CA2455878A1

CA2455878A1 - Production of l-lysine by genetically modified corynebacterium glutamicum strains

Info

Publication number: CA2455878A1
Application number: CA002455878A
Authority: CA
Inventors: Brigitte Bathe; Caroline Reynen; Bettina Moeckel; Georg Thierbach
Original assignee: Individual
Current assignee: Evonik Operations GmbH
Priority date: 2001-08-06
Filing date: 2002-07-30
Publication date: 2003-05-15
Also published as: WO2003040373A3; CN100554426C; BR0211723A; CN1539015A; AU2002325923A1; EP1414970A2; WO2003040373A2

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(1):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 sequences) which imparts) 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, a1) 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.orr 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 sequences) which imparts) 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 compounds) in the fermentation broth andlor 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 provides 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 compounds) 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 5 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 B1 (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 lilium 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 and mutants or strains, such as are known from the prior art, produced therefrom which produce chemical compounds.
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 lysCFBR 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, Nm, 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 5 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 10 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~- 2.0o.
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 a1. (Journal of Bacteriology 172, 1663-1666 (1990) and Gene 145, 69-73 (1994)) and in Schwarzer and Piihler (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°-C 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, lysCF$R, lysE, msiK, opcA, oxyR, ppc, ppcFBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigh, tal, thyA, tkt, tpi, zwal, zwf and zwf A213T.
These are summarized and explained in Table 1.
These include, in particular, the lysCFBR alleles which code for a "feed back" resistant aspartate kinase. Various lysCFBR alleles are summarized and explained in Table 2.
The following lysCFBR 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 N0: 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 N0: 2, by aspartic acid), lysC R320G
(replacement of arginine at position 320 of the aspartate kinase protein coded, according to SEQ ID N0: 2, by glycine), lysC T311I (replacement of threonine at position 311 of.the aspartate kinase protein coded, according to SEQ

ID N0: 2, by isoleucine), lysC S381F (replacement of serine at position 381 of the aspartate kinase protein coded, according to SEQ ID N0: 2, by phenylalanine).
The lysCF$R allele lysC T311I (replacement of threonine at position 311 of the aspartate kinase protein coded, according to SEQ ID N0: 2, by isoleucine), the nucleotide sequence of which is shown as SEQ ID N0:3, is particularly preferred; the amino acid sequence of the aspartate kinase protein coded is shown as SEQ ID N0:4.
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, ~.-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 5 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 10 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, 15 3rd 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 Name Description of the coded enzymeReference Access or protein Number accBC Acyl-CoA Carboxylase Jager et al. U35023 EC 6.3.4.14 Archives of (acyl-CoA carboxylase) ' Microbiology (1996) 166:76-EP1108790; AX123524 accDA Acetyl-CoA Carboxylase EP1055725 EC 6.4.1.2 EP1108790 AX121013 (acetyl-CoA carboxylase) W00100805 AX066443 cstA Carbon Starvation Protein A EP1108790 AX120811 (carbon starvation protein A) W00100804 AX066109 cysD Sulfate Adenylyltransferase EP1108790 AX123177 sub-unit II

EC 2.7.7.4 (sulfate adenylyltransferase small chain ) cysE Serine Acetyltransferase EP1108790 AX122902 EC 2.3.1.30 W00100843 AX063961 (serine acetyltransferase) cysH 3'-Phosphoadenyl Sulfate ReductaseEP1108790 AX123178 EC 1.8.99.4 W00100842 AX066001 (3'-phosphoadenosine 5'-phosphosulfate reductase) cysK. Cysteine Synthase EP1108790 AX122901 EC 4.2.99.8 W00100843 AX063963 (cysteine synthase) cysN Sulfate Adenylyltransferase EP1108790 AX123176 sub-unit I AX127152 EC 2.7.7.4 (sulfate adenylyltransferase) cysQ Transport Protein CysQ EP1108790 AX127145 (transporter cysQ) W00100805 AX066423 dapA Dihydrodipicolinate Synthase Bonnassie et X53993 EC 4.2.1.52 al. Nucleic (dihydrodipicolinate synthase) Acids Research 18:6421 (1990) Pisabarro et al., Journal of Bacteriology X21502 175:2743-2749(1993) dapB Dihydrodipicolinate Reductase EP1108790 AX127149 EC 1.3.1.26 W00100843 AX063753 (dihydrodipicolinate reductase)EP1067192 AX137723 Pisabarro X67737 et al., Journal 221502 of Bacteriology 175:2743-2749(1993) dapC N-Succinyl Aminoketopimelate EP1108790 AX127146 Transaminase W00100843 AX064219 EC 2.6.1.17 EP1136559 (N-succinyl diaminopimelate transaminase) dapD Tetrahydrodipicolinate SuccinylaseEP1108790 AX127146 EC 2.3.1.117 W00100843 AX063757 (tetrahydrodipicolinate Wehrmann et AJ004934 al.

succinylase) Journal of Bacteriology 180:3159-3165(1998) dapE N-Succinyl Diaminopimelate EP1108790 AX127146 Desuccinylase W00100843 AX063749 EC 3.5.1.18 Wehrmann et X81379 al.

(N-succinyl diaminopimelate Microbiology desuccinylase) 140:3349-3356 (1994) dapF Diaminopimelate Epimerase EP1108790 AX127149 EC 5.1.1.7 W00100843 AX063719 (diaminopimelate epimerase) EP1085094 AX137620 ddh Diaminopimelate Dehydrogenase EP1108790 AX127152 EC 1.4.1.16 W00100843 AX063759 (diaminopimelate dehydrogenase)Ishino et Y00151 al., Nucleic Acids Research 15:3917-3917(1987) Kim et al., D87976 Journal of Microbiology and Biotechnology 5:250-256(1995) dps DNA Protection Protein EP1108790 AX127153 (protection during starvation protein) eno Enolase EP1108790 AX127146 EC 4.2.1.11 W00100844 AX064945 (enolase) EP1090998 AX136862 Hermann et al., Electrophoresis 19:3217-3221 (1998) gap Glyceraldehyde 3-Phosphate EP1108790 AX127148 Dehydrogenase W00100844 AX064941 EC 1.2.1.12 Eikmanns et X59403 (glyceraldehyde 3-phosphate al., Journal of dehydrogenase) ~ Bacteriology 174:6076-6086(1992) gap2 Glyceraldehyde 3-Phosphate EP1108790 AX127146 Dehydrogenase W00100844 AX064939 EC 1.2.1.12 (glyceraldehyde 3-phosphate dehydrogenase 2) gdh Glutamate Dehydrogenase EP1108790 AX127150 EC 1.4.1.4 W00100844 AX063811 (glutamate dehydrogenase) Boermann et X59404 al., Molecular Microbiology 6:317-326 (1992) .

Guyonvarch X72855 et al., NCBI

gnd 6-Phosphogluconate DehydrogenaseEP1108790 AX127147 EC 1.1.1.44 AX121689 (6-phosphogluconate dehydrogenase)W00100844 . AX065125 lysC Aspartate Kinase EP1108790 AX120365 EC 2.7.2.4 ~ W00100844 AX063743 (aspartate kinase) Italinowski X57226 et al., Molecular Microbiology 5:119.7-204 (1991) lysC~R Aspartate Kinase feedback resistantsee Table 2 ( fbr) EC 2.7.2.4 (aspartate kinase fbr) lysE Lysine Exporter EP1108790 AX123539 (lysine exporter protein) W00100843 AX123539 Vrljic et al.,X96471 Molecular Microbiology 22:815-826 (1996) msiK Sugar Importer EP1108790 AX120892 (multiple sugar import protein) opcA Glucose 6-phosphate DehydrogenaseW00104325 AX076272 (subunit of glucose 6-phosphate dehydrogenase) oxyR Transcription Regulator EP1108790 AX122198 (transcriptional regulator) AX127149 ppcF$R Phosphoenol Pyruvate CarboxylaseEP0723011 feedback resistant W00100852 EC 4.1.1.31 (phosphoenol pyruvate carboxylase feedback resistant) ppc Phosphoenol Pyruvate CarboxylaseEP1108790 AX127148 EC 4.1.1.31 AX123554 (phosphoenol pyruvate carboxylase)O'Reagan et M25819 al., Gene 77(2):237-251(1989) pgk Phosphoglycerate Kinase EP1108790 AX121838 EC 2.7.2.3 AX127148 (phosphoglycerate kinase) W00100844 AX064943 Eikmanns, X59403 Journal of Bacteriology 174:6076-6086 (1992) pknA Protein Kinase A EP1108790 AX120131 (protein kinase A) AX120085 pknB Protein Kinase B EP1108790 AX120130 (protein kinase B) AX120085 pknD Protein Kinase D EP1108790 AX127150 (protein kinase D) AX122469 pknG Protein Kinase G EP1108790 AX127152 (protein kinase G) AX123109 ppsA Phosphoenol Pyruvate Synthase EP1108790 AX127144 EC 2.7.9.2 AX120700 (phosphoenol pyruvate synthase) AX122469 ptsH Phosphotransferase System ProteinEP1108790 AX122210 H

EC 2.7'.1.69 AX127149 (phosphotransferase system W00100844 AX069154 component H) ptsI Phosphotransferase System EnzymeEP1108790 AX122206 I

