CN1749390B - Method for producing L-amino acids by fermentation using bacteria having enhanced expression of xylose utilization genes - 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

Use xylose utilization genetic expression enhanced bacterium to ferment to produce L-amino acid whose method

Technical field

The present invention relates to produce the amino acid whose method of L-by pentose fermentation, relate more specifically to use xylose utilization genetic expression enhanced bacterium, produce the amino acid whose method of L thereby ferment as carbon source with the mixture of glucose with pectinose and/or wood sugar.At commercial production L-amino acid, during as L-Histidine, L-Threonine, L-Methionin, L-L-glutamic acid and L-tryptophane, can use cheap carbon source, it comprises segmental hexose of hemicellulose and pentose mixture from cellulose biomass (cellulosic biomass).

Background technology

General, L-amino acid utilizes the different microorganisms bacterial strain to carry out suitability for industrialized production by fermentation process.Employed fermention medium generally includes the various carbon sources and the nitrogenous source of sufficient quantity in this method.

Routinely, various carbohydrate such as hexose, pentose, triose; Various organic acids and alcohol are used as carbon source.Hexose comprises glucose, fructose, seminose, sorbose, semi-lactosi or the like.Pentose comprises pectinose, wood sugar, ribose or the like.Yet above-mentioned carbohydrate and other are used for the conventional carbon source of industry now, and be all quite expensive as syrup, cereal, sugarcane, starch, its hydrolysate etc.Therefore, need find out cheap more alternative carbon source and be used for the amino acid whose production of L-.

Cellulose biomass is a kind of suitable raw material that is used for the production of L-amino acid, because it is easy to obtain and is more cheap than carbohydrate, cereal, sugarcane or other carbon source.The convention amount of Mierocrystalline cellulose, hemicellulose and xylogen is Mierocrystalline cellulose, the hemicellulose of 20-40%, the xylogen of 10-25% and other composition of 10% of about 40-60% in cellulose biomass.Cellulose components is by hexose, and normally the polymkeric substance of glucose is formed.Hemicellulose components comprises wood sugar and pectinose composition mainly by pentose.The composition of various cellulose biomass raw materials (http://www.ott.doe.gov/biofuels/understanding_biomass.html) as shown in table 1.

Table 1

Be summarized in " raw-material composition of biomass and character data storehouse " (http://www.ott.doe.gov/biofuels/progs/searchl.cgi) about surpassing the more detailed information of 150 kinds of biomass (biomass) sample composition.

Can effectively cellulose biomass be converted into the proferment material but hope exploitation recently is a kind of, be generally the commercial run of the mixture of carbohydrate.Therefore, wish in the recent period to increase utilize reproducible energy derive such as Mierocrystalline cellulose and hemicellulose produce useful compound (AristidouA., Pentila.M., Curr.Opin, Biotech nol, 2000, Apr., 11:2,187-198).Yet, the overwhelming majority article and the patents delivered, or patent application, record be to utilize cellulose biomass to produce ethanol by biological catalyst (bacterium and yeast), it is supposed to become effective fuel substitute.This method comprises uses different zymomonas mobilis (Zymomonas mobilis) improved strain (Deanda K. etc., Appl.Environ.Microbiol., 1996 Dec., 62:12,4465-70; Mohagheghi A. etc., Appl.Biochem.Biotechnol., 2002,98-100:885-98; Lawford H.G., RousseauJ.D., Appl.Biochem.Biotechnol, 2002,98-100:429-48; PCT applies for WO95/28476, WO98/50524), intestinal bacteria (Escherichia coli) improved strain (Dien B.S. etc., Appl.Biochem.Biotechnol, 2000,84-86:181-96; Nichols N.N. etc., Appl.Microbiol.Biotechnol., 2001 Jul, 56:1-2,120-5; United States Patent (USP) 5,000,000) carries out the fermentation of cellulose biomass.Utilize candida tropicalis bacterium (Candida tropicalis) to the wood sugar from hemicellulose sugar ferment can produce Xylitol (Walthers T. etc., Appl.Biochem.Biotechnol., 2001,91-93:423-35).Use intestinal bacteria (Escherichia coli) bacterial strain of recombinating that pectinose, fructose, semi-lactosi, glucose, lactose, maltose, sucrose, wood sugar and its composition are fermented and to produce 1,2-propylene glycol (United States Patent (USP) 6,303,352).Also have report can obtain 3-dehydrogenation shikimic acid by using coli strain that glucose/wood sugar/pectinose mixture is fermented.Compare as carbon source with independent employing wood sugar or glucose, concentration and output the highest (KaiLi and J.W.Frost, Biotechnol.Prog., 1999 of the 3-dehydrogenation shikimic acid that when using glucose/wood sugar/pectinose mixture, is obtained as carbon source, 15,876-883).

There is report to claim that intestinal bacteria can utilize pentose such as L-arabinose and D-wood sugar (Escherichiacoli and Salmonella, Second Edition, Editor in Chief:F.C.Neidhardt, ASMPress, Washingtong D.C., 1996).L-arabinose is transported into, and cell is by two kinds of induction type systems: the low-affinity permease (K of (1) araE genes encoding _mAbout 0.1mM) and the high affinity (K of (2) araFG operon coding _mBe 1 to 3 μ M) system.The araF genes encoding has the kytoplasm conjugated protein (306 amino acid) on every side of chemoattractant receptor function, araG locus coding inner membrane protein.The metabolism of sugar is one group of enzyme by araBAD operon coding: isomerase (araA genes encoding), and its reversible is converted into the L-ribulose with aldose; Kinases (araB genes encoding), it turns to L-ribulose 5-phosphoric acid salt with ketose phosphoric acid; With L-ribulose-5-phosphoric acid-4-epimerase (araD genes encoding), the phosphatic formation of its catalysis D-wood sugar-5-(Escherichia coli and Salmonella, SecondEdition, Editor in Chief:F.C.Neidhardt, ASM Press, Washingtong D.C., 1996).

Most of coli strain can be grown in the D-wood sugar, but the K-12 bacterial strain just can be grown in this compound after need making a variation.The utilization of this pentose is the approach that suppresses by induction type and catabolite, comprising utilizing two kinds of induction type permeases (to D-ribose or D-pectinose non-activity) transhipment passing through cytoplasmic membrane, isomery turns to the D-xylulose, and ATP dependent form phosphorylation ketopentose produces D-xylulose 5-phosphate salt.High affinity (K _mBe 0.3 to 3 μ M) movement system depend on every side kytoplasm conjugated protein (37,000Da) and may drive by high energy compound.Low-affinity (K _mAbout 170 μ M) system is activated by prime mover between proton.The transfer system of this D-wood sugar-proton-in the same way is by the xylE genes encoding.Oligogene group corresponding to the D-xylose utilization is xylAB (RT).XylA genes encoding isomerase (54,000Da), xylB genes encoding kinases (52,000Da).Operon comprises two transcription initiation sites, and one of them is inserted into xylB opening code-reading frame upstream.Because the low-affinity permease is corresponding to unconnected xylE, the xylT locus, be also referred to as xylF (xylFGHR), the high affinity movement system of may encoding and therefore contain at least two genes (cytoplasmic protein around the coding, the membranin that coding is complete) (Escherichia coli and Salmonella, Second Edition, Editor inChief:F.C.Neidhardt, ASM Press, Washingtong D.C., 1996).This xylFGHR genes encoding wood sugar abc transport albumen, wherein the xylF genes encoding wood sugar of inferring is conjugated protein, and the ATP that the xylG genes encoding is inferred is conjugated protein, the film component that the xylH genes encoding is inferred, xylR genes encoding wood sugar transcription activating protein.

Import above-mentioned coding L-arabinose isomerase, L-ribulokinase, L-ribulose 5-phosphatase 24-epimerase, xylose isomerase and xylulokinase, add the E.coli gene of transaldolase and transketolase, can make microorganism, as zymomonas mobilis, with pectinose and xylose metabolism be ethanol (WO/9528476, WO98/50524).On the contrary, the fermentation single cell bacterium gene of coding ethanol dehydrogenase (ADH) and pyruvic carboxylase (PDH) also can be used for alcohol production (Dien B.S. etc., Appl.Biochem.Biotechnol, 2000, the 84-86:181-96 of coli strain; United States Patent (USP) 5,000,000).

Author of the present invention disclosed a kind of by glucose fermentation and pentose in the past, mixture as pectinose and wood sugar is produced L-amino acid, as the method (Russian patent application 2003105269) of L-Isoleucine, L-Histidine, L-Threonine and L-tryptophane.

Yet, not about the xylose utilization gene,, or use these genes from hexose and pentose mixture, to produce the amino acid whose report of L-so far as the genetic expression enhanced bacterium of xylABFGHR locus.

Summary of the invention

The objective of the invention is to strengthen the output of L-amino acid preparation strain, provide xylose utilization genetic expression enhanced L-amino acid to produce bacterium, and provide this bacterium of use from hexose, as glucose, and pentose, as producing the amino acid whose method of L-in the mixture of wood sugar or pectinose.The proferment material that obtains from cellulose biomass can be used as the carbon source of substratum.The realization of this purpose is based on finds that the xylABFGHR locus of being cloned on the low copy carrier can strengthen L-amino acid, for example the output of L-Histidine, L-Threonine, L-Methionin, L-L-glutamic acid and L-tryptophane.Employed microorganism can grow in the proferment material and effectively produce L-amino acid.Should form by wood sugar and pectinose and glucose as the proferment material of carbon source.The L-amino acid preparation strain be exemplified as coli strain.Finish the present invention thus.

The purpose of this invention is to provide the bacterium that produces the amino acid whose enterobacteriaceae of L-(Enterobacteriaceae) family, it has any xylose utilization enzyme of increased activity.

Further aim of the present invention provides above-mentioned bacterium, and wherein this bacterium belongs to Escherichia (Escherichia).

Further aim of the present invention provides above-mentioned bacterium, and wherein this bacterium belongs to general Pseudomonas (Pantoea).

Further aim of the present invention provides above-mentioned bacterium, and wherein said xylose utilization enzymic activity strengthens by the expression amount that increases the xylABFGHR locus.

Further aim of the present invention provides above-mentioned bacterium, and copy number or the expression regulation sequence of modifying factor of wherein said xylose utilization enzymic activity by increasing the xylABFGHR locus improves with the expression of enhancing gene.

Further aim of the present invention provides above-mentioned bacterium, and wherein copy number is by increasing with the low copy carrier transform bacteria that contains the xylABFGHR locus.

Further aim of the present invention provides above-mentioned bacterium, and wherein the xylABFGHR locus is derived from the bacterium that belongs to Escherichia (Escherichia).

Further aim of the present invention provides the amino acid whose method of a kind of production L-, and it is included in cultivates above-mentioned bacterium in the substratum that contains glucose and pentose mixture, and collects L-amino acid from substratum.

Further aim of the present invention provides aforesaid method, and wherein pentose is pectinose and wood sugar.

Further aim of the present invention provides aforesaid method, and wherein Tang mixture is the mixture of raw material of the sugar that obtains from cellulose biomass.

Further aim of the present invention provides aforesaid method, and wherein the L-amino acid of Sheng Chaning is the L-Histidine.

Further aim of the present invention provides aforesaid method, and wherein said bacterium strengthens expresses L-Histidine biosynthesis related genes.

Further aim of the present invention provides aforesaid method, and wherein the L-amino acid of Sheng Chaning is the L-Threonine.

Further aim of the present invention provides aforesaid method, and wherein said bacterium strengthens expresses L-Threonine biosynthesis related genes.

Further aim of the present invention provides aforesaid method, and wherein the L-amino acid of Sheng Chaning is L-Methionin.

Further aim of the present invention provides aforesaid method, and wherein said bacterium strengthens expresses L-Methionin biosynthesis related genes.

Further aim of the present invention provides aforesaid method, and wherein the L-amino acid of Sheng Chaning is L-L-glutamic acid.

Further aim of the present invention provides aforesaid method, and wherein said bacterium strengthens expresses L-L-glutamic acid biosynthesis related genes.

Further aim of the present invention provides aforesaid method, and wherein the L-amino acid of Sheng Chaning is the L-tryptophane.

Further aim of the present invention provides aforesaid method, and wherein said bacterium strengthens expresses L-tryptophane biosynthesis related genes.

