KR100824551B1 - Nucleotide sequences which code for the lysR2 gene - Google Patents

Abstract

a) a polynucleotide at least 70% identical to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, b) a poly encoding a polypeptide comprising an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO: 2 Nucleotides, c) polynucleotides complementary to the polynucleotides of a) or b), and d) polynucleotides comprising 15 or more consecutive nucleotides of the polynucleotide sequence of a), b) or c). A method of preparing fermentation of L-amino acids using isolated polynucleotides comprising selected polynucleotide sequences, and coryneform bacteria in which at least the lysR2 gene is present in attenuated form, and the use of said polynucleotide sequences as hybridization probes.

lysR2 gene, L-lysine, L-valine, coryneform bacteria, Corynebacterium glutamicum

Description

Nucleotide sequences which code for the lysR2 gene}             

The present invention provides nucleotide sequences from coryneform bacteria encoding the lysR2 gene, and methods for the fermentation of amino acids, in particular L-lysine and L-valine, by lysR2 gene attenuation. The lysR2 gene encodes the LysR2 protein, a transcriptional regulator of the LysR family.

L-amino acids, in particular L-lysine and L-valine, are used in human medicine and the pharmaceutical industry, the food industry, and very particularly in animal feed.

Amino acids are known to be produced by fermentation of coryneform bacterial strains, in particular Corynebacterium glutamicum . Due to the importance of amino acids, efforts are constantly being made to improve the manufacturing process. Improvements to this method include fermentation methods such as agitation and oxygenation, or composition of the nutrient medium such as sugar concentration during fermentation, or post-treatment of the product form, for example by ion exchange chromatography. Or to the inherent production properties of the microorganisms themselves.

Mutagenesis methods, selection, and mutant selection are used to improve the production properties of these microorganisms. Strains that are resistant to antimetabolic products, nutritionally required for regulatory critical metabolites and produce amino acids are obtained in this way.

In addition, methods of recombinant DNA technology have been used for years to improve strains of Corynebacterium strains producing L-amino acids.

Object of the present invention

We aim to provide a novel method for improved fermentation preparation of amino acids, in particular L-lysine and L-valine.

The present invention,

a) a polynucleotide comprising the amino acid sequence of SEQ ID NO: 2, preferably at least 70% identical to a polynucleotide encoding a polypeptide having a transcriptional regulator LysR2 activity,

b) a polynucleotide comprising an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO: 2, preferably encoding a polypeptide having a transcriptional regulator LysR2 activity,

c) a polynucleotide complementary to the polynucleotide of a) or b), and                 

d) isolated from a coryneform bacterium comprising a polynucleotide sequence encoding a lysR2 gene selected from the group consisting of polynucleotides comprising at least 15 consecutive nucleotides of the polynucleotide sequence of a), b) or c) above Polynucleotides are provided.

In addition, the present invention

(i) the nucleotide sequence of SEQ ID NO: 1, or

(ii) one or more sequences corresponding to sequence (i) within the scope of degeneration gene code, or

(iii) one or more sequences that hybridize with sequences complementary to sequence (i) or (ii), and optionally

(iv) The above-mentioned polynucleotide is provided which is preferably replicable DNA, comprising a sense mutation of neutralization in (i) above which does not modify protein / polypeptide activity.

The present invention also provides

a) a polynucleotide comprising at least 15 contiguous nucleotides selected from the nucleotide sequence between positions 1 and 231 of SEQ ID NO: 1,

b) a polynucleotide comprising at least 15 contiguous nucleotides selected from the nucleotide sequences between positions 232 and 1161 of SEQ ID NO: 1,

c) providing a polynucleotide comprising at least 15 consecutive nucleotides selected from the nucleotide sequences between positions 1162 and 1364 of SEQ ID NO: 1.

The present invention also provides

Polynucleotides, in particular DNA, comprising and replicating a nucleotide sequence as set forth in SEQ ID NO: 1;

Polynucleotides encoding a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 2;

As part of a polynucleotide according to the invention, a vector containing at least 15 consecutive nucleotides of a claimed sequence, and

Provides coryneform bacteria in which the lysR2 gene is attenuated, in particular by insertion or deletion.

The invention also relates to hybridization of a corresponding gene library comprising a complete gene having a polynucleotide sequence corresponding to SEQ ID NO: 1 using a probe comprising the mentioned polynucleotide sequence according to SEQ ID NO: 1 or a fragment thereof. Polynucleotides are provided that substantially comprise a polynucleotide sequence obtainable by screening and separation of the mentioned DNA sequences.

Polynucleotide sequences according to the present invention are hybrids for RNA, cDNA, and DNA for isolation of full-length nucleic acids or polynucleotides or genes encoding LysR2 protein, or for separating nucleic acids or polynucleotides or genes with high similarity to the lysR2 gene sequence. It is suitable as a ignition probe. They are also suitable for incorporation into so-called "arrays", "microarrays" or "DNA chips" in order to detect and measure corresponding polynucleotides.

The polynucleotide sequence according to the present invention is further suitable as a primer for preparing DNA of genes encoding LysR2 protein by polymerase chain reaction (PCR).

Such oligonucleotides used as probes or primers have at least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or 24, very particularly preferably 15, 16, 17, 18 or more than 19 consecutive nucleotides. Oligonucleotides of 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more, or 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 or more nucleotides in length Is also suitable. Also suitable are oligonucleotides, optionally having a length of at least 100, 150, 200, 250 or 300 nucleotides.

"Isolated" means to be separated from its natural environment.

Generally "polynucleotide" relates to polyribonucleotides and polydeoxyribonucleotides, which may be non-modified RNA or DNA or modified RNA or DNA.

The polynucleotides according to the invention are at least 70% and at least 80%, preferably at least 81%, of the polynucleotides according to SEQ ID NO: 1 or fragments prepared therefrom, and in particular the polynucleotides according to SEQ ID NO: 1 or fragments prepared therefrom At least 85%, particularly preferably at least 86% to 90% and very particularly preferably at least 91%, 93%, 95%, 97% or 99% identical.                 

A "polypeptide" is understood to mean a peptide or protein comprising two or more amino acids joined via peptide bonds.

The polypeptide according to the invention has the biological activity of the polypeptide according to SEQ ID NO: 2, in particular LysR2 protein, and also from 70% to 80% or more, preferably 81% to 85% with the polypeptide according to SEQ ID NO: 2. Above, particularly preferably at least 86% to 90% and very particularly preferably at least 91%, 93%, 95%, 97% or 99% identical and include those having the stated activity.

