KR20050018797A - Production of L-lysine by genetically modified Corynebacterium glutamicum strains - Google Patents

Abstract

The present invention, in addition to an open reading frame (ORF) encoding the synthesis of a protein or RNA, one or more copies present in the natural site (locus) of a gene or allele, in each case such an open reading frame (ORF) By coryneform bacteria containing the second, optionally third or fourth copy of the gene or allele in the chromosome, respectively, in the second, optionally third or fourth site, and by fermentation of these bacteria The present invention relates to a method for producing a chemical compound.

Description

Production of L-lysine by genetically modified Corynebacterium glutamicum strains

The base pair numbers described are approximate values obtained in terms of reproducibility of the measurements.

1 is a map of plasmid pK18mobsacBglu1_1.

The abbreviations or symbols used have the following meanings:

KanR: Kanamycin Resistance Gene

HindIII: cleavage site of the restriction enzyme HindIII

BamHI: cleavage site of the restriction enzyme BamHI

lysC: lysC ^FBR allele, lysC T311I

'gluA: the 3' terminal fragment of the gluA gene

gluB ': 5' terminal fragment of the gluB gene

'gluB: the 3' terminal fragment of the gluB gene

gluC ': 5' terminal fragment of the gluC gene

sacB: sacB gene

RP4mob: mob region with a replica (oriT) for delivery

oriV: Replica V

2 is a map of plasmid pK18mobsacBaecD1_1.

The abbreviations or symbols used have the following meanings:

KanR: Kanamycin Resistance Gene

SalI: cleavage site of restriction enzyme SalI

lysC: lysC ^FBR allele, lysC T311I

aecD ': 5' terminal fragment of the aecD gene

'aecD: the 3' terminal fragment of the aecD gene

sacB: sacB gene

RP4mob: mob region with a replica (oriT) for delivery

oriV: Replica V

3 is a map of plasmid pK18mobsacBpck1_1.

The abbreviations or symbols used have the following meanings:

KanR: Kanamycin Resistance Gene

BamHI: cleavage site of the restriction enzyme BamHI

lysC: lysC ^FBR allele, lysC T311I

pck ': 5' terminal fragment of the pck gene

'pck: the 3' terminal fragment of the pck gene

sacB: sacB gene

RP4mob: mob region with a replica (oriT) for delivery

oriV: Replica V

4 is a map of plasmid pK18mobsacBgluB2_1.

The abbreviations or symbols used have the following meanings:

KanR: Kanamycin Resistance Gene

SalI: cleavage site of restriction enzyme SalI

EcoRI: cleavage site of restriction enzyme EcoRI

BamHI: cleavage site of the restriction enzyme BamHI

ddh: ddh gene

gluA: the gluA gene

gluB ': 5' terminal fragment of the gluB gene

'gluB: the 3' terminal fragment of the gluB gene

gluC: the gluC gene

gluD ': 5' terminal fragment of the gluD gene

sacB: sacB gene

RP4mob: mob region with a replica (oriT) for delivery

oriV: Replica V

5 is a map of plasmid pK18mobsacBaecD2_1.

The abbreviations or symbols used have the following meanings:

KanR: Kanamycin Resistance Gene

EcoRI: cleavage site of restriction enzyme EcoRI

SalI: cleavage site of restriction enzyme SalI

dapA: dapA gene

aecD ': 5' terminal fragment of the aecD gene

'aecD: the 3' terminal fragment of the aecD gene

sacB: sacB gene

RP4mob: mob region with a replica (oriT) for delivery

oriV: Replica V

6 is a map of pK18mobsacBpck1_3.

The abbreviations or symbols used have the following meanings:

KanR: Kanamycin Resistance Gene

pyc: pyc allele, pyc P458S

pck ': 5' terminal fragment of the pck gene

'pck: the 3' terminal fragment of the pck gene

sacB: sacB gene

RP4mob: mob region with a replica (oriT) for delivery

oriV: Replica V

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 A depositary certificate of the original deposit issued in accordance with Rule 7.1 of the following international depositary bodies:

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I. Identification of Microorganisms Identification designated by the depositary: DSM 12866glu :: lysC Accession No. by International Depository: DSM 15039 II. Scientific nature and / or taxonomic position The microorganisms in column I are accompanied by a document describing the following matters: (x) Scientific properties (x) Taxonomic location (marked with x if applicable) III. Receipt and Entrustment The international depositary authority shall be entrusted with the microorganisms of the column I received on June 5, 2002 (original deposit) ^1. IV. Receipt of Transition Request The microorganisms in column I were received by the International Depositary on (Date of Deposit) and the request for conversion of the original deposits to deposits under the Budapest Treaty was received by the Agency on the date of receipt of the conversion request. V. International Depositary Organizations Name: DSMZ-Doyeck Samrong von Microorganics Menmount Zellkulenten Gembeha Address: De-38124 Brownshoe Bikes Signature (s) of person (s) or certificate officer (s) having authority to represent the international depositary organization: Signature date: 6 June 2002

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I. Depositary II. Microbial Identification Real Name: Degusa Age Address: 33790 Halle (West) Kantstrasse 2 Accession No. by DSM 15039 Deposit Date or Transfer Date ¹ : June 5, 2002 III. Survival statement Microbial viability of the column II was tested on June 5, 2002. ² The microorganism is (×) ³ viable on test day. () ³ No longer viable. IV. Conditions at the time of survival test ⁴ V. International Depositary Organizations Name: DSMZ-Doyeck Samrong Pon Microorganism Menmount Zellkulenten Gembeha Address: De-38124 Braunschweig Signature (s) of person (s) or certificate officer (s) having authority to represent the international depositary organization: Signature date: 6 June 2002

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Budapest Treaty on International Approval of Microbial Deposits in Patent Proceedings

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 A depositary certificate of the original deposit issued in accordance with Rule 7.1 of the following international depositary bodies:

 Degusa Age

 33790 Halle (West) Kantstrasse 2

I. Identification of Microorganisms Identification indication decided by depositary: DH5alphamcr / pK18mobsacBaecD1_1 Accession No. by International Depository: DSM 15040 II. Scientific nature and / or taxonomic position The microorganisms in column I are accompanied by a document describing the following matters: (x) Scientific properties (x) Taxonomic location (marked with x if applicable) III. Receipt and Entrustment The international depositary authority shall be entrusted with the microorganisms of the column I received on June 5, 2002 (original deposit) ^1. IV. Receipt of Transition Request The microorganisms in column I were received by the International Depositary on (Date of Deposit) and the request for conversion of the original deposits to deposits under the Budapest Treaty was received by the Agency on the date of receipt of the conversion request. V. International Depositary Organizations Name: DSMZ-Doyeck Samrong von Microorganics Menmount Zelkulturen GmbH Signature (s) of person (s) or certificate officer (s) having authority to represent the international depositary organization: Signature date: 6 June 2002

 1. Where Rule 6.4 (d) applies, this date shall be the date on which the International Depositary Authority is certified.

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Survival Statement issued in accordance with Rule 10.2 set forth by the following International Depositaries:

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I. Depositary II. Microbial Identification Real Name: Degusa Age Address: 33790 Halle (West) Kantstrasse 2 Accession No. by DSM 15040 Deposited Date or Transfer Date ¹ : June 5, 2002 III. Survival statement Microbial viability of the column II was tested on June 5, 2002. ² The microorganism is (×) ³ viable on test day. () ³ No longer viable. IV. Conditions at the time of survival test ⁴ V. International Depositary Organizations Name: DSMZ-Doyeck Samrong Pon Microorganism Menmount Zellkulenten Gembeha Address: De-38124 Braunschweig Signature (s) of person (s) or certificate officer (s) having authority to represent the international depositary organization: Signature date: 6 June 2002

1.The date of original deposit or, if a new deposit or transfer has been made, indicates the most recent date (new or previous deposit date)

2. Where Rule 10.2 (a) (ii) and (iii) applies, the most recent survival test.

3. Mark the cross with a cross.

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 A depositary certificate of the original deposit issued in accordance with Rule 7.1 of the following international depositary bodies:

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I. Identification of Microorganisms Identification indication decided by depositary: DH5alphamcr / pK18mobsacBglu1_1 Accession No. by International Depository: DSM 14243 II. Scientific nature and / or taxonomic position The microorganisms in column I are accompanied by a document describing the following matters: (x) Scientific properties (x) Taxonomic location (marked with x if applicable) III. Receipt and Entrustment The international depositary authority shall be entrusted with the microorganisms of the column I received on April 20, 2001 (original deposit) ^1. IV. Receipt of Transition Request The microorganisms in column I were received by the International Depositary on (Date of Deposit) and the request for conversion of the original deposits to deposits under the Budapest Treaty was received by the Agency on the date of receipt of the conversion request. V. International Depositary Organizations Name: DSMZ-Doyeck Samrong von Microorganics Menmount Zellkulenten Gembeha Address: De-38124 Brownshoe Bikes Signature (s) of person (s) or certificate officer (s) having authority to represent the international depositary organization: Signature date: April 26, 2001

 1. Where Rule 6.4 (d) applies, this date shall be the date on which the International Depositary Authority is certified.

Budapest Treaty on International Approval of Microbial Deposits in Patent Proceedings

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Survival Statement issued in accordance with Rule 10.2 set forth by the following International Depositaries:

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33790 Halle / Quincebeck Kantstrasse 2

I. Depositary II. Microbial Identification Real Name: Degusa Age Address: 33790 Halle / Quinsebeck Kantstrasse 2 Accession No. by DSM 14243 Deposited Date or Transfer Date ¹ : April 20, 2001 III. Survival statement Microbial viability of the column II was tested on April 20, 2001. ² The microorganism is (×) ³ viable on test day. () ³ No longer viable. IV. Conditions at the time of survival test ⁴ V. International Depositary Organizations Name: DSMZ-Doyeck Samrong Pon Microorganism Menmount Zellkulenten Gembeha Address: De-38124 Braunschweig Signature (s) of person (s) or certificate officer (s) having authority to represent the international depositary organization: Signature date: April 26, 2001

1.The date of original deposit or, if a new deposit or transfer has been made, indicates the most recent date (new or previous deposit date)

2. Where Rule 10.2 (a) (ii) and (iii) applies, the most recent survival test.

3. Mark the cross with a cross.

4. If information is required and the test result is negative, enter it.

Chemical compounds are to be understood in particular to mean amino acids, vitamins, nucleosides and nucleotides. Biosynthetic pathways of these compounds are known in the art and available.

The amino acids are preferably L-amino acids, in particular L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine, L-cysteine, L-valine, L Group consisting of methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-proline and L-arginine and salts thereof, in particular L Means a proteingenic L-amino acid selected from the group consisting of lysine, L-methionine and L-threonine. Very particular preference is given to L-lysine.

Protein source amino acids are understood to mean natural proteins, ie amino acids found in proteins of microorganisms, plants, animals and humans.

Vitamins, in particular, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B12 (cyanocobalamin), nicotinic acid / nicotinamide, vitamin M (folic acid) and vitamin E ( Tocopherol) and salts thereof, with pantothenic acid being preferred.

Nucleosides and nucleotides mean, in particular, S-adenosylmethionine, inosine-5'-monophosphate and guanosine-5'-monophosphate and salts thereof.

Coryneform bacteria means, in particular, bacteria of the genus Corynebacterium. Among the genus Corynebacterium, species of Corytebacterium glutamicum, Corynebacterium ammoniagenes and Corynebacterium thermoaminogenes are preferred. Information on the classification of strains of this group of bacteria is found in particular in Kampfer and Kroppenstedt (Canadian Jounal of Microbiology 42, 989-1005 (1996)) and in US-A-5, 250, 434. can see.

Suitable strains of Corynebacterium glutamicum species (C. glutamicum) are particularly known wild type strains, namely

Corynebacterium glutamicum ATCC 13032,

Corynebacterium acetoglutamicum ATCC 15806,

Corynebacterium acetoacidophilum ATCC 13870,

Corynebacterium lilium ATCC15990,

Corynebacterium melasesecola ATCC17965,

Corynebacterium herculis ATCC13868,

Arthrobacter sp. ATCC243,

Brevibacterium chang-fua ATCC14017,

Brevibacterium flaum ATCC 14067,

Brevibacterium lactofermentum ATCC 13869,

Brevibacterium divaricatum ATCC14020,

Brevibacterium Taipei (Taipei ATCC13744) and

Microbacterium ammoniaphilum ATCC21645; And

There are mutations or strains that produce chemical compounds, as known from the prior art, produced therefrom.

Suitable strains of Corynebacterium ammonia genes (C. ammonia genes) are particularly known wild type strains, namely

Brevibacterium ammonia genes ATCC6871,

Brevibacterium ammonia genes ATCC15137 and

Corynebacterium spp. ATCC21084; And

There are mutations or strains that produce chemical compounds, as known from the prior art, produced therefrom.

Suitable strains of Corynebacterium mother amino genes (C. mother amino genes) are particularly known wild type strains, namely

Corynebacterium mother aminogenes FERM BP-1539,

Corynebacterium mother amino genes FERM BP-1540,

Corynebacterium mother aminogenes FERM BP-1541 and

Corynebacterium mother aminoaminoses FERM BP-1542; And

There are mutations or strains that produce chemical compounds, as known from the prior art, produced therefrom.

Strains with the name "ATCC" can be obtained from the International Deposit Organization (Manassas, Va., USA). Strains with the name "FERM" can be obtained from the National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan). The mentioned Corynebacterium mother aminogenes strains (FERM BP-1539, FERM BP-1540, FERM BP-1541 and FERM BP-1542) are described in US-A 5, 250, 434. have.

An open reading frame (ORF) represents a portion of a nucleotide sequence capable of encoding or encoding a protein or polypeptide or ribonucleic acid, according to the prior art, in which no function can be specified.

After the function for that nucleotide sequence portion is specified, it is generally referred to as a gene.

Alleles are generally understood to mean another type of a given gene. This type is distinguished by a difference in nucleotide sequence.

In the context of the present invention, endogenous, ie species-specific, open reading frames, genes or alleles are preferably used. These are understood to mean open reading frames, genes or alleles or nucleotide sequences thereof which exist within a population of species such as, for example, Corynebacterium glutamicum.

In the context of the present invention, "an open reading frame (ORF), a gene or an allele of a gene present at a natural site (gene locus)" means a contiguous ORF as present in the corresponding wild type or the corresponding parent or starting organism or It is understood to mean the position or site of the ORF or gene or allele relative to the gene or allele.

Thus, for example, the natural site of the lysC gene or lysC ^FBR allele, which encodes a "feedback" resistant aspartate kinase from Corynebacterium glutamicum, is in one aspect an orfX and leuA gene or an open type. LysC site or lysC locus or lysC gene site immediately adjacent the reading frame and immediately adjacent to the asd gene on the other side.

As compared with the wild type, "feedback" resistant aspartate kinase is aspartate with low sensitivity to inhibition by a mixture of lysine and threonine or a mixture of AEC (aminoethylcysteine) and threonine or lysine alone or AEC alone. It is understood to mean a kinase. Strains producing L-lysine typically contain this "feedback" resistant or desensitized aspartate kinase.