The amino acid whose method of this production L-comprises by glucose fermentation and pentose, produces the L-Histidine as the mixture of pectinose and wood sugar.And the amino acid whose method of this production L-comprises by glucose fermentation and pentose, produces the L-Threonine as the mixture of pectinose and wood sugar.And the amino acid whose method of this production L-comprises by glucose fermentation and pentose, produces L-Methionin as the mixture of pectinose and wood sugar.And the amino acid whose method of this production L-comprises by glucose fermentation and pentose, produces L-L-glutamic acid as the mixture of pectinose and wood sugar.And the amino acid whose method of this production L-comprises by glucose fermentation and pentose, produces the L-tryptophane as the mixture of pectinose and wood sugar.Can as cellulose biomass, obtain in the hydrolysate of preferred cellulose from the plant biomass resource of underusing as raw-material above-mentioned glucose of fermentation and pentose mixture.

The accompanying drawing summary

Fig. 1 shows the structure of xylABFGHR locus on the karyomit(e) of coli strain MG1655.The position of arrow indication PCR primer among the figure.

The explanation of preferred embodiment

Among the present invention, " L-amino acid is produced bacterium " represents a kind of bacterium, and it can cause the amino acid whose accumulation of L-in the substratum when microbial culture of the present invention is in substratum.Can give or strengthen the amino acid whose throughput of L-by breeding.Compare with wild-type or parental strain at this term " L-amino acid produce bacterium " also expression and can produce and the amino acid whose bacterium of the more L-of accumulation in substratum, preferred this microorganism of expression can produce and accumulate in substratum and be no less than 0.5g/L, more preferably is no less than the purpose L-amino acid of 1.0g/L." L-amino acid " comprises L-L-Ala, L-arginine, altheine acid, L-aspartic acid, L-halfcystine, L-L-glutamic acid, L-glutaminate, L-Padil, L-Histidine, L-Isoleucine, L-leucine, L-Methionin, L-methionine(Met), L-phenylalanine, L-proline(Pro), L-Serine, L-Threonine, L-tryptophane, L-tyrosine and L-Xie Ansuan.

Enterobacteriaceae (enterobacteriaceae) family comprises and belongs to Escherichia (Escherichia), enterobacter (Enterobacter), erwinia belongs to (Erwinia), Klebsiella (Klebsiella), general Pseudomonas (Pantoea), Photorhabdus, Pu Luweidengsi bacillus (Providencia), salmonella (Salmonella), Serratia (Serratia), Shigella (Shigella), Morganella (Morganella), the bacterium that yersinia's genus (Yersinia) etc. belong to.Particularly, can use according to classification (the http://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/wgetorg in NCBI (the National Center for Biotechnology Information) database? mode=Tree﹠amp; Id=1236﹠amp; 1v1=3﹠amp; Keep=1﹠amp; Srchmode=1﹠amp; Unlock) classify bacterium in enterobacteriaceae.The bacterium of preferred Escherichia or general Pseudomonas.

Term " Escherichia bacterium " represents that this bacterium classifies in Escherichia according to the known classification of microbial technology field routine techniques personnel.The example that is used for Escherichia bacterium of the present invention includes, but are not limited to intestinal bacteria (E.coli).

Can be used in Escherichia bacterium of the present invention and do not do special restriction, but for example, Neidhardt, F.C. wait the bacterium (Escherichia coli and Salmonella, Second Edition, the Editor in Chief:F.C.Neidhardt that are recorded and narrated, ASM Press, Washingtong D.C., 1208, table 1) be contained among the present invention.Term " general Pseudomonas bacterium " represents that this bacterium classifies in general Pseudomonas according to the known classification of microbial technology field routine techniques personnel.According to the nucleotide sequence analysis to 16S rRNA etc., the kind of some enterobacter agglomerans (Enterobacter agglomerans) reclassifies among pantoea agglomerans (Pantoeaagglomerans), Pantoea ananatis, the Pantoea Stewartii etc. now.

Term " xylose utilization enzyme enhanced activity " represents that the activity of enzyme in each cell is higher than the bacterial strain of unmodified, as wild type strain.Example comprises in each cell wherein that the enzyme molecular amounts increases and the activity specific of each enzyme molecule increases or the like.The measurement of gene coded protein quantity can be adopted currently known methods, carries out immunoblotting assay (Western blotting analysis) etc. after comprising SDS-PAGE.In addition, comprise with the wild type strain that compares, for example e. coli k-12.Result as strengthening xylose utilization enzyme intracellular reactive can observe L-amino acid, as L-Histidine, L-Threonine, L-Methionin, L-L-glutamic acid and or the L-tryptophane in substratum, accumulate.

" xylose utilization enzyme " comprises the enzyme and the modulin of wood sugar transhipment, xylose isomerase and wood sugar phosphorylation.This enzyme comprises xylose isomerase, xylulokinase, wood sugar translocator and wood sugar transcription activating protein.Xylose isomerase enzyme catalysis D-wood sugar is to the isomerization reaction of D-xylulose.Xylulokinase catalysis D-xylulose uses ATP to generate the phosphorylation reaction of D-xylulose-5-phosphoric acid salt and ADP.The xylose utilization enzyme depends on the complementary action of the negative intestinal bacteria mutant of corresponding xylose isomerase feminine gender or xylulokinase respectively as xylose isomerase, the active existence of xylulokinase.

Term " Escherichia bacterium " represents that this bacterium classifies in Escherichia according to the microbiology field known classification of routine techniques personnel.An example that is used for Escherichia microorganism belonging to genus of the present invention is intestinal bacteria (E.coli).

Term " increase gene expression amount " expression expression of gene amount is higher than the bacterial strain of unmodified, as wild type strain.The example of this modification comprises the quantity that increases expressing gene in each cell, increases expression of gene level etc.Quantitatively can passing through of expressing gene copy number for example uses the probe based on gene order to carry out southern blotting technique analysis (Sourthn blotting) behind the restriction enzyme digestion chromosomal DNA, fluorescence in situ hybridization (HSH) etc. is measured.Gene expression dose can adopt multiple different methods, comprises rna blot analysis (Northern blotting), mensuration such as quantitative RT-PCT.In addition, the wild type strain that can be used as contrast comprises, for example e. coli k-12.Result as strengthening xylose utilization enzyme intracellular reactive can observe L-amino acid, as L-Histidine, L-Threonine, L-Methionin, L-L-glutamic acid and or the L-tryptophane containing pentose, accumulate in the substratum as wood sugar.

The enhancing of xylose utilization enzymic activity can realize by the expression that increases described enzyme coding gene in the bacterial cell.The relevant gene of xylose utilization comprises any gene that derives from enterobacteriaceae family bacterium, and derive from other bacterium such as thermophilic bacteria (thermophilic Bacillus sp) gene (Biochem, Mol.Bio.Int., 1996,39 (5), 1049-1062).Preferably derive from the gene of Escherichia bacterium.

The encoding gene of intestinal bacteria xylose isomerase (EC numbers 5.3.1.5) is known and is marked as xylA (Nucleotide numbering 3727072 to 3728394 in the sequence of GenBank accession NC_000913.1, gi:16131436).The encoding gene of xylulokinase (EC numbers 2.7.1.17) is known and is marked as xylB (Nucleotide numbering 3725546 to 3727000 in the sequence of GenBank accession NC_000913.1, gi:16131435).The encoding gene of the conjugated protein movement system of wood sugar is known and is marked as xylF (Nucleotide numbering 3728760 to 3729752 in the sequence of GenBank accession NC_000913.1, gi:16131437).The protein-bonded encoding gene of wood sugar movement system ATP of inferring is known and is marked as xylG (Nucleotide numbering 3729830 to 3731371 in the sequence of GenBank accession NC_000913.1, gi:16131438).The encoding gene of ABC class wood sugar movement system permease component is known and is marked as xylH (Nucleotide numbering 3731349 to 3732530 in the sequence of GenBank accession NC_000913.1, gi:16131439).The encoding gene of xyl operon transcriptional regulation protein is known and is marked as xylR (Nucleotide numbering 3732608 to 3733786 in the sequence of GenBank accession NC_000913.1, gi:16131440).Therefore, said gene can be carried out PCR (polymerase chain reaction by using the primer based on gene nucleotide series; With reference to White, T.J. etc., TrendsGenet., 5,185 (1989)) and obtain.

The xylose utilization enzyme coding gene of other microorganism can similarly obtain.

Encode following albumen (A) or DNA (B) is the example of intestinal bacteria xylA gene:

(A) has the albumen of the aminoacid sequence shown in the SEQ ID NO:2; Or

(B) have in the aminoacid sequence shown in the SEQ ID NO:2, lack, replace, insert or increase one or several amino acid after the aminoacid sequence of gained, and have the active albumen of xylose isomerase.

Encode following albumen (C) or DNA (D) is the example of intestinal bacteria xylB gene:

(C) has the albumen of the aminoacid sequence shown in the SEQ ID NO:4; Or

(D) have in the aminoacid sequence shown in the SEQ ID NO:4, lack, replace, insert or increase one or several amino acid after the aminoacid sequence of gained, and have the active albumen of xylulokinase.

Encode following albumen (E) or DNA (F) is the example of intestinal bacteria xylF gene:

(E) has the albumen of the aminoacid sequence shown in the SEQ ID NO:6; Or

(F) have in the aminoacid sequence shown in the SEQ ID NO:6, lack, replace, insert or increase one or several amino acid after the albumen of aminoacid sequence of gained, it has when the protein content of protein content and xylAB and xylGHR genes encoding in the L-amino acid production bacterium increases, increase L-amino acid in the substratum, as L-Histidine, L-Threonine, L-Methionin, L-L-glutamic acid and or the activity of L-tryptophane.

Encode following albumen (G) or DNA (H) is the example of intestinal bacteria xylG gene:

(G) has the albumen of the aminoacid sequence shown in the SEQ ID NO:8; Or

(H) have in the aminoacid sequence shown in the SEQ ID NO:8, lack, replace, insert or increase one or several amino acid after the albumen of aminoacid sequence of gained, it has when the protein content of protein content and xylAB and xylFHR genes encoding in the L-amino acid production bacterium increases, increase L-amino acid in the substratum, as L-Histidine, L-Threonine, L-Methionin, L-L-glutamic acid and or the activity of L-tryptophane.

Encode following albumen (I) or DNA (J) is the example of intestinal bacteria xylH gene:

(I) has the albumen of the aminoacid sequence shown in the SEQ ID NO:10; Or

(J) have in the aminoacid sequence shown in the SEQ ID NO:10, lack, replace, insert or increase one or several amino acid after the albumen of aminoacid sequence of gained, it has when the protein content of protein content and xylAB and xylFGR genes encoding in the L-amino acid production bacterium increases, increase L-amino acid in the substratum, as L-Histidine, L-Threonine, L-Methionin, L-L-glutamic acid and or the activity of L-tryptophane.

Encode following albumen (K) or DNA (L) is the example of intestinal bacteria xylR gene:

(K) has the albumen of the aminoacid sequence shown in the SEQ ID NO:12; Or

(L) have in the aminoacid sequence shown in the SEQ ID NO:12, lack, replace, insert or increase one or several amino acid after the albumen of aminoacid sequence of gained, it has when the protein content when protein content and xylAB and xylFGH genes encoding in the L-amino acid production bacterium increases, increase L-amino acid in the substratum, as L-Histidine, L-Threonine, L-Methionin, L-L-glutamic acid and or the activity of L-tryptophane.

The DNA of coding xylose isomerase comprises and is coded in the albumen (A) one or more topagnosises, replacement, insertion or increases one or several amino acid, but do not lose the proteic DNA of protein-active.Though the quantity of " several " amino acid difference depends on the position and the type of amino-acid residue in protein three-dimensional structure, it can be 2 to 50 for albumen (A), and is preferred 2 to 20, more preferably 2 to 10.This is can not cause big influence to proteic three-dimensional structure and activity because some amino acid have these the amino acid whose replacements of high homology each other.Therefore, albumen (B) has with respect to the whole aminoacid sequence of xylose isomerase and is no less than 30 to 50% preferred 50 to 70% more preferably 70-90%, also more preferably surpasses 90% and most preferably above 95% homology, and has the xylose isomerase enzymic activity.Same method and homology are given birth to judgement can be applied to other albumen (C), (E), (G), (I) and (K).

