JP2006238830A - New lactic acid dehydrogenase gene and method for producing lactic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene encoding a new lactic acid dehydrogenase, and to provide a method for producing lactic acid with the gene. <P>SOLUTION: This lactic acid dehydrogenase gene having high homology to the lactic acid dehydrogenase gene of Rhizopus oryzae known to have a lactic acid-producing ability, and originated from a genus Aspergillus microorganism. The method for producing the lactic acid comprises culturing a genus Aspergillus microorganism having the increased activity of the gene and then recovering the produced lactic acid. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel lactate dehydrogenase gene and a method for producing lactic acid.

Lactic acid is used as an acidulant and preservative in the manufacture of processed foods and beverages, and in raw materials for chemical products and pharmaceuticals. Furthermore, it is an important substance as a raw material for polylactic acid, which has been most developed as a green (plant) plastic, which has been attracting attention with increasing interest in environmental issues in recent years.
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 been known for a long time to specifically produce lactic acid, and is involved in the production of yogurt and the like. Lactic acid has D-type and L-type, and lactic acid bacteria generally produce a mixture of D-type and L-type lactic acid. However, as a raw material for polylactic acid for making plant plastic, lactic acid with high optical purity consisting only of L-type or D-type is required.

Rhizopus oryzae strains have been well known as filamentous fungi having the ability to produce lactic acid (for example, Wang, CW, Z. Lu, and GT Tsao. 1995. Lactic acid production by pellet-form Rhizopus oryzae in a Submerged system. Appl. Biochem. Biotechnol. 51/52: 57-71., Christopher D. Skory Isolation and expression of lactate dehydrogenase genes from Rhizopus oryzae. Appl Environ Microbiol. 2000 Jun; 66 (6): 2343-8.) . However, in order to produce lactic acid with this strain, it is necessary to add a neutralizing agent such as calcium carbonate, or it is necessary to inoculate with mycelium grown by seed culture in liquid culture prior to solid culture.
On the other hand, in the production of citric acid, which is the same organic acid, production is carried out in koji molds suitable for solid culture. However, no microorganism of the genus Aspergillus that produces a large amount of lactic acid has been known so far. In addition, a lactate dehydrogenase gene derived from the genome of an Aspergillus microorganism has not yet been found.

Wang, C. W., Z. Lu, and G. T. Tsao. 1995. Lactic acid production by pellet-form Rhizopus oryzae in a submerged system. Appl. Biochem. Biotechnol. 51/52: 57-71. Christopher D. Skory Isolation and expression of lactate dehydrogenase genes from Rhizopus oryzae.Appl Environ Microbiol. 2000 Jun; 66 (6): 2343-8. T. Yoneda et al. J. Protein Chem., 16, 597-605, 1997

  The present invention provides an enzyme that catalyzes a reaction for converting pyruvate to lactic acid, a gene encoding the enzyme, and a new method for producing lactic acid, derived from the genome of Aspergillus microorganisms.

In one aspect, the present invention is a method for producing lactic acid, comprising culturing a microorganism having an improved activity of catalyzing a reaction for converting pyruvic acid into lactic acid in a medium and producing lactic acid in the culture. In this method, the activity is improved by the expression amplification of the DNA according to any one of the following (a) to (j).
(A) DNA having the base sequence described in SEQ ID NO: 1
(B) DNA encoding the amino acid sequence set forth in SEQ ID NO: 2;
(C) DNA having the base sequence set forth in SEQ ID NO: 3;
(D) DNA having the base sequence set forth in SEQ ID NO: 4;
(E) DNA encoding the amino acid sequence set forth in SEQ ID NO: 5;
(F) DNA having the base sequence set forth in SEQ ID NO: 6;
(G) DNA having the base sequence set forth in SEQ ID NO: 7;
(H) DNA encoding the amino acid sequence set forth in SEQ ID NO: 8;
(I) DNA having the base sequence set forth in SEQ ID NO: 9; or
(J) encodes a protein that can hybridize with the DNA of any one of (a) to (i) under stringent conditions and has an activity of catalyzing a reaction of converting pyruvic acid into lactic acid DNA.

Further, the present invention provides the method according to the above method, wherein the microorganism increases the copy number of the DNA according to any one of the following (a) to (j), or modifies the expression control sequence of the DNA. It is also a method for producing lactic acid modified so that expression is enhanced.
(A) DNA having the base sequence described in SEQ ID NO: 1
(B) DNA encoding the amino acid sequence set forth in SEQ ID NO: 2;
(C) DNA having the base sequence set forth in SEQ ID NO: 3;
(D) DNA having the base sequence set forth in SEQ ID NO: 4;
(E) DNA encoding the amino acid sequence set forth in SEQ ID NO: 5;
(F) DNA having the base sequence set forth in SEQ ID NO: 6;
(G) DNA having the base sequence set forth in SEQ ID NO: 7;
(H) DNA encoding the amino acid sequence set forth in SEQ ID NO: 8;
(I) DNA having the base sequence set forth in SEQ ID NO: 9; or
(J) encodes a protein that can hybridize with the DNA of any one of (a) to (i) under stringent conditions and has an activity of catalyzing a reaction of converting pyruvic acid into lactic acid DNA.

In particular, the present invention is also a method for producing lactic acid, in which a microorganism is a transformant introduced with a DNA capable of expressing the DNA described in any of (a) to (g) below.
(A) DNA having the base sequence described in SEQ ID NO: 1
(B) DNA encoding the amino acid sequence set forth in SEQ ID NO: 2;
(C) DNA having the base sequence set forth in SEQ ID NO: 3;
(D) DNA having the base sequence set forth in SEQ ID NO: 4;
(E) DNA encoding the amino acid sequence set forth in SEQ ID NO: 5;
(F) DNA having the base sequence set forth in SEQ ID NO: 6;
(G) DNA having the base sequence set forth in SEQ ID NO: 7;
(H) DNA encoding the amino acid sequence set forth in SEQ ID NO: 8;
(I) DNA having the base sequence set forth in SEQ ID NO: 9; or
(J) encodes a protein that can hybridize with the DNA of any one of (a) to (i) under stringent conditions and has an activity of catalyzing a reaction of converting pyruvic acid into lactic acid DNA.

