CN1749390A - Method for producing L-amino acid by fermenting xylose using bacteria with enhanced gene expression - Google Patents

Abstract

A method for producing an L-amino acid, such as L-histidine, L-threonine, L-lysine, L-glutamic acid, and L-tryptophan, using bacterium belonging to the genus Escherichia which has increased expression of genes, such as those of the xylABFGHR locus, which encode the xylose utilization enzymes, is disclosed. The method includes cultivating the L-amino acid producing bacterium in a culture medium containing xylose, and collecting the L-amino acid from the culture medium.

Description

Method for producing L-amino acid by fermentation using xylose using bacteria with enhanced gene expression

technical field

The present invention relates to a method for the production of L-amino acids by fermentation of pentose sugars, and more particularly to production of L-amino acids by fermenting arabinose and/or a mixture of xylose together with glucose as a carbon source using bacteria with enhanced expression of xylose utilization genes Methods. In the commercial production of L-amino acids, such as L-histidine, L-threonine, L-lysine, L-glutamic acid, and L-tryptophan, inexpensive carbon sources can be used, including those from A mixture of hexose and pentose sugars in the hemicellulose fraction of cellulosic biomass.

Background technique

Generally, L-amino acids are industrially produced by fermentation using different microbial strains. Fermentation media used in this method generally include sufficient amounts of various carbon and nitrogen sources.

Conventionally, various carbohydrates such as hexoses, pentoses, trioses; various organic acids and alcohols are used as carbon sources. Hexoses include glucose, fructose, mannose, sorbose, galactose, and the like. Pentose sugars include arabinose, xylose, ribose, and the like. However, the above-mentioned carbohydrates, as well as other conventional carbon sources that are now used in industry, such as molasses, grains, cane sugar, starch, its hydrolysates, etc., are quite expensive. Therefore, it is necessary to find out cheaper alternative carbon sources for the production of L-amino acids.

Cellulosic biomass is a suitable feedstock for L-amino acid production because it is readily available and less expensive than carbohydrates, grains, sugar cane or other carbon sources. Typical amounts of cellulose, hemicellulose and lignin in cellulosic biomass are about 40-60% cellulose, 20-40% hemicellulose, 10-25% lignin and 10% other components . The cellulosic component consists of polymers of hexose sugars, usually glucose. The hemicellulose fraction is mainly composed of pentose sugars, including xylose and arabinose. The composition of various cellulosic biomass feedstocks is shown in Table 1 (http://www.ott.doe.gov/biofuels/understanding_biomass.html).

Table 1 Material six-carbon sugar five-carbon sugar lignin Ash hardwood 39-50% 18-28% 15-28% 0.3-1.0% cork 41-57% 8-12% 24-27% 0.1-0.4%

More detailed information on more than 150 biomass (biomass) sample components is compiled in the "Database on Composition and Properties of Biomass Raw Materials" (http://www.ott.doe.gov/biofuels/progs/searchl.cgi).

It has recently been desired to develop an industrial process capable of efficiently converting cellulosic biomass into a mixture of fermentable raw materials, usually carbohydrates. Therefore, in the near future, it is hoped to increase the utilization of renewable energy sources such as cellulose and hemicellulose to produce useful compounds (AristidouA., Pentila.M., Curr.Opin, Biotech nol, 2000, Apr., 11:2, 187-198 ). However, the vast majority of published articles and patents, or patent applications, describe the production of ethanol from cellulosic biomass by means of biocatalysts (bacteria and yeast), which is expected to be an effective alternative fuel. The method involves the use of different strains of Zymomonas mobilis (Deanda K. et al., Appl. Environ. Microbiol., 1996 Dec., 62:12, 4465-70; Mohagheghi A. et al., Appl. Biochem. .Biotechnol., 2002,98-100:885-98; Lawford H.G., RousseauJD., Appl.Biochem.Biotechnol, 2002,98-100:429-48; PCT application WO95/28476, WO98/50524), Escherichia coli ( Escherichia coli) improved strain (Dien B.S. et al., Appl.Biochem.Biotechnol, 2000, 84-86:181-96; Nichols N.N. et al., Appl.Microbiol.Biotechnol., 2001 Jul, 56:1-2, 120-5; US Patent 5,000,000) for the fermentation of cellulosic biomass. Xylitol is produced by fermenting xylose from hemicellulose sugars with Candida tropicalis (Walthers T. et al., Appl. Biochem. Biotechnol., 2001, 91-93:423-35). 1,2-propanediol is produced by fermenting arabinose, fructose, galactose, glucose, lactose, maltose, sucrose, xylose, and combinations thereof using recombinant strains of Escherichia coli (US Patent 6,303,352). It has also been reported that 3-dehydroshikimic acid can be obtained by fermentation of a glucose/xylose/arabinose mixture using E. coli strains. Compared with xylose or glucose alone as carbon source, the highest concentration and yield of 3-dehydroshikimic acid was obtained when glucose/xylose/arabinose mixture was used as carbon source (KaiLi and J.W.Frost, Biotechnol.Prog ., 1999, 15, 876-883).

It has been reported that Escherichia coli can utilize pentose sugars such as L-arabinose and D-xylose (Escherichiacoli and Salmonella, Second Edition, Editor in Chief: FC Neidhardt, ASMPress, Washington DC, 1996). L-arabinose is transported into cells through two inducible systems: (1) a low-affinity permease (K _m about 0.1 mM) encoded by the araE gene, and (2) a high-affinity (K m) encoded by the araFG operon _m is 1 to 3 μM) system. The araF gene encodes a periplasmic binding protein (306 amino acids) with chemotactic receptor function, and the araG locus encodes an inner membrane protein. Sugar metabolism is encoded by the araBAD operon - group of enzymes: isomerase (encoded by araA gene), which reversibly converts aldose to L-ribulose; kinase (encoded by araB gene), which phosphorylates ketose is L-ribulose 5-phosphate; and L-ribulose-5-phosphate-4-epimerase (encoded by the araD gene), which catalyzes the formation of D-xylose-5-phosphate (Escherichia coli and Salmonella, Second Edition, Editor in Chief: FC Neidhardt, ASM Press, Washington DC, 1996).

Most E. coli strains can grow in D-xylose, but the K-12 strain needs to be mutated to grow in this compound. Utilization of this pentose is via an inducible and catabolite-inhibited pathway, which involves transport across the plasma membrane by two inducible permeases (inactive on D-ribose or D-arabinose), isomerizing to D-xylulose, and ATP-dependent phosphorylation of ketopentose to produce D-xylulose 5-phosphate. The high affinity ( _Km of 0.3 to 3 μΜ) transport system is dependent on surrounding cytoplasmic binding proteins (37,000 Da) and is likely driven by energetic compounds. The low affinity (K _m about 170 μM) system is activated by an interproton motive force. The D-xylose-proton-symport system is encoded by the xylE gene. The major gene group corresponding to D-xylose utilization is xylAB(RT). The xylA gene encodes an isomerase (54,000 Da) and the xylB gene encodes a kinase (52,000 Da). The operon includes two transcription initiation sites, one of which is inserted upstream of the xylB open reading frame. Since the low-affinity permease corresponds to the unlinked xylE, the xylT locus, also known as xylF (xylFGHR), likely encodes a high-affinity transport system and thus contains at least two genes (one encoding the peripheral cytosolic protein, one encoding the Intact Membrane Protein) (Escherichia coli and Salmonella, Second Edition, Editor in Chief: FCNeidhardt, ASM Press, Washington DC, 1996). The xylFGHR gene encodes a xylose ABC transporter, wherein the xylF gene encodes a putative xylose-binding protein, the xylG gene encodes a putative ATP-binding protein, the xylH gene encodes a putative membrane component, and the xylR gene encodes a xylose transcriptional activator protein.

Introduce the above codes for L-arabinose isomerase, L-ribulose kinase, L-ribulose 5-phosphate 4-epimerase, xylose isomerase and xylulokinase, plus transaldolase E. coli genes for enzymes and transketolases that allow microorganisms, such as Zymomonas mobilis, to metabolize arabinose and xylose to ethanol (WO/9528476, WO98/50524). Conversely, the Zymomonas genes encoding alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDH) can also be used for ethanol production in E. coli strains (Dien B.S. et al., Appl. Biochem. Biotechnol, 2000, 84-86: 181-96; US Patent 5,000,000).

The authors of the present invention previously disclosed a method for the production of L-amino acids such as L-isoleucine, L-histidine, L-threonine and L - The method of tryptophan (Russian patent application 2003105269).

However, there have been no reports so far of bacteria with enhanced gene expression of xylose utilization genes, such as the xylABFGHR locus, or the production of L-amino acids from mixtures of hexose and pentose sugars using these genes.

Summary of the invention

The object of the present invention is to enhance the yield of L-amino acid producing strains, provide L-amino acid producing bacteria with enhanced expression of xylose utilization genes, and provide the use of the bacterium to obtain the L-amino acid producing bacteria from hexoses, such as glucose, and pentoses, such as xylose or arabinose The method for producing L-amino acid in the mixture of. Fermentation raw material obtained from cellulosic biomass can be used as a carbon source for the culture medium. The realization of this objective is based on the discovery that the xylABFGHR locus cloned on a low copy vector enhances L-amino acids such as L-histidine, L-threonine, L-lysine, L-glutamic acid and L- Production of tryptophan. The microorganisms used are capable of growing in fermented raw materials and efficiently producing L-amino acids. The fermented raw material used as carbon source consists of xylose and arabinose and glucose. Examples of L-amino acid producing strains are Escherichia coli strains. The present invention has thus been completed.

An object of the present invention is to provide an L-amino acid-producing bacterium of the family Enterobacteriaceae having any xylose-utilizing enzyme with enhanced activity.

A further object of the present invention is to provide the above-mentioned bacterium, wherein the bacterium belongs to the genus Escherichia.

A further object of the present invention is to provide the above-mentioned bacterium, wherein the bacterium belongs to the genus Pantoea.

A further object of the present invention is to provide the above bacteria, wherein the xylose utilization enzyme activity is enhanced by increasing the expression level of the xylABFGHR locus.

A further object of the present invention is to provide the above bacteria, wherein the xylose utilization enzyme activity is increased by increasing the copy number of the xylABFGHR locus or modifying the expression regulatory sequence of the gene to enhance the expression of the gene.

A further object of the present invention is to provide the above-mentioned bacteria, wherein the copy number is increased by transforming the bacteria with a low copy vector containing the xylABFGHR locus.

A further object of the present invention is to provide the above-mentioned bacterium, wherein the xylABFGHR locus is derived from a bacterium belonging to the genus Escherichia.

A further object of the present invention is to provide a method for producing L-amino acid, which comprises culturing the above-mentioned bacteria in a medium containing a mixture of glucose and pentose sugar, and collecting L-amino acid from the medium.

A further object of the present invention is to provide the above method, wherein the pentoses are arabinose and xylose.

It is a further object of the present invention to provide the above method, wherein the mixture of sugars is a raw material mixture of sugars obtained from cellulosic biomass.

A further object of the present invention is to provide the above method, wherein the L-amino acid produced is L-histidine.

A further object of the present invention is to provide the above method, wherein the bacterium enhances the expression of genes related to L-histidine biosynthesis.

A further object of the present invention is to provide the above method, wherein the L-amino acid produced is L-threonine.

A further object of the present invention is to provide the above method, wherein the bacterium enhances the expression of genes related to L-threonine biosynthesis.

A further object of the present invention is to provide the above method, wherein the L-amino acid produced is L-lysine.

A further object of the present invention is to provide the above method, wherein the bacterium enhances the expression of genes related to L-lysine biosynthesis.

A further object of the present invention is to provide the above method, wherein the L-amino acid produced is L-glutamic acid.

A further object of the present invention is to provide the above method, wherein the bacterium enhances the expression of genes related to L-glutamic acid biosynthesis.

A further object of the present invention is to provide the above method, wherein the L-amino acid produced is L-tryptophan.

A further object of the present invention is to provide the above method, wherein the bacteria enhance the expression of genes related to L-tryptophan biosynthesis.

The method for producing L-amino acids involves producing L-histidine by fermenting a mixture of glucose and pentoses, such as arabinose and xylose. Also, the method for producing L-amino acid involves producing L-threonine by fermenting a mixture of glucose and pentoses such as arabinose and xylose. Also, the method for producing L-amino acid involves producing L-lysine by fermenting a mixture of glucose and pentose sugars, such as arabinose and xylose. Also, the method for producing L-amino acid involves producing L-glutamic acid by fermenting a mixture of glucose and pentose sugars, such as arabinose and xylose. Also, the method for producing L-amino acid involves producing L-tryptophan by fermenting a mixture of glucose and pentoses such as arabinose and xylose. The above-mentioned glucose and pentose sugar mixtures used as fermentation raw materials can be obtained from underutilized plant biomass resources, such as cellulosic biomass, preferably hydrolysates of cellulose.

Brief description of the drawings

Figure 1 shows the structure of the xylABFGHR locus on the chromosome of E. coli strain MG1655. Arrows in the figure indicate the positions of PCR primers.

Description of the preferred embodiment

In the present invention, "L-amino acid producing bacteria" means a bacterium capable of causing accumulation of L-amino acid in a medium when the bacterium of the present invention is cultured in the medium. The ability to produce L-amino acids can be conferred or enhanced by breeding. Herein, the term "L-amino acid producing bacteria" also means a bacterium capable of producing and accumulating more L-amino acids in a culture medium than wild-type or parental strains, and preferably means that the microorganism is capable of producing and accumulating a lot of L-amino acids in a culture medium. The target L-amino acid is less than 0.5g/L, more preferably not less than 1.0g/L. "L-amino acid" includes L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamic acid Salt, L-Glycine, L-Histidine, L-Isoleucine, L-Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Pro amino acid, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

The enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus , Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia and other genera bacteria. Specifically, according to the taxonomy in the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/wgetorg?mode=Tree&id=1236&1v1=3&keep=1&srchmode= 1&unlock) are classified as bacteria in the family Enterobacteriaceae. Bacteria of the genus Escherichia or Pantoea are preferred.

The term "bacterium of the genus Escherichia" means that the bacterium is classified in the genus Escherichia according to the classification method known to those skilled in the art of microbiology. Examples of bacteria of the genus Escherichia used in the present invention include, but are not limited to, Escherichia coli (E. coli).

