EP1414970A2

EP1414970A2 - Production of l-lysine by genetically modified corynebacterium glutamicum strains

Info

Publication number: EP1414970A2
Application number: EP02760293A
Authority: EP
Inventors: Brigitte Bathe; Caroline Kreutzer; Bettina Möckel; Georg Thierbach; Caroline Reyen
Original assignee: Degussa GmbH
Current assignee: Evonik Operations GmbH
Priority date: 2001-08-06
Filing date: 2002-07-30
Publication date: 2004-05-06
Also published as: CN1539015A; WO2003040373A3; WO2003040373A2; CN100554426C; AU2002325923A1; BR0211723A; CA2455878A1

Abstract

The invention relates to coryneform bacteria which have, in addition to at least one copy, present at the natural site (locus), of an open reading frame (ORF), gene or allele which codes for the synthesis of a protein or an RNA, in each case a second, optionally third or fourth copy of this open reading frame (ORF), gene or allele at in each case a second, optionally third or fourth site in a form integrated into the chromosome and processes for the preparation of chemical compounds by fermentation of these bacteria.

Description

Coryneform Bacteria which Produce Chemical Compounds I

Prior Art

Chemical compounds, which means, in particular, L-amino acids, vitamins, nucleosides and nucleotides and D-amino acids, are used in human medicine, in the pharmaceuticals industry, in cosmetics, in the foodstuffs industry and in animal nutrition.

Numerous of these compounds are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and which produce the particular compounds are obtained in this manner.

Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains, by amplifying individual biosynthesis genes and investigating the effect on production.

A common method comprises amplification of certain biosynthesis genes in the particular microorganism by means of episomally replicating plasmids. This procedure has the disadvantage that during the fermentation, which in industrial processes is in general associated with numerous generations, the plasmids are lost spontaneously (segregational instability) .

Another method comprises duplicating certain biosynthesis genes by means of plasmids which do not replicate in the particular microorganism. In this method, the plasmid, including the cloned biosynthesis gene, is integrated into the chromosomal biosynthesis gene of the microorganism (Reinscheid et al . , Applied and Environmental Microbiology 60(1), 126-132 (1994); Jetten et al . , Applied Microbiology and Biotechnology 43(l):76-82 (1995)). A disadvantage of this method is that the nucleotide sequences of the plasmid and of the antibiotic resistance gene necessary for the selection remain in the microorganism. This is a disadvantage, for example, for the disposal and utilization of the biomass. Moreover, the expert expects such strains to be unstable as a result of disintegration by "Campbell type cross over" in a corresponding number of generations such as are usual in industrial fermentations.

Object of the Invention

The inventors had the object of providing new measures for improved fermentative preparation chemical compounds using coryneform bacteria.

Summary of the Invention

Coryneform bacteria which produce chemical compounds, characterised in that these have, in addition to at least one copy, present at the natural site (locus) , of an open reading frame (ORF) , gene or allele which codes for the synthesis of a protein or an RNA, a second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele in question at a second, optionally third or fourth site in a form integrated into the chromosome, no nucleotide sequence which is capable of/enables episomal replication or transposition in microorganisms and no nucleotide sequence (s) which impart (s) resistance to antibiotics being present at the second, optionally third or fourth site, and the second, optionally third or fourth site not relating to open reading frames (ORF) , genes or alleles which are essential for the growth of the bacteria and the production of the desired compound.

The invention also provides processes for the preparation of one or more chemical compounds, in which the following steps are carried out:

a) fermentation of coryneform bacteria, al) which have, in addition to at least one copy, present at the natural site (locus) , of an open reading frame (ORF) , gene or allele which codes for the synthesis of a protein or an RNA, a second, optionally third or fourth copy of this open reading frame (ORF) , gene or allele at a second, optionally third or_ fourth site_in a. form integrated into the chromosome, no nucleotide sequence which is capable of/enables episomal replication or transposition in microorganisms and no nucleotide sequence (s) which impart (s) resistance to antibiotics being present at the second, optionally third or fourth site, and the second, optionally third or fourth site not relating to open reading frames (ORF) , genes or alleles which are essential for the growth of the bacteria and the production of the desired compound, and a2) in which the intracellular activity of the corresponding protein is increased, in particular the nucleotide sequence which codes for this protein is over-expressed, b) concentration of the chemical compoun (s) in the fermentation broth and/or in the cells of the bacteria,

c) isolation of the chemical compound(s), optionally

d) with constituents from the fermentation broth and/or the biomass to the extent of > (greater than) 0 to 100 wt.%.

The invention also pr.ovides processes for the preparation of one or more chemical compounds, which comprise the following steps:

a) fermentation of coryneform bacteria, in particular of the genus Corynebacterium, which have, in addition to the copy of an open reading frame (ORF) , gene or allele present at the natural site (locus) , in each case a second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele in question at in each case a second, optionally third or fourth site in integrated form, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics being present at the particular second, optionally third or fourth site,

under conditions which allow expression of the said open reading frames (ORF) , genes or alleles

b) concentration of the chemical compound(s) in the fermentation broth and/or in the cells of the bacteria,

c) isolation of the chemical compound(s) , optionally d) with constituents from the fermentation broth and/or the biomass to the extent of > (greater than) 0 to 100%.

Detailed Description of the Invention

Chemical compounds are to be understood, in particular, as meaning amino acids, vitamins, nucleosides and nucleotides. The biosynthesis pathways of these compounds are known and are available in the prior art.

Amino acids mean, preferably, L-amino acids, in particular the proteinogenic L-amino acids, chosen from the group consisting of L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine, L-cysteine, L-valine, L-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-lysine, L-methionine and L- threonine. L-Lysine is very particularly preferred.

Proteinogenic amino acids are understood- as meaning the amino acids which occur in natural proteins, that is to say in proteins of microorganisms, plants, animals and humans.

Vitamins mean, in particular, vitamin Bl (thiamine) , vitamin B2 (riboflavin) , vitamin B5 (pantothenic acid) , vitamin B6 (pyridoxines) , vitamin B12 (cyanocobalamin) , nicotinic acid/nicotinamide, vitamin M (folic acid) and vitamin E (tocopherol) and salts thereof, pantothenic acid being preferred.

Nucleosides and nucleotides mean, inter alia, S-adenosyl- methionine, inosine-5 ' -monophosphoric acid and guanosine- 5 ' -monophosphoric acid and salts thereof.

The coryneform bacteria are, in particular, those of the genus Corynebacterium. of the genus Corynebacterium, the species Corynebacterium glutamicum, Corynebacterium ammoniagenes and Corynebacterium thermoaminogenes are preferred. Information on the taxonomic classification of strains of this group of bacteria is to be found, inter alia, in Kampfer and Kroppenstedt (Canadian Journal of Microbiology 42, 989-1005 (1996)) and in US-A-5,250, 434.

Suitable strains of the species Corynebacterium glutamicum (C. glutamicum) are, in particular, the known wild-type strains

Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806

Corynebacterium acetoacidophilum ATCC13870 Corynebacterium 1ilium ATCC15990 Corynebacterium melassecola ATCC17965 Corynebacterium herculis ATCC13868 Arthrobacter sp. ATCC243

Brevibacterium chang-fua ATCC14017 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 Brevibacterium divaricatum ATCC14020 Brevibacterium taipei ATCC13744 and

Microbacterium ammoniaphilum ATCC21645

and mutants or strains, such as are known from the prior art, produced therefrom which produce chemical compounds.

Suitable strains of the species Corynebacterium ammoniagenes (C. ammoniagenes) are, in particular, the known wild-type strains

Brevibacterium ammoniagenes ATCC6871 Brevibacterium ammoniagenes ATCC15137 and Corynebacterium sp. ATCC21084

and mutants or strains, such as are known from the prior art, produced therefrom which produce chemical compounds. Suitable strains of the species Corynebacterium thermoaminogenes (C. thermoaminogenes) are, in particular, the known wild-type strains

Corynebacterium thermoaminogenes FERM BP-1539 Corynebacterium thermoaminogenes FERM BP-1540

Corynebacterium thermoaminogenes FERM BP-1541 and Corynebacterium thermoaminogenes FERM BP-1542

Strains with the designation "ATCC" can be obtained from the American Type Culture Collection (Manassas, VA, USA) . Strains with the designation "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 strains of Corynebacterium thermoaminogenes mentioned (FERM BP-1539, FERM BP-1540, FERM BP-1541 and FERM BP-1542) are described in US-A 5,250,434.

Open reading frame (ORF) describes a section of a nucleotide sequence which codes or can code for a protein or polypeptide or ribonucleic acid to which no function can be assigned according to the prior art.

After assignment of a function to the nucleotide sequence section in question, it is in general referred to as a gene.

Alleles are in general understood as meaning alternative forms of a given gene. The forms are distinguished by differences in the nucleotide sequence.

In the context of the present invention, endogenous, that is to say species-characteristic, open reading frames, genes or alleles are preferably used. These are understood as meaning the open reading frames, genes or alleles or nucleotide sequences thereof present in the population of a species, such as, for example, Corynebacterium glutamicum.

"A copy of an open reading frame (ORF) , a gene or allele present at the natural site (locus) " in the context of this invention is understood as meaning the position or situation of the ORF or gene or allele in relation to the adjacent ORFs or genes or alleles such as exists in the corresponding wild-type or corresponding parent organism or starting organism.

Thus, for example, the natural site of the lysC gene or of an lysC^FBR allele, which codes for a "feed back" resistant aspartate kinase from Corynebacterium glutamicum is the lysC site or lysC locus or lysC gene site with the directly adjacent genes or open reading frames orfX and leuA on one flank and the asd gene on the other flank.

"Feed back" resistant aspartate kinase is understood as meaning aspartate kinases which, compared with the wild- type form, have a lower sensitivity to inhibition by mixtures of lysine and threonine or mixtures of AEC (aminoethylcysteine) and threonine or lysine by itself or AEC by itself. Strains which produce L-lysine typically contain such "feed back" resistant or desensitized aspartate kinases .

The nucleotide sequence of the chromosome of Corynebacterium glutamicum is known and can be found in Patent Application EP-A-1108790 and Access Number (Accession No.) AX114121 of the nucleotide sequence databank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK) . The nucleotide sequences of orfX, the leuA gene and the asd gene have the Access Numbers AX120364 (orfX) , AX123517 (leuA) and AX123519 (asd) . Further databanks, such as, for example, that of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) or that of the Swiss Institute of Bioinformatics (Swissprot, Geneva, Switzerland) or that of the Protein Information Resource Database (PIR, Washington, DC, USA) can also be used.

"In each case a second, optionally third or fourth site" is understood as meaning a site which differs from the "natural site". It is also called a "target site" or "target sequence" in the following. It can also be called an "integration site" or "transformation site". This second, optionally third or fourth site, or the nucleotide sequence present at the corresponding sites, is preferably in the chromosome and is in general not essential for growth and for production of the desired chemical compounds .

To produce the coryneform bacteria according to the invention, the nucleotide sequence of the desired ORF, gene or allele, optionally including expression and/or regulation signals, is isolated and provided with nucleotide sequences of the target site at the ends, these are then transferred into the desired coryneform bacterium, preferably with the aid of vectors which do not replicate or replicate to only a limited extent in coryneform bacteria, and those bacteria in which the desired ORF, gene or allele is incorporated at the target site are isolated, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remaining at the target site.