Nucleotide sequences of the chromosomes of Corynebacterium glutamicum are known from patent application EP-A-1108790 and are entrusted to the nucleotide sequence databank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK). It can be found at number AX114121. The nucleotide sequences of the orfX, leuA gene and asd gene have accession numbers AX120364 (orfX), AX123517 (leuA) and AX123519 (asd).

Also, for example, an institution [National Center for Biotechnology Information (NCBI, Bethesda, MD, USA)] or an institution [Swiss Institute of Bioinformatics (Swissprot, Geneva, Switzerland)] or an institution [Protein Information Resource Database (PIR, Washington, DC, USA)].

"In each case a second, optionally third or fourth site" is understood to mean a site that is different from the "natural site". It is also referred to hereinafter as "target site" or "target sequence". It is also called "integration site" or "transformation site". The nucleotide sequence present at this second, optionally third or fourth site, or corresponding site, is preferably in the chromosome and is generally not essential for growth and production of the desired chemical compound.

To produce coryneform bacteria according to the present invention, the nucleotide sequences of the desired ORF, gene or allele, optionally comprising expression and / or regulatory signals, are isolated, and the nucleotide sequences of the target sites are attached to the ends; They are preferably transferred to the desired coryneform bacteria using vectors that do not replicate in the coryneform bacteria or only at limited levels; The desired ORF, gene or allele is introduced at the target site, and the nucleotide sequence enables / enables episomal replication in the microorganism, the nucleotide sequence enables / disables translocation, and the nucleotide sequence confers resistance to antibiotics. These bacteria which do not remain at this target site are separated.

Thus, the present invention also provides

a) isolating nucleotide sequences of one or more desired ORFs, genes or alleles, optionally comprising expression and / or regulatory signals,

b) attaching the nucleotide sequence of the target site to the 5 'and 3' ends of the ORF, gene or allele,

c) Preferably, the nucleotide sequence of the desired ORF, gene or allele to which the nucleotide sequence of the target site is attached is introduced into a vector which does not replicate in coryneform bacteria or only at a limited level,

d) transferring the nucleotide sequence according to b) or c) into coryneform bacteria,

e) the nucleotide sequence (s) according to a) is introduced at the target site and the nucleotide sequence enables / enables episomal replication in the microorganism, the nucleotide sequence (s) that enables / enable translocation and resistance to antibiotics Provided are methods for producing coryneform bacteria that produce one or more chemical compounds, including isolating coryneform bacteria that do not remain at the target site.

Preferably, residues of the sequence of the vector or species-foreign DNA used, such as restriction cleavage sites, do not remain at the target site. Up to 24, preferably up to 12, particularly preferably up to 6 nucleotides upstream or downstream of said DNA of the introduced ORF, gene or allele are optionally left at the target site.

By means according to the invention, the productivity of coryneform bacteria or the fermentative method for producing chemical compounds is determined by the concentration (chemical compound formed on the basis of unit volume), yield (chemical compound formed on the basis of consumed carbon source). ) And at least 0.5 to 1.0% or at least 1.0 to 1.5% or at least 1.5 to 2.0% in terms of one or more properties selected from the group consisting of) and product formation rates (chemical compounds formed over time).

For example, guidance on conventional genetic engineering methods such as isolation of chromosomal DNA and plasmid DNA, handling of restriction enzymes, and the like can be found in Sambrook et al. (Molecular Cloning-A Laboratory Manual (1989) Cold Spring Harbor Laboratory Press). Guidance on transformation and conjugation in coryneform bacteria is described, in particular, in Therbach et al., Applied Microbiology and Biotechnology 29, 356-362 (1998); Schafer et al., Jounal of Bacteriology 172, 1663-1666 (1990) and Gene 145, 69-73 (1994); and Schwarzer and Puhler, Bio / Technology 9, 84-87 (1991).

Vectors capable of replicating only at limited levels are understood to mean plasmid vectors that do or do not replicate due to the action of the conditions under which the host or carrier is cultured. Thus, temperature-sensitive plasmids for coryneform bacteria that can only replicate at temperatures below 31 ° C. are described in Nakamura et al., US-A-6,303, 383.

In addition, the present invention, in addition to one or more copies of an open reading frame (ORF), gene or allele for the production of lysine present at a native site (gene locus), includes the open reading frame (ORF), gene or allele. A nucleotide sequence, a translocation, containing a second, optionally third, or fourth copy of the gene, respectively, in an integrated form, at the second, optionally third, or fourth site, wherein episomal replication is enabled / enable in the microorganism. Coryneform bacteria, especially bacteria of the genus Corynebacterium, characterized in that the nucleotide sequence which enables / enables the nucleotide sequence and the nucleotide sequence conferring resistance to antibiotics are not present at a particular second, optionally third or fourth site. to provide.

In addition, the present invention is the following steps:

a) in addition to one or more copies of an open reading frame (ORF), gene or allele for the production of lysine present in the natural site (gene locus), in each case the open reading frame (ORF), gene or allele A second, optionally a third, or a fourth copy of, respectively, in an integrated form, at the second, optionally third, or fourth site, wherein the nucleotide sequence, translocation, allows / enables episomal replication in the microorganism. Open reading of coryneform bacteria, in particular Corynebacterium glutamicum, where possible / enable nucleotide sequences and nucleotide sequences conferring resistance to antibiotics are not present at a particular second, optionally third or fourth site Fermentation under conditions that allow expression of the frame (ORF), gene or allele;

b) concentrating L-lysine in the fermentation broth;

c) separating L-lysine from the fermentation broth, optionally

d) further providing a process for producing L-lysine, comprising the step of from 0 to 100% by weight of constituents from the fermentation broth and / or biomass.

An "open reading frame (ORF), gene or allele for copying lysine production" is any, preferably endogenous, open reading frame, gene or allele that may have the effect of improving lysine production when enhanced / overexpressed. It is understood to mean a gene. Enhancement is understood to mean an increase in the intracellular concentration or activity of a particular gene product, protein or enzyme.

These include, in particular, the following open reading frames, genes or alleles: accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysC ^FBR , lysE, msiK, opcA, oxyR, ppc, ppc ^FBR , pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T. These are summarized and described in Table 1.

These include, in particular, the lysC ^FBR allele encoding the "feedback" resistant aspartate kinase. Various lysC ^FBR alleles are summarized and described in Table 2.

The following lysC ^FBR allele is preferred: lysC A279T (substituted alanine for threonine at position 279 of aspartate kinase protein encoded according to SEQ ID NO: 2), lysC A279V (aspartate encoded according to SEQ ID NO: 2) Alanine substituted with valine at position 279 of the kinase protein, lysC S301F (serine substituted with phenylalanine at position 301 of the aspartate kinase protein encoded according to SEQ ID NO: 2), lysC T308I (as encoded according to SEQ ID NO: 2) Threonine is replaced by isoleucine at position 308 of the partate kinase protein, lysC S301Y (substituted with tyrosine at position 308 of the aspartate kinase protein encoded according to SEQ ID NO: 2), lysC G345D (SEQ ID NO: 2) Glycine is substituted for aspartic acid at position 345 of the aspartate kinase protein encoded according to lysC R320G (SEQ ID NO: Arginine is substituted for glycine at position 320 of the aspartate kinase protein encoded according to No. 2, lysC T311I (SEQ ID NO: 2). According to which threonine is substituted for isoleucine at position 311 of the aspartate kinase protein encoded), lysC S381F (serine is substituted for phenylalanine at position 381 of the aspartate kinase protein encoded according to SEQ ID NO: 2).

Particular preference is given to the lysC ^FBR allele lysC T311I, wherein the nucleotide sequence is set forth in SEQ ID NO: 3 with threonine substituted with isoleucine at position 311 of the aspartate kinase protein encoded according to SEQ ID NO: 2; The amino acid sequence of the encoded aspartate kinase protein is shown in SEQ ID NO: 4.

An open reading frame (ORF), gene or allele of the allele for the production of the desired lysine may in each case be incorporated into the second, optionally third or fourth site. The following open reading frames (ORFs), genes or nucleotide sequences can be used for this purpose in particular: aecD, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, mqo, pck, pgi, poxB and zwa2, in particular aecD, gluA, gluB, gluC, gluD and pck. These are summarized and described in Table 3.

Of course, the sites mentioned are not only the coding region of the mentioned open reading frame or gene, but also the regions or nucleotide sequences located upstream involved in expression and regulation, such as ribosomal binding sites, promoters, binding sites of regulatory proteins, regulation Binding sites and attenuators of ribonucleic acid. These regions are generally in the range of 1 to 800, 1 to 600, 1 to 400, 1 to 200, 1 to 100 or 1 to 50 nucleotides upstream of the coding region. Likewise included are regions located downstream, for example a transfer terminator. These regions are generally in the range of 1 to 400, 1 to 200, 1 to 100, 1 to 50, or 1 to 25 nucleotides downstream of the coding region.

Intergenic regions in the chromosome, in other words nucleotide sequences that do not have coding functions, can also be used. Finally, either phage or defective phages contained within the chromosome can be used for this purpose.

Prophage is understood to mean a bacteriophage, in particular its genome, which replicates with the genome of the host and does not form infectious particles. Defective phages are understood as meaning those phages, in particular their genomes, which, as a result of various mutations, have lost the ability to form infectious particles. Defect phage is also called cryptic. Prophage and defective phage are often present in integrated forms within the chromosomes of these hosts. Further, the description is a conventional technology, for example described in reference: Edward A. Birge, Bacterial and Bacteriophage Genetics, 3 ^rd ed, Springer-Verlag, New York, USA, 1994; or S. Klaus et al., Bakterienviren, Gustav Fischer Verlag, Jena, Germany, 1992.

Thus, the present invention also provides

a) isolating nucleotide sequences of one or more desired ORFs, genes or alleles for lysine production, optionally comprising expression and / or regulatory signals,

b) attaching the nucleotide sequence of the target site to the 5 'and 3' ends of the ORF, gene or allele for lysine production,

c) Preferably, the nucleotide sequence of the desired ORF, gene or allele to which the nucleotide sequence of the target site is attached is introduced in a vector which does not replicate in coryneform bacteria or only at a limited level,

d) transferring the nucleotide sequence according to b) or c) into coryneform bacteria,

e) the nucleotide sequence according to a) is introduced at the target site and the nucleotide sequence capable of / capable episomal replication in the microorganism, the nucleotide sequence capable of / transferable and the nucleotide sequence conferring resistance to antibiotics Provided are methods for producing coryneform bacteria that produce L-lysine, including isolating coryneform bacteria that do not remain at the target site.

The present invention also provides an open reading frame (ORF) for methionine production or threonine production present at a natural site (gene locus), one or more copies of a gene or allele, in each case the desired open reading frame ( ORF), a second, optionally third or fourth copy of a gene or allele, in an integrated form at a second, optionally third or fourth site, wherein episomal replication is possible / possible in a microorganism Coryneform bacteria, in particular Cory, characterized in that no nucleotide sequence, a nucleotide sequence that allows / enables translocation, and a nucleotide sequence that confers resistance to antibiotics are present at a particular second, optionally third or fourth site Provide bacteria from the genus Nebacterium.

In addition, the present invention is the following steps:

a) an open reading frame (ORF) for the production of methionine or threonine present in the natural site (gene locus), gene or allele of the allele desired in addition to one or more copies of the allele, in each case the desired open reading frame (ORF), gene Or a nucleotide sequence containing a second, optionally third or fourth copy of an allele in an integrated form at a second, optionally third or fourth site, wherein episomal replication is / is enabled within the microorganism. Coryneform bacteria, in particular Corynebacterium glutamicum, wherein the nucleotide sequence that enables / enable translocation and the nucleotide sequence conferring resistance to antibiotics are not present at a particular second, optionally third or fourth site, Fermentation under conditions that permit expression of said open reading frame (ORF), gene or allele;

b) concentrating L-methionine and / or L-threonine in the fermentation broth;

c) separating L-methionine and / or L-threonine from the fermentation broth, optionally

d) further providing a process for producing L-methionine and / or L-threonine, comprising the step of from 0 to 100% by weight of constituents from the fermentation broth and / or biomass.

An "open reading frame (ORF), gene or allele for copying methionine" is any, preferably endogenous, open reading frame, gene or allele that can have the effect of improving methionine production when enhanced / overexpressed. It is understood to mean a gene.

These include, in particular, the following open reading frames, genes or alleles: accBC, accDA, aecD, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dps, eno, fda, gap, gap2, gdh , gnd, glyA, hom, hom ^FBR , lysC, lysC ^FBR , metA, metB, metE, metH, metY, msiK, opcA, oxyR, ppc, ppc ^FBR , pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T. These are summarized and described in Table 4. These include, in particular, the lysC ^FBR allele encoding the "feedback" resistant aspartate kinase (see Table 2) and the hom ^FBR allele encoding the "feedback" resistant homoserine dehydrogenase.

A second, optionally third or fourth copy of an open reading frame (ORF), gene or allele for the production of the desired methionine may in each case be incorporated into a second, optionally third or fourth site. The following open reading frames, genes or nucleotide sequences can in particular be used for this: brnE, brnF, brnQ, ccpA1, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, metD, metK, pck, pgi, poxB and zwa2. These are summarized and described in Table 5.

Of course, the sites mentioned are not only the coding regions of the open reading frames or genes mentioned, but also the regions or nucleotide sequences located upstream involved in expression and regulation, such as ribosome binding sites, promoters, binding sites for regulatory proteins, Binding sites and attenuators for regulatory ribonucleic acid. These regions are generally in the range of 1 to 800, 1 to 600, 1 to 400, 1 to 200, 1 to 100 or 1 to 50 nucleotides upstream of the coding region. Likewise included are regions located downstream, for example a transfer terminator. These regions are generally in the range of 1 to 400, 1 to 200, 1 to 100, 1 to 50, or 1 to 25 nucleotides downstream of the coding region.

Intergenic regions in the chromosome, in other words nucleotide sequences that do not have coding functions, can also be used. Finally, either phage or defective phages contained within the chromosome can be used for this purpose.

"Open reading frame (ORF), gene or allele for copying threonine production" is understood to mean any open reading frame, gene or allele that may have the effect of enhancing threonine production when enhanced / overexpressed. do.

These include, in particular, the following open reading frames, genes or alleles: accBC, accDA, cstA, cysD, cysE, cysH, cysI, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd , hom, hom ^FBR , lysC, lysC ^FBR , msiK, opcA, oxyR, ppc, ppc ^FBR , pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, , sigH, sigM, tal, thyA, tkt, tpi, thrB, thrC, thrE, zwa1, zwf and zwf A213T. These are summarized and described in Table 6. These include, in particular, the lysC ^FBR allele encoding the "feedback" resistant aspartate kinase (see Table 2) and the hom ^FBR allele encoding the "feedback" resistant homoserine dehydrogenase.

A second, optionally third or fourth copy of an open reading frame (ORF), gene or allele for the production of the desired threonine may in each case be incorporated into the second, optionally third or fourth site. The following open reading frames, genes or nucleotide sequences can be used for this in particular: ccpA1, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, glyA, ilvA, ilvBN, ilvC, ilvD, luxR, luxS, lysR1, lysR2, lysR3, mdh, menE, metA, metD, pck, poxB, sigB and zwa2. These are summarized and described in Table 7.

