# Patent

## Title
Method for producing an organic acid

## Abstract

An organic acid is produced by allowing a bacterium which has an ability to produce an organic acid and has been modified so that expression of yidE gene is enhanced, or a product obtained by processing the bacterium, to act on an organic raw material in a reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas to produce the organic acid, and collecting the organic acid.

## Background
This application is a continuation under 35 U.S.C. §120 of PCT Patent Application No. PCT/JP2008/057397, filed Apr. 16, 2008, which claims priority under 35 U.S.C. §119 to Japanese Patent Application No. 2007-106909, filed on Apr. 16, 2007, and Japanese Patent Application No. 2007-242866, filed on Sep. 19, 2007, which are incorporated in their entireties by reference. The Sequence Listing in electronic format filed herewith is also hereby incorporated by reference in its entirety (File Name: US-411_Seq_List; File Size: 179 KB; Date Created: Oct. 15, 2009).


BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to a method for producing an organic acid such as succinic acid using a bacterium.
2. Background Art
For the production of non-amino organic acids, including succinic acid, by fermentation, anaerobic bacteria are typically used, including bacteria belonging to the genus Anaerobiospirillum or Actinobacillus (U.S. Pat. Nos. 5,142,834 and 5,504,004, International Journal of Systematic Bacteriology (1999), 49, 207-216). Although such anaerobic bacteria provide high yields of products, many nutrients are required for their proliferation, and therefore it is necessary to add large amounts of organic nitrogen sources such as corn steep liquor (CSL) into the culture medium. The addition of large amounts of sources of organic nitrogen results in not only an increase in cost for the culture medium, but also an increase in the purification costs for isolating the product, and therefore it is not economical.
In addition, methods are known in which aerobic bacteria such as coryneform bacteria are cultured once under aerobic conditions to proliferate the bacterial cells, then the cells are harvested, washed, and allowed to rest so that a non-amino organic acid is produced without having to supply oxygen (Japanese Patent Laid-open (KOKAI) Nos. 11-113588 and 11-196888). These methods are economical, since organic nitrogen can be added in a smaller amount for proliferation of the bacterial cells, and the bacteria can sufficiently grow in a simple culture medium. However, there is still a room for improvement in terms of production amounts, concentration, and production rate per cell of the target organic acids as well as simplification of the production process, and the like. Furthermore, production of a non-amino organic acid by fermentation using a bacterium in which phosphoenolpyruvate carboxylase activity is enhanced (for example, Japanese Patent Laid-open No. 11-196887), and the like, has also been reported.
Furthermore, in the facultative anaerobic gram negative bacterium Escherichia coli, methods are known for producing a non-amino organic acid by culturing it once under aerobic conditions to allow for the growth of cells, and then culturing the resting cells without supplying oxygen to anaerobically produce the non-amino organic acid (Journal of Industrial Microbiology and Biotechnology (2002), 28 (6), 325-332), which is similar to the methods using coryneform bacteria. Also, the Escherichia coli bacteria can be aerobically cultured to aerobically produce the non-amino organic acid (U.S. Patent Published Application No. 20050170482). However, since Escherichia coli is a gram negative bacterium, it is vulnerable to osmotic pressure, and there remains room for improvement in productivity per cell etc.
The entire genome sequence of Escherichia coli, which belongs to the Enterobacteriaceae family, has been reported, and functions of putative protein-coding sequences have been predicted. The yidE gene is one of these putative protein-coding sequences, and although it is thought to code for a transporter, the actual function of this gene has not been clarified. Finally, the role of this gene in organic acid production has not been previously reported Nucleic Acid Research (2006), 34 (1), 1-9; Applied and Environmental Microbiology (2005), 71 (12), 8008-8015).
SUMMARY OF THE INVENTION
An aspect of the present invention is to provide a more efficient method for producing an organic acid using a bacterium, wherein higher yields are obtained.
It has been found that when using a bacterium which has been modified to enhance expression of the yidE gene, or a product obtained by processing the bacterium, the consumption rate of an organic raw material, generation rate of organic acid, or yield of organic acid increases.
It is an aspect of the present invention to provide a method for producing an organic acid comprising A) allowing a substance to act on an organic raw material in a reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas, wherein the substance is selected from the group consisting of: i) a bacterium which has an ability to produce an organic acid and has been modified to have enhanced expression of the yidE gene is enhanced, ii) a product obtained by processing the bacterium of i), and iii) combinations thereof, and B) collecting the organic acid.
It is a further aspect of the present invention to provide the method as described above, wherein the enhanced expression is obtained by a method selected from the group consisting of: i) increasing the copy number of the yidE gene, ii) modifying an expression control sequence of the yidE gene, and iii) combinations thereof.
It is a further aspect of the present invention to provide the method as described above, wherein the yidE gene is selected from the group consisting of:
(a) a DNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 15, 25, 27, 30 and 46, and
(b) a DNA which hybridizes with a nucleotide sequence complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 15, 25, 27, 30 and 46 under stringent conditions, and said DNA improves the ability of the bacterium to produce an organic acid when expression of the DNA is enhanced in the bacterium.
It is a further aspect of the present invention to provide the method as described above, wherein the yidE gene codes for a protein selected from the group consisting of: A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 16, 24, 26, 28, 29, 31, 47, 71 and 72, and B) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 16, 26, 28, 31 and 47, but which includes one or more substitutions, deletions, insertions or additions of one or several amino acid residues.
It is a further aspect of the present invention to provide the method as described above, wherein the bacterium belongs to the family Enterobacteriaceae.
It is a further aspect of the present invention to provide the method as described above, wherein the bacterium belongs to a genus selected from the group consisting of Escherichia, Enterobacter, Raoultella, Klebsiella, and Pantoea. 
It is a further aspect of the present invention to provide the method as described above, wherein the bacterium is a rumen bacterium.
It is a further aspect of the present invention to provide the method as described above, wherein the bacterium is Mannheimia succiniciproducens. 
It is a further aspect of the present invention to provide the method as described above, wherein the bacterium has been further modified to decrease an enzymatic activity selected from the group consisting of lactate dehydrogenase activity, alcohol dehydrogenase activity, pyruvate formate lyase activity, and combinations thereof.
It is a further aspect of the present invention to provide the method as described above, wherein the bacterium has been further modified so that pyruvate carboxylase activity is enhanced.
It is a further aspect of the present invention to provide the method as described above, wherein the organic acid is succinic acid.
It is an aspect of the present invention to provide a method for producing a succinic acid-containing polymer comprising A) producing succinic acid by a method as described above, and B) polymerizing the obtained succinic acid.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the effect of yidE amplification in Escherichia bacterium MG1655ΔldhAΔlldD (change of succinic acid accumulation over time).
FIG. 2 shows the effect of yidE amplification in Enterobacter bacterium AJ110637 strain (change of succinic acid accumulation over time).
FIG. 3 shows the structure of RSF-Red-TER.
FIG. 4 shows the construction scheme of RSF-Red-TER.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter, embodiments of the presently disclosed subject matter will be explained in detail.
<1> Bacterium
The bacterium used in the method of the presently disclosed subject matter can be a bacterium which has an ability to produce an organic acid and has been modified so that expression of the yidE gene is enhanced. The term “organic acid-producing ability” can mean an ability of the bacterium to produce and accumulate an organic acid in a reaction mixture or in the cells of the bacterium to such a degree that the organic acid can be collected from the reaction mixture or cells when the bacterium is cultured in the reaction mixture. For example, the bacterium can accumulate a target organic acid in a medium or a reaction mixture in an amount of, for example, 0.5 g/L or more, or 1.0 g/L or more in another example. Such a bacterium can be obtained by modifying a parent bacterial strain that has an organic acid-producing ability to enhance expression of the yidE gene. When the parent strain does not have a native ability to produce an organic acid, the ability to produce an organic acid can be imparted to the parent strain and then modified so that expression of the yidE gene is enhanced. Furthermore, the ability to produce an organic acid can be imparted to a strain which has already been modified to enhance expression of the yidE gene. The organic acid-producing ability can be native to the chosen bacterium, or can be obtained by modifying the bacterium, such as those mentioned below, using mutation techniques or recombinant DNA techniques.
The organic acid can be a metabolic intermediate of the TCA cycle, and examples include, for example, succinic acid, malic acid, fumaric acid, citric acid, isocitric acid, cis-aconitic acid, and the like.
Although the parent strain of the bacterium which can be used to derive the bacterium as described in the presently disclosed subject matter is not particularly limited, bacteria belonging to the family Enterobacteriaceae, bacteria classified as rumen bacteria, and coryneform bacteria are examples.
The family Enterobacteriaceae encompasses bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Raoultella, Salmonella, Serratia, Shigella, Morganella, Yersinia, and the like. In particular, bacteria classified into the family Enterobacteriaceae according to the taxonomy used by the NCBI (National Center for Biotechnology Information) database (www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) are examples.
A “bacterium belonging to the genus Escherichia” can mean that the bacterium is classified into the genus Escherichia according to the classification known to a person skilled in the art of microbiology, although the bacterium is not particularly limited. Examples of the bacterium belonging to the genus Escherichia include but are not limited to, Escherichia coli (E. coli).
The bacterium belonging to the genus Escherichia is not particularly limited. However, examples include, for example, the bacteria of the phyletic groups described in the work of Neidhardt et al. (Neidhardt F. C. Ed., 1996, Escherichia coli and Salmonella: Cellular and Molecular Biology/Second Edition, pp. 2477-2483, Table 1, American Society for Microbiology Press, Washington, D.C.). Specific examples include Escherichia coli W3110 (ATCC 27325), Escherichia coli MG1655 (ATCC 47076) and the like, and others derived from the prototype wild-type strain, K12 strain.
These strains are available from, for example, the American Type Culture Collection (Address: 12301 10801 University Boulevard, Manassas, Va. 20108, United States of America). That is, registration numbers are given to each of the strains, and the strains can be ordered by using these numbers. The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection.
Pantoea bacteria, Erwinia bacteria, and Enterobacter bacteria are classified as γ-proteobacteria, and they are taxonomically very close to one another (J. Gen. Appl. Microbiol., 1997, 43, 355-361; Int. J. Syst. Bacteriol., 1997, 43, 1061-1067). In recent years, some bacteria belonging to the genus Enterobacter were reclassified as Pantoea agglomerans, Pantoea dispersa, or the like, on the basis of DNA-DNA hybridization experiments etc. (Int. J. Syst. Bacteriol., 1989, 39:337-345). Furthermore, some bacteria belonging to the genus Erwinia were reclassified as Pantoea ananas or Pantoea stewartii (Int. J. Syst. Bacteriol., 1993, 43:162-173).
Examples of the Enterobacter bacteria include Enterobacter agglomerans, Enterobacter aerogenes, and the like. Specifically, the strains exemplified in European Patent Application Laid-open No. 952221 can be used. Typical strains of the genus Enterobacter include Enterobacter agglomerans ATCC 12287, Enterobacter aerogenes ATCC 13048, Enterobacter aerogenes NBRC 12010 (Biotechnol Bioeng., 2007, Mar. 27; 98(2):340-348), Enterobacter aerogenes AJ110637 (FERM ABP-10955), and the like. The AJ110637 strain was obtained from soil of seashore at Susuki Kaigan, Makinohara-shi, Shizuoka-ken on March, 2006 by cumulative liquid culture using glycerol as the carbon source. The full length 16S rDNA sequence was then determined, and was found to be 99.9% homologousto that of the Enterobacter aerogenes NCTC 10006. Moreover, also in a physiological test using an API kit, this strain gave results similar to the prototype species of Enterobacter aerogenes, and therefore was identified as Enterobacter aerogenes. This strain was deposited at International Patent Organism Depository, Agency of Industrial Science and Technology (Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Aug. 22, 2007, and assigned an accession number of FERM P-21348. Then, the deposit was converted to an international deposit based on the Budapest Treaty on Mar. 13, 2008, and assigned a receipt number of FERM ABP-10955.
Typical strains of the Pantoea bacteria include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specific examples include the following strains:
Pantoea ananatis AJ13355 (FERM BP-6614, European Patent Laid-open No. 0952221)
Pantoea ananatis AJ13356 (FERM BP-6615, European Patent Laid-open No. 0952221)
Although these strains are described as Enterobacter agglomerans in European Patent Laid-open No. 0952221, they are currently classified as Pantoea ananatis on the basis of nucleotide sequence analysis of the 16S rRNA etc., as described above.
Examples of the Erwinia bacteria include Erwinia amylovora and Erwinia carotovora, examples of the Klebsiella bacteria include Klebsiella planticola, and examples of the Raoultella bacteria include Raoultella planticola. Specific examples include the following strains:
Erwinia amylovora ATCC 15580 strain
Erwinia carotovora ATCC 15713 strain
Klebsiella planticola AJ13399 strain (FERM BP-6600, European Patent Laid-open No. 955368)