The bacteria of the genus Escherichia that can be used in the present invention are not particularly limited, but for example, the bacteria described by Neidhardt, F.C., etc. (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F.C.Neidhardt, ASM Press, Washington D.C., 1208 , Table 1) are included in the present invention. The term "Pantoea bacterium" means that the bacterium is classified in the genus Pantoea according to taxonomy well known to those of ordinary skill in the art of microbiology. According to the nucleotide sequence analysis of 16S rRNA, etc., some species of Enterobacter agglomerans are now reclassified into Pantoea agglomerans, Pantoea ananatis, Pantoea Stewartii, etc.

The term "enhanced activity of a xylose-utilizing enzyme" means that the activity of the enzyme per cell is higher than that of an unmodified strain, such as a wild-type strain. Examples include wherein the number of enzyme molecules per cell is increased and the specific activity of each enzyme molecule is increased, among others. Known methods can be used to measure the amount of gene-encoded protein, including Western blotting analysis after SDS-PAGE and the like. In addition, wild-type strains used as controls include, for example, Escherichia coli K-12. As a result of enhanced intracellular activity of xylose-utilizing enzymes, L-amino acids such as L-histidine, L-threonine, L-lysine, L-glutamic acid and or L-tryptophan can be observed Accumulated in culture medium.

"Xylose utilization enzymes" include enzymes and regulatory proteins of xylose transport, xylose isomerization, and xylose phosphorylation. The enzymes include xylose isomerase, xylulokinase, xylose transporter and xylose transcriptional activator. Xylose isomerase catalyzes the isomerization of D-xylose to D-xylulose. Xylulokinase catalyzes the phosphorylation of D-xylulose using ATP to generate D-xylulose-5-phosphate and ADP. The presence of xylose-utilizing enzymes such as xylose isomerase and xylulokinase activity depends on the complementation of the corresponding xylose isomerase-negative or xylulokinase-negative E. coli mutants, respectively.

The term "bacterium of the genus Escherichia" means that the bacterium is classified in the genus Escherichia according to classifications well known to those of ordinary skill in the art of microbiology. One example of the microorganism belonging to the genus Escherichia used in the present invention is Escherichia coli (E. coli).

The term "increased gene expression level" means that the gene expression level is higher than that of an unmodified strain, such as a wild-type strain. Examples of such modifications include increasing the number of expressed genes per cell, increasing the expression level of a gene, and the like. Quantification of the copy number of expressed genes can be carried out, for example, by restriction enzyme digestion of chromosomal DNA, Southern blotting analysis (Southern blotting), fluorescence in situ hybridization (FISH) and the like using gene sequence-based probes. Gene expression levels can be determined by a variety of methods, including Northern blotting, quantitative RT-PCT, and the like. In addition, wild-type strains that can be used as controls include, for example, Escherichia coli K-12. As a result of enhanced intracellular activity of xylose-utilizing enzymes, L-amino acids such as L-histidine, L-threonine, L-lysine, L-glutamic acid and or L-tryptophan can be observed Accumulates in media containing pentose sugars such as xylose.

Enhancement of the activity of xylose-utilizing enzymes in bacterial cells can be achieved by increasing the expression of genes encoding said enzymes. Genes related to xylose utilization include any genes derived from bacteria of the Enterobacteriaceae family, as well as genes derived from other bacteria such as thermophilic Bacillus sp (Biochem, Mol. Bio. Int., 1996, 39(5) , 1049-1062). Genes derived from bacteria of the genus Escherichia are preferred.

The gene encoding E. coli xylose isomerase (EC number 5.3.1.5) is known and designated xylA (nucleotide numbers 3727072 to 3728394, gi: 16131436 in the sequence of GenBank accession NC_000913.1). The gene encoding xylulokinase (EC number 2.7.1.17) is known and indicated as xylB (nucleotide numbers 3725546 to 3727000, gi: 16131435 in the sequence of GenBank accession NC_000913.1). The gene encoding the xylose-binding protein transport system is known and designated as xylF (nucleotide numbers 3728760 to 3729752, gi: 16131437 in the sequence of GenBank accession NC_000913.1). The gene encoding the putative ATP-binding protein of the xylose transport system is known and designated xylG (nucleotide numbers 3729830 to 3731371, gi: 16131438 in the sequence of GenBank accession NC_000913.1). The gene encoding the permease component of the ABC-like xylose transport system is known and designated xylH (nucleotide numbers 3731349 to 3732530 in the sequence of GenBank accession NC_000913.1, gi: 16131439). The gene encoding the transcriptional regulatory protein of the xyl operon is known and designated xylR (nucleotide numbers 3732608 to 3733786 in the sequence of GenBank accession NC_000913.1, gi: 16131440). Therefore, the above-mentioned gene can be obtained by performing PCR (polymerase chain reaction; refer to White, T.J. et al., Trends Genet., 5, 185 (1989)) using primers based on the nucleotide sequence of the gene.

Genes encoding xylose-utilizing enzymes of other microorganisms can be similarly obtained.

DNA encoding the following protein (A) or (B) is an example of the E. coli xylA gene:

(A) a protein having the amino acid sequence shown in SEQ ID NO: 2; or

(B) A protein having an amino acid sequence obtained after deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2, and having xylose isomerase activity.

DNA encoding the following protein (C) or (D) is an example of the E. coli xylB gene:

(C) a protein having the amino acid sequence shown in SEQ ID NO: 4; or

(D) A protein having an amino acid sequence obtained after deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 4, and having xylulokinase activity.

DNA encoding the following protein (E) or (F) is an example of the E. coli xylF gene:

(E) a protein having the amino acid sequence shown in SEQ ID NO: 6; or

(F) A protein having an amino acid sequence obtained after deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 6, which has the same protein content as xylAB and xylAB in the L-amino acid producing bacteria When the amount of protein encoded by the xylGHR gene increases, increase the content of L-amino acids in the medium, such as L-histidine, L-threonine, L-lysine, L-glutamic acid and or L-tryptophan content active.

DNA encoding the following proteins (G) or (H) is an example of the E. coli xylG gene:

(G) a protein having the amino acid sequence shown in SEQ ID NO: 8; or

(H) A protein having an amino acid sequence obtained after deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 8, which has the same protein content as xylAB and xylAB in L-amino acid producing bacteria When the amount of protein encoded by the xylFHR gene increases, increase the content of L-amino acids in the medium, such as L-histidine, L-threonine, L-lysine, L-glutamic acid and or L-tryptophan active.

DNA encoding the following protein (I) or (J) is an example of the Escherichia coli xylH gene:

(1) a protein having the amino acid sequence shown in SEQ ID NO: 10; or

(J) A protein having an amino acid sequence obtained after deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 10, which has the same protein content as xylAB and xylAB in L-amino acid producing bacteria When the amount of protein encoded by the xylFGR gene increases, increase the content of L-amino acids in the medium, such as L-histidine, L-threonine, L-lysine, L-glutamic acid and or L-tryptophan active.

DNA encoding the following protein (K) or (L) is an example of the E. coli xylR gene;

(K) a protein having the amino acid sequence shown in SEQ ID NO: 12; or

(L) A protein having an amino acid sequence obtained after deletion, substitution, insertion or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 12, which has the same protein amount as xylAB in the L-amino acid producing bacteria When the amount of protein encoded by the xylFGH gene increases, increase the content of L-amino acids in the medium, such as L-histidine, L-threonine, L-lysine, L-glutamic acid and or L-tryptophan activity.

The DNA encoding xylose isomerase includes the DNA encoding the protein (A) in which one or more positions are deleted, replaced, inserted or increased by one or several amino acids, but the protein activity is not lost. Although the number of "several" amino acid differences depends on the position and type of amino acid residues in the three-dimensional structure of the protein, it may be 2 to 50, preferably 2 to 20, more preferably 2 to 10 for protein (A). This is because some amino acids have high homology with each other, and the substitution of these amino acids will not have a large impact on the three-dimensional structure and activity of the protein. Therefore, protein (B) has not less than 30 to 50%, preferably 50 to 70%, more preferably 70-90%, still more preferably more than 90% and most preferably more than 95% of the entire amino acid sequence of xylose isomerase. % homology, and has xylose isomerase activity. The same method and homology judgment can be applied to other proteins (C), (E), (G), (I) and (K).

To calculate the homology level of proteins or DNA, several calculation methods can be used, such as BLAST search, FASTA search and ClustalW.

BLAST (Basic Local Alignment Search Tool) is a heuristic search algorithm used by the programs blastp, blastn, blastx, megablast, tblastn, and tblastx; these programs use the statistical methods of Karlin, Samuel, and Stephen F. Altschul to measure the significance of their search results ("Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes". Proc. Natl. Acad. Sci. USA, 1990, 87: 2264-68; "Applications and statistics for multiple high-scoring quantitative segments in molecular" USA, 1993, 90:5873-7). The FASTA search method is described by W.R. Pearson ("Rapid and Sensitive Sequence Comparison with FASTP and FASTA", Methods in Enzymology, 1990 183:63-98). The ClustalW method was described by Thompson J.D., Higgins D.G. and Gibson T.J. (“CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight martrix choice” Nucleic Acids4-92 Res. 1 4680).

Changes to proteins defined in (A), such as those described above, are generally conservative changes that preserve protein activity. Substitutional changes include changes in which at least one residue within the amino acid sequence is removed and a different residue is inserted in its place. The original amino acids in the above proteins can be replaced, and examples of amino acids that are considered conservative substitutions include: Ala replaced by ser or thr; arg replaced by gln, his or lys; asn replaced by glu, gln, lys, his, asp; asp replaced asn, glu or gln; cys is replaced by ser or ala; gln is replaced by asn, glu, lys, his, asp or arg; glu is replaced by ash, gln, lys or asp; gly is replaced by pro; his is replaced by asn, lys, gln, arg, tyr; ile is replaced by leu, met, val, phe; leu is replaced by ile, met, val, phe; lys is replaced by ash, glu, gln, his, arg; met is replaced by ile, leu, val, phe; phe replaced by trp, tyr, met, ile, or leu; ser replaced by thr, ala; thr replaced by ser or ala; trp replaced by phe, tyr; tyr replaced by his, phe, or trp; and val replaced For met, ile, leu.

Obtaining DNA encoding a protein substantially identical to the protein defined in (A) can be achieved, for example, by modifying the nucleotide sequence encoding the protein defined in (A) by point mutations resulting in one or more amino acid residues Deletion, substitution, insertion or addition. The modified DNA can be obtained by a conventional method using a mutagenic reagent or conditional treatment. The treatment includes treating the protein-encoding DNA of the present invention with hydroxylamine, or treating DNA-containing bacteria with UV radiation or reagents such as N-methyl-N'-nitroso-N-nitrosoguanidine or nitrous acid.

The DNA encoding xylose isomerase includes variants found in different strains of bacteria of the genus Escherichia due to natural diversity. The DNA encoding the variant can be obtained by isolating a DNA that hybridizes to the xylA gene or a part of the gene under stringent conditions and encodes a protein having xylose isomerase activity. The term "stringent conditions" herein includes conditions under which so-called specific hybridization is formed while non-specific hybridization is not formed. For example, stringent conditions include conditions under which DNA having high homology, for example, a mutual homology of not less than 70%, preferably not less than 80%, more preferably not less than 90%, most preferably not less than 95% of the DNA hybridized. Alternatively, conditions as examples of stringent conditions include conventional conditions for Southern hybridization elution, such as 60°C, 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS. The duration of the elution process depends on the type of membrane used for blotting and the standard time recommended by the manufacturer. For example, the recommended elution duration for a Hybond ^™ N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. Preferably, elution can be performed 2 to 3 times. Partial sequences of the nucleotide sequence SEQ ID NO: 1 can also be used as probes for variant-encoding DNA and hybridize to the xylA gene. The probe can be prepared by PCR, wherein an oligonucleotide produced according to the nucleotide sequence of SEQ ID NO: 1 is used as a primer, and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1 is used as a template. When a DNA fragment with a length of about 300 bp is used as a probe, the elution conditions for hybridization may be, for example, 50° C., 2×SSC and 0.1% SDS.

A method similar to that described above for obtaining xylose isomerase can be used to obtain DNA encoding a protein substantially identical to other xylose-utilizing enzymes.

Transforming bacteria with DNA encoding a protein means introducing DNA into bacterial cells by, for example, conventional methods to increase the expression of the gene encoding the protein of the present invention and enhance the activity of the protein in bacterial cells.

The bacterium of the present invention also includes a kind of bacterium, the activity of the protein of the present invention in this bacterium is by using coding (A) or (B), (C) or (D), (E) or (F), (G) or (H ), (I) or (J), (K) or (L) to transform said bacteria with DNA of the protein defined in (I) or (J), (K) or (L), or to obtain enhancement by changing the expression control sequence of said DNA in the bacterial chromosome.

Methods to enhance gene expression include increasing gene copy number. Introduction of a gene into a vector capable of functioning in bacteria of the genus Escherichia increases the copy number of the gene. For this purpose, multi-copy vectors are preferably used. Preferably, low copy vectors are used. Examples of low copy vectors are pSC101, pMW118, pMW119 and the like. The term "low copy vector" is used to denote a vector having a copy number of up to 5 per cell. Methods of transformation include any known to those skilled in the art. For example, treatment of recipient cells with calcium chloride to increase cell permeability to DNA has been reported to be useful in Escherichia coli K-12 (Mandel, M.And Higa, A., J.Mol.Biol., 53, 159 (1970)) and is available here.

Enhancement of gene expression can also be achieved by introducing multiple copies of the gene into the bacterial chromosome by, for example, homologous recombination, Mu integration, and the like. For example, one round of Mu integration can introduce up to 3 copies of a gene into a bacterial chromosome.

On the other hand, the enhancement of gene expression can also be achieved by replacing the natural promoter controlling the DNA of the present invention with a more efficient promoter. The strength of the promoter is measured by the frequency of initiation of RNA synthesis. Deuschle, U., Kammerer, W., Gentz, R., Bujard, H describe methods for measuring promoter strength and examples of efficient promoters (Promoters in Escherichia coli: a hierarchy of in vivo strength indicates alternate structures. EMBO J. 1986, 5, 2987-2994). For example, the _PR promoter is a well known efficient constitutive promoter. Other well-known strong promoters are the _PL promoter, lac promoter, trp promoter, trc promoter, etc. of lambda phage.

