Method For Producing An Acidic Substance Having A Carboxyl Group

Abstract

An acidic substance having a carboxyl group is produced by culturing in a medium a microorganism which has been modified to enhance expression of the ybjL gene, and collecting the acidic substance having a carboxyl group from the medium.

Description

[0001] This application is a continuation under 35 U.S.C. .sctn.120 of PCT Patent Application No. PCT/JP2008/057478, filed Apr. 17, 2008, which claims priority under 35 U.S.C. .sctn.119 to Japanese Patent Application No. 2007-108631, filed on Apr. 17, 2007, and Japanese Patent Application No. 2007-242859, 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-408_Seq_List; File Size: 240 KB; Date Created: Oct. 15, 2009).

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a method for producing an acidic substance having a carboxyl group. L-Glutamic acid and L-aspartic acid are widely used as raw materials in making seasonings and so forth. Succinic acid is widely used as a raw material in making seasonings and biodegradable plastics.

[0004] 2. Brief Description of the Related Art

[0005] L-Glutamic acid is mainly produced by fermentation utilizing L-glutamic acid-producing bacteria of the so-called coryneform bacteria belonging to the genus Brevibacterium, Corynebacterium or Microbacterium, or their mutant strains (see, for example, Kunihiko Akashi et al., Amino Acid Fermentation, Japan Scientific Societies Press (Gakkai Shuppan Center), pp. 195-215, 1986). Methods are known for producing L-glutamic acid by fermentation using other bacterial strains, including microorganisms belonging to the genera Bacillus, Streptomyces, Penicillium or the like (see, for example, U.S. Pat. No. 3,220,929), microorganisms belonging to the genera Pseudomonas, Arthrobacter, Serratia, Candida or the like (see, for example, U.S. Pat. No. 3,563,857), microorganisms belonging to the genera Bacillus, Pseudomonas, Serratia, Aerobacter aerogenes (currently referred to as Enterobacter aerogenes) or the like (see, for example, Japanese Patent Publication (KOKOKU) No. 32-9393), a mutant strain of Escherichia coli (see, for example, Japanese Patent Laid-open (KOKAI) No. 5-244970), and the like. In addition, methods for producing L-glutamic acid have also been disclosed using microorganisms belonging to the genera Klebsiella, Erwinia, Pantoea or Enterobacter (see, for example, Japanese Patent Laid-open No. 2000-106869 (U.S. Pat. No. 6,682,912), Japanese Patent Laid-open No. 2000-189169 (U.S. Patent Published Application No. 2001009836), Japanese Patent Laid-open No. 2000-189175 (U.S. Pat. No. 7,247,459)).

[0006] Furthermore, various techniques have been disclosed for increasing the L-glutamic acid-producing ability by enhancing the L-glutamic acid biosynthetic enzymes using recombinant DNA techniques. For example, it has been reported for Corynebacterium or Brevibacterium bacteria that introduction of a gene coding for citrate synthase derived from Escherichia coli or Corynebacterium glutamicum was effective to enhance the L-glutamic acid-producing ability of coryneform bacteria (see, for example, Japanese Patent Publication No. 7-121228). Furthermore, it has also been reported that introduction of a citrate synthase gene derived from a coryneform bacterium into enterobacteria belonging to the genera Enterobacter, Klebsiella, Serratia, Erwinia or Escherichia was effective to enhance the bacteria's L-glutamic acid-producing ability (see, for example, Japanese Patent Laid-open No. 2000-189175 (U.S. Pat. No. 7,247,459)).

[0007] Methods for improving the production of target substances such as amino acids are also known, including by modifying the uptake or secretion systems of target substances. Such methods include, for example, by deleting or attenuating the system for uptake of a target substance into cells. Specifically, known methods to improve production of L-glutamic acid include deleting the gluABCD operon, or a part thereof, to eliminate or attenuate uptake of L-glutamic acid into cells (see, for example, European Patent Application Laid-open No. 1038970), and enhancing production of purine nucleotides by attenuating uptake of purine nucleotides into cells (see, for example, European Patent Application Laid-open No. 1004663), and the like.

[0008] Furthermore, methods of enhancing the secretion system for a target substance, and methods of deleting or attenuating the secretion system for an intermediate or substrate in the biosynthetic system of a target substance are known. Known methods of enhancing the secretion system of a target substance include, for example, production of L-lysine by utilizing a Corynebacterium strain in which the L-lysine secretion gene (lysE) is enhanced (see, for example, WO2001/5959), and production of L-glutamic acid by using an enterobacterium in which the L-glutamic acid secretion system gene (yhfK) is enhanced (see, for example, Japanese Patent Laid-open No. 2005-278643 (U.S. Patent Published Application No. 2005196846)). Furthermore, methods for producing an L-amino acid using the rhtA, B, C genes, which have been suggested to be involved in the secretion of L-amino acids, have also been reported (see, for example, Japanese Patent Laid-open No. 2000-189177 (U.S. Patent Published Application No. 2005239177)). Known methods for, for example, deleting a secretion system for an intermediate or substrate in a biosynthesis system of a target substance include, for L-glutamic acid, mutating or disrupting the 2-oxoglutarate permease gene to attenuate secretion of 2-oxoglutarate, which is an intermediate of the target substance (see, for example, WO97/23597).

[0009] Furthermore, use of the gene coding for the ATP binding cassette superfamily (ABC transporter), which is involved in transportation of substances through cell membranes, in the breeding of microorganisms in which transmembrane transportation of amino acids is modified has been suggested (see, for example, WO00/37647).

[0010] Furthermore, it has also been reported that L-glutamic acid production efficiency can be improved in Escherichia bacteria by enhancing the expression of genes thought to participate in secretion of L-amino acids such as yfiK (see, for example, Japanese Patent Laid-open No. 2000-189180 (U.S. Pat. No. 6,979,560)). Moreover, it has also been reported that L-glutamic acid-producing ability can be improved by enhancing expression of the yhfK gene (see, for example, Japanese Patent Laid-open No. 2005-278643 (U.S. Patent Published Application No. 2005196846)).

[0011] Moreover, methods are known for producing L-glutamic acid by culturing a microorganism under acidic conditions to precipitate the L-glutamic acid (see, for example, Japanese Patent Laid-open No. 2001-333769 (U.S. Patent Published Application No. 2007134773)). When the pH is kept low, L-glutamic acid is precipitated, and the ratio of L-glutamic acid in free form with no electrical charge increases. As a result, the L-glutamic acid easily penetrates cell membranes. When L-glutamic acid is taken up into cells, it is converted into an intermediate of the TCA cycle, 2-oxoglutaric acid, in one step by glutamate dehydrogenase, and therefore it is generally thought that L-glutamic acid taken up into cells is easily metabolized. However, 2-oxoglutarate dehydrogenase activity can be deleted or attenuated, or the like, in the fermentative production of L-glutamic acid (for example, Japanese Patent Laid-open No. 2001-333769 (U.S. Patent Published Application No. 2007134773), Japanese Patent Laid-open No. 7-203980 (U.S. Pat. No. 5,573,945)), but then 2-oxoglutaric acid is not degraded, intracellular 2-oxoglutaric acid concentration increases which inhibits the growth of the microorganism. Thus, the culture fails. Therefore, as described in Japanese Patent Laid-open No. 2001-333769 (U.S. Patent Published Application No. 2007134773), a strain was bred using a mutation that is deficient in 2-oxoglutarate dehydrogenase activity and can produce L-glutamic acid accompanying precipitation, and this strain can be used for the production of L-glutamic acid.

[0012] For fermentative production of non-amino organic acids, including succinic acid, anaerobic bacteria including those belonging to the genus Anaerobiospirillum or Actinobacillus are usually used (U.S. Pat. Nos. 5,142,834 and 5,504,004, Guettler, M. V. et al., 1999, International Journal of Systematic Bacteriology, 49:207-216). Although the use of such anaerobic bacteria provides high product yields, many nutrients are required for sufficient 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 organic nitrogen sources can result in not only an increase in the cost of the culture, but also an increase in the cost for isolating or purifying the product, and therefore, their use is not economical.

[0013] In addition, methods are known in which aerobic bacteria such as coryneform bacteria are cultured once under aerobic conditions, then harvested, washed, and allowed to rest, producing a non-amino organic acid in the absence of supplied oxygen (Japanese Patent Laid-open Nos. 11-113588 and 11-196888). These methods are economical since a smaller amount of organic nitrogen can be added, and the bacteria will sufficiently grow in a simple culture medium. However, there is still a room for improvement in terms of production amount, concentration, and production rate per cell of the target organic acids, as well as simplification of the production process, and the like. Furthermore, the 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 have also been reported.

[0014] Furthermore, as for Escherichia coli, which is a facultative anaerobic gram negative bacterium, methods for producing a non-amino organic acid by culturing it once under aerobic conditions, and then allowing the cells to rest in the absence of supplied oxygen, resulting in an anaerobically produced non-amino organic acid (Vemuri G. N. et al., 2002, Journal of Industrial Microbiology and Biotechnology, 28(6):325-332). This is similar to the methods using coryneform bacteria. Alternatively, E. coli 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.

[0015] The ybjL gene is located on the genome of Escherichia coli (see, for example, Blattner, F. R. et al., 1997, Science, 277(5331):1453-74), and it is also thought to code for a transporter on the basis of the motifs, topology etc. of the deduced amino acid sequence. However, cloning of the gene, as well as expression of the gene and analysis of the expression product have not been reported, and the actual functions of the gene remained unknown.

SUMMARY OF THE INVENTION

[0016] An aspect of the present invention is to provide a bacterial strain that can efficiently produce an acidic substance having a carboxyl group, especially L-glutamic acid, L-aspartic acid, and succinic acid, and to provide a method for efficiently producing an acidic substance having a carboxyl group by using such a strain.

[0017] The ybjL gene was isolated and shown to be involved in L-glutamic acid resistance. Also, it has been found that when the expression of the ybjL gene is enhanced, L-glutamic acid fermentation yield is improved and the production rate or yield of succinic acid is improved.

[0018] It is an aspect of the present invention to provide a microorganism that has an ability to produce an acidic substance having a carboxyl group and has been modified to enhance expression of the ybjL gene.

[0019] It is a further aspect of the present invention to provide the aforementioned microorganism, wherein enhanced expression is obtained by a method selected from the group consisting of: A) increasing copy number of the ybjL gene, B) modifying an expression control sequence of the ybjL gene, and C) combinations thereof.

[0020] It is a further aspect of the present invention to provide the aforementioned microorganism, wherein the ybjL gene encodes a protein: (A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4 and 87; (B) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4 and 87, but wherein one or several amino acid residues are substituted, deleted, inserted or added, and the protein improves the ability of the microorganism to produce an acidic substance having a carboxyl group when expression of the gene encoding the protein is enhanced in the microorganism.

[0021] It is a further aspect of the present invention to provide the aforementioned microorganism, wherein the ybjL gene encodes the protein selected from the group consisting of SEQ ID NO: 5 and 88.

[0022] It is a further aspect of the present invention to provide the aforementioned microorganism, wherein the microorganism is a bacterium belonging to the family Enterobacteriaceae.

[0023] It is a further aspect of the present invention to provide the aforementioned microorganism, wherein the bacterium belongs to a genus selected from the group consisting of Escherichia, Enterobacter, Raoultella, Pantoea, and Klebsiella.

[0024] It is a further aspect of the present invention to provide the aforementioned microorganism, wherein the microorganism is a rumen bacterium.

[0025] It is a further aspect of the present invention to provide the aforementioned microorganism, wherein the microorganism is Mannheimia succiniciproducens.

[0026] It is a further aspect of the present invention to provide the aforementioned microorganism, wherein the acidic substance is an organic acid selected from the group consisting of succinic acid, fumaric acid, malic acid, oxalacetic acid, citric acid, isocitric acid, .alpha.-ketoglutaric acid, and combinations thereof.

[0027] It is a further aspect of the present invention to provide the aforementioned microorganism, wherein the acidic substance is L-glutamic acid and/or L-aspartic acid.

[0028] It is a further aspect of the present invention to provide a method for producing an acidic substance having a carboxyl group comprising culturing the aforementioned microorganism in a medium to produce and accumulate the acidic substance having a carboxyl group in the medium, and collecting the acidic substance having a carboxyl group from the medium.

[0029] It is a further aspect of the present invention to provide the aforementioned method, wherein the acidic substance is an organic acid selected from the group consisting of succinic acid, fumaric acid, malic acid, oxalacetic acid, citric acid, isocitric acid, .alpha.-ketoglutaric acid, and combinations thereof.

[0030] It is a further aspect of the present invention to provide the aforementioned method, wherein the acidic substance is L-glutamic acid and/or L-aspartic acid.

[0031] It is a further aspect of the present invention to provide a method for producing an acidic substance having a carboxyl group 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) the microorganism as described above, ii) a product obtained by processing the microorganism of i), and iii) combinations thereof, and collecting the acidic substance having a carboxyl group.

[0032] It is a further aspect of the present invention to provide the aforementioned method, wherein the acidic substance is an organic acid selected from the group consisting of succinic acid, fumaric acid, malic acid, oxalacetic acid, citric acid, isocitric acid, .alpha.-ketoglutaric acid, and combinations thereof.

[0033] It is a further aspect of the present invention to provide a method for producing a polymer comprising succinic acid comprising A) producing succinic acid by the aforementioned method, and B) polymerizing the succinic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 shows the structure of helper plasmid RSF-Red-TER.

[0035] FIG. 2 shows construction of helper plasmid RSF-Red-TER.

[0036] FIG. 3 shows the growth of the ybjL-amplified strain in the presence of a high concentration L-glutamic acid.

[0037] FIG. 4 shows accumulation of succinic acid obtained with the ybjL-amplified strain of Escherichia bacterium.

[0038] FIG. 5 shows accumulation of succinic acid obtained with the ybjL-amplified strain of Enterobacter bacterium.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Hereinafter, the present invention will be explained in detail.

[0040] <1> Microorganism Having Ability to Produce Acidic Substance Having a Carboxyl Group

[0041] The microorganism in accordance with the presently disclosed subject matter has an ability to produce an acidic substance having a carboxyl group and has been modified so that expression of the ybjL gene is enhanced. The term "ability to produce an acidic substance having a carboxyl group" can mean the ability of a microorganism to produce and cause accumulation of an acidic substance having a carboxyl group in a medium or cells to such a degree that the acidic substance having a carboxyl group can be collected from the cells or medium when the microorganism of the present invention is cultured in the medium. The microorganism can originally have the ability to produce an acidic substance having a carboxyl group, or the ability to produce an acidic substance can be obtained by modifying a microorganism such as those described below using mutation or recombinant DNA techniques. Also, a microorganism can be imparted with the ability to produce an acidic substance having a carboxyl group, or the ability to produce an acidic substance having a carboxyl group can be enhanced by introducing the gene described herein.

[0042] In the present invention, the "acidic substance having a carboxyl group" can mean an organic compound having one or more carboxyl groups and which is acidic when the substance is in the free form, and not in the salt form. Specifically, examples include organic acids and L-amino acids having two carboxyl groups, for example, acidic amino acids. Examples of the L-amino acids include L-glutamic acid and L-aspartic acid, and examples of the organic acids include succinic acid, fumaric acid, malic acid, oxalacetic acid, citric acid, isocitric acid, .alpha.-ketoglutaric acid, and the like.

[0043] The parent strain which can be used to derive the microorganism in accordance with the presently disclosed subject matter is not particularly limited, and can include bacteria, for example, Enterobacteriaceae, rumen bacteria, and coryneform bacteria.

[0044] The Enterobacteriaceae family 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 (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) are examples. Among these, bacteria belonging to the genus Escherichia, Enterobacter, Raoultella, Pantoea, Klebsiella, or Serratia are particular examples.

[0045] A "bacterium belonging to the genus Escherichia" means 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). Other 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, the K12 strain.

[0046] 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.

[0047] Pantoea, Erwinia, and Enterobacter bacteria are classified as .gamma.-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 (refer to Int. J. Syst. Bacteriol., 1993, 43:162-173).

[0048] 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), and Enterobacter aerogenes AJ110637 (FERM ABP-10955), and the like. The AJ110637 strain was obtained from the soil at the seashore of 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 it was found to be 99.9% homologous to that from Enterobacter aerogenes NCTC 10006. Moreover, in a physiological test using an API kit, the strain gave results similar to the prototype species of Enterobacter aerogenes, and therefore it 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 an accession number of FERM ABP-10955.

[0049] Typical strains of the Pantoea bacteria include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specific examples include the following strains:

[0050] Pantoea ananatis AJ13355 (FERM BP-6614, European Patent Laid-open No. 0952221)

[0051] Pantoea ananatis AJ13356 (FERM BP-6615, European Patent Laid-open No. 0952221)

[0052] 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.

[0053] 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:

[0054] Erwinia amylovora ATCC 15580 strain

[0055] Erwinia carotovora ATCC 15713 strain

[0056] Klebsiella planticola AJ13399 strain (FERM BP-6600, European Patent Laid-open No. 955368)

[0057] Klebsiella planticola AJ13410 strain (FERM BP-6617, European Patent Laid-open No. 955368).

[0058] Raoultella planticola ATCC 33531 strain

[0059] 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).

[0060] Examples of rumen bacteria include Mannheimia, Actinobacillus, Anaerobiospirillum, Pyrobacterium, and Selenomonas. Bacteria including Mannheimia succiniciproducens, Actinobacillus succinogenes, Selenomonas ruminantium, Veillonella parvula, Wolnella succinogenes, Anaerobiospirillum succiniciproducens, and the like, can be used. Specific strains include Mannheimia sp. 55E (KCTC0769BP strain, U.S. Patent Published Application No. 20030113885, International Patent Publication WO2005/052135).

[0061] <1-1> Imparting an Ability to Produce an Acidic Substance Having a Carboxyl Group

[0062] Hereinafter, methods for imparting an ability to produce an acidic substance having a carboxyl group to bacteria, or methods for enhancing the ability to produce an acidic substance having a carboxyl group are described.

[0063] To impart an ability to produce an acidic substance having a carboxyl group, 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 biosynthesis of acidic substances having a carboxyl group. In the breeding of bacteria that produce an acidic substance having a carboxyl group, one or more properties, such as auxotrophic mutation, analogue resistance, or metabolic regulation mutation, can be imparted. The expression of one or two or more enzymes involved in the biosynthesis of an acidic substance having a carboxyl group can be enhanced. Furthermore, the impartation of properties such as auxotrophic mutation, analogue resistance, or metabolic regulation mutation can be combined with the enhancement of the biosynthetic enzymes.

[0064] A mutant strain auxotrophic for an acidic substance having a carboxyl group, a strain resistant to an analogue of an acidic substance having a carboxyl group, or a metabolic regulation mutant strain 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 bacteria which exhibit an autotrophy, analogue resistance, or metabolic regulation mutation and which also have the ability to produce an acidic substance having a carboxyl group.

[0065] Methods for imparting an amino acid- or organic acid-producing ability to microorganisms, and amino acid- or organic acid-producing bacteria will be specifically exemplified below.

[0066] <L-Glutamic Acid-Producing Bacteria>

[0067] Examples of the method of modifying a microorganism to impart L-glutamic acid-producing ability to the microorganism include, for example, enhancing expression of a gene coding for an enzyme involved in L-glutamic acid biosynthesis. Examples of an enzyme involved in L-glutamic acid biosynthesis include glutamate dehydrogenase ("GDH")(gdh), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase ("CS") (gltA), methylcitrate synthase (hereinafter also referred to as "PRPC" (prpC), phosphoenolpyruvate carboxylase ("PEPC") (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA, pgml), phosphoglycerate kinase (pgk), glyceraldehyde-3-phophate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), and glucose phosphate isomerase (pgi), and the like. The capital letter abbreviations in parentheses after the enzyme names are the enzyme abbrevation, while the lower case abbreviations indicate the name of the gene encoding the enzyme.

[0068] Methods for modifying a microorganism to increase expression of a target gene will be explained below.

[0069] The first method is to increase the copy number of the target gene by cloning the target gene on an appropriate plasmid and transforming the chosen host bacterium with the obtained plasmid. For example, when the target gene is the gene coding for CS (gltA gene), the gene coding for PEPC (ppc gene) or the gene coding for GDH (gdhA gene), nucleotide sequences of these genes from Escherichia bacteria and Corynebacterium bacteria have already been reported (Ner, S. et al., 1983, Biochemistry, 22:5243-5249; Fujita, N. et al., 1984, J. Biochem., 95:909-916; Valle, F. et al., 1984, Gene, 27:193-199; Microbiology, 140:1817-1828, 1994; Eikmanns, B. J. et al., 1989, Mol. Gen. Genet., 218:330-339; Bormann, E. R. et al., 1992, Molecular Microbiology, 6:317-326), and therefore they can be obtained by synthesizing primers based on their respective nucleotide sequences, and performing PCR using chromosomal DNA as the template.

[0070] Examples of plasmids which can be used for transformation include a plasmid which autonomously replicates in the host bacterium belonging to the family Enterobacteriaceae, such as pUC19, pUC18, pBR322, RSF1010, pHSG299, pHSG298, pHSG399, pHSG398, pSTV28, pSTV29 (pHSG and pSTV are available from Takara Bio), pMW119, pMW118, pMW219, pMW218 (pMW vectors are available from Nippon Gene), and the like. Moreover, a phage DNA can also be used as the vector instead of a plasmid. Examples of plasmids for simultaneously enhancing the activities of CS or PRPC, PEPC, and GDH as described above include RSFCPG which includes the gltA gene, ppc gene, and gdhA gene (refer to European Patent Laid-open No. 0952221), and RSFPPG which is the same as RSFCPG, but the gltA gene is replaced with the prpC gene.

[0071] Examples of transformation methods include treating recipient cells with calcium chloride so to increase permeability for DNA, which has been reported for Escherichia coli K-12 (Mandel, M. and Higa, A., 1970, J. Mol. Biol., 53:159-162), and preparing competent cells from cells which are in the growth phase, followed by transformation with DNA, which has been reported for Bacillus subtilis (Duncan, C. H., et al., 1977, Gene, 1:153-167). Alternatively, a method of making potential host cells into protoplasts or spheroplasts, which can easily take up recombinant DNA, followed by introducing the recombinant DNA into the cells. This method is known to be applicable to Bacillus subtilis, actinomycetes and yeasts (Chang, S, and Choen, S. N., 1979, Molec. Gen. Genet., 168:111-115; Bibb, M. J. et al., 1978, Nature, 274:398-400; Hinnen, A., et al., 1978, Proc. Natl. Sci., USA, 75:1929-1933). In addition, microorganisms can also be transformed by the electric pulse method (Japanese Patent Laid-open No. 2-207791).

[0072] The copy number of a target gene can also be increased by introducing multiple copies of the gene into the chromosomal DNA of the microorganism. Multiple copies of a gene can be introduced into the chromosomal DNA by homologous recombination (Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory) using multiple copies of a sequence as targets in the chromosomal DNA. Sequences, which are present in multiple copies on the chromosomal DNA, repetitive DNAs, and inverted repeats present at the end of a transposable element, are exemplary. Also, as disclosed in Japanese Patent Laid-open No. 2-109985, it is possible to incorporate a target gene into a transposon, and allow it to be transferred to introduce multiple copies of the gene into the chromosomal DNA. The target gene can also be introduced into the bacterial chromosome by using Mu phage (Japanese Patent Laid-open No. 2-109985).

[0073] The second method is to increase expression of the target gene by replacing an expression regulatory sequence of the target gene, such as promoter, on the chromosomal DNA or plasmid with a stronger promoter. For example, the lac promoter, trp promoter, trc promoter, etc. are known as strong promoters. Moreover, it is also possible to substitute several nucleotides in the promoter region of a gene so that the promoter is stronger, or to modify the SD sequence as disclosed in International Patent Publication WO00/18935. Examples of methods for evaluating the strength of promoters and strong promoters are described in the article of Goldstein et al. (Goldstein, M. A., and Doi, R. H., 1995, Biotechnol. Annu. Rev., 1:105-128), etc.

[0074] Substitution of an expression regulatory sequence can be performed, for example, in the same manner as for gene substitution using a temperature-sensitive plasmid. Examples of vectors having a temperature-sensitive replication origin and are usable for Escherichia coli and Pantoea ananatis include, for example, the pMAN997 plasmid described in International Publication WO99/03988, and the like. Furthermore, an expression regulatory sequence can also be substituted by the method called "Red-driven integration" using Red recombinase of .lamda. phage (Datsenko, K. A. and Wanner, B. L., 2000, Proc. Natl. Acad. Sci. USA. 97:6640-6645), or by combining the Red-driven integration method and the .lamda. phage excisive system (Cho, E. H., et al., 2002, J. Bacteriol., 184:5200-5203) (WO2005/010175), and the like. Modification of an expression regulatory sequence can be combined with increasing the gene copy number.

[0075] As shown in Reference Example 1, a strain resistant to a .lamda. Red gene product, for example, Pantoea ananatis SC17(0), can be used for the Red driven integration. The SC17(0) strain was deposited at the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetika (Russia, 117545 Moscow 1, Dorozhny proezd. 1) on Sep. 21, 2005 under an accession number of VKPM B-9246.

[0076] Examples of microorganisms modified by the method described above so that expression of citrate synthase gene, methylcitrate synthase gene, phosphoenolpyruvate carboxylase gene and/or glutamate dehydrogenase gene are enhanced include the microorganisms disclosed in Japanese Patent Laid-open Nos. 2001-333769, 2000-106869, 2000-189169, 2000-333769, 2006-129840, WO2006/051660, and the like.

[0077] Furthermore, L-glutamic acid-producing ability can also be imparted by enhancing the 6-phosphogluconate dehydratase activity, 2-keto-3-deoxy-6-phosphogluconate aldolase activity, or both. Examples of the microorganism in which 6-phosphogluconate dehydratase activity and 2-keto-3-deoxy-6-phosphogluconate aldolase activity are increased include the microorganism disclosed in Japanese Patent Laid-open No. 2003-274988.

[0078] L-glutamic acid-producing ability can also be imparted by decreasing or eliminating the activity of an enzyme that catalyzes a reaction that branches off from the L-glutamic acid biosynthesis pathway, producing a compound other than L-glutamic acid. Examples of these include 2-oxoglutarate dehydrogenase (sucA), isocitrate lyase (aceA), phosphate acetyltransferase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB), 1-pyrroline-5-carboxylate dehydrogenase (putA), and the like. Gene names are indicated in the parentheses after the enzyme names. The activity of 2-oxoglutarate dehydrogenase can be decreased or completely eliminated.

[0079] In order to decrease or eliminate the activities of the aforementioned enzymes, methods similar to the method for decreasing or eliminating the activity of lactate dehydrogenase (LDH) described later can be used.

[0080] A decrease in the intracellular activity of the target enzyme and the degree by which the activity is decreased, including if it is completely eliminated, can be confirmed by measuring the enzyme activity of a cell extract or a purified fraction thereof obtained from a candidate strain and comparing it with that of a wild-type strain. For example, 2-oxoglutarate dehydrogenase activity can be measured by the method of Reed et al. (Reed L. J. and Mukherjee B. B., 1969, Methods in Enzymology, 13, pp. 55-61).

[0081] Methods of eliminating or decreasing the 2-oxoglutarate dehydrogenase activity in Escherichia bacteria are disclosed in Japanese Patent Laid-open Nos. 5-244970, 7-203980 and the like. A method of eliminating or decreasing 2-oxoglutarate dehydrogenase activity in coryneform bacteria is disclosed in International Patent Publication WO95/34672. Furthermore, such a method for Enterobacter bacteria is disclosed in Japanese Patent Laid-open No. 2001-333769.

[0082] Specific examples of Escherichia bacteria which are deficient in 2-oxoglutarate dehydrogenase activity or in which the 2-oxoglutarate dehydrogenase activity is decreased include the following strains (U.S. Pat. Nos. 5,378,616 and 5,573,945).

[0083] E. coli W3110sucA::Kmr

[0084] E. coli AJ12624 (FERM BP-3853)

[0085] E. coli AJ12628 (FERM BP-3854)

[0086] E. coli AJ12949 (FERM BP-4881)

[0087] E. coli W3110sucA::Kmr is obtained by disrupting the 2-oxoglutarate dehydrogenase gene (sucA gene) of Escherichia coli W3110. This strain is completely deficient in 2-oxoglutarate dehydrogenase.

[0088] Other specific examples of bacteria wherein the activity of 2-oxoglutarate dehydrogenase is deleted or decreased include the following strains:

[0089] Pantoea ananatis AJ13601 (FERM BP-7207, European Patent Laid-open No. 1078989)

[0090] Pantoea ananatis AJ13356 (FERM BP-6615, U.S. Pat. No. 6,331,419)

[0091] Pantoea ananatis SC17sucA (FERM BP-8646, WO2005/085419)

[0092] Klebsiella planticola AJ13410 strain (FERM BP-6617, U.S. Pat. No. 6,197,559)

[0093] Brevibacterium lactofermentum AS strain (refer to International Patent Publication WO95/34672)

[0094] The SC17sucA strain is obtained by obtaining a low phlegm production mutant strain (SC17) from the AJ13355 strain, which was isolated from nature as a strain that could proliferate in a medium containing L-glutamic acid and a carbon source at low pH, and disrupting the 2-oxoglutarate dehydrogenase gene (sucA) in the mutant strain. The AJ13601 strain is obtained by introducing the plasmid RSFCPG containing the gltA, ppc and gdhA genes derived from Escherichia coli and the plasmid pSTVCB containing the gltA gene derived from Brevibacterium lactofermentum into the SC17sucA strain to obtain the SC17sucA/RSFCPG+pSTVCB strain, and selecting a high concentration L-glutamic acid-resistant strain at a low pH and a strain showing a high proliferation degree and a high L-glutamic acid-producing ability (European Patent Laid-open No. 0952221). The AJ13356 strain was obtained by deleting the .alpha.KGDH-E1 subunit gene (sucA) from the AJ13355 strain.

[0095] The SC17sucA strain was assigned a private number of AJ417, deposited in the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postal code: 305-8566) on Feb. 26, 2004, and assigned an accession number of FERM BP-08646.

[0096] The AJ13410 strain was deposited in the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postal code: 305-8566) on Feb. 19, 1998, and assigned an accession number of FERM P-16647. The deposit was then converted to an international deposition under the provisions of Budapest Treaty on Jan. 11, 1999 and assigned an accession number of FERM BP-6617.

[0097] The aforementioned Pantoea ananatis AJ13355, AJ13356, and AJ13601 strains and the Klebsiella planticola AJ13399 strain have an ability to accumulate L-glutamic acid in a concentration which exceeds the saturation concentration of L-glutamic acid in a liquid medium when it is cultured under acid conditions.

[0098] Furthermore, in order to improve the L-glutamic acid-producing ability of Enterobacteriaceae bacteria, the method of deleting the arcA gene (U.S. Pat. No. 7,090,998), and the method of amplifying the yhfK gene, which is a glutamic acid secretion gene (WO2005/085419) can also be used.

[0099] Other than the above, examples of coryneform bacteria having L-glutamic acid-producing ability include the following strains:

[0100] Brevibacterium flavum AJ11573 (FERM P-5492, refer to Japanese Patent Laid-open No. 56-151495)

[0101] Brevibacterium flavum AJ12210 (FERM P-8123, refer to Japanese Patent Laid-open No. 61-202694)

[0102] Brevibacterium flavum AJ12212 (FERM P-8123, refer to Japanese Patent Laid-open No. 61-202694)

[0103] Brevibacterium flavum AJ12418 (FERM-BP2205, refer to Japanese Patent Laid-open No. 2-186994)

[0104] Brevibacterium flavum DH18 (FERM P-11116, refer to Japanese Patent Laid-open No. 3-232497)

[0105] Corynebacterium melassecola DH344 (FERM P-11117, refer to Japanese Patent Laid-open No. 3-232497)

[0106] Corynebacterium glutamicum AJ11574 (FERM P-5493, refer to Japanese Patent Laid-open No. 56-151495)

[0107] Microorganisms having L-glutamic acid-producing ability include the Brevibacterium lactofermentum AJ13029 strain (FERM BP-5189, refer to WO96/06180), which was made to be able to produce L-glutamic acid in a medium containing an excessive amount of biotin in the absence of biotin action inhibitor, such as surfactant, by introducing a mutation which imparts temperature sensitivity to the biotin action inhibitor. Examples further include microorganisms that belong to the genus Alicyclobacillus and are resistant to a L-glutamic acid antimetabolite (Japanese Patent Laid-open No. 11-262398).

[0108] Furthermore, the microorganism having a L-glutamic acid producing ability can also be unable to degrade L-glutamic acid, or express the maleate synthase (aceB).cndot.isocitrate lyase (aceA).cndot.isocitrate dehydrogenase kinase/phosphatase (aceK) operon (henceforth abbreviated as "ace operon") constitutively. Examples of such microorganisms include, for example, the following:

[0109] Escherichia coli AJ12628 (FERM BP-3854)

[0110] Escherichia coli AJ12624 (FERM BP-3853)

[0111] The former is a mutant strain in which 2-oxoglutarate dehydrogenase activity is decreased and expression of the ace operon has become constitutive. The latter is a mutant strain in which 2-oxoglutarate dehydrogenase activity is decreased and the ability to degrade L-glutamic acid is decreased (refer to French Patent No. 2680178).

[0112] When a Pantoea bacterium is used, a mutation can be imparted that results in production of less extracellular phlegm as compared to a wild-type strain when it is cultured in a medium containing a saccharide. In order to introduce such a mutation, bacterial strains can be screened for their ability to produce viscous materials on the solid medium (Japanese Patent Laid-open No. 2001-333769), and the ams operon that is involved in polysaccharide synthesis can be disrupted. The nucleotide sequence of the ams operon is shown in SEQ ID NO: 66, and the amino acid sequences of AmsH, I, A, C and B encoded by this operon are shown in SEQ ID NOS: 67, 68, 69, 70 and 71, respectively.

[0113] <Succinic Acid-Producing Bacteria>

[0114] As succinic acid-producing bacteria, strains which 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).

[0115] Strains deficient in their abilities to form acetic acid, lactic acid, ethanol and formic acid can be obtained by using a strain that cannot assimilate acetic acid and lactic acid in a minimal medium, or by decreasing the activities of the lactic acid or acetic acid biosynthetic enzymes described below (International Patent Publication WO2005/052135).

[0116] Moreover, such strains as described above can also be obtained by imparting monofluoroacetic acid resistance (U.S. Pat. No. 5,521,075).

[0117] Other examples of methods for obtaining a strain with improved succinic acid-producing ability include culturing a strain which is deficient in both formate lyase and lactate dehydrogenase and cannot assimilate pyruvic acid in a glucose-enriched medium under anaerobic conditions, and isolating a mutant strain having the ability to assimilate pyruvic acid (International Patent Publication WO97/16528).

[0118] The ability to produce succinic acid can also be imparted by amplifying a gene encoding an enzyme which is involved in the succinic acid biosynthesis system described below, or deleting a gene encoding an enzyme which catalyzes a reaction which branches away from the succinic acid biosynthesis system to produce another compound.

[0119] The ability to produce succinic acid can also be imparted by modifying a microorganism 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 microorganisms can have both L-lactate dehydrogenase and D-lactate dehydrogenase, and can be modified to decrease the activity of either one of the enzymes, or the activities of both the enzymes, in another example.

[0120] The ability to produce succinic acid can also be imparted by modifying a microorganism 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. et al., 1998, Appl. Biochem. Biotechnol., 70-72:187-198).

[0121] The ability to produce succinic acid can also be imparted by modifying a microorganism to decrease the enzymatic activities of phosphate acetyltransferase (PTA), acetate kinase (ACK), pyruvate oxidase (POXB), 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).

[0122] The ability to produce succinic acid can also be enhanced by modifying a microorganism to decrease the enzymatic activity of alcohol dehydrogenase (ADH), which is an ethanol biosynthesis system enzyme (refer to International Patent Publication WO2006/031424).

[0123] 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, WO2007007933, Japanese Patent Laid-open No. 2005-168401). The gene names are indicated in the parentheses following the enzyme names.

[0124] The ability to produce succinic acid can also be imparted by enhancing a biosynthesis system enzyme involved in the succinic acid production.

[0125] The ability to produce succinic acid can also be enhanced by increasing the 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). Increasing the enzymatic activities of these target enzymes can be performed by referring to the methods for enhancing expression of a target gene described herein, and the methods for enhancing expression of ybjL gene described later.

[0126] Specific examples of succinic acid-producing bacteria belonging to the family Enterobacteriaceae include the following strains:

[0127] Escherichia coli SS373 strain (International Patent Publication WO99/06532)

[0128] Escherichia coli AFP111 strain (International Patent Publication WO97/16528)

[0129] Escherichia coli NZN111 strain (U.S. Pat. No. 6,159,738)

[0130] Escherichia coli AFP184 strain (International Patent Publication WO2005/116227)

[0131] Escherichia coli SBS100MG strain, SBS110MG strain, SBS440MG strain, SBS550MG strain, and SBS660MG strain (International Patent Publication WO2006/031424)

[0132] Examples of succinic acid-producing bacteria belonging to coryneform bacteria include the following strains.

[0133] Brevibacterium flavum AB-41 strain (Japanese Patent Laid-open No. 11-113588)

[0134] Brevibacterium flavum AB-41 strain (PC-amplified strain, Japanese Patent Laid-open No. 11-196888)

[0135] Corynebacterium glutamicum AJ110655 strain (FERM BP-10951)

[0136] Brevibacterium flavum MJ233.DELTA.ldh strain (International Patent Publication WO2005/021770)

[0137] Brevibacterium lactofermentum (Corynebacterium glutamicum) 2256.DELTA.(ldh, ach, pta, ack) (International Patent Publication WO2005/113744)

[0138] Brevibacterium lactofermentum 2256.DELTA.(ldh, pta, ack, poxB) (International Patent Publication WO2005/113745)

[0139] Corynebacterium glutamicum Rldh-/pCRB-1 PC strain (International Patent Publication WO2005/010182)

[0140] Examples of succinic acid-producing rumen bacteria include the following strains.

[0141] Mannheimia succiniciproducens LPK, LPK7 and LPK4 (International Patent Publication WO2005/052135)

[0142] Actinobacillus succinogenes 130Z (U.S. Pat. No. 5,504,004)

[0143] Anaerobiospirillum succiniciproducens FZ10 (U.S. Pat. No. 5,521,075)

[0144] Anaerobiospirillum succiniciproducens FZ53 (U.S. Pat. No. 5,573,931)

[0145] <1-2> Enhancement of Expression of ybjL Gene

[0146] A microorganism that has an ability to produce an acidic substance having a carboxyl group can be modified such as those described above so that expression of the ybjL gene is enhanced. However, the modification to enhance expression of the ybjL gene is performed first, and then the ability to produce an acidic substance having a carboxyl group can be imparted. Furthermore, the microorganism can already have the ability to produce an acidic substance having a carboxyl group, or the ability to produce an acidic substance having a carboxyl group can be enhanced by amplification of the ybjL gene.

[0147] The "ybjL gene" refers to the ybjL gene of Escherichia coli or a homologue thereof, the ybjL gene of Pantoea ananatis or a homologue thereof, or the ybjL gene of Enterobacter aerogenes or a homologue thereof. Examples of the ybjL gene of Escherichia coli include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 4, for example, a gene having the nucleotide sequence of the nucleotides 101 to 1783 in SEQ ID NO: 3. Furthermore, examples of the ybjL gene derived from Pantoea ananatis include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 2, for example, a gene having the nucleotide sequence of the nucleotides 298 to 1986 in SEQ ID NO: 1. Furthermore, examples of the ybjL gene derived from the Enterobacter aerogenes AJ110673 strain include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 87, for example, a gene having the nucleotide sequence of the nucleotides 19 to 1704 in SEQ ID NO: 86. Furthermore, examples of the ybjL gene derived from Salmonella typhimurium include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 25 (SEQ ID NO: 24). Examples of the ybjL gene derived from Yersinia pestis include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 27 (SEQ ID NO: 26). Examples of the ybjL gene derived from Erwinia carotovora include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 29 (SEQ ID NO: 28). Examples of the ybjL gene derived from Vibrio cholerae include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 31 (SEQ ID NO: 30). Examples of the ybjL gene derived from Aeromonas hydrophila include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 33 (SEQ ID NO: 32). Examples of the ybjL gene derived from Photobacterium profundum include a gene coding for a protein having the amino acid sequence of SEQ ID NO: 35 (SEQ ID NO: 34). Furthermore, the ybjL gene can be cloned from a coryneform bacterium such as Corynebacterium glutamicum and Brevibacterium lactofermentum, Pseudomonas bacterium such as Pseudomonas aeruginosa, Mycobacterium bacterium such as Mycobacterium tuberculosis or the like, on the basis of homology to the genes exemplified above. The amino acid sequences of the proteins encoded by the ybjL genes of Salmonella typhimurium, Yersinia pestis, Erwinia carotovora, Vibrio cholerae, Aeromonas hydrophila and Photobacterium profundum described above are 96%, 90%, 88%, 64%, 60% and 68% homologous to the amino acid sequence of SEQ ID NO: 4, respectively, and 86%, 90%, 84%, 63%, 60% and 67% homologous to the amino acid sequence of SEQ ID NO: 2, respectively. The amino acid sequences of SEQ ID NOS: 2 and 4 are 86% homologous. The consensus sequence of SEQ ID NOS: 2 and 4 is shown in SEQ ID NO: 5. The amino acid sequences of SEQ ID NOS: 4 and 87 re 92% homologous, and the amino acid sequences of SEQ ID NOS: 2 and 87 are 83% homologous. The sequence of the ybjL gene from the Enterobacter aerogenes AJ110637 strain (SEQ ID NO: 86) and the encoded amino acid sequence (SEQ ID NO: 87) have not been previously reported. The consensus sequence for the amino acid sequences of SEQ ID NOS: 2, 4 and 87 is shown in SEQ ID NO: 88.

[0148] The term "ybjL gene homologue" refers to a gene derived from a microorganism other than Escherichia coli, Pantoea ananatis, and Enterobacter aerogenes, which exhibits high structural similarity to the ybjL gene of Escherichia coli, Pantoea ananatis, or Enterobacter aerogenes and codes for a protein that improves an ability of a microorganism to produce an acidic substance having a carboxyl group when expression of the gene is enhanced in the microorganism. Examples of ybjL gene homologues include genes coding for a protein having a homology of 70% or more, 80% or more in another example, 90% or more in another example, 95% or more in another example, 97% or more in another example, to the entire amino acid sequence of SEQ ID NO: 2, 4 or 87 or the amino acid sequence of SEQ ID NO: 2, 4 or 87, and which improves an ability to produce an acidic substance having a carboxyl group of a microorganism when expression of the gene is enhanced in the microorganism. The ybjL gene homologue can code for a protein having a homology of 70% or more, 80% or more in another example, 90% or more in another example, 95% or more in another example, or 97% or more in another example, to any of the aforementioned amino acid sequences, and the consensus sequences of the SEQ ID NOS: 5 or 88. Homology of the 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 were developed on the basis of the algorithm BLAST (refer to www.ncbi.nlm.nih.gov). The term "homology" can also be used to mean "identity".

[0149] The ybjL gene can have one or more conservative mutations, and can be artificially modified, so long as enhancement of the tbjL gene's expression results in an improved ability to produce a target substance by a microorganism. That is, the ybjL gene can encode for an amino acid sequence of a known protein, for example, a protein having an amino acid sequence of SEQ ID NO: 2, 4, 25, 27, 29, 31, 33, 35 or 87, but which includes a conservative mutation, specifically, substitution, deletion, insertion or addition, of one or several amino acid residues at one or several positions. Although the number meant by the term "several" can differ depending on the position in the three-dimensional structure of the protein, or the type of amino acid residue. For example, it can be 1 to 20, 1 to 10 in another example, 1 to 5 in another example. Furthermore, the ybjL gene can encode for a protein having a homology of 70% or more, 80% or more in another example, 90% or more in another example, 95% or more in another example, or 97% or more in another example, to the entire amino acid sequence of SEQ ID NO: 2, 4, 25, 27, 29, 31, 33, 35 or 87, and which improves an ability to produce a target substance by a microorganism when expression of the gene is enhanced in the microorganism.

[0150] The aforementioned conservative substitution can be neutral, in that the function of the protein is not changed. The conservative mutation can take place mutually among Phe, Tip 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 substitution considered conservative substitution 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 H is; 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 Tip for Tyr; and substitution of Met, Ile or Leu for Val.