  In particular, the present invention is also a method for producing the above-mentioned lactic acid, wherein the microorganism is a filamentous fungus, particularly a filamentous fungus suitable for solid culture, or an Escherichia bacterium.

Furthermore, the present invention is also a DNA according to any one of the following (a) to (j).
(A) DNA having the base sequence set forth in SEQ ID NO: 1 or its complementary sequence
(B) DNA encoding the amino acid sequence set forth in SEQ ID NO: 2;
(C) DNA having the base sequence set forth in SEQ ID NO: 3 or its complementary sequence;
(D) DNA having the base sequence set forth in SEQ ID NO: 4 or a complementary sequence thereof;
(E) DNA encoding the amino acid sequence set forth in SEQ ID NO: 5;
(F) DNA having the base sequence set forth in SEQ ID NO: 6 or its complementary sequence;
(G) DNA having the base sequence set forth in SEQ ID NO: 7 or its complementary sequence;
(H) DNA encoding the amino acid sequence set forth in SEQ ID NO: 8;
(I) a DNA having the base sequence set forth in SEQ ID NO: 9 or a complementary sequence thereof; or
(J) encodes a protein that can hybridize with the DNA of any one of (a) to (i) under stringent conditions and has an activity of catalyzing a reaction of converting pyruvic acid into lactic acid DNA.

Moreover, this invention is also a protein in any one of following (a)-(f).
(A) a protein having the amino acid sequence of SEQ ID NO: 2;
(B) a protein having the amino acid sequence set forth in SEQ ID NO: 5;
(C) a protein having the amino acid sequence set forth in SEQ ID NO: 8;
(D) A reaction comprising an amino acid sequence comprising one or several amino acid substitutions, deletions, additions, insertions, or inversions in the amino acid sequence shown in SEQ ID NO: 2 and converting pyruvic acid to lactic acid. A protein with catalytic activity;
(E) a reaction comprising the amino acid sequence of SEQ ID NO: 5 comprising an amino acid sequence containing substitution, deletion, addition, insertion, or inversion of one or several amino acids, and converting pyruvic acid into lactic acid A protein with catalytic activity;
(F) a reaction comprising an amino acid sequence comprising one or several amino acid substitutions, deletions, additions, insertions or inversions in the amino acid sequence set forth in SEQ ID NO: 8, and converting pyruvic acid into lactic acid Protein with catalytic activity.

  INDUSTRIAL APPLICABILITY According to the present invention, a novel gene encoding an enzyme (lactate dehydrogenase) that catalyzes a reaction for converting pyruvate into lactic acid, derived from Aspergillus (Aspergillus) microorganisms, is provided. Lactic acid can be produced at low cost by introducing the present gene into microorganisms such as filamentous fungi, particularly filamentous fungi suitable for solid-state culture and bacteria belonging to the genus Escherichia. In particular, introduction into a microorganism of the genus Aspergillus that is frequently used by a solid fermentation method that enables inexpensive and simple fermentation production enables production of lactic acid by an microorganism of the genus Aspergillus that has not been known so far. The newly obtained lactic acid-producing microorganism belonging to the genus Aspergillus is one that enhances the expression of genes inherently possessed by the microorganism belonging to the genus Aspergillus, and is not a recombinant microorganism, so that it can be fermented by ordinary equipment and methods.

In order to produce lactic acid, the presence of a lactate dehydrogenase that changes the precursor pyruvic acid to lactic acid is indispensable. The present inventors have discovered the enzyme that can be expressed in filamentous fungi, in particular bacteria belonging to the genus Aspergillus, and bacteria belonging to the genus Escherichia typified by Escherichia coli (Escherichia coli), to enhance the expression thereof, We focused on the possibility of creating microorganisms with high lactic acid-producing ability, especially Aspergillus microorganisms.
Specifically, the present inventors searched for a lactate dehydrogenase gene derived from the genome of an Aspergillus microorganism and introduced it into a host microorganism, particularly an Aspergillus microorganism, together with a high expression vector to enhance the expression of the gene. As a result, it was thought that microorganisms suitable for lactic acid fermentation, especially Aspergillus spp., Especially Aspergillus oryzae and Aspergillus niger suitable for solid culture, could be constructed. The present inventors discovered the gene of the present invention and completed the present invention. Accordingly, the present invention enhances the expression of this gene by introducing a gene construct containing an exogenous lactate dehydrogenase gene, and enhances the expression of the endogenous lactate dehydrogenase gene. This embodiment is also included.
In this specification, when the term “DNA” is used in relation to “expression”, “gene” and “DNA” are synonymous in the context unless otherwise specified. Accordingly, expressions such as “DNA expression amplification” and “DNA expression regulation” are synonymous with “gene expression amplification” and “gene expression regulation”, respectively. In the present specification, unless otherwise specified, “lactate dehydrogenase” includes both L-lactate dehydrogenase and D-lactate dehydrogenase. Further, as used herein, “DNA” includes both single-stranded DNA and double-stranded DNA depending on the context, and thus “DNA sequence” is directly described depending on the context. As well as complementary sequences thereof.

  Lactic acid dehydrogenase genes have been found in lactic acid bacteria, some fungi (such as Rhizopus oryzae), and higher organisms, and genome information has already been revealed for Aspergillus microorganisms based on the nucleotide sequence information. By comparing and searching with the base sequence information of Aspergillus nidulans or Aspergillus oryzae, it is possible to find a base sequence that seems to be a lactate dehydrogenase gene candidate. Based on the amino acid sequence read from the base sequence, enzyme protein models can be constructed by protein engineering techniques, and the possibility that these proteins have the function of lactate dehydrogenase can be confirmed. Based on the obtained candidate nucleotide sequence, cDNA is prepared according to a conventional method, incorporated into an appropriate vector, and introduced into a microorganism generally used for cloning, for example, Escherichia coli. Lactic acid dehydrogenase obtained by examining whether or not the microorganisms of the above produce lactic acid that is not originally produced, or by producing an enzyme protein encoded by the sequence and checking whether the protein has lactate dehydrogenation activity It can be confirmed that the nucleotide sequence of the gene candidate is actually the target lactate dehydrogenase gene.