The invention accordingly also provides a process for the production of coryneform bacteria which produce one or more chemical compounds, which comprises a) isolating the nucleotide sequence of at least one desired ORF, gene or allele, optionally including the expression and/or regulation signals,

b) providing the 5' and the 3' end of the ORF, gene or allele with nucleotide sequences of the target site,

c) preferably incorporating the nucleotide sequence of the desired ORF, gene or allele provided with nucleotide sequences of the target site into a vector which does not replicate or replicates to only a limited extent in coryneform bacteria,

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

e) isolating coryneform bacteria in which the nucleotide sequence according to a) is incorporated at the target site, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remaining at the target site.

Preferably, also, no residues of sequences of the vectors used or species-foreign DNA, such as, for example, restriction cleavage sites, remain at the target site. A maximum of 24, preferably a maximum of 12, particularly preferably a maximum of 6 nucleotides of such DNA upstream or downstream of the ORF, gene or allele incorporated optionally remain at the target site.

By the measures according to the invention, the productivity of the coryneform bacteria or of the fermentative processes for the preparation of chemical compounds is improved in respect of one or more of the features chosen from the group consisting of concentration (chemical compound formed, based on the unit volume) , yield (chemical compound formed, based on the source of carbon consumed) and product formation rate (chemical compound formed, based on the time) by at least 0.5 - 1.0% or at least 1.0 to 1.5% or at least 1.5 - 2.0%.

Instructions on conventional genetic engineering methods, such as, for example, isolation of chromosomal DNA, plasmid DNA, handling of restriction enzymes etc., are found in Sambrook et al . (Molecular Cloning - A Laboratory Manual

(1989) Cold Spring Harbor Laboratory Press) . Instructions on transformation and conjugation in coryneform bacteria are found, inter alia, in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), in Schafer et al. (Journal of Bacteriology 172, 1663-1666

(1990) and Gene 145, 69-73 (1994)) and in Schwarzer and Pϋhler (Bio/Technology 9, 84-87 (1991)).

Vectors which replicate to only a limited extent are understood as meaning plasmid vectors which, as a function of the conditions under which the host or carrier is cultured, replicate or do not replicate. Thus, a temperature-sensitive plasmid for coryneform bacteria which can replicate only at temperatures below 31^SC has been described by Nakamura et al. (US-A-6, 303, 383) .

The invention furthermore provides coryneform bacteria, in particular of the genus Corynebacterium, which produce L- lysine, characterized in that these have, in addition to at least one of the copy of an open reading frame (ORF) , gene or allele of lysine production present at the natural site (locus) , in each case a second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele in question at in each case a second, optionally third or fourth site in integrated form, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics being present at the particular second, optionally third or fourth site.

The invention also furthermore provides a process for the preparation of L-lysine, which comprises the following steps:

a) fermentation of coryneform bacteria, in particular Corynebacterium glutamicum, characterized in that these have, in addition to at least one of the copy of an open reading frame (ORF) , gene or allele of lysine production present at the natural site

(locus) , in each case a second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele in question at in each case a second, optionally third or fourth site in integrated form, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics being present at the particular second, optionally third or fourth site,

under conditions which allow expression of the said open reading frames (ORF) , genes or alleles,

b) concentration of the L-lysine in the fermentation broth,

c) isolation of the L-lysine from the fermentation broth, optionally

d) with constituents from the fermentation broth and/or the biomass to the extent of > (greater than) 0 to 100%.

A "copy of an open reading frame (ORF) , gene or allele of lysine production" is to be understood as meaning all the, preferably endogenous, open reading frames, genes or alleles of which enhancement/over-expression can have the effect of improving lysine production. Enhancement is understood as meaning an increase in the intracellular concentration or activity of the particular gene product, protein or enzyme.

These include, inter alia, 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, ptsl, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwal, zwf and zwf A213T. These are summarized and explained in Table 1.

These include, in particular, the lysC^FBR alleles which code for a "feed back" resistant aspartate kinase. Various lysC^FBR alleles are summarized and explained in Table 2.

The following lysC^FBR alleles are preferred: lysC A279T (replacement of alanine at position 279 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by threonine) , lysC A279V (replacement of alanine at position 279 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by valine), lysC S301F (replacement of serine at position 301 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by phenylalanine), lysC T308I (replacement of threonine at position 308 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by isoleucine) , lysC S301Y (replacement of serine at position 308 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by tyrosine), lysC G345D (replacement of glycine at position 345 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by aspartic acid), lysC R320G (replacement of arginine at position 320 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by glycine) , lysC T311I (replacement of threonine at position 311 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by isoleucine), lysC S381F (replacement of serine at position 381 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by phenylalanine).

The lysC^FBR allele lysC T311I (replacement of threonine at position 311 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by isoleucine), the nucleotide sequence of which is shown as SEQ ID NO: 3, is particularly preferred; the amino acid sequence of the aspartate kinase protein coded is shown as SEQ ID NO : 4.

The second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele of lysine production in question can be integrated at in each case a second, optionally third or fourth site. The following open reading frames, genes or nucleotide sequences, inter alia, can be used for this: aecD, ccpAl, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysRl, lysR2, lysR3 , menE, mqo, pck, pgi, poxB and zwa2, in particular the genes aecD, gluA, gluB, gluC, gluD and pck. These are summarized and explained in Table 3.

The sites mentioned include, of course, not only the coding regions of the open reading frames or genes mentioned, but also the regions or nucleotide sequences lying upstream which are responsible for expression and regulation, such as, for example, ribosome binding sites, promoters, binding sites for regulatory proteins, binding sites for regulatory ribonucleic acids and attenuators. These regions in general lie in a range of 1-800, 1-600, 1-400, 1-200, 1-100 or 1-50 nucleotides upstream of the coding region. In the same way, regions lying downstream, such as, for example, transcription terminators, are also included. These regions in general lie in a range of 1-400, 1-200, 1-100, 1-50 or 1-25 nucleotides downstream of the coding region.

Intergenic regions in the chromosome, that is to say nucleotide sequences without a coding function, can furthermore be used. Finally, prophages or defective phages contained in the chromosome can be used for this .

A prophage is understood as meaning a bacteriophage, in particular the genome thereof, where this is replicated together with the genome of the host and the formation of infectious particles does not take place. A defective phage is understood as meaning a prophage, in particular the genome thereof, which, as a result of various mutations, has lost the ability to form so-called infectious particles. Defective phages are also called cryptic. Prophages and defective phages are often present in integrated form in the chromosome of their host. Further details exist in the prior art, for example in the textbook by Edward A. Birge (Bacterial and Bacteriophage Genetics, 3^rd ed., Springer-Verlag, New York, USA, 1994) or in the textbook by S. Klaus et al . (Bakterienviren, Gustav Fischer Verlag, Jena, Germany, 1992) .

Table 1

Open reading frames, genes and alleles of lysine production

Table 2 lysC alleles which code for eed back resistant aspartate kinases

Table 3

Target sites for integration of open reading frames, genes and alleles of lysine production

The invention accordingly also provides a process for the production of coryneform bacteria which produce L-lysine, which comprises

a) isolating the nucleotide sequence of at least one desired ORF, gene or allele of lysine production, optionally including the expression and/or regulation signals,

b) providing the 5' and the 3' end of the ORF, gene or allele of lysine production with nucleotide sequences of the target site,

The invention furthermore provides coryneform bacteria, in particular of the genus Corynebacterium, which produce L- methionine and/or L-threonine, characterized in that these have, in addition to at least one of the copy of an open reading frame (ORF) , gene or allele of methionine production or threonine production present at the natural site (locus) , in each case a second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele in question at in each case a second, optionally third or fourth site in integrated form, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics being present at the particular second, optionally third or fourth site.

The invention also furthermore provides a process for the preparation of L-methionine and/or L-threonine, which comprises the following steps:

a) fermentation of coryneform bacteria, in particular Corynebacterium glutamicum, characterized in that these have, in addition to at least one of the copy of an open reading frame (ORF) , gene or allele of methionine production or threonine production present at the natural site (locus) , in each case a second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele in question at in each case a second, optionally third or fourth site in integrated form, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics being present at the particular second, optionally third or fourth site,

b) concentration of the L-methionine and/or L-threonine in the fermentation broth,

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

A "copy of an open reading frame (ORF) , gene or allele of methionine production" is to be understood as meaning all the, preferably endogenous, open reading frames, genes or alleles of which enhancement/over-expression can have the effect of improving methionine production.

These include, inter alia, 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, horn, hom^FBR, lysC, lysC^FBR, metA, metB, metE, metH, etY, msiK, opcA, oxyR, ppc, ppc^FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsl, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwal, zwf and zwf A213T. These are summarized and explained in Table 4. These include, in particular, the lysC^FBR alleles which code for a "feed back" resistant aspartate kinase (see Table 2) and the hom^FBR alleles which code for a "feed back" resistant ho oserine dehydrogenase.

The second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele of methionine production in question can be integrated at in each case a second, optionally third or fourth site. The following open reading frames, genes or nucleotide sequences, inter alia, can be used for this: brnE, brnF, brnQ, ccpAl, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, luxR, luxS, lysRl, lysR2, lysR3, menE, etD, metK, pck, pgi, poxB and zwa2. These are summarized and explained in Table 5.

The sites mentioned include, of course, not only the coding regions of the open reading frames or genes mentioned, but also the regions or nucleotide sequences lying upstream which are responsible for expression and regulation, such as, for example, ribosome binding sites, promoters, binding sites for regulatory proteins, binding sites for regulatory ribonucleic acids and attenuators . These regions in general lie in a range of 1-800, 1-600, 1-400, 1-200, 1-100 or 1-50 nucleotides upstream of the coding region. In the same way, regions lying downstream, such as, for example, transcription terminators, are also included. These regions in general lie in a range of 1-400, 1-200, 1-100, 1-50 or 1-25 nucleotides downstream of the coding region.

Intergenic regions in the chromosome, that is to say nucleotide sequences without a coding function, can furthermore be used. Finally, prophages or defective phages contained in the chromosome can be used for this . Table 4

Open reading frames, genes and alleles of methionine production

Table 5

Target sites for integration of open reading frames, genes and alleles of methionine production

A "copy of an open reading frame (ORF) , gene or allele of threonine production" is to be understood as meaning all the open reading frames, genes or alleles of which enhancement/over-expression can have the effect of improving threonine production.

These include, inter alia, the following open reading frames, genes or alleles: accBC, accDA, cstA, cysD, cysE, cysH, cysl, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd, horn, hom^FBR, lysC, lysC^FBR, msiK, opcA, oxyR, ppc, ppc^FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsl, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, thrB, thrC, thrE, zwal, zwf and zwf A213T. These are summarized and explained in Table 6. These include, in particular, the lysC^BR alleles which code for a "feed back" resistant aspartate kinase (See Table 2) and the hom^FBR alleles which code for a "feed back" resistant homoserine dehydrogenase.

The second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele of threonine production in question can be integrated at in each case a second, optionally third or fourth site. The following open reading frames, genes or nucleotide sequences, inter alia, can be used for this: ccpAl, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, glyA, ilvA, ilvBN, ilvC, ilvD, luxR, luxS, lysRl, lysR2, lysR3 , mdh, menE, metA, metD, pck, poxB, sigB and zwa2. These are summarized and explained in Table 7.

The sites mentioned include, of course, not only the coding regions of the open reading frames or genes mentioned, but also the regions or nucleotide sequences lying upstream which are responsible for expression and regulation, such as, for example, ribosome binding sites, promoters, binding sites for regulatory proteins, binding sites for regulatory ribonucleic acids and attenuators . These regions in general lie in. a range of 1-800, 1-600, 1-400, 1-200, 1-100 or 1-50 nucleotides upstream of the coding region. In the same way, regions lying downstream, such as, for example, . transcription terminators, are also included. These regions in general lie in a range of 1-400, 1-200, 1-100, 1-50 or 1-25 nucleotides downstream of the coding region.