The invention further relates to the use of L-asparagine, L-threonine, L-serine, L-glutamate, particularly with coryneform bacteria which already produce amino acids and whose nucleotide sequences encoding the lysR2 gene are attenuated, in particular eliminated or expressed at low levels. , L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and A method for preparing fermentation of L-arginine, especially amino acids selected from the group consisting of L-lysine and L-valine.

In this context the term “attenuation” refers to, for example, using a weak promoter, encoding a corresponding enzyme with low activity, or using a gene or allele that inactivates the corresponding gene or allele or enzyme (protein) and optionally Combining these methods indicates the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in the microorganism encoded by the corresponding DNA.

Microorganisms provided by the present invention can produce amino acids, especially L-lysine and L-valine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They may be representative of the coryneform bacteria, in particular the Corynebacterium genus. Among the genus Corynebacterium, mention may be made in particular of Corynebacterium glutamicum species, which are known among experts for their ability to produce L-amino acids.

Suitable strains of the genus Corynebacterium, in particular Corynebacterium glutamicum (C. glutamicum) species, include the following wild type strains,

Corynebacterium glutamicum ATCC13032,

Corynebacterium acetoglutamicum ATCC15806,

Corynebacterium acetonitrile evil attempts pilrum (Corynebacterium acetoacidophilum) ATCC13870,

Corynebacterium melassecola ATCC17965

Corey's Corynebacterium thermo amino Ness (Corynebacterium thermoaminogenes) FERM BP-1539 ,

Brevibacterium flavum ATCC14067,

Brevibacterium lactofermentum ATCC13869 and

Brevibacterium divaricatum ATCC14020, or an L-amino acid producing mutant or strain produced therefrom, for example, an L-lysine producing strain

Corynebacterium glutamicum FERM-P 1709,                 

Brevibacterium plaboom FERM-P 1708,

Brevibacterium lactofermentum FERM-P 1712,

Corynebacterium glutamicum FERM-P 6463,

Corynebacterium glutamicum FERM-P 6464

Corynebacterium glutamicum DM58-1

Corynebacterium glutamicum DG52-5

Corynebacterium glutamicum DSM 5715 and

Corynebacterium glutamicum DSM12866, or, for example, L-valine producing strain

Corynebacterium glutamicum DSM 12455

Corynebacterium glutamicum FERM-P 9325

Brevibacterium lactofermentum FERM-P 9324

Brevibacterium lactofermentum FERM-BP 1763.

We found that Mr. LysR2 protein encodes a protein. Successful isolation of the novel lysR2 gene of glutamicum is a transcriptional regulator of the LysR family.

Seed. To isolate the lysR2 gene or other genes of glutamicum, the genetic library of the microorganism is first set up in Escherichia coli (E. coli). The establishment of gene libraries is generally described in known textbooks and handbooks. See, for example, Winnacker: Gene and Klone, Eine Einfuhrung in die Gentechnologies (Genes and Clones, An Introduction to Genetic Engineering), Verlag Chemie, Weinheim, Germany, 1990, or handbooks [Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989. Well-known gene libraries are described in E. coli in the λ vector by Kohara et al., Cell 50, 495-508 (1987). From E. coli K-12 strain W3110. See Bathe et al., Molecular and General Genetics, 252: 255-265, 1996. A gene library of glutamicum ATCC13032 is described, which is described in E. coli. Among coli K-12 strain NM554 [Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575] cosmid vector SuperCos I [Wahl et al., 1987, Proceeding of the National Academy of Sciences USA, 84: 2160-2164]. In turn, Bormann et al., Molecular Microbiology 6 (3), 317-326 (1992) describe seeds using cosmid pHC79 (Hohn and Collins, 1980, Gene 11, 291-298). . Describes a genetic library of glutamicum ATCC13032.

this. Mr. E. Coli. To prepare a gene library of glutamicum, pBR322 [Bolivar, 1979, Life Sciences, 25, 807-818] or pUC9 [Vieira et al., 1982, Gene, 19: 259-268] It is also possible to use plasmids such as Suitable hosts are particularly restriction- and recombinant-defective, for example strain DH5α [Ref. Jeffrey H. Miller: "A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria", Cold Spring Such as Harbor Laboratory Press, 1992. E. coli strains.

Long chain DNA fragments cloned using cosmids or other λ-vectors may then be subcloned into conventional vectors suitable for DNA sequencing.

DNA sequencing methods are described in particular in Sanger et al., Proceedings of the National Academy of Sciences of the United States of America USA, 74: 5463-5467, 1977.

The obtained DNA sequence is then described, for example, in Staden (Nucleic Acids Research 14, 217-232 (1986)), Mark (Nucleic Acids Research 16, 1829-1836 (1988). Can be investigated using known algorithms, such as the programs of), or GCG programs of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)).

A seed encoding the lysR2 gene and constituting the present invention as shown in SEQ ID NO: 1. A novel DNA sequence of glutamicum was obtained in this manner. The amino acid sequence of the corresponding protein was further derived from this DNA sequence by the method described above. The amino acid sequence of the obtained lysR2 gene product is shown in SEQ ID NO: 2. It is known that endogenous enzymes in the host can remove the N-terminal amino acid methionine or formylmethionine of the prepared protein.

The coding DNA sequence resulting from SEQ ID NO: 1 due to the degeneracy of the genetic code also constitutes the present invention. In the same manner, a DNA sequence which hybridizes with SEQ ID NO 1 or a portion of SEQ ID NO 1 constitutes the present invention. Conservative amino acid exchanges, for example, the exchange of glycine to alanine or aspartic acid to glutamic acid, do not result in a fundamental change in protein activity, ie known as "sense mutations" of neutralizing function among experts. It is done. In addition, it is known that a change on the N and / or C terminus of a protein can cause substantially no damage or rather stabilize its function. Information on this content can be found by experts, in particular, in Ben-Bassat et al., Journal of Bacteriology 169: 751-757 (1987); O'Regan et al., Gene 77: 237-251 (1989); Sahin-Toth et al., Protein Sciences 3: 240-247 (1994); Hochuli et al., Bio / Technology 6: 1321-1325 (1988) and known textbooks of genetics and molecular biology. The amino acid sequences obtained in a corresponding manner from SEQ ID NO: 2 also constitute a subject of the invention.

Finally, DNA sequences prepared by polymerase chain reaction (PCR) using primers resulting from SEQ ID NO: 1 constitute the present invention. The oligonucleotides typically have a length of at least 15 nucleotides.

Guidance for identifying DNA sequences by hybridization can be found by experts, in particular in handbooks [The DIG System Users Guide for Filter Hybridization, Boehringer Mannheim GmbH, Mannheim, Germany, 1993] and Liebl et al. , International Journal of Systematic Bacteriology 41: 255-260 (1991). Instructions for amplifying DNA sequences with the help of polymerase chain reaction (PCR) are provided by experts, in particular in handbooks (Gait, Oligonucleotide Synthesis, A Practical Approach, IRL Press, Oxford, UK, 1984) and in literature. Newton and Graham, PCR, Spektrum Akademischer Verlag, Heidelberg, Germany, 1994.