Examples of embodiments of the present invention will be specifically described below.
1) Search for lactate dehydrogenase gene candidate sequences Appropriate homology to the genome information of Aspergillus microorganisms in the database based on the nucleotide sequence information of known lactate dehydrogenase (L-type or D-form) genes A search program such as BLAST can be used to search for sequences having a certain sequence homology, for example, 40% homology or more. Known lactate dehydrogenase (L-type or D-type) gene sequences include Rhizopus oryzae lactate dehydrogenase (L-type) gene and Escherichia coli and Nostock (Cyanobacteria) lactate dehydrogenase (D-type). ) The base sequence of the gene can be used. Examples of Aspergillus microorganisms to be searched for include, but are not limited to, Aspergillus nidulans and Aspergillus oryzae. A homology search program such as BLAST is provided by the National Center for Biotechnology Information (NCBI) (http // www.ncbi.nlm.nih.gov / blast /), for example. In this way, a candidate sequence gene for a lactate dehydrogenase gene of an Aspergillus microorganism can be obtained.

2) Acquisition of Lactate Dehydrogenase Gene Candidate Sequence Genes As a result of such a search, microorganisms belonging to the genus Aspergillus, for example, A. nidulans FGSCA4 (Fungal Genetics Stock Center, School) of Biologycal Sciences, University of Missouri, Kansas City 5007 Rockhill Rd, Kansas City, Missouri, 64110 USA), A.oryzae RIB40 strain (Independent Administrative Institution Liquor Research Institute, 739-0046 Kagamiyama, Higashihiroshima City, 739-0046 -7-1) is grown using an appropriate medium such as a YPM medium consisting of yeast extract, peptone, maltose, the cells are crushed, and PCR is performed using genomic DNA extracted according to a conventional method as a template. The target candidate gene can be actually cloned by the method.
Molecular genetic techniques including genomic DNA extraction from Aspergillus microorganisms are well known to those skilled in the art, such as Nick Talbot (ed.) Molecular and Cellular Biology of Filamentus Fungi; A Practical Approach, Oxford University Press (2001). Can be referred to. More general molecular biology techniques are described in, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, FM Ausubel et alo. eds), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

3) Preparation of cDNA from Lactate Dehydrogenase Gene Candidate Sequences Aspergillus microorganisms, for example, A. nidulans FGSCA4 strain and A. oryzae RIB40 strain, whose presence of lactate dehydrogenase gene candidate sequences has been clarified, For example, it is grown using a YPM medium consisting of yeast extract, peptone, maltose, the cells are pulverized, and the target cDNA is obtained by reverse transcriptase (RT) PCR using RNA extracted according to a conventional method. be able to.

4) Estimation of enzyme activity by protein homology modeling based on the obtained cDNA sequence The nucleotide sequence of the obtained cDNA can be determined, and the amino acid sequence can be determined based on this information. Regarding the three-dimensional structure of the protein predicted from the obtained amino acid sequence, the three-dimensional structure of a known lactate dehydrogenase using homology modeling techniques (T.Yoneda et al., J. Protein Chem., 16, 597-605, 1997). Analyzing the homology with the structure, for example, preferably 30% or more of the homology is estimated to have a basic element of lactate dehydrogenase activity expression, the resulting cDNA is lactate dehydrogenase activity Can be selected as a candidate gene encoding a protein having

5) Confirmation that a lactate dehydrogenase candidate gene is actually a lactate dehydrogenase gene A microorganism prepared by an appropriate method, for example, the method of Hanahan, which is often used for gene cloning in this field. A cDNA linked to a commercially available cloning vector can be introduced into a competent cell of E. coli prepared according to a conventional method.
Microorganisms into which cDNA has been introduced, such as E. coli, are cultured with shaking in an appropriate medium (for example, LB medium in the case of E. coli) at 28 ° C to 40 ° C, preferably about 37 ° C overnight, collecting only the cells and disrupting them. The presence of lactic acid dehydrogenation activity can be confirmed using the extract obtained in this manner. The activity of lactate dehydrogenase can be measured, for example, according to a method well known to those skilled in the art using pyruvate and NADH as substrates (see, for example, Enzyme Handbook, p10-11, Asakura Shoten). Furthermore, it can be confirmed that L-lactic acid and / or D-lactic acid is produced in the enzyme reaction solution by analyzing the culture solution from which the microorganisms have been removed by a liquid chromatography method for organic acid analysis.

6) Acquisition of homologous genes Genes having functions equivalent to the genes thus obtained are also within the scope of the present invention. Such a gene can be obtained as a gene encoding a protein that hybridizes with a nucleotide corresponding to the above-described gene under stringent conditions and has an activity of catalyzing a reaction of converting pyruvate to lactic acid. Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify this condition clearly, for example, DNAs with high homology, for example, DNAs with homology of 70% or more preferentially hybridize and are more homologous. The conditions are such that DNAs with low DNA do not hybridize significantly, or washing conditions for normal Southern hybridization are 50 ° C, 2xSSC, 0.1% SDS, preferably 1xSSC, 0.1% SDS, more preferably 0.1xSSC, 0.1% SDS The conditions for hybridizing at the corresponding salt concentration can be mentioned. Conditions equivalent to these will be readily apparent to those skilled in the art (eg, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994)). Among the genes that hybridize under these conditions, there are those in which a stop codon has occurred in the middle and those that have lost the active center mutation. These are linked to a commercially available active expression vector, as described above. It can be easily removed by measuring lactate dehydrogenase activity by a simple method. The protein encoded by such a gene is generally a protein having the same amino acid sequence as that of the above-mentioned gene, or substitution, deletion, addition, insertion of one or several amino acids in the amino acid sequence, Alternatively, it may be a protein having an amino acid sequence containing an inversion and having an activity of catalyzing a reaction for converting pyruvic acid to lactic acid. Such a gene and the protein encoded by the gene are also within the scope of the present invention.