Klebsiella planticola AJ13410 strain (FERM BP-6617, European Patent Laid-open No. 955368).
Raoultella planticola ATCC 33531 strain
Although the AJ13399 strain and the AJ13410 strain were classified as Klebsiella planticola at the time of the deposit, Klebsiella planticola is currently classified into Raoultella planticola (Drancourt, M., 2001, Int. J. Syst. Evol. Microbiol, 51:925-32).
Examples of rumen bacteria can include Mannheimia bacteria, Actinobacillus bacteria, Anaerobiospirillum bacteria, Pyrobacterium bacteria, and Selenomonas bacteria. Bacteria including Mannheimia succiniciproducens, Actinobacillus succinogenes, Selenomonas ruminantium, Veillonella parvula, Wolnella succinogenes, and the like can be used. Specific strains of Mannheimia succiniciproducens can include Mannheimia sp. 55E strain (KCTC0769BP strain, U.S. Patent Published Application No. 20030113885, International Patent Publication WO2005/052135).
<1-1> Impartation of Organic Acid-Producing Ability
Hereinafter, methods for imparting an organic acid-producing ability to bacteria or methods for enhancing an organic acid-producing ability of bacteria are described.
To impart an ability to produce an organic acid, methods conventionally employed in the breeding of bacteria for producing substances by fermentation (see “Amino Acid Fermentation”, Japan Scientific Societies Press, 1st Edition, published May 30, 1986, pp. 77-100) can be applied. Such methods include the acquisition of an auxotrophic mutant, an analogue-resistant strain, or a metabolic regulation mutant, or construction of a recombinant strain having enhanced expression of an enzyme involved in the biosynthesis of an organic acid. When breeding bacteria that produce an organic acid, one or more properties, such as an auxotrophic mutation, analogue resistance, or metabolic regulation mutation, can be imparted. The expression of one or more enzymes involved in the biosynthesis of an organic acid can be enhanced. Furthermore, imparting properties such as auxotrophy, analogue resistance, or metabolic regulation can be combined with the enhancing biosynthetic enzymes.
An auxotrophic mutant strain, a strain resistant to an organic acid analogue, or a metabolic regulation mutant strain which is able to produce an organic acid can be obtained by subjecting a parent or wild-type strain to a conventional mutagenesis, such as exposure to X-rays or UV irradiation, or a treatment with a mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine, and then selecting the bacteria which exhibit an auxotrophy, analogue resistance, or metabolic regulation mutation and which also have the ability to produce an organic acid.
Methods for imparting the ability to produce succinic acid to bacteria, and succinic acid-producing bacteria, will be specifically exemplified below.
Succinic Acid-Producing Bacteria
As succinic acid-producing bacteria, strains that are unable to form acetic acid, lactic acid, ethanol and formic acid can be used, and specific examples include the Escherichia coli SS373 strain (International Patent Publication WO99/06532).
Strains that are unable to form acetic acid, lactic acid, ethanol and formic acid can be obtained by obtaining a strain that cannot assimilate acetic acid and lactic acid in a minimal medium, or by decreasing the activities of the lactic acid biosynthesis enzymes and acetic acid biosynthesis enzymes mentioned below (International Patent Publication WO2005/052135).
Moreover, such strains as described above can also be obtained by imparting resistance to monofluoroacetic acid (U.S. Pat. No. 5,521,075).
The ability to produce succinic acid can also be improved by imparting a glucose-assimilating ability under anaerobic conditions to a strain that is unable to produce both formic acid and lactic acid (International Patent Publication WO97/16528).
The ability to produce succinic acid can also be imparted by amplifying a gene which encodes an enzyme which is involved in the succinic acid biosynthesis system, or deleting a gene which encodes an enzyme which catalyzes a reaction which branches off from the succinic acid biosynthesis system to produce another compound.
The ability to produce succinic acid can also be imparted by modifying a bacterium to decrease the enzymatic activity of lactate dehydrogenase (LDH), which is a lactic acid biosynthesis system enzyme (International Patent Publications WO2005/052135, WO2005/116227, U.S. Pat. No. 5,770,435, U.S. Patent Published Application No. 20070054387, International Patent Publication WO99/53035, Alam, K. Y. and Clark, D. P., 1989, J. Bacteriol., 171:6213-6217). Some bacteria can have L-lactate dehydrogenase and D-lactate dehydrogenase, and such bacteria can be modified to decrease the activity of either one, or both, of the enzymes.
The ability to produce succinic acid can also be imparted by modifying a bacterium to decrease the enzymatic activity of the formic acid biosynthesis system enzyme, pyruvate-formate lyase (PFL) (U.S. Patent Published Application No. 20070054387, International Patent Publications WO2005/116227, WO2005/52135, Donnelly, M. I., Millard, C. S., Clark, D. P., Chen, M. J., Rathke, J. W., 1998, Appl. Biochem. Biotechnol., 70-72, 187-198.).
The ability to produce succinic acid can also be imparted by modifying a bacterium to decrease the enzymatic activities of phosphate acetyltransferase (PTA), acetate kinase (ACK), pyruvate oxidase (PDXB), acetyl-CoA synthetase (ACS) and acetyl-CoA hydrolase (ACH), which are acetic acid biosynthesis system enzymes (U.S. Patent Published Application No. 20070054387, International Patent Publications WO2005/052135, WO99/53035, WO2006/031424, WO2005/113745, and WO2005/113744).
The ability to produce succinic acid can also be enhanced by modifying a bacterium to decrease the enzymatic activity of alcohol dehydrogenase (ADH), which is an ethanol biosynthesis system enzyme (refer to International Patent Publication WO2006/031424).
The ability to produce succinic acid can also be enhanced by decreasing the activities of pyruvate kinase, glucose PTS (ptsG), ArcA protein, IclR protein (iclR), glutamate dehydrogenase (gdh) and/or glutamine synthetase (glnA), and glutamate synthase (gltBD) (International Patent Publication WO2006/107127, U.S. Patent Published Application No. 2007007933, Japanese Patent Laid-open No. 2005-168401). The gene abbreviations are in the parentheses following the enzyme names.
The ability to produce succinic acid can also be imparted by enhancing a biosynthesis system enzyme involved in succinic acid production.
The ability to produce succinic acid can also be enhanced by enhancing enzymatic activities of pyruvate carboxylase, malic enzyme, phosphoenolpyruvate carboxylase, fumarase, fumarate reductase, malate dehydrogenase and phosphoenolpyruvate carboxykinase (Japanese Patent Laid-open No. 11-196888, International Patent Publication WO99/53035, Hong, S. H., and S. Y. Lee, 2001, Biotechnol. Bioeng., 74:89-95, Millard, C. S. et al., 1996, Appl. Environ. Microbiol., 62:1808-1810, International Patent Publication WO2005/021770, Japanese Patent Laid-open No. 2006-320208, Kim, P. et al., 2004, Appl. Environ. Microbiol., 70:1238-1241). Enhancing the enzymatic activities of these target enzymes can be performed by referring to the methods for enhancing expression of the yidE gene described later.
Specific examples of succinic acid-producing bacteria belonging to the family Enterobacteriaceae include the following strains:
Escherichia coli S S373 strain (International Patent Publication WO99/06532)
Escherichia coli AFP111 strain (International Patent Publication WO97/16528)
Escherichia coli NZN111 strain (U.S. Pat. No. 6,159,738)
Escherichia coli AFP184 strain (International Patent Publication WO2005/116227)
Escherichia coli SBS100MG strain, SBS110MG strain, SBS440MG strain, SBS550MG strain, and SBS660MG strain (International Patent Publication WO2006/031424)
Examples of succinic acid-producing bacteria belonging to coryneform bacteria include the following strains:
Brevibacterium flavum AB-41 strain (Japanese Patent Laid-open No. 11-113588)
Brevibacterium flavum AB-41 strain (PC-amplified strain, Japanese Patent Laid-open No. 11-196888)
Corynebacterium glutamicum AJ110655 strain (FERM BP-10951)
Brevibacterium flavum MJ233Δldh strain (International Patent Publication WO2005/021770)
Brevibacterium lactofermentum (Corynebacterium glutamicum) 2256Δ(ldh, ach, pta, ack) (International Patent Publication WO2005/021770)
Brevibacterium lactofermentum 2256Δ(ldh, pta, ack, poxB) (International Patent Publication WO2005/113745)
Corynebacterium glutamicum Rldh-/pCRB-1 PC strain (International Patent Publication WO2005/010182)
Examples of succinic acid-producing bacteria belonging to rumen bacteria include the following strains:
Mannheimia succiniciproducens LPK, LPK7 and LPK4 (International Patent Publication WO2005/052135)
Actinobacillus succinogenes 130Z (U.S. Pat. No. 5,504,004)
Anaerobiospirillum succiniciproducens FZ10 (U.S. Pat. No. 5,521,075)
Anaerobiospirillum succiniciproducens FZ53 (U.S. Pat. No. 5,573,931)
<1-2> Enhancing Expression of the yidE Gene
The bacterium in accordance with the presently disclosed subject matter can be obtained by modifying a bacterium having an organic acid-producing ability such as those described above so that expression of the yidE gene is enhanced. However, the modification to enhance expression of the yidE gene is performed first, and then the ability to produce an organic acid can be imparted.
The yidE gene is thought to encode a kind of transporter (Riley, M. et al., 2006, Nucleic Acid Res., 34(1):1-9; Torres A. G. et al., 2005, Applied and Environmental Microbiology, 71 (12):8008-8015), and in addition, this gene codes for a protein which enhances an organic acid-producing ability of a bacterium when expression of this gene is enhanced. The degree of the enhancement of an organic acid-producing ability is not particularly limited, so long as productivity of the organic acid is improved as compared to an unmodified strain. However, the organic acid-producing ability is improved, for example, by 10% or more, 20% or more in another example, or 30% or more in another example, as compared to an unmodified strain.
The increase in expression of the yidE gene as compared to that of, for example, a wild-type or unmodified strain can be confirmed by comparing the amount of mRNA of yidE with that of the wild-type or unmodified strain.
To confirm the expression, exemplary methods include Northern hybridization and reverse transcriptase PCR(RT-PCR, Sambrook, J., and Russell, D. W., Molecular Cloning A Laboratory Manual/Third Edition, New York: Cold Spring Harbor Laboratory Press (2001)). The expression level of yidE can be increased to any level so long as the level is increased as compared to that of a wild-type or unmodified strain, and for example, it can be increased, for example, not less than 1.5 times, not less than 2 times in another example, or not less than 3 times in another example, as compared to that of, for example, a wild-type or an unmodified strain. Moreover, the increase in expression can also be confirmed on the basis of an increase in the amount of the target protein as compared to that in an unmodified or a wild-type strain, and it can be detected by, for example, Western blotting using an antibody (Sambrook, J., and Russell, D. W., Molecular Cloning A Laboratory Manual/Third Edition, New York: Cold Spring Harbor Laboratory Press (2001)).
Examples of the yidE gene can include yidE genes derived from enterobacteria, yidE genes derived from rumen bacteria, and homologues of these yidE genes. Examples can include yidE genes derived from Escherichia bacteria, yidE genes derived from Enterobacter bacteria, yidE genes derived from Salmonella bacteria, and the MS0288 or MS0289 gene derived from Mannheimia bacteria. Specific examples can also include the yidE gene derived from Escherichia coli MG1655 (SEQ ID NO: 13), the yidE gene derived from Salmonella typhimurium LT2 (SEQ ID NO: 15), the yidE gene derived from Shigella flexneri (SEQ ID NO: 25), the yidE gene derived from Yersinia pestis (SEQ ID NO: 27), the MS0288 gene derived from Mannheimia succiniciproducens (SEQ ID NO: 30), and the yidE gene derived from Enterobacter aerogenes AJ110637 (SEQ ID NO: 46). The yidE gene derived from Enterobacter aerogenes AJ110637 (SEQ ID NO: 46) and its encoded amino acid sequence (SEQ ID NO: 47) are novel sequences which have not been previously reported.
The yidE gene of Escherichia coli MG1655 is registered as b3685 in the genome sequence registered as GenBank Accession No. NC—000913 (amino acid sequence is GenBank Accession No. NP—418140). The yidE gene of Salmonella typhimurium LT2 is registered as STM3807 in the genome sequence registered as GenBank Accession No. NC—003197 (amino acid sequence is GenBank Accession No. NP—462707), and the yidE gene of Mannheimia succiniciproducens MBEL55E is registered as MS0288 or MS0289 in the genome sequence registered as GenBank Accession No. NC—006300 (amino acid sequence is GenBank Accession No. YP087480 (MS0288) or YP087481 (MS0289)). The yidE gene of the Shigella flexneri 8401 is registered as SFV3826 in the genome sequence registered as GenBank Accession No. NC—008258 (amino acid sequence is GenBank Accession No. YP—691142). The yidE gene of Yersinia pestis C092 is registered as YPO4083 in the genome sequence registered as GenBank Accession No. NC—003143 (amino acid sequence is GenBank Accession No. YP—407507).
A yidE gene homologue is a gene that can be derived from another microorganism, shows high homology to the yidE gene of Escherichia coli, and improves the ability to produce an organic acid when it is introduced into a host. The conserved sequence of the yidE proteins derived from Salmonella typhimurium and Escherichia coli is shown in SEQ ID NO: 24, and the conserved sequence of the yidE proteins derived from Salmonella typhimurium, Escherichia coli, and Enterobacter aerogenes AJ110637 is shown in SEQ ID NO: 71. The conserved sequence of the yidE proteins derived from Escherichia coli, Yersinia pestis, and Shigella flexneri is shown in SEQ ID NO: 29, and the conserved sequence of the yidE proteins derived from Salmonella typhimurium, Escherichia coli, Yersinia pestis, Shigella flexneri, and Enterobacter aerogene AJ110637 is shown in SEQ ID NO: 72. Examples of yidE gene homologues include genes coding for a protein having the amino acid sequence of SEQ ID NO: 24, 29, 71 or 72, or a protein having a homology of, for example, 80% or more, 90% or more in another example, 95% or more in another example, 98% or more in another example, or 99% or more in a another example, to the amino acid sequence of SEQ ID NO: 14, 16, 26, 28, 31 or 47, and which improves the ability to produce an organic acid of a host when it is introduced into the host. The yidE gene homologues coding for a protein having a homology of, for example, 80% or more, 90% or more in another example, 95% or more in another example, 98% or more in another example, or 99% or more in a another example, to the amino acid sequence of SEQ ID NO: 14, 16, 26, 28, 31 or 47 can have the consensus sequence of SEQ ID NOS: 24, 29, 71 or 72. Homology of amino acid sequences and nucleotide sequences can be determined by using, for example, the algorithm BLAST of Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA (Methods Enzymol., 183, 63 (1990)). Programs called BLASTN and BLASTX have been developed on the basis of this algorithm BLAST (refer to www.ncbi.nlm.nih.gov). In this specification, the term “homology” can also refer to “identity”.