EC 2.7.3.9 AX127149 (phosphotransferase system enzyme I) ptsM Glukose-specific Phosphotransferase.Lee et al., L18874 System Enzyme II FEMS

EC 2.7.1.69 Microbiology (glucose phosphotransferase Letters 119 system enzyme II) (1-2):137-145 (1994) pyc Pyruvate Carboxylase W09918228 A97276 EC 6.4.1.1 Peters-WendischY09548 (pyruvate carboxylase) et al., Microbiology 144:915-927 (1998) pyc Pyruvate Carboxylase EP1108790 P458S EC 6.4.1.1 (pyruvate carboxylase) amino acid exchange P458S

sigC Sigma Factor C EP1108790 AX120368 EC 2.7.7.6 AX120085 (extracytoplasmic function alternative sigma factor C) sigD RNA Polymerase Sigma Factor EP1108790 AX120753 D

EC 2.7.7.6 AX127144 (RNA polymerase sigma factor) sigE Sigma Factor E EP1108790 AX127146 EC 2.7.7.6 AX121325 (extracytoplasmic function alternative sigma factor E) sigH Sigma Factor H EP1108790 AX127145 EC 2.7.7.6 AX120939 (sigma factor SigH) sigh Sigma Factor M EP1108790 AX123500 EC 2.7.7.6 AX127145 (sigma factor Sigh) tal Transaldolase W00104325 AX076272 EC 2.2.1.2 (transaldolase) thyA Thymidylate Synthase EP1108790 AX121026 EC 2.1.1.45 AX127145 (thymidylate synthase) tkt Transketolase Ikeda et al., AB023377 EC 2.2.1.1 NCBI

(transketolase) tpi Triose Phosphate Isomerase Eikmanns, X59403 EC 5.3.1.1 Journal of (triose phosphate isomerase) Bacteriology 174:6076-6086 (1992) zwal Cell Growth Factor 1 EP1111062 AX133781 (growth factor 1) zwf Glucose 6-phosphate 1-DehydrogenaseEP1108790 AX127148 EC 1.1.1.49 AX121827 (glucose 6-phosphate 1- W00104325 AX076272 dehydrogenase) zwf Glucose 6-phosphate 1-DehydrogenaseEP1108790 A213T EC 1.1.1.49 (glucose 6-phosphate 1-dehydrogenase) amino acid exchange A213T

Table 2 lysCF$R alleles which code for feed back resistant aspartate kinases Name of the Further Reference Access Number allele information lysCFaa-E05108 JP 1993184366-A E05108 (sequence 1) lysCFax-E06825 lysC A279T JP 1994062866-A E06825 (sequence 1) lysCFBa-E06826 lysC A279T JP 1994062866-A E06826 (sequence 2) lysCFax-E06827 JP 1994062866-A E06827 (sequence 3) lysCFax-E08177 JP 1994261766-A E08177 (sequence 1) lysCFax-E08178 lysC A279T JP 1994261766-A E08178 (sequence 2) lysCFax-E08179 lysC A279V JP 1994261766-A E08179 (sequence 3) lysCFaR-E08180 lysC 5301F JP 1994261766-A E08180 (sequence 4) lysCFaR-E08181 lysC T308I JP 1994261766-A E08181 (sequence .5) lysC~-E08182 JP 1994261766-A E08182 (sequence 6) lysCFaR-E12770 JP 1997070291-A E12770 (sequence 13) lysCFSa-E14514 JP 1997322774-A E14514 (sequence 9) lysCFaR-E16352 JP 1998165180-A E16352 (sequence 3) lysCFax-E16745 JP 1998215883-A E16745 (sequence 3) lysCF'BR-E16746 JP 1998215883-A E16746 (sequence 4) lysC~R-174588 US 5688671-A I74588 (sequence 1) lysCFaR-174589 lysC A279T US 5688671-A I74589 (sequence 2) lysCFHR_I74590 US 5688671-A ~ 174590 (sequence 7) lysCFaR-174591 lysC A279T US 5688671-A I74591 (sequence 8) lysCFaR_I74592 US 5688671-A I74592 (sequence 9) lysCFaR-174593 lysC A279T US 5688671-A I74593 (sequence 10) lysCFaR-174594 US 5688671-A I74594 (sequence 11) lysCFax-174595 lysC A279T US 5688671-A I74595 (sequence 12) lysCFaR-174596 US 5688671-A 174596 (sequence 13) lysCFHR-174597 lysC A279T US 5688671-A I74597 (sequence 14) lysCFSR-X57226 lysC S301Y EP0387527 X57226 Kalinowski et al., Molecular and General Genetics 224:317-324 (1990) lysCFHR-L16848 lysC G345D Follettie and L16848 Sinskey NCBI Nucleotide Database (1990) lysCFBR-L27125 lysC R320G Jetten et al., L27125 lysC G345D Applied Microbiology Biotechnology 43:76-82 (1995) lysCFBR lysC T311I W00063388 (sequence 17) lysCF$R lysC S301F US3732144 lysCF$R lysC S381F

lysC~R JP6261766 (sequence 1) lysC~R lysC A279T JP6261766 (sequence 2) lysCF$R lysC A279V JP6261766 (sequence 3) __ lysC S301F JP6261766 lysC~R

(sequence 4) lysC~R lysC T308I JP6261766 (sequence 5) Table 3 Target sites for integration of open reading frames, genes and alleles of lysine production Gene Description of the Ref erence Access coded name enzyme or protein Number aecD beta C-S Lyase Rossol et al., JournalM89931 EC 2.6.1.1 of Bacteriology (beta C-S lyase) 174(9):2968-77 (1992) ccpAl Catabolite Control W00100844 AX065267 Protein EP1108790 AX127147 (catabolite control protein A1) ccpA2 Catabolite Control W00100844 AX065267 Protein EP1108790 AX121594 (catabolite control protein A2) citA Sensor Kinase CitA EP1108790 AX120161 (sensor kinase CitA) _ cit8 Transcription RegulatorEP1108790 AX120163 CltB

(tranSCriptiOn regulator citB) citE Citrate Lyase W00100844 AX065421 EC 4.1.3.6 EP1108790 AX127146 (citrate lyase) fda Fructose Bisphosphate von der Osten et al.,X17323 Aldolase Molecular Microbiology EC 4.1.2.13 3(11):2625-37 (1989) (fructose 1,6-bisphosphate aldolase) gluA Glutamate Transport Kronemeyer et al., X8119 binding Protein Journal of Bacteriology (glutamate transport 177(5):1152-8 (1995) ATP-binding protein) gluB Glutamate-binding Kronemeyer et al., X81191 Protein Journal of Bacteriology (glutamate-binding 177(5):1152-8 (1995) protein) gluC Glutamate Transport Kronemeyer et al., X81191 Permease Journal of Bacteriology (glutamate transport 177(5):1152-8 (1995) system permease) gluD Glutamate Transport Kronemeyer et al., X81191 Permease Journal of Bacteriology (glutamate transport 177(5):1152-8 (1995) system permease) luxR Transcription RegulatorW00100842 AX065953 LuxR EP1108790 AX123320 (transcription regulator LuxR) luxS Histidine Kinase LuxS EP1108790 AX123323 (histidine kinase LuxS) AX127145 lysR1 Transcription RegulatorEP1108790 AX064673 LysR1 AX127144 (transcription regulator LysR1) lysR2 Transcription ActivatorEP1108790 AX123312 LysR2 (transcription regulator LysR2) lysR3 Transcription RegulatorW00100842 AX065957 LysR3 EP1108790 AX127150 (transcription regulator LysR3) menE 0-Succinylbenzoic AcidW00100843 AX064599 CoA Lipase EP1108790 AX064193 EC 6.2.1.26 AX127144 (0-succinylbenzoate CoA

lipase) mqo Malate-Quinone Molenaar et al., Eur.AJ224946 Oxidoreductase Journal of Biochemistry (malate-quinone- 1;254(2):395-403 (1998) oxidoreductase) pck Phosphoenol Pyruvate W00100844 AJ269506 Carboxykinase Ax065053 (phosphoenol pyruvate carboxykinase) pgi Glucose 6-phosphate EP1087015 AX136015 Isomerase EP1108790 AX127146 EC 5.3.1.9 (glucose 6-phosphate isomerase) poxB Pyruvate Oxidase ' W00100844 AX064959 EC 1.2.3.3 EP1096013 AX137665 (pyruvate oxidase) zwa2 Cell Growth Factor EP1106693 AX113822 (growth factor 2) EP1108790 AX127146 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, 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) 5 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 10 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 15 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 20 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 25 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, under conditions which allow expression of the said open reading frames (ORF), genes or alleles, 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 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 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, cysI~, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd, glyA, hom, homFBR, lysC, lysCF$R, metA, metB, metE, metes, metY, msiK, opcA, oxyR, ppc, ppcFBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigh, tal, thyA, tkt, tpi, zwal, zwf and zwf A213T, These are summarized and explained in Table 4.
These include, in particular, the lysCFBR alleles which code for a "feed back" resistant aspartate kinase (see Table 2) and the hom~BR alleles which code for a "feed back"
resistant homoserine dehydrogenase.
The second, optionally third or fourth copy of the opera.
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, metD, 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 Name Description of the coded enzymeReference Access or protein Number AccBC Acyl-CoA Carboxylase Jager et al. U35023 EC 6.3.4.14 Archives of (acyl-CoA carboxylase) , Microbiology (1996) 166:76-82 EP1108790; AX123524 AccDA Acetyl-CoA Carboxylase EP1055725 EC 6.4.1.2 EP1108790 AX121013 (acetyl-CoA carboxylase) W00100805 AX066443 AecD Cystathionine beta-Lyase Rossol et al., M89931 EC 4.4.1.8 Journal of (cystathionine beta-lyase) Bacteriology 174:2968-2977 (1992) CstA Carbon Starvation Protein A EP2108790 AX220811 (carbon starvation protein A) W00100804 AX066109 CysD Sulfate Adenylyltransferase EP1108790 AX123177 sub-unit II