7) Expression of lactate dehydrogen gene in microorganisms Microorganisms that can be used to express the lactate dehydrogen gene of the present invention include filamentous fungi including Aspergillus microorganisms and bacteria including Escherichia bacteria (for example, Escherichia coli). . Filamentous fungi include Aspergillus oryzae, Aspergillus niger, Aspergillus genus such as Aspergillus nidulans, Neurospora crassa such as Neurospora crassa, Rhizomucor miehei, etc. Examples include, but are not limited to, microorganisms belonging to the genus Rhizomucor. In particular, filamentous fungi suitable for solid culture are preferable for expressing the lactate dehydrogenase gene of the present invention. Filamentous fungi suitable for solid culture include Aspergillus oryzae, Aspergillus soya, Aspergillus niger, Aspergillus awamori, Aspergillus inui, etc., especially Aspergillus genus microorganisms suitable for solid culture include Aspergillus oryzae Examples include, but are not limited to, Aspergillus niger and other strains that have a high spore engraftment ability, a high growth rate, and do not require the addition of a neutralizing agent such as calcium carbonate. More specifically, Aspergillus oryzae and Aspergillus niger are particularly preferable.
The gene of the present invention can be expressed not only in bacteria such as E. coli but also in various microorganisms such as filamentous fungi including Aspergillus microorganisms by selecting an appropriate promoter according to the host. For example, a selection marker for collecting transgenic strains by performing mutation operations on filamentous fungi containing Aspergillus spp., Particularly Aspergillus oryzae (A. oryzae) or Aspergillus niger (A.niger) For example, niaD ⁻ (nitrate assimilation deficiency) strain, sC ⁻ (ATP sulfurylase deficiency) strain, argB ⁻ (arginine requirement) strain etc. are prepared and combined with an appropriate vector in these microorganisms. Can be expressed. On the other hand, gene constructs in which the above-described cDNA is linked to a high expression promoter suitable for host microorganisms can be prepared and introduced into these microorganisms. Various vectors are available for that purpose.

As vectors used for Aspergillus microorganisms, pUNG (Lee, BR et al., Appl. Microbiol. Biotechnol., 44, 425-431 (1995)), pMARG (Tsuchiya, K. et al., Appl. Microbiol. Biotechnol., 40, 327-332 (1993)), pUSC (Gomi, K. et al., Agric. Biol. Chem. 51, 2549-2555 (1987)). pUNG the niaD ^- a marker complementing (nitrate assimilation deficient), PMARG the argB ^- a marker complementing (arginine request), PUSC is sC ^- a marker complementing (ATP sulfurylase deficiency), have respectively Yes.
Among such vectors, when a vector containing a promoter is used, the lactate dehydrogenase gene can be expressed by inserting a DNA molecule encoding lactate dehydrogenase in a frame downstream of the promoter. I can do it. For example, pUNG and pMARG have a glucoamylase gene (glaA) promoter and an α-amylase gene (amyB terminator), so that DNA encoding lactate dehydrogenase is inserted downstream of the promoter. Lactate dehydrogenase can be expressed under the control of the promoter. In addition, when a vector not containing a promoter, such as pUCS, is used, lactate dehydrogenase is introduced into the host microorganism by co-transformation of a plasmid such as pUC19 inserted with the above DNA and pUSC. Can be expressed. In particular, vectors, promoters and markers described in the literature shown in Table 1 below can also be used depending on the host filamentous fungus. In addition, an endogenous lactate dehydrogenase gene promoter can also be used. In Table 1, the name of the promoter is shown using the enzyme name encoded by the gene under its control in nature.

Table 1. Examples of available promoters

  A method for introducing the prepared gene construct into filamentous fungi, for example, Aspergillus microorganisms including A. oryzae and A. niger is not particularly limited. For example, the protoplast-PEG method described in the following examples can be used. . The transformed strain is layered with a soft agar medium on a suitable medium, for example, a minimal medium plate containing salt, and cultured on that medium at 30 ° C. for 5 days to collect vigorous colonies. Thus, a microorganism into which the gene of the present invention has been introduced, for example, A.oryzae or A.niger into which the gene of the present invention has been introduced can be obtained.

8) Production of lactic acid By culturing microorganisms into which the gene of the present invention has been introduced, for example, Aspergillus microorganisms or Escherichia bacteria according to a general culture method, these microorganisms can produce lactic acid. The general culture methods for these microorganisms are well known to those skilled in the art. When culturing Aspergillus microorganisms by the method of the present invention to produce lactic acid, Aspergillus microorganisms into which the gene of the present invention has been introduced, for example, A. oryzae or A. niger, are preferably 25 to 40 ° C., preferably in an appropriate medium. Incubate at 30-35 ° C for several days. The number of culture days is not essential in the present invention, and may be a period in which lactic acid is sufficiently produced. For example, by culturing at the aforementioned temperature for 2 to 30 days, preferably 3 to 10 days, particularly preferably 4 to 7 days, L-lactic acid or D-lactic acid is produced depending on the introduced gene. Those skilled in the art will be able to set optimized culture conditions depending on the selected host. The medium is not particularly limited as long as it is a medium in which Aspergillus microorganisms such as A. oryzae or A. niger can grow healthy and / or produce lactic acid. For example, for Aspergillus microorganisms, a medium containing rice and soybean flour, a solid medium using sweet potato starch and rice bran as substrates, or a liquid medium such as wheat bran can be used.