In the study of the present invention, it was confirmed that coryneform bacteria produce amino acids, in particular L-lysine and L-valine, in an improved method after lysR2 gene attenuation.

To achieve attenuation, the catalytic properties of lysR2 gene expression or enzyme protein can be reduced or eliminated. The two methods can be arbitrarily combined.

Reduction of gene expression may be caused by genetic modification (mutation) of the signal structure of appropriate culture or gene expression. Signal structures of gene expression are, for example, inhibitor genes, activator genes, operators, promoters, attenuators, ribosomal binding sites, initiation codons and terminators. The expert is described, for example, in Patent Application WO 96/15246, Boyd and Murphy., Journal of Bacteriology 170: 5949 (1988), Voskuil and Chambliss., Nucleic Acids Research 26: 3548 (1998) , Jensen and Hammer., Biotechnology and Bioengineering 58: 191 (1998), Patek et al., Microbiology 142: 1297 (1996), Vasicova et al., Journal of Bacteriology 181: 6188 (1999) and, for example, textbooks [Reference: Knippers., "Molekulare Genetik [Molecular Genetics]", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995 or Winnacker., "Gene und Klone [Genes and Clones]", VCH Verlagsgesellschaft, Weinheim, Germany, Known genetic and molecular biology textbooks, such as 1990, can be found.

Mutations that induce changes or decreases in the catalytic properties of enzyme proteins are known in the art; Examples that may be mentioned include Qiu and Goodman., Journal of Biological Chemistry 272: 8611-8617 (1997), Sugimoto et al., Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997) and Mockel., "Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der allosterischen Regulation und Struktur des Enzyms [Threonine dehydratase from Corynebacterium glutamicum: Cancelling the allosteric regulation and structure of the enzyme]", Report [Reference: Julich Research Centre, Jul-2906, ISSN09442952, Julich , Germany, 1994]. Comprehensive descriptions can be found, for example, in known genetics and molecular biology textbooks such as textbooks (Hagemann., "Allgemeine Genetik [General Genetics]", Gustav Fischer Verlag, Stuttgart, 1986).

Possible mutations are translocations, inversions, insertions and deletions. Depending on the effect of amino acid substitutions on enzymatic activity, missense mutations or nonsense mutations are mentioned. Insertion or deletion of one or more base pairs (bp) in a gene results in frame shift mutations, as a result of incorporation of erroneous amino acids or immature cessation of translation. Several codon deletions typically result in complete loss of enzyme activity. A description of the occurrence of such mutations is given in the prior art, for example in textbooks [Knippers., "Molekulare Genetik [Molecular Genetics]", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995, Winnacker., Known genetics and molecular biology textbooks such as "Gene und Klone [Genes and Clones]", VCH Verlagsgesellschaft, Weinheim, Germany, 1990, or Hagemann., "Allgemeine Genetik [General Genetics]", Gustav Fischer Verlag, Stuttgart, 1986. Can be identified at

Seed. A common method of mutating a gene of glutamicum is the method of gene disruption and gene substitution described in Schwarzer and Puhler., Bio / Technology 9, 84-87 (1991).

Blunt[참고문헌: Invitrogen, Groningen, The Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)] 또는 pEM1[참고문헌: Schrumpf et al, 1991, Journal of Bacteriology 173: 4510-4516]이다. 이어서, 유전자 암호화 영역의 중심 부분을 함유하는 플라스미드 벡터는 접합 또는 형질전환에 의해 목적하는 균주 씨. 글루타미쿰내로 전달된다. 접합 방법은 예를 들어, 문헌[참고문헌: Schafer et al., Applied and Environmental Microbiology 60, 756-759 (1994)]에 기재된다. 형질전환 방법은 예를 들어, 문헌[참고문헌: Thierbach et al., Applied Microbiology and Biotechnology 29, 356-362 (1988), Dunican and Shivnan., Bio/Technology 7, 1067-1070 (1989) 및 Tauch et al., FEMS Microbiological Letters 123, 343-347 (1994)]에 기재된다. "교차" 이벤트에 의한 상동 재조합후, 당해 유전자의 암호화 영역은 벡터 서열에 의해 중단되고, 3' 말단이 없는 것과, 5' 말단이 없는 두개의 불완전한 대립유전자가 수득된다. 이러한 방법은 예를 들어, 문헌[참고문헌: Fitzpatrick et al., Applied Microbiology and Biotechnology 42, 575-580 (1994)]에 씨. 글루타미쿰의 recA 유전자를 제거하기 위해 사용되었다.In gene disruption methods, the central portion of the coding region of a gene of interest can replicate in a host (typically E. coli), but C. In glutamicum, it is cloned into a plasmid vector that cannot be replicated. Possible vectors are described, for example, in pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob [Schafer et al., Gene 145, 69-73 (1994). )], pK18mobsacB or pK19mobsacB [Reference: Jager et al., Journal of Bacteriology 174: 5462-65 (1992)], pGEM-T [Reference: Promega corporation, Madison, WI, USA], pCR2.1-TOPO [Reference: Shuman (1994). Journal of Biological Chemistry 269: 32678-84; US Patent Application No. 5,487,993], pCR

Blunt [Ref. Invitrogen, Groningen, The Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)] or pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173: 4510-4516). The plasmid vector containing the central portion of the gene coding region is then strained by the strain of interest. Delivered into glutamicum. Conjugation methods are described, for example, in Schafer et al., Applied and Environmental Microbiology 60, 756-759 (1994). Transformation methods are described, for example, in Thierbach et al., Applied Microbiology and Biotechnology 29, 356-362 (1988), Dunican and Shivnan., Bio / Technology 7, 1067-1070 (1989) and Tauch et. al., FEMS Microbiological Letters 123, 343-347 (1994). After homologous recombination by "crossover" events, the coding region of the gene is interrupted by the vector sequence, resulting in two incomplete alleles without the 3 'end and without the 5' end. Such methods are described, for example, in Fitzpatrick et al., Applied Microbiology and Biotechnology 42, 575-580 (1994). It was used to remove the glutamicum recA gene.

1 shows by way of illustration the plasmid vector pCR2.1lysR2int, which is used to destroy or eliminate the lysR2 gene.

