 strain, MT-10934 / pGlyldA strain, MT-10934 / pGLPldh strain, MT-10934 / pGPAldh strain were inoculated separately, and overnight at 30 ° C. Agitation culture was performed at 120 rpm to obtain a preculture solution.

  All the preculture liquids prepared previously were inoculated into 4 L culture vessels (cultivation apparatus BMJ-01, manufactured by ABLE) containing 475 g of the medium shown in (Table 3). The main culture was performed at atmospheric pressure for 120 hours at an aeration rate of 0.5 vvm, a stirring speed of 200 rpm, a culture temperature of 31 ° C., and a pH of 6.8 (adjusted with NaOH). Table 4 shows the results of quantification of D-lactic acid in the obtained main culture solution after completion of the culture, as determined by HPLC according to a conventional method.

Example 4 (D-lactic acid production by D-lactic acid producing bacteria MT-10934 / pGlyldA, MT-10934 / pGLPldh, MT-10934 / pGPAldh (addition effect of corn steep liquor))
In the medium composition of the main culture, corn steep liquor 50.0 g / L was added instead of casamino acid 5.0 g / L, and the culture time of the main culture was set to 50 hours. The results are shown in Table 5. The main culture medium contains 0.34% reducing sugars derived from corn steep liquor, 0.31% D-lactic acid, 0.31% L-lactic acid, 0.33% free amino acid, and a trace amount. Various organic acids are included.

Claims (11)

  Escherichia coli introduced with a gene encoding D-lactate dehydrogenase (except for a gene encoding D-lactate dehydrogenase derived from Escherichia coli), which produces D-lactic acid when cultured under aerated conditions. E. coli characterized by   2. The method according to claim 1, which is obtained by introducing a gene encoding D-lactate dehydrogenase (excluding those derived from Escherichia coli) into Escherichia coli having the property of accumulating pyruvic acid in the medium. The described E. coli.   The Escherichia coli according to claim 1 or 2, which has lipoic acid requirement.   The Escherichia coli according to any one of claims 1 to 3, wherein the function of pyruvate formate lyase is deleted.   The gene encoding D-lactate dehydrogenase is linked to a promoter of a gene that controls expression of a protein involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis. E. coli according to one item.   The E. coli according to any one of claims 1 to 5, wherein the gene encoding D-lactate dehydrogenase is derived from lactic acid bacteria.   6. The gene encoding D-lactate dehydrogenase is derived from Lactobacillus plantarum (Lactobacillus plantarum) and / or Pediococcus acidilactici. E. coli according to any one of the above.   A method for producing D-lactic acid, comprising culturing E. coli according to any one of claims 1 to 7 under aeration conditions using a medium, and producing and accumulating D-lactic acid in the medium.   The method for producing D-lactic acid according to claim 8, wherein the pH of the medium during the culture is 4 to 8.   The method for producing D-lactic acid according to claim 9, wherein the pH of the medium during the culture is 6-7. The cells were cultured using a culture tank, the culture aeration conditions the volumetric oxygen transfer coefficient k _L a is 1h ^-1 or 400h ^-1 or less become like air water temperature 30 ° C. In operating conditions normal pressure at that time The lactic acid production method according to any one of claims 8 to 10, wherein the lactic acid production conditions are equivalent to the oxygen supply amount that can be achieved when the tank is supplied.