For calculating the homology level of albumen or DNA, can use several method of calculation, as blast search, FASTA search and ClustalW.

BLAST (Basic Local Alignment Search Tool) is the employed heuristic search algorithm of program blastp, blastn, blastx, megablast, tblastn and tblastx; These programs adopt the statistical method of Karlin, Samuel and Stephen F.Altschul to weigh significance (" the Methodsfor assessing the statistical significance of molecular sequence features by usinggeneral sco of its Search Results

To the proteic variation of definition in (A), for example above-mentioned those, the conservative variation that normally can keep protein-active.Replace to change the position that comprises in the variation that at least one residue in its aminoacid sequence is removed and be inserted into it by different residues.Can replace original amino acid in the above-mentioned albumen, and be considered to the conservative amino acid example of replacing and comprise: Ala replaces with ser or thr; Arg replaces with gln, his or lys; Asn replaces with glu, gln, lys, his, asp; Asp replaces with asn, glu or gln; Cys replaces with ser or ala; Gln replaces with asn, glu, lys, his, asp or arg; Glu replaces with asn, gln, lys or asp; Gly replaces with pro; His replaces with asn, lys, gln, arg, tyr; Ile replaces with leu, met, val, phe; Leu replaces with ile, met, val, phe; Lys replaces with asa, glu, gln, his, arg; Met replaces with ile, leu, val, phe; Phe replaces with trp, tyr, met, ile or leu; Ser replaces with thr, ala; Thr replaces with ser or ala; Trp replaces with phe, ty; Tyr replaces with his, phe or trp; And val replaces with met, ile, leu.

Obtaining the proteic coding DNA substantially the same with the albumen of definition in (A) can adopt, for example, by point mutation the proteic nucleotide sequence of definition in the coding (A) is modified, caused one or more amino-acid residue disappearances, replace, insert or increase.The DNA of this modification can obtain by the ordinary method that the reagent or the condition of use generation sudden change are handled.This processing comprises with azanol handles encoding histone DNA among the present invention, perhaps uses UV radiation or reagent, handles the bacterium that contains DNA as N-methyl-N '-nitrogen-N-nitrosoguanidine or nitrous acid.

The DNA of coding xylose isomerase comprises because the natural varient of finding in Escherichia bacterium different strains that diversity caused.By being separated under the stringent condition and the hybridization of xylA gene or this a gene part, and the DNA of coding with xylose isomerase zymoprotein can obtain the to encode DNA of this varient.Comprise condition at this term " stringent condition ", under this condition, can form so-called specific hybrid, do not form non-specific hybridization simultaneously.For example, stringent condition comprises condition, under this condition, has the DNA of high homology, and for example each other homology is no less than 70%, preferably is no less than 80%, more preferably is no less than 90%, most preferably is no less than 95% DNA and hybridizes.Perhaps, comprise the normal condition of Southern hybridization elution as the condition of stringent condition example, for example 60 ℃, 1 * SSC, 0.1%SDS, preferred 0.1 * SSC, 0.1%SDS.The time length of elution process is depended on the type of the film that is used for trace and the standard times of manufacturer recommendation.For example, Hybond under stringent condition ^TMThe recommendation wash-out time length of N+ nylon membrane (Amersham) is 15 minutes.Preferably, wash-out can carry out 2 to 3 times.The partial sequence of nucleotide sequence SEQ ID NO:1 also can be used as the varient coding DNA probe and with the xylA gene recombination.This probe can prepare by PCR, wherein with the oligonucleotide produced according to SEQ ID NO:1 nucleotide sequence as primer, be template with the dna fragmentation that contains SEQ ID NO:1 nucleotide sequence.When the dna fragmentation that is about 300bp with length during as probe, the elution requirement of hybridization can for, for example, 50 ℃, 2 * SSC and 0.1%SDS.

Obtain coding and can adopt the method that is similar to above-mentioned acquisition xylose isomerase with the substantially the same proteic DNA of other xylose utilization enzyme.

With the DNA transform bacteria of proteins encoded represent by, for example ordinary method imports in the bacterial cell DNA to increase coding proteic expression of gene of the present invention and to strengthen proteic activity in the bacterial cell.

Bacterium of the present invention also comprises a kind of bacterium, in this bacterium the proteic activity of the present invention by with coding (A) or (B), (C) or (D), (E) or (F), (G) or (H), (I) or (J), (K) or (L) in the proteic DNA of definition transform described bacterium, or be enhanced by the expression regulation sequence that changes DNA described in the bacterial chromosome.

The method of reinforcing gene expression comprises the increase gene copy number.Gene is imported the copy number that the carrier that can bring into play function in the Escherichia bacterium can increase gene.Be this purpose, preferably use multi-copy vector.Preferably, use low copy carrier.The example of low copy carrier is pSC101, pMW118, pMW119 etc.Term " low copy carrier " is used for being illustrated in each cell copy number and mostly is 5 carrier most.The method that transforms comprises any method well known by persons skilled in the art.For example, handle recipient cell to increase cell the perviousness method of DNA be can be used in the e. coli k-12 (Mandel, M.And Higa, A., J.Mol.Biol., 53,159 (1970)) by report and can use with calcium chloride at this.

By, for example, methods such as homologous recombination, Mu integration import the enhancing that also can realize genetic expression in the bacterial chromosome with the multiple copy of gene.For example, one take turns Mu integrate can the gene importing bacterial chromosome of maximum 3 copies in.

On the other hand, the natural promoter of control DNA of the present invention is replaced by the enhancing that more effective promotor also can realize genetic expression.The intensity of promotor is weighed with the frequency of the synthetic initiation of RNA.Deuschle, U., Kammerer, W., Gentz, R., Bujard, H described the method for measuring promotor intensity and effectively start example (Promoters in Escherichia coli:a hierarchy of in vivo strengthindicates alternate structures.EMBO J.1986,5,2987-2994).For example, P _RPromotor is known effective constitutive promoter.Other known strong promoter is the P of lambda particles phage _LPromotor, lac promotor, trp promotor, trc promotor etc.

By the enhancing that more effective Shine-Dalgarno sequence (SD sequence) importing DNA of the present invention can be realized with alternative natural SD sequence translate.The SD sequence is the upstream region of mRNA initiator codon, itself and ribosomal 16S RNA interact (Shine J.and DalgarnoL., Proc.Natl.Acad.Sci.USA, 1974,71,4,1342-6).

Adopt more effective promotor to be used in combination with the method that increases gene copy number.

Perhaps, by, for example, in promotor, import sudden change and can strengthen promotor, thereby improve the expression of gene level that is positioned at the promotor downstream.In addition, known replacement is positioned at the several amino acid between ribosome bind site (RBS) and initiator codon, and especially, upstream from start codon next-door neighbour's sequence greatly influences the translating property of mRNA.For example, find the expression level of 20 times of scopes depend on the character of three Nucleotide in initiator codon front (Gold etc., Annu.Rev.Microbiol., 35,365-403,1981; Hui etc., EMBO J., 3,623-629,1984).

Preparation chromosomal DNA, hybridization, PCR, preparation plasmid DNA, digestion and be connected DNA transform, select the oligonucleotide can be for well known to a person skilled in the art ordinary method as the method for primer etc.These methods are recorded in Sambrook, J., and Russell D., " Molecular Cloning A LaboratoryManual, Third Edition ", Cold Spring Harbor Laboratory Press (2001) etc.

Can obtain bacterium of the present invention by aforementioned DNA importing itself being had the bacterium that produces the L-ability of amino acid.Perhaps, can obtain bacterium of the present invention by producing the bacterium that the amino acid whose ability of L-gives to contain this DNA.

The example of the L-amino acid production bacterium of Escherichia is as described below.

The L-Histidine is produced bacterium

The example of the bacterium with L-Histidine throughput of Escherichia comprises that the L-Histidine of Escherichia produces bacterium, as E.coli bacterial strain 24 (VKPM B-5945, RU2003677); E.coli bacterial strain 80 (VKPM B-7270, RU2119536); E.coli bacterial strain NRRL B-12116-B12121 (US4388405); E.coli bacterial strain H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) are (US6344347); E.coli bacterial strain H-9341 (FERM BP-6674) (EP1085087); E.coli strains A I80/pFM201 (US6258554) etc.

Preferably, bacterium of the present invention is further modified to strengthen the L-Histidine produces histidine operon expression of gene in the bacterium, the hisG gene that it preferably includes the coding ATP phosphoribosyltransferase makes the feedback inhibition sensitivity to the L-Histidine reduce (Russ P 2003677 and 2119536).

The L-Threonine is produced bacterium

The example that the L-Threonine of the present invention of deriving is produced the parental strain of bacterium comprises, but be not limited to, the L-Threonine of Escherichia is produced bacterium, as E.coli bacterial strain TDH-6/pVIC40 (VKPM B-3996) (United States Patent (USP) 5,175,107, United States Patent (USP) 5,705,371), E.coli bacterial strain NRRL-21593 (United States Patent (USP) 5,939,307), E.coli bacterial strain FERM BP-3756 (United States Patent (USP) 5,474,918), E.coli bacterial strain FERM BP-3519 and FERM BP-3520 (United States Patent (USP) 5,376,538), E.coli bacterial strain MG442 (Gusyatiner etc., Genetika (in Russian), 14,947-956 (1978)), E.coli bacterial strain VL643 and VL2055 (EP 1148811A) etc.

Bacterial strain TDH-6 lacks the thrC gene, and the sucrose assimilative capacity, and the ilvA gene has leaky mutation.Also there is variation in this bacterial strain at the rhtA gene, and it causes that high density Threonine or homoserine are had resistance.Bacterial strain B-3996 contains plasmid pVIC40, and it is that thrA*BC operon by the thrA gene that will contain variation is inserted in the RSF1010 derivative vector and obtains.The thrA genes encoding E.C. 2.7.2.4. homoserine dehydrogenase I of variation, it is insensitive to the feedback inhibition of Threonine substantially.Bacterial strain B-3996 is deposited in full alliance antibiotic class scientific center (Nagatinskaya Street 3-A, 117105Moscow, Russian Federation) on November 19th, 1987, is numbered RIA1867.This bacterial strain also is deposited in Russian state-run industrial microorganism preservation center (VKPM) (Dorozhny proezd.1 on April 7th, 1987, Moscow117545, Russian Federation), be numbered B-3996, classification called after Escherichia coli Tur6 pVIC40.

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

The thrA gene of-variation, the E.C. 2.7.2.4. homoserine dehydrogenase I that its coding has resistance to the feedback inhibition of Threonine;

-thrB gene, its homoserine kinase of encoding;

-thrC gene, its threonine synthase of encoding;

-rhtA gene, the transmembrane protein that its coding is inferred;

-asd gene, its aspartate-of encoding; And

-aspC gene, its aspartate aminotransferase of encoding;

The thrA gene of coding intestinal bacteria E.C. 2.7.2.4. homoserine dehydrogenase I is open, and (Nucleotide numbers 337 to 2799 in the sequence of GenBank accession NC_000913.2, gi:49175990).This thrA gene is on the E.coli K-12 karyomit(e) between thrL and the thrB gene.The thrB gene of coding intestinal bacteria homoserine kinase is open, and (Nucleotide numbers 2801 to 3733 in the sequence of GenBank accession NC_000913.2, gi:49175990).This thrB gene is on the E.coli K-12 karyomit(e) between thrA and the thrC gene.The thrC gene of coding intestinal bacteria threonine synthase is open, and (Nucleotide numbers 3734 to 5020 in the sequence of GenBank accession NC_000913.2, gi:49175990).This thrB gene is on the E.coli K-12 karyomit(e) between thrB gene and the yaaX opening code-reading frame.All these three kinds of gene actings in conjunction become an independently threonine operon.

Coding has the thrA gene of variation of the E.C. 2.7.2.4. homoserine dehydrogenase I of resistance to the feedback inhibition of Threonine, and thrB and thrC gene can be used as an operon and obtain from known plasmid pVIC40, and this plasmid is present in the intestinal bacteria Threonine and produces among the bacterial strain VKPM B-3996.Plasmid pVIC40 write up is in United States Patent (USP) 5,705, in 371.