[0151] Such a gene can be obtained by modifying the nucleotide sequence of the nucleotides 298 to 1986 in SEQ ID NO: 1, the nucleotide sequence of the nucleotides 101 to 1783 in SEQ ID NO: 3, the nucleotide sequence of the nucleotides 19 to 1704 in SEQ ID NO: 86, or the nucleotide sequence of SEQ ID NO: 24, 26, 28, 30, 32 or 34 by, for example, site-specific mutagenesis, so that substitution, deletion, insertion or addition of an amino acid residue or residues occurs at a specific site in the encoded protein. Furthermore, such a gene can also be obtained by a known mutation treatment, examples of which include treating a gene having any of the nucleotide sequences described above with hydroxylamine or the like in vitro, and treating a microorganism, for example, an Escherichia bacterium, containing the gene with ultraviolet ray irradiation or a mutagen used in a usual mutation treatment, such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or EMS (ethyl methanesulfonate). The mutation for the substitutions, deletions, insertions, additions, inversions or the like of amino acid residues described above also includes a naturally occurring mutation based on individual difference, difference in species of microorganisms that contain the ybjL gene (mutant or variant), and the like.

[0152] The ybjL gene can also be a DNA hybridizable with a complementary strand of DNA having the nucleotide sequence of the nucleotides 298 to 1986 in SEQ ID NO: 1, a DNA having the nucleotide sequence of the nucleotides 101 to 1783 in SEQ ID NO: 3, a DNA having the nucleotide sequence of the nucleotides 19 to 1704 in SEQ ID NO: 86, or a DNA having the nucleotide sequence of SEQ ID NO: 1, 3, 24, 26, 28, 30, 32, 34 or 86, or a probe that can be prepared from the DNAs having these sequences under stringent conditions and which codes for a protein which improves an ability to produce a substance of a microorganism when expression of the gene is enhanced in the microorganism.

[0153] The "stringent conditions" can be conditions under which a so-called specific hybrid is formed, and non-specific hybrid is not formed. It is difficult to clearly express this condition by using any numerical value. However, the stringent conditions include, for example, when DNAs having high homology to each other, for example, DNAs having 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 another example, hybridize with each other, and DNAs having homology lower than the above level do not hybridize with each other, and when washing in ordinary Southern hybridization, i.e., washing once, twice or three times in another example, at salt concentrations and temperature of 1.times.SSC, 0.1% SDS at 60.degree. C., 0.1.times.SSC, 0.1% SDS at 60.degree. C. in another example, 0.1.times.SSC, 0.1% SDS at 65.degree. C., 0.1.times.SSC in another example, or 0.1% SDS at 68.degree. C. in another example.

[0154] The probe can have a partial sequence of the ybjL gene. Such a probe can be produced by PCR using oligonucleotides prepared on the basis of the nucleotide sequence of the gene and a DNA fragment including the gene as the template and performed in a manner well known to those skilled in the art. When a DNA fragment having a length of about 300 by is used as the probe, the washing conditions after hybridization can be exemplified by 2.times.SSC, 0.1% SDS at 50.degree. C.

[0155] The aforementioned descriptions concerning the gene homologue and conservative mutations can be similarly applied to the other enzyme genes described herein.

[0156] The expression of the ybjL gene can be enhanced by increasing the copy number of the ybjL gene, modifying an expression control sequence of the ybjL gene, amplifying a regulator that increases expression of the ybjL gene, or deleting or attenuating a regulator that decreases expression of the ybjL gene. Expression can be enhanced or increased using transformation or homologous recombination performed in the same manner as the methods for enhancing expression of a target gene described above for L-glutamic acid-producing bacteria.

[0157] In the microorganism, an activity for secreting an acidic substance having a carboxyl group can be improved by enhancing the expression of the ybjL gene. Whether "the activity for secreting an acidic substance having a carboxyl group is improved" can be confirmed by comparing the amount of the acidic substance having a carboxyl group which has been secreted into the medium in which the microorganism is cultured with the amount obtained by culturing a control microorganism into which the ybjL gene is not introduced. That is, the "improvement in the activity for secreting the acidic substance having a carboxyl group" is observed as an increase in the concentration of the acidic substance having a carboxyl group in the medium in which the microorganism is cultured as compared to the concentration obtained with a control microorganism. Furthermore, the "improvement in the activity for secreting an acidic substance having a carboxyl group" is also observed as a decrease in intracellular concentration of the acidic substance having a carboxyl group in the microorganism. As for the improvement of the "activity for secreting an acidic substance having a carboxyl group", the intracellular concentration of the acidic substance having a carboxyl group can be decreased by 10% or more, 20% or more in another example, 30% or more in another example, as compared to the concentration in a strain in which expression of the ybjL gene is not enhanced. The absolute "activity for secreting an acidic substance having a carboxyl group" of a microorganism can be detected by measuring the difference between the intracellular and extracellular concentrations of the acidic substance having a carboxyl group. Furthermore, the "activity for secreting an acidic substance having a carboxyl group" can also be detected by measuring the activity for taking up amino acids into the cells using reverted membrane vesicles with a radioisotope (J. Biol. Chem., 2002, vol. 277, No. 51, pp. 49841-49849). For example, the activity can be measured by preparing reverted membrane vesicles from cells in which the ybjL gene is expressed, adding a substrate which can act as a driving force such as ATP, and measuring the uptake activity for RI-labeled glutamic acid. Moreover, the activity can also be measured by detecting an exchange reaction rate of a labeled acidic substance having a carboxyl group and unlabeled acidic substance having a carboxyl group in live bacteria.

[0158] The microorganism can have an ability to accumulate L-glutamic acid in the liquid medium in an amount which is more than the amount at the saturation concentration of L-glutamic acid when it is cultured under acidic conditions (this ability is also referred to as the "L-glutamic acid accumulation ability under acidic conditions"). Such a microorganism can have the L-glutamic acid accumulation ability under acidic conditions by enhancing the expression of the ybjL gene. Alternatively, the microorganism can have a native ability to accumulate L-glutamic acid under acidic conditions.

[0159] Specific examples of microorganisms having L-glutamic acid accumulation ability under acidic conditions include the aforementioned Pantoea ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207) (for these, refer to Japanese Patent Laid-open No. 2001-333769), and the like. The Pantoea ananatis AJ13355 and AJ13356 strains were deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology, Address: Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Feb. 19, 1998 and given accession numbers of FERM P-16644 and FERM P-16645. The deposits were then converted to international deposits under the provisions of Budapest Treaty on Jan. 11, 1999 and given accession numbers of FERM BP-6614 and FERM BP-6615. The AJ13601 strain was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently, International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology) on Aug. 18, 1999 and given an accession number of FERM P-17516. The deposit was converted to an international deposit under the provisions of the Budapest Treaty on Jul. 6, 2000 and given an accession number of FERM BP-7207. These strains were identified as Enterobacter agglomerans when they were isolated and deposited as Enterobacter agglomerans AJ13355, AJ13356, and AJ13601 strains. However, they were recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and the like.

[0160] When an organic acid, especially succinic acid, is produced, using a microorganism which is modified to decrease the activities of one or more enzymes among lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), and pyruvate formate lyase (PFL), in addition to increasing the expression of the ybjL gene, is more effective.

[0161] 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 can be decreased to 10% per cell or lower as compared to that of a lactate dehydrogenase-unmodified strain. 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 the 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 microorganism with decreased lactate dehydrogenase activity and enhanced expression of the ybjL gene can be obtained by, for example, preparing a microorganism in which the LDH gene is disrupted, and transforming this microorganism with a recombinant vector containing the ybjL gene, as described in Example 1. However, either the modification for decreasing the LDH activity or the modification for enhancing expression of the ybjL 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: 36, the amino acid sequence encoded thereby is shown in SEQ ID NO: 37, the DNA sequence of the lldD gene is shown in SEQ ID NO: 38, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 39.

[0162] In order to decrease or delete activity of LDH, a mutation that decreases or deletes the intracellular activity of LDH can be introduced into the LDH gene on the chromosome by a known 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 the Shine-Dalgarno (SD) sequence can be modified by gene recombination. Furthermore, a mutation can also be introduced to cause 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 coding region of the LDH on the chromosome, or a part of, 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 deleting the coding region of the LDH gene in a DNA construct, and replacing the normal LDH gene with the DNA construct on the chromosome by homologous recombination or the like, or by introducing a transposon or IS factor into the gene.

[0163] In order to introduce a mutation which results in decreasing or deleting the LDH activity by genetic recombination, for example, the following methods are used. The chromosomal LDH gene 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 an enzyme that can function normally, and transforming a bacterium with a DNA containing the mutant gene to cause homologous recombination between the mutant gene and the gene on a chromosome. Such site-specific mutagenesis based on gene substitution utilizing homologous recombination has been already reported, and include a method called Red driven integration developed by Datsenko and Wanner (Datsenko, K. A, and Wanner, B. L., 2000, Proc. Natl. Acad. Sci. USA, 97:6640-6645), a method of using a linear DNA such as by utilizing Red driven integration in combination with an excision system derived from .lamda. phage (Cho, E. H., et al., 2002, J. Bacteriol., 184:5200-5203), a method of 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 by using a plasmid that is unable to replicate in a host.

[0164] 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 alcohol dehydrogenase is unmodified. The alcohol dehydrogenase activity can be decreased to 10% per cell or lower as compared to an alcohol dehydrogenase-unmodified strain. The alcohol dehydrogenase activity can also be completely deleted. Decrease in the alcohol dehydrogenase activity can be confirmed by measuring the alcohol dehydrogenase activity by a known method (Lutstorf, U. M. et al., 1970, Eur. J. Biochem., 17:497-508). 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. The microorganism in which alcohol dehydrogenase activity is decreased and expression of the ybjL gene is enhanced can be obtained by, for example, preparing a microorganism in which the gene coding for alcohol dehydrogenase (ADH) is disrupted, and transforming this microorganism with a recombinant vector containing the ybjL gene. However, either the modification for decreasing the ADH activity or the modification for enhancing expression of the ybjL gene can be performed first. The alcohol dehydrogenase activity can be decreased by a method similar to that for decreasing the lactate dehydrogenase activity described above. The nucleotide sequence (partial sequence) of the ADH gene from the Enterobacter aerogenes AJ110637 strain (FERM ABP-10955) is shown in SEQ ID NO: 74. 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 this partial sequence.

[0165] 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 strain in which the pyruvate formate lyase is unmodified. The pyruvate formate lyase activity can be decreased to 10% per cell or lower as compared to a pyruvate formate lyase-unmodified strain. The pyruvate formate lyase activity can also be completely deleted. A decrease in 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 microorganism in which pyruvate formate lyase activity is decreased and expression of the ybjL gene is enhanced can be obtained by, for example, preparing a microorganism in which the PFL gene is disrupted, and transforming this microorganism with a recombinant vector containing the ybjL gene. However, either the modification for decreasing the PFL activity or the modification for enhancing expression of ybjL 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.

[0166] A bacterium modified so that the pyruvate carboxylase (PC) activity is enhanced, in addition to the enhanced the expression of the ybjL gene, can also be used to produce an organic acid, especially succinic acid. 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, a method of measuring a decrease of NADH as described later.

[0167] The nucleotide sequence of the PC gene can be a reported sequence or can be obtained by isolating a DNA fragment encoding a protein having the PC activity from the chromosome of a microorganism, animal, plant, or the like, and then the nucleotide sequence can be determined. After the nucleotide sequence is determined, a gene synthesized on the basis of that sequence can also be used.

[0168] The PC gene can be derived from a coryneform bacterium, such as Corynebacterium glutamicum (Peters-Wendisch, P. G. et al., 1998, Microbiology, vol. 144:915-927). Furthermore, so long as the functions of the encoded PC protein, such as its involvement in carbon dioxide fixation, are not substantially degraded, nucleotides in the PC gene can be replaced with other nucleotides or deleted, or other nucleotides can be inserted into the sequence. Alternatively, a part of the nucleotide sequence can be transferred.

[0169] PC genes obtained from bacteria other than Corynebacterium glutamicum, including from other microorganisms, animals, and plants, can also be used. In particular, the sequences of PC genes derived from other microorganisms, animals and plants described below are known (references are indicated below), and they can be obtained by hybridization or amplification by PCR of the ORF portions in the same manner as described above.

[0170] Human [Biochem. Biophys. Res. Comm., 202, 1009-1014, (1994)]

[0171] Mouse [Proc. Natl. Acad. Sci. USA., 90, 1766-1779, (1993)]

[0172] Rat [GENE, 165, 331-332, (1995)]

[0173] Yeast: Saccharomyces cerevisiae [Mol. Gen. Genet., 229, 307-315, (1991)], Schizosaccharomyces pombe [DDBJ Accession No.; D78170]

[0174] Bacillus stearothermophilus [GENE, 191, 47-50, (1997)]

[0175] Rhizobium etli [J. Bacteriol., 178, 5960-5970, (1996)]

[0176] The PC gene can be enhanced in the same manner as when enhancing expression of a target gene described above for L-glutamic acid-producing bacteria and enhancing expression of the ybjL gene.

[0177] <2> Method for Producing an Acidic Substance Having a Carboxyl Group

[0178] An acidic substance having a carboxyl group can be produced by culturing the microorganism in a medium to produce and cause accumulation of an acidic substance having a carboxyl group in the medium and collecting the substance from the medium. Furthermore, the acidic substance having a carboxyl group can also be produced by allowing the microorganism, or a product obtained by processing the microorganism, to act on an organic raw material in a reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas to produce the acidic substance having a carboxyl group, and collecting the substance. The former method is exemplary for producing an acidic amino acid. The latter method is exemplary for producing an organic acid. Hereinafter, further examples methods for producing an acidic amino acid and an organic acid will be exemplified.

[0179] <2-1> Production of Acidic Amino Acid

[0180] An acidic amino acid can be produced by culturing the microorganism in a medium to produce and cause accumulation of an acidic amino acid in the medium and collecting the acidic amino acid from the medium.

[0181] As the medium used for the culture, a known medium containing a carbon source, nitrogen source, and inorganic salts, as well as trace amounts of organic nutrients such as amino acids and vitamins as required can be used. Either a synthetic medium or natural medium can be used. The carbon source and nitrogen source used in the medium can be of any type so long as substances that can be utilized by the chosen strain to be cultured.

[0182] As the carbon source, saccharides such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysate and molasses can be used. In addition, alcohols such as ethanol can be used independently or in combination with another carbon source. Furthermore, organic acids such as acetic acid and citric acid other than the target substance can also be used as the carbon source. As the nitrogen source, ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate and ammonium acetate, nitrates and the like can be used. As the trace amount organic nutrients, amino acids, vitamins, fatty acids, nucleic acids, those containing these substances such as peptone, casamino acid, yeast extract and soybean protein decomposition products can be used. When an auxotrophic mutant strain that requires an amino acid or the like for growth is used, the required nutrient can be supplemented. As mineral salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like can be used.

[0183] The culture can be performed as an aeration culture, while the fermentation temperature can be controlled to be 20 to 45.degree. C., and pH to be 3 to 9. When pH drops during the culture, the medium can be neutralized by addition of, for example, calcium carbonate, or with an alkali such as ammonia gas. An acidic amino acid can accumulate in the culture broth, for example, after 10 to 120 hours of culture under such conditions as described above.

[0184] When the acidic amino acid is L-glutamic acid, L-glutamic acid can precipitate into the medium by using a liquid medium, the pH of which is adjusted so that L-glutamic acid precipitates. L-glutamic acid precipitates around, for example, pH 5.0 to 4.0, pH 4.5 to 4.0 in another example, pH 4.3 to 4.0 in another example, or pH 4.0 in another example.

[0185] After completion of the culture, L-glutamic acid can be collected from the culture medium by any known collection method. For example, after cells are removed from the culture medium, L-glutamic acid can be collected by concentrating the culture medium so it crystallizes, or by ion exchange chromatography, or the like. When the culture is performed so that L-glutamic acid precipitates, the L-glutamic acid that has precipitated in the medium can be collected by centrifugation, filtration, or the like. In this case, it is also possible to crystallize L-glutamic acid that has dissolved in the medium, and then collect the precipitated L-glutamic acid in the culture broth together with the crystallized L-glutamic acid.

[0186] <2-2> Production of Organic Acid

[0187] An organic acid can be produced by allowing the microorganism, or a product obtained by processing the microorganism, to act on an organic raw material in a reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas, and collecting the organic acid.

[0188] In a first example, by culturing the microorganism in a medium containing carbonate ions, bicarbonate ions, or carbon dioxide gas, and an organic raw material, proliferation of the microorganism and production of the organic acid simultaneously occur. In this example, the medium can be the reaction mixture. Proliferation of the microorganism and production of the organic acid can simultaneously occur, or there can be a period during the culture in which proliferation of the microorganism mainly occurs, and a period in which production of the organic acid mainly occurs.

[0189] In a second example, by allowing cells that have proliferated in the medium to coexist with a reaction mixture containing carbonate ions, bicarbonate ions, or carbon dioxide gas, and an organic raw material, and thereby allowing the microorganism to act on the organic raw material in the reaction mixture, an organic acid can be produced. In this example, a product obtained by processing the cells of the microorganism can also be used. Examples of the product obtained by processing cells include, for example, immobilized cells obtained with acrylamide, carragheenan, or the like, a disrupted cellular product, a centrifugation supernatant of the disrupted product, a fraction obtained by partial purification of the supernatant by ammonium sulfate treatment or the like, and the like.

[0190] Although the bacteria can be obtained on a solid medium such as an agar medium by slant culture, bacteria previously cultured in a liquid medium (seed culture) are examples.

[0191] A medium usually used for culture of microorganisms can be used. For example, a typical medium obtained by adding natural nutrients such as meat extract, yeast extract and peptone, to a composition comprising inorganic salts such as ammonium sulfate, potassium phosphate and magnesium sulfate can be used.

[0192] In the aforementioned first example, the carbon source added to the medium also serves as the organic raw material for the production of the organic acid.

[0193] In the aforementioned second example, the cells after the culture are collected by centrifugation, membrane separation, or the like, and used for the organic acid production reaction.

[0194] The organic raw material is not particularly limited so long as the chosen microorganism can assimilate it to produce succinic acid. 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 typically used. Glucose, fructose and glycerol are examples, and glucose is a particular example. 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.

[0195] Furthermore, a saccharified starch solution, molasses, or the like containing the aforementioned fermentable carbohydrates can also be used. The fermentable carbohydrates can be used independently or in combination. Although concentration of the aforementioned organic raw material is not particularly limited, it is more advantageous that the concentration is as high as possible and within such a range that the culture of the microorganism and production of the organic acid are not inhibited. In the aforementioned first example, the 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, it may be necessary to add additional organic raw material as the concentration of the organic raw material decreases with the progress of the culture or reaction.

[0196] 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 typical medium for culturing microorganisms, 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 the chosen microorganism can assimilate it 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 during the reaction, an appropriate amount of a commercially available antifoam can be added to the medium.

[0197] The 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 can be adjusted to be within the aforementioned range with an alkaline substance, carbonate, urea, or the like, even during the reaction, if needed.

[0198] Water, buffer, medium or the like can be used as the reaction mixture, but a medium is a 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. Magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate, or potassium bicarbonate can be the source of the carbonate or bicarbonate ions, and these 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 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 in an amount of 50 mg to 25 g, 100 mg to 15g in another example, or 150 mg to 10g in another example, per liter of the solution.

[0199] The optimal growth temperature of the chosen microorganism is generally in the range of 25 to 40.degree. C. Therefore, the reaction temperature is generally in the range of 25 to 40.degree. C., or in the range of 30 to 37.degree. C. in another example. The amount of bacterial cells in the reaction mixture is, 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 batchwise or on a column.

[0200] The culture of the bacteria can be performed under aerobic conditions. On the other hand, the organic acid production reaction can be performed under aerobic conditions, microaerobic conditions, or anaerobic conditions. When performed under microaerobic conditions or anaerobic conditions, the reaction can be performed in a sealed reaction vessel without aeration, with a supplied inert gas such as nitrogen gas, or with a supplied inert gas containing carbon dioxide gas, and the like.

[0201] The organic acid can accumulate in the reaction mixture (culture medium) and 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, then the resulting solution can be desalted with an ion exchange resin or the like, and the organic acid can be separated and purified from the solution by crystallization or column chromatography.

[0202] Furthermore, when the target organic acid is succinic acid, and after the production, a polymerization reaction can be carried out by using the produced 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. The produced 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 obtained by polymerizing a diol such as butanediol and ethylene glycol and succinic acid, succinic acid polyamides obtained by polymerizing a diamine such as hexamethylenediamine and succinic acid, and the like. In addition, succinic acid and succinic acid-containing polymers obtained by the production method described herein, and compositions containing these can be used for food additives, pharmaceutical agents, cosmetics, and the like.

EXAMPLES

[0203] Hereinafter, the present invention will be explained more specifically with reference to the following non-limiting examples.

Reference Example 1

Construction of Pantoea ananatis Strain Resistant to .lamda. Red gene Product

[0204] In order to disrupt the sdhA gene in Pantoea ananatis, a recipient strain for efficiently carrying out the method called "Red-driven integration" or "Red-mediated integration" (Datsenko, K A. and Wanner, B. L., 2000, Proc. Natl. Acad. Sci. USA., 97, 6640-6645) was constructed.

[0205] First, the novel helper plasmid RSF-Red-TER which expresses the gam, bet and exo genes of .lamda. (".lamda. Red genes") was constructed (FIG. 1). The details of this construction are described in Reference Example 2.

[0206] This plasmid can be used in a wide range of hosts having different genetic backgrounds. This is because 1) this plasmid has the replicon of the RSF1010 wide host spectrum plasmid (Scholz, et al., 1989, Gene, 75:271-288; Buchanan-Wollaston et al., 1987, Nature, 328:172-175), which is stably maintained by many types of gram negative and gram positive bacteria, and even plant cells, 2) the .lamda. Red genes, gam, bet and exo, are under the control of the PlacUV5 promoter, which is recognized by the RNA polymerases of many types of bacteria (for example, Brunschwig, E. and Darzins, A., 1992, Gene, 111, 1, 35-41; Dehio, M. et al, 1998, Gene, 215, 2, 223-229), and 3) the autoregulation factor PlacUV5-lacI and the p-non-dependent transcription terminator (TrrnB) of the rrnB operon of Escherichia coli lower the basal expression level of the .lamda. Red genes (Skorokhodova, A. Y. et al, 2004, Biotekhnologiya (Rus), 5, 3-21). Furthermore, the RSF-Red-TER plasmid contains the levansucrase gene (sacB), and by the expression of this gene, the plasmid can be collected from cells in a medium containing sucrose.

[0207] In Escherichia coli, the frequency of integration of a PCR-generated DNA fragment along with the short flanking region provided by the RSF-Red-TER plasmid is as high as the frequency obtained when using the pKD46 helper plasmid (Datsenko, K. A., Wanner, B. L., 2000, Proc. Natl. Acad. Sci. USA, 97, 6640-6645). However, expression of the .lamda. Red genes is toxic to Pantoea ananatis. Cells transformed with the RSF-Red-TER helper plasmid grow extremely slowly in LB medium containing IPTG (isopropyl-.beta.-D-thiogalactopyranoside, 1 mM) and an appropriate antibiotic (25 .mu.g/ml of chloramphenicol or 40 .mu.g/ml of kanamycin), and the efficiency of .lamda. Red-mediated recombination is extremely low (10.sup.-8), if observed at all.

[0208] A variant strain of Pantoea ananatis which is resistant to expression of all three of the .lamda. Red genes was selected. For this purpose, the RSF-Red-TER plasmid was introduced into the Pantoea ananatis SC17 strain (U.S. Pat. No. 6,596,517) by electroporation. After an 18-hour culture, about 10.sup.6 transformants were obtained, and among these, 10 clones formed colonies of a large size, and the remainder formed extremely small colonies. After an 18 hour culture, the large colonies were about 2 mm, and the small colonies were about 0.2 mm. While the small colonies did not grow any more even when the culture was extended up to 24 hours, the large colonies continued to grow. One of the large colony Pantoea ananatis mutant strains which was resistant to expression of all three of the .lamda. Red genes (gam, bet, and exo) was used for further analysis.

[0209] The RSF-Red-TER plasmid DNA was isolated from one clone of the large colony clones, and from several clones of the small colony clones, and transformed again into Escherichia coli MG1655 to examine the ability of the plasmid to synthesize an active Red gene product. A control experiment for Red-dependent integration in the obtained transformants was used to demonstrate that only the plasmid isolated from the large colony clone induced expression of the .lamda. Red genes required for the Red-dependent integration. In order to investigate whether the Red-mediated integration occurs in the selected large colony clone, electroporation was performed using a linear DNA fragment produced by PCR. This fragment was designed so that it contains a Km.sup.R marker and a flanking region of 40 by homologous to the hisD gene, and is integrated into the hisD gene of Pantoea ananatis at the SmaI recognition site. Two small colony clones were used as control. The nucleotide sequence of the hisD gene of Pantoea ananatis is shown in SEQ ID NO: 40. For PCR, the oligonucleotides of SEQ ID NOS: 41 and 42 were used as primers, and the pMW118-(.lamda.att-Km.sup.r-.lamda.att) plasmid was used as the template. The two small colony clones which were not resistant to the Red genes were used as the control. Construction of the pMW118-(.lamda.attL-Km.sup.r-.lamda.attR) plasmid will be explained in detail in Reference Example 3.

[0210] The RSF-Red-TER plasmid can induce expression of the Red genes by the lad gene carried on the plasmid. Two kinds of induction conditions were investigated. In the first group, IPTG (1 mM) was added 1 hour before the electroporation, and in the second group, IPTG was added at the start of the culture to prepare cells in which electroporation is possible. The growth rate of the progeny of the SC17 strain harboring RSF-Red-TER derived from the large colony clone was not significantly lower than that of a strain not having the RSF-Red-TER plasmid. The addition of IPTG only slightly decreased the growth rate of these cultures. On the other hand, the RSF-Red-TER-introduced SC17 strain derived from the progeny of the small colony clones grew extremely slowly even without the addition of IPTG, and after induction, growth was substantially arrested. After electroporation of RSF-Red-TER isolated from the cells of the progeny of the large colony clone, many Km.sup.R clones grew (18 clones after a short induction time, and about 100 clones after an extended induction time). All of the 100 clones that were investigated had a His.sup.- phenotype, and about 20 clones were confirmed by PCR to have the expected structure of the chromosome in the cells. On the other hand, even when electroporation was performed with RSF-Red-TER isolated from cells of the progeny of the small colony clones, an integrated strain was not obtained.

[0211] The large colony clone was grown on a plate containing 7% sucrose to eliminate the plasmid, and transformed again with RSF-Red-TER. The strain without the plasmid was designated SC17(0). This strain was deposited at the Russian National Collection of Industrial Microorganisms (VKPM), GNII Genetica (Address: 1 Dorozhny proezd., 1 Moscow 117545, Russia) on Sep. 21, 2005, and assigned an accession number of VKPM B-9246.

[0212] All the clones which grew after the aforementioned re-transformation were large like the parent strain clone SC17(0). The Red-mediated integration experiment was performed in the SC17(0) strain which had been re-transformed with the RSF-Red-TER plasmid. Three of the independent transformants obtained were investigated using the same DNA fragment as that used for the previous experiment. A short induction time (1 hour before electroporation) was employed. Km.sup.R clones exceeding ten clones grew in each experiment. All the examined clones had the His.sup.- phenotype. In this way, a mutant strain designated SC17(0) which is resistant to the expression of the .lamda. Red genes was selected. This strain can be used as a recipient strain suitable for the Red-dependent integration into the Pantoea ananatis chromosome.

Reference Example 2

Construction of Helper Plasmid RSF-Red-TER

[0213] The scheme for constructing the helper plasmid RSF-Red-TER is shown in FIG. 2.

[0214] As the first step in 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: 43 and 44, and 45 and 46, respectively. These oligonucleotides contained the BglII, Sad, XbaI and BamHI restriction enzyme sites in the 5' end regions. These sites are required and convenient for further cloning. The obtained sacB fragment of 1.5 kb was cloned into the previously obtained pMW119-P.sub.laclacI vector at the XbaI-BamHI site. This vector was constructed in the same manner as that described for the pMW118-P.sub.laclacI 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.

[0215] Then, the aforementioned cat fragment of 1.0 kb was treated with BglII and Sad, and cloned into the RSF-P.sub.laclacIsacB plasmid obtained in the previous step at the BamHI-SacI site. The obtained plasmid pMW-P.sub.laclacIsacBcat contained the PlacUV5-lacI-sacB-cat fragment. In order to subclone this fragment into the RSF1010 vector, pMW-P.sub.laclacIsacBcat was digested with BglII, blunt-ended with DNA polymerase I Klenow fragment, and successively digested with Sad. A 3.8 kb BglII-SacI fragment of the pMWP.sub.laclacIsacBcat plasmid was eluted from a 1% agarose gel, and ligated with the RSF1010 vector which had been 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: 47 and 48 as primers and the pMW119-P.sub.laclacI plasmid as a template. The obtained fragment of 146 by was digested with SacI and NotI, and ligated with the SacI-NotI large fragment of the RSFsacB plasmid. Then, by PCR using the oligonucleotides of SEQ ID NOS: 49 and 50 as primers, and the pKD46 plasmid (Datsenko, K. A., Wanner, B. L., 2000, Proc. Natl. Acad. Sci. USA, 97, 6640-6645) as a template, a DNA fragment of 2.3 kb containing the .lamda.Red.alpha..beta..gamma. 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.

[0216] In order to eliminate read through transcription of the Red genes, a .rho.-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: 51 and 48 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: 48 and 52 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 the pMW118-(.lamda.attL-Km.sup.r-.lamda.attR) Plasmid

[0217] The pMW118-(.lamda.attL-Km.sup.r-.lamda.attR) plasmid was constructed from the pMW118-attL-Tc-attR (WO2005/010175) plasmid by replacing the tetracycline resistance marker gene with the kanamycin resistance gene of the pUC4K plasmid. For that purpose, the EcoRI-HindIII large fragment from pMW118-attL-Tc-attR was ligated to two fragments from the pUC4K plasmid: the HindIII-PstI fragment (676 bp) and EcoRI-HindIII fragment (585 bp). Basic pMW118-attL-Tc-attR was obtained by ligation of the following four fragments.

[0218] 1) The BglII-EcoRI fragment (114 bp) including attL (SEQ ID NO: 55) which was obtained by PCR amplification of the region corresponding to attL of the Escherichia coli W3350 (containing .lamda. prophage) chromosome using the primers P1 and P2 (SEQ ID NOS: 53 and 54) (these primers contained the subsidiary recognition sites for BglII and EcoRI).

[0219] 2) The PstI-HindIII fragment (182 bp) including attR (SEQ ID NO: 58) which was obtained by PCR amplification of the region corresponding to attR of the Escherichia coli W3350 (containing .lamda. prophage) chromosome using the primers P3 and P4 (SEQ ID NOS: 56 and 57) (these primers contained the subsidiary recognition sites for PstI and HindIII).

[0220] 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: [0221] 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 digested with AatII; [0222] 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: 59 and 60) (these primers contained the subsidiary recognition sites for PstI, AatII and BglII); [0223] 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: 61 and 62) (these primers contained the subsidiary recognition sites for PstI, BglII and PstI).

[0224] 4) The small EcoRI-PstI fragment (1388 bp) (SEQ ID NO: 63) 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: [0225] 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: 64 and 65) (these primers contained the subsidiary recognition sites for PstI, XbaI and BamHI); [0226] 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 ligated 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).

Example 1

Search of L-Glutamic Acid Secretion Gene

[0227] A search for an L-glutamic acid secretion gene was performed as follows. Since L-glutamic acid is converted into an intermediate of the tricarboxylic acid cycle, 2-oxoglutarate, in one step by glutamate dehydrogenase, L-glutamic acid is thought to be easily metabolized in many microorganisms having glutamate dehydrogenase or the tricarboxylic acid cycle. However, since a strain in which 2-oxoglutarate dehydrogenase is deleted cannot degrade L-glutamic acid, growth of the cells is inhibited in the presence of a high concentration glutamic acid. In this example, the SC17sucAams strain derived from the Pantoea ananatis SC17sucA strain (refer to Japanese Patent Laid-open No. 2001-333769) is deficient in the extracellular polysaccharide biosynthesis system, and is also deficient in 2-oxoglutarate dehydrogenase. Therefore, this strain was used to try to obtain an L-glutamic acid excretion gene utilizing resistance to a high concentration of L-glutamic acid as a marker.

[0228] Since the SC17sucA strain produced a marked amount of extracellular polysaccharides when grown on an agar medium containing a sugar source, the handling of this strain is extremely difficult. Therefore, by deleting the ams operon coding for the extracellular polysaccharide biosynthesis system genes, production of extracellular polysaccharides was suppressed. PCR was performed by using pMW118-.alpha.attL-Km.sup.r-.lamda.attR as the template and the primers of SEQ ID NOS: 6 and 7 to amplify a gene fragment containing a kanamycin resistance gene, attL and attR sequences of .lamda. phage at the both ends of the resistance gene, and 50 by upstream sequence of amsI and 50 by downstream sequence of the amsC gene added to the outer ends of the .lamda. phage sequences. This fragment was purified by using Wizard PCR Prep DNA Purification System (Promega).

[0229] Then, the SC17(0) strain was transformed with RSF-Red-TER to obtain an SC17(0)/RSF-Red-TER strain. This strain was cultured overnight in L medium (10 g of Bacto tryptone, 5 g of yeast extract and 5 g of NaCl in 1 L of pure water, pH 7.0) containing 25 mg/L of chloramphenicol, and then the culture medium after the overnight culture was inoculated in 1/100 volume into 100 mL of the L medium containing 25 mg/L of chloramphenicol and 1 mM isopropyl-.beta.-D-thiogalactopyranoside, and culture was performed at 34.degree. C. for 3 hours. The cells prepared as described above 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 100 ng of the PCR fragment prepared in the above section was introduced into the cells by using GENE PULSER II (BioRad) with a field strength of 18 kV/cm, capacitor capacity of 25 .mu.F and resistance of 200.OMEGA.. Ice-cooled SOC medium (20 g/L of Bacto tryptone, 5 g/L of yeast extract, 0.5 g/L of NaCl, and 10 g/L of glucose) was added to the cell suspension, and culture was performed at 34.degree. C. for 2 hours with shaking. The culture was applied to a medium prepared by adding ingredients of minimal medium (5 g of glucose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride and 6 g of disodium phosphate in 1 L) and 40 mg/L of kanamycin to the L medium (10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH 7.0). The colonies that appeared were purified with the same medium, and then it was confirmed by PCR that the ams gene had been replaced with the kanamycin resistance gene.

[0230] The chromosome was extracted from the ams gene-deficient strain using a Bacterial Genomic DNA Purification Kit (Edge Biosystems). Separately, the SC17sucA strain was cultured overnight on an agar medium obtained by adding the ingredients of the minimal medium described above to the L medium. The cells were scraped with a loop, washed three times with ice-cooled 10% glycerol, and finally suspended in 10% glycerol to a final volume of 500 .mu.l. The suspended cells were used as competent cells, and 600 ng of the aforementioned chromosome DNA was introduced into the competent cells using GENE PULSER II (BioRad) with a field strength of 17.5 kV/cm, capacitor capacity of 25 .mu.F and resistance of 200.OMEGA.. Ice-cooled SOC medium was added to the cell suspension, and culture was performed at 34.degree. C. for 2 hours with shaking. Then, the culture was applied on an agar medium prepared by adding ingredients of the minimal medium described above and 40 mg/L of kanamycin to the L medium. The colonies that appeared were purified with the same medium, and then it was confirmed by PCR that the ams gene had been replaced with the kanamycin resistance gene. This strain was designated as SC17sucAams.

[0231] Chromosomal DNA extracted from the Pantoea ananatis AJ13355 strain was partially digested with the restriction enzyme Sau3AI. Then, fragments of about 10 kb were collected and introduced into the BamHI site of pSTV28 (Takara Bio) to prepare a plasmid library. This plasmid library was introduced into competent cells of the SC17sucAams strain prepared in a conventional manner by electroporation.

[0232] Selection was performed for the SC17sucAams strain which had been introduced with a plasmid library on a plate of the L medium (10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH 7.0) added with ingredients of minimal medium (5 g of glucose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride and 6 g of disodium phosphate in 1 L of pure water) using chloramphenicol resistance as a marker to obtain transformants. These transformants were plated on a glucose minimal medium containing a high concentration L-glutamic acid (the glucose minimal medium mixed with 0.2 M L-glutamic acid, 100 mg/L each of lysine, methionine and diaminopimelic acid as final concentrations), in which SC17sucAams cannot form colonies.

[0233] The transformants were cultured at 34.degree. C. for 3 days, and 64 clones among the colonies that appeared were allowed to again form single colonies on the same plate. As a result, 11 clones were found to form colonies after 48 hours, and the remaining colonies formed colonies after 72 hours.

[0234] Then, the genes inserted into the vectors harbored by the transformants were analyzed. Plasmids were extracted from the transformants, and nucleotide sequences were determined. It was found that among the 11 clones that formed colonies within 48 hours, 10 clones had the same loci, and all contained genes showing homology to ybjL, which was an Escherichia coli gene of unknown function. In addition, this ybjL gene could not be obtained under the same conditions that the glutamic acid secretion system gene described in WO2005/085419, yhfK, was obtained, and conversely, the yhfK gene could not be obtained under the selection conditions used in this example.

[0235] In order to confirm that the factor which imparts glutamic acid resistance is ybjL, the ybjL gene was cloned. PCR was performed using the chromosomal DNA of AJ13355 strain as the template and oligonucleotides ybjL-F1 and ybjL-R2 shown in SEQ ID NOS: 8 and 9 to amplify the fragment of about 1.8 kb containing the ybjL gene of P. ananatis. This fragment was purified using Wizard PCR Prep DNA Purification System (Promega), and then treated with the restriction enzymes KpnI and SphI, and the product was ligated with pSTV28 (Takara Bio) which had been treated with the same enzymes to obtain pSTV-PanybjL. The SC17sucA strain was transformed with this pSTV-PanybjL plasmid, and using pSTV28 as a control for comparison (Takara Bio) to construct the SC17sucA/pSTV-PanybjL and SC17sucA/pSTV28 strains.

[0236] The SC17sucA/pSTV-ybjL strain was plated on minimal medium (5 g of glucose or sucrose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride, 6 g of disodium phosphate, 0.2 M sodium L-glutamate, 100 mg/L each of lysine, methionine and diaminopimelic acid and 15 g of agar in 1 L of pure water) containing glutamic acid. Culture was performed at 34.degree. C. for 2 days. As a result, it was confirmed that whereas the control vector-introduced strain, SC17sucA/pSTV28, could not form colonies, SC17sucA/pSTV-ybjL could form colonies on the minimal medium.

[0237] Then, the SC17sucA/pSTV-PanybjL strain was cultured in liquid minimal medium (5 g of glucose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride, 6 g of disodium phosphate, 0.2 M sodium L-glutamate, 100 mg/L each of lysine, methionine and diaminopimelic acid in 1 L of pure water) containing glutamic acid to examine growth of the strain in the presence of a high concentration of L-glutamic acid.

[0238] The results are shown in FIG. 3. It was found that growth of SC17sucA/pSTV-PanybjL in which the ybjL gene had been enhanced was markedly improved in the presence of high concentration L-glutamic acid as compared to the control strain SC17sucA/pSTV28. Accordingly, it was confirmed that ybjL is a factor which imparts resistance to L-glutamic acid.

Example 2

Effect of ybjL Gene Amplification on L-Glutamic Acid Production at Neutral pH

[0239] Then, in order to examine the effect of this gene on L-glutamic acid production, the plasmid for ybjL amplification, pSTV-PanybjL, was introduced into the L-glutamic acid producing bacterium SC17sucA/RSFCPG having the plasmid for L-glutamic acid production, RSFCPG, shown in SEQ ID NO: 10 (refer to Japanese Patent Laid-open No. 2001-333769), and L-glutamic acid productivity thereof was examined.

[0240] pSTV-PanybjL and the control plasmid, pSTV28 (Takara Bio), were each introduced into SC17sucA/RSFCPG by electroporation, and transformants were obtained using chloramphenicol resistance as a marker. After confirmation of the presence of the plasmids, the strain with the plasmid for ybjL amplification was designated SC17sucA/RSFCPG+pSTV-PanybjL, and the strain with pSTV29 was designated SC17sucA/RSFCPG+pSTV28.

[0241] Then, SC17sucA/RSFCPG+pSTV-PanybjL and the control strain SC17sucA/RSFCPG+pSTV28 were cultured to examine the L-glutamic acid producing ability of the strains. The medium had the following composition:

[0242] Composition of Culture Medium:

TABLE-US-00001 section A: Sucrose 30 g/L MgSO.sub.4.cndot.7H.sub.2O 0.5 g/L [[Group B: KH.sub.2PO.sub.4 2.0 g/L Yeast Extract 2.0 g/L FeSO.sub.4.cndot.7H.sub.2O 0.02 g/L MnSO.sub.4.cndot.5H.sub.2O 0.02 g/L L-Lysine hydrochloride 0.2 g/L DL-Methionine 0.2 g/L Diaminopimelic acid 0.2 g/L (adjusted to pH 7.0 with KOH) Group C: CaCO.sub.3 20 g/L

[0243] The ingredients of groups A and B were sterilized at 115.degree. C. for 10 minutes by autoclaving, and the ingredient of group C was sterilized at 180.degree. C. for 3 hours with dry heat. Then, the ingredients of the three groups were mixed, and 12.5 mg/L of tetracycline hydrochloride and 25 mg/L of chloramphenicol were added to the mixture.

[0244] SC17sucA/RSFCPG+pSTV29 and SC17sucA/RSFCPG+pSTV-PanybjL were each precultured on a medium obtained by adding ingredients of the minimal medium (medium containing 5 g of glucose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride and 6 g of disodium phosphate in 1 L of pure water), 25 mg/L of chloramphenicol and 12.5 mg/L tetracycline to the L medium (medium containing 10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH 7.0), and inoculated into an appropriate amount to 5 ml of the aforementioned medium in a test tube.

[0245] The cells were cultured for 17.5 hours, and then cell density, L-glutamic acid concentration, and the amount of residual saccharide in the culture medium were measured. The cell density was examined by measuring turbidity at 620 nm of the medium diluted 51 times using a spectrophotometer (U-2000A, Hitachi). The L-glutamic acid concentration was measured in culture supernatant appropriately diluted with water by using Biotech Analyzer (AS-210, Sakera SI). The results are shown in Table 1. L-Glutamic acid accumulation was increased by about 3 g/L, and the yield based on saccharide increased by about 9% in the ybjL-amplified strain, SC17sucA/RSFPCPG+pSTV-PanybjL, as compared to the control strain, SC17sucA/RSFCPG+pSTV28.

TABLE-US-00002 TABLE 1 OD 620 nm L-Glutamic Yield based on (.times.51) acid (g/L) saccharide (%) SC17sucA/ 0.385 15.0 44.5 RSFCPG + pSTV28 SC17sucA/ 0.280 18.0 53.7 RSFCPG + pSTV-PanybjL

Example 3

Effect of Amplification of the ybjL Gene Derived from Escherichia coli

[0246] Then, the ybjL gene from Escherichia coli was introduced into the Pantoea ananatis SC17sucA/RSFCPG strain, and the effect of this amplification on glutamic acid production was examined.

[0247] PCR was performed using the oligonucleotides shown in SEQ ID NOS: 11 and 12 prepared on the basis of the sequence of the ybjL of Escherichia con registered at GeneBank as AP009048 (SEQ ID NO: 3) and the chromosome from the Escherichia coli W3110 strain (ATCC 27325) as the template to obtain a fragment of about 1.7 kb containing the ybjL gene. This fragment was treated with SphI and KpnI, and ligated with pSTV28 (Takara Bio) at the corresponding site. The plasmid for amplification of ybjL of Escherichia coli was designated as pSTV-EcoybjL.

[0248] The plasmid pSTV-EcoybjL for ybjL amplification was introduced into the aforementioned SC17sucA/RSFCPG strain by electroporation, and a transformant was obtained using chloramphenicol resistance as a marker. The obtained Escherichia coli ybjL gene-amplified strain was designated as SC17sucA/RSFCPG+pSTV-EcoybjL.

