WO2007037460A1 - An l-amino acid-producing bacterium and a method for producing l-amino acids - Google Patents

Abstract

A method for producing an L-amino acid is provided which includes culturing in a medium a microorganism of the Enterobacteriaceae family which has an ability to produce an L-amino acid and which has been modified so as to enhance the ß-glucoside PTS activity, and collecting the L-amino acid from the medium or cells.

Description

DESCRIPTION

AN L AMINO ACID -PRODUCING BACTERIUM AND A ME

PRODUCINGL-AMINO ACIDS

Technical Field

The present invention relates to a method for producing an L-a microorganism and more specifically to^{^}a method for producing an L as L-Iy sine L-threonine and L glutamic acid etc L-Iy sine and L-thre used as animal feed additives health food ingredients amino acid infus glutamic acid is typically used as a seasoning Therefore these are ind amino acids

Background Art

L-amino acids are industrially produced employing fermentatio microorganisms of the genera Brevibactenum Corynebacterium and (EP0857784 0999267 1170358 JP1 1-192088A WO00/53726 WO96 WO03/04674) Wild type microorganisms artificial mutants of said b and microorganisms which have been modified in such a way that the a amino acid biosynthesis enzymes is enhanced by recombinant DNA te typically used for L-amino acid production

Known methods for enhancing the ability of the various strains amino acid include modifying the L-amino acid uptake or export For One of the methods for modifying the export of an L-amino aci reduce the export of an L-amino acid biosynthetic intermediate and an strengthen the L-amino acid export For the former if the target amino glutamic acid a known method is to reduce the export of α-ketoglutara intermediate in the biosynthesis of L glutamic acid by mutating or disr ketoglutarate permease gene (WOOl /005959)

To delete or reduce the export of an L-amino acid biosynthetic i methods for overexpressing the L amino acid export have been reporte producing L-lysine (WO97/23597) or L arginine using a bacterial strai microorganism of the genus Corynebacterium in which the expression L-arginine export gene (LysE) (Journal of Molecular Microbiology Bio Microbiol Biotechnol) 1999 Nov 1 (2) 327-36) is enhanced Furthermor producing an L-amino acid using a bacteria of the genus Escherichia in expression of the rhtA B and C genes (US6 303 348) or the yfiK yah been reported (EP 1013765)

Aside from modifying the L-amino acid biosynthesis pathway a uptake and export of the L-amino acid as described above another exa for improving the ability to produce an L-amino acid is to modify the a sugar For example the phosphoenolpyruvate carbohydrate phosphotr (hereinafter also referred to as PTS phosphotransferase) is widely kno transporter to uptake sugar Furthermore PTS is classified as a substra common system EI (encoded by ptsl) HPr (encoded by ptsH) or subst component EH Glucose-specific Eu is encoded by ptsG and err with a part of an operon with ptsH and ptsl One known method for produci No 2 p462-468 Biochemistry 1998 VoI 37 pi 7040- 17047 Bioche 37 p8714-8723) but there have been no reports that a gene encoding glucose PTS have been used for the production of an L-amino acid

SUMMARY OF THE INVENTION

An object of the present invention is to provide a bacterial strai of efficiently producing an L amino acid and to also provide a method L amino acid using said bacterial strain

In order to resolve the above mentioned problem it has been discovere acid can be effectively produced using a microorganism belonging to t Enterobacteriaceae which has been modified to increase β-glucoside P

That is the present invention is as follows

It is an object of the present invention to provide a method for p amino acid comprising culturing in a medium a microorganism of the family which has an ability to produce an L-amino acid and which has as to enhance the β-glucoside PTS activity and collecting the L-amin medium or cells

It is an object of the present invention to provide the method des wherein the microorganism has been modified to enhance the expressio encoding the β-glucoside PTS by increasing the number of copies of th modifying the expression regulatory sequence of said gene or combina It is an object of the present invention to provide the method des wherein said bglF gene is selected from the group consisting of

(a) a DNA which comprises a nucleotide sequence of SEQ ID No 5 It is an object of the present invention to provide a method descri wherein said L-amino acid is selected from a group consisting of L-lysi L-glutamic acid and combinations thereof

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows construction of the plasmid pMWl 18-attL Tc-a Figure 2 shows construction of the plasmid pMWl 18-attL Cm- Figure 3 shows construction of the plasmid pMW intxis-ts

DETAILED DESCRIPTION OF THE PREFERRED EMBO

Hereinafter the present invention will be explained in detail <1> The microorganism of the present invention

The microorganism of the present invention is a microorganism Enterobacteriaceae family which has an ability to produce an L-amino has been modified so as to enhance the β glucoside PTS activity The to produce an L-amino acid means the ability to produce and cause ac L-amino acid in a medium or in the cells of the microorganism when th of the present invention is cultured in a medium The microorganism o invention may have the ability to produce multiple L-amino acids The may inherently possesses the ability to produce an L-amino acid or ma mutagenesis or recombinant DNA techniques to impart the ability to pr acid such as those described below L-tryptophan sulfur-containing L-amino acids such as L-cysteine L cy methionine and acidic L-amino acid such as L glutamic acid L asparti acidic L-amino acid such as L-glutamine L-asparagine etc The micr present invention may have the ability to produce two or more amino a <1-1> Imparting L-amino acid producing ability

The following examples includes a description of the method fo amino acid-producing ability along with examples of microorganisms amino acid-producing ability which can be used in the present inventio microorganisms of the present invention are not limited to these but ca as they have an L-amino acid-producing ability

There is no particular limitation on the microorganism used in t invention as long as it belongs to the family Enterobacteriaceae such Escherichia Enterobacter Pantoea Klebsiella Serratia Erwinia Sal Morganella etc and it has an L-ammo acid-producing ability Specifi microorganism belonging to the family Enterobacteriaceae as classifie (National Center for Biotechnology Information) database may be used (http //www genome ad jp/dbget-bin/get_htext?0rganisms+-e+L+A+ s f+F+A2_A#L2)

As a parent strain of the family Enterobacteriaceae which can b particularly desirable to use bacteria which belong to the genera Escher Enterobacter or Pantoea

The parent strain of the bacteria of the genus Escherichia used t bacteria of the present invention is not particularly limited but strains li et al may be used (Neidhardt F C et al Escherichia coli and Salmon These are available for example from the American Type Cult (address P O Box 1549 Manassas VA 20108 USA) They are availab accession number given to each bacterial strain (see http /www atcc or numbers correspond to each bacterial strain and are listed in the Ameri Collection s catalogue

Examples of bacteria of the genus Enterobacter include Entero agglomerans and Enterobacter aerogenes An example of a bacterium Pantoea is Pantoea ananatis In recent years based on 16S rRNA nucl analysis Enterobacter agglomerans has on occasion been reclassifϊed agglomerans Pantoea ananatis and Pantoea stewartu For the presen bacterium classified in the family Enterobacteriaceae whether under t Enterobacter or Pantoea may be employed The strains Pantoea anan (FERM BP-6614) AJ13356 (FERM BP-6615) AJ13601 (FERM BP-7 derivative thereof may be employed to breed Pantoea ananatis by gene methods When isolated these strains were identified and deposited as agglomerans As stated above by analysis using 16S rRNA nucleotide bacteria have been reclassified as Pantoea ananatis For the present in bacterium belonging to the genus Enterobacter or Pantoea may be use bacterium is classified in the family Enterobacteriaceae

The following is a description of methods for imparting an L-a producing ability to a microorganism which belongs to the Enterobacte

To impart the ability to produce an L amino acid an auxotrophi analog resistant strain or a metabolic regulation mutant can be obtaine recombinant strain having enhanced expression of an L-amino acid bio mutation may be imparted Enhancing the expression of one or more L biosynthesis enzymes may also be employed Furthermore imparting auxotrophic mutation analog resistance or metabolic regulation mutati performed in combination with enhancing the activity of biosynthesis e