Of course, the sites mentioned are not only the coding regions of the open reading frames or genes mentioned, but also the regions or nucleotide sequences located upstream involved in expression and regulation, such as ribosome binding sites, promoters, binding sites for regulatory proteins, Binding sites and attenuators for regulatory ribonucleic acid. These regions are generally in the range of 1 to 800, 1 to 600, 1 to 400, 1 to 200, 1 to 100 or 1 to 50 nucleotides upstream of the coding region. Likewise included are regions located downstream, for example a transfer terminator. These regions are generally in the range of 1 to 400, 1 to 200, 1 to 100, 1 to 50, or 1 to 25 nucleotides downstream of the coding region.

Intergenic regions in the chromosome, in other words nucleotide sequences that do not have coding functions, can also be used. Finally, either phage or defective phages contained within the chromosome can be used for this purpose.

Thus, the present invention also provides

a) isolating nucleotide sequences of one or more desired ORFs, genes or alleles, optionally comprising expression and / or regulatory signals,

b) attaching the nucleotide sequence of the target site to the 5 'and 3' ends of the ORF, gene or allele,

c) Preferably, the nucleotide sequence of the desired ORF, gene or allele to which the nucleotide sequence of the target site is attached is introduced in a vector which does not replicate in coryneform bacteria or only at a limited level,

d) transferring the nucleotide sequence according to b) or c) into coryneform bacteria,

e) the nucleotide sequence according to a) is introduced at the target site and the nucleotide sequence capable of / capable episomal replication in the microorganism, the nucleotide sequence capable of / transferable and the nucleotide sequence conferring resistance to antibiotics Provided are methods for producing coryneform bacteria that produce L-methionine and / or L-threonine, including isolating coryneform bacteria that do not remain at the target site.

The present invention is in addition to one or more copies of an open reading frame (ORF), gene or allele for valine production present at a native site (gene locus), in each case the desired open reading frame (ORF), gene or A nucleotide sequence containing a second, optionally third or fourth copy of an allele in an integrated form at a second, optionally third or fourth site, wherein episomal replication is possible / enabled in microorganisms, Coryneform bacteria, particularly bacteria of the genus Corynebacterium, are provided in which the nucleotide sequence that enables / enable translocation and the nucleotide sequence conferring resistance to antibiotics are not present at a particular second, optionally third or fourth site.

In addition, the present invention is the following steps:

a) in addition to one or more copies of the open reading frame (ORF), gene or allele for valine production present at the natural site (gene locus), in each case the desired open reading frame (ORF), gene or allele A second, optionally third or fourth copy of the gene is contained in an integrated form at the second, optionally third or fourth site, wherein the nucleotide sequence, translocation, enables / disables episomal replication in the microorganism. Coryneform bacteria, in particular Corynebacterium glutamicum, characterized in that the nucleotide sequences that make / possible / enable resistance to antibiotics are not present in a particular second, optionally third or fourth site Fermenting under conditions permitting expression of said open reading frame (ORF), gene or allele;

b) concentrating L-valine in the fermentation broth;

c) separating L-valine from fermentation broth, optionally

d) further providing a process for producing L-valine, comprising the step of from 0 to 100% by weight of constituents from the fermentation broth and / or biomass.

"Open reading frame (ORF), gene or allele for copying valine production" is understood to mean any open reading frame, gene or allele that may have the effect of improving valine production when enhanced / overexpressed. do.

These include, in particular, the following open reading frames, genes or alleles: brnE, brnF, brnEF, cstA, cysD, dps, eno, fda, gap, gap2, gdh, ilvB, ilvN, ilvBN, ilvC, ilvD , ilvE msiK, pgk, ptsH, ptsI, ptsM, sigC, sigD, sigE, sigH, sigM, tpi, zwa1. These are summarized and described in Table 8. These include in particular acetolactate synthase, which encodes valine resistance.

A second, optionally third or fourth copy of an open reading frame (ORF), gene or allele for the production of the desired threonine may in each case be incorporated into the second, optionally third or fourth site. The following open reading frames, genes or nucleotide sequences can in particular be used for this: aecD, ccpA1, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, glyA, ilvA, luxR, lysR1, lysR2, lysR3, panB, panC, poxB and zwa2. These are summarized and described in Table 9.

Of course, the sites mentioned are not only the coding regions of the open reading frames or genes mentioned, but also the regions or nucleotide sequences located upstream involved in expression and regulation, such as ribosome binding sites, promoters, binding sites for regulatory proteins, Binding sites and attenuators for regulatory ribonucleic acid. These regions are generally in the range of 1 to 800, 1 to 600, 1 to 400, 1 to 200, 1 to 100 or 1 to 50 nucleotides upstream of the coding region. Likewise included are regions located downstream, for example a transfer terminator. These regions are generally in the range of 1 to 400, 1 to 200, 1 to 100, 1 to 50, or 1 to 25 nucleotides downstream of the coding region.

Intergenic regions in the chromosome, in other words nucleotide sequences that do not have coding functions, can also be used. Finally, either phage or defective phages contained within the chromosome can be used for this purpose.

Thus, the present invention also provides

a) isolating nucleotide sequences of one or more desired ORFs, genes or alleles for valine production, optionally comprising expression and / or regulatory signals,

b) attaching the nucleotide sequence of the target site to the 5 'and 3' ends of the ORF, gene or allele,

c) Preferably, the nucleotide sequence of the desired ORF, gene or allele to which the nucleotide sequence of the target site is attached is introduced in a vector which does not replicate in coryneform bacteria or only at a limited level,

d) transferring the nucleotide sequence according to b) or c) into coryneform bacteria,

e) the nucleotide sequence according to a) is introduced at the target site and the nucleotide sequence capable of / capable episomal replication in the microorganism, the nucleotide sequence capable of / transferable and the nucleotide sequence conferring resistance to antibiotics Provided are methods for producing coryneform bacteria that produce L-valine, including isolating coryneform bacteria that do not remain at the target site.

During the work of the present invention, the nucleotide sequence that enables / enables episomal replication in the microorganism, the nucleotide sequence that enables / enable translocation, and the nucleotide sequence that confers resistance to antibiotics does not remain in the gluB gene region. A second copy of the lysC ^FBR allele was introduced into the gluB gene of Nebacterium glutamicum. This strain, called DSM13994glu :: lysC, retains the lysC ^FBR allele lysC T311I at its native lysC site and the second copy of the lysC ^FBR allele lysC T311I at the second site (target site), ie the gluB gene. Hold. A plasmid into which the lysC ^FBR allele can be introduced into the gluB gene is shown in FIG. 1. It is named pK18mobsacBglu1_1.

During the work of the present invention, the nucleotide sequence that enables / enables episomal replication in the microorganism, the nucleotide sequence that enables / enable translocation, and the nucleotide sequence that confers resistance to antibiotics does not remain in the gluB gene region. A copy of the lysC ^FBR allele could also be introduced at the target site of the gluB gene of Nebacterium glutamicum. This strain, called DSM12866glu :: lysC, retains the wild type of the lysC gene in its native lysC site, and the second copy of the lysC gene in the form of the lysC ^FBR allele lysC T311I, the second site (target site), ie the gluB gene. Hold on. It was deposited with DSM15039 at the International Depository Deutsche Sammlung fur Mikroorganismen und Zellkulturen (German Collection of Microbial and Cell Cultures). A plasmid into which the lysC ^FBR allele can be introduced into the gluB gene is shown in FIG. 1. It is named pK18mobsacBglu1_1.

During the work of the present invention, the nucleotide sequence that enables / enables episomal replication in the microorganism, the nucleotide sequence that enables / enable translocation, and the nucleotide sequence that confers resistance to antibiotics does not remain at the aecD gene site. A copy of the lysC ^FBR allele could also be introduced at the target site of the aecD gene of Nebacterium glutamicum. This strain, called DSM12866aecD :: lysC, retains the wild type of the lysC gene in its native lysC site and the second copy of the lysC gene in the form of the lysC ^FBR allele lysC T311I, the second site (target site), namely the aecD gene. Hold on. A plasmid into which the lysC ^FBR allele can be introduced into the aecD gene is shown in FIG. 2. It is named pK18mobsacBaecD1_1.

During the work of the present invention, the nucleotide sequence that enables / enables episomal replication in the microorganism, the nucleotide sequence that enables / enable translocation, and the nucleotide sequence that confers resistance to antibiotics does not remain in the pck gene region. A copy of the lysC ^FBR allele could also be introduced at the target site of the pck gene of Nebacterium glutamicum. This strain, called DSM12866pck :: lysC, retains the wild type of the lysC gene in its native lysC site and the second copy of the lysC gene in the form of the lysC ^FBR allele lysC T311I, the second site (target site), namely the pck gene. Hold on. Plasmids that can be introduced into the pck gene are shown in FIG. 3. It is named pK18mobsacBpck1_1.

During the work of the present invention, the nucleotide sequence that enables / enables episomal replication in the microorganism, the nucleotide sequence that enables / enable translocation, and the nucleotide sequence that confers resistance to antibiotics does not remain in the gluB gene region. A copy of the ddh gene could also be introduced at the target site of the gluB gene of Nebacterium glutamicum. This strain, called DSM12866glu :: ddh, holds a copy of the ddh gene in its native ddh site and a second copy of ddh in a second site (target site), ie the gluB gene. A plasmid into which the ddh gene can be introduced into the gluB gene is shown in FIG. 4. It is named pK18mobsacBgluB2_1.

During the work of the present invention, the nucleotide sequence that enables / enables episomal replication in the microorganism, the nucleotide sequence that enables / enable translocation, and the nucleotide sequence that confers resistance to antibiotics does not remain at the aecD gene site. A copy of the dapA gene could also be introduced at the target site of the aecD gene of Nebacterium glutamicum. This strain, called DSM12866aecD :: dapA, retains a copy of the dapA gene at its native dapA site and a second copy of dapA at a second site (target site), namely the aecD gene. A plasmid into which the dapA gene can be introduced into the aecD gene is shown in FIG. 5. It is named pK18mobsacBaecD2_1.

During the work of the present invention, the nucleotide sequence that enables / enables episomal replication in the microorganism, the nucleotide sequence that enables / enable translocation, and the nucleotide sequence that confers resistance to antibiotics does not remain in the pck gene region. A copy of the pyc allele could also be introduced at the target site of the pck gene of Nebacterium glutamicum. This strain, called DSM12866pck :: pyc, carries a wild-type copy of the pyc gene at its native pyc site, and the second copy of the pyc gene in the form of the pyc allele pyc P458S at the second site (target site), i.e. holds in the pck gene. A plasmid into which the pck allele can be introduced into the pck gene is shown in FIG. 6. It is named pK18mobsacBpck1_3.

In order to prepare chemical compounds, coryneform bacteria produced according to the present invention can be cultured continuously or discontinuously in a batch process (batch culture), or in a fed-batch culture (feeding process) or a repetitive feeding process (repeated feeding process). . An overview of known culture methods can be found in Chmiel, Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991); and Storhas, Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994).

The culture medium used should meet the needs of the particular strain in a suitable manner. Culture media for various microorganisms are described in "Manual of Methods for General Bacteriology" The American Society for Bacteriology (Washington D.C., USA, 1981).

Sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and oils (e.g. soybean oil, sunflower oil, peanut oil and coconut oil), fatty acids (e.g. palmitic acid, Stearic acid and linoleic acid), alcohols such as glycerol and ethanol, and organic acids such as acetic acid or lactic acid can be used as the carbon source. These materials can be used individually or as a mixture.

Organic compounds containing nitrogen (e.g. peptone, yeast extract, meat extract, malt extract, corn deposit, soy flour and urea), or inorganic compounds (e.g. 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, dipotassium hydrogen phosphate, or the corresponding sodium-containing salts can be used as the phosphorus source. In addition, the culture medium should contain metal salts necessary for growth, such as magnesium sulfate or iron sulfate. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the substances mentioned above. In addition, suitable precursors may be added to the culture medium. The starting materials mentioned can be added to the medium in single batch form or supplied in a suitable manner during the culture.

In order to adjust the pH of the medium, a basic compound (such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water), or an acid compound (such as phosphoric acid or sulfuric acid) may be suitably used. Antifoams such as fatty acid polyglycol esters can be used to control foam formation. In order to maintain the stability of the plasmid, a suitable substance which optionally acts, for example antibiotics, can be added to the medium. In order to maintain an aerobic state, oxygen or an oxygen-containing gas mixture (eg air) may be introduced into the medium. The temperature of the medium is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. Incubation is continued until the desired chemical compound is produced to the maximum. This goal can typically be achieved in 10 to 160 hours.

It has been found that coryneform bacteria according to the invention, especially coryneform bacteria producing L-lysine, unexpectedly have a high degree of stability. They are 10-20 times, 20-30 times, 30-40 times, 40-50 times or more, preferably 50-60 times, 60-70 times, 70-80 times and 80-90 times or more generation or cell division cycles. Stable for a while.

The following microorganisms have been deposited:

Strain Corynebacterium glutamicum DSM 12866glu :: lysC is in the form of pure cultures and is in accordance with the Budapest Treaty (Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microbial and Cell Cultures, Braunschweig, Germany)). Deposited No. DSM15039, dated 5 June 2002.

Plasmid pK18mobsacBglu1_1 is in the form of a pure culture of the strain E. coli DH5αmcr / pK18mobsacBglu1_1 (= DH5alpha mcr / pK18mobsacBglu1_1) according to the Budapest Treaty [Deutsche Sammlung fur Mikroorganismen und Zellkulturen (schweiz, Germany); Deposited as DSM14243 on April 20, 2001.

Plasmid pK18mobsacBaecD1_1 is in the form of a pure culture of the strain E. coli DH5αmcr / pK18mobsacBaecD1_1 (= DH5alphamcr / pK18mobsacBaecD1_1) according to the Budapest Treaty [Deutsche Sammlung fur Mikroorganismen und MellZwei, Ze., D. Z. Zellkulturen; Deposited as DSM15040, dated June 5, 2002.

Example 1

LysC into the chromosome of strain DSM13994 and into the chromosome of strain DSM12866 ^FBR  Introduction of the second copy of the allele

Corynebacterium glutamicum strain DSM13994 was obtained from Corynebacterium glutamicum ATCC13032 by several non-directed mutagenesis, selection and mutant selection methods. The strain is resistant to lysine homologue S- (2-aminoethyl) -L-cysteine and has a feed-bag resistant aspartate kinase that is insensitive to inhibition by a mixture of lysine and threonine (25 mM each). . The nucleotide sequence of the lysC ^FBR allele of this strain is shown in SEQ ID NO: 3. It is also referred to below as lysC T311I. The amino acid sequence of the encoded aspartate kinase protein is shown in SEQ ID NO: 4. Pure cultures of the strains were deposited on 16 January 2001 by the International Depositary Organization (Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microbial and Cell Cultures, Braunschweig, Germany)) in accordance with the Budapest Treaty.