[0249] Then, SC17sucA/RSFCPG+pSTV-EcoybjL and the control strain, SC17sucA/RSFCPG+pSTV28, were cultured to examine their L-glutamic acid producing ability. The medium had the following composition.

[0250] Composition of Culture Medium:

TABLE-US-00003 Group A: Sucrose 30 g/L MgSO.sub.4.cndot.7H.sub.2O 0.5 g/L Group B: KH.sub.2PO.sub.4 2.0 g/L Yeast Extract 2.0 g/L FeSO.sub.4.cndot.7H.sub.2O 0.02 g/L MnSO.sub.4.cndot.5H.sub.2O 0.02 g/L L-Lysine hydrochloride 0.2 g/L DL-Methionine 0.2 g/L Diaminopimelic acid 0.2 g/L (adjusted to pH 7.0 with KOH) Group C: CaCO.sub.3 20 g/L

[0251] The ingredients of groups A and B were sterilized at 115.degree. C. for 10 minutes by autoclaving, and the ingredient of group C was sterilized at 180.degree. C. for 3 hours with dry heat. Then, the ingredients of the three groups were mixed, and 12.5 mg/L of tetracycline hydrochloride and 25 mg/L of chloramphenicol were added to the mixture.

[0252] SC17sucA/RSFCPG+pSTV28 and SC17sucA/RSFCPG+pSTV-EcoybjL were each precultured on a medium obtained by adding ingredients of the minimal medium (5 g of glucose, 2 mM magnesium sulfate, 3 g of monopotassium phosphate, 0.5 g of sodium chloride, 1 g of ammonium chloride and 6 g of disodium phosphate in 1 L of pure water), 25 mg/L of chloramphenicol, and 12.5 mg/L tetracycline to the L medium (10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH 7.0), and inoculated in an appropriate amount to 5 ml of the aforementioned medium in a test tube.

[0253] The cells were cultured for 17.5 hours, and then cell density and L-glutamic acid concentration in the culture medium were measured in the same manner as shown in Example 2. The results are shown in Table 2. L-Glutamic acid accumulation was increased by about 2 g/L, and the yield based on saccharide increased by about 7% in the ybjL-amplified strain, SC17sucA/RSFCPG+pSTV-EcoybjL, as compared to the control strain, SC17sucA/RSFCPG+pSTV28.

TABLE-US-00004 TABLE 2 OD 620 nm L-Glutamic Yield based on (.times.51) acid (g/L) saccharide (%) SC17sucA/ 0.385 15.0 44.5 RSFCPG + pSTV28 SC17sucA/ 0.348 17.2 51.2 RSFCPG + pSTV-EcoybjL

Example 4

Effect of Amplification of ybjL Gene on L-Amino Acid Production in Escherichia coli

[0254] Then, the ybjL gene derived from Pantoea ananatis and the ybjL gene derived from Escherichia coli were each introduced into Escherichia coli, and the effect of the amplification was examined.

[0255] The aforementioned vector for amplification of the ybjL gene derived from Pantoea ananatis, pSTV-PanybjL, vector for amplification of ybjL gene derived from Escherichia coli, pSTV-EcoybjL, and the control plasmid pSTV28 were each introduced into an Escherichia coli wild-type strain, W3110, by electroporation to obtain transformants resistant to chloramphenicol. The strain in which ybjL derived from Pantoea ananatis had been amplified was designated W3110/pSTV-PanybjL, the strain in which ybjL derived from Escherichia coli had been amplified was designated W3110/pSTV-EcoybjL, and the control strain with pSTV28 was designated W3110/pSTV28.

[0256] Then, the ability to produce L-glutamic acid of the ybjL-amplified strains, W3110/pSTV-PanybjL and W3110/pSTV-EcoybjL, and the control strain W3110/pSTV28 were examined. W3110/pSTV-PanybjL, W3110/pSTV-EcoybjL, and W3110/pSTV28 were each precultured on L medium (10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH 7.0) containing chloramphenicol, and one loop of cells were inoculated by using a 1-mL volume loop (Nunc) to 5 mL of a medium having the following composition in a test tube, and cultured at 37.degree. C. for 11.5 hours with shaking. Cell density and L-glutamic acid concentration in the culture medium were measured in the same manners as those in Example 2. The results are shown in Table 3.

[0257] Composition of Culture Medium:

TABLE-US-00005 Group A: Glucose 30 g/L MgSO.sub.4.cndot.7H.sub.2O 0.5 g/L Group B: (NH.sub.4).sub.2SO.sub.4 20 g/L KH.sub.2PO.sub.4 2.0 g/L Yeast Extract 2.0 g/L FeSO.sub.4.cndot.7H.sub.2O 20 mg/L MnSO.sub.4.cndot.5H.sub.2O 20 mg/L (adjusted to pH 7.0 with KOH) Group C: Calcium carbonate 20 g/L

[0258] The ingredients of groups A and B were sterilized at 115.degree. C. for 10 minutes by autoclaving, and the ingredient of group C was sterilized at 180.degree. C. for 3 hours with dry heat. Then, the ingredients of the three groups were mixed, and 25 mg/L of chloramphenicol was added to the mixture.

TABLE-US-00006 TABLE 3 OD 620 nm L-Glutamic Yield based on (.times.51) acid (g/L) saccharide (%) W3110/pSTV28 0.341 1.2 6.5 W3110/pSTV-PanybjL 0.303 5.7 28.8 W3110/pSTV-EcoybjL 0.310 4.8 25.2

[0259] The L-glutamic acid producing ability was markedly improved in the Escherichia coli W3110/pSTV-PanybjL strain, which is a Pantoea ananatis in which the ybjL gene has been amplified, and the Escherichia coli W3110/pSTV-EcoybjL strain, which is an Escherichia coli in which the ybjL gene has been amplified, as compared to the control strain W3110/pSTV28 strain.

[0260] Then, the ability of the ybjL-amplified strain to produce other amino acids was examined. W3110/pSTV-PanybjL, and the control strain W3110/pSTV28 were precultured in L medium (10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH 7.0) containing chloramphenicol, and one loop of cells were inoculated by using a 5-mL volume loop (Nunc) to 5 mL of a medium having the following composition in a test tube, and cultured at 37.degree. C. for 7 hours with shaking. Cell densities and L-amino acid concentrations in the culture medium were measured in the same manner as in Example 2. The results are shown in Table 4.

[0261] Composition of Culture Medium:

TABLE-US-00007 Group A: Glucose 40 g/L MgSO.sub.4.cndot.7H.sub.2O 0.5 g/L Group B: (NH.sub.4).sub.2SO.sub.4 20 g/L KH.sub.2PO.sub.4 2.0 g/L Yeast Extract 2.0 g/L FeSO.sub.4.cndot.7H.sub.2O 20 mg/L MnSO.sub.4.cndot.5H.sub.2O 20 mg/L (adjusted to pH 7.0 with KOH) Group C: Calcium carbonate 30 g/L

[0262] The ingredients of groups A and B were sterilized at 115.degree. C. for 10 minutes by autoclaving, and the ingredient of group C was sterilized at 180.degree. C. for 3 hours with dry heat. Then, the ingredients of the three groups were mixed, and 25 mg/L of chloramphenicol was added to the mixture.

TABLE-US-00008 TABLE 4 W3110/ W3110/pSTV- MS pSTV28 PanybjL medium* Asp 26.69 62.35 40.21 Thr 0.00 0.00 36.23 Ser 0.00 0.00 56.03 Glu 1258.75 3702.74 127.50 Gly 0.00 0.00 27.28 Ala 5.49 9.79 77.61 (Cys).sub.2 16.49 13.72 10.13 Val 3.30 8.02 59.32 Met 0.00 0.00 85.06 Ile 0.00 0.00 50.51 Leu 0.00 0.00 80.02 Tyr 48.20 0.00 38.80 Phe 2.88 4.47 70.49 Lys 8.07 8.00 50.50 His 0.00 0.00 20.95 Arg 0.00 0.00 35.80 OD 620 nm (.times.51) 0.245 0.197 -- Residual glucose (g/L) 28.9 28.3 40.0 *Blank inoculated with no cells

[0263] Not only did the amount of L-glutamic acid increase, but the L-aspartic acid amount also increased in the Escherichia coli W3110/pSTV-PanybjL, which is a ybjL gene-amplified strain, as compared to the vector-introduced control strain, W3110/pSTV28 strain.

Example 5

Effect of Amplification of the ybjL Gene on L-Glutamic Acid Production in Klebsiella planticola

[0264] The ybjL gene derived from Pantoea ananatis was introduced into Klebsiella planticola, and the effect of the amplification of the gene was examined.

[0265] The aforementioned vector for amplification of ybjL gene derived from Pantoea ananatis, pSTV-PanybjL, and the control plasmid pSTV28 were each introduced into a Klebsiella planticola L-glutamic acid-producing strain, AJ13410 (Japanese Patent Application No. 11-68324), by electroporation to obtain transformants which are resistant to chloramphenicol. The strain in which ybjL derived from Pantoea ananatis had been amplified was designated as AJ13410/pSTV-PanybjL, and the control strain with pSTV28 was designated as AJ13410/pSTV28.

[0266] Then, the ability to produce L-glutamic acid of the ybjL-amplified strain, AJ13410/pSTV-PanybjL, and the control strain AJ13410/pSTV28 were examined. AJ13410/pSTV-PanybjL and AJ13410/pSTV28 were precultured on L medium (10 g of Bacto tryptone, 5 g of yeast extract, 5 g of NaCl and 15 g of agar in 1 L of pure water, pH 7.0) containing chloramphenicol, and one loop of cells were inoculated by using a 1-mL volume loop (Nunc) to 5 mL of a medium having the following composition in a test tube, and cultured at 37.degree. C. for 17 hours with shaking. Cell density and L-glutamic acid concentration in the culture medium were measured in the same manner as in Example 2. The results are shown in Table 5.

[0267] Composition of Culture Medium:

TABLE-US-00009 Group A: Sucrose 30 g/L MgSO.sub.4.cndot.7H.sub.2O 0.5 g/L Group B: KH.sub.2PO.sub.4 2.0 g/L Yeast Extract 2.0 g/L FeSO.sub.4.cndot.7H.sub.2O 0.02 g/L MnSO.sub.4.cndot.5H.sub.2O 0.02 g/L L-Lysine hydrochloride 0.2 g/L DL-Methionine 0.2 g/L Diaminopimelic acid 0.2 g/L (adjusted to pH 7.0 with KOH) Group C: CaCO.sub.3 20 g/L

[0268] The ingredients of groups A and B were sterilized at 115.degree. C. for 10 minutes by autoclaving, and the ingredient of group C was sterilized at 180.degree. C. for 3 hours with dry heat. Then, the ingredients of the three groups were mixed, and 25 mg/L of chloramphenicol was added to the mixture.

TABLE-US-00010 TABLE 5 OD 620 nm L-Glutamic Yield based on (.times.51) acid (g/L) saccharide (%) AJ13410/ 0.236 5.3 23.3 pSTV28 AJ13410/ 0.220 12.1 48.7 pSTV-PanybjL

[0269] L-glutamic acid-producing ability was markedly improved in the Klebsiella planticola AJ13410/pSTV-PanybjL, which is a Pantoea ananatis ybjL gene-amplified strain, as compared to the control strain, AJ13410/pSTV28.

Reference Example 4

Construction of an Escherichia coli Strain Deficient in the L,D-Lactate Dehydrogenase Gene

[0270] 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 .lamda. 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 designed in the 5' end and a part of an antibiotic resistance gene is designed in the 3' end. Furthermore, by using the .lamda. phage excision system in combination, the antibiotic resistance gene which is integrated into the gene-disrupted strain can be removed.

[0271] <1-1> Construction of a Strain deficient in the ldhA Gene Coding for D-Lactate Dehydrogenase

[0272] 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 and sequences corresponding to both ends of attL and attR of .lamda. phage at the 3' end as primers and plasmid pMW118-attL-Cm-attR as the template. pMW118-attL-Cm-attR is a plasmid obtained by inserting attL and attR genes, which are the attachment sites for .lamda. phage, and the cat gene, which is an antibiotic 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. 13 and 14. 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 the deletion of the ldhA gene was confirmed by PCR. PCR was performed by using the synthetic oligonucleotides shown in SEQ ID NOS: 15 and 16 as primers. Whereas the PCR product obtained for the parent strain was about 1.2 kb, the deficient strain was about 1.9 kb.

[0273] To eliminate the att-cat gene introduced into the ldhA gene, the strain was transformed with helper plasmid pMW-intxis-ts, and an ampicillin resistant strain was selected. The pMW-intxis-ts contains the .lamda. phage integrase (Int) gene and excisionase (Xis) gene and shows temperature-sensitive replication. Then, the strain in which att-cat and pMW-intxis-ts had been 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: 15 and 16 as primers. Whereas the PCR product obtained for the strain in which att-cat remained was 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 MG1655.DELTA.ldhA strain.

[0274] <1-2> Construction of a Strain Deficient in the lldD Gene Coding for L-Lactate Dehydrogenase

[0275] A strain was constructed in the same manner as that of the construction of the ldhA gene-deficient strain. PCR was performed using synthetic oligonucleotides having sequences corresponding to parts of the lldD gene in the 5' end and sequences corresponding to both ends of attL and attR of .lamda. phage in the 3' end 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. 17 and 18. The amplified PCR product was purified on an agarose gel and introduced into the Escherichia coli MG1655.DELTA.ldhA strain containing plasmid pKD46 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: 19 and 20 as primers. Whereas the PCR product obtained for the parent strain was about 1.4 kb, a band of about 1.9 kb was observed for the deficient strain.

[0276] To eliminate the att-cat gene introduced into the lldD gene, the strain was transformed with helper plasmid pMW-intxis-ts, and an ampicillin resistant strain was selected. Then, the strain from which att-cat and pMW-intxis-ts had been eliminated was obtained and 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: 19 and 20 as primers. Whereas the PCR product obtained for the strain in which att-cat remained was 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 MG1655.DELTA.ldhA.DELTA.lldD strain.

Example 6

Construction of ybjL Gene-Enhanced Strain of Succinic Acid-Producing Bacterium

[0277] <6-1> Construction of Plasmid for Gene Amplification

[0278] In order to amplify the ybjL gene, plasmid pMW219::Pthr was used. This plasmid corresponds to the vector pMW219 (Nippon Gene) having the promoter region of the threonine operon (thrLABC) in the genome of the Escherichia coli shown in SEQ ID NO: 21 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.

[0279] <6-2> Construction of Plasmid for Enhancing ybjL

[0280] PCR was performed by using the synthetic oligonucleotide having a BamHI site shown in SEQ ID NO: 22 as a 5' primer, and the synthetic oligonucleotide having a BamHI site shown in SEQ ID NO: 23 as a 3' primer, which were prepared on the basis of the nucleotide sequence of the ybjL gene in the genome sequence of Escherichia coli (GenBank Accession No. U00096), and the genomic DNA of Escherichia coli MG1655 strain as the template. The product was treated with the restriction enzyme BamHI to obtain a gene fragment containing the ybjL gene. The PCR product was purified and ligated with the vector pMW219::Pthr which had been treated with BamHI to construct plasmid pMW219::Pthr::ybjL for ybjL amplification.

[0281] <6-3> Production of ybjL-Amplified Strain

[0282] pMW219::Pthr::ybjL obtained in <6-2> described above and pMW219 were used to transform the Escherichia coli MG1655.DELTA.ldhA.DELTA.lldD strain by the electric pulse method, and the transformants were applied to the LB agar medium containing 25 .mu.g/ml of kanamycin, and cultured at 37.degree. 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 MG1655.DELTA.ldhA.DELTA.lldD/pMW219::Pthr:ybjL and MG1655.DELTA.ldhA.DELTA.lldD/pMW219, respectively. The Enterobacter aerogenes AJ110637 strain was also transformed with pMW219::Pthr::ybjL and pMW219 by the electric pulse method, and the transformants were applied to the LB agar medium containing 50 .mu.g/ml of kanamycin, and cultured at 37.degree. 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::ybjL and AJ110637/pMW219, respectively.

Example 7

Effect of ybjL Amplification in Succinic Acid-Producing Strain of Escherichia bacterium

[0283] MG1655.DELTA.ldhA.DELTA.lldD/pMW219::Pthr:ybjL and MG1655.DELTA.ldhA.DELTA.lldD/pMW219 were each uniformly applied to an LB plate containing 25 .mu.g/ml of kanamycin, and cultured at 37.degree. C. for 16 hours. Then, each plate was incubated at 37.degree. C. for 16 hours under an anaerobic condition by using Anaeropack (Mitsubishi Gas Chemical). The 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 gland a production medium (10 g/l of glucose, 10 g/l 2Na malate, 45.88 g/l of TES, 6 g/l of Na.sub.2HPO.sub.4, 3 g/l of KH.sub.2PO.sub.4, 1 g/l of NH.sub.4Cl, adjusted to pH 7.3 with KOH and filtered) in a volume of 1.3 ml in which the 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.degree. C. for 10 hours by using a stirrer for micro tubes. After the culture, the amount of succinic acid which had 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.degree. 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 .mu.M EDTA, and succinic acid was quantified by measuring electric conductivity with CDD-10AD (Shimadzu).

[0284] The amounts of succinic acid which had accumulated after 10 hours are shown in Table 6, and the change in succinic acid accumulation is shown in FIG. 4.

[0285] Succinic acid accumulation was markedly increased in the ybjL gene-amplified strain MG1655.DELTA.ldhA.DELTA.lldD/pMW219::Pthr::ybjL as compared to the control strain MG1655.DELTA.ldhA.DELTA.lldD/pMW219.

TABLE-US-00011 TABLE 6 Effect of ybjL amplification in succinic acid-producing strain, MG1655.DELTA.ldhA.DELTA.lldD Strain Succinate (g/L) MG1655.DELTA.ldhA.DELTA.lldD/pMW219 1.91 (.+-.0.13) MG1655.DELTA.ldhA.DELTA.lldD/pMW219::Pthr::ybjL 2.34 (.+-.0.01)

Example 8

Effect of ybjL Amplification in a Succinic Acid-Producing Strain of Enterobacter Bacterium

[0286] AJ110637/pMW219::Pthr::ybjL and AJ110637/pMW219 were each uniformly applied to an LB plate containing 50 .mu.g/ml of kanamycin, and cultured at 37.degree. C. for 16 hours. Then, each plate was incubated at 37.degree. C. for 16 hours under an anaerobic condition by using Anaeropack (Mitsubishi Gas Chemical). The 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 1000 and a production medium (10 g/l of glucose, 10 g/l 2Na malate, 45.88 g/l of TES, 6 g/l of Na.sub.2HPO.sub.4, 3 g/l of KH.sub.2PO.sub.4, 1 g/l of NH.sub.4Cl, adjusted to pH 7.3 with KOH and filtered) in a volume of 1.3 ml in which the 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.degree. C. for 6 hours by using a stirrer for micro tubes. After the culture, the amount of succinic acid which had 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.degree. 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 .mu.M EDTA, and succinic acid was quantified by measuring electric conductivity with CDD-10AD (Shimadzu).

[0287] The amounts of succinic acid which had accumulated after 6 hours are shown in Table 7, and change of succinic acid accumulation is shown in FIG. 5.

[0288] Succinic acid accumulation was markedly increased in the ybjL gene-amplified strain AJ110637/pMW219::Pthr::ybjL as compared to the control strain AJ110637/pMW219.

TABLE-US-00012 TABLE 7 Effect of ybjL amplification in AJ110637 strain Strain Succinate (g/L) AJ110637/pMW219 1.81 (.+-.0.04) AJ110637/pMW219::Pthr::ybjL 2.11 (.+-.0.02)

Example 9

<9-1> Construction of an adhE-Deficient Strain of Enterobacter aerogenes AJ110637

[0289] 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 the generation of ethanol.

[0290] A gene fragment for deleting 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: 72 and 73 as primers. pMW118-attL-Tc-attR is a plasmid obtained by inserting attL and attR genes, which are the attachment sites of .lamda. 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. By PCR described above, a gene fragment containing a tetracycline resistance gene, attL and attR sites of .lamda. phage at the both ends of the resistance gene, and 60 by upstream sequence and 59 by downstream sequence of the adhE gene added to the outer ends of the .lamda. phage sequences was amplified. This fragment was purified by using Wizard PCR Prep DNA Purification System (Promega).

[0291] Then, the Enterobacter aerogenes AJ110637 strain was transformed with RSF-Red-TER to obtain the Enterobacter aerogenes AJ110637/RSF-Red-TER strain. This strain was cultured overnight in LB medium containing 40 .mu.g/mL of chloramphenicol, the culture medium was inoculated in a 1/100 volume to 50 mL of the LB medium containing 40 .mu.g/mL of chloramphenicol and 0.4 mM isopropyl-.beta.-D-thiogalactopyranoside, and culture was performed at 31.degree. 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 .mu.F and resistance of 200.OMEGA.. Ice-cooled LB medium was added to the cell suspension, and culture was performed at 31.degree. C. for 2 hours with shaking. Then, the culture was applied to a LB plate containing 25 .mu.g/mL of tetracycline. The colonies that appeared were purified with the same plate, and then it was confirmed by PCR that the adhE gene had been replaced with the tetracycline resistance gene.

<9-2> Construction of Pyruvate Carboxylase Gene-Enhanced Strain of AJ110637.DELTA.adhE Strain

[0292] The ability to produce succinic acid was imparted to the Enterobacter aerogenes AJ110637.DELTA.adhE strain by amplifying pyc coding for pyruvate carboxylase derived from Corynebacterium glutamicum.

[0293] In order to express pyc derived from Corynebacterium glutamicum in the AJ110637.DELTA.adhE strain, it was attempted to obtain a threonine operon promoter fragment of the Escherichia coli MG1655 strain. The total genomic sequence of Escherichia coli (Escherichia coli K-12 strain) has already been elucidated (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 SacI site shown in SEQ ID NO: 75 as a 5' primer, the synthetic oligonucleotide shown in SEQ ID NO: 76 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: 77).

[0294] 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: 78 as a 5' primer, the synthetic oligonucleotide having an SacI site shown in SEQ ID NO: 79 as a 3' primer, and the genomic DNA of the Corynebacterium glutamicum 2256 strain (ATCC 13869) as a template to obtain a pyc gene fragment (B) (SEQ ID NO: 80).

[0295] PCR was performed by using the fragments (A) and (B) as templates, the primers of SEQ ID NOS: 75 and 79 to obtain a gene fragment (C) including the fragments (A) and (B) ligated to each other. This gene fragment (C) was treated with the restriction enzyme SacI, and purified, and the product was ligated with the plasmid vector pSTV28 (Takara Bio) which had been digested with the restriction enzyme Sad to construct a plasmid pSTV28::Pthr::pyc for pyc amplification.

[0296] The plasmid pSTV28::Pthr::pyc for pyc amplification was introduced into the aforementioned Enterobacter aerogenes AJ110637.DELTA.adhE strain by electroporation to obtain a transformant which are resistant to tetracycline and chloramphenicol. This pyc-amplified strain of Enterobacter aerogenes AJ110637.DELTA.adhE was designated as AJ110637.DELTA.adhE/pSTV28::Pthr::pyc.

<9-3> Construction of Enterobacter aerogenes ybjL Gene-Enhanced Strain of AJ110637.DELTA.adhE/pSTV28::Pthr::pyc

[0297] 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: 81 as a 5' primer, the synthetic oligonucleotide shown in SEQ ID NO: 82 as a 3' primer, and the genomic DNA of Escherichia coli MG1655 strain (ATCC 47076, ATCC 700926) as a template to obtain a threonine operon promoter fragment (A) (SEQ ID NO: 83).

[0298] Furthermore, in order to clone the ybjL gene of the Enterobacter aerogenes AJ110637 strain, PCR was performed by using the synthetic oligonucleotide shown in SEQ ID NO: 84 as a 5' primer, the synthetic oligonucleotide shown in SEQ ID NO: 85 as a 3' primer, and the genomic DNA of the Enterobacter aerogenes AJ110637 strain as the template to obtain a ybjL gene fragment (B) (SEQ ID NO: 86).

[0299] PCR was performed by using the fragments (A) and (B) as templates, and the primers of SEQ ID NOS: 81 and 85 to obtain a gene fragment (C) with 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-ybjL for ybjL amplification.

[0300] The aforementioned vector pMW218::Pthr::Ent-ybjL for amplification of the ybjL gene derived from the Enterobacter aerogenes AJ110637 strain, and the control plasmid pMW218 were each introduced into the Enterobacter aerogenes AJ110637.DELTA.adhE/pSTV28::Pthr::pyc strain by electroporation to obtain transformants which are resistant to tetracycline, chloramphenicol and kanamycin. The ybjL-amplified strain derived from the Enterobacter aerogenes AJ110637.DELTA.adhE/pSTV28::Pthr::pyc strain was designated as AJ110637.DELTA.adhE/pSTV28::Pthr::pyc/pMW218::Pthr::Ent-ybjL, and the control strain as with pMW218 was designated as AJ110637.DELTA.adhE/pSTV28::Pthr:pyc/pMW218.

<9-4> Effect of Amplification of ybjL Derived from Enterobacter aerogenes in Enterobacter Bacterium

[0301] AJ110637.DELTA.adhE/pSTV28::Pthr::pyc/pMW218::Pthr::Ent-ybjL, and AJ110637.DELTA.adhE/pSTV28::Pthr:pyc/pMW218 were each uniformly applied to an LB plate containing 50 .mu.g/ml of kanamycin, 25 .mu.g/ml of tetracycline, and 40 .mu.g/ml of chloramphenicol, and cultured at 31.5.degree. 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) in a test tube, and cultured at 31.5.degree. 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.degree. C. for 24 hours with shaking. After the culture, the amount of succinic acid which had 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.degree. 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 .mu.M EDTA, and succinic acid was quantified by measuring electric conductivity with CDD-10AD (Shimadzu).

[0302] The amounts of succinic acid which had accumulated after 24 hours are shown in Table 8.

[0303] Succinic acid accumulation and succinic acid yield based on consumed glucose were markedly increased in the ybjL gene-amplified strain

[0304] 110637.DELTA.adhE/pSTV28::Pthr::pyc/pMW218::Pthr::Ent-ybjL as compared to control AJ110637.DELTA.adhE/pSTV28::Pthr::pyc/pMW218.

TABLE-US-00013 TABLE 8 AJ110637.DELTA.adhE/ AJ110637.DELTA.adhE/ pSTV28::Pthr::pyc/ pSTV28::Pthr::pyc/ pMW218 pMW218::Pthr::Ent-ybjL Consumed glucose 9.53 (.+-.1.85) 9.10 (.+-.0.66) amount (g/L) OD (600 nm) 8.90 (.+-.0.18) 8.72 (.+-.0.95) Succinic acid 3.40 (.+-.0.56) 6.37 (.+-.0.51) accumulation (g/L) Succinic acid yield 35.80 (.+-.1.82) 69.70 (.+-.1.79) based on consumed glucose (%)

[0305] Explanation of Sequence Listing

[0306] SEQ ID NO: 1: ybjL gene of Pantoea ananatis

[0307] SEQ ID NO: 2: YbjL of Pantoea ananatis

[0308] SEQ ID NO: 3: ybjL gene of Escherichia coli

[0309] SEQ ID NO: 4: YbjL of Escherichia coli

[0310] SEQ ID NO: 5: Consensus sequence of YbjL of Pantoea ananatis and Escherichia coli

[0311] SEQ ID NO: 6: Primer for ams gene disruption

[0312] SEQ ID NO: 7: Primer for ams gene disruption

[0313] SEQ ID NO: 8: Primer for amplification of ybjL of P. ananatis

[0314] SEQ ID NO: 9: Primer for amplification of ybjL of P. ananatis

[0315] SEQ ID NO: 10: Sequence of RSFCPG plasmid

[0316] SEQ ID NO: 11: Primer for amplification of ybjL of E. coli W3110

[0317] SEQ ID NO: 12: Primer for amplification of ybjL of E. coli W3110

[0318] SEQ ID NO: 13: Primer for deletion of ldhA

[0319] SEQ ID NO: 14: Primer for deletion of ldhA

[0320] SEQ ID NO: 15: Primer for confirming deletion of ldhA

[0321] SEQ ID NO: 16: Primer for confirming deletion of ldhA

[0322] SEQ ID NO: 17: Primer for deletion of lldD

[0323] SEQ ID NO: 18: Primer for deletion of lldD

[0324] SEQ ID NO: 19: Primer for confirming deletion of lldD

[0325] SEQ ID NO: 20: Primer for confirming deletion of lldD

[0326] SEQ ID NO: 21: Threonine promoter sequence

[0327] SEQ ID NO: 22: Primer for amplification of ybjL of E. coli MG1655

[0328] SEQ ID NO: 23: Primer for amplification of ybjL of E. coli MG1655

[0329] SEQ ID NO: 24: ybjL gene of Salmonella typhimurium

[0330] SEQ ID NO: 25: YbjL of Salmonella typhimurium

[0331] SEQ ID NO: 26: ybjL gene of Yersinia pestis

[0332] SEQ ID NO: 27: YbjL of Yersinia pestis

[0333] SEQ ID NO: 28: ybjL gene of Erwinia carotovora

[0334] SEQ ID NO: 29: YbjL of Erwinia carotovora

[0335] SEQ ID NO: 30: ybjL gene of Vibrio cholerae

[0336] SEQ ID NO: 31: YbjL of Vibrio cholerae

[0337] SEQ ID NO: 32: ybjL gene of Aeromonas hydrophia

[0338] SEQ ID NO: 33: YbjL of Aeromonas hydrophia

[0339] SEQ ID NO: 34: ybjL gene of Photobacterium profundum

[0340] SEQ ID NO: 35: YbjL of Photobacterium profundum

[0341] SEQ ID NO: 36: ldhA gene of Escherichia coli

[0342] SEQ ID NO: 37: LdhA of Escherichia coli

[0343] SEQ ID NO: 38: lldD gene of Escherichia coli

[0344] SEQ ID NO: 39: LldD of Escherichia coli

[0345] SEQ ID NO: 40: Nucleotide sequence of hisD gene of Pantoea ananatis

[0346] SEQ ID NO: 41: Primer for amplification of fragment for incorporation of Km.sup.r gene into hisD gene

[0347] SEQ ID NO: 42: Primer for amplification of fragment for incorporation of Km.sup.r gene into hisD gene

[0348] SEQ ID NO: 43: Primer for amplification of cat gene

[0349] SEQ ID NO: 44: Primer for amplification of cat gene

[0350] SEQ ID NO: 45: Primer for amplification of sacB gene

[0351] SEQ ID NO: 46: Primer for amplification of sacB gene

[0352] SEQ ID NO: 47: Primer for amplification of DNA fragment containing PlacUV5 promoter

[0353] SEQ ID NO: 48: Primer for amplification of DNA fragment containing PlacUV5 promoter

[0354] SEQ ID NO: 49: Primer for amplification of DNA fragment containing .lamda.Red.alpha..beta..gamma. gene and tL3

[0355] SEQ ID NO: 50: Primer for amplification of DNA fragment containing .lamda.Red.alpha..beta..gamma. gene and tL3

[0356] SEQ ID NO: 51: Primer for amplification of DNA fragment containing PlacUV5 promoter and TrrnB

[0357] SEQ ID NO: 52: Primer for amplification of DNA fragment containing PlacUV5 promoter and TrrnB

[0358] SEQ ID NO: 53: Primer for amplification of attL

[0359] SEQ ID NO: 54: Primer for amplification of attL

[0360] SEQ ID NO: 55: Nucleotide sequence of attL

[0361] SEQ ID NO: 56: Primer for amplification of attR

[0362] SEQ ID NO: 57: Primer for amplification of attR

[0363] SEQ ID NO: 58: Nucleotide sequence of attR

[0364] SEQ ID NO: 59: Primer for amplification of DNA fragment containing bla gene

[0365] SEQ ID NO: 60: Primer for amplification of DNA fragment containing bla gene

[0366] SEQ ID NO: 61: Primer for amplification of DNA fragment containing ter_rrnB

[0367] SEQ ID NO: 62: Primer for amplification of DNA fragment containing ter_rrnB

[0368] SEQ ID NO: 63: Nucleotide sequence of the DNA fragment containing ter_thrL terminator

[0369] SEQ ID NO: 64: Primer for amplification of DNA fragment containing ter_thrL terminator

[0370] SEQ ID NO: 65: Primer for amplification of DNA fragment containing ter_thrL terminator

[0371] SEQ ID NO: 66: ams operon of Pantoea ananatis

[0372] SEQ ID NO: 67: AmsH of Pantoea ananatis

[0373] SEQ ID NO: 68: AmsI of Pantoea ananatis

[0374] SEQ ID NO: 69: AmsA of Pantoea ananatis

[0375] SEQ ID NO: 70: AmsC of Pantoea ananatis

[0376] SEQ ID NO: 71: AmsB of Pantoea ananatis

[0377] SEQ ID NO: 72: Nucleotide sequence of primer for deletion of adhE

[0378] SEQ ID NO: 73: Nucleotide sequence of primer for deletion of adhE

[0379] SEQ ID NO: 74: Nucleotide sequence of adhE of Enterobacter aerogenes AJ110637 partial sequence)

[0380] SEQ ID NO: 75: Primer for amplification of threonine promoter

[0381] SEQ ID NO: 76: Primer for amplification of threonine promoter

[0382] SEQ ID NO: 77: Threonine promoter gene fragment

[0383] SEQ ID NO: 78: Primer for amplification of pyruvate carboxylase gene

[0384] SEQ ID NO: 79: Primer for amplification of pyruvate carboxylase gene

[0385] SEQ ID NO: 80: Pyruvate carboxylase gene fragment

[0386] SEQ ID NO: 81: Primer for amplification of threonine promoter

[0387] SEQ ID NO: 82: Primer for amplification of threonine promoter

[0388] SEQ ID NO: 83: Threonine promoter gene fragment

[0389] SEQ ID NO: 84: Nucleotide sequence of primer for amplification of ybjL of Enterobacter aerogenes AJ110637

[0390] SEQ ID NO: 85: Nucleotide sequence of primer for amplification of ybjL of Enterobacter aerogenes AJ110637

[0391] SEQ ID NO: 86: Nucleotide sequence of ybjL of Enterobacter aerogenes AJ110637

[0392] SEQ ID NO: 87: Amino acid sequence of YbjL of Enterobacter aerogenes AJ110637

[0393] SEQ ID NO: 88: Consensus sequence of YbjL of Escherichia, Pantoea and Enterobacter

INDUSTRIAL APPLICABILITY

[0394] Production efficiency of an acidic substance having a carboxyl group can be improved by using the microorganism of the present invention.

[0395] While the invention has been described in detail with reference to preferred 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.