Enhanced translation can be achieved by introducing a more efficient Shine-Dalgarno sequence (SD sequence) into the DNA of the invention in place of the native SD sequence. The SD sequence is the upstream region of the mRNA start codon, which interacts with the 16S RNA of the ribosome (Shine J. and Dalgamo L., Proc. Natl. Acad. Sci. USA, 1974, 71, 4, 1342-6).

The use of more efficient promoters can be used in conjunction with methods to increase gene copy number.

Alternatively, the promoter can be enhanced by, for example, introducing a mutation into the promoter, thereby increasing the expression level of a gene located downstream of the promoter. Furthermore, substitution of a few amino acids located between the ribosome binding site (RBS) and the start codon, in particular, the sequence immediately upstream of the start codon is known to greatly affect the translatability of mRNA. For example, expression levels in the 20-fold range were found to be dependent on the nature of the three nucleotides preceding the start codon (Gold et al., Annu. Rev. Microbiol., 35, 365-403, 1981; Hui et al., EMBO J. , 3, 623-629, 1984).

Methods for preparing chromosomal DNA, hybridization, PCR, preparing plasmid DNA, digesting and ligating DNA, transforming, selecting oligonucleotides as primers, etc. may be conventional methods known to those skilled in the art. These methods are described in Sambrook, J., and Russell D., "Molecular Cloning A Laboratory Manual, Third Edition", Cold Spring Harbor Laboratory Press (2001) et al.

The bacterium of the present invention can be obtained by introducing the aforementioned DNA into a bacterium which itself has the ability to produce L-amino acid. Alternatively, the bacterium of the present invention can be obtained by imparting the ability to produce L-amino acid to a bacterium already containing the DNA.

Examples of L-amino acid-producing bacteria of the genus Escherichia are as follows.

L-histidine producing bacteria

The example of the bacterium that has the L-histidine producing ability of Escherichia includes the L-histidine producing bacterium of Escherichia, as E.coli bacterial strain 24 (VKPM B-5945, RU2003677); E. coli strain 80 (VKPM B-7270, RU2119536); Ecoli strain NRRL B-12116-B12121 (US4388405); E. coli strains H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (US6344347) ; E.coli strain H-9341 (FERM BP-6674) (EP1085087); E.coli strain AI80/pFM201 (US6258554) and so on.

Preferably, the bacterium of the present invention is further modified to enhance the expression of the histidine operon gene in the L-histidine producing bacterium, which preferably includes the hisG gene encoding ATP phosphoribosyltransferase, so that the expression of the L-histidine Reduced feedback inhibition sensitivity (Russian patents 2003677 and 2119536).

L-threonine producing bacteria

Examples of parent strains from which the L-threonine-producing bacteria of the present invention are derived include, but are not limited to, L-threonine-producing bacteria of the genus Escherichia, such as E. coli strain TDH-6/pVIC40 (VKPM B- 3996) (US Patent 5,175,107, US Patent 5,705,371), E. coli strain NRRL-21593 (US Patent 5,939,307), E. coli strain FERM BP-3756 (US Patent 5,474,918), E. coli strains FERM BP-3519 and FERM BP -3520 (US Pat. No. 5,376,538), E.coli bacterial strain MG442 (Gusyatiner et al., Genetika (in Russian), 14,947-956 (1978)), E.coli bacterial strain VL643 and VL2055 (EP 1148811A) etc.

Strain TDH-6 lacks the thrC gene, as well as the ability to assimilate sucrose, and has a leaky mutation in the ilvA gene. This strain also has a mutation in the rhtA gene, which results in resistance to high concentrations of threonine or homoserine. Strain B-3996 contains plasmid pVIC40, which was obtained by inserting the thrA*BC operon containing the mutated thrA gene into the RSF1010-derived vector. The mutated thrA gene encodes the aspartokinase homoserine dehydrogenase I, which is largely insensitive to feedback inhibition by threonine. Bacterial strain B-3996 was deposited on November 19, 1987 at the All-Union Antimicrobial Science Center (Nagatinskaya Street 3-A, 117105Moscow, Russian Federation), numbered RIA1867. This bacterial strain is also deposited in Russian National Industrial Microorganism Collection Center (VKPM) (Dorozhny proezd.l, Moscow 117545, Russian Federation) on April 7, 1987, numbered as B-3996, and classified as Escherichia coli Tur 6\p VIC40.

Preferably, the bacterium of the present invention is further modified to enhance the expression of one or more of the following genes:

- a variant thrA gene encoding the aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine;

- thrB gene, which encodes a homoserine kinase;

- thrC gene, which encodes threonine synthase;

- the rhtA gene, which encodes a putative transmembrane protein;

- the asd gene, which encodes aspartate-beta-semialdehyde dehydrogenase; and

- aspC gene, which encodes aspartate aminotransferase;

The thrA gene encoding Escherichia coli aspartokinase homoserine dehydrogenase I has been published (nucleotide numbers 337 to 2799 in the sequence of GenBank accession NC_000913.2, gi: 49175990). The thrA gene is located between the thrL and thrB genes on the E.coli K-12 chromosome. The thrB gene encoding Escherichia coli homoserine kinase has been published (nucleotide numbers 2801 to 3733 in the sequence of GenBank accession NC_000913.2, gi: 49175990). The thrB gene is located between the thrA and thrC genes on the Ecoli K-12 chromosome. The thtC gene encoding E. coli threonine synthase has been published (nucleotide numbers 3734 to 5020 in the sequence of GenBank accession NC_000913.2, gi: 49175990). The thrB gene is located between the thrB gene and the yaaX open reading frame on the E. coli K-12 chromosome. All three genes act together as an independent threonine operon.

The thrA gene encoding a variant of the aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine, and the thrB and thrC genes can be obtained as an operon from the well-known plasmid pVIC40, which exists at coli threonine-producing strain VKPM B-3996. Plasmid pVIC40 is described in detail in US Patent No. 5,705,371.

The rhtA gene is located on the E. coli chromosome 18min close to the glnHPQ operon, which encodes the components of the glutamate transport system, and the rhtA gene is consistent with ORF1 (ybiF gene, GenBank accession number AAA218541 sequence number 764 to 1651, gi: 440181), Located between the pexB and ompX genes. The unit expressing the protein encoded by ORF1 is indicated as rhtA (rht: resistance to homoserine and threonine) gene. Moreover, it has been found that the variation of the rhtA23 gene is an A-for-G substitution at position -1 relative to the ATG start codon (ABSTRACTS of 17 ^th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP1013765A).

The asd gene of Escherichia coli has been published (nucleotide numbers 3572511 to 3571408 in the sequence of GenBank accession NC_000913.1, gi: 16131307), and PCR (polymerase chain reaction; ref. White, TJ. et al., Trends Genet., 5, 185 (1989)). The asd genes of other microorganisms can be obtained in a similar way.

In addition, the aspC gene of Escherichia coli has been published (nucleotide numbers 983742 to 984932 in the sequence of GenBank accession NC_000913.1, gi: 16128895), and can be obtained by PCR. The aspC gene of other microorganisms can be obtained in a similar manner.

L-throlysine producing bacteria

Examples of L-lysine-producing bacteria of the genus Escherichia include mutants having resistance to L-lysine analogues. L-lysine analogues inhibit the growth of Escherichia bacteria, but this inhibition is completely or partially desensitized when L-lysine is present in the medium. Examples of L-lysine analogs include, but are not limited to, lysosin, lysine hydroxamate, S-(2-aminoethanol)-L-cysteine (AEC), γ-methyllysine Amino acid, α-chlorocaprolactam, etc. Mutants resistant to lysine analogues can be obtained by conventional artificial mutagenesis treatment of bacteria belonging to the genus Escherichia. Specific examples of strains usable for the production of L-lysine include Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Patent 4,346,170) and Escherichia coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is desensitized.

Strain WC196 can be used as an L-lysine producer of Escherichia coli. This strain was bred to confer resistance to the strain W3110AEC derived from Escherichia coli K-12. The obtained strain was named Escherichia coli AJ13069 strain, and was deposited on December 6, 1994 at the National Institute of Biological Sciences and Human Technology, Industrial Science and Technology Agency (now National Institute of Advanced Industrial Science and Technology, International Patent Depository Unit , Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan), the deposit number is FERM P-14690. Then, according to the Budapest Treaty, it was converted to an international deposit on September 29, 1995, and the deposit number is FERM BP-5252 (US Patent 5,827,698).

L-glutamic acid producing bacteria

Examples of parent strains from which the L-glutamic acid-producing bacteria of the present invention are derived include, but are not limited to, L-glutamic acid-producing bacteria of the genus Escherichia, such as E. coli strain VL334thrC ⁺ (EP 1172433). E. coli strain VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotrophic strain with mutations in thrC and ilvA genes (US Patent No. 4,278,765). The wild-type allele of the thrC gene was transferred by the usual transduction method using phage P1 inoculated in cells grown in wild-type E. coli strain K12 (VKPM B-7). As a result, an L-isoleucine auxotrophic strain VL334thrC ⁺ (VKPM B-8961) was obtained. This strain is capable of producing L-glutamic acid.

Examples of parent strains from which the L-glutamic acid-producing bacteria of the present invention are derived include mutants lacking a-ketoglutarate dehydrogenase activity or having attenuated a+ ketoglutarate dehydrogenase activity. Bacteria of the genus Escherichia lacking a-ketoglutarate dehydrogenase activity or having attenuated a-ketoglutarate dehydrogenase activity and methods of obtaining them are described in US Pat. Nos. 5,378,616 and 5,573,945. In particular, these strains include the following:

E.coli W3110sucA∷Kmr

E.coli AJ12624(FERM BP-3853)

E.coli AJ12628(FERM BP-3854)

E.coli AJ12949(FERM BP-4881)

E. coli W3110 sucA::Kmr was obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred to as "sucA gene") of E. coli W3110. This strain completely lacks a-ketoglutarate dehydrogenase.

Examples of other L-glutamic acid producing bacteria, including Pantoea mutant strains lacking a-ketoglutarate dehydrogenase activity or having attenuated a-ketoglutarate dehydrogenase activity, obtained as above mentioned. Such strains include Pantoea ananatis AJ13356 (US Patent 6,331,419). Pantoea ananatisAJ13356 was deposited on February 19, 1998 at the National Institute of Biological Sciences and Human Technology, Industrial Science and Technology Agency (now National Institute of Advanced Industrial Science and Technology, International Patent Depository, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan), with accession number FERM P-16645. Then, according to the Budapest Treaty, it was converted to an international deposit on January 11, 1999, with the deposit number FERM BP-6615. Pantoea ananatis AJ13356 lacks a-ketoglutarate dehydrogenase activity due to disruption of the aKGDH-E1 subunit gene (sucA). When the above strain was isolated and preserved as Enterobacter agglomerans AJ13356, it was identified as Enterobacter agglomerans. However, it is now reclassified to Pantoea ananatis based on nucleotide sequencing results of 16S rRNA, etc. Although AJ13356 is deposited as Enterobacter agglomerans in the aforementioned depository, they are described as Pantoea ananatis in this specification.

L-tryptophan producing bacteria

Examples of parent strains from which the L-glutamic acid-producing bacteria of the present invention are derived include, but are not limited to, L-tryptophan-producing bacteria of the genus Escherichia, such as tryptophan-tRNA synthase lacking the trpS mutant gene encoding Ecoli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) (US Patent 5,756,345); E. coli SV164 (pGH5) with serA allele not subject to feedback inhibition by serine (US Patent 6,180,373); lacks tryptophanase E.coli AGX17(pGX44)(NRRL B-12263) and AGX6(pGX50)aroP(NRRL B-12264) (US Patent 4,371,614); E.coli AGX17/pGX50 with enhanced phosphoenolpyruvate production, pACKG4 -pps (WO9708333, US Patent 6,319,696) and the like.

The yddG gene was identified in the past to encode a membrane protein that is not involved in any L-amino acid biosynthetic pathway. Furthermore, it is known that the wild-type allele of the yddG gene confers resistance to L-phenylalanine and several amino acid analogs in microorganisms when amplified in a multi-copy vector within the microorganism. Furthermore, the yddG gene can increase the production of L-phenylalanine or L-tryptophan when an extra copy is introduced into the cells of the corresponding production strain (WO03044192). Therefore, it is preferable to further modify the L-tryptophan producing bacteria so as to enhance the expression of the yddG open reading frame.

L-arginine producing bacteria

Examples of parental strains from which the L-arginine-producing bacteria of the present invention are derived include, but are not limited to, L-arginine-producing bacteria such as E. coli strain 237 (VKPM B-7925) (US Patent Application US2002058315) and other Derivative strains containing mutated N-acetylglutamate synthase (Russian patent application No. 2001112869), E.coli strain 382 (VKPM B-7926) (European patent application EP1170358), introducing the code N-acetylglutamate Arginine-producing strains of the argA gene of acid synthase (JP 57-5693A) and the like.

L-phenylalanine producing bacteria

Examples of parent strains from which the L-phenylalanine-producing bacteria of the present invention are derived include, but are not limited to, L-phenylalanine-producing bacteria of the genus Escherichia, such as E.coli AJ12739 (tyrA::Tn10, tyrR) (VKPMB-8197); Ecoli HW1089 (ATCC 55371) (U.S. Patent 5,354,672) containing the pheA34 gene; Ecoli MWEC101-b (KR8903681); E. coli NRRL B-12141, NRRL B-12145, NRRL B-12RLL6 and N B-12147 (U.S. Patent 4,407,952). In addition, as parent strains, E.coli K-12[W3110(tyrA)/pPHAB](FERM BP-3566), E.coli K-12[W3110(tyrA)/pPHAD](FERM BP-12659), E.coli K-12[W3110(tyrA)/pPHATerm](FERM BP-12662) and E.coli K-12[W3110(tyrA)/pBR-aroG4,pACMAB]( EP 488424 B1). In addition, L-phenylalanine-producing bacteria of the genus Escherichia whose activity of the protein encoded by the yedA gene or the yddG gene has been enhanced (US Patent Application Nos. 2003/0148473 A1 and 2003/0157667 A1 ) can be used.