The rhtA gene is positioned at the close glnHPQ operon of 18min on the escherichia coli chromosome, the component of its coding glutamate transport system, this rhtA gene and ORF1 (ybiF gene, numbering 764 to 1651 in the sequence of GenBank accession numberAAA218541, gi:440181) unanimity is between pexB and ompX gene.The cell list of expressing the ORF1 proteins encoded is shown rhtA (rht: homoserine and Threonine resistance) gene.And it is replacement (the ABSTRACTS of 17 that A on the position-1 changes G that the relative ATG initiator codon of variation of finding to show the rhtA23 gene is arranged ^ThInternational Congress of Biochemistryand Molecular Biology in conjugation with 1997 Annual Meeting of the AmericanSociety for Biochemistry and Molecular Biology, San Francisco, California August 24-29,1997, abstruct No.457, EP1013765A).

Open (the Nucleotide numbering 3572511 to 3571408 in the sequence of GenBank accession NC_000913.1 of colibacillary asd gene, and can carry out PCR (polymerase chain reaction gi:16131307), by using primer according to the gene nucleotide series design; With reference to White, T.J. etc., TrendsGenet, 5,185 (1989)) and obtain.The asd gene of other microorganism can obtain with similar method.

In addition, openly (Nucleotide numbering 983742 to 984932 gi:16128895), and can obtain by PCR colibacillary aspC gene in the sequence of GenBank accession NC_000913.1.The aspC gene of other microorganism can obtain with similar method.

L-Methionin is produced bacterium

The example that the L-Methionin of Escherichia is produced bacterium comprises the mutant with L-lysine analogues resistance.The L-lysine analogues suppresses the growth of Escherichia bacterium, but when L-Methionin was present in the substratum, this inhibition was desensitized wholly or in part.The example of L-lysine analogues includes, but not limited to bacteriolysin, Methionin hydroxamate, S-(2-amino-ethyl)-L-halfcystine (AEC), γ-methyllysine, a-chlorine hexanolactam etc.The mutant that lysine analogues is had resistance can obtain by the Escherichia bacterium being carried out conventional induced mutations processing.The concrete example that can be used for producing the bacterial strain of L-Methionin comprises intestinal bacteria AJ11442 (FERM BP-1543, NRRL B-12185; See U.S.Patent 4,346,170) and intestinal bacteria VL611.In these microorganisms, L-Methionin is desensitized to the feedback inhibition of E.C. 2.7.2.4..

Bacterial strain WC196 can produce bacterium as colibacillary L-Methionin.The breeding of this bacterial strain is the bacterial strain W3110AEC resistance of giving derived from e. coli k-12.The bacterial strain called after intestinal bacteria AJ13069 bacterial strain that obtains, and be preserved in bio-science and institute of human technology country on December 6th, 1994, industrial science and technical body (present institute of advanced industrial Science and Technology country, international monopoly depositary institution, Tsukuba Central 6,1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan), preserving number is FERM P-14690.Then, transfer international preservation to September 29 nineteen ninety-five according to budapest treaty, preserving number is FERM BP-5252 (United States Patent (USP) 5,827,698).

The L-glutamate producing bacterium

The example of parental strain of L-glutamate producing bacterium of the present invention of deriving includes, but not limited to the L-glutamate producing bacterium of Escherichia, as E.coli bacterial strain VL334thrC ⁺(EP 1172433).E.coli bacterial strain VL334 (VKPM B-1641) is L-Isoleucine and the L-Threonine auxotrophic strain (United States Patent (USP) 4,278,765) that thrC and ilvA gene are undergone mutation.The wild-type allele of thrC gene shifts by transduction method commonly used, and the phage P1 that this method is used in the cell that grows in wild-type E.coli bacterial strain K12 (VKPM B-7) inoculates.As a result, obtain L-Isoleucine auxotrophic strain VL334thrC ⁺(VKPM B-8961).This bacterial strain can be produced L-L-glutamic acid.

The example of parental strain of L-glutamate producing bacterium of the present invention of deriving comprises the mutant that lacks a-ketoglutaric acid salt dehydrogenase activity or have the a-ketoglutaric acid salt dehydrogenase activity that weakens.The method that lacks a-ketoglutaric acid salt dehydrogenase activity or have the Escherichia bacterium of the a-ketoglutaric acid salt dehydrogenase activity that weakens and obtain them is recorded in United States Patent (USP) 5,378,616 and 5,573,945.Especially, these bacterial strains comprise following:

E.coli?W3110sucA::Kmr

E.coli?AJ12624(FERM?BP-3853)

E.coli?AJ12628(FERM?BP-3854)

E.coli?AJ12949(FERM?BP-4881)

Obtain E.coli W3110sucA::Kmr by the a-ketoglutaric acid salt dehydrogenase gene (following table is shown " sucA gene ") that destroys E.coli W3110.This bacterial strain lacks a-ketoglutaric acid salt desaturase fully.

The example of other L-glutamate producing bacteriums comprises the general Pseudomonas mutant strain that lacks a-ketoglutaric acid salt dehydrogenase activity or have the a-ketoglutaric acid salt dehydrogenase activity that weakens, and its preparation method as mentioned above.This bacterial strain comprises Pantoea ananatis AJ13356 (United States Patent (USP) 6,331,419).Pantoea ananatisAJ13356 is preserved in bio-science and institute of human technology country on February 19th, 1998, industrial science and technical body (present institute of advanced industrial Science and Technology country, international monopoly depositary institution, Tsukuba Central 6,1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan), preserving number is FERM P-16645.Then, transfer international preservation according to budapest treaty on January 11st, 1999, preserving number is FERM BP-6615.Because aKGDH-E1 subunit gene (sucA) is destroyed, Pantoea ananatis AJ13356 lacks a-ketoglutaric acid salt dehydrogenase activity.When the separated and preservation of above-mentioned bacterial strains was enterobacter agglomerans AJ13356, it was accredited as enterobacter agglomerans (Enterobacteragglomerans).Yet its nucleotide sequencing result according to 16S rRNA etc. reclassifies Pantoea ananatis now.Though AJ13356 preservation in aforementioned depositary institution is an enterobacter agglomerans, in this specification sheets, they are recited as Pantoea ananatis.

The L-tryptophane is produced bacterium

The example of parental strain of L-glutamate producing bacterium of the present invention of deriving comprises, but be not limited to, the L-tryptophane of Escherichia is produced bacterium, as lack the E.coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (the DSM10123) (United States Patent (USP) 5 of the tryptophane-tRNA synthase of trpS mutator gene coding, 756,345); Have serA allelotrope and be not subjected to the E.coli SV164 (pGH5) (United States Patent (USP) 6,180,373) of Serine feedback inhibition; The E.coli AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) (United States Patent (USP) 4,371,614) that lack tryptophanase; Phosphoenolpyruvic acid throughput enhanced E.coli AGX17/pGX50, pACKG4-pps (WO9708333, United States Patent (USP) 6,319,696) etc.

Identify yddG genes encoding membranin in the past, it does not participate in the amino acid whose biosynthetic pathway of any L-.In addition, known when increasing in the multi-copy vector of wild-type allele in microorganism of yddG gene, it makes microorganism have the resistance to L-phenylalanine and several amino acid analogues.In addition, when extra copy was imported into the cell of corresponding production bacterial strain, the yddG gene can increase the output (WO03044192) of L-phenylalanine or L-tryptophane.Thereby the therefore preferred L-tryptophane of further modifying is produced the expression that bacterium strengthens the yddG opening code-reading frame.

The L-arginine is produced bacterium

The example that the L-arginine of the present invention of deriving is produced the parental strain of bacterium comprises; but be not limited to; the L-arginine is produced bacterium, as E.coli bacterial strain 237 (VKPM B-7925) (U.S. Patent application US2002058315) and its contain the N-ethanoyl glutamate synthase of sudden change derivative strain (Russ P application No.2001112869), E.coli bacterial strain 382 (VKPM B-7926) (European patent application EP 1170358), import the arginine production bacterial strain (JP 57-5693A) etc. of the argA gene of coding N-ethanoyl glutamate synthase.

The L-phenylalanine is produced bacterium

The example that the L-phenylalanine of the present invention of deriving is produced the parental strain of bacterium includes, but not limited to the L-phenylalanine of Escherichia and produces bacterium, as E.coli AJ12739 (tyrA::Tn10, tyrR) (VKPMB-8197); The E.coli HW1089 (ATCC 55371) (U.S.Patent5,354,672) that contains the pheA34 gene; E.coli MWEC101-b (KR8903681); E.coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (U.S.Patent 4,407,952).In addition, as parental strain, can use 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] E.coli K-12[W3110 (tyrA)/pBR-aroG4 of (FERM BP-12662) and called after AJ12604 (FERM BP-3579), pACMAB] (EP 488424B1).In addition, can use the L-phenylalanine of the Escherichia of yedA gene or yddG gene coded protein increased activity to produce bacterium (U.S. Patent application 2003/0148473A1 and 2003/0157667A1).

The L-halfcystine is produced bacterium

The example that the L-halfcystine of the present invention of deriving is produced the parental strain of bacterium comprises, but be not limited to, the L-halfcystine of Escherichia is produced bacterium, the E.coli bacterial strain JM15 (United States Patent (USP) 6 that transforms as cysE allelotrope with different encoder feedback resistance Serine acyltransferases, 218,168, Russ P application 2003121601); Cross the E.coli bacterial strain W3110 (United States Patent (USP) 5,972,663) of the gene of expressing Codocyte toxicity secretory protein; E.coli bacterial strain (JP11-155571A) with low halfcystine devulcanization enzymic activity; Strengthen the positive transcription regulatory protein active E.coli bacterial strain W3110 of halfcystine regulon (WO0127307A1) of cysB genes encoding etc.

The L-leucine is produced bacterium

The example that the L-leucine of the present invention of deriving is produced the parental strain of bacterium comprises, but be not limited to, the L-leucine of Escherichia is produced bacterium, as the leucine analogue being had the E.coli bacterial strain of resistance, described analogue comprises β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoro leucine (JP 62-34397B and JP 08-70879A); The E.coli bacterial strain that utilizes the described gene engineering method of WO96/06926 to obtain; E.coli bacterial strain H-9068 (JP 08-70879A) etc.

Can improve bacterium of the present invention by strengthening the biosynthetic expression of gene of the one or more L-of relating to leucines.Example comprises the gene of leuABCD operon, is preferably the leuA gene of sudden change, and it is encoded not by the isopropylmalate synthase of L-leucine feedback inhibition (United States Patent (USP) 6,403,342).In addition, can improve bacterium of the present invention by strengthen expressing one or more proteic encoding genes that L-amino acid is secreted bacterial cell.The example of this gene comprises b2682 and b2683 gene (ygaZH gene) (Russ P application 2001117632).

The L-proline(Pro) is produced bacterium

The example that the L-proline(Pro) of the present invention of deriving is produced the parental strain of bacterium comprises, but be not limited to, the L-proline(Pro) of Escherichia is produced bacterium, as E.coli bacterial strain 702ilvA (VKPM B-8012), it lacks the ilvA gene, and can produce L-proline(Pro) (EP 1172433).Can improve bacterium of the present invention by strengthening the biosynthetic expression of gene of the one or more L-of relating to proline(Pro).The example that the L-proline(Pro) is produced this gene in the bacterium comprises the proB gene, and it is encoded to the insensitive Glutamate kinase of L-proline(Pro) feedback inhibition (DE patent 3127361).In addition, can improve bacterium of the present invention by strengthen expressing one or more proteic encoding genes that L-amino acid is secreted bacterial cell.The example of this gene comprises b2682 and b2683 gene (ygaZH gene) (EP1239041A2).

Have the example of producing the active Escherichia bacterium of L-proline(Pro) and comprise following E.coli bacterial strain: NRRL B-12403 and NRRL B-12404 (GB patent 2075056), VKPM B-8012 (Russ P application 2000124295), (The 15 to be recorded in the plasmid encoding mutant body that plasmid encoding mutant body in the DE patent 3127361, Bloom F.R. etc. describe ^ThMiami winter symposium, 1983, p.34) etc.

Above-mentioned L-amino acid preparation strain can further adopt and well known to a person skilled in the art that the whole bag of tricks modifies, to strengthen the pentose assimilation ratio or to strengthen L-amino acid bio synthesis capability.