Sequence CWU 1

1

8812229DNAPantoea ananatisCDS(298)..(1986) 1acgccacctg atacgcaggt tgcccagaaa taataacgct gcggcataac tccccactca 60ttaaaaacat gccaaaactt cgcctttgac cttgccgagc ctggtggaaa taaaaagaaa 120cagtcgtttt gagaacgaat gaaagtcgcc aatggctgag aatgattttt ctgattagaa 180tattggccgc gttacctttt cagacatcag tgatagctgg aggtaatctt acggcctgct 240caagatagcg aaaaacaggc ctctcttcaa taggttacaa gtaaacaaga agttaaa 297atg ttg aat gtt aac atc gca gaa ttg tta aga ggt aat gac att ctg 345Met Leu Asn Val Asn Ile Ala Glu Leu Leu Arg Gly Asn Asp Ile Leu1 5 10 15tta tta ttt ttc gta ctg gca ctg gga ctt tgc ctg gga aaa tta cgt 393Leu Leu Phe Phe Val Leu Ala Leu Gly Leu Cys Leu Gly Lys Leu Arg 20 25 30ttt ggc tcg ata caa ctc gga aat tcc att ggc gta tta gtg gtt tca 441Phe Gly Ser Ile Gln Leu Gly Asn Ser Ile Gly Val Leu Val Val Ser 35 40 45tta tta tta ggt cag caa cac ttc tcg atg aat acg gat gcc ctg agc 489Leu Leu Leu Gly Gln Gln His Phe Ser Met Asn Thr Asp Ala Leu Ser 50 55 60ctg ggt ttc atg ttg ttt att ttc tgc gtc ggc att gaa gca ggc ccc 537Leu Gly Phe Met Leu Phe Ile Phe Cys Val Gly Ile Glu Ala Gly Pro65 70 75 80aac ttt ttt tcc att ttc ttt cgg gat ggc aaa aat tat ttc atg ctc 585Asn Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn Tyr Phe Met Leu 85 90 95gcc att gtg atg gtg gtc agc gcc ctg ctg ctg gcc ctg gga ttc ggc 633Ala Ile Val Met Val Val Ser Ala Leu Leu Leu Ala Leu Gly Phe Gly 100 105 110aag ctt ttt ggc tgg gac atc ggc ctg acc gca ggc gta ctg gcc ggc 681Lys Leu Phe Gly Trp Asp Ile Gly Leu Thr Ala Gly Val Leu Ala Gly 115 120 125tcc atg acg tcc acg ccg gtc ctg gtt ggc gcg ggt gat acg ctg cgc 729Ser Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly Asp Thr Leu Arg 130 135 140cag agc atc agc gat ccc aga cag ctg agc atc atg cag gat caa ctc 777Gln Ser Ile Ser Asp Pro Arg Gln Leu Ser Ile Met Gln Asp Gln Leu145 150 155 160agc ctg ggt tat gcc gta acc tat ctt gtc ggg tta gtc agc ctg att 825Ser Leu Gly Tyr Ala Val Thr Tyr Leu Val Gly Leu Val Ser Leu Ile 165 170 175ttc ggc gca cgc tat ctg ccc cgt tta caa cat cag gac tta ccc acc 873Phe Gly Ala Arg Tyr Leu Pro Arg Leu Gln His Gln Asp Leu Pro Thr 180 185 190tgc gcc cag cag att gcg cgc gaa cgc ggt ctt gac gct gac agc cag 921Cys Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp Ala Asp Ser Gln 195 200 205cgc aaa gtg ttt ctg ccg gtg att cgc gct tac cgc gtt ggc ccg gaa 969Arg Lys Val Phe Leu Pro Val Ile Arg Ala Tyr Arg Val Gly Pro Glu 210 215 220ctg gtg gcg tgg agc ggc ggc aaa aac ttg cgc gag ctc ggt att tac 1017Leu Val Ala Trp Ser Gly Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr225 230 235 240cgg cag acc ggc tgc tat atc gaa cga att cgc cgc aat ggc atc ctt 1065Arg Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu 245 250 255gcc agc ccg gat ggt gat gcg gtg ctg caa ctg ggg gac gat att tcc 1113Ala Ser Pro Asp Gly Asp Ala Val Leu Gln Leu Gly Asp Asp Ile Ser 260 265 270ctg gtg ggc tat ccg gat gcc cac gcg cgc ctg gat gcc agc ttt cgt 1161Leu Val Gly Tyr Pro Asp Ala His Ala Arg Leu Asp Ala Ser Phe Arg 275 280 285aac ggc aaa gaa gtt ttt gac cgc gat ctg ctg gat atg cgg att gtg 1209Asn Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp Met Arg Ile Val 290 295 300acg gaa gag atc gtg gtg aaa aac cac aat gcc gtc aac aaa cgg cta 1257Thr Glu Glu Ile Val Val Lys Asn His Asn Ala Val Asn Lys Arg Leu305 310 315 320agc aaa ctc aac ctt acc gat cac ggc tgc ttt ctc aac cgc gtg att 1305Ser Lys Leu Asn Leu Thr Asp His Gly Cys Phe Leu Asn Arg Val Ile 325 330 335cgc agc cag atc gag atg cct att gat gac aac atc atg ctg aac aaa 1353Arg Ser Gln Ile Glu Met Pro Ile Asp Asp Asn Ile Met Leu Asn Lys 340 345 350ggt gat gtg ttg cag gtc agc ggc gag gcg cga cgg gtg aag agc gta 1401Gly Asp Val Leu Gln Val Ser Gly Glu Ala Arg Arg Val Lys Ser Val 355 360 365gcg gat cgc atc ggc ttt gtg gcg att cac agt cag atg acc gac ctg 1449Ala Asp Arg Ile Gly Phe Val Ala Ile His Ser Gln Met Thr Asp Leu 370 375 380ctg gca ttc tgt gct ttt ttt atc atc ggt ctg atg gtc ggg tta att 1497Leu Ala Phe Cys Ala Phe Phe Ile Ile Gly Leu Met Val Gly Leu Ile385 390 395 400acg ttc cag ttc agc aac ttt agc ttt ggc atc ggt aat gcc gca ggg 1545Thr Phe Gln Phe Ser Asn Phe Ser Phe Gly Ile Gly Asn Ala Ala Gly 405 410 415ttg ttg ttt gcc ggc att atg ctg ggc ttc ctg cgc gct aac cat cct 1593Leu Leu Phe Ala Gly Ile Met Leu Gly Phe Leu Arg Ala Asn His Pro 420 425 430acc ttt ggc tat att ccg cag ggt gcg ctg acg atg gtg aaa gag ttc 1641Thr Phe Gly Tyr Ile Pro Gln Gly Ala Leu Thr Met Val Lys Glu Phe 435 440 445ggg ctg atg gta ttc atg gcg ggc gtc ggg ctg agc gcc ggc agc ggt 1689Gly Leu Met Val Phe Met Ala Gly Val Gly Leu Ser Ala Gly Ser Gly 450 455 460atc gac cac ggt ata tcg ggt aac ggc gca ctg atg ctc ttg tgc ggc 1737Ile Asp His Gly Ile Ser Gly Asn Gly Ala Leu Met Leu Leu Cys Gly465 470 475 480ctg ctg gtc agc ctg tta ccg gtg gtg att tgc tac ctg ttt ggc gcc 1785Leu Leu Val Ser Leu Leu Pro Val Val Ile Cys Tyr Leu Phe Gly Ala 485 490 495tat gtt ctg cgc atg aac cgc gcg ctg ctg ttt ggc gcc att atg ggt 1833Tyr Val Leu Arg Met Asn Arg Ala Leu Leu Phe Gly Ala Ile Met Gly 500 505 510gca cgc acc tgc gcg cca gcc atg gag ata atc agc gac aca gct cgc 1881Ala Arg Thr Cys Ala Pro Ala Met Glu Ile Ile Ser Asp Thr Ala Arg 515 520 525agt aac atc ccg gca ctc ggc tac gcc ggg acc tat gct atc gcg aac 1929Ser Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn 530 535 540gtt ttg cta acg ctg gcg gga aca tta atc gtt att atc tgg ccg tta 1977Val Leu Leu Thr Leu Ala Gly Thr Leu Ile Val Ile Ile Trp Pro Leu545 550 555 560ttg ccc tta taaaattttt ttactgacgt ccagaacttt ttttcccgac 2026Leu Pro Leucacagtctga ataagtgcca ctgcttttct ttgaaatccc caaattgtgg agcccgctgg 2086tctttttccc agcgggtttt tttatgcctt tctcctgccc gccctgcctg gacactaaaa 2146catcattaaa cttaaaccgt tttgcaacct taacgcagat taagaacctt gtgatggact 2206ctctgaccgt ctgcacgcag gat 22292563PRTPantoea ananatis 2Met Leu Asn Val Asn Ile Ala Glu Leu Leu Arg Gly Asn Asp Ile Leu1 5 10 15Leu Leu Phe Phe Val Leu Ala Leu Gly Leu Cys Leu Gly Lys Leu Arg 20 25 30Phe Gly Ser Ile Gln Leu Gly Asn Ser Ile Gly Val Leu Val Val Ser 35 40 45Leu Leu Leu Gly Gln Gln His Phe Ser Met Asn Thr Asp Ala Leu Ser 50 55 60Leu Gly Phe Met Leu Phe Ile Phe Cys Val Gly Ile Glu Ala Gly Pro65 70 75 80Asn Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn Tyr Phe Met Leu 85 90 95Ala Ile Val Met Val Val Ser Ala Leu Leu Leu Ala Leu Gly Phe Gly 100 105 110Lys Leu Phe Gly Trp Asp Ile Gly Leu Thr Ala Gly Val Leu Ala Gly 115 120 125Ser Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly Asp Thr Leu Arg 130 135 140Gln Ser Ile Ser Asp Pro Arg Gln Leu Ser Ile Met Gln Asp Gln Leu145 150 155 160Ser Leu Gly Tyr Ala Val Thr Tyr Leu Val Gly Leu Val Ser Leu Ile 165 170 175Phe Gly Ala Arg Tyr Leu Pro Arg Leu Gln His Gln Asp Leu Pro Thr 180 185 190Cys Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp Ala Asp Ser Gln 195 200 205Arg Lys Val Phe Leu Pro Val Ile Arg Ala Tyr Arg Val Gly Pro Glu 210 215 220Leu Val Ala Trp Ser Gly Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr225 230 235 240Arg Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu 245 250 255Ala Ser Pro Asp Gly Asp Ala Val Leu Gln Leu Gly Asp Asp Ile Ser 260 265 270Leu Val Gly Tyr Pro Asp Ala His Ala Arg Leu Asp Ala Ser Phe Arg 275 280 285Asn Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp Met Arg Ile Val 290 295 300Thr Glu Glu Ile Val Val Lys Asn His Asn Ala Val Asn Lys Arg Leu305 310 315 320Ser Lys Leu Asn Leu Thr Asp His Gly Cys Phe Leu Asn Arg Val Ile 325 330 335Arg Ser Gln Ile Glu Met Pro Ile Asp Asp Asn Ile Met Leu Asn Lys 340 345 350Gly Asp Val Leu Gln Val Ser Gly Glu Ala Arg Arg Val Lys Ser Val 355 360 365Ala Asp Arg Ile Gly Phe Val Ala Ile His Ser Gln Met Thr Asp Leu 370 375 380Leu Ala Phe Cys Ala Phe Phe Ile Ile Gly Leu Met Val Gly Leu Ile385 390 395 400Thr Phe Gln Phe Ser Asn Phe Ser Phe Gly Ile Gly Asn Ala Ala Gly 405 410 415Leu Leu Phe Ala Gly Ile Met Leu Gly Phe Leu Arg Ala Asn His Pro 420 425 430Thr Phe Gly Tyr Ile Pro Gln Gly Ala Leu Thr Met Val Lys Glu Phe 435 440 445Gly Leu Met Val Phe Met Ala Gly Val Gly Leu Ser Ala Gly Ser Gly 450 455 460Ile Asp His Gly Ile Ser Gly Asn Gly Ala Leu Met Leu Leu Cys Gly465 470 475 480Leu Leu Val Ser Leu Leu Pro Val Val Ile Cys Tyr Leu Phe Gly Ala 485 490 495Tyr Val Leu Arg Met Asn Arg Ala Leu Leu Phe Gly Ala Ile Met Gly 500 505 510Ala Arg Thr Cys Ala Pro Ala Met Glu Ile Ile Ser Asp Thr Ala Arg 515 520 525Ser Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn 530 535 540Val Leu Leu Thr Leu Ala Gly Thr Leu Ile Val Ile Ile Trp Pro Leu545 550 555 560Leu Pro Leu31886DNAEscherichia coliCDS(101)..(1783) 3ggtgcgctga atgaatctgc gccctgaatt ctggtaaaaa acattatcgt aaattaccat 60ttctttcaac agcttactag taaacaagaa gttagcctcc gtg aat ata aac gtc 115 Val Asn Ile Asn Val 1 5gcc gaa ttg tta aat ggg aat tac att ctg tta tta ttt gtg gtc ctc 163Ala Glu Leu Leu Asn Gly Asn Tyr Ile Leu Leu Leu Phe Val Val Leu 10 15 20gcg ctt ggg cta tgt ctc gga aag tta cga ctt ggt tcg atc caa ctg 211Ala Leu Gly Leu Cys Leu Gly Lys Leu Arg Leu Gly Ser Ile Gln Leu 25 30 35ggt aat tcc att ggc gtt tta gtc gta tcg ctg tta tta ggc caa caa 259Gly Asn Ser Ile Gly Val Leu Val Val Ser Leu Leu Leu Gly Gln Gln 40 45 50cat ttc agc att aac acc gat gcg ctt aat ctt ggc ttt atg ctg ttt 307His Phe Ser Ile Asn Thr Asp Ala Leu Asn Leu Gly Phe Met Leu Phe 55 60 65att ttc tgc gtc ggg gtc gaa gcc gga ccg aac ttt ttt tcc att ttt 355Ile Phe Cys Val Gly Val Glu Ala Gly Pro Asn Phe Phe Ser Ile Phe70 75 80 85ttt cgc gat ggg aaa aat tac cta atg tta gca ctg gtg atg gtt ggc 403Phe Arg Asp Gly Lys Asn Tyr Leu Met Leu Ala Leu Val Met Val Gly 90 95 100agt gcg ctg gtg atc gcc tta ggg tta ggt aag ctg ttt ggc tgg gat 451Ser Ala Leu Val Ile Ala Leu Gly Leu Gly Lys Leu Phe Gly Trp Asp 105 110 115att ggc ctg acg gcc ggt atg tta gca ggc tct atg acg tcg aca ccg 499Ile Gly Leu Thr Ala Gly Met Leu Ala Gly Ser Met Thr Ser Thr Pro 120 125 130gtt ctg gtc ggt gct ggc gat aca ctg cgt cat tcc ggc atg gaa agc 547Val Leu Val Gly Ala Gly Asp Thr Leu Arg His Ser Gly Met Glu Ser 135 140 145agg cag ctc tca ctg gca ctg gat aat ctg agc ctc ggg tat gcc tta 595Arg Gln Leu Ser Leu Ala Leu Asp Asn Leu Ser Leu Gly Tyr Ala Leu150 155 160 165acc tat tta atc ggt ctg gtg agt ttg att gtt ggt gcg cgt tac ttg 643Thr Tyr Leu Ile Gly Leu Val Ser Leu Ile Val Gly Ala Arg Tyr Leu 170 175 180ccg aaa ttg cag cat cag gac tta cag acc agc gcc cag caa atc gcc 691Pro Lys Leu Gln His Gln Asp Leu Gln Thr Ser Ala Gln Gln Ile Ala 185 190 195cgc gaa cgt ggc ctg gac act gat gcc aac cgt aag gtt tat tta ccg 739Arg Glu Arg Gly Leu Asp Thr Asp Ala Asn Arg Lys Val Tyr Leu Pro 200 205 210gtg atc cgc gcc tat cgc gtc ggc ccg gaa ctg gtg gcc tgg acc gac 787Val Ile Arg Ala Tyr Arg Val Gly Pro Glu Leu Val Ala Trp Thr Asp 215 220 225ggc aaa aat ctg cgt gaa ctg ggt att tat cga caa acc ggc tgc tac 835Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr Arg Gln Thr Gly Cys Tyr230 235 240 245att gaa cgt att cga cgt aac ggg att ctg gca aat cca gac ggt gat 883Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu Ala Asn Pro Asp Gly Asp 250 255 260gcc gtg cta caa atg ggc gat gaa ata gcg ttg gta ggc tat ccc gac 931Ala Val Leu Gln Met Gly Asp Glu Ile Ala Leu Val Gly Tyr Pro Asp 265 270 275gcc cat gcc cga ctc gat ccc agc ttc cgt aac ggt aaa gaa gtt ttc 979Ala His Ala Arg Leu Asp Pro Ser Phe Arg Asn Gly Lys Glu Val Phe 280 285 290gat cgt gac ctt ctc gac atg cgt atc gtc act gaa gaa gtg gtc gtt 1027Asp Arg Asp Leu Leu Asp Met Arg Ile Val Thr Glu Glu Val Val Val 295 300 305aaa aac cat aac gct gta ggt aaa cgt ctc gca caa ctg aag ttg acc 1075Lys Asn His Asn Ala Val Gly Lys Arg Leu Ala Gln Leu Lys Leu Thr310 315 320 325gat cac ggt tgc ttc ctt aac cgc gtc att cgt agc cag att gag atg 1123Asp His Gly Cys Phe Leu Asn Arg Val Ile Arg Ser Gln Ile Glu Met 330 335 340ccg ata gat gac aac gtc gtg ctt aac aaa ggt gac gtt tta caa gtc 1171Pro Ile Asp Asp Asn Val Val Leu Asn Lys Gly Asp Val Leu Gln Val 345 350 355agc ggc gat gcc cgc cgc gta aaa acc atc gcc gat cgc atc ggc ttt 1219Ser Gly Asp Ala Arg Arg Val Lys Thr Ile Ala Asp Arg Ile Gly Phe 360 365 370atc tcg att cac agc cag gtc act gac ctg ctg gca ttc tgc gcc ttc 1267Ile Ser Ile His Ser Gln Val Thr Asp Leu Leu Ala Phe Cys Ala Phe 375 380 385ttt gtt att ggg ctg atg atc ggg atg atc acc ttc cag ttc agc aca 1315Phe Val Ile Gly Leu Met Ile Gly Met Ile Thr Phe Gln Phe Ser Thr390 395 400 405ttc agt ttc ggc atg ggg aac gct gcc ggg ttg tta ttc gcc gga att 1363Phe Ser Phe Gly Met Gly Asn Ala Ala Gly Leu Leu Phe Ala Gly Ile 410 415 420atg ctg ggc ttt atg cgt gct aac cac ccg acc ttc ggt tac att ccg 1411Met Leu Gly Phe Met Arg Ala Asn His Pro Thr Phe Gly Tyr Ile Pro 425 430 435cag ggt gca tta agc atg gtg aaa gag ttc ggc ttg atg gtg ttt atg 1459Gln Gly Ala Leu Ser Met Val Lys Glu Phe Gly Leu Met Val Phe Met 440 445 450gca ggc gtt ggt ctg agc gcc ggt agc ggt att aat aac ggc ctg ggc 1507Ala Gly Val Gly Leu Ser Ala Gly Ser Gly Ile Asn Asn Gly Leu Gly 455 460 465gcg att ggc ggt cag atg ttg att gcc gga ttg att gtc agt ctg gtg 1555Ala Ile Gly Gly Gln Met Leu Ile Ala Gly Leu Ile Val Ser Leu Val470 475 480 485ccc gtg gtt atc tgt ttc ttg ttc ggt gct tat gta ttg cga atg aac 1603Pro Val Val Ile Cys Phe Leu Phe Gly Ala Tyr Val Leu Arg Met Asn 490 495 500cgc gcg ctg ttg ttc ggc gca atg atg ggc gca cgt acc tgc gcg ccg 1651Arg Ala Leu Leu Phe Gly Ala Met Met Gly Ala Arg Thr Cys Ala Pro 505 510 515gca atg gag atc atc agt gat aca gct cgc agt aac atc ccg gcg ctg 1699Ala Met Glu Ile Ile Ser Asp Thr Ala Arg Ser Asn Ile Pro Ala Leu 520 525 530ggc tat gcg ggc acc tat gca atc gcc aac gtc ctg ctg acg ctg gca 1747Gly Tyr Ala Gly Thr Tyr Ala

Ile Ala Asn Val Leu Leu Thr Leu Ala 535 540 545ggg aca atc atc gtc atg gta tgg cca gga tta gga taaaactgaa 1793Gly Thr Ile Ile Val Met Val Trp Pro Gly Leu Gly550 555 560gttgccctga aaatgaaatt tttttgcaca accgcagaac ttttccgcag ggcatcagtc 1853ttaattagtg ccactgcttt tctttgatgt ccc 18864561PRTEscherichia coli 4Val Asn Ile Asn Val Ala Glu Leu Leu Asn Gly Asn Tyr Ile Leu Leu1 5 10 15Leu Phe Val Val Leu Ala Leu Gly Leu Cys Leu Gly Lys Leu Arg Leu 20 25 30Gly Ser Ile Gln Leu Gly Asn Ser Ile Gly Val Leu Val Val Ser Leu 35 40 45Leu Leu Gly Gln Gln His Phe Ser Ile Asn Thr Asp Ala Leu Asn Leu 50 55 60Gly Phe Met Leu Phe Ile Phe Cys Val Gly Val Glu Ala Gly Pro Asn65 70 75 80Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn Tyr Leu Met Leu Ala 85 90 95Leu Val Met Val Gly Ser Ala Leu Val Ile Ala Leu Gly Leu Gly Lys 100 105 110Leu Phe Gly Trp Asp Ile Gly Leu Thr Ala Gly Met Leu Ala Gly Ser 115 120 125Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly Asp Thr Leu Arg His 130 135 140Ser Gly Met Glu Ser Arg Gln Leu Ser Leu Ala Leu Asp Asn Leu Ser145 150 155 160Leu Gly Tyr Ala Leu Thr Tyr Leu Ile Gly Leu Val Ser Leu Ile Val 165 170 175Gly Ala Arg Tyr Leu Pro Lys Leu Gln His Gln Asp Leu Gln Thr Ser 180 185 190Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp Thr Asp Ala Asn Arg 195 200 205Lys Val Tyr Leu Pro Val Ile Arg Ala Tyr Arg Val Gly Pro Glu Leu 210 215 220Val Ala Trp Thr Asp Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr Arg225 230 235 240Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu Ala 245 250 255Asn Pro Asp Gly Asp Ala Val Leu Gln Met Gly Asp Glu Ile Ala Leu 260 265 270Val Gly Tyr Pro Asp Ala His Ala Arg Leu Asp Pro Ser Phe Arg Asn 275 280 285Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp Met Arg Ile Val Thr 290 295 300Glu Glu Val Val Val Lys Asn His Asn Ala Val Gly Lys Arg Leu Ala305 310 315 320Gln Leu Lys Leu Thr Asp His Gly Cys Phe Leu Asn Arg Val Ile Arg 325 330 335Ser Gln Ile Glu Met Pro Ile Asp Asp Asn Val Val Leu Asn Lys Gly 340 345 350Asp Val Leu Gln Val Ser Gly Asp Ala Arg Arg Val Lys Thr Ile Ala 355 360 365Asp Arg Ile Gly Phe Ile Ser Ile His Ser Gln Val Thr Asp Leu Leu 370 375 380Ala Phe Cys Ala Phe Phe Val Ile Gly Leu Met Ile Gly Met Ile Thr385 390 395 400Phe Gln Phe Ser Thr Phe Ser Phe Gly Met Gly Asn Ala Ala Gly Leu 405 410 415Leu Phe Ala Gly Ile Met Leu Gly Phe Met Arg Ala Asn His Pro Thr 420 425 430Phe Gly Tyr Ile Pro Gln Gly Ala Leu Ser Met Val Lys Glu Phe Gly 435 440 445Leu Met Val Phe Met Ala Gly Val Gly Leu Ser Ala Gly Ser Gly Ile 450 455 460Asn Asn Gly Leu Gly Ala Ile Gly Gly Gln Met Leu Ile Ala Gly Leu465 470 475 480Ile Val Ser Leu Val Pro Val Val Ile Cys Phe Leu Phe Gly Ala Tyr 485 490 495Val Leu Arg Met Asn Arg Ala Leu Leu Phe Gly Ala Met Met Gly Ala 500 505 510Arg Thr Cys Ala Pro Ala Met Glu Ile Ile Ser Asp Thr Ala Arg Ser 515 520 525Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn Val 530 535 540Leu Leu Thr Leu Ala Gly Thr Ile Ile Val Met Val Trp Pro Gly Leu545 550 555 560Gly5563PRTArtificialConcensus between E.coli and P.ananatis 5Met Xaa Asn Xaa Asn Xaa Ala Glu Leu Leu Xaa Gly Asn Xaa Ile Leu1 5 10 15Leu Leu Phe Xaa Val Leu Ala Leu Gly Leu Cys Leu Gly Lys Leu Arg 20 25 30Xaa Gly Ser Ile Gln Leu Gly Asn Ser Ile Gly Val Leu Val Val Ser 35 40 45Leu Leu Leu Gly Gln Gln His Phe Ser Xaa Asn Thr Asp Ala Leu Xaa 50 55 60Leu Gly Phe Met Leu Phe Ile Phe Cys Val Gly Xaa Glu Ala Gly Pro65 70 75 80Asn Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn Tyr Xaa Met Leu 85 90 95Ala Xaa Val Met Val Xaa Ser Ala Leu Xaa Xaa Ala Leu Gly Xaa Gly 100 105 110Lys Leu Phe Gly Trp Asp Ile Gly Leu Thr Ala Gly Xaa Leu Ala Gly 115 120 125Ser Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly Asp Thr Leu Arg 130 135 140Xaa Ser Xaa Xaa Xaa Xaa Arg Gln Leu Ser Xaa Xaa Xaa Asp Xaa Leu145 150 155 160Ser Leu Gly Tyr Ala Xaa Thr Tyr Leu Xaa Gly Leu Val Ser Leu Ile 165 170 175Xaa Gly Ala Arg Tyr Leu Pro Xaa Leu Gln His Gln Asp Leu Xaa Thr 180 185 190Xaa Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp Xaa Asp Xaa Xaa 195 200 205Arg Lys Val Xaa Leu Pro Val Ile Arg Ala Tyr Arg Val Gly Pro Glu 210 215 220Leu Val Ala Trp Xaa Xaa Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr225 230 235 240Arg Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu 245 250 255Ala Xaa Pro Asp Gly Asp Ala Val Leu Gln Xaa Gly Asp Xaa Ile Xaa 260 265 270Leu Val Gly Tyr Pro Asp Ala His Ala Arg Leu Asp Xaa Ser Phe Arg 275 280 285Asn Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp Met Arg Ile Val 290 295 300Thr Glu Glu Xaa Val Val Lys Asn His Asn Ala Val Xaa Lys Arg Leu305 310 315 320Xaa Xaa Leu Xaa Leu Thr Asp His Gly Cys Phe Leu Asn Arg Val Ile 325 330 335Arg Ser Gln Ile Glu Met Pro Ile Asp Asp Asn Xaa Xaa Leu Asn Lys 340 345 350Gly Asp Val Leu Gln Val Ser Gly Xaa Ala Arg Arg Val Lys Xaa Xaa 355 360 365Ala Asp Arg Ile Gly Phe Xaa Xaa Ile His Ser Gln Xaa Thr Asp Leu 370 375 380Leu Ala Phe Cys Ala Phe Phe Xaa Ile Gly Leu Met Xaa Gly Xaa Ile385 390 395 400Thr Phe Gln Phe Ser Xaa Phe Ser Phe Gly Xaa Gly Asn Ala Ala Gly 405 410 415Leu Leu Phe Ala Gly Ile Met Leu Gly Phe Xaa Arg Ala Asn His Pro 420 425 430Thr Phe Gly Tyr Ile Pro Gln Gly Ala Leu Xaa Met Val Lys Glu Phe 435 440 445Gly Leu Met Val Phe Met Ala Gly Val Gly Leu Ser Ala Gly Ser Gly 450 455 460Ile Xaa Xaa Gly Xaa Xaa Xaa Xaa Gly Xaa Xaa Met Leu Xaa Xaa Gly465 470 475 480Leu Xaa Val Ser Leu Xaa Pro Val Val Ile Cys Xaa Leu Phe Gly Ala 485 490 495Tyr Val Leu Arg Met Asn Arg Ala Leu Leu Phe Gly Ala Xaa Met Gly 500 505 510Ala Arg Thr Cys Ala Pro Ala Met Glu Ile Ile Ser Asp Thr Ala Arg 515 520 525Ser Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn 530 535 540Val Leu Leu Thr Leu Ala Gly Thr Xaa Ile Val Xaa Xaa Trp Pro Xaa545 550 555 560Leu Xaa Leu680DNAArtificialprimer 6agcggcgtat tcgcctctat caaacattaa ctgatagcga agatctaaat tgaagcctgc 60ttttttatac taagttggca 80780DNAArtificialprimer 7ccaccagata accttccggt agcgttaaaa cgtcacgaca ttcaatagaa cgctcaagtt 60agtataaaaa agctgaacga 80845DNAArtificialprimer 8gatcggtacc cagacatcag tgatagctgg aggtaatctt acggc 45950DNAArtificialprimer 9gatcgcatgc gacgtcagta aaaaaatttt ataagggcaa taacggccag 501016214DNAArtificialRSFCPG 10gaattccgcc agaaccttca tcagcagcat aaacaggtgc agtgaacagc agagatacgg 60ccagtgcggc caatgttttt tgtcctttaa acataacaga gtcctttaag gatatagaat 120aggggtatag ctacgccaga atatcgtatt tgattattgc tagtttttag ttttgcttaa 180aaaatattgt tagttttatt aaattggaaa actaaattat tggtatcatg aattgttgta 240tgatgataaa tatagggggg atatgataga cgtcattttc atagggttat aaaatgcgac 300taccatgaag tttttaattc aaagtattgg gttgctgata atttgagctg ttctattctt 360tttaaatatc tatataggtc tgttaatgga ttttattttt acaagttttt tgtgtttagg 420catataaaaa tcaagcccgc catatgaacg gcgggttaaa atatttacaa cttagcaatc 480gaaccattaa cgcttgatat cgcttttaaa gtcgcgtttt tcatatcctg tatacagctg 540acgcggacgg gcaatcttca taccgtcact gtgcatttcg ctccagtggg cgatccagcc 600aacggtacgt gccattgcga aaatgacggt gaacatggaa gacggaatac ccatcgcttt 660caggatgata ccagagtaga aatcgacgtt cgggtacagt ttcttctcga taaagtacgg 720gtcgttcagc gcgatgtttt ccagctccat agccacttcc agcaggtcat ccttcgtgcc 780cagctctttc agcacttcat ggcaggtttc acgcattacg gtggcgcgcg ggtcgtaatt 840tttgtacacg cggtgaccga agcccatcag gcggaaagaa tcatttttgt ctttcgcacg 900acgaaaaaat tccggaatgt gtttaacgga gctgatttct tccagcattt tcagcgccgc 960ttcgttagca ccgccgtgcg caggtcccca cagtgaagca atacctgctg cgatacaggc 1020aaacgggttc gcacccgaag agccagcggt acgcacggtg gaggtagagg cgttctgttc 1080atggtcagcg tgcaggatca gaatacggtc catagcacgt tccagaatcg gattaacttc 1140atacggttcg cacggcgtgg agaacatcat attcaggaag ttaccggcgt aggagagatc 1200gttgcgcggg taaacaaatg gctgaccaat ggaatacttg taacacatcg cggccatggt 1260cggcattttc gacagcaggc ggaacgcggc aatttcacgg tgacgaggat tgttaacatc 1320cagcgagtcg tgatagaacg ccgccagcgc gccggtaata ccacacatga ctgccattgg 1380atgcgagtcg cgacggaaag catggaacag acgggtaatc tgctcgtgga tcatggtatg 1440acgggtcacc gtagttttaa attcgtcata ctgttcctga gtcggttttt caccattcag 1500caggatgtaa caaacttcca ggtagttaga atcggtcgcc agctgatcga tcgggaaacc 1560gcggtgcagc aaaatacctt catcaccatc aataaaagta attttagatt cgcaggatgc 1620ggttgaagtg aagcctgggt caaaggtgaa cacacctttt gaaccgagag tacggatatc 1680aataacatct tgacccagcg tgcctttcag cacatccagt tcaacagctg tatccccgtt 1740gagggtgagt tttgcttttg tatcagccat ttaaggtctc cttagcgcct tattgcgtaa 1800gactgccgga acttaaattt gccttcgcac atcaacctgg ctttacccgt tttttatttg 1860gctcgccgct ctgtgaaaga ggggaaaacc tgggtacaga gctctgggcg cttgcaggta 1920aaggatccat tgatgacgaa taaatggcga atcaagtact tagcaatccg aattattaaa 1980cttgtctacc actaataact gtcccgaatg aattggtcaa tactccacac tgttacataa 2040gttaatctta ggtgaaatac cgacttcata acttttacgc attatatgct tttcctggta 2100atgtttgtaa caactttgtt gaatgattgt caaattagat gattaaaaat taaataaatg 2160ttgttatcgt gacctggatc actgttcagg ataaaacccg acaaactata tgtaggttaa 2220ttgtaatgat tttgtgaaca gcctatactg ccgccagtct ccggaacacc ctgcaatccc 2280gagccaccca gcgttgtaac gtgtcgtttt cgcatctgga agcagtgttt tgcatgacgc 2340gcagttatag aaaggacgct gtctgacccg caagcagacc ggaggaagga aatcccgacg 2400tcggggatcc tctagagctt tagcgtctga ggttatcgca atttggttat gagattactc 2460tcgttattaa tttgctttcc tgggtcattt ttttcttgct taccgtcaca ttcttgatgg 2520tatagtcgaa aactgcaaaa gcacatgaca taaacaacat aagcacaatc gtattaatat 2580ataagggttt tatatctatg gatcagacat attctctgga gtcattcctc aaccatgtcc 2640aaaagcgcga cccgaatcaa accgagttcg cgcaagccgt tcgtgaagta atgaccacac 2700tctggccttt tcttgaacaa aatccaaaat atcgccagat gtcattactg gagcgtctgg 2760ttgaaccgga gcgcgtgatc cagtttcgcg tggtatgggt tgatgatcgc aaccagatac 2820aggtcaaccg tgcatggcgt gtgcagttca gctctgccat cggcccgtac aaaggcggta 2880tgcgcttcca tccgtcagtt aacctttcca ttctcaaatt cctcggcttt gaacaaacct 2940tcaaaaatgc cctgactact ctgccgatgg gcggtggtaa aggcggcagc gatttcgatc 3000cgaaaggaaa aagcgaaggt gaagtgatgc gtttttgcca ggcgctgatg actgaactgt 3060atcgccacct gggcgcggat accgacgttc cggcaggtga tatcggggtt ggtggtcgtg 3120aagtcggctt tatggcgggg atgatgaaaa agctctccaa caataccgcc tgcgtcttca 3180ccggtaaggg cctttcattt ggcggcagtc ttattcgccc ggaagctacc ggctacggtc 3240tggtttattt cacagaagca atgctaaaac gccacggtat gggttttgaa gggatgcgcg 3300tttccgtttc tggctccggc aacgtcgccc agtacgctat cgaaaaagcg atggaatttg 3360gtgctcgtgt gatcactgcg tcagactcca gcggcactgt agttgatgaa agcggattca 3420cgaaagagaa actggcacgt cttatcgaaa tcaaagccag ccgcgatggt cgagtggcag 3480attacgccaa agaatttggt ctggtctatc tcgaaggcca acagccgtgg tctctaccgg 3540ttgatatcgc cctgccttgc gccacccaga atgaactgga tgttgacgcc gcgcatcagc 3600ttatcgctaa tggcgttaaa gccgtcgccg aaggggcaaa tatgccgacc accatcgaag 3660cgactgaact gttccagcag gcaggcgtac tatttgcacc gggtaaagcg gctaatgctg 3720gtggcgtcgc tacatcgggc ctggaaatgc cacaaaacgc tgcgcgcctg ggctggaaag 3780ccgagaaagt tgacgcacgt ttgcatcaca tcatgctgga tatccaccat gcctgtgttg 3840agcatggtgg tgaaggtgag caaaccaact acgtgcaggg cgcgaacatt gccggttttg 3900tgaaggttgc cgatgcgatg ctggcgcagg gtgtgattta agttgtaaat gcctgatggc 3960gctacgctta tcaggcctac aaatgggcac aattcattgc agttacgctc taatgtaggc 4020cgggcaagcg cagcgccccc ggcaaaattt caggcgttta tgagtattta acggatgatg 4080ctccccacgg aacatttctt atgggccaac ggcatttctt actgtagtgc tcccaaaact 4140gcttgtcgta acgataacac gcttcaagtt cagcatccgt taactttctg cggactcacg 4200cgcgcagcac tatgccagta aagaaatccc atttgactat ttttttgata atcttcttcg 4260ctttcgaaca actcgtgcgc ctttcgagaa gctagagtcg actcgccaat caccagcact 4320aaagtgcgcg gttcgttacc cgattcatct ttgaaattag ccagtggcgg caaggcatta 4380ttttcattca gtaactttgt tagcgagttt agttgctgac gatactgata atagccggtc 4440aggaattgcc acggtgcggc aggctccata cgcgaggcca ggttatccaa cgttttctca 4500aacggcttgt ttttgataaa cgtattcatg gcgatcggat gcagaatcaa gccataaagc 4560agggcaaaag agacaacata acgccacggc tttggaatat agaccgggcg caggcgtgtc 4620cacagcagaa ctgccaccgc cgtataggcc agcgcgataa gcacaatttt caggctgaaa 4680tactggctta aatactcgct ggcttcgttg gtgttggttt cgaacatcac aaacagaacg 4740ctctgcgaga actcctgacc gtagatgacg tagtagcaca gcgccgccag agaggccgcc 4800catagcacca cgccgattac tgcggcaata attttaatcc gcttcggaaa gaggaatacc 4860gggatcaacc acagcgaact gaataacagc gagtcgcgaa tgccgttagt gccactataa 4920ccactgatgt aaataatggc ctgtagcaga gtagagaaaa accaaaagta gagcagtgcc 4980caacccaggg ctttccagct aaaaagaggt ttagcctgga cttctgtgga atgcatagta 5040agaacctgtc ttgaaaaaat atcgccgaat gtaacgacaa ttccttaagg atatctgaag 5100gtatattcag aatttgaata aaatgcagac agaaatatat tgaaaacgag ggtgttagaa 5160cagaagtatt tcagaaaacc ctcgcgcaaa agcacgaggg tttgcagaag aggaagatta 5220gccggtatta cgcatacctg ccgcaatccc ggcaatagtg accattaacg cttgttcgac 5280gcgaggatcc ggttcctggc cttctttttc tgcctggcgg gagcggtgca gcaactcggc 5340ctgcaatacg ttcagcgggt cggtgtaaat attccgtagc tgaatagact ctgcaatcca 5400cggcagatcg gccatcagat gggaatcgtt ggcaatcgcc agcaccactt tgatgtcttc 5460ttcttgcagg ttgcgtaact ctttacctaa cggccacagt gctttgtcta ccaggcgttg 5520gtcatagtat tccgccagcc acaggtctgc tttggcgaag accatctcca gcatgccgag 5580acgcgtcgag aagaatggcc aatcgcggca catagcctcc agctcgctct gtttgccgtc 5640ttcgaccact ttttgcagcg ccgtacctgc acccagccag gcggggagca tcagacggtt 5700ttgcgtccag gcgaagatcc acggaatggc gcgtagtgac tcgacgccgc cggttgggcg 5760acgtttcgcc ggacgtgaac ccaacggcag tttgcccagt tcttgttccg gcgtagcgga 5820gcggaagtaa ggcacaaaat ctttgttttc acgtacgtag ccgcggtaga catcgcagga 5880gatgactgac agttcatcca taatgcgacg ccagctctct ttcggctccg gcggtggcag 5940caggttggct tccagaatcg ccccggtata aagcgacagg ctgctgacgg tgatttctgg 6000cagaccatat ttaaagcgga tcatctcgcc ctgttcggtt acgcgcaggc cgcctttcag 6060gcttcctggc ggttgtgaca gcagcgccgc atgagcaggt gcgccgccgc gaccaatgga 6120accgccgcga ccgtggaaca acgtcagctc aatacccgct ttttcgcagg ttttgattaa 6180tgcatcctgt gcctgatatt gcgcccagga agctgccatc actcccgcat cttttgctga 6240gtcggaatag ccaatcatca ccatctgttt gccctgaatc aggccacgat accagtcaat 6300attgagcagc tgggtcatga catcgttggc gttgttcaga tcatcgaggg tttcaaacag 6360cggagcaacc ggcatcgcaa acccgatacc cgcttctttc agcagcaggt ggacagccag 6420tacgtcggac ggcgttttcg ccatcgagat cacgtaggcg gcaatggagc cttgcggtgc 6480ttcggcaatc acctggcagg tatcgagcac ttcgcgcgtt tcggcgcttg gttgccagtt 6540gcgcggcaga agcggacgtt tggagttcag ttcgcggatc aggaacgcct gtttgtcggc 6600ctctgaccag ctttcgtagt cgccgatacc gaggtagcgg gtcagctcgc ccagcgcttc 6660ggtatgacgc gtgctctcct gacggatatc aatacggacc agcggtacgc cgaaacattt 6720cacgcggcgc agggtgtcga gcagatcgcc gttggcgata atacccatgc cacacgcctg 6780aagtgactgg tagcaagcgt agagcggttc ccacagttct tcgttttgtg tcagcaggcc 6840ttctggtttt ggcagttctt cgcctttcag gcgcgcttcc agccatgcct gtgtcgccat 6900caggcgagaa cgcaggtttt tcatcagata gcgatacggt tctgcggcac cttcttcgcc 6960aaccagcgcc agcagttcag gggtcgcttc aaccatcgac agttcagaaa ccagcacctg 7020aatatctttc aggaacaaat cggtggcttt ccagcggctg agtagcagga cgtggcgggt 7080gatatcggca gtgacgttcg ggttgccgtc gcggtcgccg cccatccacg aagtaaaacg 7140gaccggaaca aattcgacgg gcagtttgta gccgaggttc tcttccagtt gttcgttcag 7200ttcgcgcagg taatttggta cgccttgcca caggctgttt tccactacgg caaagcccca 7260tttggcttca tctaccgggc ttggacgcag cttacggatt tcatcggtat gccatgactg 7320ggcgatcaac tggcgcaggc gacgcatcag