  When cultivating Escherichia bacteria to produce lactic acid, they are commonly used in the culture of these bacteria, usually used in these bacteria, a medium containing a carbon source, nitrogen source, inorganic ions, and vitamins as needed. The temperature, pH and other culture conditions that can be used can also be employed in the present invention. For example, in the case of Escherichia coli, the cells are cultured in such a medium at a temperature of 25 ° C. to 45 ° C., preferably 30 ° C. to 40 ° C., during which lactic acid is sufficiently produced. The culture period is not particularly limited as long as lactic acid is sufficiently produced. For example, in the case of Escherichia coli, it is generally 12 to 72 hours, preferably 18 to 48 hours, particularly preferably 18 to 36 hours. Even when an Escherichia bacterium is used as a host, those skilled in the art will be able to set more optimized culture conditions depending on the specific host selected.

  In addition to introducing an exogenous gene construct containing a lactate dehydrogenase gene in order to cause Aspergillus microorganisms to efficiently produce lactic acid according to the present invention, in another aspect of the present invention, Increase the copy number of the lactate dehydrogenase gene endogenous to these microorganisms, add expression control sequences such as enhancers to the endogenous lactate dehydrogenase gene, increase the number of promoters, and / or become more active The activity of lactate dehydrogenase can be improved in these microorganisms by a method such as modification to be high and / or replacement of all or part of a regulatory sequence including a promoter. Methods for increasing the copy number of a gene are well known to those skilled in the art. For example, the DNA molecule of the present invention is incorporated into a transposon and transferred to introduce multiple copies onto chromosomal DNA, or The number of copies of the lactate dehydrogenase gene can also be increased in the cell by inserting multiple copies of the DNA molecule of the present invention into the cosmid vector in tandem and introducing it into the cell and integrating it into the chromosome. . Further, homologous recombination targeting the upstream of the coding sequence of the endogenous gene based on the coding sequence of the nuclear gene described in the sequence listing and / or the genomic sequence, for example, the information described in SEQ ID NOs: 1, 4 and 7 It is also possible to insert a more powerful promoter, or to introduce a plurality of additional regions on the chromosome, including the coding region and its upstream and / or downstream regions. In this embodiment of the invention, the enhancer capable of functioning in Aspergillus microorganisms, in particular A. oryzae and / or A. niger, for example A. oryzae amylolytic enzyme (α-amylase, glucoamylase, α-glucosidase) gene The sequence (Region III) (Minetaki et al., Applied Microbiology and Biotechnology, 50, 459-467 (1998)) that is commonly present in the promoters of In addition to the endogenous promoter of the gene of the present invention, the promoters already described in Table 1 above can also be used in this aspect of the present invention.

Search for Lactate Dehydrogenase Gene Candidate Sequences Published on the National Center for Biotechnology Information (NCBI) mold genome database (http // www.ncbi.nlm.nih.gov / sutils / genom_table.cgi? Organism = fungi) Based on the genome information of Aspergillus nidulans and the genome information of Aspergillus oryzae, NCBI's blast search system (blastx) (http // www.ncbi.nlm.nih.gov / blast /) A DNA base sequence encoding a protein having high homology with the amino acid sequence of L-type lactate dehydrogenase (LdhA) of oryzae was searched. As a result, a sequence showing the highest homology (41%) in the genome of Aspergillus nidulans was obtained as an L-LDH homolog. The nucleotide sequence of the genomic region encoding the L-LDH homolog protein is shown in SEQ ID NO: 1, the amino acid sequence of the L-LDH homolog protein is shown in SEQ ID NO: 2, and the cDNA coding region nucleotide sequence is shown in SEQ ID NO: 3. In addition, the homology between L-LDH homolog protein and L-type lactate dehydrogenase (LdhA) of Rhizopus oryzae is ClustalW (http://www.ddbj.nig.ac.jp/search/clustalw-j.html) FIG. 1 shows the result of the investigation.

In the same manner as L-LDH homologues, DNA sequences encoding proteins homologous to the amino acid sequence of Escherichia coli (E. coli) D-type lactate dehydrogenase in Aspergillus nidulans genome data are searched with blastx. As a result, the sequence showing the highest homology was obtained as a D-LDH homolog. The nucleotide sequence of the genomic region encoding the D-LDH homolog protein is shown in SEQ ID NO: 4, the amino acid sequence of the encoded D-LDH homolog protein is shown in SEQ ID NO: 5, and the cDNA coding region nucleotide sequence is shown in SEQ ID NO: 6. . Based on this amino acid sequence, a blastp search against the protein database of all NCBI organisms revealed that D-LDH (registration number BAB77582) of cyanobacteria Nostoc sp. It was found that the homology was high (51%). The results of examining the homology between D-LDH homolog protein and D-LDH of cyanobacteria by ClustalW are shown in FIG.
Next, as a result of a blastx search for a DNA sequence that encodes a protein homologous to the amino acid sequence of the Aspergillus oryzae genome data and the D-LDH homologue of Aspergillus nidulans obtained earlier, the highest homology was obtained. A sequence showing sex (71%) was obtained as a D-LDH homolog of Aspergillus oryzae. The nucleotide sequence of the genomic region encoding the D-LDH homolog protein is shown in SEQ ID NO: 7, the amino acid sequence of the D-LDH homolog protein is shown in SEQ ID NO: 8, and the nucleotide region sequence of the cDNA is shown in SEQ ID NO: 9. FIG. 3 shows the result of examining the homology between Aspergillus oryzae and Aspergillus nidulans D-LDH homolog protein by ClustalW.