By amplification pentose assimilation gene, the araFG and the araBAD gene of corresponding pectinose, perhaps pass through the sudden change of glucose assimilation system (PTS and non-PTS), sudden change (Nichols N.N. etc. as ptsG, Appl.Microbiol.Biotechnol., 2001, Jul.56:1-2 120-5) can further increase the pentose utilization ratio.

Method of the present invention comprises the amino acid whose method of L-of producing, it is included in and cultivates L-amino acid production bacterium in the substratum, L-amino acid is accumulated in substratum, and from substratum, collected the amino acid whose step of L-, wherein contain the mixture of glucose and pentose in the substratum.In addition, method of the present invention comprises the method for producing the L-Histidine, and it is included in cultivates L-Histidine production bacterium of the present invention in the substratum, the L-Histidine is accumulated in substratum, and the step of from substratum, collecting the L-Histidine, wherein contain the mixture of glucose and pentose in the substratum.In addition, method of the present invention comprises the method for producing the L-Threonine, and it is included in cultivates L-Threonine production bacterium of the present invention in the substratum, the L-Threonine is accumulated in substratum, and the step of from substratum, collecting the L-Threonine, wherein contain the mixture of glucose and pentose in the substratum.In addition, method of the present invention comprises the method for producing L-Methionin, and it is included in cultivates L-Methionin production bacterium of the present invention in the substratum, L-Methionin is accumulated in substratum, and the step of from substratum, collecting L-Methionin, wherein contain the mixture of glucose and pentose in the substratum.In addition, method of the present invention comprises the method for producing L-L-glutamic acid, and it is included in cultivates L-glutamate producing bacterium of the present invention in the substratum, L-L-glutamic acid is accumulated in substratum, and the step of from substratum, collecting L-L-glutamic acid, wherein contain the mixture of glucose and pentose in the substratum.In addition, method of the present invention comprises the method for producing the L-tryptophane, it is included in and cultivates L-tryptophane production bacterium of the present invention in the substratum, the L-tryptophane is accumulated in substratum, and the step of from substratum, collecting L-tryptophane, wherein contain the mixture of glucose and pentose in the substratum.

Pentose as wood sugar and pectinose, with hexose, can obtain from the biomass resource of underusing as the mixture of glucose.By steam and/or spissated acidolysis, weak acid separate, enzymic hydrolysis, as using cellulase or alkaline purification, from plant biomass, discharge glucose, wood sugar, pectinose and other carbohydrate.When substrate was cellulose materials, Mierocrystalline cellulose can be hydrolyzed to sugar at the same time or separately and also ferment and be L-amino acid.Because hemicellulose is than the easier sugar that is hydrolyzed to of Mierocrystalline cellulose, preferred prehydrolysis cellulose materials separates pentose and then utilizes steam, acid, alkali, cellulase or its bonded treatment process hydrocellulose to form glucose.

The mixture that this research uses glucose/wood sugar/pectinose of different ratios to form, the glucose that may from plant hydrolyzed thing, obtain with simulation and the composition (referring to the embodiment part) of pentose mixture of raw material.

In the present invention, the method for employings such as the amino acid whose cultivation of L-, collection and purifying is similar to conventional microbial fermentation and produces proteic method in the substratum.The substratum that is used to cultivate can be synthetic medium or natural medium, as long as contain carbon source, nitrogenous source and mineral in this substratum, and if desired, contains the required nutrition of microorganism growth of appropriate amount.

Carbon source can comprise the multiple carbohydrate that can be used as carbon source by L-amino acid production bacterium, as glucose, sucrose, pectinose, wood sugar and other pentose and hexose.Glucose, wood sugar, pectinose and other carbohydrate can be the parts of the mixture of raw material of the sugar that obtains from cellulose biomass.

The pentose that is fit to fermentation among the present invention includes, but are not limited to wood sugar and pectinose.

As nitrogenous source, can use the organism of fermentation of multiple ammonium salt such as ammoniacal liquor and ammonium sulfate, other nitrogenous composition such as amine, natural nitrogenous source such as peptone, soya hydrolysate and assimilation.Mineral can use a potassiumphosphate, sal epsom, sodium-chlor, ferric sulfate, manganous sulfate, calcium chloride etc.Can in substratum, add extra nutrition when needing.For example, if microbial growth needs proline(Pro) (proline(Pro) defective type), when cultivating, can in substratum, add the proline(Pro) of capacity.

Preferably, cultivate and for example to carry out stir culture under the shake-flask culture and ventilation condition under the condition of aerobic, culture temperature is 20 to 40 ℃, preferred 30 to 38 ℃.The pH value of substratum is usually between 5 to 9, preferably between 6.5 to 7.2.The pH value of substratum can be regulated with ammoniacal liquor, lime carbonate, multiple acid, multiple alkali and damping fluid.Usually, cultivation can produce the amino acid whose accumulation of purpose L-in the liquid medium within 1 to 5 day.

After the cultivation, from liquid nutrient medium, remove solid matter such as cell, then adopt ion-exchange, concentrate and crystallization method collection and purifying purpose L-amino acid by centrifugal or membrane filtration.

Embodiment

The present invention will be by carrying out more definite explanation about following limiting examples.

Embodiment 1: from the karyomit(e) clone xylBFGHR locus of E.coli bacterial strain MG1655

According to the genome analysis of E.coli bacterial strain MG1655, gene xylABFGHR can be cloned in whole 556 HidIII chromosome segments one, and independently HindIII fragment (13.1kb) is (Fig. 1).For this purpose, pUC19 has set up gene library with carrier, and this carrier that contains this size inset can be survived in E.coli.

For setting up such library, the chromosomal DNA of MG1655 digests with the XbaI restriction enzyme digestion with digestion of HindIII restriction enzyme digestion and pUC19 carrier.Bacterial strain MG1655 (ATCC47076, ATCC700926) can from American type culture collection (10801 University Boulevard, Manassas, VA., 20110-2209 U.S.A) obtains.

In order to prevent that the sticky end of two DNA products is subsequently all by Klenow fragment polishing (two bases are filled up) from connecting.After Connection Step, the pUC19 plasmid storehouse that obtains recombinating.The size in storehouse is greater than 200000 clones.Gene pool uses with plasmid sequence complementary primer with clone's chromosome segment complementary primer and analyzes by PCR.In the PCR product, do not have to find to have the dna fragmentation of suitable molecular weight, omit and the corresponding fragment of xylABFGHR operon in the storehouse that its explanation is set up.This possibility of result is that it also is present in the HindIII purpose fragment owing to the negative impact of malS gene, yiaA and yiaB ORFs (unknown its function).The possible reason of the negative selection of another one is that the Xyl-locus is too big.In order to overcome this problem, set up new gene pool based on the pUC19 plasmid of modifying.Main method is that clone Xyl-locus is as the one group of fragment that does not contain adjacent malS gene and yiaA and yiaB ORFs.

For this purpose, modify the polylinker of plasmid pUC19 by inserting dna fragmentation that synthetic contains the MluI restriction site.Set up two gene pools with the pUC19 cloning vector of modifying.The foundation in first storehouse is by then being connected with the chromosomal DNA of MluI Restriction Enzyme digestion bacterial strain MG1655 and the pUC19 of modification with HindIII.A clone surplus the capacity 4,000 in storehouse.Gene pool is used with plasmid sequence complementary primer with the primer 1 (SEQ ID NO:13) and 2 (the SEQID NO:14) of the xylABFG fragment complementation of xyl locus and is carried out pcr analysis.In the PCR product, found to have the expection dna fragmentation of suitable molecular weight.Following step is with the saturated gene pool of purpose fragment.After this, there is not restriction site in the DNA endonuclease digestion that obtains from the gene pool of source in its purpose fragment.Eco1471, KpnI, MlsI, Bst1107I are arranged.The frequency of the purpose plasmid in the storehouse after enrichment is 1/800 clone.Storehouse after the enrichment pcr analysis that passes through as indicated above.After the enrichment of five successive storehouse cell masses, 10 clones that contain the xylABFG gene have only been found.The resulting segmental plasmid called after of the HindIII-MluIDNA pUC19/xylA-G that contains gene xylABFG.The HindIII-Mph1103I fragment that contains yiaA and yiaB ORFs is subsequently removed from plasmid pUC19/xylA-G; Insert the synthetic linker that contains the EcoRI restriction site by Klenow fragment polishing sticky end and by ligation.Like this, obtained plasmid pUC19/xylA-G-2.Then, the pUC19/xylA-G-2 plasmid of gained cuts off with the EheI Restriction Enzyme; Insert the synthetic linker that contains the HindIII restriction site by Klenow fragment polishing sticky end and by ligation.Obtained pUC19/xylA-G-3 like this.The HindIII restriction site inserts the remaining dna fragmentation that contains the xylHR gene, obtains complete xyl locus.

The foundation in second storehouse is by then being connected with the chromosomal DNA of MluI Restriction Enzyme digestion bacterial strain MG1655 and the pUC19 of modification with PstI.A clone surplus the capacity 6,000 in storehouse.Gene pool is used with plasmid sequence complementary primer with chromosome segment clone complementary primer 3 (SEQ ID NO:15) and 4 (SEQ ID NO:16) and is carried out pcr analysis.In the PCR product, found to have the expection dna fragmentation of suitable molecular weight.Following step is by gene pool the continuous segmentation on cell mass of pcr analysis to known dimensions.The cell mass that contains gene xylHR after seven continuous segmentations in storehouse comprises only ten clones.In this group, found target DNA fragment by restriction analysis.The plasmid of the HindIII-MluI dna fragmentation of the resulting xylHR of containing gene is named as pUC19/xylHR.Subsequently, on the pUC19/xylA-G-3 plasmid of having handled by HindIII and MluI Restriction Enzyme before the HindIII-MluI dna fragmentation that obtains from plasmid pUC19/xylHR is connected to.At last, obtain the complete xyl locus of bacterial strain MG1655.The multiple copied plasmid called after pUC19/xylA-R that contains complete xylABFGHR locus that obtains.

The HindIII-EcoRI dna fragmentation that obtains from the pUC19/xylA-R plasmid then is cloned into the low copy carrier pMW119mod that has digested with HindIII and EcoRI Restriction Enzyme again, obtains containing the low copy plasmid pMW119mod-xylA-R of complete xylABFGHR locus.Low copy carrier pMW119mod obtains by removing the PvuII-PvuII fragment from commercially available pMW119 carrier.This fragment contains multiple clone site and is the main position of lacZ gene.The LacZ gene contains the site of lacI inhibition, is the insertion that contains the synthetic linker in EcoRI and HindIII site thereafter, and its insertion for xylABFGHR locus from the pUC19/xylA-R plasmid is necessary.

Embodiment 2: use the L-Histidine to produce bacterium and carry out fermentative production L-Histidine in glucose and pentose mixture

It is the bacterial strains of fermentative production L-Histidine that are used for the mixture of glucose and pentose that the L-Histidine is produced E.coli bacterial strain 80.E.coli bacterial strain 80 (VKPM B-7270) in Russ P RU2119536, describe in detail and on October 15th, 1999 at Russian National Collection of IndustrialMicroorganisms (Russia, 113545Moscow, 1 ^StDorozhny proezd, 1) with sequence number VRPMB-7270 preservation.Subsequently, it transferred international preservation on January 12nd, 2004 according to being specified in of budapest treaty.Transform bacterial strain 80 by ordinary method with the pMW119mod-xylA-R plasmid, obtain bacterial strain 80/pMW119mod-xylA-R.

Be to obtain inoculum, bacterial strain 80 and 80/pMW119mod-xylA-R are all at the 40ml test tube of the L-meat soup of the Streptomycin sulphate that contains 1g/l that contains 2ml In, 27 ℃ in the last growth of gyrate shaker (250rpm) 6 hours.To bacterial strain 80/pMW119mod-xylA-R, add the Ampicillin Trihydrate of 100mg/l.Subsequently, the seed material with 2ml (5%) is seeded in the fermention medium.In containing the 40ml test tube of 2ml fermention medium, go up growth 65 hours in gyrate shaker (250rpm) at 27 ℃.

After the cultivation, the amount that is accumulated in the L-Histidine in the substratum is measured by paper chromatography.Moving phase composed as follows: butanols: acetic ester: water=4: 1: 1 (v/v).With the acetone soln (0.5%) of triketohydrindene hydrate as developer.The result is presented in the table 2.