An auxotrophic mutant strain L amino acid analog-resistant str regulation mutant strain with the ability to produce an L-amino acid ca subjecting a parent or wild-type strain to a conventional mutation treat treating with X-rays or UV radiation or treating with a mutagenic agen methyl-N -nitro-N-nitrosoguanidine etc ^"then selecting those which ex autotrophic mutation analog resistance or metabolic regulation mutati have the ability to produce an L-amino acid

Examples of an L-lysine analog-resistant strain or metabolic reg include but are not limited to the Escherichia coll AJl 1442 strain (FE NRRL B-12185 JP56-18596A and U S Patent No 4 346 170) Esche strain (EP1016710A) etc The Escherichia coll WC196 strain (WO96 be used as a bacterium which produces L-lysine The WCl 96 strain w imparting AEC (S (2-aminoethyl) cysteine) resistance to the W3110 str Escherichia coli K-12 This strain was named Escherichia coll AJ1306 deposited on December 6 1994 with the National Institute of Bioscienc Technology of the Agency of Industrial Science and Technology (curre Patent Organism Depositary National Institute of Advanced Industrial Technology Chuo 6 1-1 Higashi 1 -chome Tsukuba-shi Ibaraki-ken 3 under Accession No FERM P- 14690 and converted to an international Budapest Treaty on September 29 1995 and given Accession No FER (WO96/40934) the phosphoenolpyruvate carboxylase gene (ppc) (JP6 aspartate aminotransferase gene (aspC) (JP6-102028A) the diammopi gene (dapF) (WO00/56858) the aspartate-semialdehyde dehydrogenas (WO00/61723) and other genes of diammopimelate pathway enzymes homoaconitate hydratase gene (JP2000 157276) and other genes of ami pathway enzymes The abbreviations for these genes are given in the p following each name

Furthermore it is known that wild -type dihydrodipicolinate sy aspartokmase (AK) are suppressed by feedback inhibition by L-lysine dapA and lysC are used it is preferable to use mutant genes encoding

synthase and aspartokmase respectively that are resistant to the feedba L lysine (EP 0733710 US5 932 453)

Examples of the DNA encoding mutant dihydrodipicolinate syn resistant to feedback inhibition by L lysine include a DNA encoding D amino acid sequence wherein the 1 18th histidine residue is substitued 5 661 012 and 6 040 160) Furthermore examples of the DNA encodin that is resistant to feedback inhibition by L-lysine include a DNA enco the amino acid sequence wherein the 352 threonine residue is substitut (U S 5 661 012 and 6 040 160) These mutant DNAs can be obtained mutagenesis using PCR or the hlςe

The following is an example of imparting an L-lysine producin introducing a gene encoding an L-lysine biosynthesis enzyme into the h recombinant DNA is prepared by hgating the gene fragment that encod biosynthesis gene with a vector that functions in the host microorganis The genes encoding the L-lysme biosynthesis enzyme are not p specified as long as they can be expressed in the host microorganism genes derived from Escherichia coli and genes derived from corynefor Because the total genome sequences of Escherichia coli and Coryneba g/utamicum have been determined it is possible to synthesize primers nucleotide sequence of these genes and obtain these genes using the PC which the chromosomal DNA of a microorganism such as Escherichia used as the template

In order to clone these gene_> plasmids that autonomously replic Enterobacteriaceae can be used Examples include pBR322 pTWV22 Inc ) pMWl 19 (Nippon Gene Co Ltd ) pUC19 pSTV29 (Takara Bio (Gene vol 75 (2) pp 271 -288 1989) etc In addition a vector of pha be used

To hgate the target gene to the above-mentioned vector the vec with a restriction enzyme matched to the end of the DNA fragment con gene The ligation is usually conducted with a hgase such as T4 DNA genes may be present on separate vectors respectively or present on th Typical methods known to those skilled in the art can be employed for hgating the DNA as well as for preparing chromosomal DNA perform preparing plasmid DNA transformation determining the oligonucleoti primers etc These methods are described in Sambrook J and Russe Cloning A Laboratory Manual/Third Edition New York Cold Spring Press (2001) etc Any method which achieves adequate transformatio be employed to introduce recombinant DNA that has been prepared as DNA of a microorganism Multiple copies of the target gene can be int chromosomal DNA of the microorganism by using a sequence in whic are present on the chromosomal DNA as a target in homologous recom site-specific introduction of mutations based on gene substitution using recombination has been described Methods employing linear DNA or containing a temperature-sensitive replication origin have been describ 6 303 383 and 5 616 480) Repetitive DNA and inverted repeats presen transposable elements can be employed as sequences in which multiple on chromosomal DNA An L-lysine biosynthesis gene may be hgated i gene which is inherently present on the chromosome or it may be intro essential region on the chromosome or a region of the gene in which th will be improved if deleted

Furthermore as disclosed in US5 595 889 the target gene may a transposon which is then transferred to introduce multiple copies into DNA With either method the number of copies of the target gene in th increases so that the enzymatic activity of the L-lysine biosynthesis in

In addition to the above-described genetic amplification an incr lysine biosynthesis enzyme activity can be achieved by replacing an ex regulatory sequence of the target gene such as a promoter etc with a s JPl 215280A) For example the lac promoter trp promoter trc promo lambda phage PR promoter PL promoter and tet promoter are all kno promoters Substitution with these promoters increases expression of t thus enhancing enzymatic activity Examples of strong promoters and evaluating the strength of promoters are described in an article by Gold bases into the -35 -10 region of the promoter of a target gene to modi Furthermore substituting several nucleotides into the spacer region bet binding site (RBS) and the start codon particularly into the sequence i upstream of the start codon is known to have a strong effect on the mR efficiency The expression regulatory regions of the target gene s pro determined by promoter probe vectors and gene analysis software such etc Substitution of expression regulatory sequences can be conducted the same manner as in the above-described gene substitution employin sensitive plasmids The Red driven integration method(WO2005/0101

Furthermore in the L lysine producing bacteria of the present i activity of an enzyme catalyzing production of a compound other than branches off from its biosynthesis pathway or the activity of an enzym negative effect on the production of L-Iy sine may be reduced or delete include homoseπne dehydrogenase (thrA) lysine decarboxylase (cadA enzyme (sfcA b2463) The strains with the reduced or deficient enzym given in WO 95/23864 WO96/17930 WO2005/010175 etc

To reduce or delete said enzyme activity in a cell mutagenesis on the gene which encodes the above-mentioned enzymes using typica methods This can be achieved for example by deleting the gene that enzyme on the chromosome using genetic recombination or by modify regulatory sequence of a promoter or a Shine— Dalgarno (SD) sequence also be achieved by introducing an amino acid substitution (missense codon (nonsense mutation) in the region encoding the enzyme on the c introducing a frameshift mutation to add or delete 1 2 bases or by dele chromosome with this by homologous recombination etc or introduci or IS element into said gene

The following methods may be used to introduce a mutation wh deletes the above-mentioned enzyme activity by genetic recombination DNA containing the target gene is mutated so that the resulting mutant produce an enzyme that functions normally Then transforming this int microorganism which belongs to the family Enterobacteriaceae using t containing said gene and generating the recombination of the mutant-t gene on the chromosome For gene substitution using this kind of hom recombination there are methods which employ linear DNA such as th Red driven integration (Proc Natl Acad Sci USA 2000 vol 97 6645) or a method combining the Red-driven integration method and t excisive system (J Bacteπol 2002 S ep 184 (18) 5200-3 Interactions and excisionase in the phage lambda excisive nucleoprotein complex RI Gardner JF) (see WO2005/010175) etc and there are methods whi plasmid containing a temperature-sensitive replication origin (Proc Na 2000 vol 97 No 12 pp 6640-6645 U S Patent No 6 303 383 or 5 6 site specific introduction of mutations via gene substitution using hom recombination as described above may also be performed using a plas have replication ability on the host