Strain DSM12866 was obtained from Corynebacterium glutamicum ATCC13032 by non-directed mutagenesis and screening of mutants with the best L-lysine accumulation. It is sensitive to methionine. Growth on minimal medium containing L-methionine can be re-established by the addition of threonine. The strain has a lysC gene of wild type shown in SEQ ID NO: 1. The corresponding amino acid sequence of wild type aspartate kinase protein is shown in SEQ ID NO: 2. Pure cultures of these strains were deposited on June 10, 1999 to the International Depository (Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany)) under the Budapest Treaty.

1.1 Isolation and Sequencing of the DNA of the lysC Allele of Strain DSM13994

From strain DSM13994, chromosomal DNA is isolated by conventional methods (Eikmanns et al., Microbiology 140: 1817-1828 (1994)). Polymerase chain reaction is used to amplify DNA fragments carrying the lysC gene or allele. Known lysC genes for Corynebacterium glutamicum [Kalinowski et al., Molecular Microbiology, 5 (5), 1197-1204 (1991); Based on the sequence of Accession Number X57226), the following primer oligonucleotides were selected for PCR:

lysC1beg (SEQ ID NO: 5):

5 'TA (G GAT CC) T CCG GTG TCT GAC CAC GGT G 3'

lysC2end: (SEQ ID NO: 6):

5 'AC (G GAT CC) G CTG GGA AAT TGC GCT CTT CC 3'

The primers presented are synthesized by MWG Biotech and PCR reactions are described in Innis et al., PCR protocol. A guide to Methods and Applications, 1990, Academic Press]. The primers can be used to amplify DNA fragments of about 1.7 kb in length that carry the lysC gene or allele. The primer also contains the sequence of the cleavage site of the restriction endonuclease BamHI, which is indicated in parentheses in the nucleotide sequence shown above.

An amplified DNA fragment of about 1.7 kb in length carrying the lysC allele of strain DSM13994 was identified by electrophoresis on a 0.8% agarose gel and isolated from the gel, using conventional methods (QIAquick Gel Extraction Kit, Qiagen, Purified by Hilden).

The fragment is then linked into the vector pCRII-TOPO using the Topo TA cloning kit (Invitrogen, Leek, The Netherlands, Cat. Number K4600-01). This ligation batch is transformed into E. coli strain TOP10 (Invitrogen, Leek, The Netherlands). Selection of plasmid-bearing cells was carried out with kanamycin (50 mg / l)-with X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside, 64 mg / l). By plating a transgenic batch on the containing LB agar.

After isolation of the DNA, the resulting plasmid is confirmed by restriction cleavage and identified on an agarose gel. The resulting plasmid is called pCRIITOPOlysC.

Nucleotide sequences of amplified DNA fragments or PCR products were subjected to dideoxy chain termination using the "ABI Prism 377" sequencing device from PE Applied Biosystems (Weiterstadt, Germany). Sanger et al., Proceedings of the National Academy of Sciences USA, 74: 5463-5467 (1977). The sequence of the coding region of the PCR product is shown in SEQ ID NO: 3. The amino acid sequence of the relevant aspartate kinase protein is shown in SEQ ID NO: 4.

Base thymidine is found at position 932 of the nucleotide sequence (SEQ ID NO: 3) of the coding region of the lysC ^FBR allele of strain DSM13994. Base cytosine is found at the corresponding position of the wild type gene (SEQ ID NO: 1).

The amino acid isoleucine is found at position 311 of the amino acid sequence (SEQ ID NO: 4) of the aspartate kinase protein of strain DSM13994. The amino acid threonine is found at the corresponding position of the wild type protein (SEQ ID NO: 2).

Since the position 932 of the coding region contains the base thymidine, the lysC allele encoding the aspartate kinase protein containing the amino acid isoleucine at position 311 of the amino acid sequence is then referred to as the lysC ^FBR allele or lysC T311I. It is called.

The plasmid pCRIITOPOlysC carrying the lysC ^FBR allele or lysC T311I was transferred to the international depositary institution [Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany)] under No. DSM14242 on April 20, 2001. It was deposited in the form of a pure culture of E. coli TOP 10 / pCRIITOPOlysC.

1.2 Construction of the replacement vector pK18mobsacBglu1_1

Corynebacterium glutamicum strain ATCC 13032 is used as donor of chromosomal DNA. From strain ATCC13032, chromosomal DNA is isolated by conventional methods (Eikmanns et al., Microbiology 140: 1817-1828 (1994)). Polymerase chain reaction is used to amplify DNA fragments bearing the gluB gene and surrounding regions. Based on the sequence of the gluABCD gene known for Corynebacterium glutamicum (Kronemeyer et al., Journal of Bacteriology, 177: 1152-1158 (1995) (Accession No. X81191)), Primer oligonucleotides were selected for PCR:

gluBgl1 (SEQ ID NO: 7):

5 'TA (A GAT CT) G TGT TGG ACG TCA TGG CAA G 3'

gluBgl2 (SEQ ID NO: 8):

5 'AC (A GAT CT) T GAA GCC AAG TAC GGC CAA G 3'

The primers presented are synthesized by MWG Biotech and PCR reactions are described in Innis et al., PCR protocol. A Guide to Methods and Applications, 1990, Academic Press]. This primer can be used to amplify a DNA fragment of about 1.7 kb that contains the gluB gene and the surrounding region. The peripheral region is about 0.33 kb of sequence portion upstream of the gluB gene representing the 3 'end of the gluA gene, and about 0.44 kb of sequence downstream of the gluB gene representing the 5' end of the gluC gene. The primer also contains the sequence of the cleavage site of the restriction endonuclease BglHI, which is indicated in parentheses in the nucleotide sequence shown above.

Amplified DNA fragments of about 1.7 kb in length carrying the gluB gene and surrounding regions were identified by electrophoresis on a 0.8% agarose gel and separated from the gel, using conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden). Purification by

The fragment is then linked into the vector pCRII-TOPO using the Topo TA cloning kit (Invitrogen, Leek, The Netherlands, Cat. Number K4600-01). This ligation batch is transformed into E. coli strain TOP10 (Invitrogen, Leek, The Netherlands). Selection of plasmid-bearing cells was carried out with kanamycin (50 mg / l) with X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside, 64 mg / l). By plating a transgenic batch on the containing LB agar.

After isolation of the DNA, the resulting plasmid is confirmed by restriction cleavage and identified on an agarose gel. The resulting plasmid is called pCRII-TOPOglu.

Plasmid pCRII-TOPOglu was digested with restriction enzyme BglII (Amersham-Pharmacia, Freiburg, Germany), isolated from agarose gel (0.8%) using QIAquick gel extraction kit (Qiagen, Hilden, Germany), and then about 1.7 kb. The gluB fragment of was isolated from agarose gel and used for ligation with the movable cloning vector pK18mobsacB (Schafer et al., Gene 14: 69-73 (1994)). It was first cleaved with restriction enzyme BamHI, dephosphorylated with Alkaline Phosphatase (Boehringer Mannheim), mixed with about 1.7 kb of gluB fragment, and the mixture was mixed with T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany).

Subsequently, E. coli strain DH5α [Grant et al .; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649, supra, in this linkage batch (Hanahan, In. DNA cloning. A practical approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989]. Selection of plasmid-bearing cells was performed by LB agar supplemented with kanamycin 50 mg / l [Sambrook et al., Molecular Cloning: a laboratory manual. 2 ^nd Ed., Cold Spring Harbor, New York, 1989].

Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep kit (Qiagen) and confirmed by restriction digestion and subsequent agarose gel electrophoresis. This plasmid is called pK18mobsacBglu1.

Plasmid DNA was isolated from strain DSM14242 (see Example 1.1) carrying plasmid pCRIITOPOlysC, digested with restriction enzyme BamHI (Amersham-Pharmacia, Freiburg, Germany) and QIAquick gel extraction kit (Qiagen, Hilden, Germany) After separation from the agarose gel (0.8%), a lysC ^FBR containing DNA fragment of about 1.7 kb in length is separated from the agarose gel and used for ligation with the vector pK18mobsacBglu1. This was first cleaved with restriction enzyme BamHI, dephosphorylated with Alkaline Phosphatase (Boehringer Mannheim), mixed with about 1.7 kb of lysC ^FBR fragment, and the mixture was mixed with T4 DNA ligase (Amersham-Pharmacia, Freiburg). , Germany).

Subsequently, the E. coli strain DH5αmcr [Life Technologies GmbH, Karlsruhe, Germany] was subjected to the ligation batch [Hanahan, In: DNA cloning. A practical approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989]. Selection of plasmid-bearing cells was performed by LB agar supplemented with kanamycin 50 mg / l [Sambrook et al., Molecular Cloning: a laboratory manual. 2 ^nd Ed., Cold Spring Harbor, New York, 1989].

Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep kit (Qiagen) and confirmed by restriction digestion and subsequent agarose gel electrophoresis. This plasmid is called pK18mobsacBglu1_1. A map of this plasmid is shown in FIG. 1.

The plasmid pK18mobsacBglu1_1 was transferred to the International Depository [Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany)] according to the Budapest Treaty on April 20, 2001 with the strain E. coli DH5αmcr / pK18mobs = H5αr = p518r It was deposited in the form of pure culture of pK18mobsacBglu1_1).

1.3 Introduction of the second copy of the lysC ^FBR allele lysC T311I into the chromosome (target site: gluB gene) of strain DSM13994 using alternative vector pK18mobsacBglu1_1

The vector pK18mobsacBglu1_1 described in Example 1.2 is transferred to the genus Corynebacterium glutamicum strain DSM13994 by conjugation according to the protocol (Schafer et al., Journal of Microbiology 172: 1663-1666 (1990)). The vector cannot replicate independently in the DSM and is only maintained when incorporated into the chromosome. Selection of clones or transconjugants containing the integrated pK18mobsacBglu1_1 was performed by LB agar supplemented with 15 mg / l kanamycin and 50 mg / l nalidixic acid (Sambrook et al., Molecular Cloning: a laboratory). manual. 2 ^nd Ed., Cold Spring Harbor, New York, 1989]. Kanamycin resistant transconjugates are plated on LB agar supplemented with kanamycin 25 mg / l and incubated at 33 ° C. for 48 hours.

For selection of the resulting mutants from which plasmids were removed as a result of the second recombination, clones were incubated in LB agar medium for 20 hours, then plated on LB agar containing 10% agarose and incubated for 48 hours. do.

Like the starting plasmid pK18mobsacB, plasmid pK18mobsacBglu1_1 contains a copy of the sacB gene encoding levan sucrase from Bacillus subtilis in addition to the kanamycin resistance gene. Expression that can be induced by sucrose leads to the formation of Levan scurase, which catalyzes the synthesis of the product levan, which is toxic to Corynebacterium glutamicum. Thus, only those clones with integrated pK18mobsacBglu1_1 deleted as a result of the second recombination grow on LB agar. Depending on where the second recombination occurred, a second copy of the lysC ^FBR allele appears after deletion and appears at the gluB locus in the chromosome, or the original gluB locus of the host is maintained.

About 40 or 50 colonies are tested for the "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" phenotypes. About 20 colonies showing the "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" phenotypes were investigated using polymerase chain reaction. At this time, the DNA fragment containing the gluB gene and the surrounding region is amplified from the chromosomal DNA of the colony. The same primer oligonucleotides as described in Example 1.2 for the construction of the integration plasmids are selected for PCR.

gluBgl1 (SEQ ID NO: 7):

5 'TA (A GAT CT) G TGT TGG ACG TCA TGG CAA G 3'

gluBgl2 (SEQ ID NO: 8):

5 'AC (A GAT CT) T GAA GCC AAG TAC GGC CAA G 3'

The primers can be used to amplify a DNA fragment of about 1.7 kb in a control clone with the original gluB locus. In clones with a second copy of lysC ^FBR at the intrachromosomal gluB locus, a DNA fragment of about 3.4 kb is amplified.

Amplified DNA fragments are identified by electrophoresis on 0.8% agarose gel.

In addition to the copies present at the lysC locus, clones with a second copy of the lysC ^FBR allele lysC T311I at the chromosomal gluB locus were identified in this manner. This clone is called strain DSM13994glu :: lysC.

1.4 Introduction of a second copy of the lysC ^FBR allele lysC T311I form of the lysC gene into the chromosome (target site: gluB gene) of strain DSM12866 using alternative vector pK18mobsacBglu1_1

As described in Example 1.3, plasmid pK18mobsacBglu1_1 is transferred into the Corynebacterium glutamicum strain DSM12866 by conjugation. In addition to the copy of the wild-type gene present at the lysC locus, a clone having a second copy of the lysC gene of the lysC ^FBR allele lysC T311I form at the gluB locus of the chromosome was identified by the method described in Example 1.3. This clone was called strain DSM12866glu :: lysC.

Corynebacterium glutamicum according to the present invention, which has a second copy of the lysC ^FBR allele in the gluB gene, is an international depository institution [Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany)], in accordance with the Budapest Treaty. Was deposited in the form of a pure culture of strain Corynebacterium glutamicum DSM12866glu :: lysC with Accession No. DSM15039 on June 5, 2002.

1.5 Construction of the replacement vector pK18mobsacBpck1_1

Corynebacterium glutamicum strain ATCC 13032 is used as donor of chromosomal DNA. From strain ATCC13032, chromosomal DNA is isolated using conventional methods (Eikmanns et al., Microbiology 140: 1817-1828 (1994)). Polymerase chain reaction is used to amplify DNA fragments bearing the pck gene and surrounding regions. Based on the known pck gene for Corynebacterium glutamicum (EP1094111 and Riedel et al., Journal of Molecular and Microbiological Biotechnology 3: 573-583 (2001) (Accession No. AJ269506)) The following primer oligonucleotides were selected for PCR:

pck_beg (SEQ ID NO: 9):

5 'TA (A GAT CT) G CCG GCA TGA CTT CAG TTT 3'

pck_end (SEQ ID NO: 10):

5 'AC (A GAT CT) G GTG GGA GCC TTT CTT GTT ATT3'

The primers presented are synthesized by MWG Biotech, and PCR reactions are described in Innis et al., PCR Protocol. A Guide to Methods and Applications, 1990, Academic Press]. The primers can be used to amplify DNA fragments of about 2.9 kb in size containing the pck gene and adjacent regions. The primer also contains the sequence of the cleavage site of the restriction endonuclease BglHI, which is indicated in parentheses in the nucleotide sequence shown above.

Amplified DNA fragments of about 2.9 kb in length carrying the pck gene and surrounding regions were identified by electrophoresis on a 0.8% agarose gel and separated from the gel, using conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden). Purification by

The fragment is then linked into the vector pCRII-TOPO using a TOPO TA cloning kit (Invitrogen, Leek, The Netherlands, Cat. Number K4600-01). This ligation batch is transformed into E. coli strain TOP10 (Invitrogen, Leek, The Netherlands). Plasmid-bearing cells are selected by plating transfection batches on LB agar containing kanamycin (50 mg / l) with X-Gal (64 μg mg / l).

After isolation of the DNA, the resulting plasmid is confirmed by restriction cleavage and identified on an agarose gel. The resulting plasmid is called pCRII-TOPOpck.