Since sequences of the yidE gene from several different sources have already been reported as described above, the gene can be obtained by PCR using primers prepared on the basis of those nucleotide sequences. For example, a region including the yidE gene of Escherichia coli and a flanking region thereof, including a control region, can be obtained by PCR (polymerase chain reaction, see White, T. J. et al., Trends Genet., 5, 185 (1989)) using the primers shown in SEQ ID NOS: 9 and 10 and chromosomal DNA of Escherichia coli as the template. Homologues of yidE from other microorganisms can also be obtained in a similar manner.
Since the nucleotide sequence of the yidE gene can differ depending on the species or strains of enterobacteria, the yidE gene is not limited to a gene coding for the amino acid sequence of SEQ ID NO: 14, 16, 24, 26, 28, 29, 31, 47, 71 or 72, and it can be a mutant or artificially modified gene that codes for a protein having a sequence of SEQ ID NO: 14, 16, 26, 28, 31, 47 or 72, but which includes a conservative mutation, specifically, substitutions, deletions, insertions, additions, etc. of one or several amino acid residues at one or more positions so long as the ability to improve organic acid-producing ability of a bacterium when expression of the gene is enhanced is maintained. Although number meant by the term “one or several” can differ depending on positions in the three-dimensional structure of the protein or types of amino acid residues, it can be 1 to 20, 1 to 10 in another example, or 1 to 5 in another example. The substitutions, deletions, insertions, additions, inversions or the like of amino acid residues described above can also include those caused by naturally occurring mutations based on individual differences, differences in species of microorganisms that contain the yidE gene (mutant or variant), or the like.
The aforementioned substitution can be a conservative substitution that is a neutral substitution, that is, not resulting in a functional change. The conservative mutation is a mutation wherein substitution takes place mutually among Phe, Trp and Tyr, if the substitution site is an aromatic amino acid; among Leu, Ile and Val, if the substitution site is a hydrophobic amino acid; between Gln and Asn, if it is a polar amino acid; among Lys, Arg and His, if it is a basic amino acid; between Asp and Glu, if it is an acidic amino acid; and between Ser and Thr, if it is an amino acid having hydroxyl group. Specific examples of substitutions considered to be conservative substitutions can include: substitution of Ser or Thr for Ala; substitution of Gln, His or Lys for Arg; substitution of Glu, Gln, Lys, His or Asp for Asn; substitution of Asn, Glu or Gln for Asp; substitution of Ser or Ala for Cys; substitution of Asn, Glu, Lys, His, Asp or Arg for Gln; substitution of Gly, Asn, Gln, Lys or Asp for Glu; substitution of Pro for Gly; substitution of Asn, Lys, Gln, Arg or Tyr for His; substitution of Leu, Met, Val or Phe for Ile; substitution of Ile, Met, Val or Phe for Leu; substitution of Asn, Glu, Gln, His or Arg for Lys; substitution of Ile, Leu, Val or Phe for Met; substitution of Trp, Tyr, Met, Ile or Leu for Phe; substitution of Thr or Ala for Ser; substitution of Ser or Ala for Thr; substitution of Phe or Tyr for Trp; substitution of His, Phe or Trp for Tyr; and substitution of Met, Ile or Leu for Val.
Furthermore, as the yidE gene, a sequence encoding a protein having a homology not less than 80% in one example, 90% in another example, not less than 95% in another example, not less than 98% in another example, or not less than 99% in a another example, to the total amino acid sequence of SEQ ID NO: 14, 16, 26, 28, 31 or 47 and coding for a protein which improves organic acid-producing ability of a bacterium when expression is enhanced in the bacterium can be used. Furthermore, the degree of degeneracy of a gene varies depending on the hosts into which the gene is introduced, and therefore codons can be replaced with those which are favorable for the chosen host of the yidE gene. Moreover, the yidE gene can encode for a protein with an elongated or deleted N- or C-terminal sequence, so long as the gene improves the organic acid-producing ability of a bacterium when expression is enhanced in the bacterium. The length of the amino acid sequence to be elongated or deleted can be 50 or less, 20 or less in another example, 10 or less in another example, or 5 or less in a another example, in terms of number of amino acid residues. More specifically, the yidE gene can be a gene coding for a protein having the amino acid sequence of SEQ ID NO: 14, 16, 26, 28, 31 or 47 with elongation or deletion of 5 to 50 amino acid residues on the N-terminal or C-terminal side. For example, the products of the MS0288 and MS0289 genes derived from Mannheimia bacteria have 278 and 297 amino acid residues, respectively, and they are both quite a bit shorter than the yidE product of Escherichia coli, which has 554 amino acid residues. However, the MS0288 gene product has 74% homology to the C-terminal sequence of the yidE gene product derived from Escherichia coli, and the MS0289 gene product has 58% homology to the N-terminal sequence of the yidE gene product derived from Escherichia coli. 
Genes homologous to the yidE gene as described above can be obtained by modifying a gene coding for the amino acid sequence of SEQ ID NO: 14, 16, 26, 28, 31 or 47 so that the protein encoded by the gene includes substitutions, deletions, insertions, or additions of amino acid residues at a specific site(s) by, for example, site-specific mutagenesis. Furthermore, homologous genes can also be obtained by conventionally known mutation treatments, such as those described below. Examples of the mutation treatments include treating the yidE gene with hydroxylamine or the like in vitro, and treating a microorganism, for example, a coryneform bacterium, containing the gene with ultraviolet ray irradiation or a mutagen typically used for mutation such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS). Furthermore, a mutation can be artificially introduced into the yidE gene by gene recombination using error-prone PCR (Cadwell, R. C., PCR Meth. Appl., 2, 28 (1992)), DNA shuffling (Stemmer, W. P., Nature, 370, 389 (1994)), or StEP-PCR (Zhao, H., Nature Biotechnol., 16, 258 (1998)) to obtain a highly active yidE gene.
Whether such homologous yidE genes code for a protein which improves organic acid-producing ability when expression is enhanced can be confirmed by, for example, introducing these genes into a wild-type strain of Escherichia coli or the like, and determining whether the organic acid-producing ability of the bacterium is improved or not. For example, by adding a reducing substance such as glucose and an organic acid such as malic acid to the medium, and comparing the amount of succinic acid or fumaric acid which is converted from the organic acid utilizing the reducing power which occurs when glucose is assimilated, the effect can be more clearly verified.
Examples of the yidE gene also include a DNA that hybridizes with a nucleotide sequence complementary to the sequence of SEQ ID NO: 13, 15, 25, 27, 30 or 46, or a probe that can be prepared from the sequences under stringent conditions, and codes for a protein which improves organic acid-producing ability of a bacterium when expression of the gene is enhanced in the bacterium. The “stringent conditions” are conditions under which a so-called specific hybrid is formed, and non-specific hybrid is not formed. Examples include, for example, conditions under which DNAs showing high homology to each other, for example, DNAs showing a homology of, for example, not less than 80%, not less than 90% in another example, not less than 95% in another example, or not less than 97% in a another example, hybridize with each other, and DNAs having homology lower than the above level do not hybridize with each other. Other examples include typical washing conditions in ordinary Southern hybridization, i.e., washing once, or twice or three times, at salt concentrations and temperatures of 1×SSC, 0.1% SDS at 60° C., 0.1×SSC, 0.1% SDS at 60° C. in another example, 0.1×SSC, 0.1% SDS at 65° C. in another example, or 0.1×SSC, 0.1% SDS at 68° C. in another example.
A partial sequence of a nucleotide sequence complementary to the sequence of SEQ ID NO: 13, 15, 25, 27, 30 or 46 can also be used as the probe. Such a probe can be prepared by PCR using oligonucleotides prepared on the basis of any one of these nucleotide sequences as primers and a DNA fragment containing any one of the sequences as the template. When a DNA fragment having a length of about 300 by is used as the probe, the washing conditions after hybridization under the aforementioned conditions can be exemplified by 2×SSC, 0.1% SDS at 50° C.
The aforementioned descriptions concerning gene homologues and conservative mutations can be similarly applied to the other enzyme genes described in this specification.
A bacterium can be modified so that the expression of the homologous yidE genes as described above is enhanced.
The expression “modified so that expression of yidE gene is enhanced” or “modified to enhance express of the yidE gene” can mean that the number of YidE protein molecules per cell is increased, or that the activity per YidE protein molecule is increased, etc., as compared to a parent strain or a wild-type strain. Examples of the wild-type strain that can be used for comparison include Escherichia coli strains MG1655, W3110, etc., which are derived from Escherichia coli K12, and Enterobacter aerogenes strains AJ110637, NBRC 12010, etc.
Expression of the yidE gene can be enhanced by increasing the copy number of the yidE gene. For example, the copy number of the gene can be increased by ligating a fragment containing the yidE gene to a vector that functions in the chosen bacterium, for example, a multi copy vector, to prepare a recombinant DNA, and transforming the bacterium which has an organic acid-producing ability as described above with the DNA. Alternatively, after such a recombinant DNA as described above is introduced into a wild-type strain of a bacterium, an organic acid-producing ability can be imparted to the transformed bacterium. The copy number of the gene can also be increased by transferring a single copy or multiple copies of the yidE gene to the bacterial chromosome. Transfer of the gene to the chromosome can be confirmed by Southern hybridization using a part of the yidE gene as a probe.
Expression of the yidE gene can also be enhanced by modifying an expression control sequence of the yidE gene. For example, the enhancement can be achieved by replacing a promoter sequence of the yidE gene with a stronger promoter, or by making a promoter sequence closer to a consensus sequence (WO00/18935).
Methods for constructing a bacterium having an organic acid-producing ability and modified so that expression of the yidE gene is enhanced are explained below. These methods can be performed as described in a manual such as Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001).
Expression of the yidE gene can be enhanced by increasing the copy number by amplifying the yidE gene using a plasmid such as those described below. First, the yidE gene is cloned from the chromosome of Escherichia coli or the like by, for example, PCR. Chromosomal DNA can be prepared from a bacterium by, for example, the method of Saito and Miura (see H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963); Text for Bioengineering Experiments, Edited by the Society for Bioscience and Bioengineering, Japan, p 97-98, Baifukan Co., Ltd., 1992) or the like. Oligonucleotides for use in PCR can be synthesized on the basis of the aforementioned known information, for example, the synthetic oligonucleotides shown in SEQ ID NOS: 9 and 10 can be used to amplify the yidE gene.
A gene fragment including the yidE gene amplified by PCR can itself be amplified by inserting the fragment into a vector having a replication origin that enables autonomous replication in the chosen bacterium, then transform the bacterium with the vector. Examples of vectors which can be used to transform Enterobacteriaceae bacteria include pUC19, pUC18, pHSG299, pHSG399, pHSG398, RSF1010, pBR322, pACYC184, pMW219, and the like. When transforming coryneform bacteria, examples of plasmids that are autonomously replicable in coryneform bacteria include plasmid pCRY30 (Japanese Patent Laid-open No. 3-210184); plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (Japanese Patent Laid-open No. 2-72876 and U.S. Pat. No. 5,185,262); plasmids pCRY2 and pCRY3 (Japanese Patent Laid-open No. 1-191686); pAM330 (Japanese Patent Laid-open No. 58-67679); pHM1519 (Japanese Patent Laid-open No. 58-77895); pAJ655, pAJ611, and pAJ1844 (Japanese Patent Laid-open No. 58-192900); pCG1 (Japanese Patent Laid-open No. 57-134500); pCG2 (Japanese Patent Laid-open No. 58-35197); pCG4, pCG11, etc. (Japanese Patent Laid-open No. 57-183799); and pVK7 (Japanese Patent Laid-open No. 10-215883).
To prepare a recombinant DNA by ligating the yidE gene to a vector that functions in the chosen bacterium, the vector is digested with a restriction enzyme suitable for the ends of the yidE gene. Such a restriction enzyme site can be introduced in advance into the synthetic oligonucleotide which is used to amplify the yidE gene. Ligation is usually performed by using a ligase such as T4 DNA ligase.
In order to introduce a recombinant plasmid prepared as described above into a bacterium, any known transformation method reported to date be employed. For example, recipient cells can be treated with calcium chloride so as to increase permeability for the DNA, which has been reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)). Also, competent cells can be prepared from growing cells and DNA can be introduced into these cells, which has been reported for Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, F. E., Gene, 1, 153 (1977)). Another method is to make DNA recipient cells into protoplasts or spheroplasts which easily take up a recombinant DNA, and a recombinant DNA can be introduced into these cells, which are known for Bacillus subtilis, actinomycetes, and yeasts (Chang, S, and Choen, S. N., Mol. Gen. Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M. and Hopwood, O. A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B. and Fink, G. R., Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)). In addition, transformation of bacteria can also be performed by the electric pulse method (Japanese Patent Laid-open No. 2-207791) or by the conjugal transfer method (Biotechnology (NY). 1991 January; 9(1):84-7).
The copy number of the yidE can also be increased by integrating multiple copies of the yidE gene into the chromosomal DNA of a bacterium, which can be accomplished by homologous recombination. This technique is performed by targeting a sequence which is present in multiple copies on the chromosomal DNA. Sequences present on the chromosomal DNA in multiple copies include a repetitive DNA or inverted repeats present at the end of a transposable element. Alternatively, as disclosed in Japanese Patent Laid-open No. 2-109985, multiple copies of the yidE gene can be introduced into a chromosomal DNA by incorporating them into a transposon and transferring it (Japanese Patent Laid-open Nos. 2-109985, 7-107976, Mol. Gen. Genet., 245, 397-405 (1994); Plasmid, 2000 November; 44(3): 285-91).