ctggttgtgt tcgtagtcag cgatatcttt 7380gttatcgagc tgttttaaac aggcgttcac ttccaccatt ttgtggatca gtgtacgacg 7440ggtaatttcg gttgggtgag ccgtgaggac cagttccagc gacagcgatt ccactgcttt 7500tttgatggtg tcttcgctca gttccggctg gtttttcagt ttacgcaggg tgcgggcgat 7560cacttccggg ttgctggcag cttcgccttt cggcgaaatg ctgtggtatt gctcggcggt 7620gttggccagg ttcaggaact gactaaacgc acgcgcaacg ggcagcagct cgtcgttcga 7680caaattttgt aaggtggtga gcaactcctg gcggttagca tcattgccag cgcgtgaaga 7740tttcgacaac ttacggatag tttctacgcg ttcaagaatg tgttctccca acgcatcctt 7800gatggtttct cccagcactt tgccgagcat actgacatta ctacgcaatg cggaatattg 7860ttcgttcata ttaccccaga caccccatct tatcgtttga tagccctgta tccttcacgt 7920cgcattggcg cgaatatgct cgggctttgc ttttcgtcgt cttttataaa gccacgtaaa 7980agcggtgacg tcaaatgctg cgaaatcgct tcagcaaacg aataaatagc aggaatttac 8040gtcattaaat tcacgacgct ttaaataagc gtaacttatg gaaatgttaa aaaatcgccc 8100caagtaacac caaaggtgta ggtcggataa gatgcgcaag tatcgcatcc gacattattg 8160cggcactgga gtttggcaac agtgccggat gcggcgcgag cgccttatcc ggcctacagt 8220tgggcatcgt ttgagtcact gtcggtcgga taagatgcgc aagtatcgca tccgacatta 8280ttgcggcact ggagtttggc aacagtgccg gatgcggcgc gagcgcctta tccggcctac 8340ggttgggcat cgtttgagtc actgtaggtc ggataagatg cgcaagcatc gcatccgaca 8400ttattgcggc actggagttt ggcaacagcg ccggatgcgg cgcgagcgcc ttatccggcc 8460tacgttttaa tgccagcaaa aatggtgaat tacctgggtt atcagttcgc gggtgggctt 8520gataaaccgt gtttccagat attcatcagg ttgatgagcc tgattaattg agccaggccc 8580caacaccagc gtcgggcata acgtttgaat aaacggcgct tcggtacagt agttcaccac 8640ttcggttttt gctccgagca atttctcaac cacttcaacc agttgatgat tcggtgggca 8700ttcatagcca gggatcggcg gatgcagctc gtcgacctgc aggagcagaa gagcatacat 8760ctggaagcaa agccaggaaa gcggcctatg gagctgtgcg gcagcgctca gtaggcaatt 8820tttcaaaata ttgttaagcc ttttctgagc atggtatttt tcatggtatt accaattagc 8880aggaaaataa gccattgaat ataaaagata aaaatgtctt gtttacaata gagtgggggg 8940ggtcagcctg ccgccttggg ccgggtgatg tcgtacttgc ccgccgcgaa ctcggttacc 9000gtccagccca gcgcgaccag ctccggcaac gcctcgcgca cccgctggcg gcgcttgcgc 9060atggtcgaac cactggcctc tgacggccag acatagccgc acaaggtatc tatggaagcc 9120ttgccggttt tgccggggtc gatccagcca cacagccgct ggtgcagcag gcgggcggtt 9180tcgctgtcca gcgcccgcac ctcgtccatg ctgatgcgca catgctggcc gccacccatg 9240acggcctgcg cgatcaaggg gttcagggcc acgtacaggc gcccgtccgc ctcgtcgctg 9300gcgtactccg acagcagccg aaacccctgc cgcttgcggc cattctgggc gatgatggat 9360accttccaaa ggcgctcgat gcagtcctgt atgtgcttga gcgccccacc actatcgacc 9420tctgccccga tttcctttgc cagcgcccga tagctacctt tgaccacatg gcattcagcg 9480gtgacggcct cccacttggg ttccaggaac agccggagct gccgtccgcc ttcggtcttg 9540ggttccgggc caagcactag gccattaggc ccagccatgg ccaccagccc ttgcaggatg 9600cgcagatcat cagcgcccag cggctccggg ccgctgaact cgatccgctt gccgtcgccg 9660tagtcatacg tcacgtccag cttgctgcgc ttgcgctcgc cccgcttgag ggcacggaac 9720aggccggggg ccagacagtg cgccgggtcg tgccggacgt ggctgaggct gtgcttgttc 9780ttaggcttca ccacggggca cccccttgct cttgcgctgc ctctccagca cggcgggctt 9840gagcaccccg ccgtcatgcc gcctgaacca ccgatcagcg aacggtgcgc catagttggc 9900cttgctcaca ccgaagcgga cgaagaaccg gcgctggtcg tcgtccacac cccattcctc 9960ggcctcggcg ctggtcatgc tcgacaggta ggactgccag cggatgttat cgaccagtac 10020cgagctgccc cggctggcct gctgctggtc gcctgcgccc atcatggccg cgcccttgct 10080ggcatggtgc aggaacacga tagagcaccc ggtatcggcg gcgatggcct ccatgcgacc 10140gatgacctgg gccatggggc cgctggcgtt ttcttcctcg atgtggaacc ggcgcagcgt 10200gtccagcacc atcaggcggc ggccctcggc ggcgcgcttg aggccgtcga accactccgg 10260ggccatgatg ttgggcaggc tgccgatcag cggctggatc agcaggccgt cagccacggc 10320ttgccgttcc tcggcgctga ggtgcgcccc aagggcgtgc aggcggtgat gaatggcggt 10380gggcgggtct tcggcgggca ggtagatcac cgggccggtg ggcagttcgc ccacctccag 10440cagatccggc ccgcctgcaa tctgtgcggc cagttgcagg gccagcatgg atttaccggc 10500accaccgggc gacaccagcg ccccgaccgt accggccacc atgttgggca aaacgtagtc 10560cagcggtggc ggcgctgctg cgaacgcctc cagaatattg ataggcttat gggtagccat 10620tgattgcctc ctttgcaggc agttggtggt taggcgctgg cggggtcact acccccgccc 10680tgcgccgctc tgagttcttc caggcactcg cgcagcgcct cgtattcgtc gtcggtcagc 10740cagaacttgc gctgacgcat ccctttggcc ttcatgcgct cggcatatcg cgcttggcgt 10800acagcgtcag ggctggccag caggtcgccg gtctgcttgt ccttttggtc tttcatatca 10860gtcaccgaga aacttgccgg ggccgaaagg cttgtcttcg cggaacaagg acaaggtgca 10920gccgtcaagg ttaaggctgg ccatatcagc gactgaaaag cggccagcct cggccttgtt 10980tgacgtataa ccaaagccac cgggcaacca atagcccttg tcacttttga tcaggtagac 11040cgaccctgaa gcgctttttt cgtattccat aaaaccccct tctgtgcgtg agtactcata 11100gtataacagg cgtgagtacc aacgcaagca ctacatgctg aaatctggcc cgcccctgtc 11160catgcctcgc tggcggggtg ccggtgcccg tgccagctcg gcccgcgcaa gctggacgct 11220gggcagaccc atgaccttgc tgacggtgcg ctcgatgtaa tccgcttcgt ggccgggctt 11280gcgctctgcc agcgctgggc tggcctcggc catggccttg ccgatttcct cggcactgcg 11340gccccggctg gccagcttct gcgcggcgat aaagtcgcac ttgctgaggt catcaccgaa 11400gcgcttgacc agcccggcca tctcgctgcg gtactcgtcc agcgccgtgc gccggtggcg 11460gctaagctgc cgctcgggca gttcgaggct ggccagcctg cgggccttct cctgctgccg 11520ctgggcctgc tcgatctgct ggccagcctg ctgcaccagc gccgggccag cggtggcggt 11580cttgcccttg gattcacgca gcagcaccca cggctgataa ccggcgcggg tggtgtgctt 11640gtccttgcgg ttggtgaagc ccgccaagcg gccatagtgg cggctgtcgg cgctggccgg 11700gtcggcgtcg tactcgctgg ccagcgtccg ggcaatctgc ccccgaagtt caccgcctgc 11760ggcgtcggcc accttgaccc atgcctgata gttcttcggg ctggtttcca ctaccagggc 11820aggctcccgg ccctcggctt tcatgtcatc caggtcaaac tcgctgaggt cgtccaccag 11880caccagacca tgccgctcct gctcggcggg cctgatatac acgtcattgc cctgggcatt 11940catccgcttg agccatggcg tgttctggag cacttcggcg gctgaccatt cccggttcat 12000catctggccg gtggtggcgt ccctgacgcc gatatcgaag cgctcacagc ccatggcctt 12060gagctgtcgg cctatggcct gcaaagtcct gtcgttcttc atcgggccac caagcgcagc 12120cagatcgagc cgtcctcggt tgtcagtggc gtcaggtcga gcaagagcaa cgatgcgatc 12180agcagcacca ccgtaggcat catggaagcc agcatcacgg ttagccatag cttccagtgc 12240cacccccgcg acgcgctccg ggcgctctgc gcggcgctgc tcacctcggc ggctacctcc 12300cgcaactctt tggccagctc cacccatgcc gcccctgtct ggcgctgggc tttcagccac 12360tccgccgcct gcgcctcgct ggcctgctgg gtctggctca tgacctgccg ggcttcgtcg 12420gccagtgtcg ccatgctctg ggccagcggt tcgatctgct ccgctaactc gttgatgcct 12480ctggatttct tcactctgtc gattgcgttc atggtctatt gcctcccggt attcctgtaa 12540gtcgatgatc tgggcgttgg cggtgtcgat gttcagggcc acgtctgccc ggtcggtgcg 12600gatgccccgg ccttccatct ccaccacgtt cggccccagg tgaacaccgg gcaggcgctc 12660gatgccctgc gcctcaagtg ttctgtggtc aatgcgggcg tcgtggccag cccgctctaa 12720tgcccggttg gcatggtcgg cccatgcctc gcgggtctgc tcaagccatg ccttgggctt 12780gagcgcttcg gtcttctgtg ccccgccctt ctccggggtc ttgccgttgt accgcttgaa 12840ccactgagcg gcgggccgct cgatgccgtc attgatccgc tcggagatca tcaggtggca 12900gtgcgggttc tcgccgccac cggcatggat ggccagcgta tacggcaggc gctcggcacc 12960ggtcaggtgc tgggcgaact cggacgccag cgccttctgc tggtcgaggg tcagctcgac 13020cggcagggca aattcgacct ccttgaacag ccgcccattg gcgcgttcat acaggtcggc 13080agcatcccag tagtcggcgg gccgctcgac gaactccggc atgtgcccgg attcggcgtg 13140caagacttca tccatgtcgc gggcatactt gccttcgcgc tggatgtagt cggccttggc 13200cctggccgat tggccgcccg acctgctgcc ggttttcgcc gtaaggtgat aaatcgccat 13260gctgcctcgc tgttgctttt gcttttcggc tccatgcaat ggccctcgga gagcgcaccg 13320cccgaagggt ggccgttagg ccagtttctc gaagagaaac cggtaagtgc gccctcccct 13380acaaagtagg gtcgggattg ccgccgctgt gcctccatga tagcctacga gacagcacat 13440taacaatggg gtgtcaagat ggttaagggg agcaacaagg cggcggatcg gctggccaag 13500ctcgaagaac aacgagcgcg aatcaatgcc gaaattcagc gggtgcgggc aagggaacag 13560cagcaagagc gcaagaacga aacaaggcgc aaggtgctgg tgggggccat gattttggcc 13620aaggtgaaca gcagcgagtg gccggaggat cggctcatgg cggcaatgga tgcgtacctt 13680gaacgcgacc acgaccgcgc cttgttcggt ctgccgccac gccagaagga tgagccgggc 13740tgaatgatcg accgagacag gccctgcggg gctgcacacg cgcccccacc cttcgggtag 13800ggggaaaggc cgctaaagcg gctaaaagcg ctccagcgta tttctgcggg gtttggtgtg 13860gggtttagcg ggctttgccc gcctttcccc ctgccgcgca gcggtggggc ggtgtgtagc 13920ctagcgcagc gaatagacca gctatccggc ctctggccgg gcatattggg caagggcagc 13980agcgccccac aagggcgctg ataaccgcgc ctagtggatt attcttagat aatcatggat 14040ggatttttcc aacaccccgc cagcccccgc ccctgctggg tttgcaggtt tgggggcgtg 14100acagttattg caggggttcg tgacagttat tgcagggggg cgtgacagtt attgcagggg 14160ttcgtgacag ttagtacggg agtgacgggc actggctggc aatgtctagc aacggcaggc 14220atttcggctg agggtaaaag aactttccgc taagcgatag actgtatgta aacacagtat 14280tgcaaggacg cggaacatgc ctcatgtggc ggccaggacg gccagccggg atcgggatac 14340tggtcgttac cagagccacc gacccgagca aacccttctc tatcagatcg ttgacgagta 14400ttacccggca ttcgctgcgc ttatggcaga gcagggaaag gaattgccgg gctatgtgca 14460acgggaattt gaagaatttc tccaatgcgg gcggctggag catggctttc tacgggttcg 14520ctgcgagtct tgccacgccg agcacctggt cgctttcagc tgtaagcgtc gcggtttctg 14580cccgagctgt ggggcgcggc ggatggccga aagtgccgcc ttgctggttg atgaagtact 14640gcctgaacaa cccatgcgtc agtgggtgtt gagcttcccg tttcagctgc gtttcctgtt 14700tggggtcgtt tgcgggaagg ggcggaatcc tacgctaagg ctttggccag cgatattctc 14760cggtgagatt gatgtgttcc caggggatag gagaagtcgc ttgatatcta gtatgacgtc 14820tgtcgcacct gcttgatcgc ggcccaaggg ttggtttgcg cattcacagt tctccgcaag 14880aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca 14940ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt 15000atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa 15060tcgccgtgac gatcagcggt ccagtgatcg aagttaggct ggtaagagcc gcgagcgatc 15120cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg 15180gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg 15240tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct 15300gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca 15360agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct 15420cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga 15480cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt 15540tgaaggctct caagggcatc ggtcgacgct ctcccttatg cgactcctgc attaggaagc 15600agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg 15660agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag 15720cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg 15780cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga 15840tccacaggac gggtgtggtc gccatgatcg cgtagtcgat agtggctcca agtagcgaag 15900cgagcaggac tgggcggcgg ccaaagcggt cggacagtgc tccgagaacg ggtgcgcata 15960gaaattgcat caacgcatat agcgctagca gcacgccata gtgactggcg atgctgtcgg 16020aatggacgat atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag 16080catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg 16140cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa 16200gctgtcaaac atga 162141140DNAArtificialprimer 11gatcggtacc ggtgcgctga atgaatctgc gccctgaatt 401240DNAArtificialprimer 12gatcgcatgc ggcaacttca gttttatcct aatcctggcc 401355DNAArtificialprimer 13gtgattcaac atcactggag aaagtcttat gaaactctga agcctgcttt tttat 551455DNAArtificialprimer 14cctggaatgc aggggagcgg caagattaaa ccagttccgc tcaagttagt ataaa 551522DNAArtificialprimer 15gcgcctacac taagcatagt tg 221622DNAArtificialprimer 16ccatcagcag gcttagcgca ac 221755DNAArtificialprimer 17gagggagaaa aacgcatgat tatttccgca gccagcgtga agcctgcttt tttat 551854DNAArtificialprimer 18gcgcaaacga ctatgccgca ttccctttcg ccatggcgct caagttagta taaa 541923DNAArtificialprimer 19gcgtaaagca atgatggcgc acc 232023DNAArtificialprimer 20gcggtgtcgt ttcagagtga ggg 2321348DNAEscherichia coli 21aagctttacg cgaacgagcc atgacattgc tgacgactct ggcagtggca gatgacataa 60aactggtcga ctggttacaa caacgcctgg ggcttttaga gcaacgagac acggcaatgt 120tgcaccgttt gctgcatgat attgaaaaaa atatcaccaa ataaaaaacg ccttagtaag 180tatttttcag cttttcattc tgactgcaac gggcaatatg tctctgtgtg gattaaaaaa 240agagtgtctg atagcagctt ctgaactggt tacctgccgt gagtaaatta aaattttatt 300gacttaggtc actaaatact ttaaccaata taggcgactc taggatcc 3482224DNAArtificialprimer 22cgggatccca gcttactagt aaac 242332DNAArtificialprimer 23gggaaaggat ccgatgggct ccacaaaatg gg 32241686DNASalmonella typhimuriumCDS(1)..(1686) 24gtg aat ata aac gtc gca gat ttg tta aat ggg aat tac atc ctg tta 48Val Asn Ile Asn Val Ala Asp Leu Leu Asn Gly Asn Tyr Ile Leu Leu1 5 10 15tta ttt gtg gtc ctg gct ctt ggc ctg tgt ctt ggc aag tta cgt ctg 96Leu Phe Val Val Leu Ala Leu Gly Leu Cys Leu Gly Lys Leu Arg Leu 20 25 30ggt tca gtc caa ctt ggt aat tcc att ggc gtt tta gtg gtc tct cta 144Gly Ser Val Gln Leu Gly Asn Ser Ile Gly Val Leu Val Val Ser Leu 35 40 45tta tta ggg cag caa cac ttc agt att aac acc gac gcg cta aat ctg 192Leu Leu Gly Gln Gln His Phe Ser Ile Asn Thr Asp Ala Leu Asn Leu 50 55 60gga ttt atg ctg ttt att ttt tgt gtc ggc gtc gag gct ggc ccc aac 240Gly Phe Met Leu Phe Ile Phe Cys Val Gly Val Glu Ala Gly Pro Asn65 70 75 80ttt ttt tcg att ttt ttt cgc gac ggt aaa aat tat ctg atg ctt gcc 288Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn Tyr Leu Met Leu Ala 85 90 95ctg gtc atg gtc ggt agc gct ctg cta atc gcc tta ggc ctt ggt aag 336Leu Val Met Val Gly Ser Ala Leu Leu Ile Ala Leu Gly Leu Gly Lys 100 105 110cta ttc ggc tgg gat atc ggc ctg acg gcc ggt atg ctg gcc ggc tca 384Leu Phe Gly Trp Asp Ile Gly Leu Thr Ala Gly Met Leu Ala Gly Ser 115 120 125atg acc tcc acg ccc gtg ctg gtt ggc gca ggc gat acc ctg cga cat 432Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly Asp Thr Leu Arg His 130 135 140tcc ggc atc gcc agc acc caa ctc tcc tcc gct ctt gat aat ctg agc 480Ser Gly Ile Ala Ser Thr Gln Leu Ser Ser Ala Leu Asp Asn Leu Ser145 150 155 160ctg ggc tat gcc ctg acc tat ctg att ggg ttg gtc agt ctg atc gtg 528Leu Gly Tyr Ala Leu Thr Tyr Leu Ile Gly Leu Val Ser Leu Ile Val 165 170 175ggc gcg cgc tac ttg cct aaa cta cag cat cag gat ttg caa acc agc 576Gly Ala Arg Tyr Leu Pro Lys Leu Gln His Gln Asp Leu Gln Thr Ser 180 185 190gcc cag caa att gct cgc gag cgc ggt ctg gat acc gat gct aat cgc 624Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp Thr Asp Ala Asn Arg 195 200 205aag gtt tat ttg ccg gtt atc cgc gcc tat cgg gtt ggc ccg gaa ctg 672Lys Val Tyr Leu Pro Val Ile Arg Ala Tyr Arg Val Gly Pro Glu Leu 210 215 220gtg gcc tgg act gac ggt aaa aac ctg cgc gag ctg ggt att tac cgc 720Val Ala Trp Thr Asp Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr Arg225 230 235 240cag aca ggc tgc tat atc gaa cgt att cgc cgt aac ggt att ctg gcg 768Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu Ala 245 250 255aac ccg gat ggc gat gcg gtg ctg caa atg ggc gat gag att gcg ctg 816Asn Pro Asp Gly Asp Ala Val Leu Gln Met Gly Asp Glu Ile Ala Leu 260 265 270gtc ggc tat ccg gac gcg cac gcc agg ctc gat ccc agc ttc cgt aat 864Val Gly Tyr Pro Asp Ala His Ala Arg Leu Asp Pro Ser Phe Arg Asn 275 280 285ggt aag gaa gta ttt gat cgc gat ctg ctt gat atg cgc atc gtg acg 912Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp Met Arg Ile Val Thr 290 295 300gaa gaa att gtg gtg aaa aac cac aat gcg gtc ggt cgc cgc ctc gct 960Glu Glu Ile Val Val Lys Asn His Asn Ala Val Gly Arg Arg Leu Ala305 310 315 320cag ctc aag ctg acc gat cat ggc tgc ttc ctt aac cgc gtc atc cgc 1008Gln Leu Lys Leu Thr Asp His Gly Cys Phe Leu Asn Arg Val Ile Arg 325 330 335agc cag atc gaa atg cct atc gac gat aac gtc gtg ctg aat aaa ggc 1056Ser Gln Ile Glu Met Pro Ile Asp Asp Asn Val Val Leu Asn Lys Gly 340 345 350gac gtg tta cag gtg agc ggc gac gcc aga cgc gta aaa acc atc gcc 1104Asp Val Leu Gln Val Ser Gly Asp Ala Arg Arg Val Lys Thr Ile Ala 355 360 365gat cgc att ggt ttt att tcg att cat agt cag gtg acg gac ctg ctt 1152Asp Arg Ile Gly Phe Ile Ser Ile His Ser Gln Val Thr Asp Leu Leu 370 375 380gcc ttc tgc gct ttt ttt atc atc ggg tta atg atc ggg atg att acc 1200Ala Phe Cys Ala Phe Phe Ile Ile Gly Leu Met Ile Gly Met Ile Thr385 390 395 400ttc cag ttc agc aat ttt agt ttc ggt atc ggc aac gcg gcc gga ttg 1248Phe Gln Phe Ser Asn Phe Ser Phe Gly Ile Gly Asn Ala Ala Gly Leu 405 410 415ctg ttc gcc ggg atc atg ctc ggt ttc ctg cgc gct aac cat cct acc 1296Leu Phe Ala Gly Ile Met Leu Gly Phe Leu Arg Ala Asn His Pro Thr 420 425 430ttt ggt tat att ccg cag ggc gcg ctt aat atg gtg aaa gaa ttc ggc 1344Phe Gly Tyr Ile Pro Gln Gly Ala Leu Asn Met Val Lys Glu Phe Gly 435 440 445ttg atg gtg ttt atg gca ggc gtt ggt tta agc gcc ggc agc ggt atc 1392Leu Met Val Phe Met Ala Gly Val Gly Leu Ser Ala Gly Ser Gly Ile 450 455 460agc aac ggt ctg ggc gcg gtc ggc gga caa atg ctg atc gcc ggg ctt 1440Ser Asn Gly Leu Gly Ala Val Gly Gly Gln Met Leu Ile Ala Gly Leu465 470 475 480gtc gtc agc ctg

gta ccg gtg gtg atc tgc ttc ttg ttt ggc gct tat 1488Val Val Ser Leu Val Pro Val Val Ile Cys Phe Leu Phe Gly Ala Tyr 485 490 495gtg ctg cgc atg aac cgg gcg ctg ctg ttc ggt gcc atg atg ggc gcg 1536Val Leu Arg Met Asn Arg Ala Leu Leu Phe Gly Ala Met Met Gly Ala 500 505 510cgt acc tgc gcc ccg gcg atg gaa atc att agc gat acg gcg cgc agt 1584Arg Thr Cys Ala Pro Ala Met Glu Ile Ile Ser Asp Thr Ala Arg Ser 515 520 525aat att cca gcg ctg ggt tat gcg ggg act tac gcc atc gcc aac gtt 1632Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn Val 530 535 540ctg cta acg ctg gcg ggg acg ctt att gtc atc atc tgg ccg ggg tta 1680Leu Leu Thr Leu Ala Gly Thr Leu Ile Val Ile Ile Trp Pro Gly Leu545 550 555 560gga taa 1686Gly25561PRTSalmonella typhimurium 25Val Asn Ile Asn Val Ala Asp Leu Leu Asn Gly Asn Tyr Ile Leu Leu1 5 10 15Leu Phe Val Val Leu Ala Leu Gly Leu Cys Leu Gly Lys Leu Arg Leu 20 25 30Gly Ser Val Gln Leu Gly Asn Ser Ile Gly Val Leu Val Val Ser Leu 35 40 45Leu Leu Gly Gln Gln His Phe Ser Ile Asn Thr Asp Ala Leu Asn Leu 50 55 60Gly Phe Met Leu Phe Ile Phe Cys Val Gly Val Glu Ala Gly Pro Asn65 70 75 80Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn Tyr Leu Met Leu Ala 85 90 95Leu Val Met Val Gly Ser Ala Leu Leu Ile Ala Leu Gly Leu Gly Lys 100 105 110Leu Phe Gly Trp Asp Ile Gly Leu Thr Ala Gly Met Leu Ala Gly Ser 115 120 125Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly Asp Thr Leu Arg His 130 135 140Ser Gly Ile Ala Ser Thr Gln Leu Ser Ser Ala Leu Asp Asn Leu Ser145 150 155 160Leu Gly Tyr Ala Leu Thr Tyr Leu Ile Gly Leu Val Ser Leu Ile Val 165 170 175Gly Ala Arg Tyr Leu Pro Lys Leu Gln His Gln Asp Leu Gln Thr Ser 180 185 190Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp Thr Asp Ala Asn Arg 195 200 205Lys Val Tyr Leu Pro Val Ile Arg Ala Tyr Arg Val Gly Pro Glu Leu 210 215 220Val Ala Trp Thr Asp Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr Arg225 230 235 240Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu Ala 245 250 255Asn Pro Asp Gly Asp Ala Val Leu Gln Met Gly Asp Glu Ile Ala Leu 260 265 270Val Gly Tyr Pro Asp Ala His Ala Arg Leu Asp Pro Ser Phe Arg Asn 275 280 285Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp Met Arg Ile Val Thr 290 295 300Glu Glu Ile Val Val Lys Asn His Asn Ala Val Gly Arg Arg Leu Ala305 310 315 320Gln Leu Lys Leu Thr Asp His Gly Cys Phe Leu Asn Arg Val Ile Arg 325 330 335Ser Gln Ile Glu Met Pro Ile Asp Asp Asn Val Val Leu Asn Lys Gly 340 345 350Asp Val Leu Gln Val Ser Gly Asp Ala Arg Arg Val Lys Thr Ile Ala 355 360 365Asp Arg Ile Gly Phe Ile Ser Ile His Ser Gln Val Thr Asp Leu Leu 370 375 380Ala Phe Cys Ala Phe Phe Ile Ile Gly Leu Met Ile Gly Met Ile Thr385 390 395 400Phe Gln Phe Ser Asn Phe Ser Phe Gly Ile Gly Asn Ala Ala Gly Leu 405 410 415Leu Phe Ala Gly Ile Met Leu Gly Phe Leu Arg Ala Asn His Pro Thr 420 425 430Phe Gly Tyr Ile Pro Gln Gly Ala Leu Asn Met Val Lys Glu Phe Gly 435 440 445Leu Met Val Phe Met Ala Gly Val Gly Leu Ser Ala Gly Ser Gly Ile 450 455 460Ser Asn Gly Leu Gly Ala Val Gly Gly Gln Met Leu Ile Ala Gly Leu465 470 475 480Val Val Ser Leu Val Pro Val Val Ile Cys Phe Leu Phe Gly Ala Tyr 485 490 495Val Leu Arg Met Asn Arg Ala Leu Leu Phe Gly Ala Met Met Gly Ala 500 505 510Arg Thr Cys Ala Pro Ala Met Glu Ile Ile Ser Asp Thr Ala Arg Ser 515 520 525Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn Val 530 535 540Leu Leu Thr Leu Ala Gly Thr Leu Ile Val Ile Ile Trp Pro Gly Leu545 550 555 560Gly261689DNAYersinia pestisCDS(1)..(1689) 26gtg aat ata aac gtc gca aat ttg tta aac ggc aac tat atc ttg ctg 48Val Asn Ile Asn Val Ala Asn Leu Leu Asn Gly Asn Tyr Ile Leu Leu1 5 10 15tta ttc gtc gtc tta gcg ttg ggg tta tgc tta ggt aaa ttg cgc ctt 96Leu Phe Val Val Leu Ala Leu Gly Leu Cys Leu Gly Lys Leu Arg Leu 20 25 30ggc tct atc caa tta ggt aac gct att ggt gta ctg gtg gta tca ctg 144Gly Ser Ile Gln Leu Gly Asn Ala Ile Gly Val Leu Val Val Ser Leu 35 40 45ttg cta ggg caa cag cat ttc gcc atc aat act gaa gcg cta aat ctg 192Leu Leu Gly Gln Gln His Phe Ala Ile Asn Thr Glu Ala Leu Asn Leu 50 55 60ggc ttt atg ttg ttt att ttt tgt gtc ggt gtt gaa gcc ggt cca aac 240Gly Phe Met Leu Phe Ile Phe Cys Val Gly Val Glu Ala Gly Pro Asn65 70 75 80ttc ttt tct att ttc ttc cgt gat ggc aaa aat tac ctg atg ctc gcc 288Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn Tyr Leu Met Leu Ala 85 90 95ttg gtt atg gtg ggc agc gcg atg att tta gcg ttg ggg ctt ggc aaa 336Leu Val Met Val Gly Ser Ala Met Ile Leu Ala Leu Gly Leu Gly Lys 100 105 110tta ttt ggt tgg gat atc ggt ctg acc gcc ggg atg ctg gca ggg tca 384Leu Phe Gly Trp Asp Ile Gly Leu Thr Ala Gly Met Leu Ala Gly Ser 115 120 125atg aca tcg acc ccc gtt ttg gtt ggg gcc ggt gat acg cta cgc cat 432Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly Asp Thr Leu Arg His 130 135 140acc atg gct aat ggt tcg tca ctg caa caa gca cag gat aat ttg agc 480Thr Met Ala Asn Gly Ser Ser Leu Gln Gln Ala Gln Asp Asn Leu Ser145 150 155 160ctc ggc tat gcc ctg act tat ctt atc ggg ctg gtt agc ctg att tta 528Leu Gly Tyr Ala Leu Thr Tyr Leu Ile Gly Leu Val Ser Leu Ile Leu 165 170 175ggt gcg cgc tat tta cct aag ctt cag cat cag gat tta ccg acc agc 576Gly Ala Arg Tyr Leu Pro Lys Leu Gln His Gln Asp Leu Pro Thr Ser 180 185 190gcc caa caa att gcc cgc gaa cga ggg ctg gac acc gat agc cag cgt 624Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp Thr Asp Ser Gln Arg 195 200 205aaa gtg tat ttg cct gtc atc cgt gct tat cgt gta ggc ccg gag ctg 672Lys Val Tyr Leu Pro Val Ile Arg Ala Tyr Arg Val Gly Pro Glu Leu 210 215 220gtt gcc tgg gca gat ggt aaa aac ttg cgt gaa tta ggg att tac cgc 720Val Ala Trp Ala Asp Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr Arg225 230 235 240caa acc ggt tgc tat atc gag cgt atc cgc cgc aac ggt att ctc gcg 768Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu Ala 245 250 255aat ccg gat ggt gac gcc gtg cta caa gtc ggt gat gaa atc tcg ttg 816Asn Pro Asp Gly Asp Ala Val Leu Gln Val Gly Asp Glu Ile Ser Leu 260 265 270gtg ggt tac cca gat gcc cat tcc cgc ctt gac ccc agc ttc cgt aat 864Val Gly Tyr Pro Asp Ala His Ser Arg Leu Asp Pro Ser Phe Arg Asn 275 280 285ggc aaa gaa gtt ttc gac cgt gat ctg ttg gat atg cgt atc gtc acc 912Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp Met Arg Ile Val Thr 290 295 300gaa gag atc gtg gtc aaa aat agc aat gct gtc ggt aaa cgc ctc agc 960Glu Glu Ile Val Val Lys Asn Ser Asn Ala Val Gly Lys Arg Leu Ser305 310 315 320cat tta aaa ctc acc gat cac ggt tgc ttc ctt aat cgg gtc att cgt 1008His Leu Lys Leu Thr Asp His Gly Cys Phe Leu Asn Arg Val Ile Arg 325 330 335agc caa att gaa atg ccc att gat gat aac gtg gtg ttg aat aaa ggt 1056Ser Gln Ile Glu Met Pro Ile Asp Asp Asn Val Val Leu Asn Lys Gly 340 345 350gat gtg ctg caa gtc agc ggt gat gcc cgg cga gtc aaa agc gtg gca 1104Asp Val Leu Gln Val Ser Gly Asp Ala Arg Arg Val Lys Ser Val Ala 355 360 365gaa aaa att ggc ttt atc tct att cac agt cag gtc act gac ctg ctg 1152Glu Lys Ile Gly Phe Ile Ser Ile His Ser Gln Val Thr Asp Leu Leu 370 375 380gcc ttc tgc tca ttc ttt att ctt ggg tta atg att ggc ctg att act 1200Ala Phe Cys Ser Phe Phe Ile Leu Gly Leu Met Ile Gly Leu Ile Thr385 390 395 400ttc cag ttc agc aat ttc agc ttc ggt att ggc aat gcc gca ggg ctg 1248Phe Gln Phe Ser Asn Phe Ser Phe Gly Ile Gly Asn Ala Ala Gly Leu 405 410 415tta tta gca ggg atc atg ctg gga ttt tta cgt gcc aac cac cct act 1296Leu Leu Ala Gly Ile Met Leu Gly Phe Leu Arg Ala Asn His Pro Thr 420 425 430ttt ggc tat atc ccg caa ggc gcc ttg aat atg gtg aaa gag ttc ggg 1344Phe Gly Tyr Ile Pro Gln Gly Ala Leu Asn Met Val Lys Glu Phe Gly 435 440 445tta atg gtc ttt atg gca ggt gtc ggg tta agt gcc ggg ggc ggc att 1392Leu Met Val Phe Met Ala Gly Val Gly Leu Ser Ala Gly Gly Gly Ile 450 455 460aat agc agc ctg ggc gcc gtc ggt gga caa atg ttg att tcc ggt ttg 1440Asn Ser Ser Leu Gly Ala Val Gly Gly Gln Met Leu Ile Ser Gly Leu465 470 475 480ata gtc agt ttg gtt ccg gtg gtg atc tgc ttt gtt ttt ggt gct tat 1488Ile Val Ser Leu Val Pro Val Val Ile Cys Phe Val Phe Gly Ala Tyr 485 490 495gtt ctg cgt atg aac cgt gca ctg ctt ttt ggt gcc att atg ggg gcc 1536Val Leu Arg Met Asn Arg Ala Leu Leu Phe Gly Ala Ile Met Gly Ala 500 505 510aga acc tgt gca ccc gcc atg gac atc atc agt gat acc gcg cgt agc 1584Arg Thr Cys Ala Pro Ala Met Asp Ile Ile Ser Asp Thr Ala Arg Ser 515 520 525aat att ccc gca tta ggc tac gcg ggg act tac gct atc gcc aac gtc 1632Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn Val 530 535 540tta ctg acc ttg gct ggc tcg ttg att gtc atc ctc tgg cca ggg ata 1680Leu Leu Thr Leu Ala Gly Ser Leu Ile Val Ile Leu Trp Pro Gly Ile545 550 555 560tta ggt tag 1689Leu Gly27562PRTYersinia pestis 27Val Asn Ile Asn Val Ala Asn Leu Leu Asn Gly Asn Tyr Ile Leu Leu1 5 10 15Leu Phe Val Val Leu Ala Leu Gly Leu Cys Leu Gly Lys Leu Arg Leu 20 25 30Gly Ser Ile Gln Leu Gly Asn Ala Ile Gly Val Leu Val Val Ser Leu 35 40 45Leu Leu Gly Gln Gln His Phe Ala Ile Asn Thr Glu Ala Leu Asn Leu 50 55 60Gly Phe Met Leu Phe Ile Phe Cys Val Gly Val Glu Ala Gly Pro Asn65 70 75 80Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn Tyr Leu Met Leu Ala 85 90 95Leu Val Met Val Gly Ser Ala Met Ile Leu Ala Leu Gly Leu Gly Lys 100 105 110Leu Phe Gly Trp Asp Ile Gly Leu Thr Ala Gly Met Leu Ala Gly Ser 115 120 125Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly Asp Thr Leu Arg His 130 135 140Thr Met Ala Asn Gly Ser Ser Leu Gln Gln Ala Gln Asp Asn Leu Ser145 150 155 160Leu Gly Tyr Ala Leu Thr Tyr Leu Ile Gly Leu Val Ser Leu Ile Leu 165 170 175Gly Ala Arg Tyr Leu Pro Lys Leu Gln His Gln Asp Leu Pro Thr Ser 180 185 190Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp Thr Asp Ser Gln Arg 195 200 205Lys Val Tyr Leu Pro Val Ile Arg Ala Tyr Arg Val Gly Pro Glu Leu 210 215 220Val Ala Trp Ala Asp Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr Arg225 230 235 240Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu Ala 245 250 255Asn Pro Asp Gly Asp Ala Val Leu Gln Val Gly Asp Glu Ile Ser Leu 260 265 270Val Gly Tyr Pro Asp Ala His Ser Arg Leu Asp Pro Ser Phe Arg Asn 275 280 285Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp Met Arg Ile Val Thr 290 295 300Glu Glu Ile Val Val Lys Asn Ser Asn Ala Val Gly Lys Arg Leu Ser305 310 315 320His Leu Lys Leu Thr Asp His Gly Cys Phe Leu Asn Arg Val Ile Arg 325 330 335Ser Gln Ile Glu Met Pro Ile Asp Asp Asn Val Val Leu Asn Lys Gly 340 345 350Asp Val Leu Gln Val Ser Gly Asp Ala Arg Arg Val Lys Ser Val Ala 355 360 365Glu Lys Ile Gly Phe Ile Ser Ile His Ser Gln Val Thr Asp Leu Leu 370 375 380Ala Phe Cys Ser Phe Phe Ile Leu Gly Leu Met Ile Gly Leu Ile Thr385 390 395 400Phe Gln Phe Ser Asn Phe Ser Phe Gly Ile Gly Asn Ala Ala Gly Leu 405 410 415Leu Leu Ala Gly Ile Met Leu Gly Phe Leu Arg Ala Asn His Pro Thr 420 425 430Phe Gly Tyr Ile Pro Gln Gly Ala Leu Asn Met Val Lys Glu Phe Gly 435 440 445Leu Met Val Phe Met Ala Gly Val Gly Leu Ser Ala Gly Gly Gly Ile 450 455 460Asn Ser Ser Leu Gly Ala Val Gly Gly Gln Met Leu Ile Ser Gly Leu465 470 475 480Ile Val Ser Leu Val Pro Val Val Ile Cys Phe Val Phe Gly Ala Tyr 485 490 495Val Leu Arg Met Asn Arg Ala Leu Leu Phe Gly Ala Ile Met Gly Ala 500 505 510Arg Thr Cys Ala Pro Ala Met Asp Ile Ile Ser Asp Thr Ala Arg Ser 515 520 525Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn Val 530 535 540Leu Leu Thr Leu Ala Gly Ser Leu Ile Val Ile Leu Trp Pro Gly Ile545 550 555 560Leu Gly281689DNAErwinia carotovoraCDS(1)..(1689) 28atg aat ata aac gtc gct gat ttg tta aac ggg aat tac att ctg ctt 48Met Asn Ile Asn Val Ala Asp Leu Leu Asn Gly Asn Tyr Ile Leu Leu1 5 10 15tta ttc gtg gtt ctt tca tta gga ctc tgt ctg ggg aaa ttg cgc ctc 96Leu Phe Val Val Leu Ser Leu Gly Leu Cys Leu Gly Lys Leu Arg Leu 20 25 30ggg cca gta caa ctc ggt aat tct att ggc gtt tta gtg gtt tct tta 144Gly Pro Val Gln Leu Gly Asn Ser Ile Gly Val Leu Val Val Ser Leu 35 40 45tta ctc ggc caa caa cat ttt tcg att aat acc gaa gca ctg agc ctc 192Leu Leu Gly Gln Gln His Phe Ser Ile Asn Thr Glu Ala Leu Ser Leu 50 55 60ggt ttt atg tta ttt att ttt tgc gtg gga gta gaa gcc ggg ccg aat 240Gly Phe Met Leu Phe Ile Phe Cys Val Gly Val Glu Ala Gly Pro Asn65 70 75 80ttc ttt tct att ttc ttc cgc gac ggg aaa aat tat ttc atg ctg gcg 288Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn Tyr Phe Met Leu Ala 85 90 95ctg gtg atg gtc ggc agc gcc atg tta ctg gcg tta ggg tta ggc aaa 336Leu Val Met Val Gly Ser Ala Met Leu Leu Ala Leu Gly Leu Gly Lys 100 105 110ctt ttc ggc tgg gga atc ggc ctg acc gcg ggg atg ctg gca gga tcc 384Leu Phe Gly Trp Gly Ile Gly Leu Thr Ala Gly Met Leu Ala Gly Ser 115 120 125atg aca tcg aca ccg gtg ctg gtc ggt gca ggc gac aca cta cgc aat 432Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly Asp Thr Leu Arg Asn 130 135 140acc gcc agt ttg ggt ggt caa ctc ggt gtt gaa caa gat cat ttg agc 480Thr Ala Ser Leu Gly Gly Gln Leu Gly Val Glu Gln Asp His Leu Ser145 150 155 160ctg ggc tat gcg ctg acg tat ctg gtc ggg ctg gtc agt ctc att ttt 528Leu Gly Tyr Ala Leu Thr Tyr Leu Val Gly Leu Val Ser Leu Ile Phe 165 170 175ggt gca cgc tat ctg ccc aaa ctt cag cat

cag gat ctt ccc acc agt 576Gly Ala Arg Tyr Leu Pro Lys Leu Gln His Gln Asp Leu Pro Thr Ser 180 185 190gcg cag caa att gca cgc gag cgc ggg ctg gat gta gac agc caa aga 624Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp Val Asp Ser Gln Arg 195 200 205aaa gtg tat ctg cca gtg att cgt gcc tat cgc gtc ggg cca gag ttg 672Lys Val Tyr Leu Pro Val Ile Arg Ala Tyr Arg Val Gly Pro Glu Leu 210 215 220gtt gac tgg gcg gct ggc aaa aac ttg cgc gaa ctg gga atc tat cgc 720Val Asp Trp Ala Ala Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr Arg225 230 235 240cag acc ggt tgc tac atc gag cgc att cga cgc aac gga att ctg gca 768Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu Ala 245 250 255aat ccc gac ggg gat gcc gtc ttg caa atc ggc gac gag atc gcg ctg 816Asn Pro Asp Gly Asp Ala Val Leu Gln Ile Gly Asp Glu Ile Ala Leu 260 265 270gtc ggt tat ccc gat gca cac tct cgt ctg aac tcc aat ttc cgc gat 864Val Gly Tyr Pro Asp Ala His Ser Arg Leu Asn Ser Asn Phe Arg Asp 275 280 285ggc aaa gaa gtt ttc gac cgt aat ttg ttg gac atg cgc atc gtg acg 912Gly Lys Glu Val Phe Asp Arg Asn Leu Leu Asp Met Arg Ile Val Thr 290 295 300gaa gag att gtc gtc aag aat cac aac gcc gtc ggt aag cgc ctc agc 960Glu Glu Ile Val Val Lys Asn His Asn Ala Val Gly Lys Arg Leu Ser305 310 315 320caa ttg aag ctg act gac cac ggc tgt ttc ctt aac cgt att gtt cgc 1008Gln Leu Lys Leu Thr Asp His Gly Cys Phe Leu Asn Arg Ile Val Arg 325 330 335agc cag ata gaa atg ccg ctc gac gat aac gtc gtg ctg aat aaa ggc 1056Ser Gln Ile Glu Met Pro Leu Asp Asp Asn Val Val Leu Asn Lys Gly 340 345 350gat gtc tta cag gtc agc ggt gac gcc cgt cgg gta aaa agc att gct 1104Asp Val Leu Gln Val Ser Gly Asp Ala Arg Arg Val Lys Ser Ile Ala 355 360 365gag cga atc ggg ttc att tct att cac agt cag gtc acc gat tta ctg 1152Glu Arg Ile Gly Phe Ile Ser Ile His Ser Gln Val Thr Asp Leu Leu 370 375 380gcc ttc tgc gcc ttt ttt gtt atc ggc gtg atg gtc ggg ctg atc acc 1200Ala Phe Cys Ala Phe Phe Val Ile Gly Val Met Val Gly Leu Ile Thr385 390 395 400atc cag ttc agc aac ttc acc ttt ggc atc ggt aac gcg gct ggg ttg 1248Ile Gln Phe Ser Asn Phe Thr Phe Gly Ile Gly Asn Ala Ala Gly Leu 405 410 415ctc ttc gcc ggt atc atg ctg ggc ttc ctg cgc gcc aac cac ccg acc 1296Leu Phe Ala Gly Ile Met Leu Gly Phe Leu Arg Ala Asn His Pro Thr 420 425 430ttc ggc tat att ccg caa ggc gcg ctt aac atg gtc aaa gag ttt ggc 1344Phe Gly Tyr Ile Pro Gln Gly Ala Leu Asn Met Val Lys Glu Phe Gly 435 440 445ctg atg gtc ttt atg gcg ggc gta gga ctg agt gcc ggt agc acc atc 1392Leu Met Val Phe Met Ala Gly Val Gly Leu Ser Ala Gly Ser Thr Ile 450 455 460aac agt agc cta gga gaa gtc ggt atc cag atg ctg gct tca ggg ctg 1440Asn Ser Ser Leu Gly Glu Val Gly Ile Gln Met Leu Ala Ser Gly Leu465 470 475 480att gtc agt ctc gtg cca gtg gtg att tgt ttt ctg ttc ggc gcg tat 1488Ile Val Ser Leu Val Pro Val Val Ile Cys Phe Leu Phe Gly Ala Tyr 485 490 495gtg ctg aaa atg aat cga gcg ctg ctg ttt ggt gcc atg atg ggc gct 1536Val Leu Lys Met Asn Arg Ala Leu Leu Phe Gly Ala Met Met Gly Ala 500 505 510cgg acc tgc gcg cca gcc atg gag atc atc agc gat acc gct cgc agt 1584Arg Thr Cys Ala Pro Ala Met Glu Ile Ile Ser Asp Thr Ala Arg Ser 515 520 525aat att cct gct ctc ggc tat gcg ggt acc tac gcc atc gct aac gtt 1632Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn Val 530 535 540ctg ctc aca tta gca ggt tca ctt atc gtg gtt atc tgg cct gaa ttg 1680Leu Leu Thr Leu Ala Gly Ser Leu Ile Val Val Ile Trp Pro Glu Leu545 550 555 560ccc ggc tga 1689Pro Gly29562PRTErwinia carotovora 29Met Asn Ile Asn Val Ala Asp Leu Leu Asn Gly Asn Tyr Ile Leu Leu1 5 10 15Leu Phe Val Val Leu Ser Leu Gly Leu Cys Leu Gly Lys Leu Arg Leu 20 25 30Gly Pro Val Gln Leu Gly Asn Ser Ile Gly Val Leu Val Val Ser Leu 35 40 45Leu Leu Gly Gln Gln His Phe Ser Ile Asn Thr Glu Ala Leu Ser Leu 50 55 60Gly Phe Met Leu Phe Ile Phe Cys Val Gly Val Glu Ala Gly Pro Asn65 70 75 80Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn Tyr Phe Met Leu Ala 85 90 95Leu Val Met Val Gly Ser Ala Met Leu Leu Ala Leu Gly Leu Gly Lys 100 105 110Leu Phe Gly Trp Gly Ile Gly Leu Thr Ala Gly Met Leu Ala Gly Ser 115 120 125Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly Asp Thr Leu Arg Asn 130 135 140Thr Ala Ser Leu Gly Gly Gln Leu Gly Val Glu Gln Asp His Leu Ser145 150 155 160Leu Gly Tyr Ala Leu Thr Tyr Leu Val Gly Leu Val Ser Leu Ile Phe 165 170 175Gly Ala Arg Tyr Leu Pro Lys Leu Gln His Gln Asp Leu Pro Thr Ser 180 185 190Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp Val Asp Ser Gln Arg 195 200 205Lys Val Tyr Leu Pro Val Ile Arg Ala Tyr Arg Val Gly Pro Glu Leu 210 215 220Val Asp Trp Ala Ala Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr Arg225 230 235 240Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu Ala 245 250 255Asn Pro Asp Gly Asp Ala Val Leu Gln Ile Gly Asp Glu Ile Ala Leu 260 265 270Val Gly Tyr Pro Asp Ala His Ser Arg Leu Asn Ser Asn Phe Arg Asp 275 280 285Gly Lys Glu Val Phe Asp Arg Asn Leu Leu Asp Met Arg Ile Val Thr 290 295 300Glu Glu Ile Val Val Lys Asn His Asn Ala Val Gly Lys Arg Leu Ser305 310 315 320Gln Leu Lys Leu Thr Asp His Gly Cys Phe Leu Asn Arg Ile Val Arg 325 330 335Ser Gln Ile Glu Met Pro Leu Asp Asp Asn Val Val Leu Asn Lys Gly 340 345 350Asp Val Leu Gln Val Ser Gly Asp Ala Arg Arg Val Lys Ser Ile Ala 355 360 365Glu Arg Ile Gly Phe Ile Ser Ile His Ser Gln Val Thr Asp Leu Leu 370 375 380Ala Phe Cys Ala Phe Phe Val Ile Gly Val Met Val Gly Leu Ile Thr385 390 395 400Ile Gln Phe Ser Asn Phe Thr Phe Gly Ile Gly Asn Ala Ala Gly Leu 405 410 415Leu Phe Ala Gly Ile Met Leu Gly Phe Leu Arg Ala Asn His Pro Thr 420 425 430Phe Gly Tyr Ile Pro Gln Gly Ala Leu Asn Met Val Lys Glu Phe Gly 435 440 445Leu Met Val Phe Met Ala Gly Val Gly Leu Ser Ala Gly Ser Thr Ile 450 455 460Asn Ser Ser Leu Gly Glu Val Gly Ile Gln Met Leu Ala Ser Gly Leu465 470 475 480Ile Val Ser Leu Val Pro Val Val Ile Cys Phe Leu Phe Gly Ala Tyr 485 490 495Val Leu Lys Met Asn Arg Ala Leu Leu Phe Gly Ala Met Met Gly Ala 500 505 510Arg Thr Cys Ala Pro Ala Met Glu Ile Ile Ser Asp Thr Ala Arg Ser 515 520 525Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn Val 530 535 540Leu Leu Thr Leu Ala Gly Ser Leu Ile Val Val Ile Trp Pro Glu Leu545 550 555 560Pro Gly301677DNAVibrio choleraeCDS(1)..(1677) 30gtg aac atc gac gtc gtt ttg ttg cta gag caa aat cca att ttg ctg 48Val Asn Ile Asp Val Val Leu Leu Leu Glu Gln Asn Pro Ile Leu Leu1 5 10 15ata ttt gtt gtc ctc gcc atc ggt ctc tct ttc ggg aaa att cgc ttt 96Ile Phe Val Val Leu Ala Ile Gly Leu Ser Phe Gly Lys Ile Arg Phe 20 25 30ggc agc ttc caa ttg ggt aat tcg att gga gtg ctc atc acc tct ctg 144Gly Ser Phe Gln Leu Gly Asn Ser Ile Gly Val Leu Ile Thr Ser Leu 35 40 45atc atg ggg cat ctt ggt ttt tcc ttt aca ccg gaa gca tta acc atc 192Ile Met Gly His Leu Gly Phe Ser Phe Thr Pro Glu Ala Leu Thr Ile 50 55 60ggc ttt atg ctc ttt att tat tgt gtc ggc att gaa gcg ggt cct aac 240Gly Phe Met Leu Phe Ile Tyr Cys Val Gly Ile Glu Ala Gly Pro Asn65 70 75 80ttc ttt ggt att ttc ttc cgc gat ggt aaa cac tac tta att cta agc 288Phe Phe Gly Ile Phe Phe Arg Asp Gly Lys His Tyr Leu Ile Leu Ser 85 90 95tta gtg gtc ttg atc act gcg act tgg att gcc tac ttt ggt ggt tac 336Leu Val Val Leu Ile Thr Ala Thr Trp Ile Ala Tyr Phe Gly Gly Tyr 100 105 110tat ctt aat ctg gat tat ggc tta gcc gcc ggg atg atg gcg ggc gca 384Tyr Leu Asn Leu Asp Tyr Gly Leu Ala Ala Gly Met Met Ala Gly Ala 115 120 125tta acc tca aca ccc gta ctg gtt ggt gct cag gat gcg ctc aat tct 432Leu Thr Ser Thr Pro Val Leu Val Gly Ala Gln Asp Ala Leu Asn Ser 130 135 140ggg ctt gcc gcg gta cca aga cat atg gat tta tca ctg att tta gat 480Gly Leu Ala Ala Val Pro Arg His Met Asp Leu Ser Leu Ile Leu Asp145 150 155 160aat gtc tcg gtc ggt tat gcg atg gcc tac ttg atc ggc ctt atc agc 528Asn Val Ser Val Gly Tyr Ala Met Ala Tyr Leu Ile Gly Leu Ile Ser 165 170 175atg atc atg ttt gct aaa tta ctc cct aag ttg caa aag caa aac ctg 576Met Ile Met Phe Ala Lys Leu Leu Pro Lys Leu Gln Lys Gln Asn Leu 180 185 190tcc gat tcg gct caa caa atc gcg caa gaa cga ggg tta ggc agt cag 624Ser Asp Ser Ala Gln Gln Ile Ala Gln Glu Arg Gly Leu Gly Ser Gln 195 200 205cgt aaa gtc tat ctg ccg atc atc cgt gcc tat cga gtc gga cct gaa 672Arg Lys Val Tyr Leu Pro Ile Ile Arg Ala Tyr Arg Val Gly Pro Glu 210 215 220 ctg att aac tgg atc gac ggt cgt aat ctg cgc gag ctg ggt atc tac 720Leu Ile Asn Trp Ile Asp Gly Arg Asn Leu Arg Glu Leu Gly Ile Tyr225 230 235 240cgt caa aca ggc tgt tat att gag cgt atc cgc cga cat ggc att ttg 768Arg Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg His Gly Ile Leu 245 250 255gcc cat cct gat ggt gat gcc att ttg caa gaa ggg gat gaa att gct 816Ala His Pro Asp Gly Asp Ala Ile Leu Gln Glu Gly Asp Glu Ile Ala 260 265 270ctc gta ggt ttc cca gat agt cac gct cgt ctt gac ccg agc ttt cgc 864Leu Val Gly Phe Pro Asp Ser His Ala Arg Leu Asp Pro Ser Phe Arg 275 280 285aat ggc aaa gaa gtg ttt gac cgt aac tta ctc gac cta cga att tca 912Asn Gly Lys Glu Val Phe Asp Arg Asn Leu Leu Asp Leu Arg Ile Ser 290 295 300gaa gaa gag atc gtg gta aaa agt gac agc att gcg gga aaa cgt ctc 960Glu Glu Glu Ile Val Val Lys Ser Asp Ser Ile Ala Gly Lys Arg Leu305 310 315 320tcc gat ctt aat ctt tcg gaa tac ggc tgc ttc ctt aac cga gtc gtt 1008Ser Asp Leu Asn Leu Ser Glu Tyr Gly Cys Phe Leu Asn Arg Val Val 325 330 335cgt gcg caa atc gaa atg ccg atg gat ctg gat atc gtg ctc gcc aaa 1056Arg Ala Gln Ile Glu Met Pro Met Asp Leu Asp Ile Val Leu Ala Lys 340 345 350ggc gat gtc ctg caa gtc agc ggc gag aaa agc aaa gta aag ggg ctg 1104Gly Asp Val Leu Gln Val Ser Gly Glu Lys Ser Lys Val Lys Gly Leu 355 360 365gca gat aaa atc ggt ttt atc tct gta cac agc caa atg gct gat ctg 1152Ala Asp Lys Ile Gly Phe Ile Ser Val His Ser Gln Met Ala Asp Leu 370 375 380ctt gct ttc tgt agc ttt ttt att ctt ggc att ctg ttt ggt ttg gtc 1200Leu Ala Phe Cys Ser Phe Phe Ile Leu Gly Ile Leu Phe Gly Leu Val385 390 395 400acc atg acc ttt ggc caa gtg tcg ttt agc tta ggt aat gcg gtg ggg 1248Thr Met Thr Phe Gly Gln Val Ser Phe Ser Leu Gly Asn Ala Val Gly 405 410 415tta ctg ctc tct ggt atc act ttg ggc ttt ttg cga gcc aac cac ccg 1296Leu Leu Leu Ser Gly Ile Thr Leu Gly Phe Leu Arg Ala Asn His Pro 420 425 430act ttc ggt tat gtt ccg caa ggc gca ctc aat atg gtc aaa gac ctt 1344Thr Phe Gly Tyr Val Pro Gln Gly Ala Leu Asn Met Val Lys Asp Leu 435 440 445ggt cta gcc atc ttc atg gtc ggt att ggg ctc aat gca ggt agc aaa 1392Gly Leu Ala Ile Phe Met Val Gly Ile Gly Leu Asn Ala Gly Ser Lys 450 455 460atg ttc cag cat ctc tcc gaa gta ggt gtg caa gtg atc ggt tta gcc 1440Met Phe Gln His Leu Ser Glu Val Gly Val Gln Val Ile Gly Leu Ala465 470 475 480ttt tta gtg agc gtt gta cct gtc gtg ttc gct tac ttg gtt ggc gcc 1488Phe Leu Val Ser Val Val Pro Val Val Phe Ala Tyr Leu Val Gly Ala 485 490 495tac att cta aag atg aac cgc gct cta ttg ttt ggt gcg att att ggc 1536Tyr Ile Leu Lys Met Asn Arg Ala Leu Leu Phe Gly Ala Ile Ile Gly 500 505 510gcg cgc acc tgt gct ccg gcg atg gat gtg gtt aat gaa tac gct aag 1584Ala Arg Thr Cys Ala Pro Ala Met Asp Val Val Asn Glu Tyr Ala Lys 515 520 525tct acc att cca gca ctg ggt tat gcg ggc acc tac gcg att gcc aat 1632Ser Thr Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn 530 535 540atc tta atg acc tta acg ggt acc atc ttt atc ttg ctc agt taa 1677Ile Leu Met Thr Leu Thr Gly Thr Ile Phe Ile Leu Leu Ser545 550 55531558PRTVibrio cholerae 31Val Asn Ile Asp Val Val Leu Leu Leu Glu Gln Asn Pro Ile Leu Leu1 5 10 15Ile Phe Val Val Leu Ala Ile Gly Leu Ser Phe Gly Lys Ile Arg Phe 20 25 30Gly Ser Phe Gln Leu Gly Asn Ser Ile Gly Val Leu Ile Thr Ser Leu 35 40 45Ile Met Gly His Leu Gly Phe Ser Phe Thr Pro Glu Ala Leu Thr Ile 50 55 60Gly Phe Met Leu Phe Ile Tyr Cys Val Gly Ile Glu Ala Gly Pro Asn65 70 75 80Phe Phe Gly Ile Phe Phe Arg Asp Gly Lys His Tyr Leu Ile Leu Ser 85 90 95Leu Val Val Leu Ile Thr Ala Thr Trp Ile Ala Tyr Phe Gly Gly Tyr 100 105 110Tyr Leu Asn Leu Asp Tyr Gly Leu Ala Ala Gly Met Met Ala Gly Ala 115 120 125Leu Thr Ser Thr Pro Val Leu Val Gly Ala Gln Asp Ala Leu Asn Ser 130 135 140Gly Leu Ala Ala Val Pro Arg His Met Asp Leu Ser Leu Ile Leu Asp145 150 155 160Asn Val Ser Val Gly Tyr Ala Met Ala Tyr Leu Ile Gly Leu Ile Ser 165 170 175Met Ile Met Phe Ala Lys Leu Leu Pro Lys Leu Gln Lys Gln Asn Leu 180 185 190Ser Asp Ser Ala Gln Gln Ile Ala Gln Glu Arg Gly Leu Gly Ser Gln 195 200 205Arg Lys Val Tyr Leu Pro Ile Ile Arg Ala Tyr Arg Val Gly Pro Glu 210 215 220Leu Ile Asn Trp Ile Asp Gly Arg Asn Leu Arg Glu Leu Gly Ile Tyr225 230 235 240Arg Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg His Gly Ile Leu 245 250 255Ala His Pro Asp Gly Asp Ala Ile Leu Gln Glu Gly Asp Glu Ile Ala 260 265 270Leu Val Gly Phe Pro Asp Ser His Ala Arg Leu Asp Pro Ser Phe Arg 275 280 285Asn Gly Lys Glu Val Phe Asp Arg Asn Leu Leu Asp Leu Arg Ile Ser 290 295 300Glu Glu Glu Ile Val Val Lys Ser Asp Ser Ile Ala Gly Lys Arg Leu305 310 315 320Ser Asp Leu Asn Leu Ser Glu Tyr Gly Cys Phe Leu Asn Arg Val Val 325 330 335Arg Ala Gln Ile Glu Met Pro Met Asp Leu Asp Ile Val Leu Ala Lys 340 345 350Gly Asp Val Leu Gln Val Ser Gly Glu Lys Ser Lys Val Lys Gly Leu 355 360 365Ala Asp Lys Ile Gly Phe Ile Ser Val His Ser Gln Met Ala Asp Leu 370 375 380Leu Ala Phe Cys Ser Phe Phe Ile Leu Gly