The composition of fermention medium (g/l):

Carbohydrate (total amount) 100.0

0.2 of Mameno TN (all nitrogen)

(soya hydrolysate)

L-proline(Pro) 0.8

(NH ₄) ₂SO ₄ 25.0

K ₂HPO ₄ 2.0

MgSO ₄·7H ₂O 1.0

FeSo ₄·7H ₂O 0.01

MnSO ₄·5H ₂O 0.01

Thiamine hydrochloride 0.001

Trimethyl-glycine 2.0

CaCO ₃ 6.0

Streptomycin sulphate 1.0

Carbohydrate (glucose, pectinose, wood sugar), L-proline(Pro), trimethyl-glycine and sal epsom are sterilized respectively.CaCO ₃Xeothermic 110 ℃ of sterilizations 30 minutes.PH is adjusted to 6.0 by KOH before the sterilization.As seen, the enhancing of xylABFGHR locus is expressed and has been promoted the L-Histidine of cultivating in containing the substratum of wood sugar to produce the productive rate of E.coli bacterial strain 80 from table 2.

Table 2

Embodiment 3. uses L-Threonine production bacterium to carry out production of L-threonine by fermentation in glucose and pentose mixture

The L-Threonine is produced the fermentative production L-Histidine that E.coli bacterial strain B-3996 is used to estimate the mixture of glucose and pentose.Respectively by using CaCl ₂Ordinary method transform bacterial strain B-3996 with pMW119mod-xylA-R plasmid and carrier pMW119, obtain bacterial strain 3996/pMW119mod-xylA-R and 3996/pMW119.

E.coli bacterial strain B-3996 and B-3996/pMW119mod-xylA-R all grew in 37 ℃ in the L-agar plate that contains Streptomycin sulphate (50mg/l) and Ampicillin Trihydrate (150mg/l) 12-15 hour.Subsequently, contain wood sugar (4%) as the fermention medium of carbon source in the inoculation one the ring bacterial strain.In 20 * 20mm test tube, in the fermention medium that contains Streptomycin sulphate (50mg/l) of 2ml, ferment.Cell was grown 65 hours under 32 ℃ of vibrations at 250rpm.

After the cultivation, the amount that is accumulated in the L-Threonine in the substratum is measured by paper chromatography, and it uses following moving phase: butanols: acetic acid: water=4: 1: 1 (v/v).With the ninhydrin solution (2%) of acetone as developer.Cut away the point that contains the L-Threonine, L-Threonine CdCl ₂0.5% aqueous solution wash-out, the amount of L-Threonine is measured at 540nm with spectrophotometry.The result is displayed in Table 3.

(g/l) is as follows for the composition of fermention medium:

Carbohydrate (total amount) 40.0

(NH ₄) ₂SO ₄ 24.0

NaCl 0.8

KH ₂PO ₄ 2.0

MgSO ₄·7H ₂O 0.8

FeSO ₄·7H ₂O 0.02

MnSO ₄·5H ₂O 0.02

Thiamine hydrochloride 0.0002

Yeast extract 1.0

CaCO ₃ 30.0

Glucose and sal epsom are sterilized respectively.CaCO ₃Xeothermic 180 ℃ of sterilizations 2 hours.PH regulator to 7.0.Sterilization back antibiotic is introduced into substratum.

Table 3

As shown in the table 3, the productive rate that the L-Threonine that has promoted to be incubated in the substratum that contains wood sugar is produced E.coli bacterial strain B-3996/pMW119 is expressed in the enhancing of xylABFGHR locus.

Embodiment 4. uses L-Methionin production bacterium to carry out fermentation production of L-lysine in glucose and pentose mixture

L-Methionin generates the fermentation production of L-lysine that E.coli bacterial strain WC196 Δ cadA Δ ldc is used to estimate the mixture of glucose and pentose.Bacterial strain WC196 Δ cadA Δ ldc is from by as United States Patent (USP) 5,827, and the deactivation of describing in 698 is by acquisition among the bacterial strain WC 196 of the lysine decarboxylase of ldcC gene and cadA genes encoding.Respectively by using CaCl ₂Ordinary method transform bacterial strain WC196 Δ cadA Δ ldc with pMW119mod-xylA-R plasmid and carrier pMW119, obtain bacterial strain WC196 Δ cadA Δ ldc/pMW119mod-xylA-R and WC196 Δ cadA Δ ldc/pMW119.

E.coli bacterial strain WC196 Δ cadA Δ ldc/pMW119 and WC196 Δ cadA Δ ldc/pMW119mod-xylA-R all grew in 37 ℃ in the L-agar plate that contains Ampicillin Trihydrate (150mg/l) 12-15 hour.Subsequently, contain wood sugar (4%) or wood sugar (2%)/glucose (2%) mixture as the fermention medium of carbon source in the inoculation one the ring bacterial strain.In 20 * 20mm test tube, in the fermention medium of 2ml, ferment.Cell was grown 25 hours under 32 ℃ of vibrations at 250rpm.

After the cultivation, the amount that is accumulated in the L-Methionin in the substratum is measured by paper chromatography, and it uses following moving phase: butanols: acetic acid: water=4: 1: 1 (v/v).With the ninhydrin solution (2%) of acetone as developer.Cut away the point that contains L-Methionin, L-Methionin CdCl ₂0.5% aqueous solution wash-out, the amount of L-Methionin is measured at 540nm with spectrophotometry.The result is displayed in Table 4.

(g/l) is as follows for the composition of fermention medium:

Carbohydrate (total amount) 40.0

(NH ₄) ₂SO ₄ 24.0

KH ₂PO ₄ 1.0

MgSO ₄·7H ₂O 1.0

FeSO ₄·7H ₂O 0.01

MnSO ₄·5H ₂O 0.01

Yeast extract 2.0

CaCO ₃ 30.0

Glucose and sal epsom are sterilized respectively.CaCO ₃Xeothermic 180 ℃ of sterilizations 2 hours.PH is adjusted to 7.0 with KOH.Sterilization back antibiotic is introduced into substratum.

As shown in the table 4, the enhancing of xylABFGHR locus is expressed and has been promoted to be incubated at the productive rate that L-Methionin in the substratum that contains wood sugar generates E.coli bacterial strain WC196 Δ cadA Δ ldc/pMW119.

Embodiment 5. uses the L-glutamate producing bacterium to carry out fermentation production of L-glutamic acid in glucose and pentose mixture.

L-L-glutamic acid generates the fermentation production of L-glutamic acid product that E.coli strains A J12624 is used to estimate the mixture of glucose and pentose.Respectively by using CaCl ₂Ordinary method transform bacterial strain AJ12624 with pMW119mod-xylA-R plasmid and carrier pMW119, obtain strains A J12624/pMW119mod-xylA-R and AJ12624/pMW119.

E.coli strains A J12624/pMW119 and AJ12624/pMW119mod-xylA-R all grew in 37 ℃ in the L-agar plate that contains Ampicillin Trihydrate (150mg/l) 12-15 hour.Subsequently, to containing the fermention medium inoculation one ring bacterial strain of wood sugar (4%) as carbon source.In 20 * 20mm test tube, in the fermention medium of 2ml, ferment.Cell was grown 48 hours under 32 ℃ of vibrations at 250rpm.

After the cultivation, the amount that is accumulated in the L-L-glutamic acid in the substratum is measured by paper chromatography, and it uses following moving phase: butanols: acetic acid: water=4: 1: 1 (v/v).With the ninhydrin solution (2%) of acetone as developer.Cut away the point that contains L-L-glutamic acid, L-L-glutamic acid CdCl ₂0.5% aqueous solution wash-out, the amount of L-L-glutamic acid is measured at 540nm with spectrophotometry.The result is displayed in Table 5.

The composition of fermention medium (g/l):

Carbohydrate 40.0

(NH ₄) ₂SO ₄ 25.0

KH ₂PO ₄ 2.0

MgSO ₄·7H ₂O 1.0

Thiamine hydrochloride 0.0001

L-Isoleucine 0.07

CaCO ₃ 25.0

Glucose and sal epsom are sterilized respectively.CaCO ₃Xeothermic 180 ℃ of sterilizations 2 hours.PH regulator to 7.2.

Table 5

As shown in the table 5, the enhancing of xylABFGHR locus is expressed and has been promoted the L-L-glutamic acid of cultivating in containing the substratum of wood sugar to produce the productive rate of E.coli strains A J12624/pMW119.

Embodiment 6. uses L-tryptophane production bacterium to carry out fermentative production L-tryptophane in glucose and pentose mixture.

The L-tryptophane generates the fermentative production L-tryptophane that E.coli bacterial strain SV164/pGH5 is used to estimate the mixture of glucose and pentose.Respectively by using CaCl ₂Ordinary method transform bacterial strain SV164/pGH5 with pMW119mod-xylA-R plasmid and carrier pMW119, obtain bacterial strain SV164/pGH5/pMW119mod-xylA-R and SV164/pGH5/pMW119.

E.coli bacterial strain SV164/pGH5/pMW119 and SV164/pGH5/pMW119mod-xylA-R all grew in 37 ℃ in the L-agar plate that contains tsiklomitsin (30mg/l) and Ampicillin Trihydrate (150mg/l) 12-15 hour.Subsequently, to containing the fermention medium inoculation one ring bacterial strain of wood sugar (4%) or wood sugar (2%)/glucose (2%) mixture as carbon source.In 20 * 20mm test tube, in the fermention medium of 2ml, ferment.Cell was grown 48 hours under 30 ℃ of vibrations at 250rpm.

After the cultivation, the amount that is accumulated in the L-tryptophane in the substratum is measured by TLC.Adopt Sorbfil silica gel (Stock CompanySorbpolymer Krasnodar, Russia) the bag quilt of the TLC plate of 10 * 15cm with the 0.11mm that does not contain fluorescent indicator.The Sorbfil plate can be used following moving phase video picture: propylene glycol: the ethene acetic ester: 25% liquid ammonia: water=40: 40: 7: 16 (v/v).With the ninhydrin solution (2%) of acetone as developer.The result is displayed in Table 7.The fermentation culture based composition and use thereof in packaging is listed in the table 5, but should organize between A, B, C, D, E, F and the H deactivation respectively, as shown, and for fear of each other side effect in inactivation process.

Table 6.

Solution	Composition	Ultimate density, G/L
			A B C D E F	KH 2PO 4 NaCl (NH 4) 2SO 4L-MET L-phenylalanine L-tyrosine Mameno (full N) glucose MgSO 4·7H 2O CaCl 2 FeSO 4·7H 2O Trisodium Citrate Na 2MoO 4·2H 2O H 3BO 3 CoCl 2·6H 2O CuSO 4·5H 2O MnCl 2·4H 2O ZnSO 4·7H 2The O thiamine hydrochloride	1.5 0.5 1.5 0.05 0.1 0.1 0.07 40.0 0.3 0.011 0.075 1.0 0.00015 0.0025 0.00007 0.00025 0.0016 0.0003 0.005

G H

CaCO 3Pyridoxol

30.0 0.03

Solution A is passed through NH ₄OH regulates pH to 7.1.

Table 7

As shown in the table 7, the enhancing of xylABFGHR locus is expressed and has been promoted the L-tryptophane of cultivating in containing the substratum of wood sugar to produce the productive rate of E.coli bacterial strain SV164/pGH5/pMW119.

Though the present invention preferred embodiment describes in detail with it, obviously can carry out various conversion to the those skilled in the art, and in the replacement that does not deviate from the equivalent under the scope of the present invention.Each aforementioned document all is incorporated by reference in this text at this and examines.

Sequence table

<110>Ajinomoto?Co.，Inc.