EC 2.7.7.4 (sulfate adenylyltransferase small chain) CysE Serine Acetyltransferase EP1108790 AX122902 EC 2.3.1.30 W00100843 AX063961 (serine acetyltransferase) CysH 3'-Phosphoadenyl Sulfate ReductaseEP1108790 AX123178 EC 1.8.99.4 W00200842 AX066001 t3'-phosphoadenosine 5'-phosphosulfate reductase) CysK Cysteine Synthase EP1108790 AX122901 EC 4.2.99.8 W00100843 AX063963 (cysteine synthase) CysN Sulfate Adenylyltransferase EP1108790 AK123176 sub-unit I AX127152 EC 2.7.7.4 tsulfate adenylyltransferase)~

CysQ Transport protein CysQ EP1108790 AX127145 (transporter cysQ) W00100805 AX066423 Dps DNA Protection Protein EP1108790 AX127153 (protection during starvation protein) Eno Enolase EP1108790 AX127146 EC 4.2.1.11 W00100844 AX064945 (enolase) EP1090998 AX136862 Hermann et al., Electrophoresis 19:3217-3221 (1998) Fda Fructose Bisphosphate Aldolase van der Osten X17313 et EC 4.1.2.13 al., Molecular (fructose bisphosphate aldolase)Microbiology 3:1625-1637 (1989) Gap Glyceraldehyde 3-Phosphate EP1108790 AX127148 Dehydrogenase W00100844 AX064941 EC 1.2.1.12 Eikmanns et X59403 al., (glyceraldehyde 3-phosphate Journal of dehydrogenase) Bacteriology 174:6076-6086(1992) gap2 Glyceraldehyde 3-Phosphate EP1108790 AX127146 Dehydrogenase W00100844 AX064939 EC 1.2.1.12 (glyceraldehyde 3-phosphate dehydrogenase 2) Gdh Glutamate Dehydrogenase EP1108790 AX127150 EC 1.4.1.4 W00100844 AX063811 (glutamate dehydrogenase) Boermann et X59404 al., Molecular Microbiology 6:317-326 (1992);

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GlyA Glycine/Serine EP1108790 AX127146 Hydroxymethyltransferase AX121194 EC 2.1.2.1 (glycine/serine hydroxymethyltransferase) Gnd 6-Phosphogluconate DehydrogenaseEP1108790 AX127147 EC 1.1.1.44 ~ AX121689 (6-phosphogluconate dehydrogenase)W00100844 AX065125 Hom Homoserine Dehydrogenase Peoples et al.,Y00546 EC 1.1.1.3 Molecular (homoserine dehydrogenase) Microbiology 2:63-72 (1988) homFBRHomoserine Dehydrogenase feedbackReinscheid et resistant (fbr) al., Journal of EC 1.1.1.3 Bacteriology (homoserine dehydrogenase fbr) 173:3228-30 (1991) LysC Aspartate Kinase EP1108790 AX120365 EC 2.7.2.4 W00100844 AX063743 (aspartate kinase) Kalinowski et X57226 al., Molecular Microbiology 5:1197-204 ( 1991 ) lysC~RAspartate Kinase feedback resistantsee Table 2 ( fbr ) EC 2.7.2.4 (aspartate kinase fbr) MetA Homoserine Acetyltransferase Park et al., AF052652 EC 2.3.1.31 Molecular Cells (homoserine acetyltransferase) 8:286-94 (1998) MetB Cystathionine y-Lyase Hwang et al., AF126953 I

EC 4.4.1.1 Molecular Cells (cystathionine gamma-synthase)9:300-308 (1999) MetE Homocysteine MethyltransferaseEP1108790 AX127146 EC 2.1.1.14 ~ AX121345 (homocysteine methyltransferase) Metes Homocysteine MethyltransferaseEP1108790 AX127148 (Vitamin B12-dependent) AX121747 EC 2.1.1.14 (homocysteine methyltransferase) MetY Acetylhomoserine SulfhydrolaseEP1108790 AX120810 (acetylhomoserine sulfhydrolase) AX127145 MsiK Sugar Importer EP1108790 AX120892 (multiple sugar import protein) OpcA Glucose 6-phosphate DehydrogenaseW00104325 AX076272 (subunit of glucose 6-phosphate dehydrogenase) OxyR Transcription Regulator EP1108790 AX122198 (transcriptional regulator) AX127149 ppc~R Phosphoenol Pyruvate CarboxylaseEP0723011 feedback resistent W00100852 EC 4.1.1.31 (phosphoenol pyruvate carboxylase feedback resistant) Ppc Phosphoenol Pyruvate CarboxylaseEP1108790 AX127148 EC 4.1.1.31 AX123554 (phosphoenol pyruvate carboxylase)O'Reagan et M25819 al., Gene 77(2):237-251(1989) Pgk Phosphoglycerate Kinase EP1108790 AX121838 EC 2.7.2.3 AX127148 (phosphoglycerate kinase) W00100844 AX064943 Eikmanns, X59403 Journal of Bacteriology 174:6076-6086 (1992 ) PknA Protein k~inase A EP1108790 AX120131 (protein kinase A) AX120085 PknB Protein Kinase B EP1108790 AX120130 (protein kinase B) AX120085 PknD Protein Kinase D EP1108790 AX127150 (protein Kinase D) AX122469 PknG Protein Kinase G EP1108790 AX127152 (protein kinase G) AX123109 PpsA Phosphoenol Pyruvate Synthase EP1108790 AX127144 EC 2.7.9.2 AX120700 (phosphoenol pyruvate synthase) AX122469 PtsH Phosphotransferase System ProteinEP1108790 AX122210 H

EC 2.7.1.69 AX127149 (phosphotransferase system W00100844 AX069154 component H) PtsI Phosphotransferase System EnzymeEP1108790 AX122206 I

EC 2.7.3.9 AX127149 (phosphotransferase system enzyme I) PtsM Glucose-specific PhosphotransferaseLee et al., L18874 FEMS

System Enzyme II Microbiology EC 2.7.1.69 Letters 119 (glucose phosphotransferase (1-2):137-145 system enzyme II) (1994) Pyc Pyruvate Carboxylase W09918228 A97276 EC 6.4.1.1 Peters-WendischY09548 (pyruvate carboxylase) et al., Microbiology 144:915-927 (1998) Pyc Pyruvate Carboxylase EP1108790 P458s EC 6.4.1.1 (pyruvate carboxylase) amino acid exchange P458S

SigC Sigma Factor C EP1108790 AX120368 EC 2.7.7.6 AX120085 (extracytoplasmic function alternative sigma factor C) SigD RNA Polymerase Sigma Factor EP1108790 AX120753 D

EC 2.7.7.6 AX127144 (RNA polymerase sigma factor) SigE Sigma Factor E EP1108790 AX127146 EC 2.7.7.6 AX121325 (extracytoplasmic function alternative sigma factor E) SigH Sigma Factor H EP1108790 AX127145 EC 2.7.7.6 AX120939 (sigma factor SigH) Sigh Sigma Factor M EP1108790 AX123500 EC 2.7.7.6 AX127153 (sigma factor Sigh) Tal Transaldolase W00104325 AX076272 EC 2.2.1.2 (transaldolase) ThyA Thymidylate Synthase EP1108790 AX121026 EC 2.1.1.45 AX127145 (thymidylate synthase) Tkt Transketolase Ikeda et al., AB023377 EC 2.2.1.1 NCBI

(transktolase) Tpi Triose Phosphate Isomerase Eikmanns, X59403 EC 5.3.1.1 Journal of (triose phosphate isomerase) Bacteriology 174:6076-6086 (1992) zwal Cell Growth Factor 1 EP1111062 AX133781 (growth factor 1) Zwf Glucose 6-phosphate 1-DehydrogenaseEP1108790 AX127148 EC 1.1.1.49 AX121827 (glucose 6-phosphate 1- W00104325 AX076272 dehydrogenase) Zwf Glucose 6-phosphate 1-DehydrogenaseEP1108790 A213T EC 1.1.1.49 (glucose 6-phosphate 1-dehydrogenase) amino acid exchange A213T