The above-mentioned method for increasing the enzyme activit lysine biosynthesis and the method for lowering the enzyme activity m used in breeding other L-amino acid producing bacteria The following methods for breeding other L-amino acid bacteria synthase (glnA) isocitrate dehydrogenase (icd) aconitate hydratase (ae synthase (gltA) phosphoenolpyruvate carboxylase (ppc) pyruvate carb pyruvate dehydrogenase (pdhA) pyruvate kinase (pykA) phosphoenol (pps) enolase (eno) phosphoglucomutase (pgm) phosphoglycerate kin glyceraldehyde 3 -phosphate dehydrogenase (gpd) tπose phosphate iso fructose-bisphosphate aldolase (fba) phosphofructokinase (pfk) glucos isomerase (gpi) etc Of these enzymes citrate synthase phosphoenolp carboxylase glutamate dehydrogenase and combinations thereof are pr use of all three is more preferable

Examples of microorganisms belonging to the family Enteroba have been modified to enhance the expression of the citrate synthase ge phosphoenolpyruvate carboxylase gene and/or glutamate dehydrogena methods described above are given in U S Patent Nos 6 197 559 & 6 3 EP0999282 WO2006/051660

Furthermore microorganisms belonging to the family Enteroba which have been modified to increase the activity of either 6-phosphogl dehydratase or the activity of 2-keto-3 deoxy-6-phosphogluconate aldol the activity of both may also be used (EP1352966B)

The microorganisms of the family Enterobacteriaceae having t produce an L glutamic acid which may be used include a bacterium in of an enzyme that catalyzes production of a compound other than L-glu which branches off from the biosynthesis pathway of L glutamic acid or lowered Examples of enzymes that catalyze production of a compo glutamic acid which branch off from the biosynthesis pathway of L gl Methods for deleting or reducing the activity of 2 oxoglutarate a microorganism belonging to the family Enterobacteriaceae are descri 5 573 945 U S Patent No 6 197 559 and U S Patent No 6 331 419 microorganisms belonging to the family Enterobacteriaceae wherein t oxoglutarate dehydrogenase has been deleted or reduced include the fol

Pantoea ananatis AJl 3601 (FERM BP-7207)

Klebsiella planticola AJ13410 strain (FERM BP-6617)

Escherichia coll AJ12949 (FERM BP 4881) and others

The AJl 2949 strain is a bacterial strain in which the α-ketogluta dehydrogenase activity has been reduced and was deposited on Decem the National Institute of Bioscience and Human Technology of the Age Science and Technology (currently International Patent Organism Dep Institute of Advanced Industrial Science and Technology Chuo 6 1-1 Tsukuba-shi Ibaraki-ken 305-8566 Japan) under Accession No FER converted to an international deposit under the Budapest Treaty on Nov and given Accession No FERM BP 4881

The L-tryptophan producing bacteria preferably used in the pre bacteria in which the activity of one or more of the following enzymes synthase (trpE) phosphoglycerate, dehydrogenase (serA) or tryptophan has been enhanced Since anthranilate synthase and phosphoglycerate both are subject to feedback inhibition by L-tryptophan and L serine th these enzymes can be increased by retaining the desensitizing mutant e (US5 618 716 US 6 180 373) For instance it is possible to obtain ba with plasmid pGH5 having a mutated serA that encodes desensitized p dehydrogenase (WO94/08301)

Bacteria transformed with recombinant DNA containing a trypt also preferable L-tryptophan producing bacteria A specific example is transformed with a tryptophan operon containing a gene encoding dese anthranilate synthase (trpAB) (Japanese Patent Application Publication Japanese Patent Application Publication No JP 62-244382 U S Patent Furthermore in the tryptophan operon it is possible to enhance the abi tryptophan by increasing the expression of the gene (trpBA) encoding t synthase Tryptophan synthase contains α and β subunits that are enco trpB respectively (WO2005/103275)

Examples of L-tryptophan-producing bacteria are Escherichia c (pGX44) [NRRL B-12263] which requires L-phenylalanine and L-tyro and AGX6 (pGX50) aroP [NRRL B- 12264] which retains plasmid pG tryptophan operon (see U S Patent No 4 371 614)

A strain wherein the tryptophan operon repressor (trpR) is defi with a mutant trpT are also desirable L-tryptophan-producing bacteria 4 371 614 WO2005/056776)

Another preferable L tryptophan-producing bacterium is the ba malate synthase (aceB) isocitrate lyase (aceA) and the isocitrate dehydrogenase/phosphatase (icl) operon (ace operon) are structurally e expression of said operon has been enhanced (WO2005/103275)

L-tryptophan L-phenylalanine and L-tyrosine are all aromatic share a biosynthesis system Examples of genes encoding biosynthesis producing ability can be improved It is known that these genes are co tyrosine repressor (tyrR) so the biosynthesis enzyme activity of an aro may also be increased by deleting the tyrR gene (EP763127)

The L- threonine-producing bacteria are preferably microorgani the family Enterobacteriaceae wherein the L threonine biosynthesis en enhanced Examples of genes encoding L-threonine biosynthesis enzy aspartokinase III gene (lysC) the aspartate-semialdehyde dehydrogena aspartokinase I gene encoding the thr operon (thrA) the homoseπne ki and the threonine synthase gene (thrC) The abbreviations for these ge parentheses following their names More than two types of these genes introduced The L threonine biosynthesis gene may be introduced into genus Escherichia wherein threonine degradation has been suppressed bacteria of the genus Escherichia wherein threonine degradation has be include the TDH6 strain wherein the threonine dehydrogenase activity (Japanese Patent Application Publication No 2001-346578) and so for

Activities of some of L-threonine biosynthesis enzymes are sup threomne that is produced Therefore in order to construct an L-threon bacterium it is preferable to modify the L-threonine biosynthesis enzy enzyme is not subject to feedback inhibition by L-threonine The abov thrB and thrC genes make up the threonine operon which is in the for structure The expression of the threonine operon is subject to inhibitio and threonine present in the culture and the expression is attenuated T of the theonine operon can be achieved by removing the leader sequenc attenuation region or the attenuator (WO 02/26993 Biotechnology Let feedback inhibition by L threonine modification of the bacteria of the can also be obtained by selecting an α-amino-β-hydroxyvaleπc acid (A bacteria strain (JP45026708B) _χ

It is preferred that the threonine operon which is modified to pr inhibition by L-threonine has an increased copy number in the host or i strong promoter In addition to amplifying the copy number of the gene the copy number of the gene can be increased by introducing the threon the chromosome using a transposon Mu phage etc

For the aspartokinase III gene (lysC) it is desirable to use a gen prevent this feedback inhibition by L-lysine A lysC gene which has b prevent feedback inhibition can be obtained using the method describe Patent No 5 932 453

Aside from the L-threonine biosynthesis enzyme it is desirable genes involved in the glycolytic system TCA cycle and respiratory ch controls gene expression and a gene which induces uptake of sugar E genes which are effective in L-threonine production include the transhy (pntAB) (EP733712) phosphoenolpyruvate carboxylase gene (ppc) (W phosphoenolpyruvate synthase gene (pps) (EP 877090) the pyruvate ca the coryneform bacteria or Bacillus bacteria (WO99/18228 EP 109277

It is also preferable to enhance the expression of a gene that imp L-threonine and a gene that imparts resistance to L-homoseπne or to i resistance and L homoseπne resistance to the host Examples of such gene (Res Microbiol 2003 Mar 154 (2) 123 35) the rhtB gene (EP 09 gene (EP1013765) and the yfiK yeaS genes (EP1016710To impart L-t National Collection of Industrial Microorganisms (VKPM) GNII Gene the VKPM B-3996 strain retains plasmid pVIC40 (WO90/04636) obtai threonine biosynthesis gene (threonine operon thrABC) into a wide-ho pAY32 including a streptomycin -resistant marker (Chistorerdov A Y Plasmid 1986 16 161-167) In this pVTC40 the feedback inhibition b of the aspartokinase I-homeseπne dehydrogenase I that the thrA in the encodes has been desensitized