L-cysteine producing bacteria

Examples of parental strains from which the L-cysteine-producing bacteria of the present invention are derived include, but are not limited to, L-cysteine-producing bacteria of the genus Escherichia , such as those with different coding feedback-resistant serine acyltransferases E.coli strain JM15 transformed with the cysE allele (US Patent 6,218,168, Russian Patent Application 2003121601); E.coli strain W3110 (US Patent 5,972,663) overexpressing genes encoding cytotoxic secreted proteins; with lower cysteine E. coli strain (JP 11-155571A) with amino acid desulfurase activity; E. coli strain W3110 (WO0127307A1) with enhanced activity of cysteine regulator positive transcription regulator protein encoded by cysB gene, etc.

L-leucine producing bacteria

Examples of the parental strains from which the L-leucine-producing bacteria of the present invention are derived include, but are not limited to, L-leucine-producing bacteria of the genus Escherichia, such as E.coli which are resistant to leucine analogs. strains, the analogs include β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397B and JP 08-70879A); Utilize the Ecoli bacterial strain obtained by the genetic engineering method described in WO96/06926; E.coli bacterial strain H-9068 (JP 08-70879A) etc.

The bacteria of the present invention can be improved by enhancing the expression of one or more genes involved in L-leucine biosynthesis. Examples include the genes of the leuABCD operon, preferably the mutated leuA gene, which encodes isopropylmalate synthase that is not feedback inhibited by L-leucine (US Patent 6,403,342). Furthermore, the bacteria of the present invention can be improved by enhancing the expression of one or more genes encoding proteins that secrete L-amino acids out of the bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) (Russian patent application 2001117632).

L-proline producing bacteria

Examples of parent strains from which the L-proline-producing bacteria of the present invention are derived include, but are not limited to, L-proline-producing bacteria of the genus Escherichia, such as E. coli strain 702ilvA (VKPM B-8012), which Lacks the ilvA gene and is able to produce L-proline (EP 1172433). The bacteria of the present invention can be improved by enhancing the expression of one or more genes involved in L-proline biosynthesis. Examples of such genes in L-proline-producing bacteria include proB gene, which encodes glutamic acid kinase insensitive to feedback inhibition by L-proline (DE patent 3127361). Furthermore, the bacteria of the present invention can be improved by enhancing the expression of one or more genes encoding proteins that secrete L-amino acids out of the bacterial cell. Examples of the gene include b2682 and b2683 genes (ygaZH gene) (EP1239041 A2).

The example of the Escherichia bacterium having the activity of producing L-proline includes the following E.coli bacterial strains: NRRL B-12403 and NRRL B-12404 (GB patent 2075056), VKPM B-8012 (Russian patent application 2000124295) , the plasmid mutant described in DE patent 3127361, the plasmid mutant described by Bloom FR et al. (The 15 ^th Miami winter symposium, 1983, p.34) and the like.

The above-mentioned L-amino acid producing strains can be further modified by various methods known to those skilled in the art to enhance the assimilation rate of pentose sugar or enhance the biosynthesis ability of L-amino acid.

By amplifying the pentose assimilation gene, araFG and araBAD genes corresponding to arabinose, or by mutation of the glucose assimilation system (PTS and non-PTS), such as the mutation of ptsG (Nichols N.N. et al. Appl. Microbiol. Biotechnol., 2001, Jul. 56:1-2, 120-5) can further increase pentose utilization.

The method of the present invention includes a method for producing L-amino acid, which includes the steps of cultivating L-amino acid producing bacteria in a medium, accumulating L-amino acid in the medium, and collecting the L-amino acid from the medium, wherein the medium Contains a mixture of glucose and pentoses. In addition, the method of the present invention includes a method for producing L-histidine, which comprises culturing the L-histidine-producing bacteria of the present invention in a medium, accumulating L-histidine in the medium, and extracting the L-histidine from the medium. The step of collecting L-histidine in a medium containing a mixture of glucose and pentose sugars. In addition, the method of the present invention includes a method for producing L-threonine, which comprises culturing the L-threonine-producing bacteria of the present invention in a medium, accumulating L-threonine in the medium, and extracting the L-threonine from the medium. The step of collecting L-threonine in a medium containing a mixture of glucose and pentose sugars. In addition, the method of the present invention includes a method for producing L-lysine, which comprises culturing the L-lysine-producing bacteria of the present invention in a medium, accumulating L-lysine in the medium, and extracting the L-lysine from the medium. The step of collecting L-lysine in a medium containing a mixture of glucose and pentose sugars. In addition, the method of the present invention includes a method for producing L-glutamic acid, which comprises culturing the L-glutamic acid-producing bacteria of the present invention in a medium, accumulating L-glutamic acid in the medium, and removing the L-glutamic acid from the medium. The step of collecting L-glutamic acid in a medium containing a mixture of glucose and pentose sugars. In addition, the method of the present invention includes a method for producing L-tryptophan, which comprises culturing the L-tryptophan-producing bacteria of the present invention in a medium, accumulating L-tryptophan in the medium, and extracting the L-tryptophan from the medium. The step of collecting L-tryptophan in a medium containing a mixture of glucose and pentose sugars.

Mixtures of pentoses, such as xylose and arabinose, and hexoses, such as glucose, can be obtained from underutilized biomass resources. Glucose, xylose, arabinose and other carbohydrates are released from plant biomass by steam and/or concentrated acid hydrolysis, mild acid hydrolysis, enzymatic hydrolysis, eg using cellulase or alkaline treatment. When the substrate is a cellulosic material, the cellulose can be hydrolyzed into sugars and also fermented into L-amino acids simultaneously or separately. Since hemicellulose is more easily hydrolyzed to sugars than cellulose, it is preferred to prehydrolyze the cellulosic material, separate the pentose sugars and then hydrolyze the cellulose to form glucose using steam, acid, alkali, cellulase or a combination of treatments.

Mixtures of glucose/xylose/arabinose compositions at different ratios were used in this study to simulate the composition of glucose and pentose starting material mixtures that might be obtained from plant hydrolysates (see Examples section).

In the present invention, the methods used for culturing, collecting and purifying L-amino acids in the culture medium are similar to the methods of conventional microbial fermentation to produce protein. The medium used for culturing may be a synthetic medium or a natural medium as long as the medium contains carbon sources, nitrogen sources and minerals, and if necessary, nutrients necessary for the growth of microorganisms in an appropriate amount.

The carbon source may include various carbohydrates that can be used as a carbon source by L-amino acid producing bacteria, such as glucose, sucrose, arabinose, xylose, and other pentose and hexose sugars. Glucose, xylose, arabinose and other carbohydrates may be part of the raw material mixture of sugars obtained from cellulosic biomass.

Pentose sugars suitable for fermentation in the present invention include, but are not limited to, xylose and arabinose.

As a nitrogen source, various ammonium salts such as ammonia water and ammonium sulfate, other nitrogen-containing compounds such as amines, natural nitrogen sources such as peptone, soybean hydrolyzate, and assimilated fermentative microorganisms can be used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like can be used. Additional nutrients can be added to the medium as needed. For example, if proline is required for the growth of the microorganism (proline-deficient type), sufficient amount of proline can be added to the medium during cultivation.

Preferably, the culture is carried out under aerobic conditions such as shake flask culture, and agitated culture under aerated conditions, and the culture temperature is 20 to 40°C, preferably 30 to 38°C. The pH of the medium is usually between 5 and 9, preferably between 6.5 and 7.2. The pH of the medium can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, culturing for 1 to 5 days can result in the accumulation of the desired L-amino acid in the liquid medium.

After culturing, solid substances such as cells are removed from the liquid medium by centrifugation or membrane filtration, and then the target L-amino acid is collected and purified by ion exchange, concentration and crystallization methods.

Example

The invention will be more precisely explained with respect to the following non-limiting examples.

Example 1. Cloning of the xvlABFGHR locus from the chromosome of E. coli strain MG1655

According to the genome analysis of E. coli strain MG1655, the gene xylABFGHR could be cloned as an independent HindIII fragment (13.1 kb) among all 556 HindIII chromosome fragments (Fig. 1). For this purpose, a gene library was created with the vector pUC19, which contains an insert of this size, which is able to survive in E. coli.

To create such a library, the chromosomal DNA of MG1655 was restricted with HindIII and the pUC19 vector was restricted with Xbal. Strain MG1655 (ATCC47076, ATCC700926) can be obtained from American Type Culture Collection (10801 University Boulevard, Manassas, VA., 20110-2209, U.S.A).

To prevent self-ligation, the cohesive ends of both DNA products were then filled in with Klenow fragments (two-base filling). After the ligation step, a library of recombinant pUC19 plasmids was obtained. The size of the library is greater than 200,000 clones. Gene banks were analyzed by PCR using primers complementary to plasmid sequences and primers complementary to cloned chromosomal fragments. No DNA fragment with appropriate molecular weight was found in the PCR product, which indicated that the fragment corresponding to the xylABFGHR operon was missing in the established library. This result may be due to the negative influence of the malS gene, yiaA and yiaB ORFs (whose function is unknown), which are also present in the HindIII target fragment. Another possible reason for negative selection is that the Xyl-locus is too large. To overcome this problem, a new gene bank was established based on the modified pUC19 plasmid. The main approach is to clone the Xyl-locus as a set of fragments that do not contain the adjacent malS gene and the yiaA and yiaB ORFs.

For this purpose, the polylinker of plasmid pUC19 was modified by inserting a synthetic DNA fragment containing an MluI restriction site. Two gene banks were established with the modified pUC19 cloning vector. The first library was created by digesting chromosomal DNA of strain MG1655 and modified pUC19 with HindIII and MluI restriction enzymes followed by ligation. The capacity of the library is more than 4,000 clones. The gene bank was analyzed by PCR with primers complementary to the plasmid sequence and primers 1 (SEQ ID NO: 13) and 2 (SEQ ID NO: 14) complementary to the xylABFG fragment of the xyl locus. The expected DNA fragment with suitable molecular weight was found in the PCR product. The next step is to saturate the gene pool with the fragment of interest. After this, the DNA obtained from the source gene pool is digested with an endonuclease in the absence of restriction sites in its fragment of interest. There are Eco1471, KpnI, MlsI, Bst1107I. The frequency of the target plasmid in the enriched library was 1/800 clones. The enriched pool was analyzed by PCR as described above. After enrichment of five consecutive pool cell populations, only 10 clones containing the xylABFG gene were found. The resulting plasmid containing the HindIII-MluI DNA fragment of the gene xylABFG was named pUC19/xylA-G. The HindIII-Mph1103I fragment containing the yiaA and yiaB ORFs was subsequently removed from plasmid pUC19/xylA-G; cohesive ends were filled in by the Klenow fragment and a synthetic linker containing an EcoRI restriction site was inserted by ligation. Thus, plasmid pUC19/xylA-G-2 was obtained. The resulting pUC19/xylA-G-2 plasmid was then cut with the EheI restriction enzyme; cohesive ends were filled in by the Klenow fragment and a synthetic linker containing a HindIII restriction site was inserted by ligation. Thus pUC19/xylA-G-3 was obtained. A HindIII restriction site was inserted into the remaining DNA fragment containing the xylHR gene, resulting in the complete xyl locus.

The second library was created by digesting chromosomal DNA of strain MG1655 and modified pUC19 with PstI and MluI restriction enzymes followed by ligation. The capacity of the library is more than 6,000 clones. The gene bank was analyzed by PCR with primers complementary to the plasmid sequence and primers 3 (SEQ ID NO: 15) and 4 (SEQ ID NO: 16) complementary to the chromosomal fragment clone. The expected DNA fragment with suitable molecular weight was found in the PCR product. The next step is the serial subdivision of the gene pool of known size on the cell population by PCR analysis. The population of cells containing the gene xylHR contained only ten clones after seven consecutive subdivisions of the pool. In this group, the DNA fragment of interest was found by restriction analysis. The resulting plasmid containing the HindIII-MluI DNA fragment of the xylHR gene was named pUC19/xylHR. Subsequently, the HindIII-MluI DNA fragment obtained from plasmid pUC19/xylHR was ligated into pUC19/xylA-G-3 plasmid that had been previously treated with HindIII and MluI restriction enzymes. Finally, the complete xyl locus of strain MG1655 was obtained. The resulting multi-copy plasmid containing the complete xylABFGHR locus was named pUC19/xylA-R.

The HindIII-EcoRI DNA fragment obtained from the pUC19/xylA-R plasmid was then recloned into the low-copy vector pMW119mod digested with HindIII and EcoRI restriction enzymes to obtain the low-copy plasmid pMW119mod-xylA-R containing the complete xylABFGHR locus . The low copy vector pMW119mod was obtained from the commercially available pMW119 vector by removing the PvuII-PvuII fragment. This fragment contains a multiple cloning site and is the major part of the lacZ gene. The LacZ gene contains a site for the lacI repressor, followed by the insertion of a synthetic linker containing EcoRI and HindIII sites, necessary for insertion of the xylABFGHR locus from the pUC19/xylA-R plasmid.

Example 2: Fermentative production using L-histidine producing bacteria in a mixture of glucose and pentose L-histidine

L-histidine producing E. coli strain 80 is a strain for the fermentation of a mixture of glucose and pentose sugars to produce L-histidine. E.coli strain 80 (VKPM B-7270) is described in detail in Russian patent RU2119536 and has been in Russian National Collection of Industrial Microorganisms (Russia, 113545 Moscow, 1 ^st Dorozhny proezd, 1) on October 15, 1999 under the serial number VRPMB- 7270 deposits. Subsequently, it was transferred to an international deposit on January 12, 2004 in accordance with the provisions of the Budapest Treaty. Strain 80 was transformed with the pMW119mod-xylA-R plasmid by a conventional method to obtain strain 80/pMW119mod-xylA-R.

To obtain seed cultures, both strains 80 and 80/pMW119mod-xylA-R were grown in 40 ml test tubes (18 mm) containing 2 ml of L-broth containing 1 g/l of streptomycin at 27°C on a rotary shaker ( 250rpm) for 6 hours. For strain 80/pMW119mod-xylA-R, 100 mg/l of ampicillin was added. Subsequently, 2 ml (5%) of the seed material were inoculated into the fermentation medium. Grow in 40 ml tubes containing 2 ml of fermentation medium for 65 hours at 27°C on a rotary shaker (250 rpm).