Table 5 Target sites for integration of open reading frames, genes and alleles of methionine production Gene Description of the Reference Access name coded enzyme or protein Number BrnE Transporter of EP1096010 AX137709 branched-chain amino AX137714 acids (branched-chain amino acid transporter) BrnF Transporter of EP1096010 AX137709 branched-chain amino AX137714 acids (branched-chain amino acid transporter) BrnQ Carrier protein of Tauch et al., ArchivesM89931 branched-chain amino of Microbiology AX066841 acids 169(4):303-12 (1998) AX127150 (branched-chain aminoW00100805 acid transport systemEP1108790 carrier protein) ccpA1 Catabolite Control W00100844 AX065267 Protein EP1108790 AX127147 (catabolite control protein A1) ccpA2 Catabolite Control W00100844 AX065267 Protein EP1108790 AX121594 (catabolite control protein A2) citA Sensor Kinase CitA EP1108790 AX120161 (sensor kinase CitA) citB Transcription RegulatorEP1108790 AX120163 CitB

(transcription regulator CitB) citE Citrate Lyase W00100844 AX065421 EC 4.1.3.6 EP1108790 AX127146 (citrate lyase) ddh Diaminopimelate Ishino et al., Nucleic507384 Dehydrogenase Acids Research 15: AX127152 EC 1.4.1.16 (1987) (diaminopimelate EP1108790 dehydrogenase) gluA Glutamate Transport Kronemeyer et al., X81191 ATP-binding Protein Journal of Bacteriology (glutamate transport 177(5):1152-8 (1995) ATP-binding protein) gluB Glutamate-binding Kronemeyer et al., X81191 Protein Journal of Bacteriology (glutamate-binding 177(5):1152-8 (1995) protein) gluC Glutamate Transport Kronemeyer et al., X81191 Permease Journal of Bacteriology (glutamate transport 177(5):1152-8 (1995) system permease) gluD Glutamate Transport Kronemeyer et al., X81191 Permease Journal of Bacteriology (glutamate transport177(5):1152-8 (1995) system permease) luxR Transcription RegulatorW00100842 AX065953 LuxR EP1108790 AX123320 (transcription regulator LuxR) luxS Histidine Kinase EP1108790 AX123323 LuxS

(histidine kinase AX127145 LuxS) lysR1 Transcription RegulatorEP1108790 AX064673 LysR1 AX127144 (transcription regulator LysR1) lysR2 Transcription ActivatorEP1108790 AX123312 LysR2 (transcription regulator LysR2) lysR3 Transcription RegulatorW00100842 AX065957 LysR3 EP1108790 AX127150 (transcription regulator LysR3) menE 0-Succinylbenzoic W00100843 AX064599 Acid CoA Lipase EP1108790 AX064193 EC 6.2.1.26 AX127144 (0-succinylbenzoate CoA

lipase) metD Transcription RegulatorEP1108790 AX123327 MetD AX127153 (transcription regulator MetD) metK Methionine Adenosyl W00100843 AX063959 Transferase EP1108790 AX127148 EC 2.5.1.6 (S-adenosylmethionine synthetase) pck Phosphoenol PyruvateW00100844 AJ269506 Carboxykinase AX065053 (phosphoenol pyruvate carboxykinase) pgi Glucose 6-Phosphate EP1087015 AX136015 Isomerase EP1108790 AX127146 EC 5.3.1.9 (glucose-6-phosphate isomerase) poxB Pyruvate Oxidase W00100844 AX064959 EC 1.2.3.3 EP1096013 AX137665 (pyruvate oxidase) zwa2 Cell Growth Factor EP1106693 AX113822 (growth factor 2) EP1108790 AX127146 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, cysI, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd, hom, homFBR, lysC, lysCFBR, msiK, opcA, oxyR, ppc, ppcFBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigh, 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 lysCFBR alleles which code for a "feed back"
resistant aspartate kinase (See Table 2) and the homF$R
alleles which code for a "feed back" resistant homoserine dehydrogenase.
The second, optionally third or fourth copy of the open reading frame (0RF), 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 ~.-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 5 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 Name Description of the coded enzymeReference Access or protein Number accBC Acyl-CoA Carboxylase Jager et al. U35023 EC 6.3.4.14 Archives of (acyl-CoA carboxylase) Microbiology 166:76-82 (1996) accDA Acetyl-CoA Carboxylase EP1055725 EC 6.4.1.2 EP1108790 AX121013 (acetyl-CoA carboxylase) W00100805 AX066443 cstA Carbon Starvation Protein A EP1108790 AX120812 (carbon starvation protein A) W00100804 AX066109 cysD Sulfate Aderiylyltransferase EP1108790 AX123177 sub-unit II

EC 2.7.7.4 (sulfate adenylyltransferase small chain) cysE Serine Acetyltransferase EP1108790 AX122902 EC 2.3.1.30 W00100843 AX063961 (serine acetyltransferase) cysH 3'-Phosphoadenyl Sulfate ReductaseEP1108790 AX123178 EC 1.8.99.4 WO0100842 AX066001 (3'-phosphoadenosine 5'-phosphosulfate reductase) cysK Cysteine Synthase EP1108790 AX122901 EC 4.2.99.8 W00100843 AX063963 (cysteine synthase) cysN Sulfate Adenylyltransferase EP1108790 AX123176 sub-unit I AX127152 EC 2.7.7.4 (sulfate adenylyltransferase) cysQ Transport protein CysQ EP1108790 AX127145 (transporter cysQ) W00100805 AX066423 dps DNA Protection Protein EP1108790 AX127153 (protection during starvation protein) eno Enolase EP1108790 AX127146 EC 4.2.2.11 W00100844 AX064945 (enolase) EP1090998 AX136862 Hermann et al., Electrophoresis 19:3217-3221 (1998) fda Fructose Bisphosphate Aldolase van der Osten X17313 et EC 4.1.2.13 al., Molecular (fructose bisphosphate aldolase)Microbiology 3:1625-1637 (1989) gap Glyceraldehyde 3-Phosphate EP11fl8790 AX127148 Dehydrogenase W00100844 AX064942 EC 1.2.1.12 Eikmanns et X59403 al., (glyceraldehyde 3-phosphate Journal of dehydrogenase) Bacteriology 174:6076-6086(1992) gap2 Glyceraldehyde 3-Phosphate EP1108790 AX127146 Dehydrogenase W00100844 AX064939 EC 1.2.1.12 (glyceraldehyde 3-phosphate dehydrogenase 2) gdh Glutamate Dehydrogenase EP1108790 AX127150 EC 1.4.1.4 W00100844 AX063811 (glutamate dehydrogenase) Boermann et X59404 al., Molecular Microbiology 6:317-326 (1992);

Guyonvarch et X72855 al., NCBI

gnd 6-Phosphogluconate DehydrogenaseEP1108790 AX127147 EC 1.1.1.44 AX121689 (6-phosphogluconate dehydrogenase)W00100844 AX065125 hom Homoserine Dehydrogenase Peoples et al.,Y00546 EC 1.1.1.3 Molecular (homoserine dehydrogenase) Microbiology 2:63-72 (1988) hom~R Homoserine Dehydrogenase feedbackReinscheid et resistant (fbr) al., Journal of EC 1.1.1.3 Bacteriology (homoserine dehydrogenase fbr)173:3228-30 (1991) lysC Aspartate Kinase EP1108790 AX120365 EC 2.7.2.4 W00100844 AX063743 (aspartate kinase) Kalinowski et X57226 al., Molecular Microbiology 5:1197-204 (1991) lysC~R Aspartate Kinase feedback resistentsee Table 2 ( fbr) EC 2.7.2.4 (aspartate kinase fbr) msiK Sugar Importer EP1108790 AX120892 (multiple sugar import protein) opcA Glucose 6-Phosphate DehydrogenaseW00104325 AX076272 (subunit of glucose 6-phosphate dehydrogenase) oxyR Transcription Regulator EP1108790 AX122198 (transcriptional regulator) AX127149 ppc~R Phosphoenol Pyruvate CarboxylaseEP0723011 feedback resistent W00100852 EC 4.1.1.31 (phosphoenol pyruvate carboxylase feedback resistant) ppc Phosphoenol Pyruvate CarboxylaseEP1108790 AX127148 EC 4.1.1.31 AX123554 (phosphoenol pyruvate carboxylase)0'Reagan et M25819 al., Gene 77(2):237-251(1989) pgk Phosphoglycerate Kinase EP1108790 AX121838 EC 2.7.2.3 AX127148 (phosphoglycerate kinase) W00100844 AX064943 Eikmanns, X59403 Journal of Bacteriology 174:6076-6086 (1992) pknA Protein Kinase A EP1108790 AX120131 (protein kinase A) AX120085 pknB Protein Kinase B EP1108790 AX120130 (protein kinase B) AX120085 pknD Protein Kinase D EP1108790 'AX127150 (protein kinase D) AX122469 pknG Protein Kinase G EP1108790 AX127152 (protein kinase G) AX123109 ppsA Phosphoenol Pyruvate Synthase EP1108790 AX127144 EC 2.7.9'.2 ' AX120700 (phosphoenol pyruvate synthase) AX122469 ptsH Phosphotransferase System ProteinEP1108790 AX122210 H

EC 2.7.1.69 AX127149 (phosphotransferase system WOU100844 AX069154 component H) ptsI Phosphotransferase System EnzymeEP1108790 AX122206 I

EC 2.7.3.9 ~ AX127149 (phosphotransferase system enzyme I) ptsM Glukose-specific PhosphotransferaseLee, et al., L18874 FEMS