Expression of the yidE gene can also be enhanced by replacing a native expression control sequence, such as a promoter, of the yidE gene on the chromosomal DNA or a plasmid with a stronger promoter. Other methods include modifying a factor involved in expression control of the yidE gene such as operator or repressor, or ligating a strong terminator (Hamilton et al., Journal of Bacteriology 171:4617-4622; WO98/004715). For example, the lac promoter, trp promoter, trc promoter, tac promoter, PR promoter derived from λ-phage, lacUV promoter, and the like are known as strong promoters. Methods for evaluating the strength of promoters and examples of strong promoters are described in the paper of Goldstein et al. (Goldstein, M. A. and Doi, R. H., 1995, Biotechnol. Annu. Rev., 1, 105-128), and the like. Furthermore, as disclosed in WO00/18935, the strength of a promoter can be increased by making several nucleotide substitutions in the promoter region of a target gene so as to make the sequence closer to a consensus sequence. For example, the −35 region can be replaced with TTGACA or TTGCCA, and the −10 region can be replaced with TATAAT or TATAAC. In addition, it is known that the translation efficiency of mRNA is significantly affected by the substitution of several nucleotides in the spacer sequence between the ribosome-binding site (RBS) and the translation initiation codon, in particular, the sequence immediately upstream of the initiation codon, and therefore, such a sequence can be modified.
Expression of a gene can also be enhanced by extending the survival time of mRNA or by preventing degradation of the encoded protein in the cells. An expression control sequence such as a promoter which is upstream of the yidE gene can also be identified by using a promoter search vector or gene analysis software such as GENETYX. Expression of the yidE gene can be enhanced by such promoter substitution or modification.
Modifying a expression control sequence can be combined with increasing the copy number of the yidE gene.
A bacterial strain that is modified to decrease the activity of one or more enzymes such as lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), and pyruvate formate lyase (PFL), in addition to increasing the expression of the yidE gene, can be more effective. The expression “modified so that lactate dehydrogenase activity is decreased” can mean that the lactate dehydrogenase activity is decreased as compared to that of a strain in which lactate dehydrogenase is unmodified. The lactate dehydrogenase activity per cell can be decreased to 10% or lower as compared to that of a strain in which lactate dehydrogenase is unmodified. The lactate dehydrogenase activity can also be completely deleted. Decrease of the lactate dehydrogenase activity can be confirmed by measuring the lactate dehydrogenase activity by a known method (Kanarek, L. and Hill, R. L., 1964, J. Biol. Chem., 239:4202). Specific examples of a method for producing a mutant strain of Escherichia coli in which the lactate dehydrogenase activity is decreased include the method described in Alam, K. Y., and Clark, D. P., 1989, J. Bacteriol., 171:6213-6217, and the like. The bacterium in which lactate dehydrogenase activity is decreased and expression of the yidE gene is enhanced can be obtained by, for example, preparing a bacterium with a disrupted LDH gene, and transforming this bacterium with a recombinant vector containing the yidE gene, as described in Example 1. However, either the modification to decrease the LDH activity or the modification to enhance expression of the yidE gene can be performed first. In Escherichia coli, LDH is encoded by the ldhA and lldD genes. The DNA sequence of the ldhA gene is shown in SEQ ID NO: 20, and the encodedamino acid sequence is shown in SEQ ID NO: 21, the DNA sequence of the lldD gene is shown in SEQ ID NO: 22, and the encoded amino acid sequence is shown in SEQ ID NO: 23.
In order to decrease or delete the activity of LDH, a mutation can be introduced into the LDH gene on the chromosome by a typical mutagenesis method. For example, the gene coding for LDH on the chromosome can be deleted, or an expression control sequence such as a promoter and/or the Shine-Dalgarno (SD) sequence can be modified by gene recombination. Furthermore, a mutation which results in an amino acid substitution (missense mutation), a stop codon (nonsense mutation), or a frame shift mutation that adds or deletes one or two nucleotides into the LDH coding region on the chromosome can be introduced, or a part of the gene, or the entire gene can be deleted (Qiu Z. and Goodman M. F., 1997, J. Biol. Chem., 272:8611-8617). Furthermore, the LDH activity can also be decreased or deleted by gene disruption, for example, by mutating or deleting the coding region of the LDH gene, and replacing the normal or native LDH gene on the chromosome with the mutant LDH gene by homologous recombination or the like. Alternatively, a transposon or IS factor can be introduced into the gene.
In order to introduce a mutation that reduces or deletes the LDH activity by genetic recombination, for example, the following methods can be used. The LDH gene on the chromosome can be replaced with a mutant gene by preparing a mutant LDH gene in which a partial sequence of the LDH gene is modified so that it does not produce a functional LDH enzyme, and transforming a bacterium with a DNA containing the mutant gene to cause homologous recombination between the mutant gene and the gene on the chromosome. Such site-specific mutagenesis based on gene substitution utilizing homologous recombination has been already reported, and includes the methods of Red driven integration developed by Datsenko and Wanner (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, pp. 6640-6645), using a linear DNA in the Red driven integration in combination with an excision system derived from λ phage (Cho, E. H., Gumport, R. I., Gardner, J. F., J. Bacteriol., 184: 5200-5203 (2002)) (refer to WO2005/010175), using a plasmid containing a temperature sensitive replication origin (U.S. Pat. No. 6,303,383, Japanese Patent Laid-open No. 05-007491, WO2005/010175), and the like. Such site-specific mutagenesis based on gene substitution using homologous recombination as described above can also be performed with a plasmid that is unable to replicate in the chosen host.
The expression “modified so that alcohol dehydrogenase activity is decreased” can mean that the alcohol dehydrogenase activity is decreased as compared to that of a strain in which the alcohol dehydrogenase is unmodified. The alcohol dehydrogenase activity per cell can be reduced to 10% or lower as compared to that of a strain in which the alcohol dehydrogenase is unmodified. The alcohol dehydrogenase activity can also be completely deleted. Reduction of the alcohol dehydrogenase activity can be confirmed by measuring the alcohol dehydrogenase activity by a known method (Lutstorf, U. M., Schurch, P. M. & von Wartburg, J. P., Eur. J. Biochem., 17, 497-508 (1970)). Specific examples of the method for producing a mutant strain of Escherichia coli in which the alcohol dehydrogenase activity is decreased include the method described in Sanchez A. M. et al., 2005, Biotechnol. Prog., 21:358-365, and the like. A bacterium in which the alcohol dehydrogenase activity is decreased and expression of the yidE gene is enhanced can be obtained by, for example, preparing a bacterium in which the gene coding for alcohol dehydrogenase (ADH) is disrupted, and transforming this bacterium with a recombinant vector containing the yidE gene. However, either the modification for decreasing the ADH activity or the modification for enhancing expression of the yidE gene can be performed first. The alcohol dehydrogenase activity can be decreased by a method similar to the method for decreasing the lactate dehydrogenase activity described above. A partial nucleotide sequence of the ADH gene from the Enterobacter aerogenes AJ110637 strain (FERM ABP-10955) is shown in SEQ ID NO: 34. The entire nucleotide sequence of this gene can be determined by, for example, isolating the ADH gene (adhE) from the chromosomal DNA of Enterobacter aerogenes on the basis of the reported partial sequence.
The expression “modified so that pyruvate formate lyase activity is decreased” can mean that the pyruvate formate lyase activity is decreased as compared to that of a a strain in which the pyruvate formate lyase is unmodified. The pyruvate formate lyase activity per cell can be decreased to 10% or lower as compared to that of a a strain in which the pyruvate formate lyase is unmodified. The pyruvate formate lyase activity can also be completely deleted. The decrease of the pyruvate formate lyase activity can be confirmed by measuring the pyruvate formate lyase activity by a known method (Knappe, J. and Blaschkowski, H. P., 1975, Meth. Enzymol., 41:508-518). The bacterium in which pyruvate formate lyase activity is decreased and expression of the yidE gene is enhanced can be obtained by, for example, preparing a bacterium in which PFL gene is disrupted, and transforming this microorganism with a recombinant vector containing the yidE gene. However, either the modification for decreasing the PFL activity or the modification for enhancing expression of yidE can be performed first. The pyruvate formate lyase activity can be decreased by a method similar to the method for decreasing the lactate dehydrogenase activity described above.
A bacterium modified so that the pyruvate carboxylase (PC) activity is enhanced, in addition to the enhanced expression of the yidE gene can also be used. Enhancing the pyruvate carboxylase activity can be combined with decreasing the lactate dehydrogenase activity, alcohol dehydrogenase activity, and/or pyruvate formate lyase activity. The expression “modified so that pyruvate carboxylase activity is enhanced” can mean that the pyruvate carboxylase activity is increased as compared to that of an unmodified strain such as a wild-type strain or parent strain. The pyruvate carboxylase activity can be measured by, for example, by measuring the decrease in NADH.
As the PC gene, a gene for which the nucleotide sequence is already determined, or a gene obtained by isolating a DNA fragment encoding a protein having the PC activity from a chromosome of a microorganism, animal, plant, or the like and determining the nucleotide sequence, can be used. After the nucleotide sequence is determined, a gene synthesized on the basis of that sequence can also be used.
As the PC gene (pyc), for example, a PC gene derived from or native to a coryneform bacterium such as Corynebacterium glutamicum or Brevibacterium flavum (Peters-Wendisch, P. G. et al., 1998, Microbiology, vol. 144:915-927) (SEQ ID NO: 17) can be used.
PC genes from bacteria other than Corynebacterium glutamicum, as well as from other microorganisms, animals and plants, can also be used. In particular, the reported sequences of PC genes derived from microorganisms, animals and plants are described below (citations are indicated in brackets), and they can be obtained by hybridization or amplification of the ORF regions by PCR in the same manner as described above.
Human [Biochem. Biophys. Res. Comm., 202, 1009-1014, (1994)]
Mouse [Proc. Natl. Acad. Sci. USA., 90, 1766-1779, (1993)]
Rat [GENE, 165, 331-332, (1995)]
Yeast: Saccharomyces cerevisiae [Mol. Gen. Genet., 229, 307-315, (1991)],
Schizosaccharomyces pombe [DDBJ Accession No.; D78170]
Bacillus stearothermophilus [GENE, 191, 47-50, (1997)]
Rhizobium etli [J. Bacteriol., 178, 5960-5970, (1996)]
The PC gene can be enhanced in the same manner as those used for enhancing expression of the yidE gene as described above.
<2> Production method
An organic acid can be produced by using a bacterium that is able to produce an organic acid, and has been modified so that expression of the yidE gene is enhanced as described above. Specifically, an organic acid can be produced by allowing the bacterium, or a product obtained by processing the bacterium, to act on an organic raw material in a reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas to produce the organic acid, and collecting the organic acid.
In one example of the method, by culturing the microorganism in a reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas, and an organic raw material, proliferation of the microorganism and production of the organic acid occur simultaneously. In this example, a medium can be the reaction mixture. Proliferation of the microorganism and production of the organic acid can be simultaneously attained, or there can be a period during the culture when proliferation of the microorganism mainly occurs, and a period in which production of the organic acid mainly occurs.
In another example, by allowing cells to proliferate in a medium in the presence of carbonate ions, bicarbonate ions, or carbon dioxide gas, and an organic raw material, and thereby allowing the cells to act on the organic raw material in the medium or reaction mixture, an organic acid can be produced. In this example, a product obtained by processing the cells of the bacterium can also be used. Examples of a product obtained by processing the cells include, for example, immobilized cells which can be obtained with acrylamide, carragheenan, or the like, disrupted cells, centrifugation supernatant of the disrupted product, fraction obtained by partial purification of the supernatant by ammonium sulfate treatment or the like.
Although the bacteria can be cultured on a solid medium such as agar medium by slant culture, bacteria previously cultured in a liquid medium (seed culture) are other examples.
As the medium used for the culture, a typical microorganism culture medium can be used. For example, a typical medium obtained by adding natural nutrients such as meat extract, yeast extract and peptone, to a composition including inorganic salts such as ammonium sulfate, potassium phosphate and magnesium sulfate can be used.
In the aforementioned first example, the carbon source that is added to the medium also serves as the organic raw material for the production of the organic acid.
In the aforementioned second example, after the culture, the cells are collected by centrifugation, membrane separation, or the like, and used for the organic acid production reaction.
The organic raw material is not particularly limited so long as a carbon source, which the chosen microorganism can assimilate to produce succinic acid, is used. However, fermentable carbohydrates including carbohydrates such as galactose, lactose, glucose, fructose, glycerol, sucrose, saccharose, starch and cellulose, polyalcohols such as glycerin, mannitol, xylitol and ribitol, and the like are usually used. When the organic acid is succinic acid, fumaric acid or the like can be added in order to efficiently produce succinic acid as described in Japanese Patent Laid-open No. 5-68576, and malic acid can be added instead of fumaric acid.