Plasmid pCRII-TOPOpck was digested with restriction enzyme BglII (Amersham-Pharmacia, Freiburg, Germany) and isolated on agarose gel (0.8%) using QIAquick gel extraction kit (Qiagen, Hilden, Germany), then about 2.9 kb The pck fragment of was isolated from an agarose gel and used for ligation with the mobile cloning vector pK18mobsacB (Schafer et al., Gene 14: 69-73 (1994)). It was first cleaved with restriction enzyme BamHI, dephosphorylated with Alkaline Phosphatase (Boehringer Mannheim), mixed with about 2.9 kb of pck fragment, and the mixture was mixed with T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany). ).

Subsequently, E. coli strain DH5α [Grant et al .; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649, supra, in this linkage batch (Hanahan, In. DNA cloning. A practical approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989]. Selection of plasmid-bearing cells was performed by LB agar supplemented with kanamycin 50 mg / l [Sambrook et al., Molecular Cloning: A Laboratory manual. 2 ^nd Ed., Cold Spring Harbor, New York, 1989].

Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep kit (Qiagen) and confirmed by restriction digestion and subsequent agarose gel electrophoresis. This plasmid is called pK18mobsacBpck1.

Plasmid DNA was isolated from strain DSM14242 (see Example 1.1) carrying plasmid pCRIITOPOlysC, digested with restriction enzyme BamHI (Amersham-Pharmacia, Freiburg, Germany) and QIAquick gel extraction kit (Qiagen, Hilden, Germany) After separation on agarose gel (0.8%), a lysC ^FBR containing DNA fragment of about 1.7 kb in length is separated from the agarose gel and used for ligation with the vector pK18mobsacBpck1 described above. This was first cleaved with restriction enzyme BamHI, dephosphorylated with Alkaline Phosphatase (Boehringer Mannheim), mixed with about 1.7 kb of lysC ^FBR fragment, and the mixture was mixed with T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany).

Subsequently, the E. coli strain DH5αmcr [Life Technologies GmbH, Karlsruhe, Germany] was subjected to the ligation batch [Hanahan, In: DNA cloning. A Practical approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989]. Selection of plasmid-bearing cells was performed by LB agar supplemented with kanamycin 50 mg / l [Sambrook et al., Molecular Cloning: a laboratory manual. 2 ^nd Ed., Cold Spring Harbor, New York, 1989].

Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep kit (Qiagen) and confirmed by restriction digestion and subsequent agarose gel electrophoresis. This plasmid is called pK18mobsacBpck1_1. A map of this plasmid is shown in FIG. 3.

1.6 Introduction of the second copy of the lysC ^FBR allele lysC T311I form of the lysC gene into the chromosome (target site: pck gene) of strain DSM12866 using alternative vector pK18mobsacBpck1_1

As described in Example 1.3, the plasmid pK18mobsacBpck1_1 described in Example 1.5 is transferred to the Corynebacterium glutamicum strain DSM12866 by conjugation. The desired recombination that occurred on the chromosome of Corynebacterium glutamicum as described in Example 1.3 is selected. Depending on the location of the second recombination, a second copy of the lysC ^FBR allele appears after deletion and appears at the pck locus in the chromosome, or the original pck locus of the host is maintained.

About 40 or 50 colonies are tested for the "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" phenotypes. About 20 colonies showing the "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" phenotypes were investigated using polymerase chain reaction. At this time, the DNA fragment containing the pck gene and the surrounding region is amplified from the chromosomal DNA of the colony. The same primer oligonucleotides as described in Example 1.5 for the construction of the integration plasmids are selected for PCR.

pck_beg (SEQ ID NO: 9):

5 'TA (A GAT CT) G CCG GCA TGA CTT CAG TTT 3'

pck_end (SEQ ID NO: 10):

5 'AC (A GAT CT) G GTG GGA GCC TTT CTT GTT ATT3'

The primers can be used to amplify a DNA fragment of about 2.9 kb in a control clone with the original pck locus. In clones with a second copy of lysC ^FBR at the intrachromosomal pck locus, a DNA fragment of about 4.6 kb is amplified.

Amplified DNA fragments are identified by electrophoresis on 0.8% agarose gel.

In addition to a copy of the wild-type gene present at the lysC locus, clones with a second copy of the lysC gene in the form of the lysC ^FBR allele lysC T311I at the pck locus in the chromosome were identified in this manner. This clone is called strain DSM12866pck :: lysC.

1.7 Construction of alternative vector pK18mobsacBaecD1_1

Corynebacterium glutamicum strain ATCC 13032 is used as donor of chromosomal DNA. From strain ATCC13032, chromosomal DNA is isolated using conventional methods (Eikmanns et al., Microbiology 140: 1817-1828 (1994)). Polymerase chain reaction is used to amplify DNA fragments bearing the aecD gene and surrounding regions. Based on the sequence of the known aecD gene for Corynebacterium glutamicum (Rossol et al., Journal of Bacteriology 174: 2968-2977 (1992) (Accession No. M89931)), the following primer oligonucleotides Was selected for PCR:

aecD_beg (SEQ ID NO: 11):

5 'GAA CTT ACG CCA AGC TGT TC 3'

aecD_end (SEQ ID NO: 12):

5 'AGC ACC ACA ATC AAC GTG AG 3'

The primers presented are synthesized by MWG Biotech, and PCR reactions are described in Innis et al., PCR Protocol. A Guide to Methods and Applications, 1990, Academic Press]. This primer can be used to amplify a DNA fragment of about 2.1 kb that contains the aecD gene and adjacent regions.

Amplified DNA fragments of about 2.1 kb in length are identified by electrophoresis on a 0.8% agarose gel, separated from the gel, and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden).

Purified DNA fragments are digested with restriction enzymes BamHI and EcoRV (Amersham-Pharmacia, Freiburg, Germany). This fragment is linked to the vector pUC18 (Norrander et al., Gene 26: 101-106 (1983)). It is first cleaved with restriction enzymes BaglII and SmaI, dephosphorylated, mixed with about 1.5 kb of aecD-bearing fragments and the mixture is treated with T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany). This ligation batch is transformed into E. coli strain TOP10 (Invitrogen, Leek, The Netherlands). Plasmid-bearing cells are selected by plating transfection batches on LB agar containing kanamycin (50 mg / l) with X-Gal (64 μg mg / l).

After isolation of the DNA, the resulting plasmid is confirmed by restriction cleavage and identified on an agarose gel. The resulting plasmid is called pUC18aecD.

Plasmid DNA is isolated from strain DSM14242 (see Example 1.1) carrying plasmid pCRIITOPOlysC, digested with restriction enzyme BamHI (Amersham-Pharmacia, Freiburg, Germany) and treated with Klenow polymerase. After separation from agarose gel (0.8%) using QIAquick gel extraction kit (Qiagen, Hilden, Germany), about 1.7kb long lysC ^FBR -containing DNA fragments were isolated from agarose gels and were isolated with the vector pUC18aecD. Used to connect. This was first cleaved with restriction enzyme StuI, dephosphorylated with Alkaline Phosphatase (Boehringer Mannheim), mixed with about 1.7 kb of lysC ^FBR fragment, and the mixture was mixed with T4 DNA ligase (Amersham-Pharmacia, Freiburg). , Germany).

Subsequently, the E. coli strain DH5α (Life Technologies GmbH, Karlsruhe, Germany) was subjected to the ligation batch [Hanahan, In: DNA cloning. A Practical approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989]. Selection of plasmid-bearing cells was performed by LB agar supplemented with kanamycin 50 mg / l [Sambrook et al., Molecular Cloning: A Laboratory manual. 2 ^nd Ed., Cold Spring Harbor, New York, 1989].

Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep kit (Qiagen) and confirmed by restriction digestion and subsequent agarose gel electrophoresis. This plasmid is called pUC18aecD1.

Plasmid pUC18aecD1 is digested with restriction enzyme KpnI and treated with Klenow polymerase. The plasmid was then digested with the restriction enzyme SalI (Amersham-Pharmacia, Freiburg, Germany) and isolated on agarose gel (0.8%) using QIAquick gel extraction kit (Qiagen, Hilden, Germany), followed by aecD and lysC A fragment of about 3.2.kb, which is retained, is separated from the agarose gel and used for ligation with the movable cloning vector pK18mobsacB (Schafer et al., Gene 14: 69-73 (1994)). It was first cleaved with restriction enzymes SmaI and SalI, dephosphorylated with Alkaline Phosphatase (Boehringer Mannheim), mixed with a fragment of about 3.2 kb with aecD and lysC and the mixture was mixed with T4 DNA ligase ( Amersham-Pharmacia, Freiburg, Germany).

Subsequently, E. coli strain DH5α [Grant et al .; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649] are transformed with this linkage batch (Hanahan, In. DNA cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989]. Selection of plasmid-bearing cells was performed by LB agar supplemented with kanamycin 50 mg / l [Sambrook et al., Molecular Cloning: a laboratory manual. 2 ^nd Ed., Cold Spring Harbor, New York, 1989].

Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep kit (Qiagen) and confirmed by restriction digestion and subsequent agarose gel electrophoresis. This plasmid is called pK18mobsacBaecD1_1. A map of this plasmid is shown in FIG.

The plasmid pK18mobsacBaecD1_1 was transferred to the International Depositary Organization (Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany)) in accordance with the Budapest Treaty No. It was deposited in the form of a pure culture of pK18mobsacBaecD1_1).

1.8 Introduction of the second copy of the lysC gene as the lysC ^FBR allele into the chromosome (target site: aecD gene) of strain DSM12866 using replacement vector pK18mobsacBaecD1_1

As described in Example 1.3, the plasmid pK18mobsacBaecD1_1 described in Example 1.4 is transferred to the Corynebacterium glutamicum strain DSM12866 by conjugation. The desired recombination that occurred on the chromosome of Corynebacterium glutamicum as described in Example 1.3 is selected. Depending on where the second recombination occurred, after deletion, a second copy of the lysC ^FBR allele appears at the aecD locus in the chromosome, or the original aecD locus of the host is maintained.

About 40 or 50 colonies are tested for the "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" phenotypes. About 20 colonies showing the "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" phenotypes were investigated using polymerase chain reaction. At this time, the DNA fragment containing the aecD gene and the surrounding region is amplified from the chromosomal DNA of the colony. The same primer oligonucleotides as described in Example 1.7 for the construction of the integration plasmid are selected for PCR.

aecD_beg (SEQ ID NO: 11):

5 'GAA CTT ACG CCA AGC TGT TC 3'

aecD_end (SEQ ID NO: 12):

5 'AGC ACC ACA ATC AAC GTG AG 3'

The primers can be used to amplify DNA fragments of about 2.1 kb in control clones with the original aecD locus. In clones with a second copy of lysC ^FBR at the intrachromosomal aecD locus, a DNA fragment of about 3.8 kb is amplified.

Amplified DNA fragments are identified by electrophoresis on 0.8% agarose gel.

In addition to a copy of the wild-type gene present at the lysC locus, clones having a second copy of the lysC gene in the form of the lysC ^FBR allele lysC T311I at the intra-chromosomal aecD locus were identified in this manner. This clone is called strain DSM12866aecD :: lysC.

Example 2

Introduction of the second copy of the ddh gene into the chromosome (target site: gluB gene) of strain DSM12866

2.1 Construction of alternative vector pK18mobsacBglu2_1

Corynebacterium glutamicum strain ATCC 13032 is used as donor of chromosomal DNA. From strain ATCC13032, chromosomal DNA is isolated using conventional methods (Eikmanns et al., Microbiology 140: 1817-1828 (1994)). Polymerase chain reaction is used to amplify DNA fragments bearing the gluB gene and surrounding regions. Known gluABCD gene population for Corynebacterium glutamicum [Kronemeyer et al., Journal of Bacteriology, 177: 1152-1158 (1995); Based on the sequence of EP1108790 (Accession Nos. X81191 and AX127149), the following primer oligonucleotides were selected for PCR:

gluA_beg (SEQ ID NO: 13):

5 'CAC GGT TGC TCA TTG TAT CC 3'

gluD_end (SEQ ID NO: 14):

5 'CGA GGC GAA TCA GAC TTC TT 3'

The primers presented are synthesized by MWG Biotech, and PCR reactions are described in Innis et al., PCR Protocol. A Guide to Methods and Applications, 1990, Academic Press]. The primers can be used to amplify DNA fragments of about 4.4 kb that contain the gluB gene and the surrounding region.

Amplified DNA fragments are identified by electrophoresis on a 0.8% agarose gel, separated from the gel, and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden).

The fragment is then linked into the vector pCRII-TOPO using a TOPO TA cloning kit (Invitrogen, Leek, The Netherlands, Cat. Number K4600-01). This ligation batch is transformed into E. coli strain TOP10 (Invitrogen, Leek, The Netherlands). Plasmid-bearing cells are selected by plating transfection batches on LB agar containing kanamycin (50 mg / l) with X-Gal (64 μg mg / l).

After isolation of the DNA, the resulting plasmid is confirmed by restriction cleavage and identified on an agarose gel. The resulting plasmid is called pCRII-TOPOglu2.

Plasmid pCRII-TOPOglu2 was digested with restriction enzymes EcoRI and SalI (Amersham-Pharmacia, Freiburg, Germany), isolated on agarose gel (0.8%) using QIAquick gel extraction kit (Qiagen, Hilden, Germany), and then A 3.7 kb gluB fragment is isolated from the agarose gel and used for ligation with the movable cloning vector pK18mobsacB (Schafer et al., Gene 14: 69-73 (1994)). It was first cleaved with restriction enzymes EcoRI and SalI, dephosphorylated with Alkaline Phosphatase (Boehringer Mannheim), mixed with about 3.7 kb of gluB fragment and the mixture was mixed with T4 DNA ligase (Amersham-Pharmacia, Freiburg). , Germany).

Subsequently, E. coli strain DH5α [Grant et al .; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649, supra, in this linkage batch (Hanahan, In. DNA cloning. A practical approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989]. Selection of plasmid-bearing cells was performed by LB agar supplemented with kanamycin 50 mg / l [Sambrook et al., Molecular Cloning: A Laboratory manual. 2 ^nd Ed., Cold Spring Harbor, New York, 1989].

Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep kit (Qiagen) and confirmed by restriction digestion and subsequent agarose gel electrophoresis. This plasmid is called pK18mobsacBglu2.

As described in Example 2.1, DNA fragments bearing the ddh gene and surrounding regions are also amplified using a polymerase chain reaction. Based on the sequence of the ddh gene cluster known for Corynebacterium glutamicum (Ishino et al., Nucleic Acids Research 15, 3917 (1987) (Accession Y00151)), Primer oligonucleotides were selected for PCR:

ddh_beg (SEQ ID NO: 15):

5 'CTG AAT CAA AGG CGG ACA TG 3'

ddh_end (SEQ ID NO: 16):

5 'TCG AGC TAA ATT AGA CGT CG 3'

The primers presented are synthesized by MWG Biotech, and PCR reactions are described in Innis et al., PCR Protocol. A Guide to Methods and Applications, 1990, Academic Press]. The primers can be used to amplify DNA fragments of about 1.6 kb in length that carry the ddh gene.

Amplified DNA fragments of about 1.6 kb in length carrying the ddh gene were identified by electrophoresis on a 0.8% agarose gel, separated from the gel, and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden). do.