System Enzyme II Microbiology EC 2.7.1.69 Letters 119 (glucose phosphotransferase-system(1-2):137-145 enzyme II) (1994) pyc Pyruvate Carboxylase W09918228 A97276 EC 6.4.1.1 Peters-WendischY09548 (pyruvate carboxylase) et al., Microbiology 144:915-927 (1998) pyc Pyruvate Carboxylase EP1108790 P458S EC 6.4.1.1 (pyruvate carboxylase) amino acid exchange P458S

sigC Sigma Factor C EP1108790 AX120368 EC 2.7.7.6 AX120085 (extracytoplasmic function alternative sigma factor C) sigD RNA Polymerase Sigma Factor EP1108790 AX120753 D

EC 2.7.7.6 AX127144 (RNA polymerase sigma factor) sigE Sigma Factor E EP1108790 AX127146 EC 2.7.7.6 AX121325 (extracytoplasmic function alternative sigma factor E) sigH Sigma Factor H EP1108790 AX127145 EC 2.7.7.6 A'X120939 (sigma factor SigH) sigh Sigma Factor M EP1108790 AX123500 EC 2.7.7.6 AX127153 (sigma factor Sigh) tal Transaldolase W00104325 AX076272 EC 2.2.1.2 (transaldolase) thrB Homoserine Kinase Peoples et al.,Y00546 EC 2.7.1.39 Molecular (homoserine kinase) Microbiology 2:63-72 (1988) thrC Threonine Synthase Han et al., X56037 EC 4.2.99.2 Molecular (threonine synthase) Microbiology 4:1693-1702 (1990) thrE Threonine Exporter EP1085091 AX137526 (threonine export carrier) thyA Thymidylate Synthase EP1108790 AX121026 EC 2.1.1.45 AX127145 (thymidylate synthase) tkt Transketolase Ikeda et al., AB023377 EC 2.2.1.1 NCBI

(transketolase) tpi Triose phosphate Isomerase Eikmanns, X59403 EC 5.3.1.1 Journal of (triose phosphate isomerase) Bacteriology 174:6076-6086 (1992) zwal Cel1 Growth Factor 1 EP1111062 AX133781 (growth factor 1) zwf Glucose 6-Phosphate 1-DehydrogenaseEP1108790 AX127148 EC 1.1.1.49 AX121827 (glucose 6-phosphate 1- W00104325 AX076272 dehydrogenase) zwf Glucose 6-Phosphate 1-DehydrogenaseEP1108790 A213T EC 1.1.1.49 (glucose 6-phosphate 1-dehydrogenase) amino acid exchange A213T

Table 7 Target sites for integration of open reading frames, genes and alleles of threonine production Gene Description of the codedReference Access name enzyme or protein Number ccpA1 Catabolite Control W00100844 AX065267 Protein EP1108790 AX127147 (catabolite control protein A1) ccpA2 Catabolite Control W00100844 AX065267 Protein EP1108790 AX121594 (catabolite control protein A2) citA Sensor Kinase CitA EP1108790 AX120161 (sensor kinase CitA) citB Transcription RegulatorEP1108790 AX120163 CitB

(transcription regulator CitB) citE Citrate Lyase W00100844 AX065421 EC 4.1.3.6 EP1108790 AX127146 (citrate lyase) ddh Diaminopimelate Ishino et al., NucleicS07384 Dehydrogenase Acids Research 15: AX127152 EC 1.4.1.16 . (1987) (diaminopimelate EP1108790 dehydrogenase) gluA Glutamate Transport Kronemeyer et al., X81191 ATP-binding Protein Journal of Bacteriology (glutamate transport 177(5):1152-8 (1995) ATP-binding protein) gluB Glutamate-binding ProteinKronemeyer et al., X81191 (glutamate-binding Journal of Bacteriology protein) 177(5):1152-8 (1995) gluC Glutamate Transport Kronemeyer et al., X81191 Permease Journal of Bacteriology (glutamate transport 177(5):1152-8 (1995) system permease) ,gluD Glutamate Transport Kronemeyer et al., X82191 Permease Journal of Bacteriology (glutamate transport 177(5):1152-8 (1995) system permease) glyA Glycine WO0100843 AX063861 Hydroxymethyltransferase AF327063 EC 2.1.2.1 (glycine hydroxymethyltransferase) ilvA Threonine Dehydratase Mockel et al., JournalA47044 EC 4.2.1.16 of Bacteriology 174 L01508 (threonine dehydratase)(24), 8065-8072 (1992)AX127150 ilvBN Acetolactate Synthase Keilhauer et al., L09232 EC 4.1.3.18 Journal of Bacteriology (acetolactate synthase)175(17):5595-603 (1993) ilvC Reductoisomerase Keilhauer et al., C48648 EC 1.1.1.86 Journal of BacteriologyAX127147 (ketol-acid 175(17):5595-603 (1993) reductoisomerase) EP1108790 ilvD Dihydroxy-acid EP1006189 AX136925 Dehydratase EC 4.2.1.9 (dihydroxy-acid dehydratase) luxR Transcription RegulatorW00100842 AX065953 LuxR EP1108790 AX123320 (transcription regulator LuxR) luxS Histidine Kinase LuxS EP1108790 AX123323 (histidine kinase LuxS) AX127153 lysR1 Transcription RegulatorEP1108790 AX064673 LysR1 AX127144 (transcription regulator LysR1) lysR2 .Transcription ActivatorEP1108790 AX123312 LysR2 (transcription regulator LysR2) lysR3 Transcription RegulatorW00100842 AX065957 LysR3 EP1108790 AX127150 (transcription regulator LysR3) mdh Malate Dehydrogenase W00100844 AX064895 EC 1.1.1.37 (malate dehydrogenase) menE O-Succinylbenzoic AcidW00100843 AX064599 CoA Ligase EP1108790 AX064193 EC 6.2.1.26 AX127144 (O-succinylbenzoate CoA

ligase) metA Homoserine 0- Park et al., MolecularAX063895 Acetyltransferase Cells 30;8(3):286-94 AX127145 EC 2.3.1.31 (1998) (homoserine 0- W00100843 acetyltransferase) EP1108790 metD Transcription RegulatorEP1108790 AX123327 MetD AX127153 (transcription regulator MetD) pck Phosphoenol Pyruvate W00100844 AJ269506 Carboxykinase AX065053 (phosphoenol pyruvate carboxykinase) poxB Pyruvate Oxidase W00100844 AX064959 EC 1.2.3.3 EP1096013 AX137665 (pyruvate oxidase) sigB RNA Polymerase EP1108790 AX127149 Transcription Factor (RNA polymerase transcription factor) zwa2 Cell Growth Factor 2 EP1106693 AX113822 (growth factor 2) EP1108790 AX127146 The invention accordingly also provides a process for the production of coryneform bacteria which produce L-methionine and/or L-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, b) providing the 5' and the 3' end of the ORF, gene or 20 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 oflenables 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 L-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, under conditions which allow expression of the said open reading frames (ORF), genes or alleles, 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 msiK, pgk, ptsH, ptsl, ptsM, sigC, sigD, sigE, sigH, sigh, 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, pang, panC, poxB and zwa2. These are summarized and explained in Table 9.
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 5 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 8 Open reading frames, genes and alleles of valine production Name Description of the coded enzymeReference Access or protein Number brnEF Export of branched-chain amino EP1096010 acids (branched chain amino acid export)Kennerknecht AF454053 et al., NCBI

cstA Carbon Starvation Protein A EP1108790 AX120811 (carbon starvation protein A) W00100804 AX066109 dps DNA Protection Protein EP1108790 AX127153 (protection during starvation protein) eno Enolase EP1108790 AX127146 EC 4.2.1.12 W00100844 AX064945 (enolase) EP1090998 AX136862 Hermann et al., Electrophoresis 19:3217-3221 (1998) fda Fructose Bisphosphate Aldolase van der Osten X17313 et EC 4.1.2.13 al., Molecular (fructose bisphosphate aldolase)Microbiology 3:1625-1637 (1989) gap Glyceraldehyde 3-Phosphate EP1108790 AX127148 Dehydrogenase W00100844 AX064941 EC 1.2.1.22 Eikmanns et X59403 al., (glyceraldehyde 3-phosphate Journal of dehydrogenase) Bacteriology 174:6076-6086(1992) gap2 Glyceraldehyde 3-Phosphate EP1108790 AX127146 Dehydrogenase W00100844 AX064939 EC 1.2.1.12 (glyceraldehyde 3-phosphate dehydrogenase 2) gdh Glutamate Dehydrogenase EP1108790 AX127150 EC 1.4.1.4 W00100844 AX063811 (glutamate dehydrogenase) Boermann et X59404 al., Molecular Microbiology 6:317-326 (1992);

Guyonvarch et X72855 al., NCBI

ilvBN Acetolactate Synthase Keilhauer et L09232 EC 4.1.3.18 al., Journal of (aeetolactate synthase) Bacteriology 175(17):5595-603 (1993) ilvC Isomeroreductase Keilhauer et C48648 EC 1.1,1.86 al., Journal AX127147 of (acetohydroxy acid Bacteriology isomeroreductase) 175(17):5595-603 (1993) ilvD Dihydroxy-acid Dehydratase EP1006189 AX136925 EC 4.2.1.9 (dihydroxy acid dehydratase) ilvE Transaminase B EP1108790 AX127150 EC 2.6.1.42 AX122498 (transaminase B) msiK Sugar Importer EP1108790 AX120892 (multiple sugar import protein) pgk Phosphoglycerate Kinase EP1108790 AX121838 EC 2.7.2.3 AX127148 (phosphoglycerate kinase) W00100844 AX064943 Eikmanns, X59403 Journal of Bacteriology 174:6076-6086 (1992) ptsH Phosphotransferase System ProteinEP1108790 AX122210 H