Ile Leu Phe Gly Leu Val385 390 395 400Thr Met Thr Phe Gly Gln Val Ser Phe Ser Leu Gly Asn Ala Val Gly 405 410 415Leu Leu Leu Ser Gly Ile Thr Leu Gly Phe Leu Arg Ala Asn His Pro 420 425 430Thr Phe Gly Tyr Val Pro Gln Gly Ala Leu Asn Met Val Lys Asp Leu 435 440 445Gly Leu Ala Ile Phe Met Val Gly Ile Gly Leu Asn Ala Gly Ser Lys 450 455 460Met Phe Gln His Leu Ser Glu Val Gly Val Gln Val Ile Gly Leu Ala465 470 475 480Phe Leu Val Ser Val Val Pro Val Val Phe Ala Tyr Leu Val Gly Ala 485 490 495Tyr Ile Leu Lys Met Asn Arg Ala Leu Leu Phe Gly Ala Ile Ile Gly 500 505 510Ala Arg Thr Cys Ala Pro Ala Met Asp Val Val Asn Glu Tyr Ala Lys 515 520 525Ser Thr Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn 530 535 540Ile Leu Met Thr Leu Thr Gly Thr Ile Phe Ile Leu Leu Ser545 550 555321686DNAAeromonas hydrophilaCDS(1)..(1686) 32atg acc ata gat ttt gtg aca ctg cta cat caa agc gac tcc ctg ctg 48Met Thr Ile Asp Phe Val Thr Leu Leu His Gln Ser Asp Ser Leu Leu1 5 10 15ctg ttc gtg gta ctg gcc ttc ggc ctg ctg ctc ggt aaa gtc agg ttc 96Leu Phe Val Val Leu Ala Phe Gly Leu Leu Leu Gly Lys Val Arg Phe 20 25 30ggc aac ttc cag atc ggc aac acc atc ggc gtg ctg ttt acc gcc ctg 144Gly Asn Phe Gln Ile Gly Asn Thr Ile Gly Val Leu Phe Thr Ala Leu 35 40 45ctg ttc ggc cag atg ggc ttc gaa ttc acc gcc acc acg gag aac gtc 192Leu Phe Gly Gln Met Gly Phe Glu Phe Thr Ala Thr Thr Glu Asn Val 50 55 60ggc ttc atg ctg ttc atc ttc tgc gtg ggg ata gaa gcg ggg ccg cac 240Gly Phe Met Leu Phe Ile Phe Cys Val Gly Ile Glu Ala Gly Pro His65 70 75 80ttc ttc agc gtc ttc ctg cgt gac ggc atc cac tac atc acc ctc acc 288Phe Phe Ser Val Phe Leu Arg Asp Gly Ile His Tyr Ile Thr Leu Thr 85 90 95 ctg gtg atc ctg ctg acc gcc ctc ttc ctc acc gtg ggg ctc gcc aag 336Leu Val Ile Leu Leu Thr Ala Leu Phe Leu Thr Val Gly Leu Ala Lys 100 105 110ttc ttc aac ctg ggg ccg ggc atg gcg gcc ggc atc ctg gcc ggc tcg 384Phe Phe Asn Leu Gly Pro Gly Met Ala Ala Gly Ile Leu Ala Gly Ser 115 120 125ctc acc tcg acc ccg gcg ctg gtg ggt gcg cag gat gcc ctg cgc agc 432Leu Thr Ser Thr Pro Ala Leu Val Gly Ala Gln Asp Ala Leu Arg Ser 130 135 140ggc ctg ctc aac ctg ccc cat cag acc gac atg cag tcg gtg ctc gac 480Gly Leu Leu Asn Leu Pro His Gln Thr Asp Met Gln Ser Val Leu Asp145 150 155 160aac atg ggc atc ggc tat gcg ttg acc tat ctg gtg ggg ctg gtc ggc 528Asn Met Gly Ile Gly Tyr Ala Leu Thr Tyr Leu Val Gly Leu Val Gly 165 170 175ctg atg ctg gtg gtg cgc tac ctg cct tcg ctg gca cgg ctc gac ctc 576Leu Met Leu Val Val Arg Tyr Leu Pro Ser Leu Ala Arg Leu Asp Leu 180 185 190aac acc gag gcg cag aag atc gcc cgt gag cgc ggc ctc tcc gac aac 624Asn Thr Glu Ala Gln Lys Ile Ala Arg Glu Arg Gly Leu Ser Asp Asn 195 200 205gag agc cgc aag acc tac ctg ccc atc atc cgt gcc tac cgg gtt ggt 672Glu Ser Arg Lys Thr Tyr Leu Pro Ile Ile Arg Ala Tyr Arg Val Gly 210 215 220ccc gag ctc gcc gcc tgg atc ggc ggc cgc acc ctg cgc gag acc ggc 720Pro Glu Leu Ala Ala Trp Ile Gly Gly Arg Thr Leu Arg Glu Thr Gly225 230 235 240atc tac ccc cac acc ggc tgc tac gtg gag cgg atc cgc cgc aac ggc 768Ile Tyr Pro His Thr Gly Cys Tyr Val Glu Arg Ile Arg Arg Asn Gly 245 250 255atc ctg gcg agc ccg gat ggc gac gcc gtc atc cag gaa ggg gac gag 816Ile Leu Ala Ser Pro Asp Gly Asp Ala Val Ile Gln Glu Gly Asp Glu 260 265 270atc gcc ctg gtg ggt tac ccc gag agc cac gag aag ctc gac gtc aac 864Ile Ala Leu Val Gly Tyr Pro Glu Ser His Glu Lys Leu Asp Val Asn 275 280 285tac cgc aac ggc aag gag gtg ttc gac cgc aac ctg ctt gac ctg cag 912Tyr Arg Asn Gly Lys Glu Val Phe Asp Arg Asn Leu Leu Asp Leu Gln 290 295 300atc gtc acc gaa gag ata gtg gtg aaa aac gat gcg gtg gtg ggt cgc 960Ile Val Thr Glu Glu Ile Val Val Lys Asn Asp Ala Val Val Gly Arg305 310 315 320cat ctg gtg gag ctc aac ctc acc gag aag ggc tgc ttc ctc aac cgg 1008His Leu Val Glu Leu Asn Leu Thr Glu Lys Gly Cys Phe Leu Asn Arg 325 330 335gtg gtg cgc tcc cag atc gag atg ccg ttc gat cgc aac atc atg ctg 1056Val Val Arg Ser Gln Ile Glu Met Pro Phe Asp Arg Asn Ile Met Leu 340 345 350caa aag ggc gac gtg ctg cag atc agc ggc gag aag cag cgg gtc aag 1104Gln Lys Gly Asp Val Leu Gln Ile Ser Gly Glu Lys Gln Arg Val Lys 355 360 365ctg ctg gcc aac aag att ggc ttc atc agc atc cac agc cag acc acg 1152Leu Leu Ala Asn Lys Ile Gly Phe Ile Ser Ile His Ser Gln Thr Thr 370 375 380gat ctg gtg gcc ttc acc acc ttc ttc gtg ctg ggg ctg ctg ata ggc 1200Asp Leu Val Ala Phe Thr Thr Phe Phe Val Leu Gly Leu Leu Ile Gly385 390 395 400tcc gtc tcc ctg gtg ttc ggc cag ctc gag ttt ggg ctg ggc aac gcc 1248Ser Val Ser Leu Val Phe Gly Gln Leu Glu Phe Gly Leu Gly Asn Ala 405 410 415gtc ggc ctg ctg ctg gcg ggg atc ctg atg ggc tac ctg cgc gcc aac 1296Val Gly Leu Leu Leu Ala Gly Ile Leu Met Gly Tyr Leu Arg Ala Asn 420 425 430cac ccc acc gtc ggc tac gtg ccg ccg ggc gcc ctg cgt ctg gcc aag 1344His Pro Thr Val Gly Tyr Val Pro Pro Gly Ala Leu Arg Leu Ala Lys 435 440 445gat ctc ggt ctg gcg gtg ttc atg gtg agc acc ggc ctc aaa gct ggc 1392Asp Leu Gly Leu Ala Val Phe Met Val Ser Thr Gly Leu Lys Ala Gly 450 455 460ggc ggc att ctg gac cac ctg agc cag gtc ggt gcc gtg gtg ctc ttt 1440Gly Gly Ile Leu Asp His Leu Ser Gln Val Gly Ala Val Val Leu Phe465 470 475 480tcc ggc atg ctg gtg acc acc ctg ccg gtg ctg gtg ggc tac ctg ttc 1488Ser Gly Met Leu Val Thr Thr Leu Pro Val Leu Val Gly Tyr Leu Phe 485 490 495gga gtc tgg gtg ctg aag atg aac ccg gcc ctg ctg ctc ggc gcc atc 1536Gly Val Trp Val Leu Lys Met Asn Pro Ala Leu Leu Leu Gly Ala Ile 500 505 510acg ggg gcc cgc acc tgc gcc ccg gcc atg gac gtg gtg aac gag gcg 1584Thr Gly Ala Arg Thr Cys Ala Pro Ala Met Asp Val Val Asn Glu Ala 515 520 525gcc aac agc tcc atc ccg gcg ctc ggc tat gcc ggc acc tat gcg gtg 1632Ala Asn Ser Ser Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Val 530 535 540gcc aac gtc atg ctg acc ttg gcg ggc tcc ttc atc atc ggc ttc tgg 1680Ala Asn Val Met Leu Thr Leu Ala Gly Ser Phe Ile Ile Gly Phe Trp545 550 555 560ttc tag 1686Phe33561PRTAeromonas hydrophila 33Met Thr Ile Asp Phe Val Thr Leu Leu His Gln Ser Asp Ser Leu Leu1 5 10 15Leu Phe Val Val Leu Ala Phe Gly Leu Leu Leu Gly Lys Val Arg Phe 20 25 30Gly Asn Phe Gln Ile Gly Asn Thr Ile Gly Val Leu Phe Thr Ala Leu 35 40 45Leu Phe Gly Gln Met Gly Phe Glu Phe Thr Ala Thr Thr Glu Asn Val 50 55 60Gly Phe Met Leu Phe Ile Phe Cys Val Gly Ile Glu Ala Gly Pro His65 70 75 80Phe Phe Ser Val Phe Leu Arg Asp Gly Ile His Tyr Ile Thr Leu Thr 85 90 95Leu Val Ile Leu Leu Thr Ala Leu Phe Leu Thr Val Gly Leu Ala Lys 100 105 110Phe Phe Asn Leu Gly Pro Gly Met Ala Ala Gly Ile Leu Ala Gly Ser 115 120 125Leu Thr Ser Thr Pro Ala Leu Val Gly Ala Gln Asp Ala Leu Arg Ser 130 135 140Gly Leu Leu Asn Leu Pro His Gln Thr Asp Met Gln Ser Val Leu Asp145 150 155 160Asn Met Gly Ile Gly Tyr Ala Leu Thr Tyr Leu Val Gly Leu Val Gly 165 170 175Leu Met Leu Val Val Arg Tyr Leu Pro Ser Leu Ala Arg Leu Asp Leu 180 185 190Asn Thr Glu Ala Gln Lys Ile Ala Arg Glu Arg Gly Leu Ser Asp Asn 195 200 205Glu Ser Arg Lys Thr Tyr Leu Pro Ile Ile Arg Ala Tyr Arg Val Gly 210 215 220Pro Glu Leu Ala Ala Trp Ile Gly Gly Arg Thr Leu Arg Glu Thr Gly225 230 235 240Ile Tyr Pro His Thr Gly Cys Tyr Val Glu Arg Ile Arg Arg Asn Gly 245 250 255Ile Leu Ala Ser Pro Asp Gly Asp Ala Val Ile Gln Glu Gly Asp Glu 260 265 270Ile Ala Leu Val Gly Tyr Pro Glu Ser His Glu Lys Leu Asp Val Asn 275 280 285Tyr Arg Asn Gly Lys Glu Val Phe Asp Arg Asn Leu Leu Asp Leu Gln 290 295 300Ile Val Thr Glu Glu Ile Val Val Lys Asn Asp Ala Val Val Gly Arg305 310 315 320His Leu Val Glu Leu Asn Leu Thr Glu Lys Gly Cys Phe Leu Asn Arg 325 330 335Val Val Arg Ser Gln Ile Glu Met Pro Phe Asp Arg Asn Ile Met Leu 340 345 350Gln Lys Gly Asp Val Leu Gln Ile Ser Gly Glu Lys Gln Arg Val Lys 355 360 365Leu Leu Ala Asn Lys Ile Gly Phe Ile Ser Ile His Ser Gln Thr Thr 370 375 380Asp Leu Val Ala Phe Thr Thr Phe Phe Val Leu Gly Leu Leu Ile Gly385 390 395 400Ser Val Ser Leu Val Phe Gly Gln Leu Glu Phe Gly Leu Gly Asn Ala 405 410 415Val Gly Leu Leu Leu Ala Gly Ile Leu Met Gly Tyr Leu Arg Ala Asn 420 425 430His Pro Thr Val Gly Tyr Val Pro Pro Gly Ala Leu Arg Leu Ala Lys 435 440 445Asp Leu Gly Leu Ala Val Phe Met Val Ser Thr Gly Leu Lys Ala Gly 450 455 460Gly Gly Ile Leu Asp His Leu Ser Gln Val Gly Ala Val Val Leu Phe465 470 475 480Ser Gly Met Leu Val Thr Thr Leu Pro Val Leu Val Gly Tyr Leu Phe 485 490 495Gly Val Trp Val Leu Lys Met Asn Pro Ala Leu Leu Leu Gly Ala Ile 500 505 510Thr Gly Ala Arg Thr Cys Ala Pro Ala Met Asp Val Val Asn Glu Ala 515 520 525Ala Asn Ser Ser Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Val 530 535 540Ala Asn Val Met Leu Thr Leu Ala Gly Ser Phe Ile Ile Gly Phe Trp545 550 555 560Phe341638DNAPhotobacterium profundumCDS(1)..(1638) 34ttg ctt ttt gtt gtt ctc gct gtt ggt cta agt ttt ggt aaa att cgt 48Leu Leu Phe Val Val Leu Ala Val Gly Leu Ser Phe Gly Lys Ile Arg1 5 10 15ttt gct aag atg caa gta ggc aat tct att ggt gtt tta ctg act gcc 96Phe Ala Lys Met Gln Val Gly Asn Ser Ile Gly Val Leu Leu Thr Ala 20 25 30ata ctg ttc ggt aat gcc ggg ttt acc ttt aat act gaa gca tta aat 144Ile Leu Phe Gly Asn Ala Gly Phe Thr Phe Asn Thr Glu Ala Leu Asn 35 40 45att ggc ttt atg ctg ttt att ttt tgt gtg ggt ata gag gct ggc ccc 192Ile Gly Phe Met Leu Phe Ile Phe Cys Val Gly Ile Glu Ala Gly Pro 50 55 60aac ttt ttt ggt att ttc ttc cga gat ggt aaa cat tac ctt tta ctc 240Asn Phe Phe Gly Ile Phe Phe Arg Asp Gly Lys His Tyr Leu Leu Leu65 70 75 80gcc ctt gtt gtg ctg ctt tcc gcc ata gct atc act tta gcc atg act 288Ala Leu Val Val Leu Leu Ser Ala Ile Ala Ile Thr Leu Ala Met Thr 85 90 95cat tat ctt ggg ctt gat ata ggt tta gcg aca ggc tta atg gct ggt 336His Tyr Leu Gly Leu Asp Ile Gly Leu Ala Thr Gly Leu Met Ala Gly 100 105 110tca ttg aca gca aca cct gtt ctt gta ggt gca aaa gac gca ttg aat 384Ser Leu Thr Ala Thr Pro Val Leu Val Gly Ala Lys Asp Ala Leu Asn 115 120 125tcc gga tta tct ggc att agc gat cct gat atc att aaa caa acc att 432Ser Gly Leu Ser Gly Ile Ser Asp Pro Asp Ile Ile Lys Gln Thr Ile 130 135 140gat agc cta agc gtt ggc tat gct atg tct tat tta atg ggc tta atc 480Asp Ser Leu Ser Val Gly Tyr Ala Met Ser Tyr Leu Met Gly Leu Ile145 150 155 160agt ctt att ttt ctc gct aaa cta atg cct aaa tta cag aaa caa gac 528Ser Leu Ile Phe Leu Ala Lys Leu Met Pro Lys Leu Gln Lys Gln Asp 165 170 175ctg gct gaa tcg tcg caa caa att gcc cgt gaa cgc ggt att ggt gaa 576Leu Ala Glu Ser Ser Gln Gln Ile Ala Arg Glu Arg Gly Ile Gly Glu 180 185 190gtg ggg caa cgt aaa gta tat ttg cca atc att cgc gct tac cgt gtt 624Val Gly Gln Arg Lys Val Tyr Leu Pro Ile Ile Arg Ala Tyr Arg Val 195 200 205ggc cct gag ctt att aac tgg att gat agt cgt aac tta cgt gag ttg 672Gly Pro Glu Leu Ile Asn Trp Ile Asp Ser Arg Asn Leu Arg Glu Leu 210 215 220ggc atc tac cgc caa aca ggc tgc tac atc gaa cgt ata cgg cga aac 720Gly Ile Tyr Arg Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn225 230 235 240ggt ata tta gct aac cca gat ggt gat gct att tta caa gaa ggt gat 768Gly Ile Leu Ala Asn Pro Asp Gly Asp Ala Ile Leu Gln Glu Gly Asp 245 250 255gag att gcc tta gtg ggt tac cca gac agc cat gca cgt ttg gat ccc 816Glu Ile Ala Leu Val Gly Tyr Pro Asp Ser His Ala Arg Leu Asp Pro 260 265 270agc ttt cga aac ggc aaa gaa gtt ttc gac cgt gat ctt ctc gac ctt 864Ser Phe Arg Asn Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp Leu 275 280 285cgt att gtt gaa gaa gag att gta gtt aag aat gac aac atc tct ggc 912Arg Ile Val Glu Glu Glu Ile Val Val Lys Asn Asp Asn Ile Ser Gly 290 295 300aaa cgc cta tct gag cta aat tta tca gaa tac ggt tgt ttc ttg aac 960Lys Arg Leu Ser Glu Leu Asn Leu Ser Glu Tyr Gly Cys Phe Leu Asn305 310 315 320cgt gtt gtt cgc gca caa ata gaa atg ccg atg gac cat aat att ctt 1008Arg Val Val Arg Ala Gln Ile Glu Met Pro Met Asp His Asn Ile Leu 325 330 335ttg aat aaa ggg gat att ttg caa gtc agt gga gaa aaa agt cga gta 1056Leu Asn Lys Gly Asp Ile Leu Gln Val Ser Gly Glu Lys Ser Arg Val 340 345 350ctt ggt ctt gct gaa cgt att ggt ttt atc tcg att cac agt caa att 1104Leu Gly Leu Ala Glu Arg Ile Gly Phe Ile Ser Ile His Ser Gln Ile 355 360 365gcc gat cta tta gca ttc tgc tgt ttt ttt atc atc ggg tta ctt atc 1152Ala Asp Leu Leu Ala Phe Cys Cys Phe Phe Ile Ile Gly Leu Leu Ile 370 375 380ggt tcg att aca tta gca ttt ggt cac gtt gca ttt ggt tta ggt agt 1200Gly Ser Ile Thr Leu Ala Phe Gly His Val Ala Phe Gly Leu Gly Ser385 390 395 400gct gca ggg ctt ctg att gcc ggt att aca tta gga ttc cta cgt gct 1248Ala Ala Gly Leu Leu Ile Ala Gly Ile Thr Leu Gly Phe Leu Arg Ala 405 410 415aac cac cct act ttt ggc tat gta ccc caa ggt gcg ctt aat atg gcg 1296Asn His Pro Thr Phe Gly Tyr Val Pro Gln Gly Ala Leu Asn Met Ala 420 425 430aaa gat ctt ggc tta atg gtc ttc atg gta gga att ggt ttg agt gcg 1344Lys Asp Leu Gly Leu Met Val Phe Met Val Gly Ile Gly Leu Ser Ala 435 440 445ggt tct aat tta ttc gat tca ttt gca cat att ggc cct atg gtc tta 1392Gly Ser Asn Leu Phe Asp Ser Phe Ala His Ile Gly Pro Met Val Leu 450 455 460gtc acc agc tta atg gtg agt gtg atc cct gtt gta ttg gca tac ctg 1440Val Thr Ser Leu Met Val Ser Val Ile Pro Val Val Leu Ala Tyr Leu465 470 475 480ttt ggt gct tac gta ctg aag atg aac cgt gca ctt tta ttt ggt gcc 1488Phe Gly Ala Tyr Val Leu Lys Met Asn Arg Ala Leu Leu Phe Gly Ala 485 490 495atc att ggt gct cga act tgt gca cct gcg atg gat atg atc aat gaa 1536Ile Ile Gly Ala Arg Thr Cys Ala Pro Ala Met Asp Met Ile Asn Glu 500

505 510cat gct cga agc aca att cca gct tta ggc tat gct ggt act tat gcc 1584His Ala Arg Ser Thr Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala 515 520 525att gcc aac gta ctg tta acg att gcc ggt acg tta atc atc atc atg 1632Ile Ala Asn Val Leu Leu Thr Ile Ala Gly Thr Leu Ile Ile Ile Met 530 535 540aat taa 1638Asn54535545PRTPhotobacterium profundum 35Leu Leu Phe Val Val Leu Ala Val Gly Leu Ser Phe Gly Lys Ile Arg1 5 10 15Phe Ala Lys Met Gln Val Gly Asn Ser Ile Gly Val Leu Leu Thr Ala 20 25 30Ile Leu Phe Gly Asn Ala Gly Phe Thr Phe Asn Thr Glu Ala Leu Asn 35 40 45Ile Gly Phe Met Leu Phe Ile Phe Cys Val Gly Ile Glu Ala Gly Pro 50 55 60Asn Phe Phe Gly Ile Phe Phe Arg Asp Gly Lys His Tyr Leu Leu Leu65 70 75 80Ala Leu Val Val Leu Leu Ser Ala Ile Ala Ile Thr Leu Ala Met Thr 85 90 95His Tyr Leu Gly Leu Asp Ile Gly Leu Ala Thr Gly Leu Met Ala Gly 100 105 110Ser Leu Thr Ala Thr Pro Val Leu Val Gly Ala Lys Asp Ala Leu Asn 115 120 125Ser Gly Leu Ser Gly Ile Ser Asp Pro Asp Ile Ile Lys Gln Thr Ile 130 135 140Asp Ser Leu Ser Val Gly Tyr Ala Met Ser Tyr Leu Met Gly Leu Ile145 150 155 160Ser Leu Ile Phe Leu Ala Lys Leu Met Pro Lys Leu Gln Lys Gln Asp 165 170 175Leu Ala Glu Ser Ser Gln Gln Ile Ala Arg Glu Arg Gly Ile Gly Glu 180 185 190Val Gly Gln Arg Lys Val Tyr Leu Pro Ile Ile Arg Ala Tyr Arg Val 195 200 205Gly Pro Glu Leu Ile Asn Trp Ile Asp Ser Arg Asn Leu Arg Glu Leu 210 215 220Gly Ile Tyr Arg Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn225 230 235 240Gly Ile Leu Ala Asn Pro Asp Gly Asp Ala Ile Leu Gln Glu Gly Asp 245 250 255Glu Ile Ala Leu Val Gly Tyr Pro Asp Ser His Ala Arg Leu Asp Pro 260 265 270Ser Phe Arg Asn Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp Leu 275 280 285Arg Ile Val Glu Glu Glu Ile Val Val Lys Asn Asp Asn Ile Ser Gly 290 295 300Lys Arg Leu Ser Glu Leu Asn Leu Ser Glu Tyr Gly Cys Phe Leu Asn305 310 315 320Arg Val Val Arg Ala Gln Ile Glu Met Pro Met Asp His Asn Ile Leu 325 330 335Leu Asn Lys Gly Asp Ile Leu Gln Val Ser Gly Glu Lys Ser Arg Val 340 345 350Leu Gly Leu Ala Glu Arg Ile Gly Phe Ile Ser Ile His Ser Gln Ile 355 360 365Ala Asp Leu Leu Ala Phe Cys Cys Phe Phe Ile Ile Gly Leu Leu Ile 370 375 380Gly Ser Ile Thr Leu Ala Phe Gly His Val Ala Phe Gly Leu Gly Ser385 390 395 400Ala Ala Gly Leu Leu Ile Ala Gly Ile Thr Leu Gly Phe Leu Arg Ala 405 410 415Asn His Pro Thr Phe Gly Tyr Val Pro Gln Gly Ala Leu Asn Met Ala 420 425 430Lys Asp Leu Gly Leu Met Val Phe Met Val Gly Ile Gly Leu Ser Ala 435 440 445Gly Ser Asn Leu Phe Asp Ser Phe Ala His Ile Gly Pro Met Val Leu 450 455 460Val Thr Ser Leu Met Val Ser Val Ile Pro Val Val Leu Ala Tyr Leu465 470 475 480Phe Gly Ala Tyr Val Leu Lys Met Asn Arg Ala Leu Leu Phe Gly Ala 485 490 495Ile Ile Gly Ala Arg Thr Cys Ala Pro Ala Met Asp Met Ile Asn Glu 500 505 510His Ala Arg Ser Thr Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala 515 520 525Ile Ala Asn Val Leu Leu Thr Ile Ala Gly Thr Leu Ile Ile Ile Met 530 535 540Asn54536990DNAEscherichia coliCDS(1)..(990) 36atg aaa ctc gcc gtt tat agc aca aaa cag tac gac aag aag tac ctg 48Met Lys Leu Ala Val Tyr Ser Thr Lys Gln Tyr Asp Lys Lys Tyr Leu1 5 10 15caa cag gtg aac gag tcc ttt ggc ttt gag ctg gaa ttt ttt gac ttt 96Gln Gln Val Asn Glu Ser Phe Gly Phe Glu Leu Glu Phe Phe Asp Phe 20 25 30ctg ctg acg gaa aaa acc gct aaa act gcc aat ggc tgc gaa gcg gta 144Leu Leu Thr Glu Lys Thr Ala Lys Thr Ala Asn Gly Cys Glu Ala Val 35 40 45tgt att ttc gta aac gat gac ggc agc cgc ccg gtg ctg gaa gag ctg 192Cys Ile Phe Val Asn Asp Asp Gly Ser Arg Pro Val Leu Glu Glu Leu 50 55 60aaa aag cac ggc gtt aaa tat atc gcc ctg cgc tgt gcc ggt ttc aat 240Lys Lys His Gly Val Lys Tyr Ile Ala Leu Arg Cys Ala Gly Phe Asn65 70 75 80aac gtc gac ctt gac gcg gca aaa gaa ctg ggg ctg aaa gta gtc cgt 288Asn Val Asp Leu Asp Ala Ala Lys Glu Leu Gly Leu Lys Val Val Arg 85 90 95gtt cca gcc tat gat cca gag gcc gtt gct gaa cac gcc atc ggt atg 336Val Pro Ala Tyr Asp Pro Glu Ala Val Ala Glu His Ala Ile Gly Met 100 105 110atg atg acg ctg aac cgc cgt att cac cgc gcg tat cag cgt acc cgt 384Met Met Thr Leu Asn Arg Arg Ile His Arg Ala Tyr Gln Arg Thr Arg 115 120 125gat gct aac ttc tct ctg gaa ggt ctg acc ggc ttt act atg tat ggc 432Asp Ala Asn Phe Ser Leu Glu Gly Leu Thr Gly Phe Thr Met Tyr Gly 130 135 140aaa acg gca ggc gtt atc ggt acc ggt aaa atc ggt gtg gcg atg ctg 480Lys Thr Ala Gly Val Ile Gly Thr Gly Lys Ile Gly Val Ala Met Leu145 150 155 160cgc att ctg aaa ggt ttt ggt atg cgt ctg ctg gcg ttc gat ccg tat 528Arg Ile Leu Lys Gly Phe Gly Met Arg Leu Leu Ala Phe Asp Pro Tyr 165 170 175cca agt gca gcg gcg ctg gaa ctc ggt gtg gag tat gtc gat ctg cca 576Pro Ser Ala Ala Ala Leu Glu Leu Gly Val Glu Tyr Val Asp Leu Pro 180 185 190acc ctg ttc tct gaa tca gac gtt atc tct ctg cac tgc ccg ctg aca 624Thr Leu Phe Ser Glu Ser Asp Val Ile Ser Leu His Cys Pro Leu Thr 195 200 205ccg gaa aac tat cat ctg ttg aac gaa gcc gcc ttc gaa cag atg aaa 672Pro Glu Asn Tyr His Leu Leu Asn Glu Ala Ala Phe Glu Gln Met Lys 210 215 220aat ggc gtg atg atc gtc aat acc agt cgc ggt gca ttg att gat tct 720Asn Gly Val Met Ile Val Asn Thr Ser Arg Gly Ala Leu Ile Asp Ser225 230 235 240cag gca gca att gaa gcg ctg aaa aat cag aaa att ggt tcg ttg ggt 768Gln Ala Ala Ile Glu Ala Leu Lys Asn Gln Lys Ile Gly Ser Leu Gly 245 250 255atg gac gtg tat gag aac gaa cgc gat cta ttc ttt gaa gat aaa tcc 816Met Asp Val Tyr Glu Asn Glu Arg Asp Leu Phe Phe Glu Asp Lys Ser 260 265 270aac gac gtg atc cag gat gac gta ttc cgt cgc ctg tct gcc tgc cac 864Asn Asp Val Ile Gln Asp Asp Val Phe Arg Arg Leu Ser Ala Cys His 275 280 285aac gtg ctg ttt acc ggg cac cag gca ttc ctg aca gca gaa gct ctg 912Asn Val Leu Phe Thr Gly His Gln Ala Phe Leu Thr Ala Glu Ala Leu 290 295 300acc agt att tct cag act acg ctg caa aac tta agc aat ctg gaa aaa 960Thr Ser Ile Ser Gln Thr Thr Leu Gln Asn Leu Ser Asn Leu Glu Lys305 310 315 320ggc gaa acc tgc ccg aac gaa ctg gtt taa 990Gly Glu Thr Cys Pro Asn Glu Leu Val 32537329PRTEscherichia coli 37Met Lys Leu Ala Val Tyr Ser Thr Lys Gln Tyr Asp Lys Lys Tyr Leu1 5 10 15Gln Gln Val Asn Glu Ser Phe Gly Phe Glu Leu Glu Phe Phe Asp Phe 20 25 30Leu Leu Thr Glu Lys Thr Ala Lys Thr Ala Asn Gly Cys Glu Ala Val 35 40 45Cys Ile Phe Val Asn Asp Asp Gly Ser Arg Pro Val Leu Glu Glu Leu 50 55 60Lys Lys His Gly Val Lys Tyr Ile Ala Leu Arg Cys Ala Gly Phe Asn65 70 75 80Asn Val Asp Leu Asp Ala Ala Lys Glu Leu Gly Leu Lys Val Val Arg 85 90 95Val Pro Ala Tyr Asp Pro Glu Ala Val Ala Glu His Ala Ile Gly Met 100 105 110Met Met Thr Leu Asn Arg Arg Ile His Arg Ala Tyr Gln Arg Thr Arg 115 120 125Asp Ala Asn Phe Ser Leu Glu Gly Leu Thr Gly Phe Thr Met Tyr Gly 130 135 140Lys Thr Ala Gly Val Ile Gly Thr Gly Lys Ile Gly Val Ala Met Leu145 150 155 160Arg Ile Leu Lys Gly Phe Gly Met Arg Leu Leu Ala Phe Asp Pro Tyr 165 170 175Pro Ser Ala Ala Ala Leu Glu Leu Gly Val Glu Tyr Val Asp Leu Pro 180 185 190Thr Leu Phe Ser Glu Ser Asp Val Ile Ser Leu His Cys Pro Leu Thr 195 200 205Pro Glu Asn Tyr His Leu Leu Asn Glu Ala Ala Phe Glu Gln Met Lys 210 215 220Asn Gly Val Met Ile Val Asn Thr Ser Arg Gly Ala Leu Ile Asp Ser225 230 235 240Gln Ala Ala Ile Glu Ala Leu Lys Asn Gln Lys Ile Gly Ser Leu Gly 245 250 255Met Asp Val Tyr Glu Asn Glu Arg Asp Leu Phe Phe Glu Asp Lys Ser 260 265 270Asn Asp Val Ile Gln Asp Asp Val Phe Arg Arg Leu Ser Ala Cys His 275 280 285Asn Val Leu Phe Thr Gly His Gln Ala Phe Leu Thr Ala Glu Ala Leu 290 295 300Thr Ser Ile Ser Gln Thr Thr Leu Gln Asn Leu Ser Asn Leu Glu Lys305 310 315 320Gly Glu Thr Cys Pro Asn Glu Leu Val 325381191DNAEscherichia coliCDS(1)..(1191) 38atg att att tcc gca gcc agc gat tat cgc gcc gca gcg caa cgc att 48Met Ile Ile Ser Ala Ala Ser Asp Tyr Arg Ala Ala Ala Gln Arg Ile1 5 10 15ctg ccg ccg ttc ctg ttc cac tat atg gat ggt ggt gca tat tct gaa 96Leu Pro Pro Phe Leu Phe His Tyr Met Asp Gly Gly Ala Tyr Ser Glu 20 25 30tac acg ctg cgc cgc aac gtg gaa gat ttg tca gaa gtg gcg ctg cgc 144Tyr Thr Leu Arg Arg Asn Val Glu Asp Leu Ser Glu Val Ala Leu Arg 35 40 45cag cgt att ctg aaa aac atg tcc gac tta agc ctg gaa acg acg ctg 192Gln Arg Ile Leu Lys Asn Met Ser Asp Leu Ser Leu Glu Thr Thr Leu 50 55 60ttt aat gag aaa ttg tcg atg ccg gtg gca ctg gct ccg gtg ggt ttg 240Phe Asn Glu Lys Leu Ser Met Pro Val Ala Leu Ala Pro Val Gly Leu65 70 75 80tgt ggc atg tat gcg cgt cgt ggc gaa gtt cag gca gcc aaa gcg gcg 288Cys Gly Met Tyr Ala Arg Arg Gly Glu Val Gln Ala Ala Lys Ala Ala 85 90 95gac gcg cat ggt att ccg ttt act ctc tcg acg gtt tcc gtt tgc ccg 336Asp Ala His Gly Ile Pro Phe Thr Leu Ser Thr Val Ser Val Cys Pro 100 105 110att gaa gaa gtc gcg cca gcc atc aag cgc cca atg tgg ttc cag ctt 384Ile Glu Glu Val Ala Pro Ala Ile Lys Arg Pro Met Trp Phe Gln Leu 115 120 125tat gta ctg cgc gat cgc ggc ttt atg cgt aac gcg ctg gag cga gca 432Tyr Val Leu Arg Asp Arg Gly Phe Met Arg Asn Ala Leu Glu Arg Ala 130 135 140aaa gca gcg ggt tgt tcg acg ctg gtt ttc acc gtg gat atg ccg aca 480Lys Ala Ala Gly Cys Ser Thr Leu Val Phe Thr Val Asp Met Pro Thr145 150 155 160ccg ggc gca cgc tac cgt gat gcg cat tca ggt atg agc ggc ccg aac 528Pro Gly Ala Arg Tyr Arg Asp Ala His Ser Gly Met Ser Gly Pro Asn 165 170 175gcg gca atg cgc cgc tac ttg caa gcg gtg aca cat ccg caa tgg gcg 576Ala Ala Met Arg Arg Tyr Leu Gln Ala Val Thr His Pro Gln Trp Ala 180 185 190tgg gat gtg ggc ctg aac ggt cgt cca cat gat tta ggt aat atc tca 624Trp Asp Val Gly Leu Asn Gly Arg Pro His Asp Leu Gly Asn Ile Ser 195 200 205gct tat ctc ggc aaa ccg acc gga ctg gaa gat tac atc ggc tgg ctg 672Ala Tyr Leu Gly Lys Pro Thr Gly Leu Glu Asp Tyr Ile Gly Trp Leu 210 215 220ggg aat aac ttc gat ccg tcc atc tca tgg aaa gac ctt gaa tgg atc 720Gly Asn Asn Phe Asp Pro Ser Ile Ser Trp Lys Asp Leu Glu Trp Ile225 230 235 240cgc gat ttc tgg gat ggc ccg atg gtg atc aaa ggg atc ctc gat ccg 768Arg Asp Phe Trp Asp Gly Pro Met Val Ile Lys Gly Ile Leu Asp Pro 245 250 255gaa gat gcg cgc gat gca gta cgt ttt ggt gct gat gga att gtg gtt 816Glu Asp Ala Arg Asp Ala Val Arg Phe Gly Ala Asp Gly Ile Val Val 260 265 270tct aac cac ggt ggc cgc cag ctg gac ggt gta ctc tct tcc gcc cgt 864Ser Asn His Gly Gly Arg Gln Leu Asp Gly Val Leu Ser Ser Ala Arg 275 280 285gca ctg cct gct att gca gat gcg gtg aaa ggt gat ata gcc att ctg 912Ala Leu Pro Ala Ile Ala Asp Ala Val Lys Gly Asp Ile Ala Ile Leu 290 295 300gcg gat agc gga att cgt aac ggg ctt gat gtc gtg cgt atg att gcg 960Ala Asp Ser Gly Ile Arg Asn Gly Leu Asp Val Val Arg Met Ile Ala305 310 315 320ctc ggt gcc gac acc gta ctg ctg ggt cgt gct ttc ttg tat gcg ctg 1008Leu Gly Ala Asp Thr Val Leu Leu Gly Arg Ala Phe Leu Tyr Ala Leu 325 330 335gca aca gcg ggc cag gcg ggt gta gct aac ctg cta aat ctg atc gaa 1056Ala Thr Ala Gly Gln Ala Gly Val Ala Asn Leu Leu Asn Leu Ile Glu 340 345 350aaa gag atg aaa gtg gcg atg acg ctg act ggc gcg aaa tcg atc agc 1104Lys Glu Met Lys Val Ala Met Thr Leu Thr Gly Ala Lys Ser Ile Ser 355 360 365gaa att acg caa gat tcg ctg gtg cag ggg ctg ggt aaa gag ttg cct 1152Glu Ile Thr Gln Asp Ser Leu Val Gln Gly Leu Gly Lys Glu Leu Pro 370 375 380gcg gca ctg gct ccc atg gcg aaa ggg aat gcg gca tag 1191Ala Ala Leu Ala Pro Met Ala Lys Gly Asn Ala Ala385 390 39539396PRTEscherichia coli 39Met Ile Ile Ser Ala Ala Ser Asp Tyr Arg Ala Ala Ala Gln Arg Ile1 5 10 15Leu Pro Pro Phe Leu Phe His Tyr Met Asp Gly Gly Ala Tyr Ser Glu 20 25 30Tyr Thr Leu Arg Arg Asn Val Glu Asp Leu Ser Glu Val Ala Leu Arg 35 40 45Gln Arg Ile Leu Lys Asn Met Ser Asp Leu Ser Leu Glu Thr Thr Leu 50 55 60Phe Asn Glu Lys Leu Ser Met Pro Val Ala Leu Ala Pro Val Gly Leu65 70 75 80Cys Gly Met Tyr Ala Arg Arg Gly Glu Val Gln Ala Ala Lys Ala Ala 85 90 95Asp Ala His Gly Ile Pro Phe Thr Leu Ser Thr Val Ser Val Cys Pro 100 105 110Ile Glu Glu Val Ala Pro Ala Ile Lys Arg Pro Met Trp Phe Gln Leu 115 120 125Tyr Val Leu Arg Asp Arg Gly Phe Met Arg Asn Ala Leu Glu Arg Ala 130 135 140Lys Ala Ala Gly Cys Ser Thr Leu Val Phe Thr Val Asp Met Pro Thr145 150 155 160Pro Gly Ala Arg Tyr Arg Asp Ala His Ser Gly Met Ser Gly Pro Asn 165 170 175Ala Ala Met Arg Arg Tyr Leu Gln Ala Val Thr His Pro Gln Trp Ala 180 185 190Trp Asp Val Gly Leu Asn Gly Arg Pro His Asp Leu Gly Asn Ile Ser 195 200 205Ala Tyr Leu Gly Lys Pro Thr Gly Leu Glu Asp Tyr Ile Gly Trp Leu 210 215 220Gly Asn Asn Phe Asp Pro Ser Ile Ser Trp Lys Asp Leu Glu Trp Ile225 230 235 240Arg Asp Phe Trp Asp Gly Pro Met Val Ile Lys Gly Ile Leu Asp Pro 245 250 255Glu Asp Ala Arg Asp Ala Val Arg Phe Gly Ala Asp Gly Ile Val Val 260 265 270Ser Asn His Gly Gly Arg Gln Leu Asp Gly Val Leu Ser Ser Ala Arg 275 280 285Ala Leu Pro Ala Ile Ala Asp Ala Val Lys Gly Asp Ile Ala Ile Leu 290 295 300Ala Asp Ser Gly Ile Arg Asn Gly Leu Asp Val Val Arg Met Ile Ala305 310 315 320Leu Gly Ala Asp Thr Val Leu Leu Gly Arg Ala Phe Leu Tyr Ala Leu 325 330 335Ala Thr Ala Gly Gln Ala Gly Val Ala Asn Leu Leu Asn Leu Ile Glu 340