After purification, the fragment carrying the ddh gene is used for ligation with pK18mobsacBglu2 described above. It is first partially cut with the restriction enzyme BamHI. The vector was treated with Klenow polymerase (Amersham-Pharmacia, Freiburg, Germany), so that the overhang of the truncated end was the blunt end, and then the vector was mixed with a DNA fragment of about 1.6 kb carrying the ddh gene. The mixture is then treated with T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany). By using Vent polymerase for PCR reactions (New England Biolabs, Frankfurt, Germany), ddh-bearing DNA fragments with smooth ends and suitable for ligation with the pretreated vector pK18mobsacBglu2 are produced.

Subsequently, the E. coli strain DH5αmcr [Life Technologies GmbH, Karlsruhe, Germany] was subjected to the ligation batch [Hanahan, In: DNA cloning. A Practical approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989]. Selection of plasmid-bearing cells was performed by LB agar supplemented with kanamycin 50 mg / l [Sambrook et al., Molecular Cloning: a laboratory manual. 2 ^nd Ed., Cold Spring Harbor, New York, 1989].

Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep kit (Qiagen) and confirmed by restriction digestion and subsequent agarose gel electrophoresis. This plasmid is called pK18mobsacBglu2_1. A map of this plasmid is shown in FIG. 4.

2.2 Introduction of a second copy of the ddh gene into the chromosome (target site: gluB gene) of strain DSM12866 using alternative vector pK18mobsacBglu2_1

As described in Example 1.3, the plasmid pK18mobsacBglu2_1 described in Example 2.1 is transferred to the Corynebacterium glutamicum strain DSM12866 by conjugation. The desired recombination that occurred on the chromosome of Corynebacterium glutamicum DSM12866 was selected as described in Example 1.3. Depending on where the second recombination occurred, after deletion, a second copy of the ddh gene appears in the chromosome gluB locus, or the host's original gluB locus is maintained.

About 40 or 50 colonies are tested for the "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" phenotypes. About 20 colonies showing the "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" phenotypes were investigated using polymerase chain reaction. At this time, the DNA fragment containing the glu gene is amplified from the chromosomal DNA of the colony. The same primer oligonucleotides as described in Example 2.1 for the construction of replacement plasmids are selected for PCR.

gluA_beg (SEQ ID NO: 13):

5 'CAC GGT TGC TCA TTG TAT CC 3'

gluD_end (SEQ ID NO: 14):

5 'CGA GGC GAA TCA GAC TTC TT 3'

The primers can be used to amplify a DNA fragment of about 4.4 kb in a control clone with the original glu locus. In clones with a second copy of the ddh gene at the intrachromosomal gluB locus, a DNA fragment of about 6 kb is amplified.

Amplified DNA fragments are identified by electrophoresis on 0.8% agarose gel.

In addition to the copies present at the ddh locus, clones with a second copy of the ddh gene at the chromosome gluB locus have been identified in this manner. This clone is called strain DSM12866glu :: ddh.

Example 3

Introduction of the second copy of the dapA gene into the chromosome (target site: aecD gene) of strain DSM12866

3.1 Construction of the replacement vector pK18mobsacBaecD2_1

Corynebacterium glutamicum strain ATCC 13032 is used as donor of chromosomal DNA. From strain ATCC13032, chromosomal DNA is isolated using conventional methods (Eikmanns et al., Microbiology 140: 1817-1828 (1994)). Polymerase chain reaction is used to amplify DNA fragments bearing the aecD gene and surrounding regions. Based on the sequence of the known aecD gene for Corynebacterium glutamicum (Rossol et al., Journal of Bacteriology 174: 2968-2977 (1992) (Accession No. M89931)), the following primer oligonucleotides Was selected for PCR:

aecD_beg (SEQ ID NO: 11):

5 'GAA CTT ACG CCA AGC TGT TC 3'

aecD_end (SEQ ID NO: 12):

5 'AGC ACC ACA ATC AAC GTG AG 3'

The primers presented are synthesized by MWG Biotech, and PCR reactions are described in Innis et al., PCR Protocol. A Guide to Methods and Applications, 1990, Academic Press]. This primer can be used to amplify a DNA fragment of about 2.1 kb that contains the aecD gene and adjacent regions.

Amplified DNA fragments of about 2.1 kb in length are identified by electrophoresis on a 0.8% agarose gel, separated from the gel, and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden).

Purified DNA fragments are digested with restriction enzymes BamHI and EcoRV (Amersham-Pharmacia, Freiburg, Germany). This fragment is linked to the vector pUC18 (Norrander et al., Gene 26: 101-106 (1983)). It is first cleaved with restriction enzymes BamHI and SmaI, dephosphorylated, mixed with about 1.5 kb of aecD-bearing fragments and the mixture is treated with T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany). This ligation batch is transformed into E. coli strain TOP10 (Invitrogen, Leek, The Netherlands). Plasmid-bearing cells are selected by plating transfection batches on LB agar containing kanamycin (50 mg / l) with X-Gal (64 μg mg / l).

After isolation of the DNA, the resulting plasmid is confirmed by restriction cleavage and identified on an agarose gel. The resulting plasmid is called pUC18aecD.

The polymerase chain reaction is used to further amplify the DNA fragment carrying the dapA gene and the surrounding region. Based on the sequence of the dapA gene known for Corynebacterium glutamicum (Bonassi et al., Nucleic Acids Research 18: 6421 (1990) (Accession Nos. X53993 and AX127149)), the following primer oligonucleotides Was selected for PCR:

dapA_beg (SEQ ID NO: 17):

5 'CGA GCC AGT GAA CAT GCA GA 3'

dapA_end (SEQ ID NO: 18):

5 'CTT GAG CAC CTT GCG CAG CA 3'

The primers presented are synthesized by MWG Biotech, and PCR reactions are described in Innis et al., PCR Protocol. A Guide to Methods and Applications, 1990, Academic Press]. The primers can be used to amplify DNA fragments of about 1.4 kb that contain the dapA gene and adjacent regions.

Amplified DNA fragments of about 1.4 kb in length are identified by electrophoresis on a 0.8% agarose gel, separated from the gel, and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden).

After purification, a dapA-containing DNA fragment of about 1.4 kb in length is used for ligation with the vector pUC18aecD described above. It is first cleaved with restriction enzyme StuI, mixed with a DNA fragment of about 1.4 kb, and the mixture is treated with T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany).

Subsequently, the E. coli strain DH5αmcr [Life Technologies GmbH, Karlsruhe, Germany] was subjected to the ligation batch [Hanahan, In: DNA cloning. A Practical approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989]. Selection of plasmid-bearing cells was performed by LB agar supplemented with kanamycin 50 mg / l [Sambrook et al., Molecular Cloning: A Laboratory manual. 2 ^nd Ed., Cold Spring Harbor, New York, 1989].

Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep kit (Qiagen) and confirmed by restriction digestion and subsequent agarose gel electrophoresis. This plasmid is called pUC18aecD2.

Plasmid pUC18aecD2 was digested with restriction enzyme SalI and partly EcoRI (Amersham-Pharmacia, Freiburg, Germany), isolated on agarose gel (0.8%) using QIAquick gel extraction kit (Qiagen, Hilden, Germany), and then aecD And a fragment of about 2.7 kb bearing dapA was isolated from the agarose gel and used for ligation with the mobile cloning vector pK18mobsacB (Schafer et al., Gene 14: 69-73 (1994)). This was first cleaved with restriction enzymes EcoRI and SalI, dephosphorylated with Alkaline Phosphatase (Boehringer Mannheim), mixed with a fragment of about 2.7 kb bearing aecD and dapA, and the mixture was mixed with T4 DNA ligase ( Amersham-Pharmacia, Freiburg, Germany).

Subsequently, E. coli strain DH5α [Grant et al .; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649] are transformed with this linkage batch (Hanahan, In. DNA cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989]. Selection of plasmid-bearing cells was performed by LB agar supplemented with kanamycin 50 mg / l [Sambrook et al., Molecular Cloning: a laboratory manual. 2 ^nd Ed., Cold Spring Harbor, New York, 1989].

Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep kit (Qiagen) and confirmed by restriction digestion and subsequent agarose gel electrophoresis. This plasmid is called pK18mobsacBaecD2_1. A map of this plasmid is shown in FIG. 5.

3.2 Introduction of the second copy of the dapA gene into the chromosome (target site: aecD gene) of strain DSM12866 using alternative vector pK18mobsacBaecD2_1

As described in Example 1.3, the plasmid pK18mobsacBaecD2_1 described in Example 3.1 is transferred to the Corynebacterium glutamicum strain DSM12866 by conjugation. The desired recombination that occurred on the chromosome of Corynebacterium glutamicum DSM12866 was selected as described in Example 1.3. Depending on the location of the second recombination, a second copy of the dapA gene appears after deletion and appears at the aecD locus in the chromosome or the original aecD locus of the host is maintained.

About 40 or 50 colonies are tested for the "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" phenotypes. About 20 colonies showing the "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" phenotypes were investigated using polymerase chain reaction. At this time, the DNA fragment containing the aecD gene and the surrounding region is amplified from the chromosomal DNA of the colony. The same primer oligonucleotides as described in Example 3.1 for the construction of the integration plasmids are selected for PCR.

aecD_beg (SEQ ID NO: 11):

5 'GAA CTT ACG CCA AGC TGT TC 3'

aecD_end (SEQ ID NO: 12):

5 'AGC ACC ACA ATC AAC GTG AG 3'

The primers can be used to amplify DNA fragments of about 2.1 kb in control clones with the original aecD locus. In clones with a second copy of the dapA gene at the intrachromosomal aecD locus, a DNA fragment of about 3.6 kb is amplified.

Amplified DNA fragments are identified by electrophoresis on 0.8% agarose gel.

In addition to the copies present at the dapA locus, clones with a second copy of the dapA gene at the intrachromosomal aecD locus have been identified in this manner. This clone is called strain DSM12866aecD :: dapA.

Example 4

Introduction of the second copy of the pyc allele of the pyc allele pycP458S into the chromosome (target site: pck gene) of strain DSM12866

4.1 Construction of the replacement vector pK18mobsacBpck1_3

The replacement vector pK18mobsacBpck1 described in Example 1.5 is used as the base vector for the insertion of the pyc allele.

As described in Example 2.1, DNA fragments bearing the pyc gene and surrounding regions are also amplified using a polymerase chain reaction. Based on the sequence of known pyc gene clusters for Corynebacterium glutamicum (Peters-Wendisch et al., Journal of Microbiology 144: 915-927 (1998) (Accession No. Y09548)). The following primer oligonucleotides were selected for PCR:

pyc_beg (SEQ ID NO: 19):

5 'TC (A CGC GT) C TTG AAG TCG TGC AGG TCA G 3'

pyc_end (SEQ ID NO: 20):

5 'TC (A CGC GT) C GCC TCC TCC ATG AGG AAG A 3'

The primers presented are synthesized by MWG Biotech, and PCR reactions are described in Innis et al., PCR Protocol. A Guide to Methods and Applications, 1990, Academic Press]. This primer can be used to amplify a DNA fragment of about 3.6 kb that contains the pyc gene. The primer also contains the sequence of the cleavage site of the restriction endonuclease MluI, which is indicated in parentheses in the nucleotide sequence shown above.

Amplified DNA fragments of about 3.6 kb in length carrying the pyc gene were identified by electrophoresis on a 0.8% agarose gel, separated from the gel, and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden). do.

After purification, the fragment carrying the pyc gene is used for ligation with pK18mobsacBpck1 described above. It was first cleaved with restriction enzyme BssHII, dephosphorylated with Alkaline Phosphatase (Boehringer Mannheim), mixed with a DNA fragment of about 3.6 kb carrying the pyc gene, and the mixture was mixed with T4 DNA ligase (Amersham- Pharmacia, Freiburg, Germany).

Subsequently, the E. coli strain DH5αmcr [Life Technologies GmbH, Karlsruhe, Germany] was subjected to the ligation batch [Hanahan, In: DNA cloning. A Practical approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989]. Selection of plasmid-bearing cells was performed by LB agar supplemented with kanamycin 50 mg / l [Sambrook et al., Molecular Cloning: a laboratory manual. 2 ^nd Ed., Cold Spring Harbor, New York, 1989].

Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep kit (Qiagen) and confirmed by restriction digestion and subsequent agarose gel electrophoresis. This plasmid is called pK18mobsacBpck1_2.

4.2 Construction of the pyc allele pyc P458S by site-specific mutagenesis of the wild-type pyc gene

Site-directed mutagenesis is performed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, USA). EP-A-1108790 describes a point mutation in the pyc gene for Corynebacterium glutamicum that can enhance L-lysine production. Following a point mutation in the nucleotide sequence where cytosine is changed to thymidine at position 1372 in the pyc gene, proline is substituted for serine at position 458 in the amino acid sequence derived therefrom. This allele is called pyc p458S. To generate the described mutations, the following primer oligonucleotides are selected and linearly amplified:

P458S-1 (SEQ ID NO: 21):

5 'GGATTCATTGCCGATCAC (TCG) CACCTCCTTCAGGCTCCA 3'

P458S-2 (SEQ ID NO: 22):

5 'TGGAGGAAGTCCGAGGT (CGA) GTGATCGGCAATGAATCC 3'

The primers presented are synthesized by MWG Biotech. The codons for serine substituting proline at position 458 are indicated in parentheses in the sequence set forth above. The plasmid pK18mobsacBpck1_2 described in Example 4.1 is used for linear amplification by Pfu Turbo DNA polymerase, with two primers each complementary to the plasmid chain. By extension of these primers, mutated plasmids with broken cyclic chains are formed. The product of linear amplification is treated with DpnI—this endonuclease specifically cleaves methylated and 1 / 2-methylated template DNA. Newly synthesized, broken, mutated vector DNA is transformed into E. coli strain XL1 Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)). After transformation, XL1 blue cells repair the break in the mutated plasmid. Transformants are selected on LB medium containing 50 mg / l kanamycin. After isolation of the DNA, the resulting plasmid is confirmed by restriction cleavage and identified on an agarose gel. DNA sequence of the mutated DNA fragment is confirmed by sequencing. The sequence of the PCR product is described by Ohnishi et al. (2002). The resulting plasmid is called pK18mobsacBpck1_3. The column of the plasmid is shown in FIG. 6.

4.3 Introduction of a second copy of the pyc allele pycP458S form of the pyc allele into the chromosome (target site: pck gene) of strain DSM12866 using alternative vector pK18mobsacBpck1_3

As described in Example 1.3, the plasmid pK18mobsacBpck1_3 described in Example 4.2 is transferred to the Corynebacterium glutamicum strain DSM12866 by conjugation. The desired recombination that occurred on the chromosome of Corynebacterium glutamicum DSM12866 was selected as described in Example 1.3. Depending on the location where the second recombination occurred, after deletion, a second copy of the pyc allele appears at the pck locus in the chromosome, or the original pck locus of the host is maintained.