EC 2.7.1.69 AX127149 (phosphotransferase system W00100844 AX069154 component H) ptsI Phosphotransferase System EnzymeEP1108790 AX122206 I

EC 2.7.3.9 AX127149 (phosphotransferase system enzyme I ) ptsM Glucose-specific PhosphotransferaseLee et al., L18874 FEMS

System Enzyme II Microbiology EC 2.7.1.69 ' Letters 119 (glucose phosphotransferase-system(1-2):137-145 enzyme II) (1994) sigC Sigma Factor C EP1108790 AX120368 EC 2.7.7.6 AX120085 (extracytoplasmic function alternative sigma factor C) sigD RNA Polymerase Sigma Factor EP1108790 AX120753 D

EC 2.7.7.6 AX127144 (RNA polymerase sigma factor) sigE Sigma Factor E EP1108790 AX127146 EC 2.7.7.6 AX121325 (extracytoplasmic function alternative sigma factor E) sigH Sigma Factor H EP1108790 AX127145 EC 2.7.7.6 AX120939 (sigma factor SigH) sigh Sigma Factor M EP1108790 AX123500 EC 2.7.7.6 AX127153 (sigma factor Sigh) tpi Triose Phosphate Isomerase Eikmanns, X59403 EC 5.3.1.1 Journal of (triose phosphate isomerase) Bacteriology 174:6076-6086 (1992) zwal Cell Growth Factor 1 EP1111062 AX133781 (growth factor 1) Table 9 Target sites for integration of open reading frames, genes and alleles of valine production Gene Description of the codedReference Access name enzyme or protein Number aecD beta C-S Lyase Rossol et al., JournalM89931 EC 2.6.1.1 of Bacteriology (beta C-S lyase) 174(9):2968-77 (1992) ccpA1 Catabolite Control W00100844 AX065267 Protein EP1108790 AX127147 (catabolite control protein A1) ccpA2 Catabolite Control W00100844 AX065267 Protein EP1108790 AX121594 (catabolite control protein A2) citA Sensor Kinase CitA EP1108790 AX120161 (sensor kinase CitA) citB Transcription RegulatorEP1108790 AX120163 CitB

(transcription regulator CitB) citE Citrate Lyase Wo0100844 AX065421 EC 4.1.3.6 EP1108790 AX227146 (citrate lyase) ddh Diaminopimelate Ishino et al., Nucleic507384 Dehydrogenase Acids Research 15: AX227152 EC 1.4.1.16 (1987) (diaminopimelate EP1108790 dehydrogenase) gluA Glutamate Transport Kronemeyer et al., X81191 ATP-binding Protein Journal of Bacteriology (glutamate transport 177(5):1152-8 (1995) ATP-binding protein) gluB Glutamate-binding ProteinKronemeyer et al., X81191 (glutamate-binding Journal of Bacteriology protein) 177(5):1152-8 (1995) gluC Glutamate Transport Kronemeyer et al., X81191 Permease Journal of Bacteriology (glutamate transport 177(5):1152-8 (1995) system permease) gluD Glutamate Transport Kronemeyer et al., X81292 Permease Journal of Bacteriology (glutamate transport 177(5):1152-8 (2995) system permease) glyA Glycine W00100843 AX063861 Hydroxymethyltransferase AF327063 EC 2.1.2,2 (glycine hydroxyme thyl t-rans f erase ) ilvA Threonine Dehydratase Mockel et al., JournalA47044 EC 4.2.1.16 of Bacteriology 174 La1508 (threonine dehydratase)(24), 8065-8072 (1992)AX127150 luxR Transcription RegulatorW00100842 AX065953 LuxR EP1108790 AX123320 (transcription regulator LuxR) lysR1 Transcription RegulatorEP1108790 AX064673 LysR1 AX127144 (transcription regulator LysR1) lysR2 Transcription ActivatorEP1108790 AX123312 LysR2 (transcription regulator LysR2) lysR3 Transcription RegulatorW00100842 AX065957 LysR3 EP1108790 AX127150 (transcription regulator LysR3 ) pang Ketopantoate US6177264 X96580 Hydroxymethyltransferase EC 2. 1. 2. 11 (ketopantoate hydroxymethyltransferase) panC Pantothenate SynthetaseUS6177264 X96580 EC 6.3.2.1 (pantothenate synthetase) poxB Pyruvate Oxidase WOOi00844 AX064959 EC 1.2.3.3 EP1096013 AX137665 (pyruvate oxidase) zwa2 Cell Growth Factor EP1106693 AX113822 (growth factor 2) EP1108790 AX127146 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 andlor regulation signals, b) providing the 5' and the 3' end of the ORF, gene or 1p 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 5 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 10 transposition and no nucleotide sequence which imparts resistance to antibiotics remaining at the target site.
During work on the present invention, it was possible to incorporate a second copy of an lysCFBR allele into the gluB
15 gene of Corynebacterium glutamicum such that no nucleotide sequence which is capable oflenables 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 20 site. This strain, which is called DSM13994g1u::lysC, carries the lysCFBR allele lysC T311I at its natural lysC
site and a second copy of the lysCF$R allele lysC T311I at a second site (target site), namely the gluB gene. A plasmid with the aid of which the incorporation of the lysCFBR
25 allele into the gluB gene can be achieved is shown in Figure 1. It carries the name pKl8mobsacBglul 1.
During work on the present invention, it was furthermore possible to incorporate a copy of an lysCFBR allele into the target site of the gluB gene of Corynebacterium glutamicum 30 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 DSM12866g1u::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 lysCFBR allele lysC T311I at a second site (target site), namely the gluB gene. It has been deposited under number DSM15039 at the Deutsche Sammlung fur Mikroorganismen and Zellkulturen (German Collection of Microorganisms and Cell Cultures). A plasmid with the aid of which the incorporation of the lys~CFBR allele into the gluB gene can be achieved is shown in Figure 1. It carries the name pKl8mobsacBglul 1.
During work on the present invention, it was furthermore possible to incorporate a copy of an lysCFBR 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 lysCFBR allele lysC T311I at a second site (target site), namely the aecD gene. A plasmid with the aid of which the incorporation of the lysCFBR allele into the aecD gene can be achieved is shown in Figure 2. It carries the name pKl8mobsacBaecD1 1.
During work on the present invention, it was furthermore possible to incorporate a copy of an lySCF~R 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 DSM12866pck::lysC, carries the wild-type form of the lysC gene at its natural l.ysC site and a second copy of the lysC gene in the form of the lysCF$~ 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 1.
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 DSM12866g1u::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 1.
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 1.
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 DSM12866pck::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 pKl8mobsacBpckl_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 (Biopro~esstechnik 1. Einfiihrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren and 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.
An.tifoams, 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 5 culture is usually 20~C to 45~C, and preferably 25pC to 40°-C. 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 10 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.
15 The following microorganisms have been deposited:
The strain Corynebacterium glutamicum DSM12866g1u::lysC was deposited in the form of a pure culture on 5th June 2002 under number DSM15039 at the Deutsche Sammlung fu.r Mikroorganismen and Zellkulturen (DSMZ = German Collection 20 of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty.
The plasmid pKl8mobsacBglul 1 was deposited in the form of a pure culture of the strain E. coli DH5amcr/pKl8mobsacBglul 1 (_ 25 DHSalphamcr/pKl.BmobsacBglul_1) on 20th April 2001 under number DSM14243 at the Deutsche Sammlung fur Mikroorganismen and Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.
The plasmid pKl8mobsacBaecD1_1 was deposited in the form of 30 a pure culture of the strain E. coli DHSamcr/pKl8mobsacBaecD1_1 (_ DHSalphamcr/pKl8mobsacBaecD1 1) on 5th June 2002 under number DSM15040 at the Deutsche Sammlung fur Mikroorganismen and Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.
Example 1 Incorporation of a second copy of the lysCFBR allele into the chromosome of the strain DSM13994 and of the strain 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 lysCF$R allele of this strain is shown as SEQ ID
N0:3. It is also called lysC T311I in the following. The amino acid sequence of the aspartate kinase protein coded is shown as SEQ ID N0:4. A pure culture of this strain was deposited on 16th January 2001 at the Deutsche Sammlung fur Mikroorganismen and 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 N0:2. A pure culture of this strain was deposited on 10th June 1999 at the Deutsche Sammlung fur Mikroorganismen and 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 ~i-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 Biosystems (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 N0:4.
The base thymine is found at position 932 of the nucleotide sequence of the coding region of the lysCFBR allele of strain DSM13994 (SEQ ID N0:3). The base cytosine is found at the corresponding position of the wild-type gene (SEQ ID
N0:1) .
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 lysCFBR allele or lysC T311I.in the following.