＜120〉use xylose utilization genetic expression enhanced bacterium to ferment to produce the amino acid whose method of L-

<130>OP-C5007

<140>

<141>

<150>RU2004107548

<151>2004-03-16

<150>US60/610545

<151>2004-09-17

<160>16

<170>PatentIn?Ver.2.1

<210>1

<211>1323

<212>DNA

＜213〉intestinal bacteria

<220>

<221>CDS

<222>(1)..(1323)

<400>1

atg?caa?gcc?tat?ttt?gac?cag?ctc?gat?cgc?gtt?cgt?tat?gaa?ggc?tca 48

Met?Gln?Ala?Tyr?Phe?Asp?Gln?Leu?Asp?Arg?Val?Arg?Tyr?Glu?Gly?Ser

1 5 10 15

aaa?tcc?tea?aac?ccg?tta?gca?ttc?cgt?cac?tac?aat?ccc?gac?gaa?ctg 96

Lys?Ser?Ser?Asn?Pro?Leu?Ala?Phe?Arg?His?Tyr?Asn?Pro?Asp?Glu?Leu

20 25 30

gtg?ttg?ggt?aag?cgt?atg?gaa?gag?cac?ttg?cgt?ttt?gcc?gcc?tgc?tac 144

Val?Leu?Gly?Lys?Arg?Met?Glu?Glu?His?Leu?Arg?Phe?Ala?Ala?Cys?Tyr

35 40 45

tgg?cac?acc?ttc?tgc?tgg?aac?ggg?gcg?gat?atg?ttt?ggt?gtg?ggg?gcg 192

Trp?His?Thr?Phe?Cys?Trp?Asn?Gly?Ala?Asp?Met?Phe?Gly?Val?Gly?Ala

50 55 60

ttt?aat?cgt?ccg?tgg?cag?cag?cct?ggt?gag?gca?ctg?gcg?ttg?gcg?aag 240

Phe?Asn?Arg?Pro?Trp?Gln?Gln?Pro?Gly?Glu?Ala?Leu?Ala?Leu?Ala?Lys

65 70 75 80

cgt?aaa?gca?gat?gtc?gca?ttt?gag?ttt?ttc?cac?aag?tta?cat?gtg?cca 288

Arg?Lys?Ala?Asp?Val?Ala?Phe?Glu?Phe?Phe?His?Lys?Leu?His?Val?Pro

85 90 95

ttt?tat?tgc?ttc?cac?gat?gtg?gat?gtt?tcc?cct?gag?ggc?gcg?tcg?tta 336

Phe?Tyr?Cys?Phe?His?Asp?Val?Asp?Val?Ser?Pro?Glu?Gly?Ala?Ser?Leu

100 105 110

aaa?gag?tac?atc?aat?aat?ttt?gcg?caa?atg?gtt?gat?gtc?ctg?gca?ggc 384

Lys?Glu?Tyr?Ile?Asn?Asn?Phe?Ala?Gln?Met?Val?Asp?Val?Leu?Ala?Gly

115 120 125

aag?caa?gaa?gag?agc?ggc?gtg?aag?ctg?ctg?tgg?gga?acg?gcc?aac?tgc 432

Lys?Gln?Glu?Glu?Ser?Gly?Val?Lys?Leu?Leu?Trp?Gly?Thr?Ala?Asn?Cys

130 135 140

ttt?aca?aac?cct?cgc?tac?ggc?gcg?ggt?gcg?gcg?acg?aac?cca?gat?cct 480

Phe?Thr?Asn?Pro?Arg?Tyr?Gly?Ala?Gly?Ala?Ala?Thr?Asn?Pro?Asp?Pro

145 150 155 160

gaa?gtc?ttc?agc?tgg?gcg?gca?acg?caa?gtt?gtt?aca?gcg?atg?gaa?gca 528

Glu?Val?Phe?Ser?Trp?Ala?Ala?Thr?Gln?Val?Val?Thr?Ala?Met?Glu?Ala

165 170 175

acc?cat?aaa?ttg?ggc?ggt?gaa?aac?tat?gtc?ctg?tgg?ggc?ggt?cgt?gaa 576

Thr?His?Lys?Leu?Gly?Gly?Glu?Asn?Tyr?Val?Leu?Trp?Gly?Gly?Arg?Glu

180 185 190

ggt?tac?gaa?acg?ctg?tta?aat?acc?gac?ttg?cgt?cag?gag?cgt?gaa?caa 624

Gly?Tyr?Glu?Thr?Leu?Leu?Asn?Thr?Asp?Leu?Arg?Gln?Glu?Arg?Glu?Gln

195 200 205

ctg?ggc?cgc?ttt?atg?cag?atg?gtg?gtt?gag?cat?aaa?cat?aaa?atc?ggt 672

Leu?Gly?Arg?Phe?Met?Gln?Met?Val?Val?Glu?His?Lys?His?LysIle?Gly

210 215 220

ttc?cag?ggc?acg?ttg?ctt?atc?gaa?ccg?aaa?ccg?caa?gaa?ccg?acc?aaa 720

Phe?Gln?Gly?Thr?Leu?Leu?Ile?Glu?Pro?Lys?Pro?Gln?Glu?Pro?Thr?Lys

225 230 235 240

cat?caa?tat?gat?tac?gat?gcc?gcg?acg?gtc?tat?ggc?ttc?ctg?aaa?cag 768

His?Gln?Tyr?Asp?Tyr?Asp?Ala?Ala?Thr?Val?Tyr?Gly?Phe?Leu?Lys?Gln

245 250 255

ttt?ggt?ctg?gaa?aaa?gag?att?aaa?ctg?aac?att?gaa?gct?aac?cac?gcg 816

Phe?Gly?Leu?Glu?Lys?Glu?Ile?Lys?Leu?Asn?Ile?Glu?Ala?Asn?His?Ala

260 265 270

acg?ctg?gca?ggt?cac?tct ttc?cat?cat?gaa?ata?gcc?acc?gcc?att?gcg 864

Thr?Leu?Ala?Gly?His?Ser?Phe?His?His?Glu?Ile?Ala?Thr?Ala?Ile?Ala

275 280 285

ctt?ggc?ctg?ttc?ggt?tct?gtc?gac?gcc?aac?cgt?ggc?gat?gcg?caa?ctg 912

Leu?Gly?Leu?Phe?Gly?Ser?Val?Asp?Ala?Asn?Arg?Gly?Asp?Ala?Gln?Leu

290 295 300

ggc?tgg?gac?acc?gac?cag?ttc?ccg?aac?agt?gtg?gaa?gag?aat?gcg?ctg 960

Gly?Trp?Asp?Thr?Asp?Gln?Phe?Pro?Asn?Ser?Val?Glu?Glu?Asn?Ala?Leu

305 310 315 320

gtg?atg?tat?gaa?att?ctc?aaa?gca?ggc?ggt?ttc?acc?acc?ggt?ggt?ctg 1008

Val?Met?Tyr?Glu?Ile?Leu?Lys?Ala?Gly?Gly?Phe?Thr?Thr?Gly?Gly?Leu

325 330 335

aac?ttc?gat?gcc?aaa?gta?cgt?cgt?caa?agt?act?gat?aaa?tat?gat?ctg 1056

Asn?Phe?Asp?Ala?Lys?Val?Arg?Arg?Gln?Ser?Thr?Asp?Lys?Tyr?Asp?Leu

340 345 350

ttt?tac?ggt?cat?atc?ggc?gcg?atg?gat?acg?atg?gca?ctg?gcg?ctg?aaa 1104

Phe?Tyr?Gly?His?Ile?Gly?Ala?Met?Asp?Thr?Met?Ala?Leu?Ala?Leu?Lys

355 360 365

att?gca?gcg?cgc?atg?att?gaa?gat?ggc?gag?ctg?gat?aaa?cgc?atc?gcg 1152

Ile?Ala?Ala?Arg?Met?Ile?Glu?Asp?Gly?Glu?Leu?Asp?Lys?Arg?Ile?Ala

370 375 380

cag?cgt?tat?tcc?ggc?tgg?aat?agc?gaa?ttg?ggc?cag?caa?atc?ctg?aaa 1200

Gln?Arg?Tyr?Ser?Gly?Trp?Asn?Ser?Glu?Leu?Gly?Gln?Gln?Ile?Leu?Lys

385 390 395 400

ggc?caa?atg?rca?ctg?gca?gat?tta?gccaaa?tat?gct?cag?gaa?cat?cat 1248

Gly?Gln?Met?Ser?Leu?Ala?Asp?Leu?Ala?Lys?Tyr?Ala?Gln?Glu?His?His

405 410 415

ttg?tct?ccg?gtg?cat?cag?agt?ggt?cgc?cag?gaa?caa?ctg?gaa?aat?ctg 1296

Leu?Ser?Pro?Val?His?Gln?Ser?Gly?Arg?Gln?Glu?Gln?Leu?Glu?Asn?Leu

420 425 430

gta?aac?cat?tat?ctg?ttc?gac?aaa?taa 1323

Val?Asn?His?Tyr?Leu?Phe?Asp?Lys

435 440

<210>2

<211>440

<212>PRT

＜213〉intestinal bacteria

<400>2

Met?Gln?Ala?Tyr?Phe?Asp?Gln?Leu?Asp?Arg?Val?Arg?Tyr?Glu?Gly?Ser

1 5 10 15

Lys?Ser?Ser?Asn?Pro?Leu?Ala?Phe?Arg?His?Tyr?Asn?Pro?Asp?Glu?Leu

20 25 30

Val?Leu?Gly?Lys?Arg?Met?Glu?Glu?His?Leu?Arg?Phe?Ala?Ala?Cys?Tyr

35 40 45

Trp?His?Thr?Phe?Cys?Trp?Asn?Gly?Ala?Asp?Met?Phe?Gly?Val?Gly?Ala

50 55 60

Phe?Asn?Arg?Pro?Trp?Gln?Gln?Pro?Gly?Glu?Ala?Leu?Ala?Leu?Ala?Lys

65 70 75 80

Arg?Lys?Ala?Asp?Val?Ala?Phe?Glu?Phe?Phe?His?Lys?Leu?His?Val?Pro

85 90 95

Phe?Tyr?Cys?Phe?His?Asp?Val?Asp?Val?Ser?Pro?Glu?Gly?Ala?Ser?Leu

100 105 110

Lys?Glu?Tyr?Ile?Asn?Asn?Phe?Ala?Gln?Met?Val?Asp?Val?Leu?Ala?Gly

115 120 125

Lys?Gln?Glu?Glu?Ser?Gly?Val?Lys?Leu?Leu?Trp?Gly?Thr?Ala?Asn?Cys

130 135 140

Phe?Thr?Asn?Pro?Arg?Tyr?Gly?Ala?Gly?Ala?Ala?Thr?Asn?Pro?Asp?Pro

145 150 155 160

Glu?Val?Phe?Ser?Trp?Ala?Ala?Thr?Gln?Val?Val?Thr?Ala?Met?Glu?Ala

165 170 175

Thr?His?Lys?Leu?Gly?Gly?Glu?Asn?Tyr?Val?Leu?Trp?Gly?Gly?Arg?Glu

180 185 190

Gly?Tyr?Glu?Thr?Leu?Leu?Asn?Thr?Asp?Leu?Arg?Gln?Glu?Arg?Glu?Gln

195 200 205

Leu?Gly?Arg?Phe?Met?Gln?Met?Val?Val?Glu?His?Lys?His?Lys?Ile?Gly

210 215 220

Phe?Gln?Gly?Thr?Leu?Leu?Ile?Glu?Pro?Lys?Pro?Gln?Glu?Pro?Thr?Lys

225 230 235 240

His?Gln?Tyr?Asp?Tyr?Asp?Ala?Ala?Thr?Val?Tyr?Gly?Phe?Leu?Lys?Gln

245 250 255

Phe?Gly?Leu?Glu?Lys?Glu?Ile?Lys?Leu?Asn?Ile?Glu?Ala?Asn?His?Ala

260 265 270

Thr?Leu?Ala?Gly?His?Ser?Phe?His?His?Glu?Ile?Ala?Thr?Ala?Ile?Ala

275 280 285

Leu?Gly?Leu?Phe?Gly?Ser?Val?Asp?Ala?Asn?Arg?Gly?Asp?Ala?Gln?Leu

290 295 300

Gly?Trp?Asp?Thr?Asp?Gln?Phe?Pro?Asn?Ser?Val?Glu?Glu?Asn?Ala?Leu

305 310 315 320

Val?Met?Tyr?Glu?Ile?Leu?Lys?Ala?Gly?Gly?Phe?Thr?Thr?Gly?Gly?Leu

325 330 335

Asn?Phe?Asp?Ala?Lys?Val?Arg?Arg?Gln?Ser?Thr?Asp?Lys?Tyr?Asp?Leu

340 345 350

Phe?Tyr?Gly?His?Ile?Gly?Ala?Met?Asp?Thr?Met?Ala?Leu?Ala?Leu?Lys

355 360 365

Ile?Ala?Ala?Arg?Met?Ile?Glu?Asp?Gly?Glu?Leu?Asp?Lys?Arg?Ile?Ala

370 375 380

Gln?Arg?Tyr?Ser?Gly?Trp?Asn?Ser?Glu?Leu?Gly?Gln?Gln?Ile?Leu?Lys

385 390 395 400

Gly?Gln?Met?Ser?Leu?Ala?Asp?Leu?Ala?Lys?Tyr?Ala?Gln?Glu?His?His

405 410 415

Leu?Ser?Pro?Val?His?Gln?Ser?Gly?Arg?Gln?Glu?Gln?Leu?Glu?Asn?Leu

420 425 430

Val?Asn?His?Tyr?Leu?Phe?Asp?Lys

435 440

<210>3

<211>1455

<212>DNA

＜213〉intestinal bacteria

<220>

<221>CDS

<222>(1)..(1455)