Acquisition of searched lactate dehydrogenase gene candidate sequence genes
Using 50 ml of YPM medium (0.5% Bacto yeast extract, 1% Bacto peptone, 2% Maltose), A. nidulans A4 strain was cultured with shaking at 30 ° C. for 2 days, and collected using a glass filter (G2) Wash well with distilled water. The bacterial cells that were well dehydrated using a paper towel were frozen using liquid nitrogen and then crushed in a mortar. This was suspended in 0.4 ml of TE buffer, 0.4 ml of EDTA solution (5 mM EDTA, 1% SDS) and 0.8 ml of phenol / chloroform were added and mixed, and the layers were separated by centrifugation to separate only the aqueous layer. I took it. To this was added 2.5 times the amount of ethanol, and the resulting precipitate was obtained by centrifugation. After redissolving this precipitate in TE buffer, add 50 μg of RNase A, hold at 37 ° C for 30 minutes, extract with phenol / chloroform twice, treat with chloroform / isoamyl alcohol, add 2.5 volumes of ethanol, and centrifuge. , A. nidulans genomic DNA precipitation was obtained. A genomic DNA of A. oryzae NS4 strain was also prepared in the same manner.
PCR was performed using these genomic DNAs as templates. 5'-TTAGCGGCCGCACCATGGACAACCTG-3 '(SEQ ID NO: 10) with the NotI recognition sequence added to the sense primer, and 5'-TTAGCGGCCGCACCATGGACAACCTG-3' (SEQ ID NO: 11) with the HindIII and NdeI recognition sequences added to the antisense primer. ) Was used. For DNA polymerase, TaKaRa Ex-Taq was used according to the attached protocol. In other words, 5 U Ex-Taq polymerase, 1/10 vol. Ex-Taq buffer, 200 mM dNTPsmix, 1 mM sense and antisense primers, and template DNA were mixed, and 94 ° C. with a thermal cycler TP240 (TaKaRa). 30 cycles of 30 seconds, 55 ° C 90 seconds, 72 ° C 120 seconds were repeated. The nucleotide sequence of the DNA fragment obtained by PCR was determined by a sequencer and confirmed to be the target candidate gene.

Preparation of cDNA of Lactate Dehydrogenase Candidate Gene The cells of Aspergillus nidulans A4 strain cultured in the same manner as in Example 2 were isolated in the same manner, frozen in liquid nitrogen, and crushed in a mortar. RNA was extracted according to the attached protocol using 1 ml of ISOGEN (manufactured by Nippon Gene Co., Ltd.) per 0.1 g of crushed cells. To 2 μg of the obtained RNA, 4.5 μg of oligo dT12-18 primer (GIBCO BRL) was added, and 15 μl was denatured at 70 ° C. for 10 minutes, and then rapidly cooled on ice. To this, 6 μl of Superscript buffer contained in Superscript II kit (manufactured by GIBCO BRL), 3 μl of DTT, 5 μl of dNTP mix (2.5 mM each) and 2 μl of Superscript II were added, and a reverse transcription reaction was performed at 42 ° C. for 2 hours. In addition, 1 μl of Superscript II was added when 1 hour had elapsed. A reverse transcriptase (RT) PCR reaction was performed using the synthesized first strand cDNA-RNA hybrid as a template to obtain the target cDNA.

Estimating enzyme activity by protein homology modeling based on information from the cDNA of lactate dehydrogenase candidate gene About the amino acid sequence of L-ldh candidate derived from Aspergillus nidulans and amino acid sequence of D-ldh candidate derived from Aspergillus oryzae PSI-BLAST (Schaffer AA et al.) Is a protein homology search software based on NCBI (Entrez) protein structure database (http://www.ncbi.nlm.nih.gov/Entrez/index.html). Bioinformatics, 12, 1000-1011, 1999), and showed a high homology of 29-31% with the three-dimensional structure of known lactate dehydrogenases such as lactate dehydrogenases such as lactic acid bacteria. . In addition, when NADH, the coenzyme of this enzyme, was applied to these three-dimensional structure models, histidine and arginine were present in the vicinity of any candidate gene, and the basic elements for the expression of lactate dehydrogenation activity were obtained. I was able to confirm that.

Introduction of lactate dehydrogenase gene and production of lactate into Escherichia coli 1) Introduction of cDNA of candidate lactate dehydrogenase gene into Escherichia coli
Transfer 100 μl of a competent cell of Escherichia coli prepared at Hanahan's method and stored at −80 ° C. to a 1.5 ml Eppendorf tube, and connect each cDNA to a commercially available T vector (TaKaRa) in advance. The resulting plasmid was added thereto, left on ice for 30 minutes, and heat shocked at 42 ° C. for 30 seconds. The mixture was cooled again in ice for 2 minutes, 0.7 ml of LB medium (1% Bacto tryptone, 0.5% Bacto yeast extract, 0.5% NaCl) was added, and cultured with shaking at 37 ° C. for 1 hour. This culture solution was spread on an LB agar plate medium containing ampicillin as a selection marker, and cultured at 37 ° C. overnight to collect colonies that appeared. Thus, an Escherichia coli strain into which cDNA corresponding to L-type lactate dehydrogenase derived from Aspergillus nidulans and cDNA corresponding to D-type lactate dehydrogenase derived from Aspergillus oryzae was introduced was obtained.

2) Confirmation of Lactate Dehydrogenase Activity of cDNA-Introduced Escherichia coli Each Escherichia coli strain into which the lactate dehydrogenase gene obtained in 1) was introduced was cultured at 37 ° C. for 24 hours using LB medium. After cultivation, only the cells were collected by centrifugation, suspended again in phosphate buffer (0.1 M, pH 7.0), and the extract obtained by ultrasonic disruption was used as the enzyme solution. Using this, lactate dehydrogenase activity was confirmed by a conventional method (Enzyme Handbook, p10-11, Asakura Shoten). The results are shown in Table 2.

Table 2. Confirmation of lactate dehydrogenase activity of cDNA-introduced Escherichia coli strains

3) Production of lactic acid in Escherichia coli Each Escherichia coli strain into which the lactate dehydrogenase gene obtained in 1) was introduced was cultured at 37 ° C. in a glucose-containing LB medium, and glucose was obtained after 18 hours. An additional 2.5% was added and the culture was continued. The culture was completed 24 hours after the start of the culture, and the culture solution was diluted 10-fold with pure water, and then a sterilized culture solution was obtained using a filter (Millipore). This was analyzed by liquid chromatography for organic acid analysis (manufactured by Hitachi), and the amount of lactic acid produced was confirmed. The results are shown in Table 3.