After culturing, the amount of L-histidine accumulated in the medium was determined by paper chromatography. The composition of the mobile phase is as follows: butanol: acetate: water = 4:1:1 (v/v). A solution of ninhydrin in acetone (0.5%) was used as a revealing agent. The results are shown in Table 2.

Composition of fermentation medium (g/l):

Sugar (total amount) 100.0

0.2 of Mameno TN (Total Nitrogen)

(soybean hydrolyzate)

L-proline 0.8

(NH ₄ ) ₂ SO ₄ 25.0

K ₂ HPO ₄ 2.0

MgSO ₄ 7H ₂ O 1.0

FeSO ₄ ·7H ₂ O 0.01

MnSO ₄ 5H ₂ O 0.01

Thiamine hydrochloride 0.001

Betaine 2.0

CaCO ₃ 6.0

Streptomycin 1.0

Carbohydrates (glucose, arabinose, xylose), L-proline, betaine and magnesium sulfate were sterilized separately. CaCO _dry heat sterilization at 110 °C for 30 min. The pH was adjusted to 6.0 by KOH before sterilization.

As can be seen from Table 2, enhanced expression of the xylABFGHR locus promoted the yield of L-histidine producing E. coli strain 80 cultured in xylose-containing medium.

Table 2 strain glucose xylose Glucose/xylose 1:1 Arabic candy Glucose/Arabinose 1:1 _OD450 Histidine, g/l _OD450 Histidine, g/l _OD450 Histidine, g/l _OD450 Histidine, g/l _OD450 Histidine, g/l 8080/pMW119mod-xylA-R 4339 8.99.3 no growth 50 0.49.6 3939 3.29.9 3736 10.310.5 4040 8.79.1

Embodiment 3. Use L-threonine producing bacteria to carry out fermentation production in glucose and pentose mixture L-threonine

L-Threonine Production E. coli strain B-3996 was used to evaluate the fermentative production of L-histidine from a mixture of glucose and pentose sugars. Strain B-3996 was transformed with pMW119mod-xylA-R plasmid and vector pMW119 by a conventional method using CaCl ₂ to obtain strains 3996/pMW119mod-xylA-R and 3996/pMW119.

Both Ecoli strains B-3996 and B-3996/pMW119mod-xylA-R were grown on L-agar plates containing streptomycin (50mg/l) and ampicillin (150mg/l) at 37°C for 12-15 hours . Subsequently, a loop of the strain was inoculated in a fermentation medium containing xylose (4%) as a carbon source. Fermentations were carried out in 2 ml of fermentation medium containing streptomycin (50 mg/l) in 20 x 20 mm tubes. Cells were grown for 65 hours at 32°C with shaking at 250 rpm.

After culturing, the amount of L-threonine accumulated in the medium was determined by paper chromatography using the following mobile phase: butanol: acetic acid: water = 4:1:1 (v/v). A solution of ninhydrin (2%) in acetone was used as a revealing agent. Spots containing L-threonine were excised, and L-threonine was eluted with a 0.5% aqueous solution of _CdCl2 , and the amount of L-threonine was determined spectrophotometrically at 540 nm. The results are shown in Table 3.

The composition (g/l) of fermentation medium is as follows:

Sugar (total) 40.0

(NH ₄ ) ₂ SO ₄ 24.0

NaCl 0.8

KH ₂ PO ₄ 2.0

MgSO ₄ 7H ₂ O 0.8

FeSO ₄ ·7H ₂ O 0.02

MnSO ₄ 5H ₂ O 0.02

Thiamine hydrochloride 0.0002

Yeast Extract 1.0

CaCO ₃ 30.0

Dextrose and magnesium sulfate were sterilized separately. CaCO _dry heat sterilization at 180 °C for 2 h. The pH was adjusted to 7.0. Antibiotics are introduced into the medium after sterilization.

table 3 strain xylose _OD540 Threonine, g/l B-3996/pMW119B-3996/pMW119mod-xylA-R 13.9±1.016.1±0.9 7.0±0.29.3±0.9

As shown in Table 3, enhanced expression of the xylABFGHR locus enhanced the yield of L-threonine producing E. coli strain B-3996/pMW119 cultured in xylose-containing medium.

Embodiment 4. Use L-lysine producing bacteria to carry out fermentation production in glucose and pentose mixture L-Lysine

The L-lysine producing E. coli strain WC196ΔcadAΔldc was used to evaluate the fermentative production of L-lysine from a mixture of glucose and pentose sugars. Strain WC196ΔcadAΔldc was obtained from strain WC196 by inactivating the lysine decarboxylase encoded by the ldcC gene and the cadA gene as described in US Patent No. 5,827,698. Strains WC196ΔcadAΔldc/pMW119mod-xylA-R and WC196ΔcadAΔldc/pMW119 were obtained by transforming strain WC196ΔcadAΔldc with pMW119mod-xylA-R plasmid and vector pMW119 by a conventional method using _CaCl2 , respectively.

E. coli strains WC196ΔcadAΔldc/pMW119 and WC196ΔcadAΔldc/pMW119mod-xylA-R were both grown on L-agar plates containing ampicillin (150 mg/l) at 37°C for 12-15 hours. Subsequently, a loop of the strain was inoculated in a fermentation medium containing xylose (4%) or a xylose (2%)/glucose (2%) mixture as carbon source. Fermentations were carried out in 2 ml of fermentation medium in 20 x 20 mm test tubes. Cells were grown for 25 hours at 32°C with shaking at 250 rpm.

After culturing, the amount of L-lysine accumulated in the medium was determined by paper chromatography using the following mobile phase: butanol:acetic acid:water=4:1:1 (v/v). A solution of ninhydrin (2%) in acetone was used as a revealing agent. Spots containing L-lysine were excised, and L-lysine was eluted with a 0.5% aqueous solution of _CdCl2 , and the amount of L-lysine was determined spectrophotometrically at 540 nm. The results are shown in Table 4.

The composition (g/l) of fermentation medium is as follows:

Sugar (total amount) 40.0

(NH ₄ ) ₂ SO ₄ 24.0

KH ₂ PO ₄ 1.0

MgSO ₄ 7H ₂ O 1.0

FeSO ₄ ·7H ₂ O 0.01

MnSO ₄ 5H ₂ O 0.01

Yeast Extract 2.0

CaCO ₃ 30.0

Dextrose and magnesium sulfate were sterilized separately. CaCO _dry heat sterilization at 180 °C for 2 h. The pH was adjusted to 7.0 with KOH. Antibiotics are introduced into the medium after sterilization. strain xylose Glucose/xylose 1:1 _OD540 Lysine, g/l _OD540 Lysine, g/l WC196 ΔcadA1dc/pMW119WC196 ΔcadAΔ1dc/pMW119mod-xylA-R 5,7±0.235.2±0.7 0.01.8±0.2 24.54±0.236.7±0.2 1.0±0.32.0±0.3

As shown in Table 4, enhanced expression of the xylABFGHR locus enhanced the yield of the L-lysine producing E. coli strain WC196ΔcadAΔldc/pMW119 grown in xylose-containing media.

Embodiment 5. Use L-glutamic acid producing bacteria to carry out fermentation production in glucose and pentose mixture L-glutamic acid.

L-glutamate producing E. coli strain AJ12624 was used to evaluate the fermentation of a mixture of glucose and pentose sugars to produce L-glutamate product. The strain AJ12624 was transformed with the pMW119mod-xylA-R plasmid and the vector pMW119 by a conventional method using CaCl ₂ to obtain strains AJ12624/pMW119mod-xylA-R and AJ12624/pMW119.

E. coli strains AJ12624/pMW119 and AJ12624/pMW119mod-xylA-R were both grown on L-agar plates containing ampicillin (150 mg/l) at 37°C for 12-15 hours. Subsequently, a loop of the strain was inoculated to a fermentation medium containing xylose (4%) as a carbon source. Fermentations were carried out in 2 ml of fermentation medium in 20 x 20 mm test tubes. Cells were grown for 48 hours at 32°C with shaking at 250 rpm.

After culturing, the amount of L-glutamic acid accumulated in the medium was determined by paper chromatography using the following mobile phase: butanol:acetic acid:water=4:1:1 (v/v). A solution of ninhydrin (2%) in acetone was used as a revealing agent. Spots containing L-glutamic acid were excised, L-glutamic acid was eluted with a 0.5% aqueous solution of _CdCl2 , and the amount of L-glutamic acid was determined spectrophotometrically at 540 nm. The results are shown in Table 5.

Composition of fermentation medium (g/l):

Sugar 40.0

(NH ₄ ) ₂ SO ₄ 25.0

KH ₂ PO ₄ 2.0

MgSO ₄ 7H ₂ O 1.0

Thiamine hydrochloride 0.0001

L-isoleucine 0.07

CaCO ₃ 25.0

Dextrose and magnesium sulfate were sterilized separately. CaCO _dry heat sterilization at 180 °C for 2 h. The pH was adjusted to 7.2.

table 5 strain xylose _OD540 Glutamic acid, g/l AJ12624/pMW119AJ12624/pMW119mod-xylA-R 8.6±0.38.0±0.2 4.5±0.25.3±0.2

As shown in Table 5, enhanced expression of the xylABFGHR locus promoted the yield of the L-glutamic acid producing E. coli strain AJ12624/pMW119 cultured in xylose-containing medium.

Example 6. Fermentative production using L-tryptophan-producing bacteria in a mixture of glucose and pentose sugars L-tryptophan.

L-Tryptophan Production The E. coli strain SV164/pGH5 was used to evaluate the fermentative production of L-tryptophan from a mixture of glucose and pentose sugars. The strain SV164/pGH5 was transformed with the pMW119mod-xylA-R plasmid and the vector pMW119 by a conventional method using CaCl ₂ to obtain strains SV164/pGH5/pMW119mod-xylA-R and SV164/pGH5/pMW119, respectively.

E. coli strains SV164/pGH5/pMW119 and SV164/pGH5/pMW119mod-xylA-R were grown on L-agar plates containing tetracycline (30mg/l) and ampicillin (150mg/l) at 37°C for 12-15 Hours. Subsequently, a loop of the strain was inoculated into a fermentation medium containing xylose (4%) or a xylose (2%)/glucose (2%) mixture as a carbon source. Fermentations were carried out in 2 ml of fermentation medium in 20 x 20 mm test tubes. Cells were grown for 48 hours at 30°C with shaking at 250 rpm.

After culturing, the amount of L-tryptophan accumulated in the medium was determined by TLC. A 10×15 cm TLC dish was coated with 0.11 mm Sorbfil silica gel (Stock Company Sorbpolymer Krasnodar, Russia) without fluorescent indicator. Sorbfil plates can be visualized using the following mobile phase: propylene glycol: ethylene acetate: 25% liquid ammonia: water = 40:40:7:16 (v/v). A solution of ninhydrin (2%) in acetone was used as a revealing agent. The results are shown in Table 7. Fermentation medium compositions are listed in Table 5, but should be inactivated separately between groups A, B, C, D, E, F and H, as indicated, in order to avoid mutual side effects during inactivation .

Table 6. the solution combination Final concentration, g/l ABCDEF KH ₂ PO ₄ NaCl(NH ₄ ) ₂ SO ₄ L-Methionine L-Phenylalanine L-Tyrosine Mameno (All N) Glucose MgSO ₄ 7H ₂ OCaCl ₂ FeSO ₄ 7H ₂ O Citric Acid Sodium Na ₂ MoO ₄ 2H ₂ OH ₃ BO ₃ CoCl ₂ 6H ₂ OCuSO ₄ 5H ₂ OMnCl ₂ 4H ₂ OZnSO ₄ 7H ₂ O Thiamine Hydrochloride 1.50.51.50.050.10.10.0740.00.30.0110.0751.00.000150.00250.000070.000250.00160.00030.005 GH CaCO ₃ pyridoxine 30.00.03

Solution A was adjusted to pH 7.1 by _NH4OH .

Table 7 strain xylose Glucose/xylose 1:1 _OD540 Tryptophan, g/l _OD540 Tryptophan, g/l SV164/pGH5/pMW119SV164/pGH5/pMW119mod-xylA-R 3.5±0.413.9±0.5 0.5±0.23.6±0.5 15.3±0.215.1±0.8 5.3±0.76.0±0.5

As shown in Table 7, enhanced expression of the xylABFGHR locus enhanced the yield of L-tryptophan producing E. coli strain SV164/pGH5/pMW119 cultured in xylose-containing media.

While the invention has been described in detail with its preferred embodiments, it will be apparent to those skilled in the art that various changes and equivalents may be substituted without departing from the scope of the invention. Each of the foregoing documents is hereby incorporated by reference in its entirety.

Sequence Listing

<110>Ajinomoto Co., Inc.