In the method of gene replacement, mutations such as, for example, deletions, insertions or base substitutions are made in the gene of interest in vitro. The manufactured allele is again seed. Cloned in a vector that does not replicate against glutamicum, and then by transformation or conjugation. Is delivered into the desired host of glutamicum. Incorporation of mutations or alleles occurs after homologous recombination by the first "crossing" event affecting the integration of the target gene or target sequence and the appropriate second "crossing" event on elimination. Such methods are described, for example, by deletions in Peters-Wendisch et al., Microbiology 144, 915-927 (1998). It was used to remove the glutamicum pyc gene.

Deletions, insertions or base substitutions can be incorporated into the lysR2 gene in this manner.

In addition, in addition to attenuation of the lysR2 gene, enhancing, in particular overexpressing, one or more enzymes of certain biosynthetic pathways, glycolysis, supplemental metabolism, pentose phosphate cycles or amino acid excretion may result in L-amino acids, in particular L-lysine and L-valine It can be advantageous for production.

In this regard, the term "enhancer" refers to the corresponding DNA, for example, by using a strong promoter or by using a gene or allele encoding a corresponding enzyme (protein) with high activity and optionally combining these methods. It indicates an increase in the intracellular activity of one or more enzymes (proteins) in the microorganism encoded by it.

Thus, for example, for the production of L-lysine, the following genes:

DapA gene (EP-B 0 197 335), which encodes a dihydrodipicolinate synthase,

Eno gene encoding enolase (DE: 19947791.4),

Zwf gene (JP-A-09224661) encoding the zwf gene product,

Pyc gene encoding pyruvate carboxylase (Peters-Wendisch et al., Microbiology 144, 915-927 (1998)),

LysE gene encoding lysine excretion (DE-A-195 48 222),

LysC gene (EP-B-0387527; EP-A-0699759) encoding feedback resistant aspartate kinase,

One or more genes selected from the group consisting of the zwal gene (DE: 199 59 328.0, DSM 13115) encoding the Zwal protein can be simultaneously enhanced, in particular overexpressed.

Thus, for example for the production of L-valine, the following genes:

IlvBN gene (Keilhauer et al., (1993) Journal of Bacteriology 175: 5595-5603), which simultaneously encodes acetohydroxy acid synthase, or

IlvD gene which simultaneously encodes dihydroxy acid dehydratase (Sahm and Eggeling (1999) Applied and Environmental Microbiology 65: 1973-1979), or

Simultaneously malate: simultaneously enhances, in particular, one or more genes or alleles selected from the group consisting of the mqo gene (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)) encoding quinone oxidoreductase Can overexpress.

In addition, for the production of amino acids, in particular L-lysine, in addition to the lysR2 gene attenuation, the following genes:

Pck gene encoding phosphoenol pyruvate carboxykinase (DE 199 50 409.1, DSM 13047),

Pgi gene encoding glucose 6-phosphate isomerase (US 09 / 396,478, DSM 12969),

PoxB gene encoding pyruvate oxidase (DE: 1995 1975.7, DSM 13114),

It may be advantageous that at least one gene selected from the group consisting of the zwa2 gene (DE: 19959327.2, DSM 13113) encoding the Zwa2 protein is simultaneously attenuated, in particular eliminated.

Finally, for L-lysine production, in addition to the lysR2 gene attenuation, the following genes:

Hom gene (EP-A-0131171) encoding homoserine dehydrogenase,

ThrB gene encoding homoserine kinase (Peoples, O.W., et al., Molecular Microbiology 2 (1988): 63-72), and

It may be advantageous that at least one gene selected from the group consisting of panD gene (EP-A-1006192) encoding aspartate decarboxylase is simultaneously attenuated, in particular eliminated.

In addition, the attenuation of homoserine dehydrogenase is particularly dependent on amino acid substitutions, e.g., the substitution of L-valine for L-alanine, L-glycine or L-leucine at position 59 of the enzyme protein, at position 104 of the enzyme protein. L-valine can be made by substitution with L-isoleucine, L-valine or L-leucine and / or by substitution of L-asparagine with L-threonine or L-serine at the enzyme protein 118 position.

In addition, the attenuation of homoserine kinases is particularly dependent on amino acid substitutions, such as the substitution of L-alanine for L-valine, L-glycine or L-leucine at position 133 of the enzyme protein and / or at position 138 of the enzyme protein. L-proline can be made by substitution with L-threonine, L-isoleucine or L-serine.

In addition, attenuation of aspartate decarboxylase can be effected in particular by amino acid substitutions, eg, by the substitution of L-alanine with L-glycine, L-valine or L-isoleucine at position 36 of the enzyme protein. have.

In addition to lysR2 gene attenuation, further elimination of undesirable side reactions may be advantageous for the production of amino acids, particularly L-lysine and L-valine. See, Nakayama, "Breeding of Amino Acid Producing Micro-organisms", Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982].

The microorganisms prepared according to the invention are continuously subjected to a batch process (batch culture) or fed-batch (feeding process) or repeated fed-batch process (repeating fed process) for the production of L-amino acids, in particular L-lysine and L-valine. Or discontinuously cultured. A summary of known culture methods can be found in the textbook [Chmiel, Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Bioprocess Technology 1. Introduction to Bioprocess Technology), Gustav Fischer Verlag, Stuttgart, 1991] or in the textbook [Storhas, Bioreaktoren und periphere Einrichtungen (Bioreactors and Peripheral Equipment), Vieweg Verlag, Braunschweig / Wiesbaden, 1994].

The culture medium used must meet the requirements of the particular strain in a suitable manner. Descriptions of culture media for various microorganisms are included in the Handbook ("Manual of Methods for General Bacteriology" of the American Society for Bacteriology, Washington D.C., USA, 1981). Sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose; Oils and fats such as soybean oil, sunflower oil, peanut oil and coconut fat; Fatty acids such as palmitic acid, stearic acid and linoleic acid; Alcohols such as glycerol and ethanol; And organic acids such as acetic acid can be used as the carbon source. These materials can be used individually or as a mixture.

Organic nitrogen-containing compounds such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, soy flour and urea; Or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used as the nitrogen source. Nitrogen sources can be used individually or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source. The culture medium should further contain the metal salts necessary for growth, for example magnesium sulfate or iron sulfate. Finally, essential growth substances such as amino acids and vitamins can be used in addition to those mentioned above. In addition, suitable precursors may be added to the culture medium. The starting materials mentioned can be added to the culture in the form of a single batch or supplied during the culture in a suitable manner.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia solution, or acidic compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture. Antifoaming agents such as fatty acid polyglycol esters may be used to control the generation of foam. Suitable materials with selective action, such as antibiotics, can be added to the medium to maintain the stability of the plasmid. To maintain aerobic conditions, oxygen or an oxygen-containing gas mixture, such as air, is introduced into the culture. The culture temperature is generally 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. Incubation is continued until a maximum amount of desired product is produced. This object is generally achieved within 10 to 160 hours.