Intergenic regions in the chromosome, that is to say nucleotide sequences without a coding function, can furthermore be used. Finally, prophages or defective phages contained in the chromosome can be used for this.

Table 6

Open reading frames, genes and alleles of threonine production

Table 7

Target sites for integration of open reading frames, genes and alleles of threonine production

The invention accordingly also provides a process for the production of coryneform bacteria which produce L- methionine and/or -threonine, which comprises

a) isolating the nucleotide sequence of at least one desired ORF, gene or allele of methionine production or threonine production, optionally including the expression and/or regulation signals,

e) isolating coryneform bacteria in which the nucleotide sequence according to a) is incorporated at the target site, no nucleotide sequence which is capable, of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remaining at the target site.

The invention furthermore provides coryneform bacteria, in particular of the genus Corynebacterium, which produce - valine, wherein these have, in addition to at least one of the copy of an open reading frame (ORF) , gene or allele of valine production present at the natural site (locus) , in each case a second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele in question at in each case a second, optionally third or fourth site in integrated form, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics being present at the particular second, optionally third or fourth site.

The invention also furthermore provides a process for the preparation of L-valine, which comprises the following steps :

a) fermentation of coryneform bacteria, in particular Corynebacterium glutamicum, characterized in that these have, in addition to at least one of the copy of an open reading frame (ORF) , gene or allele of valine production present at the natural site (locus) , in each case a second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele in question at in each case a second, optionally third or fourth site in integrated form, no nucleotide sequence which is capable of/enables episomal replication in microorganisms , no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics being present at the particular second, optionally third or fourth site,

b) concentration of the L-valine in the fermentation broth,

c) isolation of the L-valine from the fermentation broth, optionally d) with constituents from the fermentation broth and/or^' the biomass to the extent of > (greater than) 0 to 100%.

A "copy of an open reading frame (ORF) , gene or allele of valine production" is to be understood as meaning all the open reading frames, genes or alleles of which enhancement/over-expression can have the effect of improving valine production.

These include, inter alia, 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 siK, pgk, ptsH, ptsl, ptsM, sigC, sigD, sigE, sigH, sigM, tpi, zwal . These are summarized and explained in Table 8. These include in particular the acetolactate synthase which codes for a valine-resistant .

The second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele of threonine production in question can be integrated at in each case a second, optionally third or fourth site. The following open reading frames, genes or nucleotide sequences, inter alia, can be used for this: aecD, ccpAl, ccpA2 , citA, citB, citE, ddh, gluA, gluB, gluC, gluD, glyA, ilvA, luxR, lysRl, lysR2, lysR3, panB, panC, poxB and zwa2. These are summarized and explained in Table 9.

Table 8

Open reading frames, genes and alleles of valine production

Table 9

Target sites for integration of open reading frames, genes and alleles of valine production

The invention accordingly also provides a process for the production of coryneform bacteria which produce L-valine, which comprises

a) isolating the nucleotide sequence of at least one desired ORF, gene or allele of valine production, optionally including the expression and/or regulation signals,

During work on the present invention, it was possible to incorporate a second copy of an lysC^FBR allele into the gluB gene of Corynebacterium glutamicum such that no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remained at the gluB gene site. This strain, which is called DSMl3994glu: :lysC, carries the lysC^FBR allele lysC T311I at its natural lysC site and a second copy of the lysC^FBR allele lysC T311I at a second site (target site) , namely the gluB gene. A plasmid with the aid of which the incorporation of the lysC^FBR allele into the gluB gene can be achieved is shown in Figure 1. It carries the name pK18mobsacBglul_l .

During work on the present invention, it was furthermore possible to incorporate a copy of an lysC^FBR allele into the target site of the gluB gene of Corynebacterium glutamicum such that no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remained at the gluB gene site. This strain, which is called DSMl2866glu: :lysC, carries the wild-type form of the lysC gene at its natural lysC site and a second copy of the lysC gene in the form of the lysC^FBR allele lysC T311I at a second site (target site) , namely the gluB gene. It has been deposited under number DSM15039 at the Deutsche Sammlung fϋr Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) . A plasmid with the aid of which the incorporation of the lys^'C^FBR allele into the gluB gene can be achieved is shown in Figure 1. It carries the name pKlδmobsacBglul_l .

During work on the present invention, it was furthermore possible to incorporate a copy of an lysC^FBR allele into the target site of the aecD gene of Corynebacterium glutamicum such that no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remained at the aecD gene site. This strain, which is called DSM12866aecD: :lysC, carries the wild-type form of the lysC gene at its natural lysC site and a second copy of the lysC gene in the form of the lysC^FBR allele lysC T311I at a second site (target site) , namely the aecD gene. A plasmid with the aid of which the incorporation of the lysC^FBR allele into the aecD gene can be achieved is shown in Figure 2. It carries the name pKl8mobsacBaecDl_l .

During work on the present invention, it was furthermore possible to incorporate a copy of an lysC^FBR allele into the target site of the pck gene of Corynebacterium glutamicum such that no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remained at the pck gene site. This strain, which is called DSMl2866pck: :lysC, carries the wild-type form of the lysC gene at its natural lysC site and a second copy of the lysC gene in the form of the lysC^FBR allele lysC T311I at a second site (target site) , namely the pck gene. A plasmid with the aid of which the incorporation into the pck gene can be achieved is shown in Figure 3. It carries the name pKl8mobsacBpckl_l .

During work on the present invention, it was furthermore possible to incorporate a copy of the ddh gene into the target site of the gluB gene of Corynebacterium glutamicum such that no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remained at the gluB gene site. This strain, which is called DSMl2866glu: :ddh, carries a copy of the ddh gene at its natural ddh site and a second copy of the ddh gene at a second site (target site) , namely the gluB gene. A plasmid with the aid of which the incorporation of the ddh gene into the gluB gene can be achieved is shown in Figure 4. It carries the name pKl8mobsacBgluB2_l .

During work on the present invention, it was furthermore possible to incorporate a copy of the dapA gene into the target site of the aecD gene of Corynebacterium glutamicum such that no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remained at the aecD gene site. This strain, which is called DSM12866aecD: :dapA, carries a copy of the dapA gene at its natural dapA site and a second copy of the dapA gene at a second site (target site), namely the aecD gene. A plasmid with the aid of which the incorporation of the dapA gene into the aecD gene can be achieved is shown in Figure 5. It carries the name pKl8mobsacBaecD2_l .

During work on the present invention, it was furthermore possible to incorporate a copy of a pyc allele into the target site of the pck gene of Corynebacterium glutamicum such that no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remained at the pck gene site. This strain, which is called DSMl2866pck: :pyc, carries a copy of the wild-type form of the pyc gene at its natural pyc site and a second copy of the pyc gene in the form of the pyc allele pyc P458S at a second site (target site) , namely the pck gene. A plasmid with the aid of which the incorporation of the pyc allele into the pck gene can be achieved is shown in Figure 6. It carries the name pK18mobsacBpckl_3.

The coryneform bacteria produced according to the invention can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of chemical compounds . A summary of known culture methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einfύhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994) ) .

The culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D. C. , USA, 1981). Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e.g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and organic acids, such as e.g. acetic acid or lactic acid, can be used as the source of carbon. These substances can be used individually or as a mixture.

Organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. The sources of nitrogen can be used individually or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium- containing salts can be used as the source of phosphorus . The culture medium must furthermore comprise salts of metals, such as e. g. magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, can be employed in addition to the above-mentioned substances. Suitable precursors can moreover be added to the culture medium. The starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during the culture in a suitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH of the culture. Antifoams, such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, such as e.g. antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as e.g. air, are introduced into the culture. The temperature of the culture is usually 20^aC to 45^aC, and preferably 25^SC to

40²C. Culturing is continued until a maximum of the desired chemical compound has formed. This target is usually reached within 10 hours to 160 hours.

It has been found that the coryneform bacteria according to the invention, in particular the coryneform bacteria which produce L-lysine, have an unexpectedly high stability. They were stable for at least 10-20, 20-30, 30-40, 40-50, preferably at least 50-60, 60-70, 70-80 and 80-90 generations or cell division cycles .

The following microorganisms have been deposited:

The strain Corynebacterium glutamicum DSMl2866glu: :lysC was deposited in the form of a pure culture on 5th June 2002 under number DSM15039 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty.

The plasmid pKl8mobsacBglul_l was deposited in the form of a pure culture of the strain E. coli DH5αmcr/pKl8mobsacBglul_l (= DH5alphamcr/pKl8mobsacBglul_l) on 20th April 2001 under number DSM14243 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.

The plasmid pKl8mobsacBaecDl_l was deposited in the form of a pure culture of the strain E. coli DH5oαncr/pKl8mobsacBaecDl_l (=

DH5alphamcr/pKl8mobsacBaecDl_l) on 5th June 2002 under number DSM15040 at the Deutsche Sammlung fiir Mikroorganismen und Zellkulturen (DSMZ , Braunschweig, Germany) in accordance with the Budapest Treaty.

Example 1

Incorporation of a second copy of the lysC^FBR allele into the chromosome of the strain DSM13994 and of the strain DSM12866

The Corynebacterium glutamicum strain DSM13994 was produced by multiple, non-directed mutagenesis, selection and mutant selection from C. glutamicum ATCC13032. The strain is resistant to the lysine analogue S- (2-aminoethyl) -L- cysteine and has a feed back-resistant aspartate kinase which is insensitive to inhibition by a mixture of lysine and threonine (in each case 25 mM) . The nucleotide sequence of the lysC^FBR allele of this strain is shown as SEQ ID NO: 3. It is also called lysC T311I in the following. The amino acid sequence of the aspartate kinase protein coded is shown as SEQ ID NO:4. A pure culture of this strain was deposited on 16th January 2001 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty.

The strain DSM12866 was produced from C. glutamicum ATCC13032 by non-directed mutagenesis and selection of the mutants with the best L-lysine accumulation. It is methionine-sensitive. Growth on minimal medium comprising L-methionine can be re-established by addition of threonine. This strain has the wild-type form of the lysC gene shown as SEQ ID N0:1. The corresponding amino acid sequence of the wild-type aspartate kinase protein is shown as SEQ ID NO: 2. A pure culture of this strain was deposited on 10th June 1999 at the Deutsche Sammlung fϋr Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty. 1.1 Isolation and sequencing of the DNA of the lysC allele of strain DSM13994

From the strain DSM13994, chromosomal DNA is isolated by the conventional methods (Eikmanns et al., Microbiology 140: 1817 - 1828 (1994)). With the aid of the polymerase chain reaction, a DNA section which carries the lysC gene or allele is amplified. On the basis of the sequence of the lysC gene known for C. glutamicum (Kalinowski et al . , Molecular Microbiology, 5 (5), 1197 - 1204 (1991); Accession Number X57226) , the following primer oligonucleotides were chosen for the PCR:

lysClbeg (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 shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al . (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) . The primers allow amplification of a DNA section of approx. 1.7 kb in length, which carries the lysC gene or allele. The primers moreover contain the sequence for a cleavage site of the restriction endonuclease BamHI, which is marked by parentheses in the nucleotide sequence shown above.

The amplified DNA fragment of approx. 1.7 kb in length which carries the lysC allele of the strain DSM13994 is identified by electrophoresis in a 0.8% agarose gel, isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .

Ligation of the fragment is then carried out by means of the Topo TA Cloning Kit (Invitrogen, Leek, The Netherlands, Cat. Number K4600-01) in the vector pCRII-TOPO. The ligation batch is transformed in the E. coli strain TOP10 (Invitrogen, Leek, The Netherlands) . Selection of plasmid- carrying cells is made by plating out the transformation batch on kanamycin (50 mg/1) -containing LB agar with X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside, 64 mg/1) .

The plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel. The resulting plasmid is called pCRIITOPOlysC .

The nucleotide sequence of the amplified DNA fragment or PCR product is determined by the dideoxy chain termination method of Sanger et al . (Proceedings of the National Academy of Sciences USA, 74:5463-5467 (1977)) using the "ABI Prism 377" sequencing apparatus of PE Applied Biosysterns (Weiterstadt, Germany) . The sequence of the coding region of the PCR product is shown in SEQ ID No : 3. The amino acid sequence of the associated aspartate kinase protein is shown in SEQ ID NO : 4.

The base thymine is found at position 932 of the nucleotide sequence of the coding region of the lysC^FBR allele of strain DSM13994 (SEQ ID NO: 3) . The base cytosine is found at the corresponding position of the wild-type gene (SEQ ID NO:l) .

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

The lysC allele, which contains the base thymine at position 932 of the coding region and accordingly codes for an aspartate kinase protein which contains the amino acid isoleucine at position 311 of the amino acid sequence, is called the lysC^FBR allele or lysC T311I in the following. The plasmid pCRIITOPOlysC, which carries the lysC^FBR allele lysC T311I, was deposited in the form of a pure culture of the strain E. coli TOP 10/pCRIITOPOlysC under number DSM14242 on 20th April 2001 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.

1.2 Construction of the replacement vector pKl8mobsacBglul_l

The Corynebacterium glutamicum strain ATCC13032 is used as the donor for the chromosomal DNA. From the strain ATCC13032, chromosomal DNA is isolated using the conventional methods (Eikmanns et al . , Microbiology 140: 1817 - 1828 (1994)). With the aid of the polymerase chain reaction, a DNA fragment which carries the gluB gene and surrounding regions is amplified. On the basis of the sequence of the gluABCD gene cluster known for C. glutamicum (Kronemeyer et al . , Journal of Bacteriology, 177: 1152 - 1158 (1995)) (Accession Number X81191) , the following primer oligonucleotides are chosen for the PCR:

gluBgll (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 TAG GGC CAA G 3^N

The primers shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) . The primers allow amplification of a DNA fragment of approx 1.7 kb in size, which carries the gluB gene and surrounding regions . The surrounding regions are a sequence section approx. 0.33 kb in length upstream of the gluB gene, which represents the 3' end of the gluA gene, and a sequence section approx. 0.44 kb in length downstream of the gluB gene, which represents the 5' end of the gluC gene. The primers moreover contain the sequence for the cleavage site of the restriction endonuclease Bglll, which is marked by parentheses in the nucleotide sequence shown above.

The amplified DNA fragment of approx. 1.7 kb in length which carries the gluB gene and surrounding regions is identified by means of electrophoresis in a 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .

The plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel. The resulting plasmid is called pCRII-TOPOglu.

The plasmid pCRII-TOPOglu is cleaved with the restriction enzyme Bglll (Amersham-Pharmacia, Freiburg, Germany) and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the gluB fragment of approx. 1.7 kb is isolated from the agarose gel and employed for ligation with the obilizable cloning vector pKlδmobsacB described by Schafer et al. (Gene 14: 69-73 (1994)). This is cleaved beforehand with the restriction enzyme BamHI and dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim), mixed with the gluB fragment of approx. 1.7 kb, and the mixture is treated with T4 DNA Ligase (Amersham- Pharmacia, Freiburg, Germany) . The E. coli strain DH5 (Grant et al . ; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989) . Selection of plasmid- carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold Spring Harbor, New York, 1989) , which is supplemented with 50 mg/1 kanamycin.

Plasmid DNA is isolated from a transfor ant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis. The plasmid is called pK18mobsacBglul.

Plasmid DNA was isolated from the strain DSM14242 (see Example 1.1), which carries the plasmid pCRIITOPOlysC, and cleaved with the restriction enzyme BamHI (Amersham- Pharmacia, Freiburg, Germany) , and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the lysC^FBR- containing DNA fragment of approx. 1.7 kb in length was isolated from the agarose gel and employed for ligation with the vector pKlδmobsacBglul described above. This is cleaved beforehand with the restriction enzyme BamHI, dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany) , mixed with the lysC^FBR fragment of approx. 1.7 kb and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany) .

The E. coli strain DH5αmcr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol.

1, ILR-Press, Cold Spring Harbor, New York, 1989).

Selection of plasmid-carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold Spring Harbor, New York, 1989) , which was supplemented with 50 mg/1 kanamycin.

Plasmid DNA is isolated from a transformant with the aid of the QlAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis. The plasmid is called pKl8mobsacBglul_l. A map of the plasmid is shown in Figure 1.

The plasmid pKl8mobsacBglul_l was deposited in the form of a pure culture of the strain E. coli DH5αmcr/pKl8mobsacBglul_l (=

DH5alphamcr/pKl8mobsacBglul_l) under number DSM14243 on 20.04.2001 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.

1.3 Incorporation of a second copy of the lysC^FBR allele lysC T311I into the chromosome (target site: gluB gene) of the strain DSM13994 by means of the replacement vector pKl8mobsacBglul_l

The vector pKl8mobsacBglul_l described in Example 1.2 is transferred by the protocol of Schafer et al. (Journal of Microbiology 172: 1663-1666 (1990)) into the C. glutamicum strain DSM13994 by conjugation. The vector cannot replicate independently in DSM13994 and is retained in the cell only if it has integrated into the chromosome. Selection of clones or transconjugants with integrated pKl8mobsacBglul_l is made by plating out the conjugation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold Spring Harbor, New York, 1989), which is supplemented with 15 mg/1 kanamycin and 50 mg/1 nalidixic acid. Kanamycin-resistant transconjugants are plated out on LB agar plates with 25 mg/1 kanamycin and incubated for 48 hours at 33°C. For selection of mutants in which excision of the plasmid has taken place as a consequence of a second recombination event, the clones are cultured for 20 hours in LB liquid medium and then plated out on LB agar with 10% sucrose and incubated for 48 hours .

The plasmid pKl8mobsacBglul_l, like the starting plasmid pKlδmobsacB, contains, in addition to the kanamycin resistance gene, a copy of the sacB gene which codes for levan sucrase from Bacillus subtilis. The expression which can be induced by sucrose leads to the formation of levan sucrase, which catalyses the synthesis of the product" levan, which is toxic to C. glutamicum. Only those clones in which the integrated pKl8mobsacBglul_l has excised as the consequence of a second recombination event therefore grow on LB agar. Depending on the position of the second recombination event, after the excision the second copy of the lysC^FBR allele manifests itself in the chromosome at the gluB locus, or the original gluB locus of the host remains.

Approximately 40 to 50 colonies are tested for the phenotype "growth in the presence of sucrose" and "non- growth in the presence of kanamycin". Approximately 20 colonies which show the phenotype "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" are investigated with the aid of the polymerase chain reaction. A DNA fragment which carries the gluB gene and surrounding regions is amplified here from the chromosomal DNA of the colonies. The same primer oligonucleotides as are described in Example 1.2 for the construction of the integration plasmid are chosen for the PCR.

gluBgll (SEQ ID NO: 7) :

5^V 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 allow amplification of a DNA fragment approx. 1.7 kb in size in control clones with the original gluB locus. In clones with a second copy of the lysC^FBR allele in the chromosome at the gluB locus, DNA fragments with a size of approx. 3.4 kb are amplified.

The amplified DNA fragments are identified by means of electrophoresis in a 0.8% agarose gel.

A clone which, in addition to the copy present at the lysC locus, has a second copy of the lysC^FRB allele lysC T311I at the gluB locus in the chromosome was identified in this manner. This clone was called strain DSM13994glu: :lysC.

1.4 Incorporation of a second copy of the lysC gene in the form of the lysC^FBR allele lysC T311I into the chromosome (target site: gluB gene) of the strain DSM12866 by means of the replacement vector pK18mobsacBglul_l

As described in Example 1.3, the plasmid pKl8mobsacBglul_l is transferred into the C. glutamicum strain DSM12866 by conjugation. A clone which, in addition to the copy of the wild-type gene present at the lysC locus, has a second copy of the lysC gene in the form of the lysC^BR allele lysC T311I at the gluB locus in the chromosome was identified in the manner described in 1.3. This clone was called strain DSMl2866glu: :lysC.

The Corynebacterium glutamicum strain according to the invention which carries a second copy of an lysC^FBR allele in the gluB gene was deposited in the form of a pure culture of the strain Corynebacterium glutamicum DSMl2866glu: : lysC on 5th June 2002 under number DSM15039 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty. 1.5 'Construction of the replacement vector pKl8mobsacBpckl_l

The Corynebacterium glutamicum strain ATCC13032 is used as the donor for the chromosomal DNA. From the strain ATCC13032, chromosomal DNA is isolated using the conventional methods (Eikmanns et al., Microbiology 140: 1817 - 1828 (1994)). With the aid of the polymerase chain reaction, a DNA fragment which carries the pck gene and surrounding regions is amplified. On the basis of the sequence of the pck gene known for C. glutamicum (EP1094111 and Riedel et al . , Journal of Molecular and Microbiological Biotechnology 3:573-583 (2001)) (Accession Number AJ269506) , the following primer oligonucleotides are chosen for the PCR:

pck_beg (SEQ ID NO: 9) :

5^N 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 shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) . The primers allow amplification of a DNA fragment of approx.2.9 kb in size, which carries the pck gene and adjacent regions. The primers moreover contain the sequence for the cleavage site of the restriction endonuclease Bglll, which is marked by parentheses in the nucleotide sequence shown above.

The amplified DNA fragment of approx. 2.9 kb in length which carries the pck gene and surrounding regions is identified by means of electrophoresis in a 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) . Ligation of the fragment is then carried out by means of the TOPO TA Cloning Kit (Invitrogen, Leek, The Netherlands, Cat. Number K4600-01) in the vector pCRII-TOPO. The ligation batch is transformed in the E. coli strain TOP10 (Invitrogen, Leek, The Netherlands) . Selection of plasmid- carrying cells is made by plating out the transformation batch on kanamycin (50 mg/1) -containing LB agar with X-Gal (64 mg/1) .

The plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel. The resulting plasmid is called pCRII-TOPOpck.

The plasmid pCRII-TOPOpck is cleaved with the restriction enzyme Bglll (Amersham-Pharmacia, Freiburg, Germany) and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the pck fragment of approx. 2.9 kb is isolated from the agarose gel and employed for ligation with the mobilizable cloning vector pKlδmobsacB described by Schafer et al . (Gene 14: 69-73 (1994)). This is cleaved beforehand with the restriction enzyme BamHI and dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim), mixed with the pck fragment of approx. 2.9 kb, and the mixture is treated with T4 DNA Ligase (Amersham- Pharmacia, Freiburg, Germany) .

The E. coli Strain DH5α (Grant et al.; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989) Selection of plasmid- carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold Spring Harbor, New York, 1989), which is supplemented with 50 mg/1 kanamycin. Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis. The plasmid is called pKlδmobsacBpckl.

Plasmid DNA was isolated from the strain DSM14242 (see

Example 1.1), which carries the plasmid pCRIITOPOlysC, and cleaved with the restriction enzyme BamHI (Amersham- Pharmacia, Freiburg, Germany) , and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the lysC^FBR- containing DNA fragment approx. 1.7 kb long was isolated from the agarose gel and employed for ligation with the vector pKlδmobsacBpckl described above. This is cleaved beforehand with the restriction enzyme BamHI, dephosphorylated with alkaline phosphatase (Alkaline

Phosphatase, Boehringer Mannheim, Germany) , mixed with the lysC^FBR fragment of approx. 1.7 kb and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany) .