About 40 or 50 colonies are tested for the "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" phenotypes. About 20 colonies showing the "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" phenotypes were investigated using polymerase chain reaction. At this time, the DNA fragment containing the pck gene and the surrounding region is amplified from the chromosomal DNA of the colony. The same primer oligonucleotides as described in Example 1.5 for the construction of replacement plasmids are selected for PCR.

pck_beg (SEQ ID NO: 9):

5 'TA (A GAT CT) G CCG GCA TGA CTT CAG TTT 3'

pck_end (SEQ ID NO: 10):

5 'AC (A GAT CT) G GTG GGA GCC TTT CTT GTT ATT3'

The primers can be used to amplify a DNA fragment of about 2.9 kb in a control clone with the original pck locus. In clones with a second copy of the pyc allele at the pck locus in the chromosome, about 6.5 kb of DNA fragment is amplified.

Amplified DNA fragments are identified by electrophoresis on 0.8% agarose gel.

In addition to a copy of the wild-type gene present at the pyc locus, a clone having a second copy of the pyc gene of the pyc allele pycP458S form at the pyc locus in the chromosome was identified in this way. This clone is called strain DSM12866pck :: pyc.

Example 5

Preparation of Lysine

Corynebacterium glutamicum strains DSM13994glu :: lysC, DSM12866glu :: lysC, DSM12866pck :: lysC, DSM12866aecD :: lysC, DSM12866glu :: ddh, DSM12866aecD :: dapA obtained in Examples 1, 2, 3, and 4 DSM12866pck :: pyc was incubated in a nutrient medium suitable for lysine production and then the lysine content in the culture supernatant was measured.

To this end, the cultures were first incubated at 33 ° C. for 24 hours on brain-heart agar agar plates (Merck, Darmstadt, Germany). Starting from this agar plate culture, precultures were seeded (10 ml medium per 100 ml conical flask). Medium MM is used as a medium for preculture. The preculture was incubated at 33 ° C. for 24 hours at 240 rpm in a shaker. Seed the main culture from the preculture so that the initial OD (660 nm) of the main culture is 0.1 OD. Medium MM is also used for main culture.

Badge MM:

CSL 5g / ℓ

MOPS 20g / ℓ

Glucose (autoclaved separately) 50 g / l

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 10mg / L

FeSO ₄ * 7H ₂ O 10 mg / l

MnSO ₄ * H ₂ O 5.0 mg / l

Biotin (sterile filtration) 0.3 mg / l

Thiamine * HCl (sterile filtration) 0.2mg / ℓ

CaCO ₃ 25g / ℓ

CSL (corn immersion), MOPS (morpholinopropanesulfonic acid) and salt solutions are adjusted to pH 7 with ammonia water and then autoclaved. Then, as well as sterile substrate and vitamin solution, CaCO ₃ autoclaved to dry state is added.

Incubation is performed at a 10 ml dose per 100 ml conical flask equipped with baffles. Incubation is performed at 33 ° C. and 80% atmospheric humidity.

After 48 hours, the OD is measured at a measurement wavelength of 660 nm using a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of lysine produced is measured by post-column derivatization using ion exchange chromatography and ninhydrin detection using an amino acid analyzer (Eppendorf-BioTronik, Hamburg, Germany).

The experimental results are shown in Table 10.

Conventional technology

Chemical compounds In particular, compounds meaning L-amino acids, vitamins, nucleosides and nucleotides and D-amino acids are used in human medicine, pharmaceutical industry, cosmetics, foodstuff industry and animal feed.

Many of these compounds are produced by fermentation from strains of Coryneform bacteria, in particular Corynebacterium glutamicum. Because of their enormous importance, work has continued to improve production methods. Improvements in the production methods can be achieved by fermentation techniques (e.g., stirring and oxygenation), composition of the nutrient medium (e.g. Inherent productivity.

Mutagenesis, selection, and mutant screening can be used to improve the productivity of these microorganisms. Strains that are resistant to anti-metabolites or that are nutritionally constitutive to regulatoryly important metabolites and produce certain compounds are obtained in this way.

Over the years, recombinant strains of Corynebacterium have been improved by studying the effects of amplifying and producing individual biosynthetic genes using recombinant DNA techniques.

Conventional methods include amplifying certain biosynthetic genes, particularly using plasmids that replicate as episomes in microorganisms. This method has the disadvantage that the plasmid is naturally lost during fermentation, which typically goes through many generations in an industrial process (separate instability).

Another method involves overlapping certain biosynthetic genes using plasmids that do not replicate in a particular microorganism. In this method, the plasmid containing the cloned biosynthetic gene is integrated into the chromosomal biosynthetic gene of the microorganism [Reinscheild et al., Applied and Environmental Microbiology 60 (1), 126-132 (1994); Jetten et al., Applied Microbiology and Biotechnology 43 (1): 76-82 (1995). A disadvantage of this method is that the nucleotide sequence of the plasmid and the nucleotide sequence of the antibiotic resistance gene required for selection remain in the microorganism. This is a disadvantage, for example in the treatment and use of biomass. Moreover, experts anticipate that such strains will be unstable as a result of decay by "Campbell type cross over" for a significant number of generations common in industrial fermentation.

Purpose of the Invention

It is an object of the present invention to provide new means for improving fermentative production of chemical compounds using coryneform bacteria.

Summary of the Invention

Coryneform bacteria that produce chemical compounds include, in addition to one or more copies of which are present in the open reading frame (ORF), the natural site (gene locus) of the gene or allele, that encodes the synthesis of a protein or RNA. An open reading frame (ORF), a second, optionally third or fourth copy of a gene or allele, is incorporated in the chromosome in the second, optionally third or fourth region, wherein the epitaxially in the microorganism is present. Nucleotide sequences that enable / enable som replication or translocation and nucleotide sequences conferring resistance to antibiotics are not present in said second, optionally third or fourth region, and said second, optionally third or fourth region Does not relate to open reading frames, genes or alleles essential for bacterial growth and production of the desired compound It shall be.

In addition, the present invention is the following steps:

a) a1) an open reading frame (ORF) encoding a synthesis of a protein or RNA, in addition to one or more copies present in the natural site (locus) of a gene or allele, such open reading frame (ORF), gene or A second, optionally third or fourth copy of the allele is contained in the second, optionally third or fourth site in an integrated form within the chromosome, wherein episomal replication or translocation is possible / possible in the microorganism. Wherein the nucleotide sequence and the nucleotide sequence conferring resistance to the antibiotic are not present in the second, optionally third or fourth site, and the second, optionally third or fourth site is used for bacterial growth and desired compound. It's not about open reading frames, genes or alleles that are essential for production,

a2) increased intracellular activity of the corresponding protein, in particular overexpressed the nucleotide sequence encoding such protein,

Fermenting coryneform bacteria;

b) concentrating the chemical compound (s) in fermentation broth and / or cells of the bacterium;

c) separating the chemical compound (s), optionally

d) providing a process for preparing one or more chemical compounds by performing a step in which the constituent from the fermentation broth and / or biomass is greater than 0 to about 100% by weight.

In addition, the present invention is the following steps:

a) in addition to a copy of an open reading frame (ORF), gene or allele present at the natural site (gene locus), in each case a second, optionally second, of the open reading frame (ORF), gene or allele A nucleotide sequence which enables or enables episomal replication in the microorganism, wherein the nucleotide sequence enables / enables episomal replication in the microorganism, respectively, in an integrated form, at the second, optionally third or fourth site The nucleotide sequence that confers resistance to the sequence and antibiotics permits the expression of the Coryneform bacteria absent at a particular second, optionally third or fourth site, the open reading frame (ORF), gene or allele. Fermentation under conditions;

b) concentrating the chemical compound (s) in fermentation broth and / or cells of the bacterium;

c) separating the chemical compound (s), optionally

d) providing a process for preparing one or more chemical compounds by performing a step in which the constituent from the fermentation broth and / or biomass is greater than 0 to about 100% by weight.

<110> Degussa AG <120> Production of L-lysine by genetically modified Corynebacterium glutamicum strains <130> 010301 BT <150> US 60 / 309,878 <151> 2001-08-06 <160> 22 <170> KopatentIn 1.71 <210> 1 <211> 1263 <212> DNA <213> Corynebacterium glutamicum <220> <221> CDS <222> (1) .. (1263) <223> lysC wild-type gene <400> 1 gtg gcc ctg gtc gta cag aaa tat ggc ggt tcc tcg ctt gag agt gcg 48 Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala 1 5 10 15 gaa cgc att aga aac gtc gct gaa cgg atc gtt gcc acc aag aag gct 96 Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala 20 25 30 gga aat gat gtc gtg gtt gtc tgc tcc gca atg gga gac acc acg gat 144 Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp 35 40 45 gaa ctt cta gaa ctt gca gcg gca gtg aat ccc gtt ccg cca gct cgt 192 Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg 50 55 60 gaa atg gat atg ctc ctg act gct ggt gag cgt att tct aac gct ctc 240 Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu 65 70 75 80 gtc gcc atg gct att gag tcc ctt ggc gca gaa gcc caa tct ttc acg 288 Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr 85 90 95 ggc tct cag gct ggt gtg ctc acc acc gag cgc cac gga aac gca cgc 336 Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg 100 105 110 att gtt gat gtc act cca ggt cgt gtg cgt gaa gca ctc gat gag ggc 384 Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly 115 120 125 aag atc tgc att gtt gct ggt ttc cag ggt gtt aat aaa gaa acc cgc 432 Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg 130 135 140 gat gtc acc acg ttg ggt cgt ggt ggt tct gac acc act gca gtt gcg 480 Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala 145 150 155 160 ttg gca gct gct ttg aac gct gat gtg tgt gag att tac tcg gac gtt 528 Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val 165 170 175 gac ggt gtg tat acc gct gac ccg cgc atc gtt cct aat gca cag aag 576 Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys 180 185 190 ctg gaa aag ctc agc ttc gaa gaa atg ctg gaa ctt gct gct gtt ggc 624 Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly 195 200 205 tcc aag att ttg gtg ctg cgc agt gtt gaa tac gct cgt gca ttc aat 672 Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn 210 215 220 gtg cca ctt cgc gta cgc tcg tct tat agt aat gat ccc ggc act ttg 720 Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu 225 230 235 240 att gcc ggc tct atg gag gat att cct gtg gaa gaa gca gtc ctt acc 768 Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr 245 250 255 ggt gtc gca acc gac aag tcc gaa gcc aaa gta acc gtt ctg ggt att 816 Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile 260 265 270 tcc gat aag cca ggc gag gct gcg aag gtt ttc cgt gcg ttg gct gat 864 Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp 275 280 285 gca gaa atc aac att gac atg gtt ctg cag aac gtc tct tct gta gaa 912 Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu 290 295 300 gac ggc acc acc gac atc acc ttc acc tgc cct cgt tcc gac ggc cgc 960 Asp Gly Thr Thr Asp Ile Thr Phe Thr Cys Pro Arg Ser Asp Gly Arg 305 310 315 320 cgc gcg atg gag atc ttg aag aag ctt cag gtt cag ggc aac tgg acc 1008 Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr 325 330 335 aat gtg ctt tac gac gac cag gtc ggc aaa gtc tcc ctc gtg ggt gct 1056 Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala 340 345 350 ggc atg aag tct cac cca ggt gtt acc gca gag ttc atg gaa gct ctg 1104 Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu 355 360 365 cgc gat gtc aac gtg aac atc gaa ttg att tcc acc tct gag att cgt 1152 Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg 370 375 380 att tcc gtg ctg atc cgt gaa gat gat ctg gat gct gct gca cgt gca 1200 Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala 385 390 395 400 ttg cat gag cag ttc cag ctg ggc ggc gaa gac gaa gcc gtc gtt tat 1248 Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr 405 410 415 gca ggc acc gga cgc 1263 Ala Gly Thr Gly Arg 420 <210> 2 <211> 421 <212> PRT <213> Corynebacterium glutamicum <400> 2 Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala 1 5 10 15 Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala 20 25 30 Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp 35 40 45 Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg 50 55 60 Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu 65 70 75 80 Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr 85 90 95 Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg 100 105 110 Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly 115 120 125 Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg 130 135 140 Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala 145 150 155 160 Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val 165 170 175 Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys 180 185 190 Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly 195 200 205 Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn 210 215 220 Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu 225 230 235 240 Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr 245 250 255 Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile 260 265 270 Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp 275 280 285 Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu 290 295 300 Asp Gly Thr Thr Asp Ile Thr Phe Thr Cys Pro Arg Ser Asp Gly Arg 305 310 315 320 Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr 325 330 335 Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala 340 345 350 Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu 355 360 365 Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg 370 375 380 Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala 385 390 395 400 Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr 405 410 415 Ala Gly Thr Gly Arg 420 <210> 3 <211> 1263 <212> DNA <213> Corynebacterium glutamicum <220> <221> CDS <222> (1) .. (1263) <223> lysC-fbr allele lysC T311I <400> 3 gtg gcc ctg gtc gta cag aaa tat ggc ggt tcc tcg ctt gag agt gcg 48 Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala 1 5 10 15 gaa cgc att aga aac gtc gct gaa cgg atc gtt gcc acc aag aag gct 96 Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala 20 25 30 gga aat gat gtc gtg gtt gtc tgc tcc gca atg gga gac acc acg gat 144 Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp 35 40 45 gaa ctt cta gaa ctt gca gcg gca gtg aat ccc gtt ccg cca gct cgt 192 Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg 50 55 60 gaa atg gat atg ctc ctg act gct ggt gag cgt att tct aac gct ctc 240 Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu 65 70 75 80 gtc gcc atg gct att gag tcc ctt ggc gca gaa gcc caa tct ttc acg 288 Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr 85 90 95 ggc tct cag gct ggt gtg ctc acc acc gag cgc cac gga aac gca cgc 336 Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg 100 105 110 att gtt gat gtc act cca ggt cgt gtg cgt gaa gca ctc gat gag ggc 384 Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly 115 120 125 aag atc tgc att gtt gct ggt ttc cag ggt gtt aat aaa gaa acc cgc 432 Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg 130 135 140 gat gtc acc acg ttg ggt cgt ggt ggt tct gac acc act gca gtt gcg 480 Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala 145 150 155 160 ttg gca gct gct ttg aac gct gat gtg tgt gag att tac tcg gac gtt 528 Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val 165 170 175 gac ggt gtg tat acc gct gac ccg cgc atc gtt cct aat gca cag aag 576 Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys 180 185 190 ctg gaa aag ctc agc ttc gaa gaa atg ctg gaa ctt gct gct gtt ggc 624 Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly 195 200 205 tcc aag att ttg gtg ctg cgc agt gtt gaa tac gct cgt gca ttc aat 672 Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn 210 215 220 gtg cca ctt cgc gta cgc tcg tct tat agt aat gat ccc ggc act ttg 720 Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu 225 230 235 240 att gcc ggc tct atg gag gat att cct gtg gaa gaa gca gtc ctt acc 768 Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr 245 250 255 ggt gtc gca acc gac aag tcc gaa gcc aaa gta acc gtt ctg ggt att 816 Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile 260 265 270 tcc gat aag cca ggc gag gct gcg aag gtt ttc cgt gcg ttg gct gat 864 Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp 275 280 285 gca gaa atc aac att gac atg gtt ctg cag aac gtc tct tct gta gaa 912 Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu 290 295 300 gac ggc acc acc gac atc atc ttc acc tgc cct cgt tcc gac ggc cgc 960 Asp Gly Thr Thr Asp Ile Ile Phe Thr Cys Pro Arg Ser Asp Gly Arg 305 310 315 320 cgc gcg atg gag atc ttg aag aag ctt cag gtt cag ggc aac tgg acc 1008 Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr 325 330 335 aat gtg ctt tac gac gac cag gtc ggc aaa gtc tcc ctc gtg ggt gct 1056 Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala 340 345 350 ggc atg aag tct cac cca ggt gtt acc gca gag ttc atg gaa gct ctg 1104 Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu 355 360 365 cgc gat gtc aac gtg aac atc gaa ttg att tcc acc tct gag att cgt 1152 Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg 370 375 380 att tcc gtg ctg atc cgt gaa gat gat ctg gat gct gct gca cgt gca 1200 Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala 385 390 395 400 ttg cat gag cag ttc cag ctg ggc ggc gaa gac gaa gcc gtc gtt tat 1248 Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr 405 410 415 gca ggc acc gga cgc 1263 Ala Gly Thr Gly Arg 420 <210> 4 <211> 421 <212> PRT <213> Corynebacterium glutamicum <400> 4 Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala 1 5 10 15 Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala 20 25 30 Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp 35 40 45 Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg 50 55 60 Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu 65 70 75 80 Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr 85 90 95 Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg 100 105 110 Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly 115 120 125 Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg 130 135 140 Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala 145 150 155 160 Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val 165 170 175 Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys 180 185 190 Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly 195 200 205 Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn 210 215 220 Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu 225 230 235 240 Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr 245 250 255 Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile 260 265 270 Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp 275 280 285 Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu 290 295 300 Asp Gly Thr Thr Asp Ile Ile Phe Thr Cys Pro Arg Ser Asp Gly Arg 305 310 315 320 Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr 325 330 335 Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala 340 345 350 Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu 355 360 365 Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg 370 375 380 Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala 385 390 395 400 Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr 405 410 415 Ala Gly Thr Gly Arg 420 <210> 5 <211> 28 <212> DNA <213> Artificial sequence <220> <221> misc_feature (222) (1) .. (28) Primer lysC1beg <400> 5 taggatcctc cggtgtctga ccacggtg 28 <210> 6 <211> 29 <212> DNA <213> Artificial sequence <220> <221> misc_feature (222) (1) .. (29) Primer lysC2end <400> 6 acggatccgc tgggaaattg cgctcttcc 29 <210> 7 <211> 28 <212> DNA <213> Artificial sequence <220> <221> misc_feature (222) (1) .. (28) Primer gluBgl1 <400> 7 taagatctgt gttggacgtc atggcaag 28 <210> 8 <211> 28 <212> DNA <213> Artificial sequence <220> <221> misc_feature (222) (1) .. (28) Primer gluBgl2 <400> 8 acagatcttg aagccaagta cggccaag 28 <210> 9 <211> 27 <212> DNA <213> Artificial sequence <220> <221> misc_feature (222) (1) .. (27) <223> Primer pck_beg <400> 9 taagatctgc cggcatgact tcagttt 27 <210> 10 <211> 30 <212> DNA <213> Artificial sequence <220> <221> misc_feature (222) (1) .. (30) <223> Primer pck_end <400> 10 acagatctgg tgggagcctt tcttgttatt 30 <210> 11 <211> 20 <212> DNA <213> Corynebacterium glutamicum <220> <221> misc_feature (222) (1) .. (20) <223> Primer aecD_beg <400> 11 gaacttacgc caagctgttc 20 <210> 12 <211> 20 <212> DNA <213> Corynebacterium glutamicum <220> <221> misc_feature (222) (1) .. (20) <223> Primer aecD_end <400> 12 agcaccacaa tcaacgtgag 20 <210> 13 <211> 20 <212> DNA <213> Corynebacterium glutamicum <220> <221> misc_feature (222) (1) .. (20) Primer gluA_beg <400> 13 cacggttgct cattgtatcc 20 <210> 14 <211> 20 <212> DNA <213> Corynebacterium glutamicum <220> <221> misc_feature (222) (1) .. (20) <223> Primer gluD_end <400> 14 cgaggcgaat cagacttctt 20 <210> 15 <211> 20 <212> DNA <213> Corynebacterium glutamicum <220> <221> misc_feature (222) (1) .. (20) <223> Primer ddh_beg <400> 15 ctgaatcaaa ggcggacatg 20 <210> 16 <211> 20 <212> DNA <213> Corynebacterium glutamicum <220> <221> misc_feature (222) (1) .. (20) <223> Primer ddh_end <400> 16 tcgagctaaa ttagacgtcg 20 <210> 17 <211> 20 <212> DNA <213> Corynebacterium glutamicum <220> <221> misc_feature (222) (1) .. (20) <223> Primer dapA_beg <400> 17 cgagccagtg aacatgcaga 20 <210> 18 <211> 20 <212> DNA <213> Corynebacterium glutamicum <220> <221> misc_feature (222) (1) .. (20) <223> Primer dapA_end <400> 18 cttgagcacc ttgcgcagca 20 <210> 19 <211> 28 <212> DNA <213> Artificial sequence <220> <221> misc_feature (222) (1) .. (28) <223> Primer pyc_beg <400> 19 tcacgcgtct tgaagtcgtg caggtcag 28 <210> 20 <211> 28 <212> DNA <213> Artificial sequence <220> <221> misc_feature (222) (1) .. (28) <223> Primer pyc_end <400> 20 tcacgcgtcg cctcctccat gaggaaga 28 <210> 21 <211> 39 <212> DNA <213> Corynebacterium glutamicum <220> <221> misc_feature (222) (1) .. (39) <223> Primer P458S-1 <400> 21 ggattcattg ccgatcactc gcacctcctt caggctcca 39 <210> 22 <211> 39 <212> DNA <213> Corynebacterium glutamicum <220> <221> misc_feature (222) (1) .. (39) <223> Primer P458S-2 <400> 22 gtggaggaag tccgaggtcg agtgatcggc aatgaatcc 39