Furthermore, a saccharified starch solution, molasses, or the like containing the fermentable carbohydrates can also be used. The fermentable carbohydrates can be used independently or in combination. Although the concentration of the aforementioned organic raw material is not particularly limited, it is more advantageous when the concentration is as high as possible within such a range that the production of the organic acid is not inhibited. In the aforementioned first example, concentration of the organic raw material in the medium is generally in the range of 5 to 30% (w/v), or 10 to 20% (w/v) in another example. Furthermore, in the aforementioned second example, the concentration of the organic raw material in the reaction mixture is generally in the range of 5 to 30% (w/v), or 10 to 20% (w/v) in another example. Furthermore, additional organic raw material can be added as its concentration decreases with progress of the reaction.
The aforementioned reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas and the organic raw material is not particularly limited, and it can be, for example, a medium for culturing bacteria, or it can be a buffer such as phosphate buffer. The reaction mixture can be an aqueous solution containing a nitrogen source, inorganic salts, and the like. The nitrogen source is not particularly limited so long as it is a nitrogen source which the chosen bacterium can assimilate to produce an organic acid, and specific examples include various organic or inorganic nitrogen compounds such as ammonium salts, nitrates, urea, soybean hydrolysate, casein degradation products, peptone, yeast extract, meat extract, and corn steep liquor. Examples of the inorganic salts include various phosphates, sulfates, and metallic salts such as those of magnesium, potassium, manganese, iron, and zinc. If necessary, growth-promoting factors including vitamins such as biotin, pantothenic acid, inositol, and nicotinic acid, nucleotides, amino acids and the like can be added. In order to suppress foaming at the time of the reaction, an appropriate amount of commercially available antifoam can be added to the medium.
pH of the reaction mixture can be adjusted by adding sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, or the like. Since the pH for the reaction is usually 5 to 10, or 6 to 9.5 in another example, the pH of the reaction mixture is adjusted to be within the aforementioned range with an alkaline substance, carbonate, urea, or the like even during the reaction, if needed.
As the reaction mixture, water, buffer, medium or the like can be used, but a medium is particular example. The medium can contain, for example, the aforementioned organic raw material, and carbonate ions, bicarbonate ions, or carbon dioxide gas, and the reaction can be performed under anaerobic conditions. The carbonate or bicarbonate ions can be supplied from magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate, or potassium bicarbonate, which can also be used as a neutralizing agent. However, if necessary, carbonate or bicarbonate ions can also be supplied from carbonic acid or bicarbonic acid or salts thereof or carbon dioxide gas. Specific examples of the salts of carbonic acid or bicarbonic acid include, for example, magnesium carbonate, ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate, potassium bicarbonate, and the like. Carbonate ions or bicarbonate ions can be added at a concentration of 0.001 to 5 M, 0.1 to 3 M in another example, or 1 to 2 M in another example. When carbon dioxide gas is present, it can be present in an amount of 50 mg to 25 g, 100 mg to 15 g in another example, or 150 mg to 10 g in another example, per liter of the solution.
The optimal growth temperature of the bacterium is generally in the range of 25 to 40° C. The reaction temperature is generally in the range of 25 to 40° C., or in the range of 30 to 37° C. in another example. The amount of bacterial cells in the reaction mixture can be, although it is not particularly limited, 1 to 700 g/L, 10 to 500 g/L in another example, or 20 to 400 g/L in another example. The reaction time can be 1 to 168 hours, or 3 to 72 hours in another example. The reaction can be performed batch-wise or on a column.
The bacterial culture can be performed under aerobic conditions. Alternatively, the organic acid production reaction can be performed under aerobic conditions, microaerobic conditions or anaerobic conditions. For the reaction under microaerobic conditions or anaerobic conditions, the reaction can be performed in a sealed reaction vessel without aeration, by supplying an inert gas such as nitrogen gas to the reaction mixture, by supplying an inert gas containing carbon dioxide gas to the reaction mixture, and the like.
The organic acid that accumulates in the reaction mixture (culture medium) can be separated and purified from the reaction mixture in a conventional manner. Specifically, solids such as bacterial cells can be removed by centrifugation, filtration, or the like, and then the resulting solution can be desalted with an ion exchange resin or the like. The organic acid can be separated and purified from the solution by crystallization or column chromatography.
Furthermore, when the target organic acid is succinic acid, after the succinic acid is produced, a polymerization reaction can be carried out by using the succinic acid as a raw material to produce a polymer containing succinic acid. In recent years, with the increase of environmentally friendly industrial products, polymers prepared from raw materials of plant origin have been attracting attention. Succinic acid can be converted into polymers such as polyesters and polyamides and used (Japanese Patent Laid-open No. 4-189822). Specific examples of succinic acid-containing polymers include succinic acid polyesters obtainable by polymerizing a diol such as butanediol, ethylene glycol, and succinic acid, succinic acid polyamides obtainable by polymerizing a diamine such as hexamethylenediamine and succinic acid, and the like. In addition, succinic acid and succinic acid-containing polymers and compositions containing these can be used for food additives, pharmaceutical agents, cosmetics, and the like.
EXAMPLES
Hereinafter, the present invention will be explained more specifically with reference to the following non-limiting examples.
Reference Example 1
Construction of L-, D-Lactate Dehydrogenase Gene-Deficient Escherichia coli Strain
This gene was deleted by using the method called “Red-driven integration” developed by Datsenko and Wanner (Datsenko, K. A. and Wanner, B. L., 2000, Proc. Natl. Acad. Sci. USA, vol. 97, No. 12, pp. 6640-6645), and the λ phage excision system (Cho, E. H., Gumport, R. I., and Gardner, J. F., 2002, J. Bacteriol., 184 (18):5200-5203). According to this method, it is possible to construct a gene-disrupted strain in a single step by using a PCR product obtained with synthetic oligonucleotide primers in which a part of the target gene is present in the 5′ end sequence and a part of an antibiotic resistance gene is present in the 3′ end sequence. Furthermore, by using the λ phage excision system in combination, the antibiotic resistance gene which is integrated into the gene-disrupted strain can be removed.
<1-1> Construction of Strain Deficient in ldhA Gene Coding for D-Lactate Dehydrogenase
According to the description of WO2005/010175, PCR was performed by using synthetic oligonucleotides having sequences corresponding to parts of the ldhA gene in the 5′ end sequences and sequences corresponding to both ends of attL and attR of λ phage at the 3′ end sequences as primers and plasmid pMW118-attL-Cm-attR as the template. pMW118-attL-Cm-attR is a plasmid obtained by inserting the attL and attR genes, which are the attachment sites of λ phage, and the cat gene, which is a chloramphenicol resistance gene, into pMW118 (Takara Bio), and the genes are inserted in the order of attL-cat-attR. The sequences of the synthetic oligonucleotides used as the primers are shown in SEQ ID NOS: 1 and 2. The amplified PCR product was purified on an agarose gel and introduced into Escherichia coli MG1655 strain containing the plasmid pKD46, which is capable of temperature-sensitive replication by electroporation. Then, an ampicillin-sensitive strain not harboring pKD46 was obtained, and deletion of the ldhA gene was confirmed by PCR. PCR was performed by using the synthetic oligonucleotides shown in SEQ ID NOS: 3 and 4 as primers. Whereas the PCR product obtained for the parent strain had a size of about 1.2 kb, the deficient strain showed a band with a size of about 1.9 kb.
To eliminate the att-cat gene introduced into the ldhA gene, the strain was transformed with the helper plasmid pMW-intxis-ts, and an ampicillin resistant strain was selected. The pMW-intxis-ts contains the λ phage integrase (Int) gene and excisionase (Xis) gene and shows temperature-sensitive replication. Then, the strain in which att-cat and pMW-intxis-ts were eliminated was confirmed by PCR on the basis of ampicillin sensitivity and chloramphenicol sensitivity. PCR was performed by using the synthetic oligonucleotides shown in SEQ ID NOS: 3 and 4 as primers. Whereas the PCR product obtained for the strain in which att-cat remained had a size of about 1.9 kb, a band of about 0.3 kb was observed for the strain in which att-cat was eliminated. The ldhA deficient strain obtained as described above was designated as MG1655ΔldhA strain.
<1-2> Construction of Strain Deficient in lldD Gene Coding for L-Lactate Dehydrogenase
A strain was constructed in the same manner as that of the construction of the ldhA gene-deficient strain. PCR was performed by using synthetic oligonucleotides having sequences corresponding to parts of the lldD gene in the 5′ end sequences and sequences corresponding to both ends of attL and attR of λ phage in the 3′ end sequences as primers and plasmid pMW118-attL-Cm-attR as the template. The sequences of the synthetic oligonucleotides used as the primers are shown in SEQ ID Nos. 5 and 6. The amplified PCR product was purified on an agarose gel and introduced into the Escherichia coli MG1655ΔldhA strain containing plasmid pKD46, which is capable of temperature-sensitive replication by electroporation. Then, an ampicillin-sensitive strain not harboring pKD46 was obtained, and the deletion of the lldD gene was confirmed by PCR. PCR was performed by using the synthetic oligonucleotides shown in SEQ ID NOS: 7 and 8 as primers. Whereas the PCR product obtained for the parent strain had a size of about 1.4 kb, a band of about 1.9 kb should be observed for the deficient strain.
To eliminate the att-cat gene introduced into the lldD gene, the strain was transformed with the helper plasmid pMW-intxis-ts, and an ampicillin resistant strain was selected. Then, the strain in which att-cat and pMW-intxis-ts were eliminated was obtained on the basis of ampicillin sensitivity and chloramphenicol sensitivity, and confirmed by PCR. PCR was performed by using the synthetic oligonucleotides shown in SEQ ID NOS: 7 and 8 as primers. Whereas the PCR product obtained for the strain in which att-cat remained had a size of about 1.9 kb, a band of about 0.3 kb was observed for the strain in which att-cat was eliminated. The lldD deficient strain obtained as described above was designated as MG1655ΔldhAΔlldD strain.
Example 1
Construction of yidE Gene-Enhanced Strain of Succinic Acid-Producing Bacterium
<1-1> Construction of a Plasmid for Gene Amplification
In order to amplify the yidE gene, plasmid pMW219::Pthr was used. This plasmid corresponds to the vector pMW219 (Nippon Gene) which has the promoter region of the threonine operon (thrLABC) from the genome of the Escherichia coli shown in SEQ ID NO: 11 at the HindIII site and BamHI site, and enables amplification of a gene by cloning the gene at a position in the plasmid downstream from that promoter.
<1-2> Construction of a Plasmid for Enhancing yidE Gene Derived from Escherichia coli 
PCR was performed by using the synthetic oligonucleotide having a BamHI site shown in SEQ ID NO: 12 as a 5′ primer, the synthetic oligonucleotide having a BamHI site shown in SEQ ID NO: 19 as a 3′ primer. These primers were prepared on the basis of the nucleotide sequence of the yidE gene in the genome sequence of Escherichia coli (GenBank Accession No. U00096 b3685). The genomic DNA of Escherichia coli MG1655 strain was used as the template, and PCR product was treated with the restriction enzyme BamHI to obtain a gene fragment containing the yidE gene. The fragment was purified and ligated with the vector pMW219::Pthr which had been previously treated with BamHI to construct plasmid pMW219::Pthr::yidE for yidE amplification.
<1-3> Production of yidE-Amplified Strain
pMW219::Pthr::yidE obtained in <1-2> described above and pMW219 were used to transform the Escherichia coli MG1655ΔldhAΔlldD strain by the electric pulse method, and the transformants were applied to the LB agar medium containing 25 μg/ml of kanamycin, and cultured at 37° C. for about 18 hours. The colonies which appeared were purified, and plasmids were extracted from them in a conventional manner to confirm introduction of the target plasmid. The obtained strains were designated as MG1655ΔldhAΔlldD/pMW219::Pthr::yidE and MG1655ΔldhAΔlldD/pMW219, respectively. The Enterobacter aerogenes AJ110637 strain was also transformed with pMW219::Pthr::yidE and pMW219 by the electric pulse method, and the transformants were applied to the LB agar medium containing 50 μg/ml of kanamycin, and cultured at 37° C. for about 18 hours. The colonies which appeared were purified, and plasmids were extracted from them in a conventional manner to confirm introduction of the target plasmid. The obtained strains were designated as AJ110637/pMW219::Pthr::yidE and AJ110637/pMW219, respectively.
Example 2
Effect of yidE Amplification
<2-1> Effect of yidE Amplification in Succinic Acid-Producing Strain of Escherichia bacterium 
MG1655ΔldhAΔlldD/pMW219::Pthr::yidE and MG1655ΔldhAΔlldD/pMW219 were each uniformly applied to an LB plate containing 25 μg/ml of kanamycin, and cultured at 37° C. for 16 hours. Then, each plate was incubated at 37° C. for 16 hours under anaerobic conditions by using Anaeropack (Mitsubishi Gas Chemical). The obtained cells on the plate were washed with 0.8% brine, and then diluted 51 times, and thereby a cell suspension having OD=1.0 (600 nm) was prepared. This cell suspension in a volume of 100 μl and a production medium (10 g/l of glucose, 10 g/12Na malate, 45.88 g/l of TES, 6 g/l of Na2HPO4, 3 g/l of KH2PO4, 1 g/l of NH4Cl, adjusted to pH 7.3 with KOH and filtrated) in a volume of 1.3 ml in which dissolved gases in the medium were replaced with nitrogen gas by bubbling nitrogen gas beforehand were put into 1.5-ml volume micro tubes, and the cells were cultured at 31.5° C. for 10 hours by using a stirrer for micro tubes. After the culture, the amount of succinic acid which accumulated in the medium was analyzed by liquid chromatography. Two of Shim-pack SCR-102H (Shimadzu) connected in series were used as the column, and a sample was eluted at 50° C. with 5 mM p-toluenesulfonic acid. The eluate was neutralized with 20 mM Bis-Tris aqueous solution containing 5 mM p-toluenesulfonic acid and 100 μM EDTA, and succinic acid was quantified by measuring electric conductivity with CDD-10AD (Shimadzu).
The amounts of succinic acid which accumulated after 10 hours are shown in Table 1, and change of succinic acid accumulation with time is shown in FIG. 1.