Methods of measuring L-amino acids are known from the prior art. Thus, the analysis may be done by anion exchange chromatography followed by ninhydrin derivatization, as described, for example, in Spackman et al., Analytical Chemistry, 30, (1958), 1190, For example, it may be performed by reverse phase HPLC as described in Lindroth et al., Analytical Chemistry (1979) 51: 1167-1174.

Pure cultures of the following microorganisms were deposited in a depository institution (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) on 28 July 2000 under the Budapest Treaty. :

Escherichia coli strain TOP10F / pCR2.1lysR2int (as DSM 13617).

The method according to the invention is used for the fermentation preparation of amino acids, in particular L-lysine and L-valine.

The invention is described in more detail below using exemplary embodiments.

Isolation of plasmid DNA from Escherichia coli and all restriction techniques, Klenow and alkaline phosphatase treatments are described in Sambrook et al., Molecular Cloning. A Laboratory Manual (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA. In addition, methods for transforming Escherichia coli are described in this handbook.

Compositions of conventional nutritional media, such as LB or TY media, can also be found in the handbook (Sambrook et al).

Example 1

Seed. Preparation of Genomic Cosmid Gene Library from Glutamicum ATCC 13032

Seed. Chromosome DNA from glutamicum ATCC 13032 was isolated as described in Tauch et al., 1995, Plasmid 33: 168-179, and the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02). DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Code no. 1758250). Cosmid vector SuperCos1 obtained from the manufacturer (Stratagene, La Jolla, USA, Product Description SuperCos1 Cosmid Vector Kit, Code no. 251301) (Wahl et al., 1987, Proceedings of the National Academy of Sciences, USA 84: 2160-2164] were digested with restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product Description XbaI, Code no. 27-0948-02) and similarly dephosphorylated with shrimp alkaline phosphatase.                 

The cosmid DNA was then digested with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04). Cosmid DNA treated in this manner is mixed with treated ATCC 13032 DNA and the batch is T4 DNA ligase (manufactured by Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no. 27-0870-04). ). The ligation mixture was then filled into phage using Gigapack II XL Packing Extract (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217).

this. E. coli strain NM554 [Raleigh et al., 1988, Nucleic Acid Res. 16: 1563-1575], the cells were placed in 10 mM MgSO ₄ and mixed with an aliquot of phage suspension. Titration of infection and cosmid libraries was performed as described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor, and cells contained +100 μg / ml ampicillin. Plated on LB agar (Lennox, 1955, Virology, 1: 190). After overnight incubation at 37 ° C., recombinant individual clones were selected.

Example 2

Isolation and Sequencing of the lysR2 Gene

Cosmid DNA of individual colonies was isolated using a Qiaprep Spin Miniprep Kit (Product No. 27106, manufactured by Qiagen, Hilden, Germany) according to the manufacturer's instructions, followed by restriction enzyme Sau3AI (manufactured by Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Part No. 27-0913-02). DNA fragments were dephosphorylated with shrimp alkaline phosphatase from Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Product No. 1758250. After gel electrophoresis, cosmid fragments ranging in size from 1500 to 2000 bp were separated with a QiaExII Gel Extraction Kit (Product No. 20021, manufactured by Qiagen, Hilden, Germany).

DNA of the sequencing vector pZero-1 obtained from the manufacturer (Invitrogen, Groningen, The Netherlands, Product Description Zero Background Cloning Kit, Product No. K2500-01) was subjected to restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI). , Product No. 27-0868-04). Linkage of cosmid fragments in the sequencing vector pZero-1 was performed as described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, and DNA mixtures were prepared using T4 ligase ( Manufacturer: Pharmacia Biotech, Freiburg, Germany). The linking mixture is then e. Electroporation into E. coli strain DH5αMCR [Grant, 1990, Proceedings of the National Academy of Sciences USA, 87: 4645-4649] [Tauch et al., 1994, FEMS Microbiol Letters, 123: 343-7] And plated on LB agar (Lennox, 1955, Virology, 1: 190) containing 50 μg / ml zeocin.                 

Plasmid preparation of recombinant clones was performed using Biorobot 9600 (Product No. 900200, manufactured by Qiagen, Hilden, Germany). Sequencing is described by the dideoxy chain termination method of Sanger et al., 1977, Proceedings of the National Academies of Sciences USA, 74: 5463-5467 (Zimmermann et al., 1990, Nucleic Acids). Research, 18: 1067]. "RR dRhodamin Terminator Cycle Sequencing Kit" (PE Applied Biosystems, Product No. 403044, Weiterstadt, Germany) was used. Analysis of isolation and sequencing reactions by gel electrophoresis was carried out using the "ABI Prism 377" sequencer (PE Applied Biosystems, Weiterstadt, Germany) using the "Rotiphoresis NF Acrylamide / Bisacrylamide" Gel (29: 1) (Product No. A124). .1, manufactured by Roth, Karlsruhe, Germany.

The raw sequence data obtained were then processed using the Staden program package (Ref. 1986, Nucleic Acids Research, 14: 217-231) version 97-0. Individual sequences of pZero1 derivatives were combined into successive contigs. Computer-assisted cryptographic domain analysis was made using the XNIP program (Staden, 1986, Nucleic Acids Research, 14: 217-231). Further analysis is provided by the "BLAST search program" for non-redundant databanks of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) [Altschul et al., 1997, Nucleic Acids Research, 25: 3389-3402. ] Was used.

The obtained nucleotide sequence is shown in SEQ ID NO: 1. Analysis of the nucleotide sequence shows an open reading frame of 933 base pairs, named lysR2 gene. The lysR2 gene encodes a polypeptide of 310 amino acids.

Example 3

Preparation of Integration Vectors for Integrative Mutagenesis of the lysR2 Gene

From strain ATCC 13032, chromosomal DNA was isolated by the method of Eikmanns et al., Microbiology 140: 1817-1828 (1994). Seed from Example 2. Based on the sequence of the lysR2 gene known for glutamicum, the following oligonucleotides were selected for polymerase chain reaction (see SEQ ID NO: 4 and SEQ ID NO: 5):

The primers shown were synthesized by MWG Biotech (Ebersberg, Germany), and the PCR reactions were standard PCR methods of Innis et al., PCR protocols., A guide to methods and applications, 1990, Academic Press. Was performed. With the help of the polymerase chain reaction, an internal fragment of the lysR2 gene of size 439 bp was isolated and represented by SEQ ID NO: 3.