Table 3. Production of lactic acid from Escherichia coli strain with cDNA

Introduction of lactate dehydrogenase gene and production of lactic acid into Aspergillus oryzae 1) Introduction of lactate dehydrogenase candidate gene cDNA into Aspergillus oryzae Spores of Aspergillus oryzae NS4 strain were added to YPD medium (0.5% Bacto yeast extract, 1% Bacto peptone) (2% Glucose) was inoculated into 50 ml using a platinum loop, and cultured with shaking at 30 ° C. for 48 hours. Only the cells are collected by filtration using a sterilized glass filter, and the cells washed with sterilized water are converted into a protoplast solution (10 mg / ml yatalase, 5 mg / ml cellulase Onoka R-10, 0.8 M NaCl, 10 mM phosphoric acid). The suspension was suspended in a buffer solution (pH 6.0) and shaken at 30 ° C. for 2 to 6 hours to obtain protoplasts. The protoplasts were further washed with 0.8 M saline solution or calcium-containing Tris buffer (0.8 M NaCl, 10 ml CaCl ₂ , 10 mM TrisHCl buffer, pH 8), and collected by centrifugation (2000 rpm, 5 minutes). A PEG solution (40% PEG6000, 50 mM CaCl ₂ , 50 mM TrisHCl buffer, pH 8) is added to the collected protoplast suspension at a ratio of 20%. 20 μL of the plasmid ligated to the vector containing P-enoA142) (pSEN142) was added and kept on ice for 30 minutes. Thereafter, 1 ml of PEG solution was added and left at room temperature for 15 minutes, and then the protoplasts collected by adding 10 ml of calcium-containing Tris buffer and centrifuging (2000 rpm, 5 minutes) were collected into Czapek-Dox medium containing 0.8 M sodium chloride. (70mM NaNO ₃ , 7mM KCl, 0.1M KH ₂ PO ₄ , 2mM MgSO ₄ , 1% Glucose, 1.5% agar) Soft agar of the same composition with agar cooled to 45 ° C reduced to 0.6% Medium was overlaid. Colonies that showed vigorous growth at 30 ° C. for 5 days were collected.

2) Production of lactic acid Each colony introduced with the lactic acid dehydrogenase gene thus obtained was transformed into maltose-containing medium (0.5% Bacto peptone, 70 mM NaNO ₃ , 7 mM KCl, 0.1 M KH ₂ PO ₄ , 2 mM MgSO ₄ , 2% Maltose) was inoculated with a spore suspension (2 × 10 ⁶ or more) and cultured at 30 ° C. for 5 days. The culture end solution was diluted 10 times with pure water, and then a sterilized culture solution was obtained using a filter (manufactured by Millipore). This was analyzed by liquid chromatography for organic acid analysis (manufactured by Hitachi), and the amount of lactic acid produced was confirmed. The results are shown in Table 4.

Table 4. Lactic acid production by cDNA introduced gonococci

Production of lactic acid in Aspergillus niger 1) Introduction of lactic acid dehydrogenase candidate gene cDNA into Aspergillus niger LR12 strain As in Example 8, protoplasts were obtained using spores of Aspergillus niger LR12 strain. A strain in which a plasmid in which cDNA was previously ligated to a vector (pNEN142) containing a high expression promoter (P-No8142) was obtained by the same method as in Example 8 was obtained.

2) Production of lactic acid Each spore suspension (2x10 ⁶ or more) of lactobacillus introduced with lactate dehydrogenase gene obtained in 1) was added to a solid medium (15g sweet starch starch cake, 5g rice bran, 50ml). Water) and cultured in a culture room at 30 ° C. for 1 week. The cultured solid medium is suspended in 100 ml of water, the solid matter is separated and removed by filtration, and the resulting filtrate is diluted 10-fold with pure water and analyzed by liquid chromatography for organic acid analysis (manufactured by Hitachi). The amount of lactic acid produced was confirmed. The results are shown in Table 5. Shown in

Table 5. Lactic acid production by cDNA-transfected black koji mold

It is the result of having investigated L-LDH homology of L-type lactate dehydrogenase (L-LDH) of A. nidulans and Rhizopus oryzae by ClustalW. AdldhA (upper) represents A. nidulans L-LDH, and RhldhA (lower) represents the L-LDH amino acid sequence of Rhizopus oryzae. It is the result of investigating the homology between A. nidulans D-type lactate dehydrogenase (D-LDH) and cyanobacterial D-LDH by ClustalW. AdldhD (upper) represents the amino acid sequence of A. nidulans D-LDH, and Nosto (lower) represents the amino acid sequence of D-LDH from Nostock (Cyanophyceae). It is the result of having investigated the homology between D-LDH of A.nidulans and A.oryzae by ClustalW. AoldhD (upper) represents the amino acid sequence of A.oryzae D-LDH, and AdldhD (lower) represents the amino acid sequence of A.nidulans D-LDH.

Sequence number 10 and 11: PCR primer

Claims (10)