A further example is the Escherichia coh B 5318 strain (see Eu 0593792) The B 5318 strain was deposited under Accession No VKP Russian National Collection of Industrial Microorganisms (VKPM) G Genetika(Russia 117545 Moscow 1 Dorozhny Proezd 1) on May 3 1 B 5318 strain is an isoleucine non auxotrophic strain and retains reco DNA constructed in such a way that the gene involved in threonine bio threonine operon wherein the attenuator region and the native transcrip region has been deleted is located downstream of the lambda phage te sensitive CI repressor PR promoter and the N-terminus of Cro protein and expression of the gene involved in the threonine biosynthesis is co lambda phage repressor and promoter

Examples of the preferred L histidine used in the present invent Escherichia coh FERM P-5038 and 5048 strains harboring vectors in information involved in L-histidine biosynthesis have been incorporate a bacterial strain into which the amino acid export gene Rht has been in (EPl 016710) the Escherichia coh 80 strain which has resistance to sul 1 2 4 -tπazole-3 -alanine and streptomycin (VKPM B-7270 Russian Pa (hisIE) phosphoπbosylformimino-5-aminoimidazole carboxamide πbo (hisA) amidotransferase (hisH) histidinol phosphate aminotransferase histidinol phosphatase gene (hisB) and histidinol dehydrogenase gene

The preferred L-cysteine producing bacteria of the present inve in which the activity of the cystathionine β-lyase has been reduced (JP2 bacteria of the genus Escherichia that retain serine acetyltransferase wi feedback inhibition by L-cysteine (JPl 1-155571)

The preferred L-proline producing bacteria of the present inven Escherichia coli 702 (VKPMB-80U) which is resistant to 3 4 dehydro azetidine-2-carboxylate and 702 llvA (VKPMB-8012 strain) which is and is derived from 702 (JP 2002-300874A)

Examples of L-phenylalamne-producing bacteria include AJl 2 tyrR) (VKPM B 8197) with tyrA tyrR deficient and strains with ampli encoding phenylalanine export proteins such as yddG and yedA

Examples of L-arginine producing bacteria include Escherichi strains which are resistant to α-methylmethionine p-fluorophenylalani arginine hydroxamic acid S (2-aminoethyl) cysteine α methyleseπne thienylalanine or sulfaguanidine (JP56- 106598) etc The Escherichia an L-arginine-producing bacterium that has a mutant which is resistant inhibition by L-arginine and that retains highly active N-acetyl glutama is also a preferable L arginine-producing strain (EPl 170361B) Said s VKPM B-7925 was deposited with the Russian National Collection of Microorganisms (VKPM) GNU Genetika on April 10 2000 and conv international deposit under the Budapest Treaty on May 18 2001 The Also as the microorganisms having L-arginine producing abilit with an improved expression of the gene encoding the enzyme involve biosynthesis may be used Examples of L-arginine biosynthesis enzym acetyl glutamate synthase (argA) N acetyl glutamyl-phosphate reduct ornithine acetyltransferase (argJ) N acetyl glutamate kinase (argB) ac transaminase (argD) acetyl ornithine deacetylase (argE) ornithine carb (argF) argininosuccinate synthase (argG) argininosuccinate lyase (arg phosphate synthase (carAB) and combinations thereof After each enz name of the gene encoding it is given in parentheses It is desirable to of the N-acetyl glutamate synthase gene (argA) in which L-arginine fee has been removed by substitution of the amino acid sequence correspo 15 to 19 in the wild-type (EP EP1170361)

The L-leucine-producing bacteria which may be used include a genus Escherichia coh in which the branched-chain amino acid transa the llvE gene has been inactivated and the activity of the aromatic amin transaminase encoded by the tyrB gene has been enhanced (EP1375655 Escherichia coh H 9068 strain (ATCC21530) which is resistant to 4-az 5 -tπfluoro leucine the Escherichia coh H-9070 strain (FERM BP-4704 coh H-9072 strain (FERM BP 4706) (U S Patent No 5 744 331) the strain in which the isopropylmalate synthase feedback inhibition by L-l desensitized (European Patent No 1067191) the Escherichia coh AJl 1 is resistant to β-2 thienylalanine and β hydroxyleucine (U S Patent No so on

L-isoleucine-producing bacteria include a 6 dimethyl aminopuπ Escherichia are bacterial strains in which the expression of the genes e isoleucine biosynthesis enzymes threonine deaminase or acetohydroxy have been increased (JP2-458A JP2-42988A JP 8-47397A) etc

An example of a parent strains for deriving L valine producing present invention include but are not limited to strains which have bee overexpress the llvGMEDA operon (U S Patent No 5 998 178) It is remove the region in the llvGMEDA operon which is required for atten expression of the operon is not attenuated by L-valine Furthermore th operon is desirably disrupted so that threonine deaminase activity is de

Examples of parent strains for deriving L-vahne producing bact invention include mutants having a mutation in the amino-acyl t-RNA Patent No 5 658 766) For example E coll VL1970 which has a mut gene encoding isoleucine tRNA synthetase can be used E coli VLl 9 deposited in the Russian National Collection of Industrial Microorganis (Russia 113545 Moscow 1 Dorozhny Proezd 1) on June 24 1988 und number VKPM B-4411

Furthermore mutants requiring lipoic acid for growth and/or la can also be used as parent strains (WO96/06926)

Aside from a gene which encodes a native biosynthesis enzyme involved in sugar uptake sugar metabolism (glycolytic system) and en may be enhanced in the L-amino acid-producing bacteria used in the pr

Examples of the genes involved in sugar metabolism are genes glycolytic enzymes or proteins which uptake sugar such as genes enco 6-phosphate isomerase gene (pgi WOO 1/02542) the phosphoenolpyruv systemsgene (esc EP149911 ) and the sucrose-assimilating genes (scr WO90/04636)

Examples of the genes involved in energy metabolism include t transhydrogenase gene (pntAB U S Patent No 5 830 716) and the cyt oxidase gene (cyoABCD EP1070376) <l-2> Method for increasing the activity of β glucoside PTS

The microorganism of the present invention can be obtained by microorganism which has the ability to produce an L-amino acid and w the Enterobactenaceae family as described above so as to increase th activity of the β-glucoside PTS However the ability to produce an L- imparted after modification to increase the enzymatic activity of the β- An increase in the enzymatic activity of the β-glucoside PTS can be ac modifying the expression of the bglF gene which encodes the β-glucosi later) This may be an increase in the expression of the endogenous bgl modification of the expression regulatory region including promoter m may be an increase in the expression of the exogenous bglF gene throu plasmid containing the bglF gene through an increase in the number of amplifying the bglF gene on the chromosome etc Furthermore a co techniques may be employed

The β-glucoside PTS in the present invention refers to a permea results in uptake of the sugar into the cytoplasm at the same time that t group in phosphoenolpyruvate (hereinafter referred to as PEP) is trans glucoside Here the β-glucoside is a glucoside with β-D-glucose as th for instance salicin which has been glucoside-linked with salicyl alcoh glucose at the same time (E coli & Salmonella 2nd Edition American ^ Microbiology)

Increase in the enzymatic activity of the β glucoside PTS can b vitro measurement of the phosphorylating activity using Chen et al s (Biochemistry 1998 37 8714-8723) (EC 2 7 1 69) Enhancement of th bglF when comparing it to the parent strain for example a wild strain strain can also be confirmed by comparing the amount of mRNA of bg wild-type or non-modified strain Northern hybridization and RT-PCR to confirm expression (Molecular Cloning (Cold Spring Harbor Labor Spring Harbor (USA) 2001)) The degree of increase in enzymatic act as long as the activity is increased as compared to that in the wild-type strain but it is desirable for example for it to be 1 5 or more times pre times or more preferably 3 or more times that of the wild or non-modi increase in the enzymatic activity can be confirmed if the target protein increased relative to that of the non-modified or wild-type strain This c for instance by Western blot using an antibody (Molecular Cloning (C Laboratory Press Cold spring Harbor (US A) 2001))