The amplified DNA fragments were prepared using the TOPO TA Cloning Kit (Invitrogen Corporation, Carlsbad, CA, USA; Catalog Number K4500-01), vector pCR2.1-TOPO [Mead et al., (1991) Bio / Technology 9: 657-663].

Then, this. E. coli strain TOP10F was transformed using ligation batches. Hanahan, DNA cloning. A practical approach. Vol. I, IRL-Press, Oxford, Washington DC, USA, 1985]. Selection of plasmid-containing cells plated the transfection batch on LB agar supplemented with 25 mg / L kanamycin (Sambrook et al., Molecular cloning: A Laboratory Press, Cold Spring Harbor, NY, 1989). It was performed by. Plasmid DNA was isolated from the transformants with the help of the QIAprep Spin Miniprep Kit (Qiagen), digested with restriction enzyme EcoRI and examined by subsequent agarose gel electrophoresis (0.8%). The plasmid was named pCR2.1lysR2int.

Example 4

Integrated Mutagenesis of the lysR2 Gene in Lysine Producer DSM 5715 and Valine Producer FERM BP-1763

The vector pCR2.1lysR2int described in Example 3 was subjected to Corynebacterium glutamicum DSM 5715 by electroporation by Tauch et al., FEMS Microbiological Letters, 123: 343-347 (1994) and Electroporation into Brevibacterium lactopfermentum FERM BP-1763. Strain DSM 5715 is an AEC-resistant lysine producer (EP-B-435 132). Strain FERM BP-1763 is a valine producer in need of isoleucine and methionine (US-A-5,188,948). The vector pCR2.1lysR2int cannot replicate independently in DSM 5715 or FERM BP-1763 and is maintained intracellular only when integrated into the chromosome of DSM 5715 or FERM BP-1763. The selection of clones with pCR2.1lysR2int integrated into the chromosome was found in LB agar supplemented with 15 mg / L kanamycin [Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 ^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY].

For integration detection, lysR2int fragments were labeled by the method of "The DIG System Users Guide for Filter Hybridization" (Boehringer Mannheim GmbH, Mannheim, Germany, 1993) using a Dig hybridization kit (Boehringer). Potential integration of chromosomal DNA was isolated by the method of Eikmanns et al., Microbiology 140: 1817-1828 (1994), and in each case were cleaved using restriction enzymes SalI, SacI and HindIII. . The formed fragments were separated by agarose gel electrophoresis and hybridized at 68 ° C. using a Dig hybridization kit (Boehringer). The plasmid pCR2.1lysR2int mentioned in Example 3 was inserted into the chromosome lysR2 gene of DSM 5715 and FERM BP-1763. The strains were named DSM5715 :: pCR2.1lysR2int and FERM BP-1763 :: pCR2.1lysR2int.

Example 5

Preparation of L-lysine and L-valine

Seed obtained in Example 4. Glutamicum and b. Lactofermentum strains DSM5715 :: pCR2.1lysR2int and FERM BP-1763 :: pCR2.1lysR2int were cultured in a nutrient medium suitable for the production of L-lysine and L-valine, and the L-lysine and L-valine content was extracted from the culture supernatant. Measured.

To this end, the strains were first incubated at 33 ° C. for 24 hours on agar plates containing the corresponding antibiotic (brain-heart agar containing kanamycin (25 mg / L)). Starting from this agar plate culture, the precultures were seeded (10 ml medium in a 100 ml conical flask). Complete medium CgIII was used as the medium for preculture.

Badge Cg III

NaCl 2.5g / ℓ

Bakto-Peptone 10g / ℓ

Bacterium-Yeast Extract 10g / ℓ

Glucose (autoclaved separately) 2% (w / v)

pH was adjusted to pH 7.4.

Kanamycin (25 mg / L) was added to the medium. The preculture was incubated at 240 ° C. for 24 hours at 240 rpm on a shaker. The main culture was seeded from the preculture so that the initial OD (660 nm) of the main culture was 0.1 OD. Medium MM was used for the main culture.

Badge MM

CSL (corn immersion liquid) 5g / ℓ

MOPS 20g / ℓ

50 g / l glucose (autoclaved separately)

salt:

(NH ₄ ) ₂ SO ₄ 25g / ℓ

KH ₂ PO ₄ 0.1 g / ℓ

MgSO ₄ * 7H ₂ O 1.0 g / ℓ

CaCl ₂ * 2H ₂ O 10 mg / l

FeSO ₄ * 7H ₂ O 10 mg / l

MnSO ₄ * H ₂ O 5.0 mg / l

Biotin (sterile-filtered) 0.3 mg / l

Thiamine * HCl (sterile-filtered) 0.2 mg / l

CaCO ₃ 25g / ℓ

CSL, MOPS and salt solutions were adjusted to pH 7 with aqueous ammonia and autoclaved. Thereafter, sterile substrate and vitamin solution as well as CaCO ₃ were added and then autoclaved in the dry state. For incubation of DSM 5715, 0.1 g / l leucine was additionally added to the medium. For incubation of FERM BP-1763, 0.1 g / l isoleucine and 0.1 g / l methionine were additionally added to the medium.

Cultivation was performed in 10 ml volumes in 100 ml conical flasks with baffles. Kanamycin (25 mg / L) was added. Cultivation was performed at 33 ° C. and 80% atmospheric humidity.

After 72 hours, OD was measured at a measurement wavelength of 660 nm using Biomek 1000 (Beckmann Instruments GmbH, Munich). The L-lysine and L-valine amounts formed were measured by an amino acid analyzer (Eppendorf-BioTronik, Hamburg, Germany) by post-column derivatization with ion exchange chromatography and ninhydrin detection.

The experimental results are shown in Table 1 and Table 2.

Strain OD (660 nm) Lysine HCl g / L DSM5715 7.5 13.01 DSM5715 :: pCR2.1lysR2int 7.2 14.68

Strain OD (660 nm) Valine g / ℓ FERM BP-1763 12.1 7.49 FERM BP-1763 :: pCR2.1lysR2int 13.4 10.90

Figure 1: Map of plasmid pCR2.1lysR2int

Abbreviations and names used have the following meanings:

KmR: kanamycin resistance gene

EcoRI: cleavage site of restriction enzyme EcoRI

lysR2int: internal fragment of the lysR2 gene

ColE1 ori: origin of replication of the plasmid ColE1

<110> Degussa AG <120> Nucleotide sequences which code for the lysR2 gene <130> 000181 BT <150> DE 100 39 047.1 <151> 2000-08-10 <150> DE 101 10 346.8 <151> 2001-03-03 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 1364 <212> DNA <213> Corynebacterium glutamicum <220> <221> CDS (232) .. (1161) <223> lysR2 gene <400> 1 cctgcgtgca ataaagacca ttgaaagcag caagaccggc ggccagcatc gcaaacacag 60 cgcgcttgta attgcgtgtt cctcgctcga tgccttcgtg gccttcgtgg ccttcgtgtg 120 cctcgacctt gctatctatt gcttggctca tggagttcat catgcgccaa cagcaaatat 180 tagtaaaatg ttagaaatag ctgtttttga ttcactttgt gcatgtaggc t gtg acc 237                                                          Met thr                                                            One atg ggc aac gac ggc gga gac ctg cga atc gac gac cta cgc agc ttc 285 Met Gly Asn Asp Gly Gly Asp Leu Arg Ile Asp Asp Leu Arg Ser Phe           5 10 15 att tca gtc gct caa tca ggc cac ctc acc gaa act gcc gaa aga tta 333 Ile Ser Val Ala Gln Ser Gly His Leu Thr Glu Thr Ala Glu Arg Leu      20 25 30 ggc atc ccg cag ccc aca ctt tcc aga cga atc agc cga gtg gaa aaa 381 Gly Ile Pro Gln Pro Thr Leu Ser Arg Arg Ile Ser Arg Val Glu Lys  35 40 45 50 cac gca ggc acc cca ctt ttc gac cgc gcc ggc cgc aaa ctc gtc ctc 429 His Ala Gly Thr Pro Leu Phe Asp Arg Ala Gly Arg Lys Leu Val Leu                  55 60 65 aac caa cga ggc cac gcc ttc ctc aac cac gcc agc gcc atc gtc gca 477 Asn Gln Arg Gly His Ala Phe Leu Asn His Ala Ser Ala Ile Val Ala              70 75 80 gaa ttc aac tcc gcc gca act gaa atc aaa cgc ctc atg gac cca gaa 525 Glu Phe Asn Ser Ala Ala Thr Glu Ile Lys Arg Leu Met Asp Pro Glu          85 90 95 aaa ggc aca atc cga ctg gac ttc atg cat tcc ttg ggc act tgg atg 573 Lys Gly Thr Ile Arg Leu Asp Phe Met His Ser Leu Gly Thr Trp Met     100 105 110 gtc ccc gaa ctt atc cga aca ttc cgc gcc gaa cac ccc aac gta gaa 621 Val Pro Glu Leu Ile Arg Thr Phe Arg Ala Glu His Pro Asn Val Glu 115 120 125 130 ttc caa ctc cac caa gcg gca gca atg ctc ctg gta gat cgt gtt ttg 669 Phe Gln Leu His Gln Ala Ala Ala Met Leu Leu Val Asp Arg Val Leu                 135 140 145 gct gat gaa act gac ctc gca tta gtt ggc ccc aaa cct gcc gag gtt 717 Ala Asp Glu Thr Asp Leu Ala Leu Val Gly Pro Lys Pro Ala Glu Val             150 155 160 ggt acc tct tta ggg tgg gcg cca ctg ctt cgt caa cga ctt gcc cta 765 Gly Thr Ser Leu Gly Trp Ala Pro Leu Leu Arg Gln Arg Leu Ala Leu         165 170 175 gct gtt ccc gca gat cac cgg ctt gcc tcc ttt tct ggc caa gga gaa 813 Ala Val Pro Ala Asp His Arg Leu Ala Ser Phe Ser Gly Gln Gly Glu     180 185 190 ttg ccg ttg att act gcg gcg gaa gaa cct ttc gtg gcg atg cga gca 861 Leu Pro Leu Ile Thr Ala Ala Glu Glu Pro Phe Val Ala Met Arg Ala 195 200 205 210 ggt ttc ggc acc cga ctc ctc atg gat gca tta gcc gaa gaa gcc ggt 909 Gly Phe Gly Thr Arg Leu Leu Met Asp Ala Leu Ala Glu Glu Ala Gly                 215 220 225 ttt gtt ccc aat gtg gtt ttc gaa tcc atg gaa ctc acc acc gtc gca 957 Phe Val Pro Asn Val Val Phe Glu Ser Met Glu Leu Thr Thr Val Ala             230 235 240 ggg ctt gtc agc gca ggt ctc ggc gtt ggt gtg gtt ccg atg gat gat 1005 Gly Leu Val Ser Ala Gly Leu Gly Val Gly Val Val Pro Met Asp Asp         245 250 255 ccg tac ctt ccc aca gtg gga atc gtg caa cgc cca ctt agt cca ccc 1053 Pro Tyr Leu Pro Thr Val Gly Ile Val Gln Arg Pro Leu Ser Pro Pro     260 265 270 gct tat agg gaa cta ggt ttg gtg tgg cga ctc aac gcg ggg ccg gca 1101 Ala Tyr Arg Glu Leu Gly Leu Val Trp Arg Leu Asn Ala Gly Pro Ala 275 280 285 290 cct gcg gtg gat aac ttc cgg aag ttc gtg gcg gga tcg agg tat gca 1149 Pro Ala Val Asp Asn Phe Arg Lys Phe Val Ala Gly Ser Arg Tyr Ala                 295 300 305 tta gaa gag ggc tgagctgtaa gtgtcgtggg tgccgtttta aggggttgag 1201 Leu Glu Glu Gly             310 ttttcccgat gactaggagt tggtccagat tgtgcgttag gggcccctag gggcgattct 1261 ggggctggtg tttttgtggc catgggggtt ggtgttaatc ctggaggctt gctgcaagat 1321 tgctgttaaa cttctcgtca cggatcgctt gggaagcctg gaa 1364 <210> 2 <211> 310 <212> PRT <213> Corynebacterium glutamicum <400> 2 Met Thr Met Gly Asn Asp Gly Gly Asp Leu Arg Ile Asp Asp Leu Arg   1 5 10 15 Ser Phe Ile Ser Val Ala Gln Ser Gly His Leu Thr Glu Thr Ala Glu              20 25 30 Arg Leu Gly Ile Pro Gln Pro Thr Leu Ser Arg Arg Ile Ser Arg Val          35 40 45 Glu Lys His Ala Gly Thr Pro Leu Phe Asp Arg Ala Gly Arg Lys Leu      50 55 60 Val Leu Asn Gln Arg Gly His Ala Phe Leu Asn His Ala Ser Ala Ile  65 70 75 80 Val Ala Glu Phe Asn Ser Ala Ala Thr Glu Ile Lys Arg Leu Met Asp                  85 90 95 Pro Glu Lys Gly Thr Ile Arg Leu Asp Phe Met His Ser Leu Gly Thr             100 105 110 Trp Met Val Pro Glu Leu Ile Arg Thr Phe Arg Ala Glu His Pro Asn         115 120 125 Val Glu Phe Gln Leu His Gln Ala Ala Ala Met Leu Leu Val Asp Arg     130 135 140 Val Leu Ala Asp Glu Thr Asp Leu Ala Leu Val Gly Pro Lys Pro Ala 145 150 155 160 Glu Val Gly Thr Ser Leu Gly Trp Ala Pro Leu Leu Arg Gln Arg Leu                 165 170 175 Ala Leu Ala Val Pro Ala Asp His Arg Leu Ala Ser Phe Ser Gly Gln             180 185 190 Gly Glu Leu Pro Leu Ile Thr Ala Ala Glu Glu Pro Phe Val Ala Met         195 200 205 Arg Ala Gly Phe Gly Thr Arg Leu Leu Met Asp Ala Leu Ala Glu Glu     210 215 220 Ala Gly Phe Val Pro Asn Val Val Phe Glu Ser Met Glu Leu Thr Thr 225 230 235 240 Val Ala Gly Leu Val Ser Ala Gly Leu Gly Val Gly Val Val Pro Met                 245 250 255 Asp Asp Pro Tyr Leu Pro Thr Val Gly Ile Val Gln Arg Pro Leu Ser             260 265 270 Pro Pro Ala Tyr Arg Glu Leu Gly Leu Val Trp Arg Leu Asn Ala Gly         275 280 285 Pro Ala Pro Ala Val Asp Asn Phe Arg Lys Phe Val Ala Gly Ser Arg     290 295 300 Tyr Ala Leu Glu Glu Gly 305 310 <210> 3 <211> 439 <212> DNA <213> Corynebacterium glutamicum <220> <223> lysR2int <400> 3 ccatcgtcgc agaattcaac tccgccgcaa ctgaaatcaa acgcctcatg gacccagaaa 60 aaggcacaat ccgactggac ttcatgcatt ccttgggcac ttggatggtc cccgaactta 120 tccgaacatt ccgcgccgaa caccccaacg tagaattcca actccaccaa gcggcagcaa 180 tgctcctggt agatcgtgtt ttggctgatg aaactgacct cgcattagtt ggccccaaac 240 ctgccgaggt tggtacctct ttagggtggg cgccactgct tcgtcaacga cttgccctag 300 ctgttcccgc agatcaccgg cttgcctcct tttctggcca aggagaattg ccgttgatta 360 ctgcggcgga agaacctttc gtggcgatgc gagcaggttt cggcacccga ctcctcatgg 420 atgcattagc cgaagaagc 439 <210> 4 <211> 20 <212> DNA <213> Corynebacterium glutamicum <220> <223> Primer lysR2intA <400> 4 ccatcgtcgc agaattcaac 20 <210> 5 <211> 20 <212> DNA <213> Corynebacterium glutamicum <220> Primer lysR2intB <400> 5 gcttcttcgg ctaatgcatc 20