The E. coli strain DH5cxmcr (Life Technologies GmbH,

Karlsruhe, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989). Selection of plasmid-carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al . , Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold Spring Harbor, New York, 1989), which was supplemented with 50 mg/1 kanamycin.

Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis. The plasmid is called pKlδmobdsacBpckl_l . A map of the plasmid is shown in Figure 3. 1.6 Incorporation of a second copy of the lysC gene in the form of the lysC^FBR allele lysC T311I into the chromosome (target site: pck gene) of the strain DSM12666 by means of the replacement vector pKlδmobsacBpckl_l

As described in Example 1.3, the plasmid pKlδmobsacBpckl_l. described in Example 1.5 is transferred into the C. glutamicum strain DSM12866 by conjugation. Selection is made for targeted recombination events in the chromosome of C. glutamicum DSMl2δ66 as described in Example 1.3. Depending on the position of the second recombination event, after the excision the second copy of the lysC^FBR allele manifests itself in the chromosome at the pck locus, or the original pck locus of the host remains.

Approximately 40 to 50 colonies are tested for the phenotype "growth in the presence of sucrose" and "non- growth in the presence of kanamycin" . Approximately 20 colonies which show the phenotype "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" are investigated with the aid of the polymerase chain reaction. A DNA fragment which carries the pck gene and surrounding regions is amplified here from the chromosomal DNA of the colonies . The same primer oligonucleotides as are described in Example 1.5 for the construction of the integration plasmid are chosen for the PCR.

pck_beg (SEQ ID NO: 9):

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

pck_end (SEQ ID NO: 10):

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

The primers allow amplification of a DNA fragment approx. 2.9 kb in size in control clones with the original pck locus. In clones with a second copy of the lysC^FBR allele in the chromosome at the pck locus, DNA fragments with a size of approx. 4.6 kb are amplified.

The amplified DNA fragments are identified by means of electrophoresis in a 0.6% agarose gel.

A clone which, in addition to the copy of the wild-type gene present at the lysC locus, has a second copy of the lysC gene in the form of the lysC^FBR allele lysC T311I at the pck locus in the chromosome was identified in this manner. This clone was called strain DSMl2δ66pck: :lysC.

1.7 Construction of the replacement vector pKlδmobsacBaecDl_l

The Corynebacterium glutamicum strain ATCC13032 is used as the donor for the chromosomal DNA. From the strain ATCC13032, chromosomal DNA is isolated using the conventional methods (Eikmanns et al . , Microbiology 140: 1817 - lδ28 (1994)). With the aid of the polymerase chain reaction, a DNA fragment which carries the aecD gene and surrounding regions is amplified. On the basis of the sequence of the aecD gene known for C. glutamicum (Rossol et al., Journal of Bacteriology 174:2968-2977 (1992)) (Accession Number M89931) , the following primer oligonucleotides are chosen for the PCR:

aecD_beg (SEQ ID NO: 11):

5^V GAA CTT ACG CCA AGC TGT TC 3^N

aecD_end (SEQ ID NO: 12) :

5^V AGC ACC ACA ATC AAC GTG AG 3 ^s

The primers shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) . The primers allow amplification of a DNA fragment of approx 2.1 kb in size, which carries the aecD gene and adjacent regions. The amplified DNA fragment of approx. 2.1 kb in length is identified by means of electrophoresis in a 0.δ% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .

The DNA fragment purified is cleaved with the restriction enzyme BamHI and EcoRV (Amersham Pharmacia, Freiburg, Germany) . The ligation of the fragment in the vector pUClδ then takes place (Norrander et al . , Gene 26:101-106 (1983)). This is cleaved beforehand with the restriction enzymes Bglll and Smal, dephosphorylated, mixed with the aecD-carrying fragment of approx. 1.5 kb, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany) . The ligation batch is transformed in the E. coli strain TOP10 (Invitrogen, Leek, The Netherlands) . Selection of plasmid-carrying cells is made by plating out the transformation batch on kanamycin (50 mg/1) -containing LB agar with X-Gal (64 mg/1).

The plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel. The resulting plasmid is called pUClδaecD.

Plasmid DNA was isolated from the strain DSM14242 (see Example 1.1) which carries the plasmid pCRIITOPOlysC and cleaved with the restriction enzyme BamHI (Amersham- Pharmacia, Freiburg, Germany) and then treated with Klenow polymerase. After separation in an agarose gel (0.δ%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the lysC^FBR-containing DNA fragment approx. 1.7 kb in length is isolated from the agarose gel and employed for ligation with the vector pUClδaecD described above. This is cleaved beforehand with the restriction enzyme Stul, dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany) , mixed with the lysC^FBR fragment of approx. 1.7 kb and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany) . The E. coli strain DH5αmcr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989) . Selection of plasmid-carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold Spring Harbor, New York, 1989), which was supplemented with 50 mg/1 kanamycin.

Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis. The plasmid is called pUClδaecDl.

The plasmid pUClδaecDl is cleaved with the restriction enzyme Kpnl and then treated with Klenow polymerase. The plasmid is then cleaved with the restriction enzyme Sail (Amersham-Pharmacia, Freiburg, Germany) and after separation in an agarose gel (0.δ%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the fragment of approx. 3.2 kb which carries aecD and lysC is isolated from the agarose gel and employed for ligation with the mobilizable cloning vector pKlδmobsacB described by Schafer et al. (Gene 14: 69-73 (1994)). This is cleaved beforehand with the restriction enzymes Smal and Sail and dephosphorylated with alkaline phosphatase (Alkaline

Phosphatase, Boehringer Mannheim) , mixed with the fragment of approx. 3.2 kb which carries aecD and lysC, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany) .

The E. coli strain DH5α (Grant et al.; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989) . Selection of plas id- carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al . , Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold Spring Harbor, New York, 19δ9) , which is supplemented with 50 mg/1 kanamycin.

Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis. The plasmid is called pKlδmobsacBaecDl_l. A map of the plasmid is shown in Figure 2.

The plasmid pKlδmobsacBaecDl__l was deposited in the form of a pure culture of the strain E. coli DH5oancr/pKlδmobsacBaecDl_l (=

DH5alphamcr/pKlδmobsacBaecDl_l) on 5th June 2002 under number DSM15040 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.

1.8 Incorporation of a second copy of the lysC gene as the lysC^FBR allele into the chromosome (target site: aecD gene) of the strain DSM12866 by means of the replacement vector pKlδmobsacBaecDl_l

As described in Example 1.3, the plasmid pKlδmobsacBaecDl_l described in Example 1.4 is transferred into the C. glutamicum strain DSM12866 by conjugation. Selection is made for targeted recombination events in the chromosome of C. glutamicum DSM12866 as described in Example 1.3. Depending on the position of the second recombination event, after the excision the second copy of the lysC^FBR allele manifests itself in the chromosome at the aecD locus, or the original aecD locus of the host remains.

Approximately 40 to 50 colonies are tested for the phenotype "growth in the presence of sucrose" and "non- growth in the presence of kanamycin" . Approximately 20 colonies which show the phenotype "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" are investigated with the aid of the polymerase chain reaction. A DNA fragment which carries the aecD gene and surrounding regions is amplified here from the chromosomal DNA of the colonies . The same primer oligonucleotides as are described in Example 1.7 for the construction of the integration plasmid are chosen for the PCR.

aecD_beg (SEQ ID NO: 11) :

5^V GAA CTT ACG CCA AGC TGT TC 3 "

aecD_end (SEQ ID NO: 12) : 5^s AGC ACC ACA ATC AAC GTG AG 3

The primers allow amplification of a DNA fragment approx. 2.1 kb in size in control clones with the original aecD locus. In clones with a second copy of the lysC^FBR allele in the chromosome at the aecD locus, DNA fragments with a size of approx. 3.8 kb are amplified.

A clone which, in addition to the copy of the wild-type gene present at the lysC locus, has a second copy of the lysC gene in the form of the lysC^FBR allele lysC T311I at the aecD locus in the chromosome was identified in this manner. This clone was called strain DSMl2δ66aecD: :lysC.

Example 2

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

2.1 Construction of the replacement vector pKlδmobsacBglu2_l

The Corynebacterium glutamicum strain ATCC13032 is used as the donor for the chromosomal DNA. From the strain ATCC13032, chromosomal DNA is isolated using the conventional methods (Eikmanns et al - , Microbiology 140: 1817 - lδ2δ (1994)). With the aid of the polymerase chain reaction, a DNA fragment which carries the gluB gene and surrounding regions is amplified. On the basis of the sequence of the gluABCD gene cluster known for C. glutamicum (Kronemeyer et al . , Journal of Bacteriology, 177: 1152 - 1156 (1995); EP1106790) (Accession Number X81191 and AX127149) , the following primer oligonucleotides are chosen for the PCR:

gluA_beg (SEQ ID NO: 13) : 5 CAC GGT TGC TCA TTG TAT CC 3

gluD_end (SEQ ID NO: 14) :

5^V CGA GGC GAA TCA GAC TTC TT 3

The primers shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and

Applications, 1990, Academic Press) . The primers allow amplification of a DNA fragment of approx 4.4 kb in size, which carries the gluB gene and surrounding regions.

The amplified DNA fragment is identified by means of electrophoresis in a 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .

Ligation of the fragment is then carried out by means of the TOPO TA Cloning Kit (Invitrogen, Leek, The Netherlands, Cat. Number K4600-01) in the vector pCRII-TOPO. The ligation batch is transformed in the E. coli strain TOP10 (Invitrogen, Leek, The Netherlands) . Selection of plasmid- carrying cells is made by plating out the transformation batch on kanamycin (50 mg/1) -containing LB agar with X-Gal (64 mg/1) .

The plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel . The resulting plasmid is called pCRII- T0P0glu2.

The plasmid pCR.Il-TOPOglu2 is cleaved with the restriction enzymes EcoRI and Sail (Amersham-Pharmacia, Freiburg, Germany) and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the gluB fragment of approx. 3.7 kb is isolated from the agarose gel and employed for ligation with the mobilizable cloning vector pKlδmobsacB described by Schafer et al. (Gene 14, 69-73 (1994)). This is cleaved beforehand with the restriction enzymes EcoRI and Sail and dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim) , mixed with the gluB fragment of approx. 3.7 kb, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany) .

The E. coli Strain DH5α (Grant et al . ; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989) . Selection of plasmid- carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al . , Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold Spring Harbor, New York, 19δ9) , which is supplemented with 50 mg/1 kanamycin.

Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis. The plasmid is called pKlδmobsacBglu2.

As described in Example 2.1, a DNA fragment which carries the ddh gene and surrounding regions is also amplified with the aid of the polymerase chain reaction. On the basis of the sequence of the ddh gene cluster known for C. glutamicum (Ishino et al . , Nucleic Acids Research 15, 3917(1987)) (Accession Number Y00151) , the following primer oligonucleotides are chosen for the PCR:

ddhjoeg (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 shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al . {PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) . The primers allow amplification of a DNA fragment of approx 1.6 kb in size, which carries the ddh gene.

The amplified DNA fragment of approx. 1.6 kb in length, which the ddh gene, is identified by means of electrophoresis in a 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .

After purification, the fragment carrying the ddh gene is employed for ligation in the vector pK18mobsacBglu2 described. This is partly cleaved beforehand with the restriction enzyme BamHI. By treatment of the vector with a Klenow polymerase (Amersham-Pharmacia, Freiburg, Germany) , the overhangs of the cleaved ends are completed to blunt ends, the vector is then mixed with the DNA fragment of approx. 1.6 kb which carries the ddh gene and the mixture is treated with T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany) . By using Vent Polymerase (New England Biolabs, Frankfurt, Germany) for the PCR reaction, a ddh- carrying DNA fragment which has blunt ends and is suitable for ligation in the pretreated vector pKl8mobsacBglu2 is generated.