The bglF gene of the present invention is a bglF gene of the bac Escherichia and their homologs For example the bglF gene of Escher encodes a protein with the amino acid sequence of SEQ ID No 6 The with Genbank NP_418178 and W31 10 s sequence is registered with G PTV3B_ECOLI [P08722] both identical to SEQ ID NO 5 The bglF ge CoIi is shown in SEQ ID No 5 and the amino acid sequence is shown i

The homologs of the bglF gene include those which are derived luminescens subsp (NP_927931) Furthermore based on the homolog given in the above examples the bglF gene may be cloned from the cor bacteria such as Corynebacterium glutamicum Brevibactenum lactofe bacteria of the genus Pseudomonas such as Pseudomonas aeruginosa of the genus Mycobacterium such as Mycobacterium tuberculosis etc example the bglF gene may be cloned using synthetic oligonucleotide and 2

The genes encoding the β-glucoside PTS used in the present inv limited to the wild-type genes and as long as the function of the encod PTS protein i e the β glucoside PTS activity is not impaired They c mutant or an artificially modified product encoding a protein which inc containing several amino acid substitutions deletions insertions additi one or multiple positions in the amino acid sequence of SEQ ID No 6 several varies with the type and position of the amino acid residues i stereostructure of the protein Specifically it means 1 to 20 preferably preferably 1 to 5 The above substitutions deletions insertions or addi several amino acids are conservative mutations that preserve the β gluc A conservative mutation is wherein substitution takes place mutually a Tyr if the substitution site is an aromatic amino acid among Leu He substitution site is a hydrophobic amino acid between GIn Asn if it is acid among Lys Arg His if it is a basic amino acid between Asp GI amino acid and between S er Thr if it is an amino acid having a hydro Typical conservative mutations are conservative substitutions Preferre substitutions include substitution of Ala by Ser or Thr the substitution Phe the substitution of Lys by Asn GIu GIn His or Arg the substituti Leu VaI or Phe the substitution of Phe by Tip Tyr Met He or Leu t Ser by Thr or Ala the substitution ofThr by Ser or Ala the substitution Tyr the substitution of Tyr by His Phe or Trp and the substitution of orLeu Substitutions deletions insertions additions or inversions and amino acids described above include ones that have naturally occurred variant) depending on the differences between species or individual di microorganisms that retain bglF genes Such genes can be obtained by for instance the site-specific mutation method the nucleotide sequence No 5 so that the site-specific amino acid residue in the protein encode substitutions deletions insertions or additions

Moreover as the bglF genes homologs can have 80% or above above more preferably 95% or above even more preferably 97% or ab with the amino acid sequence of SEQ No 6 Since the degenerate code gene vary with the host into which the gene is introduced a gene substi that are more readily utilized by the host is desirable Likewise as long carries the function of the β-glucoside PTS the N terminal or C termin be extended or removed For example the length of the extension or re or less preferably 20 or less more preferably 10 or less and even mor less amino acid residues More specifically a gene which encodes a pr extension or removal of from 50 to 5 amino acids of SEQ ID No 6 fro the extension or removal of from 50 to 5 amino acids from the C termi

Also the variant of the gene can be obtained by the following c mutation treatment One of the mutation treatment methods is the in vi protein that has β-glucoside PTS activity can be confirmed for exampl these genes in the appropriate cells and investigating if the ability to u has been increased or investigating the phosphorylating activity in vitro et al s method (Biochemistry 1998 37 8714-8723)

The bglF gene can also be DNA that hybridizes under stringent nucleotide sequences complementary to the nucleotide sequences of S with a probe prepared from these sequences Here the term stringent to conditions under which so-called specific hybrids are formed and no are not formed Although it is difficult to clearly express such conditio these can be exemplified as conditions under which highly homologous DNA for example DNA having homology no less than 80% 90% or with each other and DNAs having homology lower than the above do n each other Alternatively stringent conditions are exemplified by condi Southern hybridization washing conditions which are to wash once or three times at a temperature and salt concentration corresponding to 6O SDS preferably 0 1 x SSC 0 1 % SDS and more preferably 68°C 0 1 SDS

DNA containing a nucleotide sequence of SEQ ID No 5 or a pa may also be used as the probe Such a probe can be prepared employin DNA fragment containing a nucleotide sequence of SEQ ID No 5 is u and an oligonucleotide prepared based on the nucleotide sequence of S primer For example when using an approx 300 bp long DNA fragme hybridization washing conditions are 50 ⁰C 2 x SSC and 0 1% SDS

A modification to enhance the expression of bglF gene can be p When the bglF gene of Escherichia coli is used the bglF gene c employing PCR (PCR polymerase chain reaction see White T J et al 185 (1989)) in which the chromosomal DNA of Escherichia coli is the primers are prepared based on the nucleotide sequence of SEQ ID No primers shown in SEQ ID Nos 1 and 2 The bglF genes of other micro belonging to the family Enterobacteriaceae can also be obtained from in those microorganisms or bglF genes in microorganisms of other spe chromosomal DNA or a chromosomal DNA library of microorganisms method wherein the primers are prepared based on the sequence inform protein or the hybridization method wherein a probe is prepared based mentioned sequence information Incidentally chromosomal DNA ca DNA donor microorganisms For example Saito and Miura s method K Miura Biochem Biophys Acta 72 619 (1963) Seibutsu Kogaku J The Society of Biotechnology Japan pp 97-98 Baifukan 1992) may

Next the recombinant DNA is prepared by ligating the bglF ge the PCR method with a vector DNA capable of functioning in the cells microorganism A vector which can function in the cells of a host micr vector which is autonomously replicable in the cells of the host microo Examples of autonomously replicable vectors in cells of Escherichia c pUC18 pHSG299 pHSG399 pHSG398 pACYC184 (pHSG and pA from Takara Bio Inc ) RSFlOlO pBR322 pMW219 (pMW is availabl Gene Co Ltd ) pSTV29 (available from Takara Bio Inc ) etc

Recombinant DNA prepared as described above may be introdu microorganism in accordance with any of the transformation methods and Young F E Gene 1 153 (1977)) Also another method is when microorganism as known in relation to Bacillus subtihs actinomycete changed into the protoplast or spheroplast state that can easily uptake t DNA which is then introduced into the DNA recipient bacteria (Chang N Molec Gen Genet 168 111 (1979) Bibb M J Ward J M and Nature 274 398 (1978) Hinnen A Hicks J B and Fink G R Proc USA 75 1929 (1978))

The copy number of the bglF gene can be increased by introduc copies of the bglF gene as described above into the chromosomal DNA microorganism Introduction of multiple copies of the bglF gene into th DNA of the microorganism is performed by homologous recombinatio sequence of which multiple copies are present on the chromosomal D Repetitive DNA and inverted repeats present on the ends of transposabl used as the sequences multiple copies of which are present on chromos these genes may be hgated in tandem with the bglF gene present on the incorporated by duplication on unnecessary genes on the chromosome be introduced using a temperature-sensitive vector or integration vector

As disclosed JP2-109985A the bglF gene can be incorporated i and the transposon transferred to incorporate multiple copies into the c Whether or not the gene has been transferred into the chromosome can performing Southern hybridization with a part of the bglF gene as a pro

Aside from increasing copy number described above expressio can also be enhanced by employing the methods described in WOOO/18 substituting the expression regulatory sequence of the bglF gene promo φlO promoter etc are all known as strong promoters It is also possib base substitution etc into the bglF gene s promoter region and SD reg greater promoter strength

Examples of methods for evaluating the strength of promoters an strong promoters are described in articles by Goldstein et al (Prokaryo biotechnology Biotechnol Annu Rev 1995 1 105-128) etc Further substitution of several nucleotides into the spacer region between the π site (RBS) and the start codon particularly into the sequence immediat the start codon is known to have a strong effect on mRNA translation can be modified The expression regulatory regions of the bglF gene s can be determined by promoter search vectors and gene analysis softw GENETYX etc Expression of the bglF gene can be strengthened by s modifications of these promoters Substitution of expression regulatory conducted for example by employing temperature-sensitive plasmids integration method (WO2005/010175)