<120> Method for producing L-amino acid by fermenting xylose using bacteria with enhanced gene expression

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100 105 110

Pro Gln Ser Arg Val Ile Thr Gly Asn Leu Met Met Pro Gly Phe Thr

115 120 125

Ala Pro Lys Leu Leu Trp Val Gln Arg His Glu Pro Glu Ile Phe Arg

130 135 140

Gln Ile Asp Lys Val Leu Leu Pro Lys Asp Tyr Leu Arg Leu Arg Met

145 150 155 160

Thr Gly Glu Phe Ala Ser Asp Met Ser Asp Ala Ala Gly Thr Met Trp

165 170 175

Leu Asp Val Ala Lys Arg Asp Trp Ser Asp Val Met Leu Gln Ala Cys

180 185 190

Asp Leu Ser Arg Asp Gln Met Pro Ala Leu Tyr Glu Gly Ser Glu Ile

195 200 205

Thr Gly Ala Leu Leu Pro Glu Val Ala Lys Ala Trp Gly Met Ala Thr

210 215 220

Val Pro Val Val Ala Gly Gly Gly Asp Asn Ala Ala Gly Ala Val Gly

225 230 235 240

Val Gly Met Val Asp Ala Asn Gln Ala Met Leu Ser Leu Gly Thr Ser

245 250 255

GlyVal Tyr Phe Ala Val Ser Glu Gly Phe Leu Ser Lys Pro Glu Ser

260 265 270

Ala Val His Ser Phe Cys His Ala Leu Pro Gln Arg Trp His Leu Met

275 280 285

Ser Val Met Leu Ser Ala Ala Ser Cys Leu Asp Trp Ala Ala Lys Leu

290 295 300

Thr Gly Leu Ser Asn Val Pro Ala Leu Ile Ala Ala Ala Gln Gln Ala

305 310 315 320

Asp Glu Ser Ala Glu Pro Val Trp Phe Leu Pro Tyr Leu Ser Gly Glu

325 330 335

Arg Thr Pro His Asn Asn Pro Gln Ala Lys Gly Val Phe Phe Gly Leu

340 345 350

Thr His Gln His Gly Pro Asn Glu Leu Ala Arg Ala Val Leu Glu Gly

355 360 365

Val Gly Tyr Ala Leu Ala Asp Gly Met Asp Val Val His Ala Cys Gly

370 375 380

Ile Lys Pro Gln Ser Val Thr Leu Ile Gly Gly Gly Ala Arg Ser Glu

385 390 395 400

Tyr Trp Arg Gln Met Leu Ala Asp Ile Ser Gly Gln Gln Leu Asp Tyr

405 410 415

Arg Thr Gly Gly Asp Val Gly Pro Ala Leu Gly Ala Ala Arg Leu Ala

420 425 430

Gln Ile Ala Ala Asn Pro Glu Lys Ser Leu Ile Glu Leu Leu Pro Gln

435 440 445

Leu Pro Leu Glu Gln Ser His Leu Pro Asp Ala Gln Arg Tyr Ala Ala

450 455 460

Tyr Gln Pro Arg Arg Glu Thr Phe Arg Arg Leu Tyr Gln Gln Leu Leu

465 470 475 480

Pro Leu Met Ala

<210>5

<211>993

<212>DNA

<213> Escherichia coli

<220>

<221> CDS

<222>(1)..(993)

<400>5

atg aaa ata aag aac att cta ctc acc ctt tgc acc tca ctc ctg ctt 48

Met Lys Ile Lys Asn Ile Leu Leu Thr Leu Cys Thr Ser Leu Leu Leu

1 5 10 15

acc aac gtt gct gca cac gcc aaa gaa gtc aaa ata ggt atg gcg att 96

Thr Asn Val Ala Ala His Ala Lys Glu Val Lys Ile Gly Met Ala Ile

20 25 30

gat gat ctc cgt ctt gaa cgc tgg caa aaa gat cga gat atc ttt gtg 144

Asp Asp Leu Arg Leu Glu Arg Trp Gln Lys Asp Arg Asp Ile Phe Val

35 40 45

aaa aag gca gaa tct ctc ggc gcg aaa gta ttt gta cag tct gca aat 192

Lys Lys Ala Glu Ser Leu Gly Ala Lys Val Phe Val Gln Ser Ala Asn

50 55 60

ggc aat gaa gaa aca caa atg tcg cag att gaa aac atg ata aac cgg 240

Gly Asn Glu Glu Thr Gln Met Ser Gln Ile Glu Asn Met Ile Asn Arg

65 70 75 80

ggt gtc gat gtt ctt gtc att att ccg tat aac ggt cag gta tta agt 288

Gly Val Asp Val Leu Val Ile Ile Pro Tyr Asn Gly Gln Val Leu Ser

85 90 95

aac gtt gta aaa gaa gcc aaa caa gaa ggc att aaa gta tta gct tac 336

Asn Val Val Lys Glu Ala Lys Gln Glu Gly Ile Lys Val Leu Ala Tyr

100 105 110

gac cgt atg att aac gat gcg gat atc gat ttt tat att tct ttc gat 384

Asp Arg Met Ile Asn Asp Ala Asp Ile Asp Phe Tyr Ile Ser Phe Asp

115 120 125

aac gaa aaa gtc ggt gaa ctg cag gca aaa gcc ctg gtc gat att gtt 432

Asn Glu Lys Val Gly Glu Leu Gln Ala Lys Ala Leu Val Asp Ile Val

130 135 140

ccg caa ggt aat tac ttc ctg atg ggc ggc tcg ccg gta gat aac aac 480

Pro Gln Gly Asn Tyr Phe Leu Met Gly Gly Ser Pro Val Asp Asn Asn

145 150 155 160

gcc aag ctg ttc cgc gcc gga caa atg aaa gtg tta aaa cct tac gtt 528

Ala Lys Leu Phe Arg Ala Gly Gln Met Lys Val Leu Lys Pro Tyr Val

165 170 175

gat tcc gga aaa att aaa gtc gtt ggt gac caa tgg gtt gat ggc tgg 576

Asp Ser Gly Lys Ile Lys Val Val Gly Asp Gln Trp Val Asp Gly Trp

180 185 190

tta ccg gaa aac gca ttg aaa att atg gaa aac gcg cta acc gcc aat 624

Leu Pro Glu Asn Ala Leu Lys Ile Met Glu Asn Ala Leu Thr Ala Asn

195 200 205

aat aac aaa att gat gct gta gtt gcc tca aac gat gcc acc gca ggt 672

Asn Asn Lys Ile Asp Ala Val Val Ala Ser Asn Asp Ala Thr Ala Gly

210 215 220

ggg gca att cag gca tta agc gcg caa ggt tta tca ggg aaa gta gca 720

Gly Ala Ile Gln Ala Leu Ser Ala Gln Gly Leu Ser Gly Lys Val Ala

225 230 235 240

atc tcc ggc cag gat gcg gat ctc gca ggt att aaa cgt att gct gcc 768

Ile Ser Gly Gln Asp Ala Asp Leu Ala Gly Ile Lys Arg Ile Ala Ala

245 250 255

ggt acg caa act atg acg gtg tat aaa cct att acg ttg ttg gca aat 816

Gly Thr Gln Thr Met Thr Val Tyr Lys Pro Ile Thr Leu Leu Ala Asn

260 265 270

act gcc gca gaa att gcc gtt gag ttg ggc aat ggt cag gaa cca aaa 864

Thr Ala Ala Glu Ile Ala Val Glu Leu Gly Asn Gly Gln Glu Pro Lys

275 280 285

gca gat acc aca ctg aat aat ggc ctg aaa gat gtc ccc tcc cgc ctc 912

Ala Asp Thr Thr Leu Asn Asn Gly Leu Lys Asp Val Pro Ser Arg Leu

290 295 300

ctg aca ccg atc gat gtg aat aaa aac aac atc aaa gat acg gta att 960

Leu Thr Pro Ile Asp Val Asn Lys Asn Asn Ile Lys Asp Thr Val Ile

305 310 315 320

aaa gac gga ttc cac aaa gag agc gag ctg taa 993

Lys Asp Gly Phe His Lys Glu Ser Glu Leu

325 330

<210>6

<211>330

<212>PRT

<213> Escherichia coli

<400>6

Met Lys Ile Lys Asn Ile Leu Leu Thr Leu Cys Thr Ser Leu Leu Leu

1 5 10 15

Thr Asn Val Ala Ala His Ala Lys Glu Val Lys Ile Gly Met Ala lle

20 25 30

Asp Asp Leu Arg Leu Glu Arg Trp Gln Lys Asp Arg Asp Ile Phe Val

35 40 45

Lys Lys Ala Glu Ser Leu Gly Ala Lys Val Phe Val Gln Ser Ala Asn

50 55 60

Gly Asn Glu Glu Thr Gln Met Ser Gln Ile Glu Asn Met Ile Asn Arg

65 70 75 80

Gly Val Asp Val Leu Val Ile Ile Pro Tyr Asn Gly Gln Val Leu Ser

85 90 95

Asn Val Val Lys Glu Ala Lys Gln Glu Gly Ile Lys Val Leu Ala Tyr

100 105 110

Asp Arg Met Ile Asn Asp Ala Asp Ile Asp Phe Tyr Ile Ser Phe Asp

115 120 125

Asn Glu Lys Val Gly Glu Leu Gln Ala Lys Ala Leu Val Asp Ile Val

130 135 140

Pro Gln Gly Asn Tyr Phe Leu Met Gly Gly Ser Pro Val Asp Asn Asn

145 150 155 160

Ala Lys Leu Phe Arg Ala Gly Gln Met Lys Val Leu Lys Pro Tyr Val

165 170 175

Asp Ser Gly Lys lle Lys Val Val Gly Asp Gln Trp Val Asp Gly Trp

180 185 190

Leu Pro Glu Asn Ala Leu Lys Ile Met Glu Asn Ala Leu Thr Ala Asn

195 200 205

Asn Asn Lys Ile Asp Ala Val Val Ala Ser Asn Asp Ala Thr Ala Gly

210 215 220

Gly Ala Ile Gln Ala Leu Ser Ala Gln Gly Leu Ser Gly Lys Val Ala

225 230 235 240

Ile Ser Gly Gln Asp Ala Asp Leu Ala Gly Ile Lys Arg Ile Ala Ala

245 250 255

Gly Thr Gln Thr Met Thr Val Tyr Lys Pro Ile Thr Leu Leu Ala Asn

260 265 270

Thr Ala Ala Glu Ile Ala Val Glu Leu Gly Asn Gly Gln Glu Pro Lys

275 280 285

Ala Asp Thr Thr Leu Asn Asn Gly Leu Lys Asp Val Pro Ser Arg Leu

290 295 300

Leu Thr Pro Ile Asp Val Asn Lys Asn Asn Ile Lys Asp Thr Val Ile

305 310 315 320

Lys Asp Gly Phe His Lys Glu Ser Glu Leu

325 330

<210>7

<211>1542

<212>DNA

<213> Escherichia coli

<220>

<221> CDS

<222>(1)..(1542)