The E. coli strain DH5oancr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989). Selection of plasmid-carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold

Spring Harbor, New York, 1989) , which was supplemented with 50 mg/1 kanamycin.

Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis. The plasmid is called pKl8mobsacBglu2_l. A map of the plasmid is shown in Figure 4.

2.2 Incorporation of a second copy of the ddh gene into the chromosome (target site: gluB gene) of the strain DSM12666 by means of the replacement vector pKl8πιobsacBglu2_l

As described in Example 1.3, the plasmid pKl8mobsacBglu2_l described in Example 2.1 is transferred into the C. glutamicum strain DSM12866 by conjugation. Selection is made for targeted recombination events in the chromosome of C. glutamicum DSM12866 as described in Example 1.3. Depending on the position of the second recombination event, after the excision the second copy of the ddh gene manifests itself in the chromosome at the gluB locus, or the original gluB locus of the host remains.

Approximately 40 to 50 colonies are tested for the phenotype "growth in the presence of sucrose" and "non- growth in the presence of kanamycin" . Approximately 20 colonies which show the phenotype "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" are investigated with the aid of the polymerase chain reaction. A DNA fragment which carries the glu region described is amplified here from the chromosomal DNA of the colonies . The same primer oligonucleotides as are described in Example 2.1 for the construction of the replacement plasmid are chosen for the PCR.

gluA oeg (SEQ ID NO: 13) :

5 CAC GGT TGC TCA TTG TAT CC 3

gluD_end (SEQ ID NO: 14) :

5^V CGA GGC GAA TCA GAC TTC TT 3 "

The primers allow amplification of a DNA fragment approx. 4.4 kb in size in control clones with the original glu locus . In clones with a second copy of the ddh gene in the chromosome at the gluB locus, DNA fragments with a size of approx. 6 kb are amplified.

The amplified DNA fragments are identified by means of electrophoresis in a 0.8% agarose gel .

A clone which, in addition to the copy present at the ddh locus, has a second copy of the ddh gene at the gluB locus in the chromosome was identified in this manner. This clone was called strain DSMl2866glu: :ddh.

Example 3

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

3.1 Construction of the replacement vector pKl8mobsacBaecD2_l

The Corynebacterium glutamicum strain ATCC13032 is used as the donor for the chromosomal DNA. From the strain ATCC13032, chromosomal DNA is isolated using the conventional methods (Eikmanns et al., Microbiology 140: 1817 - 1828 (1994)). With the aid of the polymerase chain reaction, a DNA fragment which carries the aecD gene and surrounding regions is amplified. On the basis of the sequence of the aecD gene known for C. glutamicum (Rossol et al., Journal of Bacteriology 174:2968-2977 (1992)) (Accession Number M89931) , the following primer oligonucleotides are chosen for the PCR:

aecD_beg (SEQ ID NO: 11) :

5^V GAA CTT ACG CCA AGC TGT TC 3

aecD_end (SEQ ID NO: 12):

5 AGC ACC ACA ATC AAC GTG AG 3^V

The primers shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al . (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) . The primers allow amplification of a DNA fragment of approx 2.1 kb in size, which carries the aecD gene and adjacent regions.

The amplified DNA fragment of approx. 2.1 kb in length is identified by means of electrophoresis in a 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .

The DNA fragment purified is cleaved with the restriction enzyme Bglll and EcoRV (Amersham Pharmacia, Freiburg, Germany) . The ligation of the fragment in the vector pUClδ then takes place (Norrander et al . , Gene 26:101-106

(1983)). This is cleaved beforehand with the restriction enzymes BamHI and Smal and dephosphorylated, mixed with the aecD-carrying fragment of approx. 1.5 kb, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany) . The ligation batch is transformed in the E. coli strain TOP10 (Invitrogen, Leek, The Netherlands) . Selection of plasmid-carrying cells is made by plating out the transformation batch on kanamycin (50 mg/1) -containing LB agar with X-Gal (64 mg/1).

The plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel. The resulting plasmid is called pUC18aecD. With the aid of the polymerase chain reaction, a further DNA fragment which carries the dapA gene and surrounding regions is amplified. On the basis of the sequence of the dapA gene known for C. glutamicum (Bonassi et al . , Nucleic Acids Research 18:6421 (1990)) (Accession Number X53993 and AX127149) , the following primer oligonucleotides are chosen for the PCR:

dapA_beg (SEQ ID NO: 17) :

5^V CGA GCC AGT GAA CAT GCA GA 3^N

dapA_end (SEQ ID NO: 18) :

5 CTT GAG CAC CTT GCG CAG CA 3^s

The primers shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al . (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) . The primers allow amplification of a DNA fragment of approx. 1.4 kb in size, which carries the dapA gene and adjacent regions.

The amplified DNA fragment of approx. 1.4 kb in length is identified by means of electrophoresis in a 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .

After purification, the dapA-containing DNA fragment approx. 1.4 kb in length is employed for ligation with the vector pUClβaecD described above. This is cleaved beforehand with the restriction enzyme Stul, mixed with the DNA fragment of approx. 1.4 kb, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany) .

The E. coli strain DH5otmcr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989). Selection of plasmid-carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold Spring Harbor, New York, 1989), which was supplemented with 50 mg/1 kanamycin.

Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis. The plasmid is called pUC18aecD2.

The plasmid pUC18aecD2 is cleaved with the restriction enzyme Sail and partly with EcoRI (Amersham-Pharmacia, Freiburg, Germany) and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the fragment of approx. 2.7 kb which carries aecD and dapA is isolated from the agarose gel and employed for ligation with the mobilizable cloning vector pKlδmobsacB described by Schafer et al . (Gene 14: 69-73 (1994).). This is cleaved beforehand with the restriction enzymes EcoRI and with Sail and dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim) , mixed with the fragment of approx. 2.7 kb which carries aecD and dapA, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany) .

The E. coli strain DH5α (Grant et al . ; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989) . Selection of plasmid- carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold Spring Harbor, New York, 1989), which is supplemented with 50 mg/1 kanamycin.

Plasmid DNA is isolated from a transformant with the aid of the QIAprep.Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis. The plasmid is called pKl8mobsacBaecD2_l. A map of the plasmid is shown in Figure 5.

3.2 Incorporation of a second copy of the dapA gene into the chromosome (target site: aecD gene) of the strain DSM12866 by means of the replacement vector pKl8mobsacBaecD2_l

As described in Example 1.3, the plasmid pKlδmobsacBaecD2_l described in Example 3.1 is transferred into the C. glutamicum strain DSM12666 by conjugation. Selection is made for targeted recombination events in the chromosome of C. glutamicum DSMl2δ66 as described in Example 1.3. Depending on the position of the second recombination event, after the excision the second copy of the dapA gene manifests itself in the chromosome at the aecD locus, or the original aecD locus of the host remains.

Approximately 40 to 50 colonies are tested for the phenotype "growth in the presence of sucrose" and "non- growth in the presence of kanamycin" . Approximately 20 colonies which show the phenotype "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" are investigated with the aid of the polymerase chain reaction. A DNA fragment which carries the aecD gene and surrounding regions is amplified here from the chromosomal DNA of the colonies. The same primer oligonucleotides as are described in Example 3.1 for the construction of the integration plasmid are chosen for the PCR.

aecD_beg (SEQ ID NO: 11) :

5^X 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 allow amplification of a DNA fragment approx. 2.1 kb in size in control clones with the original aecD locus . In clones with a second copy of the dapA gene in the chromosome at the aecD locus, DNA fragments with a size of approx. 3.6 kb are amplified.

The amplified DNA fragments are identified by means of electrophoresis in a 0.δ% agarose gel.

A clone which, in addition to the copy present at the dapA locus, has a second copy of the dapA gene at the aecD locus in the chromosome was identified in this manner. This clone was called strain DSMl2866aecD: :dapA.

Example 4

Incorporation of a second copy of the pyc gene in the form of the pyc allele pycP458S into the chromosome (target site: pck gene) of the strain DSM12866

4.1 Construction of the replacement vector pKl8mobsacBpckl_3

The replacement vector pKlδmobsacBpckl described in

Example 1.5 is used as the base vector for insertion of the pyc allele.

As described in Example 2.1, a DNA fragment which carries the pyc gene and surrounding regions is also amplified with the aid of the polymerase chain reaction. On the basis of the sequence of the pyc gene cluster known for C. glutamicum ( eters-Wendisch et al . , Journal of Microbiology 144: 915-927 (1998)) (Accession Number Y09548) , the following primer oligonucleotides are chosen for the 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 shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al . (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) . The primers allow amplification of a DNA fragment of approx 3.6 kb in size, which carries the pyc gene. The primers moreover contain the sequence for the cleavage site of the restriction endonuclease Mlul, which is marked by parentheses in the nucleotide sequence shown above.

The amplified DNA fragment of approx. 3.6 kb in length, which carries the pyc gene, is cleaved with the restriction endonuclease Mlul, identified by means of electrophoresis in a 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .

After purification, the fragment carrying the pyc gene is employed for ligation in the vector pKlδmobsacBpckl described. This is cleaved beforehand with the restriction enzyme BssHII , dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany) , mixed with the DNA fragment of approx. 3.6 kb which carries the pyc gene, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany) .

The E. coli strain DHδαmcr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation batch (Hanahan_; In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989) .

Selection of plasmid-carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold Spring Harbor, New York, 1989), which was supplemented with 50 mg/1 kanamycin.

Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis. The plasmid is called pKl8mobsacBpckl_2. 4.2 Construction of the pyc allele pyc P458S by means of site-specific mutagenesis of the wild-type pyc gene

The site-directed mutagenesis is carried out with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, USA) . EP-A-1108790 describes a point mutation in the pyc gene for C. glutamicum which allows improved L-lysine production. On the basis of the point mutation in the nucleotide sequence of cytosine to thymine in the pyc gene at position 1372, replacement in the amino acid sequence derived therefrom of proline for serine at position 458 results. The allele is called pyc P458S. To generate the mutation described, the following primer oligonucleotides are chosen for the linear amplification:

P458S-1 (SEQ ID NO: 21) : 5' GGATTCATTGCCGATCAC (TCG) CACCTCCTTCAGGCTCCA 3'

P456S-2 (SEQ ID NO: 22):

5'GTGGAGGAAGTCCGAGGT (CGA) GTGATCGGCAATGAATCC 3'

The primers shown are synthesized by MWG Biotech. The codon for serine, which is to replace the proline at position 45δ, is marked by parentheses in the nucleotide sequence shown above. The plasmid pKlδmobsacBpckl_2 described in Example 4.1 is employed with the two primers, which are each complementary to a strand of the plasmid, for linear amplification by means of Pfu Turbo DNA polymerase. By this lengthening of the primers, a mutated plasmid with broken circular strands is formed. The product of the linear amplification is treated with Dpnl - this endonuclease cleaves the methylated and half-methylated template DNA specifically. The newly synthesized broken, mutated vector DNA is transformed in the E. coli strain XLl Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)). After the transformation, the XLl Blue cells repair the breaks in the mutated plasmids . Selection of the transformants was carried out on LB medium with kanamycin 50 mg/1. The plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel. The DNA sequence of the mutated DNA fragment ±_. checked by sequencing. The sequence of the PCR product coincides with the sequence described Ohnishi et al. (2002). The resulting plasmid is called pKl8mobsacBpckl_3. A map of the plasmid is shown in Figure 6.