In order to increase the activity of a protein encoded by bglF which increases the activity of a β-glucoside PTS may also be introduc gene Examples of mutations which increase the activity of the protein bglF gene include a mutation of the promoter sequence which increase of the bglF gene and a mutation within the coding region of the gene the specific activity of the BglF protein <2> Method for producing L-amino acid

The method for producing L-amino acid of the present inventio cultuπng the microorganism of the present invention in a medium the components as needed may be used Carbon sources include a sugar s sucrose lactose galactose fructose a starch hydrolysase etc an alco glycerol solbitol etc an organic acid such as fumaric acid citric aci Of these it is preferable to use glucose as the carbon source Nitrogen inorganic ammonium salt such as ammonium sulfate ammonium chlo phosphate etc an organic nitrogen such as a soybean hydrolysis prod gas ammonia water etc It is desirable for the organic micronutπent so an appropriate amount of the auxotrophic substances such as vitamin etc or yeast extract etc In addition to these according to necessity s potassium phosphate magnesium sulfate iron ions manganese ions et The medium used in the present invention may be either a natural or sy long as it contains a carbon source nitrogen source inorganic ions an organic micronutrients

It is recommended that the culture be performed under aerobic co days at a culture temperature of 24 °C-37 ⁰C with a pH during the cult adjust the pH an inorganic or organic acidic or alkali substance and a the like may be used L-amino acid can be collected from the ferment using a combination of a conventional ion-exchange resin method a pr and other known methods If the L-amino acid accumulates inside the microorganism the cells can be be crushed by ultrasound etc then re centrifugal separation to obtain the supernatant from which the L-ami collected using an ion-exchange resin method etc

It is also possible to use a liquid medium appropriate for produc acid of with precipitation and to perform the culture while the L gluta employing a concentration crystallization method after removing the c culture solution or via ion-exchange chromatography etc When cultu glutamic acid producing conditions the L-glutamic acid with precipitat solution can also be collected via centrifugal separation filtering etc glutamic acid dissolved in the culture may be crystallized and then isol

Furthermore an animal feed additive based on fermentation bro this invention can be prepared by following separation method L-ami methods such as centπfuging filtering decanting flocculating or a co can be used to remove or reduce biomass^"

The broth obtained by this invention can be concentrated using such as a rotary evaporator thin layer evaporator reverse osmosis or (FR8613346B US4 997 754 EP410005B JP1073646B)

The concentrated broth is then processed using the methods of spray-drying spray granulation or any other process to give a preferab finely divided powder to be used as an animal feed additive This free-f divided powder can be converted into a coarse-grained very free flowi largely dust-free product by using suitable compacting or granulating p Altogether more than 90% of the water is removed in this way so that concentration of the feed animal additive is less than 10% preferably l weight

The protein content of the feed additive can be less than 10% p 5% by weight and the concentration of L threonine can be more than 5 more than 85% more preferably more than 95% (US5 431 933 JP121 US4 956 471 US4 777 051 US4946654 US 5 840358 US6 238 714 The present invention will be explained more specifically belo the following non-limiting examples

Reference example 1 Construction of an L-lysine-producing ba

<1-1> Construction of a strain in which the cadA and ldcC gene lysine decarboxylase are disrupted

First a strain which does not produce lysine decarboxylase was Red-driven integration method described in WO WO2005/010175 and system (J Bacteπol 2002 Sep 184 (18) 5200-3 Interactions between excisionase in the phage lambda excisive nucleoprotein complex Cho Gardner JF) were used to construct a strain in which lysine decarboxyl disrupted Lysine decarboxylase is encoded by the cadA gene (Genban NP_418555 SEQ ID No 42) and the ldcC gene (Genbank Accession SEQ ID No 44) (WO96/17930) The WC 196 strain was used as the pa WC 196 strain was named Escherichia coll AJ 13069 and deposited on with the National Institute of Bioscience and Human Technology of the Industrial Science and Technology (currently International Patent Org National Institute of Advanced Industrial Science and Technology Ch 1 -chome Tsukuba-shi Ibaraki-ken 305 8566 Japan) under Accession 14690 and converted to an international deposit under the Budapest Tre 29 1995 and given Accession No FERM BP-5252

The cadA and ldcC genes encoding lysine decarboxylase were method called Red driven integration which was initially developed Wanner (Proc Natl Acad Sci USA 2000 vol 97 No 12 pp 6640- resistant gene Furthermore via λ phage excision the antibiotic-resista integrated into chromosome can be removed from the strain

(1) Disruption of the cad A gene

The pMWl 18-attL Cm-attR plasmid described below was used template pMWl 18 attL-Cm-attR was obtained by inserting the the att attachment site of λ phage and the cat gene which is an antibiotic-resis pMW118 (Takara Bio Inc ) in the order of attL-cat-attR (see WO2005 attL sequence is shown in SEQ ID No 1 1 and the attR sequence is sho 12

PCR was conducted using as primers the synthetic oligonucleoti SEQ ID Nos 46 and 47 wherein a sequence corresponding to both end was at the primer s 3 end and a sequence corresponding to part of the target gene was at the primer s 5 end

The amplified PCR product was purified with an agarose gel th electroporation to an Escherichia coll WC 196 strain containing plasmi has a temperature-sensitive replication ability Plasmid pKD46 (Proc USA 2000 vol 97 No 12 pp 6640-6645) includes the λ phage DNA bases) and which includes the genes (γ β and exo) that encode Red re Red homologous recombination system under the control of the arabin promoter (GenBank/EMBL Accession No J02459 31088th - 33241s

Competent cells for electroporation were prepared as follows T coli WC 196 strain which was cultured overnight at 30 ⁰C in an LB med 100 mg/L ampicillin was diluted 100 times m a 5mL SOB medium con (20 mg/L) and L-arabinose (1 mM) (Molecular Cloning Lab Manual 2 et al Cold Spring Harbor Laboratory Press (1989)) was added and cult 2 5 hours then cultured on a plate medium of L-agar containing Cm (c (25 mg/L) at 37 ⁰C and the the Cm resistant recombinants were selecte remove the pKD46 plasmid cells were subcultured twice on an L-agar containing Cm at 42 ⁰C the ampicillin resistance of the colony obtaine an ampicillin-sensitive strain without pKD46 was obtained

Deletion of the cadA gene in the mutant identified by the chlora resistant gene was confirmed using PCR The cadA deficient strain was WC196ΔcadA att-cat

Next to remove the att-cat gene which is introduced into the ca plasmid pMW-intxis-ts described below was used pMW-intxis contai ID No 13) that encodes λ phage integrase (Int) and a gene (SEQ ID No excisionase (Xis) and has temperature-sensitive replication ability By i intxis-ts attL (SEQ ID No 1 1 ) and attR (SEQ ID No 12) on the chrom recognized causing recombination and the genes between attL and att leaving only the attL or attR sequence on the chromosome

Competent cells of the WC196ΔcadA att-cat strain obtained as were prepared using a typical method and were transformed with helpe mtxis ts cultured on a plate medium of L agar containing 50 mg/L am thus selecting the ampicilhn-resistant strain Next to remove the pMW the transformants were subcultured on an L-agar medium at 42 ⁰C the resistance and the chloramphenicol resistance of the colony obtained w chloramphenicol- and ampicillin-sensitive strain from which the att cat ts were removed was obtained This strain was designated WC196Δca (3) Preparation of the PCR template and helper plasmid *

The PCR template pMWl 18-attL Cm attR and helper plasmid were prepared as follows

(3 l) pMW1 18-attL-Cm-attR pMWl 18-attL-Tc-attR was constructed based on pMWl 18-attL following four DNA fragments were prepared

1) Bgiπ-EcoRI DNA fragment (120 bp) (SEQ ID No 1 1) conta obtained by PCR amplification of the sequence corresponding to the ch E coli W3350 strain (ATCC31278 containing λ prophage) using oligo and P2 (SEQ ID Nos 17 & 18) as primers (these primers additionally c recognition sites of the Bgiπ and EcoRI endonucleases)