TABLE 1







Effect of yidE amplification in succinic acid-producing strain,


MG1655ΔldhAΔlldD










Strain
Succinate (g/L)







MG1655ΔldhAΔlldD/pMW219
1.91 (±0.13)



MG1655ΔldhAΔlldD/pMW219::Pthr::yidE
2.32 (±0.09)










Succinic acid accumulation was markedly increased in the yidE gene-amplified strain MG1655ΔldhAΔlldD/pMW219::Pthr::yidE as compared to the control MG1655ΔldhAΔlldD/pMW219, and on the basis of this result, the effect of enhancement of yidE expression on succinic acid production was confirmed.
<2-2> Effect of yidE Amplification in Enterobacter Bacterium
AJ110637/pMW219::Pthr::yidE and AJ110637/pMW219 were each uniformly applied to an LB plate containing 50 μg/ml of kanamycin, and cultured at 37° C. for 16 hours. Then, each plate was incubated at 37° C. for 16 hours under anaerobic conditions by using Anaeropack (Mitsubishi Gas Chemical). The obtained cells on the plate were washed with 0.8% brine, and then diluted 51 times, and thereby a cell suspension having OD=1.0 (600 nm) was prepared. This cell suspension in a volume of 100 μl and a production medium (10 g/l of glucose, 10 g/l 2Na malate, 45.88 g/l of TES, 6 g/l of Na2HPO4, 3 g/l of KH2PO4, 1 g/l of NH4Cl, adjusted to pH 7.3 with KOH and filtrated) in a volume of 1.3 ml in which dissolved gases in the medium were replaced with nitrogen gas by bubbling nitrogen gas beforehand were put into 1.5-ml volume micro tubes, and the cells were cultured at 31.5° C. for 6 hours by using a stirrer for micro tubes. After the culture, the amount of succinic acid which accumulated in the medium was analyzed by liquid chromatography. Two Shim-pack SCR-102H (Shimadzu) connected in series were used as the column, and a sample was eluted at 50° C. with 5 mM p-toluenesulfonic acid. The eluate was neutralized with 20 mM Bis-Tris aqueous solution containing 5 mM p-toluenesulfonic acid and 100 μM EDTA, and succinic acid was quantified by measuring electric conductivity with CDD-10AD (Shimadzu).
The amounts of succinic acid which accumulated after 6 hours are shown in Table 2, and the change of succinic acid accumulation with time is shown in FIG. 2.
Succinic acid accumulation was markedly increased in the yidE gene-amplified strain AJ110637/pMW219::Pthr::yidE as compared to AJ110637/pMW219 as the control.