4.3 Incorporation of a second copy of the pyc gene in the form of the pyc allele pycP45δS into the chromosome (target site pck gene) of the strain DSM12866 by means of the replacement vector pkl8mobsacBpckl_3

The plasmid pKlδmobsacBpckl_3 described in Example 4.2 is transferred as described in Example 1.3 into the C. glutamicum strain DSM12866 by conjugation. Selection is made for targeted recombination events in the chromosome of C. glutamicum DSM12866 as described in Example 1.3. Depending on the position of the second recombination event, after de excision the second copy of the pyc allele manifests itself in the chromosome at the pck locus, or the original pck locus of the host remains .

Approximately 40 to 50 colonies are tested for the phenotype "growth in the presence of sucrose" and "non- growth in the presence of kanamycin" . Approximately 20 colonies which show the phenotype "growth in the presence of sucrose" and "non-growth in the presence of kanamycin" are investigated with the aid of the polymerase chain reaction. A DNA fragment which carries the pck gene and surrounding regions is amplified here from the chromosomal DNA of the colonies. The same primer oligonucleotides as are described in Example 1.5 for the construction of the replacement plasmid are chosen for the PCR.

pck_beg (SEQ ID NO: 9) :

5^X 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 ^N

The primers allow amplification of a DNA fragment approx. 2.9 kb in size in control clones with the original pck locus . In clones with a second copy of the pyc allele in the chromosome at the pck locus, DNA fragments with a size of approx. 6.5 kb are amplified.

A clone which, in addition to the copy of the wild-type gene present at the pyc locus, has a second copy of the pyc gene in the form of the pyc allele pycP458S at the pck locus in the chromosome was identified in this manner. This clone was called strain DSMl2866pck: :pyc.

Example 5

Preparation of Lysine

The C. glutamicum strains DSMl3994glu: : lysC, DSMl2866glu: :lysC, DSMl2866pck: : lysC, DSMl2866aecD: : lysC, DSMl2866glu: :ddh, DSMl2866aecD: :dapA and DSMl2866pck: :pyc obtained in Example 1, 2, 3 and 4 are cultured in a nutrient medium, suitable for the production of lysine and the lysine concent in the culture supernatant was determined.

For this, the cultures are first incubated on a brain-heart agar plate (Merck, Darmstadt, Germany) for 24 hours at

33 ^aC. Starting from this agar plate culture, a preculture is seeded (10 ml medium in a 100 ml conical flask) . The medium MM is used as the medium for the preculture. The preculture is incubated for 24 hours at 33^aC at 240 rpm on a shaking machine. A main culture is seeded from this preculture such that the initial OD (660 nm) of the main culture is 0.1 OD. The Medium MM is also used for the main culture.

Medium MM

CSL 5 g/1

MOPS 20 g/1

Glucose (autociaved separately) 50 g/1

Salts:

(NH₄)₂S0₄ 25 g/1

KH₂P0₄ 0.1 g/1

MgS0₄ * 7 H₂0 1.0 g/1

CaCl₂ * 2 H₂0 10 mg/1

FeS0₄ * 7 H₂0 10 mg/1

MnS0₄ * H₂0 5.0 mg/1

Biotin (sterile-filtered) 0.3 mg/1

Thiamine * HCl (sterile-filtered) 0.2 mg/1

CaC0₃ 25 g/1

The CSL (corn steep liquor) , MOPS (morpholinopropanesulfonic acid) and the salt solution are brought to pH 7 with aqueous ammonia and autociaved. The sterile substrate and vitamin solutions, as well as the CaC0₃ autociaved in the dry state, are then added.

Culturing is carried out in a 10 ml volume in a 100 ml conical flask with baffles. Culturing is carried out at 33 ^aC and 80% atmospheric humidity. After 48 hours, the OD is determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich) . The amount of lysine formed is determined wich an amino acid analyzer from Eppendorf- BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivation with ninhydrin detection.

The result of the experiment is shown in Table 10.

Table 10

Brief Description of the Figures :

The base pair numbers stated are approximate values obtained in the context of reproducibility of measurements.

Figure 1: Map of the plasmid pKl8mobsacBglul_l .

The abbreviations and designations used have the following meaning:

KanR: Kanamycin resistance gene HindiII : Cleavage site of the restriction enzyme

Hindlll

BamHI: Cleavage site of the restriction enzyme

BamHI

lysC: lysC^FBR allele, lysC T311I

'gluA: 3 ' terminal fragment of the gluA gene

gluB ' : 5 ' terminal fragment of the gluB gene

' gluB: 3 ' terminal fragment of the gluB gene

gluC : 5 ' terminal fragment of the gluC gene

sacB: sacB gene

RP4mob: mob region with the replication origin for the transfer (oriT)

oriV: Replication origin V

Figure 2: Map of the plasmid pKlδmobsacBaecDl_l.

The abbreviations and designations used have the following meaning:

KanR: Kanamycin resistance gene

Sail: Cleavage site of the restriction enzyme Sail

lysC : lysC^FBR allele, lysC T311I

aecD' : 5 ' terminal fragment of the aecD gene

' aecD: 3 ' terminal fragment of the aecD gene

sacB: sacB gene

RP4mob: mob region with the replication origin for the transfer (oriT) oriV: Replication origin V

Figure 3: Map of the plasmid pKlδmobsacBpckl_l.

The abbreviations and designations used have the following meaning:

KanR: Kanamycin resistance gene

BamHI: Cleavage site of the restriction enzyme

BamHI

lysC: lysC*^BK allele, lysC T311I

pck' : 5 ' terminal fragment of the pck gene

'pck: 3 ' terminal fragment of the pck gene

sacB: sacB gene

RP4mob: mob region with the replication origin for the transfer (oriT)

oriV: Replication origin V

Figure 4: Map of the plasmid pKl8mobsacBgluB2_l.

The abbreviations and designations used have the following meaning:

KanR: Kanamycin resistance gene

Sail Cleavage site of the restriction enzyme Sail

EcoRI Cleavage site of the restriction enzyme

EcoRI

BamHI: Cleavage site of the restriction enzyme BamHI

ddh: ddh gene gluA gluA gene

gluB¹ : 5 ' terminal fragment of the gluB gene

' gluB : 3 ' terminal fragment of the gluB gene

gluC gluC gene

gluD¹ : 5 ' terminal fragment of the gluD gene

sacB: sacB gene

RP4mob: mob region with the replication origin for the transfer (oriT)

oriV: Replication origin V

Figure 5: Map of the plasmid pKlδmobsacBaecD2_l.

The abbreviations and designations used have the following meaning:

KanR: Kanamycin resistance gene

EcoRI Cleavage site of the restriction enzyme EcoRI

Sail: Cleavage site of the restriction enzyme Sail

dapA: dapA gene

aecD' : 5 ^! terminal fragment of the aecD gene

' aecD: 3 ' terminal fragment of the aecD gene

sacB: sacB gene

RP4mob: mob region with the replication origin for the transfer (oriT)

oriV: Replication origin V

Figure 6: Map of the plasmid pK18mobsacBpckl_3 The abbreviations and designations used have the following meaning:

KanR: Kanamycin resistance gene

pyc: pyc allele, pyc P458S

pck' : 5 ' terminal fragment of the pck gene

'pck: 3 ' terminal fragment of the pck gene

sacB: sacB gene

RP4mob: mob region with the replication origin for the transfer (oriT)

oriV: Replication origin V

BUDAPEST TREATY ON THE INTERNATIONAL Sammlung von

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Mikroorganismen und

FOR THE PURPOSES OF PATENT PROCEDURE Zellkulturen GmbH

INTERNATIONAL FORM

Degussa AG Kantstr. 2

33790 Halle (Westf.) RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

I. IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the DEPOSITOR: Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY:

DSM12866glu::lysC

DSM 15039

π. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I. above was accompanied by:

( X ) a scientific description

( X ) a proposed taxonomic designation

(Mark with a cross where applicable).

in. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2002-06-05 (Date of the original deposit)¹.

IV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of person(s) having the power to represent the MIKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authorized officials):

Address: Mascheroder eg lb D-₃81 4 Braunschweig ⁽A,

Date: 2002-06-06

¹ Where Rule _A (d) applies, such date is the date on which the status of international depositary authority was acquired. Form DSMZ-BP/ (sole-page) 12 2001 BUDAPEST TREATY ON THE INTERNATIONAL Deutsche Sammlung von

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Mikroorganismen und

FOR THE PURPOSES OF PATENT PROCEDURE Zellkulturen GmbH

INTERNATIONAL FORM

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Kantstr. 2

33790 Halle (Westf.)

VIABILITY STATEMENT issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

I. DEPOSITOR π. IDENTIFICATION OF THE MICROORGANISM

Name: Degussa AG Accession number given by the

Kantstr. 2 INTERNATIONAL DEPOSITARY AUTHORITY;

Address: 33790 Halle (Westf.)

DS 15039

Date of the deposit or the transfer':

2002-06-05 m. VIABILITY STATEMENT

The viability of the microorganism identified under II above was tested on 2002-06-05 On that date, the said microorganism was

(_x)³ viable

( )³ no longer viable

IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED⁴

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of person(s) having the power to represent the

MIKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authorized offιcial(s):

Address: Mascheroder Weg lb D-38124 Braunschweig

Date: 2002-06-06

¹ Indicate the date of original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer).

² In the cases referred to in Rule 10.2(a) (ii) and (iii), refer to the most recent viability test.

³ Mark with a cross the applicable box.

⁴ Fill in if the information has been requested and if the results of the test were negative.

Form DSMZ-BP/9 (sole page) 12/2001 BUDAPEST TREATY ON THE INTERNATIONAL Deutsche Sammlung von

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Mikroorganismen und

FOR THE PURPOSES OF PATENT PROCEDURE Zellkulturen GmbH

INTERNATIONAL FORM

Degussa AG

Kantstr. 2

I. IDENTIFICATION OF THE MICROORGANISM

DH5alphamcr/pK18mobsacBaecDl_l

DSM 15040

π. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I. above was accompanied by:

( X ) a scientific description

( ) a proposed taxonomic designation

(Mark with a cross where applicable).

m. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2002-06-05 (Date of the original deposit)'.

^TV. RECEIPT OF REQUEST FOR CONVERSION

V. INTERNATIONAL DEPOSITARY AUTHORITY

MIKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authorized official(s):

Address: Mascheroder Weg lb D-₃8124 Braunschweig

Date: 2002-06-06

¹ Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired. Form DSMZ-BP/4 (sole page) 12/2001 BUDAPEST TREAT ON THE INTERNATIONAL Deutsche Sammlung von

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Mikroorganismen und

FOR THE PURPOSES OF PATENT PROCEDURE Zellkulturen GmbH

INTERNATIONAL FORM

Degussa AG

Kantstr. 2

33790 Halle (Westf.)

I. DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM

Name: Degussa AG Accession number given by the

Kantstr. 2 INTERNATIONAL DEPOSITARY AUTHORITY:

Address: 33790 Halle (Westf.)

DSM 15040

Date of the deposit or the transfer':

2002-06-05 rπ. VIABILITY STATEMENT

The viability of the microorganism identified under H above was tested on 2002-06-05 On that date, the said microorganism was

(_x)³ viable

( )³ no longer viable fv. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED⁴

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature^) of personCs) having the power to represent the

MIKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authorized officials):

Address: Mascheroder Weg lb D- 812 Braunschweig

Date: 2002-06-06

' Indicate the date of original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer).

³ Mark with a cross the applicable box.