2) Pstl-Hindm DNA fragment (182 bp) (SEQ ID No 12) contai obtained by PCR amplification of the sequence corresponding to the ch E coh W3350 strain (containing λ prophage) using oligonucleotides P Nos 19 & 20) as primers (these primers additionally contained the reco the Pstl and Hindiπ endonucleases)

3) Bgiπ Hindm large fragment (3916 bp) of pMWl 18-ter_rrnB ter_rrnB was obtained by hgating the following three fragments i) A large fragment (2359 bp) containing an AatH EcoRIpol fra pMWl 18 obtained by digesting t|ie pMWl 18 with an EcoRI restriction treating it with a Kl enow fragment of DNA polymerase I then digestin with an AatII restriction endonuclease ii) An AatH-Bgiπ small fragment (1194 bp) of pUC19 containi resistant (Ap^R) bla gene obtained by PCR amplifying the sequence corr E coh MGl 655 strain using oligonucleotides P7 and P8 (SEQ ID Nos* primers (these primers additionally contained the recognition sites of th endonucleases)

4) A small EcoRI-Pstl fragment (1388 bp) (SEQ ID No 29) of containing a tetracycline-resistant gene and transcription terminator ter Tc-ter_thrL was obtained as follows

A pML-MSC (MoI Biol (Mosk) 2005 Sep-Oct 39(5) 823-31 B (Russian) No 5 3-20 )) was digested with Xbal and BamHI restriction and a large fragment of this (3342 bp) was hgated with an Xbal-BamHI that contained the terminator ter thrL The Xbal-BamHI fragment (68 to the choromosome of E coh MGl 655 and was obtained by PCR amp oligonucleotides P9 and PlO (SEQ ID Nos 25 & 26) as primers (these additionally contained the recognition sites of the Xbal and BamHI end hgated reaction product was designated plasmid pML ter_thrL

The pML-ter thrL was digested with Kpnl and Xbal restriction treated with a Klenow fragment of DNA polymerase I then hgated wit Van91I fragment (1317 bp) of pBR322 containing the tetracycline resis pBR322 which was digested with EcoRI and Van911 restriction endon treated with a Klenow fragment of DNA polymerase I) The product o designated plasmid pML-Tc ter_φrL

Next the pMWl 18-attL-Cm-attR was constructed by ligation o Xbal fragment (4413 bp) a PA2 promoter (initial promoter of T7 phag chloramphenicol-resistant (CmR) cat gene an artificial BglE-Xbal DN bp) containing transcription terminator ter thrL and attR The artificia corresponding to T7 phage DNA using oligonucleotides Pl 1 and Pl 2 ( & 28) as primers (these primers additionally contained the recognition and Xbal endonucleases) by PCR The product of the ligation was desi pML PA2-MCS

The Xbal site was removed from pML PA2-MCS The product plasmid pML-PA2-MCS(XbaI )

A small Bglu-Hindlll fragment (928 bp) of pML-PA2-MCS(X PA2 promoter (initial promoter of T7 phage) and chloramphenicol resi gene was ligated with a small Hindm-Hindiπ fragment (234 bp) of pM attR which contained the transcription terminator ter_thrL and attR

The target artificial DNA fragment (1156 bp) was obtained by of the ligation mixture using oligonucleotides P9 and P4 (SEQ DD Nos primers (these primers contained the recognition sites of the HindIH an endonucleases)

(3-2) pMW intxis-ts

First two DNA fragments were amplified based on λ phage DN the template The first fragment consisted of a region of nt 37168 3804 λ phage DNA (SEQ ID No 39) and contained a cl repressor Prm and the leader sequence of cro gene This fragment was obtained by amplifi oligonucleotides Pl and P2 (SEQ ID Nos 31 &-32) as primers The s consisted of a region of nt 27801 -29100 of the genome of λ phage DN 40) which contained the xis-int gene from λ phage DNA This fragmen PCR using oligonucleotides P3 and P4 (SEQ ID Nos 33 & 34) as pri primers contained proper endonuclease recognition sites fragment of DNA polymerase I and then digested with a Pstl restrictio vector fragment of pMWPlaclacI-ts was eluted from an agarose gel and cut PCR-amphfied fragment

The plasmid pMWPlaclacI-ts is a derivative of pMWPlaclacI c following parts 1) an artificial Bglll-Hindlll DNA fragment containing promoter and the lad gene under control of the RBS of the bacteriopha an AatH-Bgiπ fragment containing the ampicillin-resistant (Ap^R) gene amplification of the region corresponding to the pUC19 plasmid using P5 and P6 (SEQ ID Nos 35 & 36) as primers (these primers containe sites of the AatII and Bgiπ endonucleases) 3) an Aatϋ-Hindlll fragme AatH-PvuI fragment of a recombinant plasmid pMWl 18-ter_rrnB The pMWl 18 ter_rrnB was constructed as follows A Pstl HindUI fragmen terminator ter rrnB was obtained by PCR amplification of the region c the chromosome of the E coh MGl 655 strain using as primers oligon P8 (SEQ ID Nos 37 & 38) which contained proper endonuclease reco Prior to ligation the pMWl 18 and ter_rrnB fragments (complementary No 41) were digested with Pvul or Pstl respectively treated with a Kl DNA polymerase I to blunt the ends and then digested with AatII or Hi endonuclease In the construction of the pMWPlaclacI-ts mutant an Aa fragment of plasmid pMWPlaclacI was substituted with an Aatϋ-EcoR plasmid pMAN997 which contained the par on and repAts genes ofth replicon (Applied and Environmental Microbiology June 2005 p 322

Example 1 Construction of plasmid for bglF overexpression chromosomal DNA of the Escherichia CoIi MGl 655 strain as the temp product was treated with restriction endonucleases HindIH and Xbal a fragment that contained the bglF genes was obtained

The purified PCR product was hgated with vector pMW219 wh digested with Hindiπ and Xbal (Nippon Gene Co Ltd ) to construct a for bglF overexpression This plasmid was under the control of a lac pr bglFgene was placed downstream of the lac promoter pM-bglF was d Hindiπ and EcoRI, the bglF gene fragments were collected and purifie vector pSTV29 which had been digested with Hindiπ and EcoRI (Tak way the plasmid pS-bglF for bglF overexpression was constructed

In the same manner as with the above-mentioned bglF gene a p expressing the ptsG gene was constructed as the control The sequence in SEQ ID No 7 and the sequence of the amino acid is given in SEQ I sequence can be obtained with reference to Genbank Accession No N as a 5 primer the synthetic oligonucleotide of SEQ ID No 3 containing and as a 3 primer the synthetic oligonucleotide of SEQ DD No 4 contai PCR was performed using the chromosomal DNA of the Escherichia as the template and the PCR fragment was treated with restriction end and Xbal and a gene fragment containing ptsG was obtained The puri was hgated with vector pMW219 which had been digested with HindI construct plasmid pM ptsG for ptsG overexpression This plasmid was of a lac promoter and the ptsG gene was placed downstream of the lac same manner as with the bglF the ptsG gene fragment was excised fro and hgated to vector pSTV29 In this way the plasmid pS-ptsG for pts As an Escherichia cob L-lysine-producing strain the WC196Δl (pCABD2) strain was used as parent strain Lys-producing plasmid pC dapA dapB and lysC genes (WO01/53459) was introduced into the W strain The WC196ΔldcCΔcadA (pCABD2) strain was transformed wit overexpression plasmid pM-bglF and the ptsG-overexpression plasmid constructed in Example 1 and the control plasmid pMW219 and kana strains were obtained After confirming that these plasmids had been in bglF overexpression plasmid pM-bglF introduced strain was designate WC196ΔldcCΔcadA (pCABD2 pM-bglF) the ptsG-overexpression pl introduced strain was designated WC196ΔldcCΔcadA (pCABD2 pM- control plasmid pMW219-introduced strain was designated WC196Δld (pCABD2 pMW219)

The strains constructed as described above were cultured in an containing 25 mg/L kanamycin at 37 ⁰C to finally become OD600 0 6 volume of a 40% glycerol solution was added to the culture and stirred amounts were pipetted and stored at -80 ⁰C This was called the glycer