Claims (22)

a coryneform consisting of a) a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, and c) a polynucleotide sequence encoding a lysR2 gene selected from the group consisting of a polynucleotide complementary to the polynucleotide of a) Polynucleotides isolated from bacteria. The polynucleotide of claim 1, which is recombinant DNA capable of replicating in coryneform bacteria. The polynucleotide of claim 1, which is RNA. The polynucleotide of claim 2, which is a replicable DNA consisting of one or more of the sequences corresponding to sequence (i) within the scope of (i) the nucleotide sequence of SEQ ID NO: 1 or (ii) the degenerate gene code. The polynucleotide of claim 2, wherein the polynucleotide consists of the nucleic acid sequence set forth in SEQ ID NO: 1. 6.1 contains an internal fragment of the citA gene of size 439 bp, 6.2 is a restriction map shown in FIG. 6.3 a depository institution [German Collection of Microorganisms and Cell Cultures]. Vector pCR2.1lysR2int, deposited as DSM 13617 in E. coli strain TOP10F / pCR2.1lysR2int. Coryneform bacteria with attenuated lysR2 gene. a) producing the desired L-amino acid and fermenting the bacterium with at least the lysR2 gene attenuated, b) concentrating the desired product in the medium or in the cells of the bacterium, and c) separating the L-amino acid A method for producing L-amino acid, comprising performing a. The method of claim 8, wherein the L-amino acids are L-lysine and L-valine. The method of claim 8, wherein the bacterium is further used to further enhance additional genes of the biosynthetic pathway of the desired L-amino acid. The method of claim 8, wherein the bacterium employs bacteria that have at least partially eliminated metabolic pathways that reduce the formation of the desired L-amino acids. The method of claim 8, wherein the expression of the polynucleotide (s) encoding the lysR2 gene is reduced. The method of claim 8, wherein the polynucleotide lysR2 reduces the regulatory properties of the polypeptide encoding. The method of claim 8, 14.1 dapA gene encoding a dihydrodipicolinate synthase, 14.2 eno gene encoding enolase, 14.3 zwf gene, which encodes a zwf gene product, 14.4 pyc gene encoding pyruvate carboxylase, 14.5 lysE gene, encoding lysine excretion 14.6 the lysC gene, which encodes a feedback resistant aspartate kinase, 14.7 A method for preparing L-lysine, wherein at least one gene selected from the group consisting of a zwa1 gene encoding a Zwa1 protein is fermented simultaneously. The method of claim 8, 15.1 pck gene encoding phosphoenol pyruvate carboxykinase, 15.2 pgi gene encoding glucose 6-phosphate isomerase, 15.3 poxB gene encoding pyruvate oxidase, 15.4 zwa2 gene, which encodes a Zwa2 protein 15.5 the hom gene encoding homoserine dehydrogenase, and 15.7 A method for simultaneously attenuating one or more genes selected from the group consisting of panD genes encoding aspartate decarboxylase. The method of claim 8, 16.1 ilvBN gene, encoding acetohydroxy acid synthase, 16.2 ilvD gene encoding dihydroxy acid dehydratase, 16.3 A method for preparing L-valine, wherein the at least one gene selected from the group consisting of mqo genes encoding maleate: quinone oxidoreductase ferment simultaneously bacteria. 17. The method according to any one of claims 8 to 16, wherein the microorganism of Corynebacterium glutamicum or Brevibacterium lactofermentum species is used. A nucleic acid encoding a transcriptional regulator LysR2 or having a high similarity with the sequence of the lysR2 gene, comprising using a polynucleotide sequence according to any one of claims 1 to 4 as a hybridization probe, or a polynucleotide or gene How to find RNA, cDNA and DNA for isolation. The method of claim 18 wherein an array, microarray or DNA chip is used. A probe or primer consisting of the polynucleotide of SEQ ID NO: 4 or SEQ ID NO: 5. delete delete

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