Form DSMZ-BP/9 (sole page) 12/2001 BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Degussa AG Kantstr. 2 33790 Halle/Kύnsebeck

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the

INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

i. IDENTIΠCAΉON OF THE MICROORGANISM

Identification reference given by the DEPOSITOR: Accession number given by the INTERNAΉONAL DEPOSITARY AUTHORITY:

DH5alphamcr/ pK18mobsacBglul 1 DSM 14243

π. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I. above was accompanied by:

(X ) a scientific description

(X ) a proposed taxonomic designation

(Mark with a cross where applicable).

πi. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2001 -04 - 20 (Date of the original deposit)¹.

IV. RECEIPT OF REQUEST FOR CONVERSION

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-DEUTSCHE SAMMLUNG VON Slgnature(s) of person(s) having the power to represent (he

MIKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authorized uffi al(s):

Address: Mascheroder Weg lb D-3S124 Braunschweig /. C*>_e.^' ,

Date: 2001-04 -26

¹ Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired. Form DSMZ-BP/4 {sole page) 0196 BUDAPEST TREATY ON THE INTERNATIONAL

^COGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Degussa AG Kantstr . 2 33790 Halle/Kϋnsebeck

I. DEPOSITOR ii. IDENΉΠCAΉON OF THE MICROORGANISM

Name: Degussa AG Accession number given by the Kantstr . 2 INTERNATIONAL DEPOSITARY AUTHORITY: Address: 33790 Halle/Kύnsebeck DSM 14243

Date of the deposit or the transfer¹: 2001 -04 -20

in. VIABILITY STATEMENT

The viability of the microorganism Identified under II above was tested on 2001 - 04 - 20 On that date, the said microorganism was

(X)' viable

( )' no longer viable

TV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED⁴

V. INTERNATIONAL DEPOSITARY AUTHORITY

Address: Mascheroder Weg lb D-38124 Braunschweig

Date: 2001- 04 -26

Indicate the date of original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer).

In the cases referred to in Rule 102(a) (ii) and (iii), refer to the most recent viability test.

Mark with a cross the applicable box.

Fill in if the information has been requested and if the results of the test were negative.

Form DSMZ-BP/9 (sole page) 0196

Claims

What is claimed is:

1. Coryneform bacteria which produce chemical compounds , wherein these have, in addition to at least one copy, present at the natural site (locus) , of an open reading frame (ORF) , gene or allele which codes for the synthesis of a protein or an RNA, a second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele in question at a second, optionally third or fourth site in a form integrated into the chromosome, no nucleotide sequence which is capable of/enables episomal replication or transposition in microorganisms and no nucleotide sequence (s) which impart (s) resistance to antibiotics being present at the second, optionally third or fourth site, and the second, optionally third or fourth site not relating to open reading frames (ORF) , genes or alleles which are essential for the growth of the bacteria and the production of the desired compound.

2. Coryneform bacteria according to claim 1 which produce chemical compounds, wherein the coryneform bacteria belong to the genus Corynebacterium.

3. Coryneform bacteria of the genus Corynebacterium according to claim 2 which produce chemical compounds, wherein these belong to the species Corynebacterium glutamicum.

4. Coryneform bacteria according to claim 1 which produce chemical compounds, wherein the chemical compound is a compound chosen from the group consisting of L-amino acids, vitamins, nucleosides and nucleotides.

5. Coryneform bacteria according to claim 1 which produce chemical compounds, wherein the chemical compound is one or more L-amino acids chosen from the group consisting of L-aspartic acid, L-asparagine, L- threonine, L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-proline and L- arginine.

6. Coryneform bacteria according to claims 1 and 4 which produce chemical compounds, wherein the L-amino acid is L-lysine, and these bacteria have, in addition to at least one copy of an open reading frame (ORF) , gene or allele of lysine production present at the natural site (locus) , in each case a second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele of lysine production in question at in each case a second, optionally third or fourth site in a form integrated into the chromosome.

7. Coryneform bacteria according to claim 6 which produce L-lysine, wherein the coryneform bacteria belong to the genus Corynebacterium.

8. Coryneform bacteria of the genus Corynebacterium according to claim 7 which produce L-lysine, wherein these belong to the species Corynebacterium glutamicum.

9. Coryneform bacteria according to claim 6 which produce L-lysine, wherein the open reading frame (ORF) , gene or allele of lysine production is one or more open reading frame(s), one or more gene(s) or allele(s) chosen from the group consisting of 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, siK, opcA, oxyR, ppc, ppc^FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsl, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwal, zwf and zwf A213T.

10. Coryneform bacteria according to claim 6 which produce L-lysine, wherein the open reading frame, gene or allele of lysine production is one or more gene(s) or allele (s) chosen from the group consisting of dapA, ddh, lysC^FBR and pyc P458S.

11. Coryneform bacteria according to claim 6 which produce L-lysine, wherein the open reading frame, gene or allele of lysine production is a lysC^FBR allele which codes for a feed back resistant form of aspartate kinase.

12. Coryneform bacteria according to claim 11 which produce L-lysine, wherein the feed back resistant form of aspartate kinase coded by the lysC^FBR allele contains an amino acid sequence according to SEQ ID NO: 2, SEQ ID NO: 2 containing one or more amino acid replacements chosen from the group consisting of A279T, A279V, S301F, T308I, S301Y, G345D, R320G, T311I and S381F.

13. Coryneform bacteria according to claim 11 which produce L-lysine, wherein the feed back resistant form of aspartate kinase coded by the lysC^FBR allele includes an amino acid sequence according to SEQ ID NO: 4.

14. Coryneform bacteria according to claim 11 which produce L-lysine, wherein the coding region of the lysC^FBR allele includes the nucleotide sequence of SEQ ID NO: 3.

15. Coryneform bacteria according to claim 6 which produce L-lysine, wherein the particular second, optionally third or fourth site is a gene chosen from the group consisting of aecD, ccpAl, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysRl, lysR2, lysR3, menE, mqo, pck, pgi and poxB.

16. Coryneform bacteria according to claim 6 which produce L-lysine, wherein the particular second, optionally third or fourth site is a site chosen from the group consisting of intergenic regions of the chromosome, prophages contained in the chromosome and defective phages contained in the chromosome.

17. Coryneform bacteria according to claim 15 which produce L-lysine, wherein the particular second, optionally third or fourth site is the aecD gene site.

18. Coryneform bacteria according to claim 15 which produce L-lysine, wherein the particular second, optionally third or fourth site is the gluB gene site.

19. Coryneform bacteria according to claim 15 which produce L-lysine, wherein the particular second, optionally third or fourth site is the pck gene site.

20. Process for the preparation of chemical compounds by fermentation of coryneform bacteria, in which the following steps are carried out:

a) fermentation of coryneform bacteria, which al) which have, in addition to at least one copy, present at the natural site (locus) , of an open reading frame (ORF) , gene or allele which codes for the synthesis of a protein or an RNA, a second, optionally third or fourth copy of this open reading frame (ORF) , gene or allele at a second, optionally third or fourth site in a form integrated into the chromosome, no nucleotide sequence which is capable of/enables episomal replication or transposition in microorganisms and no nucleotide sequence (s) which impart (s) resistance to antibiotics being present at the second, optionally third or fourth site, and the second, optionally third or fourth site not relating to open reading frames (ORF) , genes or alleles which are essential for the growth of. the bacteria and the production of the desired compound, and

a2) in which the intracellular activity of the corresponding protein is increased, in particular the nucleotide sequence which codes for this protein is over-expressed,

c) concentration of the chemical compound(s) in the fermentation broth and/or in the cells of the bacteria,

d) isolation of the chemical compound(s) , optionally

e) with constituents from the fermentation broth and/or the biomass to the extent of > (greater than) 0 to 100 wt.%.

21. Process according to claim 20, wherein the coryneform bacteria belong to the genus Corynebacterium.

22. Process according to claim 20, wherein the coryneform bacteria of the genus Corynebacterium belong to the species Corynebacterium glutamicum.

23. Process according to claim 20, wherein the chemical compound is a compound chosen from the group consisting of L-amino acids, vitamins, nucleosides and nucleotides .

24. Process according to claim 20, wherein the chemical compound is one or more L-amino acids chosen from the group consisting of L-aspartic acid, L-asparagine, L- threonine, L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-proline and L- arginine.

25. Process according to claim 24, wherein the chemical compound is L-lysine.

26. Process for the preparation of L-lysine, which comprises the following steps:

a) fermentation of coryneform bacteria which have, in addition to at least one copy of an open reading frame (ORF) , gene or allele of lysine production present at the natural site (locus) , in each case a second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele of lysine production in question at in each case a second, optionally third or fourth site in a form integrated into the chromosome

under conditions which allow expression of the said open reading frames (ORF) , genes or alleles mentioned.

27. Process for the preparation of L-lysine according to claim 26, wherein the open reading frame (ORF), gene or allele of lysine production is an open reading frame, a gene or allele chosen from the group consisting of 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, ρρc^FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsl, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwal, zwf and zwf A213T.

28. Process for the preparation of L-lysine according to claim 26, wherein the open reading frame (ORF), gene or allele of lysine production is a gene or allele chosen from the group consisting of dapA, ddh, lysC^FBR and pyc P458S.

29. Process for the preparation of L-lysine according to claim 26, wherein the open reading frame (ORF), gene or allele of lysine production is a lysC^FBR allele which codes for a feed back resistant form of aspartate kinase.

30. Process for the preparation of L-lysine according to claim 29, wherein the feed back resistant form of aspartate kinase coded by the lysC^FBR allele contains an amino acid sequence according to SEQ ID NO: 2, SEQ ID NO: 2 containing one or more amino acid replacements chosen from the group consisting of A279T, A279V, S301F, T308I, S301Y, G345D, R320G, T311I and S381F.

31. Process for the preparation of L-lysine according to claim 29, wherein the feed back resistant form of aspartate kinase coded by the lysC^FBR allele includes an amino acid sequence according to SEQ ID NO: 4.

32. Process for the preparation of L-lysine according to claim 29, wherein the coding region of the lysC^FBR allele includes the nucleotide sequence of SEQ ID NO: 3

33. Process for the preparation of L-lysine according to claim 26, wherein the particular second, optionally third or fourth site is a site chosen from the group consisting of aecD, ccpAl, ccpA2 , citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysRl, lysR2, lysR3 , menE, mqo, pck, pgi and poxB.

34. Process for the preparation of L-lysine according to claim 26, wherein the second, optionally third or fourth site is the aecD gene site.

35. Process for the preparation of L-lysine according to claim 26, wherein the second, optionally third or fourth site is the gluB gene site.

36. Process for the preparation of L-lysine according to claim 26, wherein the second, optionally third or fourth site is the pck gene site.

37. Process for the production of coryneform bacteria which produce one or more chemical compounds, which comprises

a) isolating the nucleotide sequence of at least one desired ORF, gene or allele which codes for a protein or an RNA, optionally including the expression and/or regulation signals, preferably from coryneform bacteria,

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

e) isolating coryneform bacteria in which the nucleotide sequence (s) according to a) is incorporated at the target site, no nucleotide sequence (s) which is (are) capable of/enable(s) episomal replication or transposition in microorganisms, and no nucleotide sequence (s) which impart (s) resistance to antibiotics remaining at the target site.

38. Plasmid pK18mobsacBglul_l shown in Figure 1 and deposited in the form of a pure culture of the strain E. coli DH5ocmcr/pKl8mobsacBglul_l (= DH5alpha mcr/pKl8mobsacBglul_l) under number DSM14243.

39. Plasmid pKl8mobsacBaecDl_l shown in Figure 2 and deposited in the form of a pure culture of the strain E. coli DH5αmcr/pKl8mobsacBaecDl_l (= DH5alphamcr/pKl8mobsacBaecDl_l) under number DSM15040,

40. Corynebacterium glutamicum strain DSMl2866glu: :lysC deposited in the form of a pure culture under number DSM15039.