After melting the glycerol stock of these strains lOOuL of each onto an L plate containing 25 mg/L kanamycin and this was cultured a hours Approx 1/8 of the cells on the plate were inoculated into a 2Om medium (shown below) with 25 mg/L kanamycin in a 500 mL Sakaguc and cultured at 37⁰C for 24 hours using a reciprocating shaking culture culturing the amount of lysine which had accumulated in the medium using a Biotech-analyzer AS 210 (Sakura Seiki)

The OD and L lysine which had accumulated at the 24th hour a data shows that overexpression of the bglF gene is more effective in lys than overexpression of the ptsG

Table 1

Medium for L lysine production

Glucose 40 g/L

Ammonium sulfate 24 g/L

Potassium Dihydrogen Phosphate 1 0 g/L

Magnesium sulfate 7-hydrate 1 0 g/L

Ferrous sulfate 4 7-hydrate 0 01 g/L

Manganese sulfate 4 7-hydrate 0 01 g/L

Yeast extract 2 0 g/L

Calcium carbonate 30 g/L

Adjusted to pH 7 0 with KOH and sterilized at 115 ⁰C for 10 mm

Glucose and MgSθ₄ 7H₂O were sterilized separately

Example 3 Effect of bglF overexpression on an L-glutamic acid-produ Escherichia bacteria

As an Escherichia coll L-glutamic acid-producing strain the AJ used as parent strain The AJl 2949 strain is a bacterial strain in which t dehydrogenase activity has been reduced and was deposited on Decem the National Institute of Bioscience and Human Technology of the Age Science and Technology (currently International-Patent Organism Dep Institute of Advanced Industrial Science and Technology Chuo 6 1-1 Tsukuba shi Ibaraki-ken 305-8566 Japan) under Accession No FER converted to an international deposit under the Budapest Treaty on No and given Accession No FERM BP-4881 AJ 12949 (pS bglF) and the the strain into which the control plasmid p introduced was designated AJl 2949 (pSTV29)

The AJl 2949 (pS-bglF) strain and the AJl 2949 (pSTV29) strai an L medium containing 20 mg/L chloramphenicol at 37 ⁰C to finally After this an equal volume of a 40% glycerol solution was added to th stirred then appropriate amounts were pipetted to obtain a glycerol sto 80 ⁰C

After melting the glycerol stock of these strains 100 μL of each spread onto an L plate containing 20 mg/L chloramphenicol and cultur hours Approx 1/8 of the cells on the plate were inoculated into a 2Om medium (described below) with 20 mg/L chloramphenicol in a 500 mL and cultured at 37 ⁰C for 40 hours using a reciprocating shaking culture cultuπng the amount of L-glutamic acid which had accumulated in the measured using a Biotech -analyzer AS210 (Sakura Seiki)

The OD and L-glutamic acid which had accumulated at the 40^th Table 2 As shown in Table 2 a large amount of L-glutamic acid had a AJ12949 (pS-bglF) strain compared to the AJ12949 (pSTV29) strain contain the bglF genes

Table 2

Ferrous sulfate 4 7-hydrate 0 01 g/L \

Manganese sulfate 4 7 hydrate 0 01 g/L

Yeast extract 2 0 g/L

Calcium carbonate 30 g/L

Adjusted to pH 7 0 with KOH sterilized at 115 ⁰C for 10 mm

Glucose and MgSO4 7H2O were sterilized separately

Also after the culture temperature was at 60 ⁰C or below a thia hydrochloride solution which had been sterilized with a DISMIC-25cs (ADVANTEC) was added to obtain the final concentration of 0 01 g/L

Example 4

Effect of bglF overexpression on an L threonine-producing strai the genus Escherichia

As the parent strain of the bglF overexpression for L-threonine- B-5318 strain was used The B-5318 strain was deposited on May 3 19 Russian National Collection of Industrial Microorganisms (VKPM) G Genetika(Russia 117545 Moscow 1 Dorozhny Proezd 1 ) under Acces B-5318 The construction of the bglF overexpression strain from B 531 using the plasmid as described in Example 1

The B-5318 strain was transformed with the bglF-amplifying pl used in Example 1 and the control plasmid pSTV29 and chlorampheni strains were obtained After confirming that the prescribed plasmids ha the strain into which bglF-overexpression plasmid pS bglF was introdu designated B-5318 (pS-bglF) and the strain into which control plasmid After melting the glycerol stock of these strains 100 μL of each spread onto an L plate containing 20 mg/L chloramphenicol and cultur hours Approx 1/8 of the cells on the plate were inoculated into a 2Om medium with 20 mg/L chloramphenicol in a 500 mL Sakaguchi shakin cultured at 37 ⁰C for 16 hours using a reciprocating shaking culture app culturing the amount of L threonine which had accumulated in the me measured using high-performance liquid chromatography

The OD and L threonine which had accumulated at the 16th ho Table 3 As shown in the table a large amount of L-threonine had accu 5318 (pS-bglF) strain compared to the B-5318 (pSTV29) strain which the bglF gene

Table 3

Bacterial strain OD600 L-threonine (g/L)

B-5318 (pSTV29) 8 0 3 6 B 5318 (pS-bglF) 10 3 4 4

Medium for L threonine-production Glucose 60 g/L

Ammonium sulfate 16 g/L

Potassium Dihydrogen Phosphate 0 7 g/L Magnesium sulfate 7-hydrate 1 0 g/L Calcium carbonate 30 g/L

Adjusted to pH 7 0 with KOH sterilized at 115 ⁰C for 10 mm

However glucose and MgSO4 7H2O were sterilized separately Potass was sterilized by dry heat at 180 ⁰C for 3 hours After the culture tempe to 60 ⁰C or lower a thiamine hydrochloride solution which had been st

DISMIC -25cs 0 2 mm filter (ADVANTEC) was added to obtain the fin of 0 2 mg/L

Example 5

Effect of bglF overexpression on an L-glutamic acid-producing strain o

As the parent strain of the bglF amplification L glutamic acid-p the Pantoea ananatis AJl 3601 strain can be used The Pantoea ananat was deposited on August 18 1999 with the National Institute of Biosci Technology Agency of Industrial Science and Technology Ministry o and Industry (1-3 Higashi 1 chome Tsukuba-shi Ibaraki-ken 305-856 Accession No FERM P-17516 and converted to an international deposi Budapest Treaty on July 6 2000 and given Accession No FERM BP amplified strains can be constructed from L glutamic acid-producing b plasmid described in Examplel

The bglF overexpressed strains are cultured in an L-glutamic ac medium and cultured using a reciprocating shaking culture apparatus amount of L-glutamic acid which had accumulated in the medium is m Biotech-analyzer AS210 (Sakura Seiki) to confirm whether the accumu glutamic acid has increased In this way the bglF overexpressed strain

Claims

Claims <

1 A method for producing an L-amino acid comprising cultuπng microorganism of the Enterobacteriaceae family which has an ability t amino acid and which has been modified so as to enhance the β-glucosi and collecting the L-amino acid from the medium or the microorganis

2 The method according to Claim 1 wherein the microorganism to enhance the expression of a bglF gene encoding the β-glucoside PTS copy number of the gene modifying an expression regulatory sequence combinations thereof

3 The method according to Claim 1 or 2 wherein said bglF gene i the group consisting of

(a) a DNA which comprises a nucleotide sequence of SEQ ID N

(b) a DNA which hybridizes with a sequence complementary to sequence of SEQ ID NO 5 or with a probe that can be prepared from s sequence under stringent conditions and which encodes a protein havin activity

4 The method according to Claiml or 2 wherein the bglF gen selected from the group consisting of

(A) a protein consisting of the amino-acid sequence of SEQ ID

(B) a protein which comprises the amino-acid sequence of SEQ including substitution deletion insertion addition or inversion of one acid residue and has β glucoside PTS activity

5 The method according to any one of Claims 1-4 wherein the mi bacterium of the genus Escherichia or genus Pantoea