345 350Lys Glu Met Lys Val Ala Met Thr Leu Thr Gly Ala Lys Ser Ile Ser 355 360 365Glu Ile Thr Gln Asp Ser Leu Val Gln Gly Leu Gly Lys Glu Leu Pro 370 375 380Ala Ala Leu Ala Pro Met Ala Lys Gly Asn Ala Ala385 390 395401308DNAPantoea ananatis 40atgagcagaa tcatgacgcc cgtgaactgg gaagcctgca gcagcgaggc gcagcaggcg 60ctgttggcac gccctgcgct cgcctcgtct gacagcatca gccagatcgt gcgcgatgtg 120ttggtcagag tgaaagagga aggcgatgcg gctttacgag aattcagcgc gcgctttgac 180aaggttgaaa cagacgacct gcgcgttacg ccacagcaga tgcaggcggc cagcgatcgc 240cttggtgacg agctgaaaca ggcgatggcc gtggccattg gcaatattga aacctttcac 300cgtgcgcaga tcctgccgcc ggtggatgtg gaaacgcagc ccggcgtgcg ctgtcagcaa 360attacgcgcc cgatgaaatc ggtgggcttg tatattccgg gcggttctgc cccgctgttt 420tctaccgttc tgatgctggc taccccggcg cggattgcgg gctgtggtcg cgtggtgctg 480tgctcgcccc cgccgattgc tgatgaaatt ctctacgcgg ccaaactttg cggtgtggaa 540gaagtgttcc aggtgggtgg atcacaggcg attgccgccc tggcttttgg caccgaaagc 600atccctaagg tagataaaat ttttggtccg ggcaacgcgt gggttaccga agccaaacgt 660caggtcagcc agcgccttga tggcgcggcg attgatatgc ccgctggccc gtcggaagtg 720ctggtgattg ccgatgaagg tgccacaccg gccttcgttg cctctgatct gctgtcgcag 780gcggaacacg gccctgactc gcaggtgatt ttactgacgc cttcgctggc gctggccgag 840cgcgtcgccg aggcggtgga ggatcagctg gcccagttgc cacgtgcggc gacagcccgc 900caggcactgg aaagcagccg cctgatcgtc gcccgggata tgcagcaatg cattgcgatc 960tccaaccgct atggtccgga gcacctgatt ctgcaaaccc gcacgccacg ggatctggtg 1020gaacagatta ccagcgccgg ttcggttttc ctgggcgact ggtcaccgga atccgcagga 1080gattatgctt cgggcaccaa ccacgtgctg ccgacctacg gctataccgc gacatgctcc 1140agcctgggcc tggccgactt tcagaaacgc atgacggtac aggagctgac gccgcagggc 1200ttcctgaacc tggcggcgac catcgaaacc ctggcggccg ctgaacagct gcacgcccac 1260aaaaatgccg tcacgttgcg cgttgccgca ctcaaggagc aagcatga 13084168DNAArtificialprimer 41ccatagcggt tggagatcgc aatgcattgc tgcatatccc tgaagcctgc ttttttatac 60taagttgg 684268DNAArtificialprimer 42gcccgccagg cactggaaag cagccgcctg atcgtcgccc cgctcaagtt agtataaaaa 60agctgaac 684333DNAArtificialprimer 43tagcgagatc tctgatgtcc ggcggtgctt ttg 334432DNAArtificialprimer 44aaaaagagct cttacgcccc gccctgccac tc 324534DNAArtificialprimer 45caggatctag aaggagacat gaacgatgaa catc 344636DNAArtificialprimer 46gataaggatc cgaaataaaa gaaaatgcca atagga 364731DNAArtificialprimer 47cctttgagct cgcgggcagt gagcgcaacg c 314848DNAArtificialprimer 48ctagagcggc cgccgatcgg gatcctcctg tgtgaaattg ttatccgc 484942DNAArtificialprimer 49ctctacgatc gaggaggtta taaaaaatgg atattaatac tg 425036DNAArtificialprimer 50tcaaagcggc cgcttcttcg tctgtttcta ctggta 365131DNAArtificialprimer 51cctttggtac cgcgggcagt gagcgcaacg c 315234DNAArtificialprimer 52aacaggaatt ctttgcctgg cggcagtagc gcgg 345340DNAArtificialprimer P1 53ctagtaagat cttgaagcct gcttttttat actaagttgg 405441DNAArtificialprimer P2 54atgatcgaat tcgaaatcaa ataatgattt tattttgact g 4155120DNAArtificialDNA fragment containing attL 55agatcttgaa gcctgctttt ttatactaag ttggcattat aaaaaagcat tgcttatcaa 60tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttga tttcgaattc 1205641DNAArtificialprimer P3 56atgccactgc agtctgttac aggtcactaa taccatctaa g 415746DNAArtificialprimer P4 57accgttaagc tttctagacg ctcaagttag tataaaaaag ctgaac 4658184DNAArtificialDNA fragment containing attR 58ctgcagtctg ttacaggtca ctaataccat ctaagtagtt gattcatagt gactgcatat 60gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga 120tatttatatc attttacgtt tctcgttcag cttttttata ctaacttgag cgtctagaaa 180gctt 1845938DNAArtificialprimer P5 59ttcttagacg tcaggtggca cttttcgggg aaatgtgc 386037DNAArtificialprimer P6 60taacagagat ctcgcgcaga aaaaaaggat ctcaaga 376146DNAArtificialprimer P7 61aacagagatc taagcttaga tcctttgcct ggcggcagta gcgcgg 466235DNAArtificialprimer P8 62ataaactgca gcaaaaagag tttgtagaaa cgcaa 35631388DNAArtificialDNA fragment containing Tc gene and ter_thrL 63gaattctcat gtttgacagc ttatcatcga taagctttaa tgcggtagtt tatcacagtt 60aaattgctaa cgcagtcagg caccgtgtat gaaatctaac aatgcgctca tcgtcatcct 120cggcaccgtc accctggatg ctgtaggcat aggcttggtt atgccggtac tgccgggcct 180cttgcgggat atcgtccatt ccgacagcat cgccagtcac tatggcgtgc tgctagcgct 240atatgcgttg atgcaatttc tatgcgcacc cgttctcgga gcactgtccg accgctttgg 300ccgccgccca gtcctgctcg cttcgctact tggagccact atcgactacg cgatcatggc 360gaccacaccc gtcctgtgga tcctctacgc cggacgcatc gtggccggca tcaccggcgc 420cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg 480ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg 540gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg 600cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgacc 660gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat 720cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc 780gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc 840gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac 900caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta 960cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc 1020ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga 1080ccatcaggga cagcttcaag gatcgctcgc ggctcttacc agcctaactt cgatcactgg 1140accgctgatc gtcacggcga tttatgccgc ctcggcgagc acatggaacg ggttggcatg 1200gattgtaggc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag 1260ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt caccactcca 1320actagaaagc ttaacacaga aaaaagcccg cacctgacag tgcgggcttt ttttttcgac 1380cactgcag 13886436DNAArtificialprimer P9 64agtaattcta gaaagcttaa cacagaaaaa agcccg 366543DNAArtificialprimer P10 65ctagtaggat ccctgcagtg gtcgaaaaaa aaagcccgca ctg 43666550DNAPantoea ananatisCDS(231)..(1370)CDS(1380)..(1814)CDS(1827)..(4004)CDS(4260)..(539- 6)CDS(5526)..(6443) 66aggttccggc gggcagattt ctctaaaaaa aagcggtacg gcattgataa accgaaatgt 60tctgctttgc tttattccgt tccgggtaaa gcgcgtgatg ggtacaagac aggagctaag 120aataatgaac gacatgttca gggcctgagt caggatagct tacttttttc gagcgcttac 180acagcggcgt attcgcctct atcaaacatt aactgatagc gaagatctaa atg att 236 Met Ile 1aca atg aaa atg aag atg ata cct gtt ctg gta tct gca act ttt atg 284Thr Met Lys Met Lys Met Ile Pro Val Leu Val Ser Ala Thr Phe Met 5 10 15gcc ggg tgt act gtc gtg ccg ggg caa aat tta tcc act tcg gga aaa 332Ala Gly Cys Thr Val Val Pro Gly Gln Asn Leu Ser Thr Ser Gly Lys 20 25 30gat gtc att gag cag cag gac agc aat ttt gac atc gac aag tac gtt 380Asp Val Ile Glu Gln Gln Asp Ser Asn Phe Asp Ile Asp Lys Tyr Val35 40 45 50aac gtt tat ccg tta acg cca ggc ctg gtg gag aaa atg cgt cct aag 428Asn Val Tyr Pro Leu Thr Pro Gly Leu Val Glu Lys Met Arg Pro Lys 55 60 65cca ctt gtg gca cag gct aat ccg tcg tta caa acc gaa att cag aac 476Pro Leu Val Ala Gln Ala Asn Pro Ser Leu Gln Thr Glu Ile Gln Asn 70 75 80tac gaa tac cgc atc ggc atc ggt gat gtt ctc acc gtc acc gtg tgg 524Tyr Glu Tyr Arg Ile Gly Ile Gly Asp Val Leu Thr Val Thr Val Trp 85 90 95gat cac ccc gaa ctc acc acg cct gcc ggt cag tac cgt agc gcc agt 572Asp His Pro Glu Leu Thr Thr Pro Ala Gly Gln Tyr Arg Ser Ala Ser 100 105 110gac acc ggt aac tgg gtc cat tcc gac ggc acg att ttc tat cct tac 620Asp Thr Gly Asn Trp Val His Ser Asp Gly Thr Ile Phe Tyr Pro Tyr115 120 125 130atc ggc cgc gtc cgt gtt gcc ggc cgt acc gta ggt gac gtg cgt aat 668Ile Gly Arg Val Arg Val Ala Gly Arg Thr Val Gly Asp Val Arg Asn 135 140 145gaa gtg gcg cgt cgt ctg gcg cag tac atc gaa agc ccc cag gtt gac 716Glu Val Ala Arg Arg Leu Ala Gln Tyr Ile Glu Ser Pro Gln Val Asp 150 155 160gtc agc att gcc tcg ttt aag tca cag aag act tac gtg acc ggt gcc 764Val Ser Ile Ala Ser Phe Lys Ser Gln Lys Thr Tyr Val Thr Gly Ala 165 170 175gtg acg acc tca ggc cag cag cct atc acc aac gta ccg ctg act atc 812Val Thr Thr Ser Gly Gln Gln Pro Ile Thr Asn Val Pro Leu Thr Ile 180 185 190ctg gat gcg att aac gca gcg ggc ggc tta gcg gcg gac gca gac tgg 860Leu Asp Ala Ile Asn Ala Ala Gly Gly Leu Ala Ala Asp Ala Asp Trp195 200 205 210cgc aac gtt atc ctg acc cac aac ggc cgt gag cag cgc gtg tca ctg 908Arg Asn Val Ile Leu Thr His Asn Gly Arg Glu Gln Arg Val Ser Leu 215 220 225cag gcc ctg atg caa aac ggc gat ctg agc cag aac cac ctg ctt tat 956Gln Ala Leu Met Gln Asn Gly Asp Leu Ser Gln Asn His Leu Leu Tyr 230 235 240ccg ggt gac atc ctg tat gtg ccg cgt aac gac gac ctg aaa gtg ttt 1004Pro Gly Asp Ile Leu Tyr Val Pro Arg Asn Asp Asp Leu Lys Val Phe 245 250 255gtc atg ggc gaa gtg aaa cag cag gcc acc ctg aaa atg gac cgc agc 1052Val Met Gly Glu Val Lys Gln Gln Ala Thr Leu Lys Met Asp Arg Ser 260 265 270ggt atg acc ctg gct gaa gcg ctg ggt aat gcg gca ggc atg gat caa 1100Gly Met Thr Leu Ala Glu Ala Leu Gly Asn Ala Ala Gly Met Asp Gln275 280 285 290acc acg tcc gac gcg acc ggg gtc ttt gtt atc cga ccg acg cgc ggc 1148Thr Thr Ser Asp Ala Thr Gly Val Phe Val Ile Arg Pro Thr Arg Gly 295 300 305tcc gat cgc agc aaa atc gcc aat atc tac cag ctg aat acc aaa gat 1196Ser Asp Arg Ser Lys Ile Ala Asn Ile Tyr Gln Leu Asn Thr Lys Asp 310 315 320gcg gca tcc atg gtg atg ggt acc gaa ttc caa ctt gaa cct tac gat 1244Ala Ala Ser Met Val Met Gly Thr Glu Phe Gln Leu Glu Pro Tyr Asp 325 330 335atc gtc tac gtg aca tcg acg ccg ctg acg cgt tgg aac cgc gtg ata 1292Ile Val Tyr Val Thr Ser Thr Pro Leu Thr Arg Trp Asn Arg Val Ile 340 345 350tcg cag ttg ata cca acc ata gcc ggt gtt aac gat gcg acg cag gct 1340Ser Gln Leu Ile Pro Thr Ile Ala Gly Val Asn Asp Ala Thr Gln Ala355 360 365 370gca cag cgc atc cgc aac tgg tcg aac taa ggtttcgcc atg att aag tcc 1391Ala Gln Arg Ile Arg Asn Trp Ser Asn Met Ile Lys Ser 375 380gtg tta gtc gtc tgc gta ggc aac atc tgc cgc tct cct acg gga gag 1439Val Leu Val Val Cys Val Gly Asn Ile Cys Arg Ser Pro Thr Gly Glu 385 390 395cgc ctg ttc aaa cgc gcg ctg ccg cac ctg cct gtg gcc tcc gcc gga 1487Arg Leu Phe Lys Arg Ala Leu Pro His Leu Pro Val Ala Ser Ala Gly400 405 410 415ctc ggc gcc ctg gtt ggg cac gcg gcg gat aaa acg gcg acg gag gtg 1535Leu Gly Ala Leu Val Gly His Ala Ala Asp Lys Thr Ala Thr Glu Val 420 425 430gca gcc gaa aaa ggc tta tcg ctg gaa ggt cat gag gct cag cag cta 1583Ala Ala Glu Lys Gly Leu Ser Leu Glu Gly His Glu Ala Gln Gln Leu 435 440 445acg gcc agc ctg tgc cgg gag tat gac ctg att ctg gtg atg gaa aag 1631Thr Ala Ser Leu Cys Arg Glu Tyr Asp Leu Ile Leu Val Met Glu Lys 450 455 460cgc cac att gaa cag gta aac cgc atc gat ccg gct gca cgc ggc aaa 1679Arg His Ile Glu Gln Val Asn Arg Ile Asp Pro Ala Ala Arg Gly Lys 465 470 475acg atg ctg ctt ggg cac tgg ctc aat cag aag gaa att gcg gat ccg 1727Thr Met Leu Leu Gly His Trp Leu Asn Gln Lys Glu Ile Ala Asp Pro480 485 490 495tac aga aaa agc cgt gag gcc ttt gaa gag gtt tac ggg tta ctg gaa 1775Tyr Arg Lys Ser Arg Glu Ala Phe Glu Glu Val Tyr Gly Leu Leu Glu 500 505 510cac gcc act cag aaa tgg gtc agc gta cta agc cga tag ttgggattat cc 1826His Ala Thr Gln Lys Trp Val Ser Val Leu Ser Arg 515 520atg aat ata aaa aac aaa gtc atg acc gcg ccg cgc gag gaa tcg aat 1874Met Asn Ile Lys Asn Lys Val Met Thr Ala Pro Arg Glu Glu Ser Asn 525 530 535ggg tgg gat ctg gcg cat ctt gtg gga cag ctt att gat cac cgc tgg 1922Gly Trp Asp Leu Ala His Leu Val Gly Gln Leu Ile Asp His Arg Trp540 545 550 555gtt atc gtc gct gtc acc gct ttt ttc atg ctg gta gca acg ctc tac 1970Val Ile Val Ala Val Thr Ala Phe Phe Met Leu Val Ala Thr Leu Tyr 560 565 570aca ctg ttt gcg acg cct att tac agc gcc gat gcc atg gtt cag gtg 2018Thr Leu Phe Ala Thr Pro Ile Tyr Ser Ala Asp Ala Met Val Gln Val 575 580 585gag cag aag aat acc agc acc gtc ctg aat gaa tta cag acg ctg atg 2066Glu Gln Lys Asn Thr Ser Thr Val Leu Asn Glu Leu Gln Thr Leu Met 590 595 600ccc acg acg cca gca tcg gat acc gaa atc cag att ctt gaa tca cgc 2114Pro Thr Thr Pro Ala Ser Asp Thr Glu Ile Gln Ile Leu Glu Ser Arg 605 610 615atg gtg ctg gga aaa acc gtt cag gat ctt ggc ctg gat gca gtg gtt 2162Met Val Leu Gly Lys Thr Val Gln Asp Leu Gly Leu Asp Ala Val Val620 625 630 635tca cag aac tat ttc ccg gtt atc ggc aaa ggt ctg tcg cgc ctg atg 2210Ser Gln Asn Tyr Phe Pro Val Ile Gly Lys Gly Leu Ser Arg Leu Met 640 645 650ggc aac aag cct ggc aat gtt gcg atc tcc cgt ctg gag att ccg cgt 2258Gly Asn Lys Pro Gly Asn Val Ala Ile Ser Arg Leu Glu Ile Pro Arg 655 660 665acg atc gag aag cgc agt gtt gag ctc gag gtt acg ggt aaa gac agc 2306Thr Ile Glu Lys Arg Ser Val Glu Leu Glu Val Thr Gly Lys Asp Ser 670 675 680tat acc gtt tct gcc gat ggc gat gag ctg ttc aaa ggc aaa gtg ggg 2354Tyr Thr Val Ser Ala Asp Gly Asp Glu Leu Phe Lys Gly Lys Val Gly 685 690 695cag ttc gaa aaa cac ggc gat gtg agc atg ctg gtc agc gat atc aat 2402Gln Phe Glu Lys His Gly Asp Val Ser Met Leu Val Ser Asp Ile Asn700 705 710 715gcg gag ccg ggc acc acg ttt acc gtc acc aaa ctc aac gat ctg cag 2450Ala Glu Pro Gly Thr Thr Phe Thr Val Thr Lys Leu Asn Asp Leu Gln 720 725 730gca att aac agc atc ctg tct aac ctg acc gtt gca gac aaa ggg aaa 2498Ala Ile Asn Ser Ile Leu Ser Asn Leu Thr Val Ala Asp Lys Gly Lys 735 740 745gac acc ggc gta ctg gga ctg cag tat gtg ggt gaa gat cag gag cag 2546Asp Thr Gly Val Leu Gly Leu Gln Tyr Val Gly Glu Asp Gln Glu Gln 750 755 760atc agc aaa gtt ctg aac cag atc gtg aat aac tac ctg ttg cag aac 2594Ile Ser Lys Val Leu Asn Gln Ile Val Asn Asn Tyr Leu Leu Gln Asn 765 770 775gtg cag cgt aaa tcc gaa cag gcc gag aaa agc ctg gag ttc ctg aaa 2642Val Gln Arg Lys Ser Glu Gln Ala Glu Lys Ser Leu Glu Phe Leu Lys780 785 790 795cag caa ctg ccg gaa gtg cgt ggc aaa ctg gat cag gcg gaa gac aaa 2690Gln Gln Leu Pro Glu Val Arg Gly Lys Leu Asp Gln Ala Glu Asp Lys 800 805 810ctt aac gcc ttc cgc cgt gag aac gag tcc gtt gac ctg tcg ctt gaa 2738Leu Asn Ala Phe Arg Arg Glu Asn Glu Ser Val Asp Leu Ser Leu Glu 815 820 825gct aag tct gcg ttg gat tca tcc gtt agc gtg cag agc cag ctt aac 2786Ala Lys Ser Ala Leu Asp Ser Ser Val Ser Val Gln Ser Gln Leu Asn 830 835 840gag ctg acg ttc cgc gaa gcc gaa gtt tcg cag ctg ttc acc aaa gat 2834Glu Leu Thr Phe Arg Glu Ala Glu Val Ser Gln Leu Phe Thr Lys Asp 845 850 855cac cct acc tat cgg gcc ctg tta gaa aag cgt aaa acg ctg gaa gat 2882His Pro Thr Tyr Arg Ala Leu Leu Glu Lys Arg Lys Thr Leu Glu Asp860 865 870 875gag cag gcg cag ctg aac aag aaa att gcg cag atg ccg aaa act cag 2930Glu Gln Ala Gln Leu Asn Lys Lys Ile Ala Gln Met Pro Lys Thr Gln 880 885 890cag gag att ctg cgt ctg acc cgc gat gtg cag tca ggt cag gaa atc 2978Gln Glu Ile Leu Arg Leu Thr Arg Asp Val Gln Ser Gly Gln Glu Ile 895 900 905tac atg cag tta ctg aac cgc cag caa gag ctg agc atc agt aag gcc 3026Tyr Met Gln Leu Leu Asn Arg Gln Gln Glu Leu Ser Ile Ser Lys Ala 910 915 920 agc acc gtg ggt gat gtt cgc

atc atc gat cag gcc gcc acg gca aac 3074Ser Thr Val Gly Asp Val Arg Ile Ile Asp Gln Ala Ala Thr Ala Asn 925 930 935tcg ccg gtt gcg ccg aaa aag ctg ctg atc atc gca gcc agc ctg att 3122Ser Pro Val Ala Pro Lys Lys Leu Leu Ile Ile Ala Ala Ser Leu Ile940 945 950 955ctg ggg ctg atg gtg tcc gtc ggt gtg gtt ctg ctt aaa gcg ctg ttg 3170Leu Gly Leu Met Val Ser Val Gly Val Val Leu Leu Lys Ala Leu Leu 960 965 970cat cac gga atc gag aac ccg gaa cag ctg gaa gag ctg ggc atg aac 3218His His Gly Ile Glu Asn Pro Glu Gln Leu Glu Glu Leu Gly Met Asn 975 980 985gtc tat gcc agc gta ccg ctc tct gag tgg cag cgt aag aag gat acc 3266Val Tyr Ala Ser Val Pro Leu Ser Glu Trp Gln Arg Lys Lys Asp Thr 990 995 1000gaa gca ctg gcg cgt cgc ggt cac aaa ggc aaa acc gat ccg cat 3311Glu Ala Leu Ala Arg Arg Gly His Lys Gly Lys Thr Asp Pro His 1005 1010 1015gag acg ctg ttg gca ctg ggt aat cca acc gac ctc tct att gaa 3356Glu Thr Leu Leu Ala Leu Gly Asn Pro Thr Asp Leu Ser Ile Glu 1020 1025 1030gcg att cgt agc ctg cgt acc agc ctg cac ttc gcc atg atg gaa 3401Ala Ile Arg Ser Leu Arg Thr Ser Leu His Phe Ala Met Met Glu 1035 1040 1045gcg aaa aac aac att ctg atg atc acc ggc gcc agc ccg ggt att 3446Ala Lys Asn Asn Ile Leu Met Ile Thr Gly Ala Ser Pro Gly Ile 1050 1055 1060ggt aaa acc ttt atc tgc gtt aac ctc gct acc ctg gtg gcc aag 3491Gly Lys Thr Phe Ile Cys Val Asn Leu Ala Thr Leu Val Ala Lys 1065 1070 1075gcc ggt cag aag gtg ttg ttt atc gat ggt gac atg cgc cgc ggt 3536Ala Gly Gln Lys Val Leu Phe Ile Asp Gly Asp Met Arg Arg Gly 1080 1085 1090tac acc cac gaa ctg ctg ggg gcg gac aat aaa tcc ggt ctg tct 3581Tyr Thr His Glu Leu Leu Gly Ala Asp Asn Lys Ser Gly Leu Ser 1095 1100 1105aac gtc ctg tct ggc aaa acc gag ttt acg ccg acc atg att cag 3626Asn Val Leu Ser Gly Lys Thr Glu Phe Thr Pro Thr Met Ile Gln 1110 1115 1120caa ggg cca tac ggt ttt gat ttc ctg ccg cgc gga cag gtt cca 3671Gln Gly Pro Tyr Gly Phe Asp Phe Leu Pro Arg Gly Gln Val Pro 1125 1130 1135ccg aac ccg tca gag ctg ttg atg cac cgc cgc atg ggc gag ctg 3716Pro Asn Pro Ser Glu Leu Leu Met His Arg Arg Met Gly Glu Leu 1140 1145 1150ctg gag tgg gca agc aaa aac tat gat tta gtc ctg att gat aca 3761Leu Glu Trp Ala Ser Lys Asn Tyr Asp Leu Val Leu Ile Asp Thr 1155 1160 1165cca ccg att ctg gcc gta acg gat gcg tcc atc atc ggt aag ctg 3806Pro Pro Ile Leu Ala Val Thr Asp Ala Ser Ile Ile Gly Lys Leu 1170 1175 1180gcc ggt acc tcg ctg atg gtg gcg cgt ttt gaa acc aac acc aca 3851Ala Gly Thr Ser Leu Met Val Ala Arg Phe Glu Thr Asn Thr Thr 1185 1190 1195aaa gaa gtg gat gtc agc ttc aaa cgc ttc gcg cag aac ggt atc 3896Lys Glu Val Asp Val Ser Phe Lys Arg Phe Ala Gln Asn Gly Ile 1200 1205 1210gaa atc aaa ggg gtt atc ctg aac gcc gtg gta cgt aaa gcg gct 3941Glu Ile Lys Gly Val Ile Leu Asn Ala Val Val Arg Lys Ala Ala 1215 1220 1225aac tcg tac ggt tat ggt tat gac tac tac gac tac gag tac ggt 3986Asn Ser Tyr Gly Tyr Gly Tyr Asp Tyr Tyr Asp Tyr Glu Tyr Gly 1230 1235 1240aaa cca acg aaa agc taa gagctgaaag aaaaggggcg ggtttgaccg 4034Lys Pro Thr Lys Ser 1245ccccttttga tccctggctc atgataacga cacgtaacga tttgcacagg tgattctgga 4094tacaacgcac aataagcgac gatctaacgt caatcatcag aattcatcat caacacgctt 4154ttgtctgtcg cgatgtcggc gcagacctgc tgagagatca acccggaagg taatcagcac 4214tggccactgt tggcatgaga acttgtggat ttaaggaatc acacc atg gct cca 4268 Met Ala Pro 1250tat tgg tat ata tca ggg ttt ttg ctg ctg att tcg ctg ttc gaa 4313Tyr Trp Tyr Ile Ser Gly Phe Leu Leu Leu Ile Ser Leu Phe Glu 1255 1260 1265atc ctg gtg aaa aaa gac gaa cgc aca aat tat att ttg acc tgg 4358Ile Leu Val Lys Lys Asp Glu Arg Thr Asn Tyr Ile Leu Thr Trp 1270 1275 1280ctg ctg tgc ttt gcg gcc att att ctg atc gta ttc ggg ggg att 4403Leu Leu Cys Phe Ala Ala Ile Ile Leu Ile Val Phe Gly Gly Ile 1285 1290 1295cgt ggc ctc ggc acc ggc atg gat gac ttc cag tac cgc agt ttc 4448Arg Gly Leu Gly Thr Gly Met Asp Asp Phe Gln Tyr Arg Ser Phe 1300 1305 1310ttt gag gac ttt gta cgg cgt att cag atc aac ggt ttt ttc aat 4493Phe Glu Asp Phe Val Arg Arg Ile Gln Ile Asn Gly Phe Phe Asn 1315 1320 1325acc gtc gcg ttt ttc cgc tac gaa ccg ctg att ttt gcg atg gcg 4538Thr Val Ala Phe Phe Arg Tyr Glu Pro Leu Ile Phe Ala Met Ala 1330 1335 1340tgg ata acg agt ctg ttt tcg cat aac gcc agc gtg ttt ctg ttc 4583Trp Ile Thr Ser Leu Phe Ser His Asn Ala Ser Val Phe Leu Phe 1345 1350 1355gtc ttc tgt gcg att gcg gta tcc att aac gcc ttc ttt ttc aga 4628Val Phe Cys Ala Ile Ala Val Ser Ile Asn Ala Phe Phe Phe Arg 1360 1365 1370aag atg tcg cct tat ccg gta ctg gcg ctg gcg ctg tat tcg gcg 4673Lys Met Ser Pro Tyr Pro Val Leu Ala Leu Ala Leu Tyr Ser Ala 1375 1380 1385cac atc ttt att aac aaa gac atc aac cag ata cgt ttt ggc tta 4718His Ile Phe Ile Asn Lys Asp Ile Asn Gln Ile Arg Phe Gly Leu 1390 1395 1400agc tcc gcc ctg ttc ctc ggc gtg ctg tgg acg atc tac ctg aag 4763Ser Ser Ala Leu Phe Leu Gly Val Leu Trp Thr Ile Tyr Leu Lys 1405 1410 1415cgt tac tgg tgg gcc ttt acc ttt ttt atc ctg tcg ttc atg agt 4808Arg Tyr Trp Trp Ala Phe Thr Phe Phe Ile Leu Ser Phe Met Ser 1420 1425 1430cac aac acc gcc gtt atg gtg ata acg ctg gtg cct ttc ctg ttt 4853His Asn Thr Ala Val Met Val Ile Thr Leu Val Pro Phe Leu Phe 1435 1440 1445att cgt gac tgg cgc tgg tgg ccg gtg gtg atc atc gtt gtc agc 4898Ile Arg Asp Trp Arg Trp Trp Pro Val Val Ile Ile Val Val Ser 1450 1455 1460ctg ccg ctc tcg gtc gtc ggg ggt tca agc ttc atc gcc ctg att 4943Leu Pro Leu Ser Val Val Gly Gly Ser Ser Phe Ile Ala Leu Ile 1465 1470 1475gcc gga cac ctc gga tcg ctg ggg gaa agg gca tcg ggc tat aac 4988Ala Gly His Leu Gly Ser Leu Gly Glu Arg Ala Ser Gly Tyr Asn 1480 1485 1490aac gat ccc tcc tat gca atg gat ggc agc atc ctg tca gtc tcg 5033Asn Asp Pro Ser Tyr Ala Met Asp Gly Ser Ile Leu Ser Val Ser 1495 1500 1505aac ctg aag aac atc atg ctg gtg ttt att ttc ttc tat ttc atg 5078Asn Leu Lys Asn Ile Met Leu Val Phe Ile Phe Phe Tyr Phe Met 1510 1515 1520ctg act gaa gaa ctg aag cgc gaa aac tat gcc cag tat cgt ctg 5123Leu Thr Glu Glu Leu Lys Arg Glu Asn Tyr Ala Gln Tyr Arg Leu 1525 1530 1535aac tac ttg ctg att tta agt ttt gct atc ggc ggg gcg atc cgc 5168Asn Tyr Leu Leu Ile Leu Ser Phe Ala Ile Gly Gly Ala Ile Arg 1540 1545 1550atc ttc ctg tat aac ttc ccg tca ggt tcg cgt ctt tcc aac tat 5213Ile Phe Leu Tyr Asn Phe Pro Ser Gly Ser Arg Leu Ser Asn Tyr 1555 1560 1565ttg caa cag gtt gaa ccc atc att ctg acc tcg ctt atc tac cag 5258Leu Gln Gln Val Glu Pro Ile Ile Leu Thr Ser Leu Ile Tyr Gln 1570 1575 1580gcc aga cgc gtc tgg aag ccg gcg ctg ttt ggc atg ctg ttt ttc 5303Ala Arg Arg Val Trp Lys Pro Ala Leu Phe Gly Met Leu Phe Phe 1585 1590 1595ttc ctg ctc tat tac ctc tac tac aac acc atc tca acc aag cag 5348Phe Leu Leu Tyr Tyr Leu Tyr Tyr Asn Thr Ile Ser Thr Lys Gln 1600 1605 1610gcg gtt gta ggg tac gaa gtg gcc cag gag ttc tgg ctg att cgg 5393Ala Val Val Gly Tyr Glu Val Ala Gln Glu Phe Trp Leu Ile Arg 1615 1620 1625taa cggacccggc ggcgcgcccc cagtgaacgg cttcacttcg cggggcggtc 5446gcctgtaaca acacaggttt ttcttcggcg tttgtcgatt gaattgttta tttttgaata 5506ctttaatgag gtgctggca atg aag gac att cgt ttc tcc atc gta att 5555 Met Lys Asp Ile Arg Phe Ser Ile Val Ile 1630 1635ccg gct tat aac gcg tca gaa tcg att gtc acg acg ctg gat tgc 5600Pro Ala Tyr Asn Ala Ser Glu Ser Ile Val Thr Thr Leu Asp Cys 1640 1645 1650gtt aaa gca caa act tat cgc cat ttc gaa gtc atc atc gtg gat 5645Val Lys Ala Gln Thr Tyr Arg His Phe Glu Val Ile Ile Val Asp 1655 1660 1665gac aaa tct gcc gat gcc gcc gcg ctg gcg gaa gtg gtg cgc agt 5690Asp Lys Ser Ala Asp Ala Ala Ala Leu Ala Glu Val Val Arg Ser 1670 1675 1680gag cgc tat cag gac ctg gac atc aat ctg gtc ttg tct gag gtt 5735Glu Arg Tyr Gln Asp Leu Asp Ile Asn Leu Val Leu Ser Glu Val 1685 1690 1695aaa ctc aac ggt gcc ggc gcg cgt aac aaa ggc att gag ctg gca 5780Lys Leu Asn Gly Ala Gly Ala Arg Asn Lys Gly Ile Glu Leu Ala 1700 1705 1710acg ggc gac tat gtc agt ttt ctg gat gcc gat gac gag tgg cat 5825Thr Gly Asp Tyr Val Ser Phe Leu Asp Ala Asp Asp Glu Trp His 1715 1720 1725gcc gac aag ctt ctg cag gtc agt gaa aag att gcg gag cta gag 5870Ala Asp Lys Leu Leu Gln Val Ser Glu Lys Ile Ala Glu Leu Glu 1730 1735 1740gcg cag ggt cag cac aat acc att atc ttc agc cag gta aat atc 5915Ala Gln Gly Gln His Asn Thr Ile Ile Phe Ser Gln Val Asn Ile 1745 1750 1755tat cag gac ggt gcg ttc ctg aag gtt atg ccg atg cag cct ccg 5960Tyr Gln Asp Gly Ala Phe Leu Lys Val Met Pro Met Gln Pro Pro 1760 1765 1770gca aaa aac gaa act gtc gca gag tat ctg ttt ggc tgt tat ggc 6005Ala Lys Asn Glu Thr Val Ala Glu Tyr Leu Phe Gly Cys Tyr Gly 1775 1780 1785ttt att caa acc agc acc atc gtg ctg aag cgt gaa gat gcc gct 6050Phe Ile Gln Thr Ser Thr Ile Val Leu Lys Arg Glu Asp Ala Ala 1790 1795 1800aaa att cag ttc gat gcg cgt ttt atc cgc cat cag gac tat gac 6095Lys Ile Gln Phe Asp Ala Arg Phe Ile Arg His Gln Asp Tyr Asp 1805 1810 1815ttc tgc atc cgt gcc gac cgc atg ggt tac cgg ttt gaa atg atc 6140Phe Cys Ile Arg Ala Asp Arg Met Gly Tyr Arg Phe Glu Met Ile 1820 1825 1830gcc gcc ccg ctg gcc aac tac cac ctg gtg acg aag ttt ggc tcc 6185Ala Ala Pro Leu Ala Asn Tyr His Leu Val Thr Lys Phe Gly Ser 1835 1840 1845aaa cac aaa ggt gaa tcg gtc aag tac tcc ctg ttc tgg ctg gat 6230Lys His Lys Gly Glu Ser Val Lys Tyr Ser Leu Phe Trp Leu Asp 1850 1855 1860acg atg aaa ccg cac ctt acc gca cga gat att cac acc tac aaa 6275Thr Met Lys Pro His Leu Thr Ala Arg Asp Ile His Thr Tyr Lys 1865 1870 1875gca ttc aag ctg ccc ctg cgc tac aaa atg gac ggt aac tcg ctg 6320Ala Phe Lys Leu Pro Leu Arg Tyr Lys Met Asp Gly Asn Ser Leu 1880 1885 1890atg gcg agc gtc agc ttc gcg cgc tac ttc ctg ctc acc aat aaa 6365Met Ala Ser Val Ser Phe Ala Arg Tyr Phe Leu Leu Thr Asn Lys 1895 1900 1905gat aat cgc acc tac ttc ctg aac cgc gtg aaa gac aaa ctg aaa 6410Asp Asn Arg Thr Tyr Phe Leu Asn Arg Val Lys Asp Lys Leu Lys 1910 1915 1920gcg cgg ttg ggt ggc gaa aag gca ctc tcc tga ttctattgaa 6453Ala Arg Leu Gly Gly Glu Lys Ala Leu Ser 1925 1930tgtcgtgacg ttttaacgct accggaaggt tatctggtgg ccccgtctat tttatttatt 6513caggtatgaa aatgaataat cactctggta ccgtcgg 655067379PRTPantoea ananatis 67Met Ile Thr Met Lys Met Lys Met Ile Pro Val Leu Val Ser Ala Thr1 5 10 15Phe Met Ala Gly Cys Thr Val Val Pro Gly Gln Asn Leu Ser Thr Ser 20 25 30Gly Lys Asp Val Ile Glu Gln Gln Asp Ser Asn Phe Asp Ile Asp Lys 35 40 45Tyr Val Asn Val Tyr Pro Leu Thr Pro Gly Leu Val Glu Lys Met Arg 50 55 60Pro Lys Pro Leu Val Ala Gln Ala Asn Pro Ser Leu Gln Thr Glu Ile65 70 75 80Gln Asn Tyr Glu Tyr Arg Ile Gly Ile Gly Asp Val Leu Thr Val Thr 85 90 95Val Trp Asp His Pro Glu Leu Thr Thr Pro Ala Gly Gln Tyr Arg Ser 100 105 110Ala Ser Asp Thr Gly Asn Trp Val His Ser Asp Gly Thr Ile Phe Tyr 115 120 125Pro Tyr Ile Gly Arg Val Arg Val Ala Gly Arg Thr Val Gly Asp Val 130 135 140Arg Asn Glu Val Ala Arg Arg Leu Ala Gln Tyr Ile Glu Ser Pro Gln145 150 155 160Val Asp Val Ser Ile Ala Ser Phe Lys Ser Gln Lys Thr Tyr Val Thr 165 170 175Gly Ala Val Thr Thr Ser Gly Gln Gln Pro Ile Thr Asn Val Pro Leu 180 185 190Thr Ile Leu Asp Ala Ile Asn Ala Ala Gly Gly Leu Ala Ala Asp Ala 195 200 205Asp Trp Arg Asn Val Ile Leu Thr His Asn Gly Arg Glu Gln Arg Val 210 215 220Ser Leu Gln Ala Leu Met Gln Asn Gly Asp Leu Ser Gln Asn His Leu225 230 235 240Leu Tyr Pro Gly Asp Ile Leu Tyr Val Pro Arg Asn Asp Asp Leu Lys 245 250 255Val Phe Val Met Gly Glu Val Lys Gln Gln Ala Thr Leu Lys Met Asp 260 265 270 Arg Ser Gly Met Thr Leu Ala Glu Ala Leu Gly Asn Ala Ala Gly Met 275 280 285Asp Gln Thr Thr Ser Asp Ala Thr Gly Val Phe Val Ile Arg Pro Thr 290 295 300Arg Gly Ser Asp Arg Ser Lys Ile Ala Asn Ile Tyr Gln Leu Asn Thr305 310 315 320Lys Asp Ala Ala Ser Met Val Met Gly Thr Glu Phe Gln Leu Glu Pro 325 330 335Tyr Asp Ile Val Tyr Val Thr Ser Thr Pro Leu Thr Arg Trp Asn Arg 340 345 350Val Ile Ser Gln Leu Ile Pro Thr Ile Ala Gly Val Asn Asp Ala Thr 355 360 365Gln Ala Ala Gln Arg Ile Arg Asn Trp Ser Asn 370 37568144PRTPantoea ananatis 68Met Ile Lys Ser Val Leu Val Val Cys Val Gly Asn Ile Cys Arg Ser1 5 10 15Pro Thr Gly Glu Arg Leu Phe Lys Arg Ala Leu Pro His Leu Pro Val 20 25 30Ala Ser Ala Gly Leu Gly Ala Leu Val Gly His Ala Ala Asp Lys Thr 35 40 45Ala Thr Glu Val Ala Ala Glu Lys Gly Leu Ser Leu Glu Gly His Glu 50 55 60Ala Gln Gln Leu Thr Ala Ser Leu Cys Arg Glu Tyr Asp Leu Ile Leu65 70 75 80Val Met Glu Lys Arg His Ile Glu Gln Val Asn Arg Ile Asp Pro Ala 85 90 95Ala Arg Gly Lys Thr Met Leu Leu Gly His Trp Leu Asn Gln Lys Glu 100 105 110Ile Ala Asp Pro Tyr Arg Lys Ser Arg Glu Ala Phe Glu Glu Val Tyr 115 120 125Gly Leu Leu Glu His Ala Thr Gln Lys Trp Val Ser Val Leu Ser Arg 130 135 14069725PRTPantoea ananatis 69Met Asn Ile Lys Asn Lys Val Met Thr Ala Pro Arg Glu Glu Ser Asn1 5 10 15Gly Trp Asp Leu Ala His Leu Val Gly Gln Leu Ile Asp His Arg Trp 20 25 30Val Ile Val Ala Val Thr Ala Phe Phe Met Leu Val Ala Thr Leu Tyr 35 40 45Thr Leu Phe Ala Thr Pro Ile Tyr Ser Ala Asp Ala Met Val Gln Val 50 55 60Glu Gln Lys Asn Thr Ser Thr Val Leu Asn Glu Leu Gln Thr Leu Met65 70 75 80Pro Thr Thr Pro Ala Ser Asp Thr Glu Ile Gln Ile Leu Glu Ser Arg 85 90 95Met Val Leu Gly Lys Thr Val Gln Asp Leu Gly Leu Asp Ala Val Val 100