<400>3

atg?tat?atc?ggg?ata?gat?ctt?ggc?acc?tcg?ggc?gta?aaa?gtt?att?ttg 48

Met?Tyr?Ile?Gly?Ile?Asp?Leu?Gly?Thr?Ser?Gly?Val?Lys?Val?Ile?Leu

1 5 10 15

ctc?aac?gag?cag?ggt?gag?gtg?gtt?gct?gcg?caa?acg?gaa?aag?ctg?acc 96

Leu?Asn?Glu?Gln?Gly?Glu?Val?Val?Ala?Ala?Gln?Thr?Glu?Lys?Leu?Thr

20 25 30

gtt?tcg?cgc?ccg?cat?cca?ctc?tgg?tcg?gaa?caa?gac?ccg?gaa?cag?tgg 144

Val?Ser?Arg?Pro?His?Pro?Leu?Trp?Ser?Glu?Gln?Asp?Pro?Glu?Gln?Trp

35 40 45

tgg?cag?gca?act?gat?cgc?gca?atg?aaa?gct?ctg?ggc?gat?cag?cat?tct 192

Trp?Gln?Ala?Thr?Asp?Arg?Ala?Met?Lys?Ala?Leu?Gly?Asp?Gln?His?Ser

50 55 60

ctg?cag?gac?gtt?aaa?gca?ttg?ggt?att?gcc?ggc?cag?atg?cac?gga?gca 240

Leu?Gln?Asp?Val?Lys?Ala?Leu?Gly?Ile?Ala?Gly?Gln?Met?His?Gly?Ala

65 70 75 80

acc?ttg?ctg?gat?gct?cag?caa?cgg?gtg?tta?cgc?cct?gcc?att?ttg?tgg 288

Thr?Leu?Leu?Asp?Ala?Gln?Gln?Arg?Val?Leu?Arg?Pro?Ala?Ile?Leu?Trp

85 90 95

aac?gac?ggg?cgc?tgt?gcg?caa?gag?tgc?act?ttg?ctg?gaa?gcg?cga?gtt 336

Asn?Asp?Gly?Arg?Cys?Ala?Gln?Glu?Cys?Thr?Leu?Leu?Glu?Ala?Arg?Val

100 105 110

ccg?caa?tcg?cgg?gtg?att?acc?ggc?aac?ctg?atg?atg?ccc?gga?ttt?act 384

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

115 120 125

gcg?cct?aaa?ttg?cta?tgg?gtt?cag?cgg?cat?gag?ccg?gag?ata?ttc?cgt 432

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

130 135 140

caa?atc?gac?aaa?gta?tta?tta?ccg?aaa?gat?tac?ttg?cgt?ctg?cgt?atg 480

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

145 150 155 160

acg?ggg?gag?ttt?gcc?agc?gat?atg?tct?gac?gca?gct?ggc?acc?atg?tgg 528

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

165 170 175

ctg?gat?gtc?gca?aag?cgt?gac?tgg?agt?gac?gtc?atg?ctg?cag?gct tgc 576

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

180 185 190

gac?tta?tct?cgt?gac?cag?atg?ccc?gca?tta?tac?gaa?ggc?agc?gaa?att 624

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

195 200 205

act?ggt?gct?ttg?tta?cct?gaa?gtt?gcg?aaa?gcg?tgg?ggt?atg?gcg?acg 672

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

210 215 220

gtg?cca?gtt?gtc?gca?ggc?ggt?ggc?gac?aat?gca?gct?ggt?gca?gtt?ggt 720

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

225 230 235 240

gtg?gga?atg?gtt?gat?gct?aat?cag?gca?atg?tta?tcg?ctg?ggg?acg?tcg 768

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

245 250 255

ggg?gtc?tat?ttt?gct?gtc?agc?gaa?ggg?ttc?tta?agc?aag?cca?gaa?agc 816

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

260 265 270

gcc?gta?cat?agc?ttt?tgc?cat?gcg?cta?ccg?caa?cgt?tgg?cat?tta?atg 864

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

275 280 285

tct?gtg?atg?ctg?agt?gca?gcg?tcg?tgt?ctg?gat?tgg?gcc?gcg?aaa?tta 912

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

290 295 300

acc?ggc?ctg?agc?aat?gtc?cca?gct?tta?atc?gct?gca?gct?caa?cag?gct 960

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

305 310 315 320

gat gaa?agt?gcc?gag?cca?gtt?tgg?ttt?ctg?cct tat?ctt?tcc?ggc?gag 1008

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

325 330 335

cgt?acg?cca?cac?aat?aat?ccc?cag?gcg?aag?ggg?gtt?ttc?ttt?ggt?ttg 1056

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

340 345 350

act?cat?caa?cat?ggc?ccc?aat?gaa?ctg?gcg?cga?gca?gtg?ctg?gaa?ggc 1104

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

355 360 365

gtg?ggt?tat?gcg?ctg?gca?gat?ggc?atg?gat?gtc?gtg?cat?gcc?tgc?ggt 1152

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

370 375 380

att?aaa?ccg?caa?agt?gtt?acg?ttg?att?ggg?ggc?ggg?gcg?cgt?agt?gag 1200

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

385 390 395 400

tac?tgg?cgt?cag?atg?ctg?gcg?gat?atc?agc?ggt?cag?cag?ctc?gat?tac 1248

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

405 410 415

cgt?acg?ggg?ggg?gat?gtg?ggg?cca?gca?ctg?ggc?gca?gca?agg?ctg?gcg 1296

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

420 425 430

cag?atc?gcg?gcg?aat?cca?gag?aaa?tcg?ctc?att?gaa?ttg?ttg?ccg?caa 1344

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

435 440 445

cta?ccg?tta?gaa?cag?tcg?cat?cta?cca?gat?gcg?cag?cgt?tat?gcc?gct 1392

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

450 455 460

tat?cag?cca?cga?cga?gaa?acg?ttc?cgt?cgc?ctc?tat?cag?caa?ctt?ctg 1440

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

465 470 475 480

cca?tta?atg?gcg?taa 1455

Pro?Leu?Met?Ala

<210>4

<211>484

<212>PRT

＜213〉intestinal bacteria

<400>4

Met?Tyr?Ile?Gly?Ile?Asp?Leu?Gly?Thr?Ser?Gly?Val?Lys?Val?Ile?Leu

1 5 10 15

Leu?Asn?Glu?Gln?Gly?Glu?Val?Val?Ala?Ala?Gln?Thr?Glu?Lys?Leu?Thr

20 25 30

Val?Ser?Arg?Pro?His?Pro?Leu?Trp?Ser?Glu?Gln?Asp?Pro?Glu?Gln?Trp

35 40 45

Trp?Gln?Ala?Thr?Asp?Arg?Ala?Met?Lys?Ala?Leu?Gly?Asp?Gln?His?Ser

50 55 60

Leu?Gln?Asp?Val?Lys?Ala?Leu?Gly?Ile?Ala?Gly?Gln?Met?His?Gly?Ala

65 70 75 80

Thr?Leu?Leu?Asp?Ala?Gln?Gln?Arg?Val?Leu?Arg?Pro?Ala?Ile?Leu?Trp

85 90 95

Asn?Asp?Gly?Arg?Cys?Ala?Gln?Glu?Cys?Thr?Leu?Leu?Glu?Ala?Arg?Val

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?IIe?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

Gly?Val?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?TyrAla?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〉intestinal bacteria

<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〉intestinal bacteria

<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?Ile

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?Ile?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〉intestinal bacteria

<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?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?aac?gaa?gtc?aaa?gcg?att 624

Ile?Ala?Cys?Ile?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?Glu?Lys?Gln?Ser

500 505 510

gtc?tga 1542

Val

<210>8

<211>513

<212>PRT

＜213〉intestinal bacteria

<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?Ile?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?Leu?Val?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?Ile?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〉intestinal bacteria

<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〉intestinal bacteria

<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

Ile?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?Ile?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〉intestinal bacteria

<220>

<221>CDS

<222>(1)..(1179)

<400>11

atg?ttt?act?aaa?cgt?cac?cgc?atc?aca?tta?ctg?ttc?aat?gcc?aat?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〉intestinal bacteria

<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?Ile?Phe?Ile?Glu?Glu?Asp?Phe?Arg?Ala?Arg

35 40 45

Ile?Asp?Lys?Ile?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?Lys?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〉explanation of artificial sequence: primer

<400>13

ggcaactatg?catatcttcg?cgc 23

<210>14

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉explanation of artificial sequence: primer

<400>14

gcgtgaatga?attggcttag?gtgg 24

<210>15

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉explanation of artificial sequence: primer

<400>15

cagacagcga?gcgaggatcg?c 21

<210>16

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉explanation of artificial sequence: primer

<400>16

tgtgcggtta?tccatcgctg?c 21

Claims (20)

1. the L-amino acid of enterobacteriaceae (Enterobacteriaceae) is produced bacterium, and this bacterium has enhanced xylose utilization enzymic activity by the expression amount that increases the xylABFGHR locus.

2. bacterium according to claim 1, wherein this bacterium belongs to Escherichia (Escherichia).

3. bacterium according to claim 2, wherein, this bacterium belongs to intestinal bacteria.

4. bacterium according to claim 1, wherein this bacterium belongs to general Pseudomonas (Pantoea).

5. bacterium according to claim 1, wherein the xylose utilization enzymic activity strengthens with the expression that strengthens the xylABFGHR locus by copy number or the modification expression regulation sequence that increases the xylABFGHR locus.

6. bacterium according to claim 5, wherein copy number is by increasing with the low copy carrier transform bacteria that contains the xylABFGHR locus.

7. bacterium according to claim 1, wherein the xylABFGHR locus is from belonging to colibacillary bacterium.

8. produce the amino acid whose method of L-, this method is included in cultivates each described bacterium in the claim 1 to 7 in the substratum that contains glucose and pentose mixture, and collects L-amino acid from substratum.

9. method according to claim 8, wherein pentose is pectinose and wood sugar.

10. method according to claim 8, wherein Tang mixture is the mixture of raw material of the sugar that obtains from cellulose biomass.

11. method according to claim 8, wherein the L-amino acid of Sheng Chaning is the L-Histidine.

12. method according to claim 11, wherein this bacterium strengthens expression L-Histidine biosynthesis related genes.

13. method according to claim 8, wherein the L-amino acid of Sheng Chaning is the L-Threonine.

14. method according to claim 13, wherein this bacterium strengthens expression L-Threonine biosynthesis related genes.

15. method according to claim 8, wherein the L-amino acid of Sheng Chaning is L-Methionin.

16. method according to claim 15, wherein this bacterium has S-(2-amino-ethyl)-L-halfcystine resistance.

17. method according to claim 8, wherein the L-amino acid of Sheng Chaning is L-L-glutamic acid.

18. method according to claim 17, wherein this bacterium lacks a-ketoglutaric acid salt dehydrogenase activity or has the a-ketoglutaric acid salt dehydrogenase activity that weakens.

19. method according to claim 8, wherein the L-amino acid of Sheng Chaning is the L-tryptophane.

20. method according to claim 19, wherein this bacterium strengthens expression L-tryptophane biosynthesis related genes.