The plasmid pCRIITOPOlysC, which carries the lysCFBR 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 and Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.
1.2 Construction of the replacement vector pKl8mobsacBglul_1 The Cor'ynebacterium 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 N0: 7):
5~ TA(A GAT CT)G TGT TGG ACG TCA TGG CAA G 3~
gluBgl2 (SEQ ID N0: 8):
5~ AC(A GAT CT)T GAA GCC AAG TAC GGC CAA G 3~
The primers shown are synthesized by MTnIG 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 BgIII, which is marked by parentheses in the nucleotide sequence shown above.
5 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).
10 Ligation of the fragment is then carried out by means of the TOPO TA Cloning Kit (Invitrogen, Leek, The Netherlands, Cat. Number K4600-O1) in the vector pCRII-TOPO. The ligation batch is transformed in the E. coli strain TOP10 (Invitrogen, Leek, The Netherlands). Selection of plasmid-15 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 ~i-D-galactopyranoside, 64 mg/1).
The plasmid obtained is checked by means of restriction 20 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 BglII (Amersham-Pharmacia, Freiburg, Germany) and after separation in an agarose gel (0.8~) with the aid of 25 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 mobilizable cloning vector pKl8mobsacB described by Schafer et a1.
(Gene 14: 69-73 (1994)), This is cleaved beforehand with 30 the restriction enzyme BamHI and dephosphoryl.ated 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 Lipase (Amersham-Pharmacia, Freiburg, Germany).

The E. coli strain DH5a, (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. 2nd 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 pKl8mobsacBglul.
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 lysCF$R-containing DNA fragment of approx. 1.7 kb in length was isolated from the agarose gel and employed for ligation with the vector pKl8mobsacBglul described above. This is cleaved beforehand with the restriction enzyme BamHI, dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany), mixed with the lysCF~R fragment of approx. 1.7 kb and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany).
The E. coli strain DH5amcr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol.
1, ILR-Press, Cold Spring Harbor, New York, 1989).
Selection of plasmid-carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratflry Manual. 2nd Ed., Cold Spring Harbor, New York, 1989), which was supplemented with 50 mg/1 kanamycin.
Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis. The plasmid is called pKlBmobsacBglul 1. A
map of the plasmid is shown in Figure 1.
The plasmid pKl8mobsacBglul 1 was deposited in the form of a pure culture of the strain E. coli DHSamcr/pKl8mobsacBglul_1 (_ DH5alphamcr/pKl8mobsacBglul 1) under number DSM14243 on 20.04.2001 at the Deutsche Sammlung fur Mikroorganismen and zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.
1.3 Incorporation of a second copy of the lysCF$R allele lysC T311I into the chromosome (target site: gluB
gene) of the strain DSM13994 by means of the replacement vector pKl8mobsacBglul_1 The vector pKl8mobsacBglul 1 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 1 is made by plating out the conjugation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual.
2nd 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_1, like the starting plasmid pKl8mobsacB, 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 pKlBmobsacBglul_1 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 lysCFBR 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 N0: 7):
5~ TA(A GAT CT)G TGT TGG ACG TCA TGG CAA G 3~
gluBgl2 (SEQ ID N0: 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 lysCFBR 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 0.8o agarose gel.
A clone which, in addition to the copy present at the lysC
locus, has a second copy of the lysCF~ allele lysC T311I at the gluB locus in the chromosome was identified in this manner. This clone was called strain DSM13994g1u::lysC.
1.4 Incorporation of a second copy of the lysC gene in the form of the lysCF$R allele lysC T311I into the chromosome (target site: gluB gene) of the strain DSM12866 by means of the replacement vector pKl8mobsacBglul 1 As described in Example 1.3, the plasmid pKl8mobsacBglul 1 is transferred into the C. glutamicum strain DSM12866 by conjugation. A clone which, in addition to the copy of the wild-type gene present at the lysC locus, has a second copy of the lysC gene in the form of the lysCFBR allele lysC
T311I at the gluB locus in the chromosome was identified in the manner described in 1.3. This clone was called strain DSM12866g1u::lysC.
The Corynebacterium glutamicum strain according to the invention which carries a second copy of an lySCF$R allele in the gluB gene was deposited in the form of a pure culture of the strain Corynebacterium glutamicum DSM12866g1u::lysC on 5th June 2002 under number DSM15039 at the Deutsche Sammlung fur Mikroorganismen and Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.

1.5 'Construction of the replacement vector pKl8mobsacBpckl_1 The Corynebacterium glutamicum strain ATCC13032 is used as the donor for the chromosomal DNA. From the strain 5 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 10 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:
15 pck_beg (SEQ ID N0: 9):
5~ TA(A GAT~CT) G CCG GCA TGA CTT CAG TTT 3~
pck_end (SEQ ID N0: 10):
5~ AC(A GAT CT) G GTG GGA GCC TTT CTT GTT ATT3 The primers shown are synthesized by MWG Biotech and the 20 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 25 primers moreover contain the sequence for the cleavage site of the restriction endonuclease BglII, 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 30 identified by means of electrophoresis in a 0.8o 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 BglII (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 pKlBmobsacB 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 DHSo~ (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. 2nd 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 pKl8mobsacBpckl.
Plasmid DNA was isolated from the strain DSM14242 (see Example 1.1), which carries the plasmid pCRIITOP0IysC, and cleaved with the restriction enzyme BamHI (Amersham-Pharmacia, Freiburg, Germany), and after separation in an agarose gel (0.8o) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the lysCF$R-containing DNA fragment approx. 1.7 kb long was isolated from the agarose gel and employed for ligation with the vector pKl8mobsacBpckl described above. This is cleaved beforehand with the restriction enzyme BamHI, 15~ dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany), mixed with the lysCFBR fragment of approx. 1.7 kb and the mixture is treated with T4 DNA Lipase (Amersham-Pharmacia, Freiburg, Germany).
The E. coli strain DH5amcr (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. 2nd 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 pKl8mobdsacBpckl 1.
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 lysCF$R allele lysC T311I into the chromosome (target site: pck gene) of the strain DSM12866 by means of the replacement vector pKl8mobsacBpckl_1 As described in Example 1.3, the plasmid pKl8mobsacBpckl_1.
described in Example 1.5 is transferred into the C.
glutamicum strain DSM12866 by conjugation. Selection is made for targeted recombination events in the chromosome of C. glutamicum DSM12866 as described in Example 1.3.
Depending on the position of the second recombination event, after the excision the second copy of the lysCFBR
allele manifests itself in the chromosome at the pck locus, or the original pck locus of the host remains.
Approximately 40 to 50 colonies are tested for the phenotype "growth in the presence of sucrose" and "non-growth in the presence of kanamycin". Approximately 20 colonies which show the phenotype "growth in the presence of sucrose" and "non-growth in the presence of kanamycin"
are investigated with the aid of the polymerase chain reaction. A DNA fragment which carries the pck gene and surrounding regions is amplified here from the chromosomal DNA of the colonies. The same primer oligonucleotides as are described in Example 1.5 for the construction of the integration plasmid are chosen for the PCR.
pck beg (SEQ ID N0: 9):
5~ TA(A GAT CT) G CCG GCA TGA CTT CAG TTT 3~
pck_end (SEQ ID NO: 10):
5~ AC(A GAT CT) G GTG GGA GCC TTT CTT GTT ATT3 The primers allow amplification of a DNA fragment approx.
2.9 kb in size in control clones with the original pck locus. In clones with a second copy of the lysCFBR allele in the chromosome at the pck locus, DNA fragments with a size of approx. 4.6 kb are amplified.
The amplified DNA fragments are identified by means of electrophoresis in a 0.8~ agarose gel.
A clone which, in addition to the copy of the wild-type gene present at the lysC locus, has a second copy of the lysC gene in the form of the lysCF$R allele lysC T311I at the pck locus in the chromosome was identified in this manner. This clone was called strain DSM12866pck::lysC.
1.7 Construction of the replacement vector pKlBmobsacBaecD1 1 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~ 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 shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et a1. (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).
5 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 pUCl8 then takes place (Norrander et al., Gene 26:101-106 (1983)). This is cleaved beforehand with the restriction 10 enzymes BglII and SmaI, dephosphorylated, mixed with the aecD-carrying fragment of approx. 1.5 kb, and the mixture is treated with T4 DNA Lipase (Amersham-Pharmacia, Freiburg, Germany). The ligation batch is transformed in the E. coli strain TOP10 (Invitrogen, Leek, The 15 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 20 agarose gel. The resulting plasmid is called pUCl8aecD.
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 25 polymerase. After separation in an agarose gel (0.8~) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the lysCF$R-containing DNA fragment approx. 1.7 kb in length is isolated from the agarose gel and employed for ligation with the vector pUCl8aecD described above. This is 30 cleaved beforehand with the restriction enzyme StuI, dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany), mixed with the lysCFBR fragment of approx. 1.7 kb and the mixture is treated with T4 DNA Lipase (Amersham-Pharmacia, Freiburg, 35 Germany).