105 110Ser Gln Asn Tyr Phe Pro Val Ile Gly Lys Gly Leu Ser Arg Leu Met 115 120 125Gly Asn Lys Pro Gly Asn Val Ala Ile Ser Arg Leu Glu Ile Pro Arg 130 135 140Thr Ile Glu Lys Arg Ser Val Glu Leu Glu Val Thr Gly Lys Asp Ser145 150 155 160Tyr Thr Val Ser Ala Asp Gly Asp Glu Leu Phe Lys Gly Lys Val Gly 165 170 175Gln Phe Glu Lys His Gly Asp Val Ser Met Leu Val Ser Asp Ile Asn 180 185 190Ala Glu Pro Gly Thr Thr Phe Thr Val Thr Lys Leu Asn Asp Leu Gln 195 200 205Ala Ile Asn Ser Ile Leu Ser Asn Leu Thr Val Ala Asp Lys Gly Lys 210 215 220Asp Thr Gly Val Leu Gly Leu Gln Tyr Val Gly Glu Asp Gln Glu Gln225 230 235 240Ile Ser Lys Val Leu Asn Gln Ile Val Asn Asn Tyr Leu Leu Gln Asn 245 250 255Val Gln Arg Lys Ser Glu Gln Ala Glu Lys Ser Leu Glu Phe Leu Lys 260 265 270Gln Gln Leu Pro Glu Val Arg Gly Lys Leu Asp Gln Ala Glu Asp Lys 275 280 285Leu Asn Ala Phe Arg Arg Glu Asn Glu Ser Val Asp Leu Ser Leu Glu 290 295 300Ala Lys Ser Ala Leu Asp Ser Ser Val Ser Val Gln Ser Gln Leu Asn305 310 315 320Glu Leu Thr Phe Arg Glu Ala Glu Val Ser Gln Leu Phe Thr Lys Asp 325 330 335His Pro Thr Tyr Arg Ala Leu Leu Glu Lys Arg Lys Thr Leu Glu Asp 340 345 350Glu Gln Ala Gln Leu Asn Lys Lys Ile Ala Gln Met Pro Lys Thr Gln 355 360 365Gln Glu Ile Leu Arg Leu Thr Arg Asp Val Gln Ser Gly Gln Glu Ile 370 375 380Tyr Met Gln Leu Leu Asn Arg Gln Gln Glu Leu Ser Ile Ser Lys Ala385 390 395 400Ser Thr Val Gly Asp Val Arg Ile Ile Asp Gln Ala Ala Thr Ala Asn 405 410 415Ser Pro Val Ala Pro Lys Lys Leu Leu Ile Ile Ala Ala Ser Leu Ile 420 425 430Leu Gly Leu Met Val Ser Val Gly Val Val Leu Leu Lys Ala Leu Leu 435 440 445His His Gly Ile Glu Asn Pro Glu Gln Leu Glu Glu Leu Gly Met Asn 450 455 460Val Tyr Ala Ser Val Pro Leu Ser Glu Trp Gln Arg Lys Lys Asp Thr465 470 475 480Glu Ala Leu Ala Arg Arg Gly His Lys Gly Lys Thr Asp Pro His Glu 485 490 495Thr Leu Leu Ala Leu Gly Asn Pro Thr Asp Leu Ser Ile Glu Ala Ile 500 505 510Arg Ser Leu Arg Thr Ser Leu His Phe Ala Met Met Glu Ala Lys Asn 515 520 525Asn Ile Leu Met Ile Thr Gly Ala Ser Pro Gly Ile Gly Lys Thr Phe 530 535 540Ile Cys Val Asn Leu Ala Thr Leu Val Ala Lys Ala Gly Gln Lys Val545 550 555 560Leu Phe Ile Asp Gly Asp Met Arg Arg Gly Tyr Thr His Glu Leu Leu 565 570 575Gly Ala Asp Asn Lys Ser Gly Leu Ser Asn Val Leu Ser Gly Lys Thr 580 585 590Glu Phe Thr Pro Thr Met Ile Gln Gln Gly Pro Tyr Gly Phe Asp Phe 595 600 605Leu Pro Arg Gly Gln Val Pro Pro Asn Pro Ser Glu Leu Leu Met His 610 615 620Arg Arg Met Gly Glu Leu Leu Glu Trp Ala Ser Lys Asn Tyr Asp Leu625 630 635 640Val Leu Ile Asp Thr Pro Pro Ile Leu Ala Val Thr Asp Ala Ser Ile 645 650 655Ile Gly Lys Leu Ala Gly Thr Ser Leu Met Val Ala Arg Phe Glu Thr 660 665 670Asn Thr Thr Lys Glu Val Asp Val Ser Phe Lys Arg Phe Ala Gln Asn 675 680 685Gly Ile Glu Ile Lys Gly Val Ile Leu Asn Ala Val Val Arg Lys Ala 690 695 700Ala Asn Ser Tyr Gly Tyr Gly Tyr Asp Tyr Tyr Asp Tyr Glu Tyr Gly705 710 715 720Lys Pro Thr Lys Ser 72570378PRTPantoea ananatis 70Met Ala Pro Tyr Trp Tyr Ile Ser Gly Phe Leu Leu Leu Ile Ser Leu1 5 10 15Phe Glu Ile Leu Val Lys Lys Asp Glu Arg Thr Asn Tyr Ile Leu Thr 20 25 30Trp Leu Leu Cys Phe Ala Ala Ile Ile Leu Ile Val Phe Gly Gly Ile 35 40 45Arg Gly Leu Gly Thr Gly Met Asp Asp Phe Gln Tyr Arg Ser Phe Phe 50 55 60Glu Asp Phe Val Arg Arg Ile Gln Ile Asn Gly Phe Phe Asn Thr Val65 70 75 80Ala Phe Phe Arg Tyr Glu Pro Leu Ile Phe Ala Met Ala Trp Ile Thr 85 90 95Ser Leu Phe Ser His Asn Ala Ser Val Phe Leu Phe Val Phe Cys Ala 100 105 110Ile Ala Val Ser Ile Asn Ala Phe Phe Phe Arg Lys Met Ser Pro Tyr 115 120 125Pro Val Leu Ala Leu Ala Leu Tyr Ser Ala His Ile Phe Ile Asn Lys 130 135 140Asp Ile Asn Gln Ile Arg Phe Gly Leu Ser Ser Ala Leu Phe Leu Gly145 150 155 160Val Leu Trp Thr Ile Tyr Leu Lys Arg Tyr Trp Trp Ala Phe Thr Phe 165 170 175Phe Ile Leu Ser Phe Met Ser His Asn Thr Ala Val Met Val Ile Thr 180 185 190Leu Val Pro Phe Leu Phe Ile Arg Asp Trp Arg Trp Trp Pro Val Val 195 200 205Ile Ile Val Val Ser Leu Pro Leu Ser Val Val Gly Gly Ser Ser Phe 210 215 220Ile Ala Leu Ile Ala Gly His Leu Gly Ser Leu Gly Glu Arg Ala Ser225 230 235 240Gly Tyr Asn Asn Asp Pro Ser Tyr Ala Met Asp Gly Ser Ile Leu Ser 245 250 255Val Ser Asn Leu Lys Asn Ile Met Leu Val Phe Ile Phe Phe Tyr Phe 260 265 270Met Leu Thr Glu Glu Leu Lys Arg Glu Asn Tyr Ala Gln Tyr Arg Leu 275 280 285Asn Tyr Leu Leu Ile Leu Ser Phe Ala Ile Gly Gly Ala Ile Arg Ile 290 295 300Phe Leu Tyr Asn Phe Pro Ser Gly Ser Arg Leu Ser Asn Tyr Leu Gln305 310 315 320Gln Val Glu Pro Ile Ile Leu Thr Ser Leu Ile Tyr Gln Ala Arg Arg 325 330 335Val Trp Lys Pro Ala Leu Phe Gly Met Leu Phe Phe Phe Leu Leu Tyr 340 345 350Tyr Leu Tyr Tyr Asn Thr Ile Ser Thr Lys Gln Ala Val Val Gly Tyr 355 360 365Glu Val Ala Gln Glu Phe Trp Leu Ile Arg 370 37571305PRTPantoea ananatis 71Met Lys Asp Ile Arg Phe Ser Ile Val Ile Pro Ala Tyr Asn Ala Ser1 5 10 15Glu Ser Ile Val Thr Thr Leu Asp Cys Val Lys Ala Gln Thr Tyr Arg 20 25 30His Phe Glu Val Ile Ile Val Asp Asp Lys Ser Ala Asp Ala Ala Ala 35 40 45Leu Ala Glu Val Val Arg Ser Glu Arg Tyr Gln Asp Leu Asp Ile Asn 50 55 60Leu Val Leu Ser Glu Val Lys Leu Asn Gly Ala Gly Ala Arg Asn Lys65 70 75 80Gly Ile Glu Leu Ala Thr Gly Asp Tyr Val Ser Phe Leu Asp Ala Asp 85 90 95Asp Glu Trp His Ala Asp Lys Leu Leu Gln Val Ser Glu Lys Ile Ala 100 105 110Glu Leu Glu Ala Gln Gly Gln His Asn Thr Ile Ile Phe Ser Gln Val 115 120 125Asn Ile Tyr Gln Asp Gly Ala Phe Leu Lys Val Met Pro Met Gln Pro 130 135 140Pro Ala Lys Asn Glu Thr Val Ala Glu Tyr Leu Phe Gly Cys Tyr Gly145 150 155 160Phe Ile Gln Thr Ser Thr Ile Val Leu Lys Arg Glu Asp Ala Ala Lys 165 170 175Ile Gln Phe Asp Ala Arg Phe Ile Arg His Gln Asp Tyr Asp Phe Cys 180 185 190Ile Arg Ala Asp Arg Met Gly Tyr Arg Phe Glu Met Ile Ala Ala Pro 195 200 205Leu Ala Asn Tyr His Leu Val Thr Lys Phe Gly Ser Lys His Lys Gly 210 215 220Glu Ser Val Lys Tyr Ser Leu Phe Trp Leu Asp Thr Met Lys Pro His225 230 235 240Leu Thr Ala Arg Asp Ile His Thr Tyr Lys Ala Phe Lys Leu Pro Leu 245 250 255Arg Tyr Lys Met Asp Gly Asn Ser Leu Met Ala Ser Val Ser Phe Ala 260 265 270Arg Tyr Phe Leu Leu Thr Asn Lys Asp Asn Arg Thr Tyr Phe Leu Asn 275 280 285Arg Val Lys Asp Lys Leu Lys Ala Arg Leu Gly Gly Glu Lys Ala Leu 290 295 300Ser3057289DNAArtificialprimer 72caagttgata agatcttccg tgccgccgct ctggccgctg cagatgctcg aatccctctc 60tgaagcctgc ttttttatac taagttggc 897389DNAArtificialprimer 73acgtgagcca ggaaatcagc ttcctgaacg ccggcttcgc gaatagattt cggaataccc 60gctcaagtta gtataaaaaa gctgaacga 89742688DNAEnterobacter aerogenesmisc_feature(663)..(663)n is a, c, g, or t 74tcgcatgctc ccggccgcca tggccgcggg attctcgttg agcgtgtaaa aaaagcccag 60cattgaatat gccagtttca ctcaagaaca agttgataag atcttccgtg ccgccgctct 120ggccgctgca gatgctcgaa tccctctcgc taaaatggct gttgccgaat ccggcatggg 180tattattgaa gataaagtga tcaaaaacca cttcgcttcc gaatatatct acaacgccta 240taaagatgaa aagacctgtg gcgtcctgtc tgaagacgac actttcggta ccatcaccat 300tgctgagcca atcggtatta tttgcggtat cgtcccgact accaacccga cttctactgc 360tatcttcaaa tcgctgatca gcctgaagac gcgtaacgcc atcatcttct ctccgcaccc 420gcgtgctgct aaagacgcga ctaacaaagc ggcggacatc gtattgcagg cagctattgc 480cgcaggcgcg ccgaaagatc tgatcggttg gatcgaccag ccttccgtag aactgtctaa 540cgcactgatg catcatcctg acatcaacct gatcctcgcc accggcggcc caggtatggt 600taaggccgct tatagctccg gtaaaccagc tatcggcgtt ggcgcgggca acacgccggt 660tgntattgat gaaacggctg atatcaaacg cgctgtggcg tccgtactga tgtcaaaaac 720cttcgataac ggcgttatct gtgcttctga acaatccgtg gtggttgtcg actccgtcta 780cgacgccgtc cgcgagcgtt ttgccagcca tggcggctac ctgctgcagg gtaaagagct 840gaaagccgtt caggacatca tcctgaaaaa tggcgcgctg aatgcggcga tcgttggtca 900accagcggca aaaatcgctg aactggcagg cttcaccgtg ccagccacca ctaagattct 960gatcggcgaa gttaccgacg ttgacgaaag cgaaccgttc gctcacgaaa aactgtctcc 1020gacgctggca atgtaccgtg cgaaagattt cgaagacgcg gtcaataaag cagaaaaact 1080ggtcgccatg ggcggtatcg gtcacacctc ttgcctgtac accgaccagg acaaccagcc 1140ggctcgcgtg gcctacttcg gccagatgat gaaaaccgcg cgtatcctga tcaacacccc 1200ggcttcccag ggtggtatcg gcgacctgta taacttcaag ctcgcacctt ccctgactct 1260gggttgtggt tcctggggtg gtaactccat ctctgaaaac gtnggtccga aacacctgat 1320caacaagaaa accgttgcta agcgagctga aaacatgttg tggcataaac ttccgaaatc 1380tatctacttc cgtcgtggct cactgccaat cgcactggat gaagtgatta ctgatggtca 1440caaacgcgcg ctgattgtga ctgaccgctt cctgttcaac aacggttacg cggaccagat 1500cacttccgta ctgaaagcgg ctggcgtaga aaccgaagtg ttctttgaag ttgaagctga 1560cccaacgctg actatcgtgc gtaaaggcgc ggatctggcc aactccttca aaccagacgt 1620aatcatcgcc ctgggcggcg gttccccgat ggatgcggca aaaatcatgt gggtcatgta 1680cgagcacccg gaaacccact tcgaagaact ggcgctgcgc tttatggata tccgtaaacg 1740tatctacaag ttcccgaaaa tgggcgtcaa agccaagatg gttgccatta ccaccacctc 1800cggtaccggt tctgaagtta ccccgttcgc cgtagtaacc gacgatgcaa ctggacagaa 1860atatccgcng gctgactacg ctctgactcc ggatatggcg attgtcgatg ccaacctggt 1920catggatatg ccgaagtctc tctgtgcctt cggtggtctg gatgccgtga ctcacgctct 1980ggaagcttac gtctccgtac tggcttctga gttctccgat ggtcaggctc tgcaggcgct 2040gaaactgctg aaagagtatc tgccggcctc ttaccatgaa ggttctaaga acccggtagc 2100ccgcgaacgt gtgcacagcg ccgccactat cgccggtatc gcgtttgcta acgccttcct 2160cggcgtgtgc cactcaatgg cgcacaaact gggctcgcag ttccacattc ctcacggtct 2220ggcaaacgcc ctgctgatca gcaacgttat ccgctataac gcgaatgaca acccgaccaa 2280gcagaccgca ttcagccagt atgaccgtcc gcaggcgcgc cgtcgctacg ctgagatcgc 2340agaccacctg ggcctgagcg ctccgggcga ccgcactgcg gcgaaaatcg agaaactgct 2400ggcatggctg gaaagcctca aagctgaact gggtattccg aaatctatcc gtgaagctgg 2460cgttcaggaa gcagacttcc tggcgaacgt ggataaactg tctgaagatg cattcgatga 2520ccagtgcacc ggcgctaacc cgcgttaccc gctgatctcc gagctgaaac agattctgct 2580ggatacctac tacggtcgtg atttatgtnn agaaggtnga aactgcagcg aagaaagaag 2640ctgctccggc taaagctgag aaaaaagcga aaaaatccgc ttaatcag 26887530DNAArtificialprimer 75aagagctccg cgaacgagcc atgacattgc 307643DNAArtificialprimer 76gtcgacacac gtcattcctc cttgtcgcct atattggtta aag 4377361DNAEnterobacter aerogenes 77aagagctccg cgaacgagcc atgacattgc tgacgactct ggcagtggca gatgacataa 60aactggtcga ctggttacaa caacgcctgg ggcttttaga gcaacgagac acggcaatgt 120tgcaccgttt gctgcatgat attgaaaaaa atatcaccaa ataaaaaacg ccttagtaag 180tatttttcag cttttcattc tgactgcaac gggcaatatg tctctgtgtg gattaaaaaa 240agagtgtctg atagcagctt ctgaactggt tacctgccgt gagtaaatta aaattttatt 300gacttaggtc actaaatact ttaaccaata taggcgacaa ggaggaatga cgtgtgtcga 360c 3617844DNAArtificialprimer 78aaggaggaat gacgtgtgtc gactcacaca tcttcaacgc ttcc 447928DNAArtificialprimer 79ttgagctctt tttacagaaa ggtttagg 28803461DNACorynebacterium glutamicum 80aaggaggaat gacgtgtgtc gactcacaca tcttcaacgc ttccagcatt caaaaagatc 60ttggtagcaa accgcggcga aatcgcggtc cgtgctttcc gtgcagcact cgaaaccggt 120gcagccacgg tagctattta cccccgtgaa gatcggggat cattccaccg ctcttttgct 180tctgaagctg tccgcattgg taccgaaggc tcaccagtca aggcgtacct ggacatcgat 240gaaattatcg gtgcagctaa aaaagttaaa gcagatgcca tttacccggg atacggcttc 300ctgtctgaaa atgcccagct tgcccgcgag tgtgcggaaa acggcattac ttttattggc 360ccaaccccag aggttcttga tctcaccggt gataagtctc gcgcggtaac cgccgcgaag 420aaggctggtc tgccagtttt ggcggaatcc accccgagca aaaacatcga tgagatcgtt 480aaaagcgctg aaggccagac ttaccccatc tttgtgaagg cagttgccgg tggtggcgga 540cgcggtatgc gttttgttgc ttcacctgat gagcttcgca aattagcaac agaagcatct 600cgtgaagctg aagcggcttt cggcgatggc gcggtatatg tcgaacgtgc tgtgattaac 660cctcagcata ttgaagtgca gatccttggc gatcacactg gagaagttgt acacctttat 720gaacgtgact gctcactgca gcgtcgtcac caaaaagttg tcgaaattgc gccagcacag 780catttggatc cagaactgcg tgatcgcatt tgtgcggatg cagtaaagtt ctgccgctcc 840attggttacc agggcgcggg aaccgtggaa ttcttggtcg atgaaaaggg caaccacgtc 900ttcatcgaaa tgaacccacg tatccaggtt gagcacaccg tgactgaaga agtcaccgag 960gtggacctgg tgaaggcgca gatgcgcttg gctgctggtg caaccttgaa ggaattgggt 1020ctgacccaag ataagatcaa gacccacggt gcagcactgc agtgccgcat caccacggaa 1080gatccaaaca acggcttccg cccagatacc ggaactatca ccgcgtaccg ctcaccaggc 1140ggagctggcg ttcgtcttga cggtgcagct cagctcggtg gcgaaatcac cgcacacttt 1200gactccatgc tggtgaaaat gacctgccgt ggttccgact ttgaaactgc tgttgctcgt 1260gcacagcgcg cgttggctga gttcaccgtg tctggtgttg caaccaacat tggtttcttg 1320cgtgcgttgc tgcgggaaga ggacttcact tccaagcgca tcgccaccgg attcattgcc 1380gatcacccgc acctccttca ggctccacct gctgatgatg agcagggacg catcctggat 1440tacttggcag atgtcaccgt gaacaagcct catggtgtgc gtccaaagga tgttgcagct 1500cctatcgata agctgcctaa catcaaggat ctgccactgc cacgcggttc ccgtgaccgc 1560ctgaagcagc ttggcccagc cgcgtttgct cgtgatctcc gtgagcagga cgcactggca 1620gttactgata ccaccttccg cgatgcacac cagtctttgc ttgcgacccg agtccgctca 1680ttcgcactga agcctgcggc agaggccgtc gcaaagctga ctcctgagct tttgtccgtg 1740gaggcctggg gcggcgcgac ctacgatgtg gcgatgcgtt tcctctttga ggatccgtgg 1800gacaggctcg acgagctgcg cgaggcgatg ccgaatgtaa acattcagat gctgcttcgc 1860ggccgcaaca ccgtgggata caccccgtac ccagactccg tctgccgcgc gtttgttaag 1920gaagctgcca gctccggcgt ggacatcttc cgcatcttcg acgcgcttaa cgacgtctcc 1980cagatgcgtc cagcaatcga cgcagtcctg gagaccaaca ccgcggtagc cgaggtggct 2040atggcttatt ctggtgatct ctctgatcca aatgaaaagc tctacaccct ggattactac 2100ctaaagatgg cagaggagat cgtcaagtct ggcgctcaca tcttggccat taaggatatg 2160gctggtctgc ttcgcccagc tgcggtaacc aagctggtca ccgcactgcg ccgtgaattc 2220gatctgccag tgcacgtgca cacccacgac actgcgggtg gccagctggc aacctacttt 2280gctgcagctc aagctggtgc agatgctgtt gacggtgctt ccgcaccact gtctggcacc 2340acctcccagc catccctgtc tgccattgtt gctgcattcg cgcacacccg tcgcgatacc 2400ggtttgagcc tcgaggctgt ttctgacctc gagccgtact gggaagcagt gcgcggactg 2460tacctgccat ttgagtctgg aaccccaggc ccaaccggtc gcgtctaccg ccacgaaatc 2520ccaggcggac agttgtccaa cctgcgtgca caggccaccg cactgggcct tgcggatcgt 2580ttcgaactca tcgaagacaa ctacgcagcc gttaatgaga tgctgggacg cccaaccaag 2640gtcaccccat cctccaaggt tgttggcgac ctcgcactcc acctcgttgg tgcgggtgtg 2700gatccagcag actttgctgc cgatccacaa aagtacgaca tcccagactc tgtcatcgcg 2760ttcctgcgcg gcgagcttgg taaccctcca ggtggctggc cagagccact gcgcacccgc 2820gcactggaag gccgctccga aggcaaggca cctctgacgg aagttcctga ggaagagcag 2880gcgcacctcg acgctgatga ttccaaggaa cgtcgcaata gcctcaaccg cctgctgttc 2940ccgaagccaa ccgaagagtt cctcgagcac cgtcgccgct tcggcaacac ctctgcgctg 3000gatgatcgtg aattcttcta cggcctggtc gaaggccgcg agactttgat ccgcctgcca 3060gatgtgcgca ccccactgct tgttcgcctg gatgcgatct ctgagccaga cgataagggt 3120atgcgcaatg ttgtggccaa cgtcaacggc cagatccgcc caatgcgtgt gcgtgaccgc

3180tccgttgagt ctgtcaccgc aaccgcagaa aaggcagatt cctccaacaa gggccatgtt 3240gctgcaccat tcgctggtgt tgtcaccgtg actgttgctg aaggtgatga ggtcaaggct 3300ggagatgcag tcgcaatcat cgaggctatg aagatggaag caacaatcac tgcttctgtt 3360gacggcaaaa tcgatcgcgt tgtggttcct gctgcaacga aggtggaagg tggcgacttg 3420atcgtcgtcg tttcctaaac ctttctgtaa aaagagctca a 34618132DNAArtificialprimer 81aagcatgcta cgcgaacgag ccatgacatt gc 328238DNAArtificialprimer 82catacgtcat tcctccttgt cgcctatatt ggttaaag 3883357DNAEscherichia coli 83aagcatgctc gcgaacgagc catgacattg ctgacgactc tggcagtggc agatgacata 60aaactggtcg actggttaca acaacgcctg gggcttttag agcaacgaga cacggcaatg 120ttgcaccgtt tgctgcatga tattgaaaaa aatatcacca aataaaaaac gccttagtaa 180gtatttttca gcttttcatt ctgactgcaa cgggcaatat gtctctgtgt ggattaaaaa 240aagagtgtct gatagcagct tctgaactgg ttacctgccg tgagtaaatt aaaattttat 300tgacttaggt cactaaatac tttaaccaat ataggcgaca aggaggaatg acgtatg 3578437DNAArtificialprimer 84gacaaggagg aatgacgtat gaatataaac gtcgcag 378530DNAArtificialprimer 85gggaaggatc catcctggga aaaagttctg 30861781DNAEnterobacter aerogenesCDS(19)..(1704) 86gacaaggagg aatgacgt atg aat ata aac gtc gca gat ttg tta aac ggg 51 Met Asn Ile Asn Val Ala Asp Leu Leu Asn Gly 1 5 10aat tac atc ctg tta ttg ttt gtt gta ctc gca ctg ggt ctt tgc ctg 99Asn Tyr Ile Leu Leu Leu Phe Val Val Leu Ala Leu Gly Leu Cys Leu 15 20 25ggg aaa ctt cgc ctc ggg tca gta caa ctt ggt aat tcc att ggc gtt 147Gly Lys Leu Arg Leu Gly Ser Val Gln Leu Gly Asn Ser Ile Gly Val 30 35 40tta gtc gtt tct tta tta tta ggt cag cag cac ttc gcc att aac aca 195Leu Val Val Ser Leu Leu Leu Gly Gln Gln His Phe Ala Ile Asn Thr 45 50 55gat gcg cta aat ctt ggc ttt atg ctg ttt att ttc tgc gtc ggc gtc 243Asp Ala Leu Asn Leu Gly Phe Met Leu Phe Ile Phe Cys Val Gly Val60 65 70 75gaa gcg ggc ccc aac ttt ttt tcg att ttt ttc cgc gac ggc aaa aac 291Glu Ala Gly Pro Asn Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn 80 85 90tat cta atg ctg gcg ctg gtg atg gtg gcc agc gcg atg tta atc gct 339Tyr Leu Met Leu Ala Leu Val Met Val Ala Ser Ala Met Leu Ile Ala 95 100 105atg ggg ctg ggc aaa ttg ttt ggc tgg gac atc ggc ctg acc gcc ggg 387Met Gly Leu Gly Lys Leu Phe Gly Trp Asp Ile Gly Leu Thr Ala Gly 110 115 120atg ctg gcg ggc gct atg acc tcc acc ccg gtg ctg gtg ggc gct ggc 435Met Leu Ala Gly Ala Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly 125 130 135gat acc cta cgc cac ttt ggc ctg ccc agc gat cag ctg gcg cag tcg 483Asp Thr Leu Arg His Phe Gly Leu Pro Ser Asp Gln Leu Ala Gln Ser140 145 150 155ctt gac cac ctg agc ctg ggt tac gcc ctg act tac ctg gtt ggc ctg 531Leu Asp His Leu Ser Leu Gly Tyr Ala Leu Thr Tyr Leu Val Gly Leu 160 165 170gtg agc ctg atc gtc ggc gcg cgc tat atg ccg aag ctg cag cat cag 579Val Ser Leu Ile Val Gly Ala Arg Tyr Met Pro Lys Leu Gln His Gln 175 180 185gat ctg cag acc agc gcc cag cag atc gcc cgc gag cgc ggt ctg gat 627Asp Leu Gln Thr Ser Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp 190 195 200acc gac tcc aag cgt aaa gtc tat ctg ccg gtt atc cgc gcc tac cgc 675Thr Asp Ser Lys Arg Lys Val Tyr Leu Pro Val Ile Arg Ala Tyr Arg 205 210 215gtc ggc ccg gaa ttg gtc gcg tgg gcg gac ggc aaa aat ctt cgt gag 723Val Gly Pro Glu Leu Val Ala Trp Ala Asp Gly Lys Asn Leu Arg Glu220 225 230 235ctg gga att tac cgc cag acc ggc tgc tat atc gag cgt att cgt cgt 771Leu Gly Ile Tyr Arg Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg 240 245 250aac ggt atc ctg gcg aac ccg gat ggc gac gcg gta ctg cag atg ggc 819Asn Gly Ile Leu Ala Asn Pro Asp Gly Asp Ala Val Leu Gln Met Gly 255 260 265gac gat atc gcg ctg gtt ggc tac ccg gac gcc cat gcc cgc ctc gac 867Asp Asp Ile Ala Leu Val Gly Tyr Pro Asp Ala His Ala Arg Leu Asp 270 275 280cca agc ttc cgt aac ggt aaa gag gtg ttt gac cgc gac ctg ctc gat 915Pro Ser Phe Arg Asn Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp 285 290 295atg cgt atc gtc acc gaa gag att gtg gtt aag aat cat aac gcc gtc 963Met Arg Ile Val Thr Glu Glu Ile Val Val Lys Asn His Asn Ala Val300 305 310 315ggc cgc cgc ctg gcg cag ctg aag ctg acc gat cac ggc tgt ttc tta 1011Gly Arg Arg Leu Ala Gln Leu Lys Leu Thr Asp His Gly Cys Phe Leu 320 325 330aac cgg gtg atc cgc agc cag att gag atg cca atc gac gat aac gtg 1059Asn Arg Val Ile Arg Ser Gln Ile Glu Met Pro Ile Asp Asp Asn Val 335 340 345gtg ctc aat aaa ggc gac gtg ctg cag gtg agc ggc gac gcc cgc cgc 1107Val Leu Asn Lys Gly Asp Val Leu Gln Val Ser Gly Asp Ala Arg Arg 350 355 360gta aaa acc gtc gct gac cgc atc ggc ttt att tcg att cac agc cag 1155Val Lys Thr Val Ala Asp Arg Ile Gly Phe Ile Ser Ile His Ser Gln 365 370 375gtg acc gat ctg ctg gct ttc tgc gcc ttc ttt atc gtc ggc ctg atg 1203Val Thr Asp Leu Leu Ala Phe Cys Ala Phe Phe Ile Val Gly Leu Met380 385 390 395atc ggc atg atc act ttc cag ttc agc tcg ttc agc ttc ggc atc ggt 1251Ile Gly Met Ile Thr Phe Gln Phe Ser Ser Phe Ser Phe Gly Ile Gly 400 405 410aac gcc gcc ggc ctg ctg ttc gcc ggc att atg ctt ggc ttc ctg cgc 1299Asn Ala Ala Gly Leu Leu Phe Ala Gly Ile Met Leu Gly Phe Leu Arg 415 420 425gcc aac cat ccg acc ttc ggc tat atc ccg cag ggc gca ttg aac atg 1347Ala Asn His Pro Thr Phe Gly Tyr Ile Pro Gln Gly Ala Leu Asn Met 430 435 440gtt aag gag ttc ggt ctg atg gtg ttt atg gcc ggg gtc ggc ctg agc 1395Val Lys Glu Phe Gly Leu Met Val Phe Met Ala Gly Val Gly Leu Ser 445 450 455gcc ggc gcg ggt att aat aac ggt ctg ggc gcc att ggc ggg caa atg 1443Ala Gly Ala Gly Ile Asn Asn Gly Leu Gly Ala Ile Gly Gly Gln Met460 465 470 475ctg gct gcc ggt ctt atc gtc agc ctg gtg ccg gtg gtg atc tgc ttc 1491Leu Ala Ala Gly Leu Ile Val Ser Leu Val Pro Val Val Ile Cys Phe 480 485 490ctg ttc ggc gcc tat gtg cta cgc atg aac cgc gcg atg ctg ttc ggc 1539Leu Phe Gly Ala Tyr Val Leu Arg Met Asn Arg Ala Met Leu Phe Gly 495 500 505gcg atg atg ggc gcg cgc acc tgc gcc ccg gcg atg gaa att atc agc 1587Ala Met Met Gly Ala Arg Thr Cys Ala Pro Ala Met Glu Ile Ile Ser 510 515 520gat acc gcg cgc agc aac att ccc gca ctc ggc tat gct ggc act tac 1635Asp Thr Ala Arg Ser Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr 525 530 535gct atc gcc aac gtg ctg ctc acc ctt gcc ggt acg cta atc gtc atc 1683Ala Ile Ala Asn Val Leu Leu Thr Leu Ala Gly Thr Leu Ile Val Ile540 545 550 555atc tgg ccg ggc ctt ggc tag ccccggccga aaatttttaa ctttttcgca 1734Ile Trp Pro Gly Leu Gly 560aaaaaatcgg tgatgaacag aactttttcc caggatggat ccttccc 178187561PRTEnterobacter aerogenes 87Met Asn Ile Asn Val Ala Asp Leu Leu Asn Gly Asn Tyr Ile Leu Leu1 5 10 15Leu Phe Val Val Leu Ala Leu Gly Leu Cys Leu Gly Lys Leu Arg Leu 20 25 30Gly Ser Val Gln Leu Gly Asn Ser Ile Gly Val Leu Val Val Ser Leu 35 40 45Leu Leu Gly Gln Gln His Phe Ala Ile Asn Thr Asp Ala Leu Asn Leu 50 55 60Gly Phe Met Leu Phe Ile Phe Cys Val Gly Val Glu Ala Gly Pro Asn65 70 75 80Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn Tyr Leu Met Leu Ala 85 90 95Leu Val Met Val Ala Ser Ala Met Leu Ile Ala Met Gly Leu Gly Lys 100 105 110Leu Phe Gly Trp Asp Ile Gly Leu Thr Ala Gly Met Leu Ala Gly Ala 115 120 125Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly Asp Thr Leu Arg His 130 135 140Phe Gly Leu Pro Ser Asp Gln Leu Ala Gln Ser Leu Asp His Leu Ser145 150 155 160Leu Gly Tyr Ala Leu Thr Tyr Leu Val Gly Leu Val Ser Leu Ile Val 165 170 175Gly Ala Arg Tyr Met Pro Lys Leu Gln His Gln Asp Leu Gln Thr Ser 180 185 190Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp Thr Asp Ser Lys Arg 195 200 205Lys Val Tyr Leu Pro Val Ile Arg Ala Tyr Arg Val Gly Pro Glu Leu 210 215 220Val Ala Trp Ala Asp Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr Arg225 230 235 240Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu Ala 245 250 255Asn Pro Asp Gly Asp Ala Val Leu Gln Met Gly Asp Asp Ile Ala Leu 260 265 270Val Gly Tyr Pro Asp Ala His Ala Arg Leu Asp Pro Ser Phe Arg Asn 275 280 285Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp Met Arg Ile Val Thr 290 295 300Glu Glu Ile Val Val Lys Asn His Asn Ala Val Gly Arg Arg Leu Ala305 310 315 320Gln Leu Lys Leu Thr Asp His Gly Cys Phe Leu Asn Arg Val Ile Arg 325 330 335Ser Gln Ile Glu Met Pro Ile Asp Asp Asn Val Val Leu Asn Lys Gly 340 345 350Asp Val Leu Gln Val Ser Gly Asp Ala Arg Arg Val Lys Thr Val Ala 355 360 365Asp Arg Ile Gly Phe Ile Ser Ile His Ser Gln Val Thr Asp Leu Leu 370 375 380Ala Phe Cys Ala Phe Phe Ile Val Gly Leu Met Ile Gly Met Ile Thr385 390 395 400Phe Gln Phe Ser Ser Phe Ser Phe Gly Ile Gly Asn Ala Ala Gly Leu 405 410 415Leu Phe Ala Gly Ile Met Leu Gly Phe Leu Arg Ala Asn His Pro Thr 420 425 430Phe Gly Tyr Ile Pro Gln Gly Ala Leu Asn Met Val Lys Glu Phe Gly 435 440 445Leu Met Val Phe Met Ala Gly Val Gly Leu Ser Ala Gly Ala Gly Ile 450 455 460Asn Asn Gly Leu Gly Ala Ile Gly Gly Gln Met Leu Ala Ala Gly Leu465 470 475 480Ile Val Ser Leu Val Pro Val Val Ile Cys Phe Leu Phe Gly Ala Tyr 485 490 495Val Leu Arg Met Asn Arg Ala Met Leu Phe Gly Ala Met Met Gly Ala 500 505 510Arg Thr Cys Ala Pro Ala Met Glu Ile Ile Ser Asp Thr Ala Arg Ser 515 520 525Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn Val 530 535 540Leu Leu Thr Leu Ala Gly Thr Leu Ile Val Ile Ile Trp Pro Gly Leu545 550 555 560Gly 88561PRTArtificialconserved sequence 88Xaa Asn Xaa Asn Xaa Ala Xaa Leu Leu Xaa Gly Asn Xaa Ile Leu Leu1 5 10 15Leu Phe Xaa Val Leu Ala Leu Gly Leu Cys Leu Gly Lys Leu Arg Xaa 20 25 30Gly Ser Xaa Gln Leu Gly Asn Ser Ile Gly Val Leu Val Val Ser Leu 35 40 45Leu Leu Gly Gln Gln His Phe Xaa Xaa Asn Thr Asp Ala Leu Xaa Leu 50 55 60Gly Phe Met Leu Phe Ile Phe Cys Val Gly Xaa Glu Ala Gly Pro Asn65 70 75 80Phe Phe Ser Ile Phe Phe Arg Asp Gly Lys Asn Tyr Xaa Met Leu Ala 85 90 95Xaa Val Met Val Xaa Ser Ala Xaa Xaa Xaa Ala Xaa Gly Xaa Gly Lys 100 105 110Leu Phe Gly Trp Asp Ile Gly Leu Thr Ala Gly Xaa Leu Ala Gly Xaa 115 120 125Met Thr Ser Thr Pro Val Leu Val Gly Ala Gly Asp Thr Leu Arg Xaa 130 135 140Xaa Xaa Met Glu Xaa Xaa Gln Leu Xaa Xaa Xaa Xaa Asp Xaa Leu Ser145 150 155 160Leu Gly Tyr Ala Xaa Thr Tyr Leu Xaa Gly Leu Val Ser Leu Ile Xaa 165 170 175Gly Ala Arg Tyr Xaa Pro Xaa Leu Gln His Gln Asp Leu Xaa Thr Xaa 180 185 190Ala Gln Gln Ile Ala Arg Glu Arg Gly Leu Asp Xaa Asp Xaa Xaa Arg 195 200 205Lys Val Xaa Leu Pro Val Ile Arg Ala Tyr Arg Val Gly Pro Glu Leu 210 215 220Val Ala Trp Xaa Xaa Gly Lys Asn Leu Arg Glu Leu Gly Ile Tyr Arg225 230 235 240Gln Thr Gly Cys Tyr Ile Glu Arg Ile Arg Arg Asn Gly Ile Leu Ala 245 250 255Xaa Pro Asp Gly Asp Ala Val Leu Gln Xaa Gly Asp Xaa Ile Xaa Leu 260 265 270Val Gly Tyr Pro Asp Ala His Ala Arg Leu Asp Xaa Ser Phe Arg Asn 275 280 285Gly Lys Glu Val Phe Asp Arg Asp Leu Leu Asp Met Arg Ile Val Thr 290 295 300Glu Glu Xaa Val Val Lys Asn His Asn Ala Val Xaa Xaa Arg Leu Xaa305 310 315 320Xaa Leu Xaa Leu Thr Asp His Gly Cys Phe Leu Asn Arg Val Ile Arg 325 330 335Ser Gln Ile Glu Met Pro Ile Asp Asp Asn Xaa Xaa Leu Asn Lys Gly 340 345 350Asp Val Leu Gln Val Ser Gly Xaa Ala Arg Arg Val Lys Xaa Xaa Ala 355 360 365Asp Arg Ile Gly Phe Xaa Xaa Ile His Ser Gln Xaa Thr Asp Leu Leu 370 375 380Ala Phe Cys Ala Phe Phe Xaa Xaa Gly Leu Met Xaa Gly Xaa Ile Thr385 390 395 400Phe Gln Phe Ser Xaa Phe Ser Phe Gly Xaa Gly Asn Ala Ala Gly Leu 405 410 415Leu Phe Ala Gly Ile Met Leu Gly Phe Xaa Arg Ala Asn His Pro Thr 420 425 430Phe Gly Tyr Ile Pro Gln Gly Ala Leu Xaa Met Val Lys Glu Phe Gly 435 440 445Leu Met Val Phe Met Ala Gly Val Gly Leu Ser Ala Gly Xaa Gly Ile 450 455 460Xaa Xaa Gly Xaa Xaa Xaa Xaa Gly Xaa Xaa Met Leu Xaa Xaa Gly Leu465 470 475 480Xaa Val Ser Leu Xaa Pro Val Val Ile Cys Xaa Leu Phe Gly Ala Tyr 485 490 495Val Leu Arg Met Asn Arg Ala Xaa Leu Phe Gly Ala Xaa Met Gly Ala 500 505 510Arg Thr Cys Ala Pro Ala Met Glu Ile Ile Ser Asp Thr Ala Arg Ser 515 520 525Asn Ile Pro Ala Leu Gly Tyr Ala Gly Thr Tyr Ala Ile Ala Asn Val 530 535 540Leu Leu Thr Leu Ala Gly Thr Xaa Ile Val Xaa Xaa Trp Pro Xaa Leu545 550 555 560Xaa

Claims

1. A microorganism that has an ability to produce an acidic substance having a carboxyl group and has been modified to enhance expression of the ybjL gene.

2. The microorganism according to claim 1, wherein said enhanced expression is obtained by a method selected from the group consisting of: A) increasing copy number of the ybjL gene, B) modifying an expression control sequence of the ybjL gene, and C) combinations thereof.

3. The microorganism according to claim 1, wherein the ybjL gene encodes 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: 2, 4, and 87; (B) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, and 87, but wherein one or several amino acid residues are substituted, deleted, inserted or added, and the protein improves the ability of the microorganism to produce an acidic substance having a carboxyl group when expression of the gene encoding the protein is enhanced in the microorganism.

4. The microorganism according to claim 1, wherein the ybjL gene encodes for a protein selected from the group consisting of SEQ ID NO: 5 and 88.

5. The microorganism according to claim 1, wherein the microorganism is a bacterium belonging to the family Enterobacteriaceae.

6. The microorganism according to claim 5, wherein the bacterium belongs to a genus selected from the group consisting of Escherichia, Enterobacter, Raoultella, Pantoea, and Klebsiella.

7. The microorganism according to claim 1, wherein the microorganism is a rumen bacterium.

8. The microorganism according to claim 7, wherein the microorganism is Mannheimia succiniciproducens.

9. The microorganism according to claim 1, wherein the acidic substance is an organic acid selected from the group consisting of succinic acid, fumaric acid, malic acid, oxalacetic acid, citric acid, isocitric acid, .alpha.-ketoglutaric acid, and combinations thereof.

10. The microorganism according to claim 1, wherein the acidic substance is L-glutamic acid and/or L-aspartic acid.

11. A method for producing an acidic substance having a carboxyl group comprising culturing the microorganism according to claim 1 in a medium to produce and accumulate the acidic substance having a carboxyl group in the medium, and collecting the acidic substance having a carboxyl group from the medium.

12. The method according to claim 11, wherein the acidic substance is an organic acid selected from the group consisting of succinic acid, fumaric acid, malic acid, oxalacetic acid, citric acid, isocitric acid, .alpha.-ketoglutaric acid, and combinations thereof.

13. The method according to claim 11, wherein the acidic substance is L-glutamic acid and/or L-aspartic acid.

14. A method for producing an acidic substance having a carboxyl group 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 said substance is selected from the group consisting of: i) the microorganism according to claim 1, ii) a product obtained by processing the microorganism of i), and iii) combinations thereof, and B) collecting the acidic substance having a carboxyl group.

15. The method according to claim 14, wherein the acidic substance is an organic acid selected from the group consisting of succinic acid, fumaric acid, malic acid, oxalacetic acid, citric acid, isocitric acid, .alpha.-ketoglutaric acid, and combinations thereof.

16. A method for producing a polymer comprising succinic acid, comprising: A) producing succinic acid by the method according to claim 12, and B) polymerizing the obtained succinic acid.