TABLE 2







Effect of yidE amplification in AJ110637 strain










Strain
Succinate (g/L)







AJ110637/pMW219
1.81 (±0.04)



AJ110637/pMW219::Pthr::yidE
1.98 (±0.05)










Example 3
<3-1> Construction of adhE-Deficient Strain of Enterobacter aerogenes AJ110637
When Enterobacter aerogenes AJ110637 is grown in a medium containing a sugar source under anaerobic conditions, it produces a marked amount of ethanol. Therefore, adhE coding for alcohol dehydrogenase was deleted to suppress generation of ethanol.
A gene fragment for deletion of adhE was prepared by PCR using the plasmid pMW-attL-Tc-attR described in WO2005/010175 as the template and oligonucleotides of SEQ ID NOS: 32 and 33 as primers. pMW118-attL-Tc-attR is a plasmid obtained by inserting the attL and attR genes, which are the attachment sites of λ phage, and the Tc gene, which is a tetracycline resistance gene, into pMW118 (Takara Bio), and the genes are inserted in the order of attL-Tc-attR (see Reference Example 3). By PCR described above, a gene fragment containing a tetracycline resistance gene, attL and attR site of λ phage at the both ends of tetracycline gene, and 60 by upstream sequence and 59 by downstream sequence of the adhE gene added to the outer ends of the λ phage sequences was amplified. This fragment was purified by using Wizard PCR Prep DNA Purification System (Promega).
Then, the Enterobacter aerogenes AJ110637 strain was transformed with RSF-Red-TER (see FIG. 3, Reference Example 2) to obtain Enterobacter aerogenes AJ110637/RSF-Red-TER strain. This strain was cultured overnight in the LB medium containing 40 μg/mL of chloramphenicol, the culture medium was inoculated in a 1/100 volume to 50 mL of the LB medium containing 40 μg/mL of chloramphenicol and 0.4 mM isopropyl-β-D-thiogalactopyranoside, and culture was performed at 31° C. for 4 hours. The cells were collected, washed three times with ice-cooled 10% glycerol, and finally suspended in 0.5 mL of 10% glycerol. The suspended cells were used as competent cells, and the PCR fragment prepared in the above section was introduced into the cells by using GENE PULSER II (BioRad) under the conditions of a field strength of 20 kV/cm, capacitor capacity of 25 μF and resistance of 200Ω. Ice-cooled LB medium was added to the cell suspension, and culture was performed at 31° C. for 2 hours with shaking. Then, the culture was applied to LB plate containing 25 μg/mL of tetracycline. The colonies that appeared were purified with the same plate, and then it was confirmed by PCR that the adhE gene was replaced with the tetracycline resistance gene.
<3-2> Construction of Pyruvate Carboxylase Gene-Enhanced Strain of AJ110637ΔadhE Strain
An ability to produce succinic acid was imparted to the Enterobacter aerogenes AJ110637ΔadhE strain by amplifying pyc coding for pyruvate carboxylase derived from Corynebacterium glutamicum in that strain.
In order to express pyc derived from Corynebacterium glutamicum in the AJ110637ΔadhE strain, it was attempted to obtain a threonine operon promoter fragment from the Escherichia coli MG1655 strain. The entire genomic nucleotide sequence of Escherichia coli (Escherichia coli K-12 strain) has already been reported (Genbank Accession No. U00096, Science, 277, 1453-1474 (1997)). On the basis of this sequence, PCR amplification of the promoter region of the threonine operon (thrLABC) was performed. PCR was performed by using the synthetic oligonucleotide having an Sad site shown in SEQ ID NO: 35 as a 5′ primer, the synthetic oligonucleotide shown in SEQ ID NO: 36 as a 3′ primer, and the genomic DNA of Escherichia coli MG1655 strain (ATCC 47076, ATCC 700926) as the template to obtain a threonine operon promoter fragment (A) (SEQ ID NO: 37).
Furthermore, a pyc gene fragment derived from the Corynebacterium glutamicum 2256 strain (ATCC 13869) was obtained. PCR was performed by using the synthetic oligonucleotide shown in SEQ ID NO: 38 as a 5′ primer, the synthetic oligonucleotide having a Sad site shown in SEQ ID NO: 39 as a 3′ primer, and the genomic DNA of the Corynebacterium glutamicum 2256 strain (ATCC 13869) as the template to obtain a pyc gene fragment (B) (SEQ ID NO: 40).
PCR was performed by using the fragments (A) and (B) as templates, the primers of SEQ ID NOS: 35 and 39 to obtain a gene fragment (C) consisting of the fragments (A) and (B) ligated to each other. This gene fragment (C) was treated with the restriction enzyme Sad, and purified, and the product was ligated with the plasmid vector pSTV28 (Takara Bio) digested with the restriction enzyme Sad to construct a plasmid pSTV28::Pthr::pyc for pyc amplification.
The plasmid pSTV28::Pthr::pyc for pyc amplification was introduced into the aforementioned Enterobacter aerogenes AJ110637ΔadhE strain by electroporation to obtain a transformant exhibiting tetracycline and chloramphenicol resistance. This pyc-amplified strain of Enterobacter aerogenes AJ110637ΔadhE was designated as AJ110637ΔadhE/pSTV28::Pthr::pyc.
<3-3> Construction of Enterobacter aerogenes yidE Gene-Enhanced Strain of AJ110637ΔadhE/pSTV28::Pthr::pyc
In the same manner as that described above, PCR amplification of the promoter region of the threonine operon (thrLABC) of Escherichia coli (Escherichia coli K-12 strain) was performed. PCR was performed by using the synthetic oligonucleotide shown in SEQ ID NO: 41 as a 5′ primer, the synthetic oligonucleotide shown in SEQ ID NO: 42 as a 3′ primer, and the genomic DNA of Escherichia coli MG1655 strain (ATCC 47076, ATCC 700926) as the template to obtain a threonine operon promoter fragment (A) (SEQ ID NO: 43).
Furthermore, in order to clone the yidE gene of the Enterobacter aerogenes AJ110637 strain, PCR was performed by using the synthetic oligonucleotide shown in SEQ ID NO: 44 as a 5′ primer, the synthetic oligonucleotide shown in SEQ ID NO: 45 as a 3′ primer, and the genomic DNA of the Enterobacter aerogenes AJ110637 strain as the template to obtain a yidE gene fragment (B) (SEQ ID NO: 46).
PCR was performed by using the fragments (A) and (B) as templates, and the primers of SEQ ID NOS: 41 and 45 to obtain a gene fragment (C) consisting of the fragments (A) and (B) ligated to each other. This gene fragment (C) was blunt-ended by using TaKaRa BKL Kit (Takara Bio), and the 5′ end was phosphorylated. Then, the fragment was digested with the restriction enzyme SmaI, and the product was ligated with the plasmid vector pMW218 dephosphorylated with alkaline phosphatase to construct a plasmid pMW218::Pthr::Ent-yidE for yidE amplification.
The aforementioned vector pMW218::Pthr::Ent-yidE for amplification of the yidE gene derived from the Enterobacter aerogenes AJ110637 strain, and the control plasmid pMW218 were each introduced into the Enterobacter aerogenes AJ110637ΔadhE/pSTV28::Pthr::pyc strain by electroporation to obtain transformants exhibiting tetracycline, chloramphenicol and kanamycin resistance. The yidE-amplified strain derived from the Enterobacter aerogenes AJ110637ΔadhE/pSTV28::Pthr::pyc strain was designated as AJ110637ΔadhE/pSTV28::Pthr::pyc/pMW218::Pthr::Ent-yidE, and the pMW218-introduced strain as a control was designated as AJ110637ΔadhE/pSTV28::Pthr::pyc/pMW218.
<3-4> Effect of Amplification of yidE Derived from Enterobacter aerogenes in Enterobacter Bacterium
AJ110637ΔadhE/pSTV28::Pthr::pyc/pMW218::Pthr::Ent-yidE, and AJ110637ΔadhE/pSTV28::Pthr::pyc/pMW218 were each uniformly applied to a LB plate containing 50 μg/ml of kanamycin, 25 μg/ml of tetracycline, and 40 μg/ml of chloramphenicol, and cultured at 31.5° C. for 16 hours. Then, the cells were inoculated into 3 ml of a seed medium (20 g/l of Bacto tryptone, 10 g/l of yeast extract, 20 g/L of NaCl) contained in a test tube, and cultured at 31.5° C. for 16 hours with shaking. A succinic acid production medium (100 g/l of glucose, 50 g/L of calcium carbonate subjected to hot air sterilization for 3 hours or more) in a volume of 3 ml was added to the medium obtained above, then the tube was sealed with a silicone stopper, and culture was performed at 31.5° C. for 24 hours with shaking. After the culture, the amount of succinic acid which accumulated in the medium was analyzed by liquid chromatography. Two Shim-pack SCR-102H (Shimadzu) connected in series were used as the column, and a sample was eluted at 50° C. with 5 mM p-toluenesulfonic acid. The eluate was neutralized with 20 mM Bis-Tris aqueous solution containing 5 mM p-toluenesulfonic acid and 100 μM EDTA, and succinic acid was quantified by measuring electric conductivity with CDD-10AD (Shimadzu).
The amounts of succinic acid which accumulated after 24 hours are shown in Table 3.
Succinic acid accumulation markedly increased in the yidE gene-amplified strain AJ110637ΔadhE/pSTV28::Pthr::pyc/pMW218::Pthr::Ent-yidE as compared to AJ110637ΔadhE/pSTV28::Pthr::pyc/pMW218 as the control.









TABLE 3






AJ110637ΔadhE/
AJ110637ΔadhE/



pSTV28::Pthr::pyc/
pSTV28::Pthr::pyc/



pMW218
pMW218::Pthr::Ent-yidE







Consumed glucose
9.53 (±1.85)
9.23 (±1.46)


amount (g/L)




OD (600 nm)
8.90 (±0.18)
8.57 (±0.56)


Succinic acid
3.40 (±0.56)
5.12 (±1.56)


accumulation (g/L)




Succinic acid yield
35.80 (±1.82) 
54.80 (±12.81)


based on consumed




glucose (%)









Reference Example 2
Construction of Helper Plasmid RSF-Red-TER
The scheme for constructing the helper plasmid RSF-Red-TER is shown in FIG. 4.
As the first step of the construction, an RSFsacBPlacMCS vector was designed. For this purpose, DNA fragments containing the cat gene of the pACYC184 plasmid and the structural region of the sacB gene of Bacillus subtilis were amplified by PCR using the oligonucleotides of SEQ ID NOS: 48 and 49, and 50 and 51, respectively. These oligonucleotides contained BglII, Sad, XbaI and BamHI restriction enzyme sites, which are required and convenient for further cloning, in the 5′ end regions, respectively. The obtained sacB fragment of 1.5 kb was cloned into the previously obtained pMW119-PlaclacI vector at the XbaI-BamHI site. This vector was constructed in the same manner as that described for the pMW118-PlaclacI vector (Skorokhodova, A. Y. et al, 2004, Biotekhnologiya (Rus), 5:3-21). However, this vector contained a polylinker moiety derived from pMW219 instead of the pMW218 plasmid.
Then, the aforementioned cat fragment of 1.0 kb was treated with BglII and Sad, and cloned into the RSF-PlaclacIsacB plasmid obtained in the previous step at the BamHI-SacI site. The obtained plasmid pMW-PlaclacIsacBcat contained the PlacUV5-lacI-sacB-cat fragment. In order to subclone this fragment into the RSF1010 vector, pMW-PlaclacIsacBcat was digested with BglII, blunt-ended with DNA polymerase I Klenow fragment, and successively digested with SacI. A 3.8 kb BglII-SacI fragment of the pMWPlaclacIsacBcat plasmid was eluted from a 1% agarose gel, and ligated with the RSF1010 vector which had been previously treated with PstI and Sad. Escherichia coli TG1 was transformed with the ligation mixture, and plated on the LB medium containing chloramphenicol (50 mg/L). The plasmids isolated from the grown clones were analyzed with restriction enzymes to obtain an RSFsacB plasmid. In order to construct an RSFsacBPlacMCS vector, a DNA fragment containing the PlacUV5 promoter was amplified by PCR using the oligonucleotides of SEQ ID NOS: 52 and 53 as primers and the pMW119-PlaclacI plasmid as the template. The obtained fragment of 146 by was digested with Sad and Nod, and ligated with the Sad-Nod large fragment of the RSFsacB plasmid. Then, by PCR using the oligonucleotides of SEQ ID NOS: 54 and 55 as primers, and the pKD46 plasmid (Datsenko, K. A., Wanner, B. L., 2000, Proc. Natl. Acad. Sci. USA, 97, 6640-6645) as the template, a DNA fragment of 2.3 kb containing the λRedαβγ genes and the transcription terminator tL3 was amplified. The obtained fragment was cloned into the RSFsacBPlacMCS vector at the PvuI-NotI site. In this way, the RSFRed plasmid was designed.
In order to eliminate read-through transcription of the Red genes, a ρ-dependent transcription terminator of the rrnB operon of Escherichia coli was inserted at a position between the cat gene and the PlacUV5 promoter. For this purpose, a DNA fragment containing the PlacUV5 promoter and the TrrnB terminator was amplified by PCR using the oligonucleotides of SEQ ID NOS: 56 and 53 as primers and the chromosome of Escherichia coli BW3350 as the template. These obtained fragments were treated with KpnI and ligated. Then, the 0.5 kb fragment containing both PlacUV5 and TrrnB was amplified by PCR using the oligonucleotides of SEQ ID NOS: 53 and 57 as primers. The obtained DNA fragment was digested with EcoRI, blunt-ended by a treatment with DNA polymerase I Klenow fragment, digested with BamHI, and ligated with the Ecl136II-BamHI large fragment of the RSFsacBPlacMCS vector. The obtained plasmid was designated RSF-Red-TER.
Reference Example 3
Construction of pMW118-attL-Tc-attR Plasmid
The pMW118-attL-Tc-attR plasmid was obtained by ligation of the following four fragments.
1) The BglII-EcoRI fragment (114 bp) including attL (SEQ ID NO: 60) which was obtained by PCR amplification of the region corresponding to attL of the Escherichia coli W3350 (containing λ prophage) chromosome using the primers P1 and P2 (SEQ ID NOS: 58 and 59) (these primers contained the subsidiary recognition sites for BglII and EcoRI).
2) The PstI-HindIII fragment (182 bp) including attR (SEQ ID NO: 63) which was obtained by PCR amplification of the region corresponding to attR of the Escherichia coli W3350 (containing λ prophage) chromosome using the primers P3 and P4 (SEQ ID NOS: 61 and 62) (these primers contained the subsidiary recognition sites for PstI and HindIII).
3) The BglII-HindIII large fragment (3916 bp) of pMW118-ter_rrnB. The plasmid pMW118-ter_rrnB was obtained by ligation of the following three DNA fragments:

    
    
        The large DNA fragment (2359 bp) including the AatII-EcoRI fragment of pMW118 that was obtained by digesting pMW118 with EcoRI, treated with DNA polymerase I Klenow fragment, and then digesting with AatII;
        The small AatII-BglII fragment (1194 bp) of pUC19 including the bla gene for ampicillin resistance (ApR), which was obtained by PCR amplification of the corresponding region of the pUC19 plasmid using the primers P5 and P6 (SEQ ID NOS: 64 and 65) (these primers contained the subsidiary recognition sites for PstI, AatII and BglII);
        The small BglII-PstI fragment (363 bp) of the transcription terminator ter_rrnB, which was obtained by PCR amplification of the corresponding region of the Escherichia coli MG1655 chromosome using the primers P7 and P8 (SEQ ID NOS: 66 and 67) (these primers contained the subsidiary recognition sites for PstI, BglII and PstI).
    
    


4) The small EcoRI-PstI fragment (1388 bp) (SEQ ID NO: 68) of pML-Tc-ter_thrL including the tetracycline resistance gene and the ter_thrL transcription terminator; the pML-Tc-ter_thrL plasmid was obtained by the following two steps:

    
    
        the pML-ter_thrL plasmid was obtained by digesting the pML-MCS plasmid (Mashko, S. V. et al., 2001, Biotekhnologiya (in Russian), no. 5, 3-20) with XbaI and BamHI, followed by ligation of the large fragment (3342 bp) with the XbaI-BamHI fragment (68 bp) carrying ter_thrL terminator obtained by PCR amplification of the corresponding region of the Escherichia coli MG1655 chromosome using the primers P9 and P10 (SEQ ID NOS: 69 and 70) (these primers contained the subsidiary recognition sites for PstI, XbaI and BamHI);
        the pML-Tc-ter_thrL plasmid was obtained by digesting the pML-ter_thrL plasmid with KpnI and XbaI followed by treatment with Klenow fragment of DNA polymerase I and ligation with the small EcoRI-Van91I fragment (1317 bp) of pBR322 including the tetracycline resistance gene (pBR322 was digested with EcoRI and Van91I and then treated with DNA polymerase I Klenow fragment).
    
    


Explanation of Sequence Listing
SEQ ID NO: 1: Primer for deletion of ldhA
SEQ ID NO: 2: Primer for deletion of ldhA
SEQ ID NO: 3: Primer for confirming deletion of ldhA
SEQ ID NO: 4: Primer for confirming deletion of ldhA
SEQ ID NO: 5: Primer for deletion of lldD
SEQ ID NO: 6: Primer for deletion of lldD
SEQ ID NO: 7: Primer for confirming deletion of lldD
SEQ ID NO: 8: Primer for confirming deletion of lldD
SEQ ID NO: 9: Primer for amplification of yidE
SEQ ID NO: 10: Primer for amplification of yidE
SEQ ID NO: 11: Threonine promoter sequence
SEQ ID NO: 12: Primer for amplification of yidE
SEQ ID NO: 13: Nucleotide sequence of yidE of Escherichia coli 
SEQ ID NO: 14: Amino acid sequence of YidE of Escherichia coli 
SEQ ID NO: 15: Nucleotide sequence of yidE of Salmonella typhimurium 
SEQ ID NO: 16: Amino acid sequence of YidE of Salmonella typhimurium 
SEQ ID NO: 17: Nucleotide sequence of PC gene coding for pyruvate arboxylase of Brevibacterium flavum 
SEQ ID NO: 18: Amino acid sequence of pyruvate carboxylase of Brevibacterium flavum 
SEQ ID NO: 19: Primer for amplification of yidE
SEQ ID NO: 20: Nucleotide sequence of ldhA of Escherichia coli 
SEQ ID NO: 21: Amino acid sequence of LdhA of Escherichia coli 
SEQ ID NO: 22: Nucleotide sequence of lldD of Escherichia coli 
SEQ ID NO: 23: Amino acid sequence of LldD of Escherichia coli 
SEQ ID NO: 24: Consensus sequence of YidE of Escherichia and Salmonella 
SEQ ID NO: 25: Nucleotide sequence of yidE of Shigella flexneri 
SEQ ID NO: 26: Amino acid sequence of YidE of Shigella flexneri 
SEQ ID NO: 27: Nucleotide sequence of yidE of Yersinia pestis 
SEQ ID NO: 28: Amino acid sequence of YidE of Yersinia pestis 
SEQ ID NO: 29: Consensus sequence of YidE of Escherichia, Salmonella, Shigella, and Yersinia 
SEQ ID NO: 30: Nucleotide sequence of yidE of Mannheimia succiniciproducens 
SEQ ID NO: 31: Amino acid sequence of YidE of Mannheimia succiniciproducens 
SEQ ID NO: 32: Nucleotide sequence of primer for deletion of adhE
SEQ ID NO: 33: Nucleotide sequence of primer for deletion of adhE
SEQ ID NO: 34: Nucleotide sequence of adhE of Enterobacter aerogenes AJ110637 (partial sequence)
SEQ ID NO: 35: Primer for amplification of threonine promoter
SEQ ID NO: 36: Primer for amplification of threonine promoter
SEQ ID NO: 37: Threonine promoter gene fragment
SEQ ID NO: 38: Primer for amplification of pyruvate carboxylase
SEQ ID NO: 39: Primer for amplification of pyruvate carboxylase
SEQ ID NO: 40: Pyruvate carboxylase gene fragment
SEQ ID NO: 41: Primer for amplification of threonine promoter
SEQ ID NO: 42: Primer for amplification of threonine promoter
SEQ ID NO: 43: Threonine promoter gene fragment
SEQ ID NO: 44: Nucleotide sequence of primer for amplification of yidE of Enterobacter aerogenes AJ110637
SEQ ID NO: 45: Nucleotide sequence of primer for amplification of yidE of Enterobacter aerogenes AJ110637
SEQ ID NO: 46: Nucleotide sequence of yidE of Enterobacter aerogenes AJ110637
SEQ ID NO: 47: Amino acid sequence of YidE of Enterobacter aerogenes AJ110637
SEQ ID NO: 48: Primer for amplification of cat gene
SEQ ID NO: 49: Primer for amplification of cat gene
SEQ ID NO: 50: Primer for amplification of sacB gene
SEQ ID NO: 51: Primer for amplification of sacB gene
SEQ ID NO: 52: Primer for amplification of DNA fragment containing PlacUV5 promoter
SEQ ID NO: 53: Primer for amplification of DNA fragment containing PlacUV5 promoter
SEQ ID NO: 54: Primer for amplification of DNA fragment containing λRedαβγ gene and tL3
SEQ ID NO: 55: Primer for amplification of DNA fragment containing λRedαβγ gene and tL3
SEQ ID NO: 56: Primer for amplification of DNA fragment containing PlacUV5 promoter and TrrnB
SEQ ID NO: 57: Primer for amplification of DNA fragment containing PlacUV5 promoter and TrrnB
SEQ ID NO: 58: Primer for amplification of attL
SEQ ID NO: 59: Primer for amplification of attL
SEQ ID NO: 60: Nucleotide sequence of attL
SEQ ID NO: 61: Primer for amplification of attR
SEQ ID NO: 62: Primer for amplification of attR
SEQ ID NO: 63: Nucleotide sequence of attR
SEQ ID NO: 64: Primer for amplification of DNA fragment containing bla gene
SEQ ID NO: 65: Primer for amplification of DNA fragment containing bla gene
SEQ ID NO: 66: Primer for amplification of DNA fragment containing ter_rrnB
SEQ ID NO: 67: Primer for amplification of DNA fragment containing ter_rrnB
SEQ ID NO: 68: Nucleotide sequence of the DNA fragment containing ter_thrL terminator
SEQ ID NO: 69: Primer for amplification of DNA fragment containing ter_thrL terminator
SEQ ID NO: 70: Primer for amplification of DNA fragment containing ter_thrL terminator
SEQ ID NO: 71: Consensus sequence of YidE of Escherichia, Salmonella, and Enterobacter 
SEQ ID NO: 72: Consensus sequence of YidE of Escherichia, Salmonella, Shigella, Yersinia, and Enterobacter 
INDUSTRIAL APPLICABILITY
According to the production method of the present invention, an organic acid can be quickly and highly efficiently produced. When the organic acid is succinic acid, the obtained succinic acid can be used for food additives, pharmaceuticals, cosmetics, and the like. Moreover, succinic acid-containing polymers can also be produced by performing a polymerization reaction using the obtained succinic acid as a raw material.
While the invention has been described in detail with reference to exemplary embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents is incorporated by reference herein in its entirety.

## Claims

1. A method for producing succinic acid comprising:
A) allowing a substance to act on an organic raw material in a reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas, wherein the substance is selected from the group consisting of:
i) a bacterium which has an ability to produce an organic acid and has been modified to have enhanced expression of the yidE gene,
ii) a product obtained by processing the bacterium of i),
iii) combinations thereof, and

B) collecting the succinic acid:

wherein the yidE gene is selected from the group consisting of:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 46, and
(b) a DNA which hybridizes with a DNA comprising a nucleotide sequence complementary to the entire nucleotide sequence of SEQ ID NO: 46 under stringent conditions comprising washing with 1×SSC, 0.1% SDS at 60° C., and said DNA improves the ability of the bacterium to produce an organic acid when expression of the DNA is enhanced in the bacterium;
wherein the enhanced expression is obtained by a method selected from the group consisting of:
i) increasing the copy number of the yidE gene,
ii) replacing a promoter sequence of the yidE gene with a stronger promoter, and
iii) combinations thereof.

2. The method according to claim 1, wherein the bacterium belongs to the family Enterobacteriaceae.

3. The method according to claim 2, wherein the bacterium belongs to a genus selected from the group consisting Escherichia, Enterobacter, Raoultella, Klebsiella, and Pantoea.

4. The method according to claim 1, wherein the bacterium is a rumen bacterium.

5. The method according to claim 4, wherein the bacterium is Mannheimia succiniciproducens.

6. The method according to claim 1, wherein the bacterium has been further modified to decrease an enzymatic activity selected from the group consisting of lactate dehydrogenase activity, alcohol dehydrogenase activity, pyruvate formate lyase activity, and combinations thereof.

7. The method according to claim 1, wherein the bacterium has been further modified to have enhanced expression of a gene encoding a protein with pyruvate carboxylase activity.

8. A method for producing a succinic acid-containing polymer comprising:
A) producing succinic acid by the method according to claim 1, and
B) polymerizing the succinic acid.

9. The method according to claim 1, wherein the yidE gene codes for a protein selected from the group consisting of:
A) a protein comprising the amino acid sequence of SEQ ID NO: 47, and
B) a protein comprising an amino acid sequence having a homology of not less than 95% to the entire amino acid sequence of SEQ ID NO: 47, and said protein has an activity of enhancing the yield of the succinic acid.