A method for producing lactic acid, comprising culturing a microorganism having an improved activity of catalyzing a reaction for converting pyruvic acid into lactic acid in a medium, and producing lactic acid in the culture, wherein the improvement in the activity comprises: The method according to any one of the following (a) to (j):
(A) DNA having the base sequence described in SEQ ID NO: 1
(B) DNA encoding the amino acid sequence set forth in SEQ ID NO: 2;
(C) DNA having the base sequence set forth in SEQ ID NO: 3;
(D) DNA having the base sequence set forth in SEQ ID NO: 4;
(E) DNA encoding the amino acid sequence set forth in SEQ ID NO: 5;
(F) DNA having the base sequence set forth in SEQ ID NO: 6;
(G) DNA having the base sequence set forth in SEQ ID NO: 7;
(H) DNA encoding the amino acid sequence set forth in SEQ ID NO: 8;
(I) DNA having the base sequence set forth in SEQ ID NO: 9; or
(J) encodes a protein that can hybridize with the DNA of any one of (a) to (i) under stringent conditions and has an activity of catalyzing a reaction of converting pyruvic acid into lactic acid DNA. The microorganism has been modified so that the expression of the DNA is enhanced by increasing the copy number of the DNA according to any of the following (a) to (j), or by modifying the expression regulatory sequence of the DNA The manufacturing method according to claim 1, wherein:
(A) DNA having the base sequence described in SEQ ID NO: 1
(B) DNA encoding the amino acid sequence set forth in SEQ ID NO: 2;
(C) DNA having the base sequence set forth in SEQ ID NO: 3;
(D) DNA having the base sequence set forth in SEQ ID NO: 4;
(E) DNA encoding the amino acid sequence set forth in SEQ ID NO: 5;
(F) DNA having the base sequence set forth in SEQ ID NO: 6;
(G) DNA having the base sequence set forth in SEQ ID NO: 7;
(H) DNA encoding the amino acid sequence set forth in SEQ ID NO: 8;
(I) DNA having the base sequence set forth in SEQ ID NO: 9; or
(J) encodes a protein that can hybridize with the DNA of any one of (a) to (i) under stringent conditions and has an activity of catalyzing a reaction of converting pyruvic acid into lactic acid DNA. The production method according to claim 1 or 2, wherein the microorganism is a transformant introduced with a form capable of expressing the DNA according to any one of the following (a) to (j):
(A) DNA having the base sequence described in SEQ ID NO: 1
(B) DNA encoding the amino acid sequence set forth in SEQ ID NO: 2;
(C) DNA having the base sequence set forth in SEQ ID NO: 3;
(D) DNA having the base sequence set forth in SEQ ID NO: 4;
(E) DNA encoding the amino acid sequence set forth in SEQ ID NO: 5;
(F) DNA having the base sequence set forth in SEQ ID NO: 6;
(G) DNA having the base sequence set forth in SEQ ID NO: 7;
(H) DNA encoding the amino acid sequence set forth in SEQ ID NO: 8;
(I) DNA having the base sequence set forth in SEQ ID NO: 9; or
(J) encodes a protein that can hybridize with the DNA of any one of (a) to (i) under stringent conditions and has an activity of catalyzing a reaction of converting pyruvic acid into lactic acid DNA. The production method according to any one of claims 1 to 3, wherein the DNA is derived from Aspergillus nidulans and is any of the following (a) to (g):
(A) DNA having the base sequence described in SEQ ID NO: 1
(B) DNA encoding the amino acid sequence set forth in SEQ ID NO: 2;
(C) DNA having the base sequence set forth in SEQ ID NO: 3;
(D) DNA having the base sequence set forth in SEQ ID NO: 4;
(E) DNA encoding the amino acid sequence set forth in SEQ ID NO: 5;
(F) DNA having the base sequence set forth in SEQ ID NO: 6; or (g) hybridizing with DNA having the base sequence set forth in any one of (a) to (f) under stringent conditions, and pyruvin DNA encoding a protein having an activity of catalyzing a reaction of converting an acid into lactic acid. The production method according to any one of claims 1 to 3, wherein the DNA has any one of the following base sequences (a) to (d) and is derived from Aspergillus oryzae:
(A) DNA having the base sequence set forth in SEQ ID NO: 7;
(B) DNA encoding the amino acid sequence set forth in SEQ ID NO: 8;
(C) DNA having the base sequence set forth in SEQ ID NO: 9;
(D) A DNA encoding a protein that hybridizes with the DNA according to any one of (a) to (c) under a stringent condition and has an activity of catalyzing a reaction of converting pyruvic acid into lactic acid.   The production method according to any one of claims 1 to 5, wherein the microorganism is a filamentous fungus or an Escherichia bacterium.   The production method according to claim 1, wherein the microorganism is a filamentous fungus suitable for solid culture.   The production method according to claim 1, wherein the microorganism is an Aspergillus microorganism. DNA according to any one of the following (a) to (j):
(A) DNA having the base sequence set forth in SEQ ID NO: 1 or its complementary sequence
(B) DNA encoding the amino acid sequence set forth in SEQ ID NO: 2;
(C) DNA having the base sequence set forth in SEQ ID NO: 3 or its complementary sequence;
(D) DNA having the base sequence set forth in SEQ ID NO: 4 or a complementary sequence thereof;
(E) DNA encoding the amino acid sequence set forth in SEQ ID NO: 5;
(F) DNA having the base sequence set forth in SEQ ID NO: 6 or its complementary sequence;
(G) DNA having the base sequence set forth in SEQ ID NO: 7 or its complementary sequence;
(H) DNA encoding the amino acid sequence set forth in SEQ ID NO: 8;
(I) a DNA having the base sequence set forth in SEQ ID NO: 9 or a complementary sequence thereof; or
(J) encodes a protein that can hybridize with the DNA of any one of (a) to (i) under stringent conditions and has an activity of catalyzing a reaction of converting pyruvic acid into lactic acid DNA. Any of the following proteins (a) to (f):
(A) a protein having the amino acid sequence of SEQ ID NO: 2;
(B) a protein having the amino acid sequence set forth in SEQ ID NO: 5;
(C) a protein having the amino acid sequence set forth in SEQ ID NO: 8;
(D) A reaction comprising an amino acid sequence comprising one or several amino acid substitutions, deletions, additions, insertions, or inversions in the amino acid sequence shown in SEQ ID NO: 2 and converting pyruvic acid to lactic acid. A protein with catalytic activity;
(E) a reaction comprising the amino acid sequence of SEQ ID NO: 5 comprising an amino acid sequence containing substitution, deletion, addition, insertion, or inversion of one or several amino acids, and converting pyruvic acid into lactic acid A protein with catalytic activity;
(F) a reaction comprising an amino acid sequence comprising one or several amino acid substitutions, deletions, additions, insertions or inversions in the amino acid sequence set forth in SEQ ID NO: 8, and converting pyruvic acid into lactic acid Protein with catalytic activity.

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