Claims (40)

In addition to the open reading frame (ORF) encoding the synthesis of the protein or RNA, one or more copies present in the natural site (gene locus) of the gene or allele, the desired open reading frame (ORF), gene or allele A second, optionally third, or fourth copy of the nucleotide sequence in an integrated form within the chromosome, at the second, optionally third, or fourth site, wherein the nucleotide sequence enables / enables episomal replication or translocation in the microorganism. And no nucleotide sequence (s) conferring resistance to antibiotics are present at said second, optionally third or fourth site, said second, optionally third or fourth site being used for bacterial growth and desired compounds. Coryneform bacteria that produce chemical compounds that are not related to open reading frames, genes or alleles essential for production. The coryneform bacterium producing a chemical compound according to claim 1, wherein the coryneform bacterium belongs to the corynebacterium genus. The coryneform bacterium of the genus Corynebacterium according to claim 2, which produces a chemical compound belonging to the corynebacterium glutamicum species. The coryneform bacterium producing a chemical compound according to claim 1, wherein the chemical compound is a compound selected from the group consisting of L-amino acids, vitamins, nucleosides and nucleotides. The chemical compound of claim 1, wherein the chemical compound is L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine, L-cysteine, L-valine, L- At least one L selected from the group consisting of methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-proline and L-arginine Coryneform bacteria producing chemical compounds that are amino acids. The method according to claim 1 or 4, wherein the L-amino acid is L-lysine and in addition to one or more copies of an open reading frame (ORF), gene or allele for production of lysine present at the natural site (gene locus). In each case an open reading frame (ORF) for the production of the desired lysine, a second, optionally third or fourth, respectively, in a form incorporating into the chromosome a second, optionally third or fourth copy of a gene or allele Coryneform bacteria which produce chemical compounds contained in four sites. The coryneform bacterium producing L-lysine according to claim 6, wherein the coryneform bacterium belongs to the corynebacterium genus. The coryneform bacterium of the genus Corynebacterium according to claim 7, which belongs to Corynebacterium glutamicum species. The method of claim 6, wherein the open reading frame (ORF), gene or allele for lysine production is accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE , dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysC ^FBR , lysE, msiK, opcA, oxyR, ppc, ppc ^FBR , pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI one or more open reading frame (s), gene (s) selected from the group consisting of ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T Coryneform bacteria producing L-lysine, or allele (s). The method of claim 6, wherein the open reading frame, gene or allele for lysine production is one or more gene (s) or allele (s) selected from the group consisting of dapA, ddh, lysC ^FBR and pyc P458S. Coryneform bacteria producing L-lysine. The coryneform bacterium producing L-lysine according to claim 6, wherein the open reading frame, gene or allele for lysine production is the lysC ^FBR allele encoding the feedback resistance type of aspartate kinase. 12. The feed bag resistant type of aspartate kinase encoded by the lysC ^FBR allele is one selected from the group consisting of A279T, A279V, S301F, T308I, S301Y, G345D, R320G, T311I and S381F. A coryneform bacterium producing L-lysine, comprising the amino acid sequence according to SEQ ID NO: 2 containing the above amino acid substituent. The coryneform bacterium producing L-lysine according to claim 11, wherein the feedback resistance type of aspartate kinase encoded by the lysC ^FBR allele comprises the amino acid sequence according to SEQ ID NO: 4. 12. The coryneform bacterium producing L-lysine of claim 11, wherein the coding region of the lysC ^FBR allele comprises the nucleotide sequence of SEQ ID NO: 3. The method of claim 6, wherein the specific second, optionally third or fourth site is aecD, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, Coryneform bacteria producing L-lysine, a gene selected from the group consisting of menE, mqo, pck, pgi and poxB. The L of claim 6, wherein the particular second, optionally third or fourth site is a site selected from the group consisting of an intergenic region of a chromosome, a propage contained within a chromosome and a defective phage contained within a chromosome. Coryneform bacteria producing lysine. 16. The coryneform bacterium producing L-lysine according to claim 15, wherein the particular second, optionally third or fourth region is an aecD gene region. 16. The coryneform bacterium producing L-lysine according to claim 15, wherein the particular second, optionally third or fourth site is the gluB gene site. 16. The coryneform bacterium producing L-lysine according to claim 15, wherein the particular second, optionally third or fourth region is a pck gene region. a) a1) an open reading frame (ORF) encoding a synthesis of a protein or RNA, in addition to one or more copies present in the natural site (locus) of a gene or allele, said open reading frame (ORF), gene or A second, optionally third or fourth copy of the allele is contained in the second, optionally third or fourth site in an integrated form within the chromosome, allowing episomal replication or translocation within the microorganism to be / enable No nucleotide sequence (s) conferring a nucleotide sequence and resistance to antibiotics are present in the second, optionally third or fourth site, and the second, optionally third or fourth site is used for bacterial growth and desired It does not relate to open reading frames, genes or alleles essential for the production of compounds, a2) increased intracellular activity of the corresponding protein, in particular overexpressed the nucleotide sequence encoding such protein, Fermenting coryneform bacteria; b) concentrating the chemical compound (s) in fermentation broth and / or cells of the bacterium; c) separating the chemical compound (s), optionally d) a process wherein the constituents from the fermentation broth and / or biomass are from greater than 0 to about 100% by weight to produce chemical compounds by fermentation of coryneform bacteria. The method of claim 20, wherein the coryneform bacteria belong to the Corynebacterium genus. The method of claim 20, wherein the Coryneform bacteria of the genus Corynebacterium belong to the Corynebacterium glutamicum species. The method of claim 20, wherein the chemical compound is a compound selected from the group consisting of L-amino acids, vitamins, nucleosides, and nucleotides. The chemical compound of claim 20, wherein the chemical compound is L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine, L-cysteine, L-valine, L- At least one L selected from the group consisting of methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-proline and L-arginine -An amino acid. The method of claim 24, wherein the chemical compound is L-lysine. a) an open reading frame (ORF) for the production of lysine, a gene or allele of one or more copies of the allele present in the natural site (gene locus), in each case an open reading frame (ORF), gene for the lysine production Or an open reading frame (ORF) comprising a coryneform bacterium containing a second, optionally third or fourth copy of an allele in the second, optionally third or fourth region, respectively, in an integrated form in a chromosome; A method of producing L-lysine, comprising the step of fermenting under conditions that allow expression of the gene or allele. The method of claim 26, wherein the open reading frame (ORF), gene or allele for lysine production is accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE , dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysC ^FBR , lysE, msiK, opcA, oxyR, ppc, ppc ^FBR , pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI open reading frame (ORF), gene or allele selected from the group consisting of ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T How to produce phosphorus, L-lysine. The production of L-lysine according to claim 26, wherein the open reading frame (ORF), gene or allele for lysine production is a gene or allele selected from the group consisting of dapA, ddh, lysC ^FBR and pyc P458S. How to. 27. The method of claim 26, wherein the open reading frame (ORF), gene or allele for lysine production is a lysC ^FBR allele encoding a feed back resistance type of aspartate kinase. The method of claim 29, wherein the feedback resistance type of aspartate kinase encoded by the lysC ^FBR allele is one selected from the group consisting of A279T, A279V, S301F, T308I, S301Y, G345D, R320G, T311I and S381F. A method for producing L-lysine, comprising the amino acid sequence according to SEQ ID NO: 2 containing the above amino acid substituent. 30. The method of claim 29, wherein the feedback resistance type of aspartate kinase encoded by the lysC ^FBR allele comprises the amino acid sequence according to SEQ ID NO: 4. The method of claim 29, wherein the coding region of the lysC ^FBR allele comprises the nucleotide sequence of SEQ ID NO: 3. 30. The method of claim 26, wherein the particular second, optionally third or fourth site is aecD, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, A method for producing L-lysine, a site selected from the group consisting of menE, mqo, pck, pgi and poxB. The method of claim 26, wherein the second, optionally third or fourth region is an aecD gene region. The method of claim 26, wherein the second, optionally third or fourth site is the gluB gene site. The method of claim 26, wherein the second, optionally third or fourth region is a pck gene region. a) separating the nucleotide sequence of one or more desired ORFs, genes or alleles encoding a protein or RNA, optionally comprising expression and / or regulatory signals, from a coryneform bacterium, b) attaching the nucleotide sequence of the target site to the 5 'and 3' ends of the ORF, gene or allele, c) Preferably, the nucleotide sequence of the desired ORF, gene or allele to which the nucleotide sequence of the target site is attached is introduced into a vector which does not replicate in coryneform bacteria or only at a limited level, d) transferring the nucleotide sequence according to b) or c) into coryneform bacteria, e) the nucleotide sequence (s) according to a) is introduced at the target site and the nucleotide sequence (s) conferring resistance to antibiotics and the nucleotide sequence (s) that enable / enable episomal replication or translocation in the microorganism. A method of producing coryneform bacteria that produce one or more chemical compounds, including isolating coryneform bacteria that do not remain at this target site. The plasmid pK18mobsacBglu1_1 shown in FIG. 1 deposited in accession No. DSM14243 in the form of a pure culture of strain E. coli DH5αmcr / pK18mobsacBglu1_1 (= DH5alphamcr / pK18mobsacBglu1_1). Plasmid pK18mobsacBaecD1_1 as shown in FIG. 2 deposited in accession no. Corynebacterium glutamicum strain DSM 12866glu :: lysC deposited under accession no.DSM15039 in the form of a pure culture.