<400>7

atg cct tat cta ctt gaa atg aag aac att acc aaa acc ttc ggc agt 48

Met Pro Tyr Leu Leu Glu Met Lys Asn Ile Thr Lys Thr Phe Gly Ser

1 5 10 15

gtg aag gcg att gat aac gtc tgc ttg cgg ttg aat gct ggc gaa atc 96

Val Lys Ala Ile Asp Asn Val Cys Leu Arg Leu Asn Ala Gly Glu Ile

20 25 30

gtc tca ctt tgt ggg gaa aat ggg tct ggt aaa tca acg ctg atg aaa 144

Val Ser Leu Cys Gly Glu Asn Gly Ser Gly Lys Ser Thr Leu Met Lys

35 40 45

gtg ctg tgt ggt att tat ccc cat ggc tcc tac gaa ggc gaa att att 192

Val Leu Cys Gly Ile Tyr Pro His Gly Ser Tyr Glu Gly Glu Ile Ile

50 55 60

ttt gcg gga gaa gag att cag gcg agt cac atc cgc gat acc gaa cgc 240

Phe Ala Gly Glu Glu Ile Gln Ala Ser His Ile Arg Asp Thr Glu Arg

65 70 75 80

aaa ggt atc gcc atc atc att cat cag gaa ttg gcc ctg gtg aaa gaa ttg 288

Lys Gly Ile Ala Ile Ile His Gln Glu Leu Ala Leu Val Lys Glu Leu

85 90 95

acc gtg ctg gaa aat atc ttc ctg ggt aac gaa ata acc cac aat ggc 336

Thr Val Leu Glu Asn Ile Phe Leu Gly Asn Glu Ile Thr His Asn Gly

100 105 110

att atg gat tat gac ctg atg acg cta cgc tgt cag aag ctg ctc gca 384

Ile Met Asp Tyr Asp Leu Met Thr Leu Arg Cys Gln Lys Leu Leu Ala

115 120 125

cag gtc agt tta tcc att tca cct gat acc cgc gtt ggc gat tta ggg 432

Gln Val Ser Leu Ser Ile Ser Pro Asp Thr Arg Val Gly Asp Leu Gly

130 135 140

ctt ggg caa caa caa ctg gtt gaa att gcc aag gca ctt aat aaa cag 480

Leu Gly Gln Gln Gln Leu Val Glu Ile Ala Lys Ala Leu Asn Lys Gln

145 150 155 160

gtg cgc ttg tta att ctc gat gaa ccg aca gcc tca tta act gag cag 528

Val Arg Leu Leu Ile Leu Asp Glu Pro Thr Ala Ser Leu Thr Glu Gln

165 170 175

gaa acg tcg att tta ctg gat att att cgc gat cta caa cag cac ggt 576

Glu Thr Ser Ile Leu Leu Asp Ile Ile Arg Asp Leu Gln Gln His Gly

180 185 190

atc gcc tgt att tat att tcg cac aaa ctc sac gaa gtc aaa gcg att 624

Ile Ala Cys IIe Tyr Ile Ser His Lys Leu Asn Glu Val Lys Ala Ile

195 200 205

tcc gat acg att tgc gtt att cgc gac gga cag cac att ggt acg cgt 672

Ser Asp Thr Ile Cys Val Ile Arg Asp Gly Gln His Ile Gly Thr Arg

210 215 220

gat gct gcc gga atg agt gaa gac gat att atc acc atg atg gtc ggg 720

Asp Ala Ala Gly Met Ser Glu Asp Asp Ile Ile Thr Met Met Val Gly

225 230 235 240

cga gag tta acc gcg ctt tac cct aat gaa cca cat acc acc gga gat 768

Arg Glu Leu Thr Ala Leu Tyr Pro Asn Glu Pro His Thr Thr Gly Asp

245 250 255

gaa ata tta cgt att gaa cat ctg acg gca tgg cat ccg gtt aat cgt 816

Glu Ile Leu Arg Ile Glu His Leu Thr Ala Trp His Pro Val Asn Arg

260 265 270

cat att aaa cga gtt aat gat gtc tcg ttt tcc ctg aaa cgt ggc gaa 864

His Ile Lys Arg Val Asn Asp Val Ser Phe Ser Leu Lys Arg Gly Glu

275 280 285

ata ttg ggt att gcc gga ctc gtt ggt gcc gga cgt acc gag acc att 912

Ile Leu Gly Ile Ala Gly Leu Val Gly Ala Gly Arg Thr Glu Thr Ile

290 295 300

cag tgc ctg ttt ggt gtg tgg ccc gga caa tgg gaa gga aaa att tat 960

Gln Cys Leu Phe Gly Val Trp Pro Gly Gln Trp Glu Gly Lys Ile Tyr

305 310 315 320

att gat ggc aaa cag gta gat att cgt aac tgt cag caa gcc atc gcc 1008

Ile Asp Gly Lys Gln Val Asp Ile Arg Asn Cys Gln Gln Ala Ile Ala

325 330 335

cag ggg att gcg atg gtc ccc gaa gac aga aag cgc gac ggc atc gtt 1056

Gln Gly Ile Ala Met Val Pro Glu Asp Arg Lys Arg Asp Gly Ile Val

340 345 350

ccg gta atg gcg gtt ggt aaa aat att acc ctc gcc gca ctc aat aaa 1104

Pro Val Met Ala Val Gly Lys Asn Ile Thr Leu Ala Ala Leu Asn Lys

355 360 365

ttt acc ggt ggc att agc cag ctt gat gac gcg gca gag caa aaa tgt 1152

Phe Thr Gly Gly Ile Ser Gln Leu Asp Asp Ala Ala Glu Gln Lys Cys

370 375 380

att ctg gaa tca atc cag caa ctc aaa gtt aaa acg tcg tcc ccc gac 1200

Ile Leu Glu Ser Ile Gln Gln Leu Lys Val Lys Thr Ser Ser Pro Asp

385 390 395 400

ctt gct att gga cgt ttg agc ggc ggc aat cag caa aaa gcg atc ctc 1248

Leu Ala Ile Gly Arg Leu Ser Gly Gly Asn Gln Gln Lys Ala Ile Leu

405 410 415

gct cgc tgt ctg tta ctt aac ccg cgc att ctc att ctt gat gaa ccc 1296

Ala Arg Cys Leu Leu Leu Asn Pro Arg Ile Leu Ile Leu Asp Glu Pro

420 425 430

acc agg ggt atc gat att ggc gcg aaa tac gag atc tac aaa tta att 1344

Thr Arg Gly Ile Asp Ile Gly Ala Lys Tyr Glu Ile Tyr Lys Leu Ile

435 440 445

aac caa ctc gtc cag cag ggt att gcc gtt att gtc atc tct tcc gaa 1392

Asn Gln Leu Val Gln Gln Gly Ile Ala Val Ile Val Ile Ser Ser Glu

450 455 460

tta cct gaa gtg ctc ggc ctt agc gat cgt gta ctg gtg atg cat gaa 1440

Leu Pro Glu Val Leu Gly Leu Ser Asp Arg Val Leu Val Met His Glu

465 470 475 480

ggg aaa cta aaa gcc aac ctg ata aat cat aac ctg act cag gag cag 1488

Gly Lys Leu Lys Ala Asn Leu Ile Asn His Asn Leu Thr Gln Glu Gln

485 490 495

gtg atg gaa gcc gca ttg agg agc gaa cat cat gtc gaa aag caa tcc 1536

Val Met Glu Ala Ala Leu Arg Ser Glu His His Val GluLys Gln Ser

500 505 510

gtc tga 1542

Val

<210>8

<211>513

<212>PRT

<213> Escherichia coli

<400>8

Met Pro Tyr Leu Leu Glu Met Lys Asn Ile Thr Lys Thr Phe Gly Ser

1 5 10 15

Val Lys Ala Ile Asp Asn Val Cys Leu Arg Leu Asn Ala Gly Glu Ile

20 25 30

Val Ser Leu Cys Gly Glu Asn Gly Ser Gly Lys Ser Thr Leu Met Lys

35 40 45

Val Leu Cys Gly Ile Tyr Pro His Gly Ser Tyr Glu Gly Glu Ile Ile

50 55 60

Phe Ala Gly Glu Glu Ile Gln Ala Ser His Ile Arg Asp Thr Glu Arg

65 70 75 80

Lys Gly Ile Ala lle Ile His Gln Glu Leu Ala Leu Val Lys Glu Leu

85 90 95

Thr Val Leu Glu Asn Ile Phe Leu Gly Asn Glu Ile Thr His Asn Gly

100 105 110

Ile Met Asp Tyr Asp Leu Met Thr Leu Arg Cys Gln Lys Leu Leu Ala

115 120 125

Gln Val Ser Leu Ser Ile Ser Pro Asp Thr Arg Val Gly Asp Leu Gly

130 135 140

Leu Gly Gln Gln Gln LeuVal Glu Ile Ala Lys Ala Leu Asn Lys Gln

145 150 155 160

Val Arg Leu Leu Ile Leu Asp Glu Pro Thr Ala Ser Leu Thr Glu Gln

165 170 175

Glu Thr Ser Ile Leu Leu Asp Ile Ile Arg Asp Leu Gln Gln His Gly

180 185 190

Ile Ala Cys Ile Tyr Ile Ser His Lys Leu Asn Glu Val Lys Ala Ile

195 200 205

Ser Asp Thr Ile Cys Val Ile Arg Asp Gly Gln His Ile Gly Thr Arg

210 215 220

Asp Ala Ala Gly Met Ser Glu Asp Asp Ile Ile Thr Met Met Val Gly

225 230 235 240

Arg Glu Leu Thr Ala Leu Tyr Pro Asn Glu Pro His Thr Thr Gly Asp

245 250 255

Glu Ile Leu Arg Ile Glu His Leu Thr Ala Trp His Pro Val Asn Arg

260 265 270

His Ile Lys Arg Val Asn Asp Val Ser Phe Ser Leu Lys Arg Gly Glu

275 280 285

Ile Leu Gly Ile Ala Gly Leu Val Gly Ala Gly Arg Thr Glu Thr Ile

290 295 300

Gln Cys Leu Phe Gly Val Trp Pro Gly Gln Trp Glu Gly Lys Ile Tyr

305 310 315 320

Ile Asp Gly Lys Gln Val Asp Ile Arg Asn Cys Gln Gln Ala Ils Ala

325 330 335

Gln Gly Ile Ala Met Val Pro Glu Asp Arg Lys Arg Asp Gly Ile Val

340 345 350

Pro Val Met Ala Val Gly Lys Asn Ile Thr Leu Ala Ala Leu Asn Lys

355 360 365

Phe Thr Gly Gly Ile Ser Gln Leu Asp Asp Ala Ala Glu Gln Lys Cys

370 375 380

Ile Leu Glu Ser Ile Gln Gln Leu Lys Val Lys Thr Ser Ser Pro Asp

385 390 395 400

Leu Ala Ile Gly Arg Leu Ser Gly Gly Asn Gln Gln Lys Ala Ile Leu

405 410 415

Ala Arg Cys Leu Leu Leu Asn Pro Arg Ile Leu Ile Leu Asp Glu Pro

420 425 430

Thr Arg Gly Ile Asp Ile Gly Ala Lys Tyr Glu Ile Tyr Lys Leu Ile

435 440 445

Asn Gln Leu Val Gln Gln Gly Ile Ala Val Ile Val Ile Ser Ser Glu

450 455 460

Leu Pro Glu Val Leu Gly Leu Ser Asp Arg Val Leu Val Met His Glu

465 470 475 480

Gly Lys Leu Lys Ala Asn Leu Ile Asn His Asn Leu Thr Gln Glu Gln

485 490 495

Val Met Glu Ala Ala Leu Arg Ser Glu His His Val Glu Lys Gln Ser

500 505 510

Val

<210>9

<211>1182

<212>DNA

<213> Escherichia coli

<220>

<221> CDS

<222>(1)..(1182)

<400>9

atg tcg aaa agc aat ccg tct gaa gtg aaa ttg gcc gta ccg aca tcc 48

Met Ser Lys Ser Asn Pro Ser Glu Val Lys Leu Ala Val Pro Thr Ser

1 5 10 15

ggt ggc ttc tcc ggg ctg aaa tca ctg aat ttg cag gtc ttc gtg atg 96

Gly Gly Phe Ser Gly Leu Lys Ser Leu Asn Leu Gln Val Phe Val Met

20 25 30

att gca gct atc atc gca atc atg ctg ttc ttt acc tgg acc acc gat 144

Ile Ala Ala Ile Ile Ala Ile Met Leu Phe Phe Thr Trp Thr Thr Asp

35 40 45

ggt gcc tac tta agc gcc cgt aac gtc tcc aac ctg tta cgc cag acc 192

Gly Ala Tyr Leu Ser Ala Arg Asn Val Ser Asn Leu Leu Arg Gln Thr

50 55 60

gcg att acc ggc atc ctc gcg gta gga atg gtg ttc gtc ata att tct 240

Ala Ile Thr Gly Ile Leu Ala Val Gly Met Val Phe Val Ile Ile Ser

65 70 75 80

gct gaa atc gac ctt tcc gtc ggc tca atg atg ggg ctg tta ggt ggc 288

Ala Glu Ile Asp Leu Ser Val Gly Ser Met Met Gly Leu Leu Gly Gly

85 90 95

gtc gcg gcg att tgt gac gtc tgg tta ggc tgg cct ttg cca ctt acc 336

Val Ala Ala Ile Cys Asp Val Trp Leu Gly Trp Pro Leu Pro Leu Thr

100 105 110

atc att gtg acg ctg gtt ctg gga ctg ctt ctc ggt gcc tgg aac gga 384

Ile Ile Val Thr Leu Val Leu Gly Leu Leu Leu Gly Ala Trp Asn Gly

115 120 125

tgg tgg gtc gcg tac cgt aaa gtc cct tca ttt att gtc acc ctc gcg 432

Trp Trp Val Ala Tyr Arg Lys Val Pro Ser Phe Ile Val Thr Leu Ala

130 135 140

ggc atg ttg gca ttt cgc ggc ata ctc att ggc atc acc aac ggc acg 480

Gly Met Leu Ala Phe Arg Gly Ile Leu Ile Gly Ile Thr Asn Gly Thr

145 150 155 160

act gta tcc ccc acc agc gcc gcg atg tca caa att ggg caa agc tat 528

Thr Val Ser Pro Thr Ser Ala Ala Met Ser Gln Ile Gly Gln Ser Tyr

165 170 175

ctc ccc gcc agt acc ggc ttc atc att ggc gcg ctt ggc tta atg gct 576

Leu Pro Ala Ser Thr Gly Phe Ile Ile Gly Ala Leu Gly Leu Met Ala

180 185 190

ttt gtt ggt tgg caa tgg cgc gga aga atg cgc cgt cag gct ttg ggt 624

Phe Val Gly Trp Gln Trp Arg Gly Arg Met Arg Arg Gln Ala Leu Gly

195 200 205

tta cag tct ccg gcc tct acc gca gta gtc ggt cgc cag gct tta acc 672

Leu Gln Ser Pro Ala Ser Thr Ala Val Val Gly Arg Gln Ala Leu Thr

210 215 220

gct atc atc gta tta ggc gca atc tgg ctg ttg aat gat tac cgt ggc 720

Ala Ile Ile Val Leu Gly Ala Ile Trp Leu Leu Asn Asp Tyr Arg Gly

225 230 235 240

gtt ccc act cct gtt ctg ctg ctg acg ttg ctg tta ctc ggc gga atg 768

Val Pro Thr Pro Val Leu Leu Leu Thr Leu Leu Leu Leu Gly Gly Met

245 250 255

ttt atg gca acg cgg acg gca ttt gga cga cgc att tat gcc atc ggc 816

Phe Met Ala Thr Arg Thr Ala Phe Gly Arg Arg Ile Tyr Ala Ile Gly

260 265 270

ggc aat ctg gaa gca gca cgt ctc tcc ggg att aac gtt gaa cgc acc 864

Gly Asn Leu Glu Ala Ala Arg Leu Ser Gly Ile Asn Val Glu Arg Thr

275 280 285

aaa ctt gcc gtg ttc gcg att aac gga tta atg gta gcc atc gcc gga 912

Lys Leu Ala Val Phe Ala Ile Asn Gly Leu Met Val Ala Ile Ala Gly

290 295 300

tta atc ctt agt tct cga ctt ggc gct ggt tca cct tct gcg gga aat 960

Leu Ile Leu Ser Ser Arg Leu Gly Ala Gly Ser Pro Ser Ala Gly Asn

305 310 315 320

atc gcc gaa ctg gac gca att gca gca tgc gtg att ggc ggc acc agc 1008

Ile Ala Glu Leu Asp Ala Ile Ala Ala Cys Val Ile Gly Gly Thr Ser

325 330 335

ctg gct ggc ggt gtg gga agc gtt gcc gga gca gta atg ggg gca ttt 1056

Leu Ala Gly Gly Val Gly Ser Val Ala Gly Ala Val Met Gly Ala Phe

340 345 350

atc atg gct tca ctg gat aac ggc atg agt atg atg gat gta ccg acc 1104

Ile Met Ala Ser Leu Asp Asn Gly Met Ser Met Met Asp Val Pro Thr

355 360 365

ttc tgg cag tat atc gtt aaa ggt gcg att ctg ttg ctg gca gta tgg 1152

Phe Trp Gln Tyr Ile Val Lys Gly Ala Ile Leu Leu Leu Ala Val Trp

370 375 380

atg gac tcc gca acc aaa cgc cgt tct tga 1182

Met Asp Ser Ala Thr Lys Arg Arg Ser

385 390

<210>10

<211>393

<212>PRT

<213> Escherichia coli

<400>10

Met Ser Lys Ser Asn Pro Ser Glu Val Lys Leu Ala Val Pro Thr Ser

1 5 10 15

Gly Gly Phe Ser Gly Leu Lys Ser Leu Asn Leu Gln Val Phe Val Met

20 25 30

lle Ala Ala Ile Ile Ala Ile Met Leu Phe Phe Thr Trp Thr Thr Asp

35 40 45

Gly Ala Tyr Leu Ser Ala Arg Asn Val Ser Asn Leu Leu Arg Gln Thr

50 55 60

Ala Ile Thr Gly Ile Leu Ala Val Gly Met Val Phe Val Ile Ile Ser

65 70 75 80

Ala Glu Ile Asp Leu Ser Val Gly Ser Met Met Gly Leu Leu Gly Gly

85 90 95

Val Ala Ala Ile Cys Asp Val Trp Leu Gly Trp Pro Leu Pro Leu Thr

100 105 110

Ile lle Val Thr Leu Val Leu Gly Leu Leu Leu Gly Ala Trp Asn Gly

115 120 125

Trp Trp Val Ala Tyr Arg Lys Val Pro Ser Phe Ile Val Thr Leu Ala

130 135 140

Gly Met Leu Ala Phe Arg Gly Ile Leu Ile Gly Ile Thr Asn Gly Thr

145 150 155 160

Thr Val Ser Pro Thr Ser Ala Ala Met Ser Gln Ile Gly Gln Ser Tyr

165 170 175

Leu Pro Ala Ser Thr Gly Phe Ile Ile Gly Ala Leu Gly Leu Met Ala

180 185 190

Phe Val Gly Trp Gln Trp Arg Gly Arg Met Arg Arg Gln Ala Leu Gly

195 200 205

Leu Gln Ser Pro Ala Ser Thr Ala Val Val Gly Arg Gln Ala Leu Thr

210 215 220

Ala Ile Ile Val Leu Gly Ala Ile Trp Leu Leu Asn Asp Tyr Arg Gly

225 230 235 240

Val Pro Thr Pro Val Leu Leu Leu Thr Leu Leu Leu Leu Gly Gly Met

245 250 255

Phe Met Ala Thr Arg Thr Ala Phe Gly Arg Arg Ile Tyr Ala Ile Gly

260 265 270

Gly Asn Leu Glu Ala Ala Arg Leu Ser Gly Ile Asn Val Glu Arg Thr

275 280 285

Lys Leu Ala Val Phe Ala Ile Asn Gly Leu Met Val Ala Ile Ala Gly

290 295 300

Leu Ile Leu Ser Ser Arg Leu Gly Ala Gly Ser Pro Ser Ala Gly Asn

305 310 315 320

Ile Ala Glu Leu Asp Ala Ile Ala Ala Cys Val Ile Gly Gly Thr Ser

325 330 335

Leu Ala Gly Gly Val Gly Ser Val Ala Gly Ala Val Met Gly Ala Phe

340 345 350

Ile Met Ala Ser Leu Asp Asn Gly Met Ser Met Met Asp Val Pro Thr

355 360 365

Phe Trp Gln Tyr Ile Val Lys Gly Ala Ile Leu Leu Leu Ala Val Trp

370 375 380

Met Asp Ser Ala Thr Lys Arg Arg Ser

385 390

<210>11

<211>1179

<212>DNA

<213> Escherichia coli

<220>

<221> CDS

<222>(1)..(1179)

<400>11

atg ttt act aaa cgt cac cgc atc aca tta ctg ttc aat gcc sat aaa 48

Met Phe Thr Lys Arg His Arg Ile Thr Leu Leu Phe Asn Ala Asn Lys

1 5 10 15

gcc tat gac cgg cag gta gta gaa ggc gta ggg gaa tat tta cag gcg 96

Ala Tyr Asp Arg Gln Val Val Glu Gly Val Gly Glu Tyr Leu Gln Ala

20 25 30

tca caa tcg gaa tgg gat att ttc att gaa gaa gat ttc cgc gcc cgc 144

Ser Gln Ser Glu Trp Asp Ile Phe Ile Glu Glu Asp Phe Arg Ala Arg

35 40 45

att gat aaa atc aag gac tgg tta gga gat ggc gtc att gcc gac ttc 192

Ile Asp Lys Ile Lys Asp Trp Leu Gly Asp Gly Val Ile Ala Asp Phe

50 55 60

gac gac aaa cag atc gag caa gcg ctg gct gat gtc gac gtc ccc att 240

Asp Asp Lys Gln Ile Glu Gln Ala Leu Ala Asp Val Asp Val Pro Ile

65 70 75 80

gtt ggg gtt ggc ggc tcg tat cac ctt gca gaa agt tac cca ccc gtt 288

Val Gly Val Gly Gly Ser Tyr His Leu Ala Glu Ser Tyr Pro Pro Val

85 90 95

cat tac att gcc acc gat aac tat gcg ctg gtt gaa agc gca ttt ttg 336

His Tyr Ile Ala Thr Asp Asn Tyr Ala Leu Val Glu Ser Ala Phe Leu

100 105 110

cat tta aaa gag aaa ggc gtt aac cgc ttt gct ttt tat ggt ctt ccg 384

His Leu Lys Glu Lys Gly Val Asn Arg Phe Ala Phe Tyr Gly Leu Pro

115 120 125

gaa tca agc ggc aaa cgt tgg gcc act gag cgc gaa tat gca ttt cgt 432

Glu Ser Ser Gly Lys Arg Trp Ala Thr Glu Arg Glu Tyr Ala Phe Arg

130 135 140

cag ctt gtc gcc gaa gaa aag tat cgc gga gtg gtt tat cag ggg tta 480

Gln Leu Val Ala Glu Glu Lys Tyr Arg Gly Val Val Tyr Gln Gly Leu

145 150 155 160

gaa acc gcg cca gag aac tgg caa cac gcg caa aat cgg ctg gca gac 528

Glu Thr Ala Pro Glu Asn Trp Gln His Ala Gln Asn Arg Leu Ala Asp

165 170 175

tgg cta caa acg cta cca ccg caa acc ggg att att gcc gtt act gac 576

Trp Leu Gln Thr Leu Pro Pro Gln Thr Gly Ile Ile Ala Val Thr Asp

180 185 190

gcc cga gcg cgg cat att ctg caa gta tgt gaa cat cta cat att ccc 624

Ala Arg Ala Arg His Ile Leu Gln Val Cys Glu His Leu His Ile Pro

195 200 205

gta ccg gaa aaa tta tgc gtg att ggc atc gat aac gaa gaa ctg acc 672

Val Pro Glu Lys Leu Cys Val Ile Gly Ile Asp Asn Glu Glu Leu Thr

210 215 220

cgc tat ctg tcg cgt gtc gcc ctt tct tcg gtc gct cag ggc gcg cgg 720

Arg Tyr Leu Ser Arg Val Ala Leu Ser Ser Val Ala Gln Gly Ala Arg

225 230 235 240

caa atg ggc tat cag gcg gca aaa ctg ttg cat cga tta tta gat aaa 768

Gln Met Gly Tyr Gln Ala Ala Lys Leu Leu His Arg Leu Leu Asp Lys

245 250 255

gaa gaa atg ccg cta cag cga att ttg gtc cca cca gtt cgc gtc att 816

Glu Glu Met Pro Leu Gln Arg Ile Leu Val Pro Pro Val Arg Val Ile

260 265 270

gaa cgg cgc tca aca gat tat cgc tcg ctg acc gat ccc gcc gtt att 864

Glu Arg Arg Ser Thr Asp Tyr Arg Ser Leu Thr Asp Pro Ala Val Ile

275 280 285

cag gcc atg cat tac att cgt aat cac gcc tgt aaa ggg att aaa gtg 912

Gln Ala Met His Tyr Ile Arg Asn His Ala Cys Lys Gly Ile Lys Val

290 295 300

gat cag gta ctg gat gcg gtc ggg atc tcg cgc tcc aat ctt gag aag 960

Asp Gln Val Leu Asp Ala Val Gly Ile Ser Arg Ser Asn Leu Glu Lys

305 310 315 320

cgt ttt aaa gaa gag gtg ggt gaa acc atc cat gcc atg att cat gcc 1008

Arg Phe Lys Glu Glu Val Gly Glu Thr Ile His Ala Met Ile His Ala

325 330 335

gag aag ctg gag aaa gcg cgc agt ctg ctg att tca acc acc ttg tcg 1056

Glu Lys Leu Glu Lys Ala Arg Ser Leu Leu Ile Ser Thr Thr Leu Ser

340 345 350

atc aat gag ata tcg caa atg tgc ggt tat cca tcg ctg caa tat ttc 1104

Ile Asn Glu Ile Ser Gln Met Cys Gly Tyr Pro Ser Leu Gln Tyr Phe

355 360 365

tac tct gtt ttt aaa aaa gca tat gac acg acg cca aaa gag tat cgc 1152

Tyr Ser Val Phe Lys Lys Ala Tyr Asp Thr Thr Pro Lys Glu Tyr Arg

370 375 380

gat gta aat agc gag gtc atg ttg tag 1179

Asp Val Asn Ser Glu Val Met Leu

385 390

<210>12

<211>392

<212>PRT

<213> Escherichia coli

<400>12

Met Phe Thr Lys Arg His Arg Ile Thr Leu Leu Phe Asn Ala Asn Lys

1 5 10 15

Ala Tyr Asp Arg Gln Val Val Glu Gly Val Gly Glu Tyr Leu Gln Ala

20 25 30

Ser Gln Ser Glu Trp Asp IIe Phe Ile Glu Glu Asp Phe Arg Ala Arg

35 40 45

Ile Asp Lys lle Lys Asp Trp Leu Gly Asp Gly Val Ile Ala Asp Phe

50 55 60

Asp Asp Lys Gln Ile Glu Gln Ala Leu Ala Asp Val Asp Val Pro Ile

65 70 75 80

Val Gly Val Gly Gly Ser Tyr His Leu Ala Glu Ser Tyr Pro Pro Val

85 90 95

His Tyr Ile Ala Thr Asp Asn Tyr Ala Leu Val Glu Ser Ala Phe Leu

100 105 110

His Leu Lys Glu Lys Gly Val Asn Arg Phe Ala Phe Tyr Gly Leu Pro

115 120 125

Glu Ser Ser Gly Lys Arg Trp Ala Thr Glu Arg Glu Tyr Ala Phe Arg

130 135 140

Gln Leu Val Ala Glu Glu Lys Tyr Arg Gly Val Val Tyr Gln Gly Leu

145 150 155 160

Glu Thr Ala Pro Glu Asn Trp Gln His Ala Gln Asn Arg Leu Ala Asp

165 170 175

Trp Leu Gln Thr Leu Pro Pro Gln Thr Gly Ile Ile Ala Val Thr Asp

180 185 190

Ala Arg Ala Arg His Ile Leu Gln Val Cys Glu His Leu His Ile Pro

195 200 205

Val Pro Glu Lys Leu Cys Val Ile Gly Ile Asp Asn Glu Glu Leu Thr

210 215 220

Arg Tyr Leu Ser Arg Val Ala Leu Ser Ser Val Ala Gln Gly Ala Arg

225 230 235 240

Gln Met Gly Tyr Gln Ala Ala Lys Leu Leu His Arg Leu Leu Asp Lys

245 250 255

Glu Glu Met Pro Leu Gln Arg Ile Leu Val Pro Pro Val Arg Val Ile

260 265 270

Glu Arg Arg Ser Thr Asp Tyr Arg Ser Leu Thr Asp Pro Ala Val Ile

275 280 285

Gln Ala Met His Tyr Ile Arg Asn His Ala Cys Lys Gly Ile Lys Val

290 295 300

Asp Gln Val Leu Asp Ala Val Gly Ile Ser Arg Ser Asn Leu Glu Lys

305 310 315 320

Arg Phe Lys Glu Glu Val Gly Glu Thr Ile His Ala Met Ile His Ala

325 330 335

Glu Lya Leu Glu Lys Ala Arg Ser Leu Leu Ile Ser Thr Thr Leu Ser

340 345 350

Ile Asn Glu Ile Ser Gln Met Cys Gly Tyr Pro Ser Leu Gln Tyr Phe

355 360 365

Tyr Ser Val Phe Lys Lys Ala Tyr Asp Thr Thr Pro Lys Glu Tyr Arg

370 375 380

Asp Val Asn Ser Glu Val Met Leu

385 390

<210>13

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Primers

<400>13

ggcaactatg catatcttcg cgc 23

<210>14

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Primers

<400>14

gcgtgaatga attggcttag gtgg 24

<210>15

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Primers

<400>15

cagacagcga gcgaggatcg c 21

<210>16

<211>21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Primers

<400>16

tgtgcggtta tccatcgctg c 21

Claims (20)

CLAIMS 1. An L-amino acid-producing bacterium of the family Enterobacteriaceae, which bacterium has enhanced activity of any xylose-utilizing enzyme. 2. The bacterium according to claim 1, wherein the bacterium belongs to the genus Escherichia. 3. The bacterium according to claim 1, wherein the bacterium belongs to the genus Pantoea. 4. The bacterium according to claim 1, wherein the xylose utilization enzyme activity is enhanced by increasing the expression level of the xyLABFGHR locus. 5. The bacterium according to claim 4, wherein the xylose utilization enzyme activity is enhanced by increasing the copy number of the xyLABFGHR locus or modifying the expression control sequence to enhance the expression of the gene. 6. The bacterium according to claim 5, wherein the copy number is increased by transforming the bacterium with a low copy vector containing the xyLABFGHR locus. 7. The bacterium according to any one of claims 1 to 6, wherein the xyLABFGHR locus is from a bacterium of the genus Escherichia. 8. A method for producing L-amino acid, which comprises culturing the bacterium according to any one of claims 1 to 7 in a medium containing a mixture of glucose and pentose sugar, and collecting L-amino acid from the medium. 9. The method of claim 8, wherein the pentoses are arabinose and xylose. 10. The method of claim 8, wherein the mixture of sugars is a raw mixture of sugars obtained from cellulosic biomass. 11. The method according to claim 8, wherein the L-amino acid produced is L-histidine. 12. The method according to claim 11, wherein the bacterium enhances the expression of L-histidine biosynthesis-related genes. 13. The method according to claim 8, wherein the L-amino acid produced is L-threonine. 14. The method according to claim 13, wherein the bacterium enhances the expression of L-threonine biosynthesis-related genes. 15. The method according to claim 8, wherein the L-amino acid produced is L-threonine. 16. The method according to claim 15, wherein the bacterium enhances the expression of L-lysine biosynthesis-related genes. 17. The method according to claim 8, wherein the L-amino acid produced is L-tryptophan. 18. The method according to claim 17, wherein the bacterium enhances the expression of L-glutamic acid biosynthesis-related genes. 19. The method according to claim 8, wherein the L-amino acid produced is L-tryptophan. 20. The method according to claim 19, wherein the bacterium enhances the expression of L-tryptophan biosynthesis-related genes.

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