The E. coli strain DH5amcr (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. 2na 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 pUCl8aecDl.
The plasmid pUCl8aecD1 is cleaved with the restriction enzyme Kpnl and then treated with Klenow polymerase. The plasmid is then cleaved with the restriction enzyme Sall (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. 3.2 kb which carries aecD and lysC is isolated from the agarose gel and employed for ligation with the mobilizable cloning vector pKl8mobsacB described by Schafex et al. (Gene 14: &9-73 (1994)). This is cleaved beforehand with the restriction enzymes Smal and Sall 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 DHSa (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. 2nd 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 pKl8mobsacBaecD1_1.
A map of the plasmid is shown in Figure 2.
The plasmid pKl8mobsacBaecD1 1 was deposited in the form of a pure culture of the strain E. coli DHSamcr/pKl8mobsacBaecD1_1 (_ DHSalphamcr/pKl8mobsacBaecD1 1) on 5th June 2002 under number DSM15040 at the Deutsche Sammlung fur Mikroorganismen and Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.
1.8 Incorporation of a second copy of the lysC gene as the lysCFBR allele into the chromosome (target site:
aecD gene) of the strain DSM12866 by means of the replacement vector pKl8mobsacBaecD1_1 As described in Example 1.3, the plasmid pKl8mobsacBaecD1 1 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 lysCFBR
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 N0: 11):
5~ GAA CTT ACG CCA AGC TGT TC 3~
aecD_end (SEQ ID N0: 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 IySCFBR allele in the chromosome at the aecD locus, DNA fragments with a size of approx. 3..8 kb are amplified.
The amplified DNA fragments are identified by means of electrophoresis in a 0.8o 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 lysCFBR allele lysC T311I at the aecD locus in the chromosome was identified in this manner. This clone was called strain DSM12866aecD::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 pKl8mobsacBglu2 1 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); EP1108790) (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~ 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 25 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. eoli 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-TOPOglu2.
The plasmid pCP.II-TOPOglu2 is cleaved with the restriction enzymes EcoRI and Sall (Amersham-Pharmacia, Freiburg, 5 Germany) and after separation in an agarose gel (0.80) 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 pKl8mobsacB described by Schafer 10 et al. (Gene 14, 69-73 (1994)). This is cleaved beforehand with the restriction enzymes EcoRI and SalI 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 15 T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany).
The E. coli Strain DH5a, (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 20 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. 2nd Ed., Cold Spring Harbor, New York, 1989), which is supplemented with 50 mg/1 kanamycin.
25 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.
As described in Example 2.1, a DNA fragment which carries 30 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:
ddh beg ( SEQ ID NO : 15 ) 5' CTG AAT CAA AGG CGG ACA TG 3' ddh_end (SEQ ID NO: 16):
5' TCG AGC TAA ATT AGA CGT CG 3' The primers 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 0.8o agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiageri, Hilden).
After purification, the fragment carrying the ddh gene is employed for ligation in the vector pKl8mobsacBglu~
described. This is partly cleaved beforehand with the restriction enzyme BamHI. By treatment of the vector with a Klenow polymerise (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 lipase (Amersham-Pharmacia, Freiburg, Germany). By using Vent Polymerise (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 DH5amcr (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. 2nd 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 1. 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 DSM12866 by means of the replacement vector pKlBmobsacBglu2 1 As described ir? Example 1.3, the plasmid pKl8mobsacBglu2_1 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 investigav~ed with the aid of the polymerase chain reaction. A DPdA fragment which carries the glu region described is amplified here from the chromosomal DNA of the colonies. The same primer oligonucheotides as are described in Example 2.1 for the construction of the replacement plasmid are chosen for the PCR.
gluA beg ( SEQ TD NO : 13 ) 5~ CAC GGT TGC TCA TTG TAT CC 3~
gluD end (SEQ ID N0: 14):
5~ CGA GGC GAA TCA GAC TTC TT 3~
The primers allow amplification of a DNA fragment approx.
4.4 kb in sire in control clones with the original glu locus. In clonE~s 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 0.8o agarose gel.
A clone which, in addition to the copy present at the ddh locus, has a sncon.d copy of the ddh gene at the gluB locus in the chromosome was identified in this manner. This clone was called strain DSM12866g1u::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_1 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 TD NO: 11):
5~ GAA CTT ACG CCA AGC TGT TC 3~
aecD_end (SEA ID NO: 12):
5~ AGC ACC ACA ATC AAC GTG AG 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 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 (QIAquvck Gel Extraction Kit, Qiagen, Hilden).
The DNA fragment purified is cleaved with the restriction enzyme BglII and EcoRV.(Amersham Pharmacia, Freiburg, Germany). The ligation of the fragment in the vector pUCl8 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 pUCl8aecD.

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 kno~,rur~ for C. glutamicum (Bonassi et al., Nucleic 5 Acids Research 18:6421 (1990)) (Accession Number X53993 and AX127149), the following primer oligonucleotides are chosen for the PCR:
dapA beg (SEQ ID N0: 17):
5~ CGA GCC AGT GAA CAT GCA GA 3~
10 dapA end (SEQ ID NO: 18):
5~ CTT GAG CAC CTT GCG CAG CA 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 15 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 20 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 pUCl8aecD described above. This is cleaved 25 beforehand with the restriction enzyme StuI, mixed with the DNA fragment of approx. 1.4 kb, and the mixture is treated with T4 DNA Lipase (Amersham-pharmacia, Freiburg, Germany).
The E. coli strain DH5amcr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation 30 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. 2nd 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 pUCl8aecD2.
The plasmid pUClBaecD2 is cleaved with the restriction enzyme Sall arid partly with EcoRI (Amersham-Pharmacies, Freiburg, Germany) and of ter separation in an agarose gel (0.8~) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the fragment of approx. ~.7 kb which carries aecD and dapA is isolated from the agarose gel and employed for ligation with the mobilizable cloning vector pKlBmobsacB described by Schafer et al. (Gene 14:
69-73 (1994).). This is cleaved beforehand with the restriction enzymes EcoRI and with SalI 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 Lipase (Amersham-Pharmacies, Freiburg, Germany).
The E. coli strain DHSa (Grant et al.; Proceedings of the National Acaderly of Sciences USA, 87 (1990) 4f45-4649) 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. 2nd 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_1.
A map of the plasmid is shown in Figure 5.
3.2 Incorpnwation 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 1 As described in Example 1.3, the plasmid pKl8mobsacBaecD2 1 described in Example 3.1 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 dapA gene manifests itself in the chromosome at the aecD locus, or the original aeeD 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 N0: 11):
5~ GAA CTT ACG CCA AGC TGT TC 3~
aecD end (SEQ ID N0: 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 ~~Vlth 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 0.8~ 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 DSM12866aecD::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 pKlBmobsacBpckl 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 (Peters-Wendisch et al., Journal of Microbiology 144: 915-927 (1998)) (Accession Number Y09548), the following prirner 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 carriea +.:he pyc gene. The primers moreover contain the sequence for the cleavage site of the restriction endonuclease MluI, which is marked by parentheses in the nucleotide sequence shown above. .
The amplified DIVA fragment of approx. 3.6 kb in length, which carries the pyc gene, is cleaved with the restriction endonuclease MluI, 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, riilden).
After purification, the fragment carrying the pyc gene is employed for ligation in the vector pKl8mobsacBpckl 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 Lipase (Amersham-Pharmacia, Freiburg, Germany).
The E. coli strain DH5amcr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation batch (Hanaha~~; 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. 2nd 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 Niiniprep 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-direr_ted mutagenesis is carried out with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La 5 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 i3i2, replacement in the amino acid sequence 10 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):
15 5° GGATTCATTGCCGATCAC (TCG) CACCTCCTTCAGGCTCCA 3' P458S-2 (SEQ ZD 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 20 458, is marked by parentheses in the nucleotide sequence shown above. The plasmid pKl8mobsacBpckl 2 described in Example 4.1 is employed with the two primers, which are each complemeW~ary to a strand of the plasmid, for linear amplification by means of Pfu Turbo DNA polymerase. By this 25 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 30 DNA is transformed in the E. coli strain XL1 Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)).
After the transformation, the XL1 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 pycP458S into the chromosome (target site pck gene) of the strain DSM12866 by means of the replacement vector pkl8mobsacBpckl 3 The plasmid pK~8mobsacBpcki._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, of ter the 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 ~.n Example 1.5 for the construction of the replacement plasmid are chosen for the PCR.
pck_beg (SEQ ID N0: 9):
5~ TA(A GA~1 C'i) 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 ,z1 _'_ow 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.
The amplified DNA fragments are identified by means of electrophoresis in a 0.8o agarose gel.
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 DSM12866pck::pyc.
Example 5 Preparation of Lysine The C. glutamicum strains DSM13994g1u::lysC, DSM12866g1u::lysC, DSM12866pck::lysC, DSM12866aecD::lysC, DSM12866g1u::ddh, DSM12866aecD::dapA and DSM12866pck::pyc obtained in Example 1, 2, 3 and 4 are cultured in a nutrient mediurz suitable for the production of lysine and the lysine content 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 33qC. Starting from this agar plate culture, a preculture is seeded (10 mi 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°-C 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 (autoc7_aved separately) 50 g/1 Salts:
(NH4) 2504 25 g/1 KH2P04 0.1 g/1 MgS04 * ~ H~0 1.0 g/1 CaCl2 * 2 H20 10 mg/ 1 FeS04 * 7 HBO 10 mg/1 MnS04 * H20 5.0 mg/1 Biotin (sterile-filtered) 0.3 mg/1 Thiamine * HC1 (sterile-filtered) 0.2 mg/1 25 g/1 CaC03 The CSL (corn steep liquor), MOPS
(morpholinopropanesulfonic acid) and the salt solution are brought to pH 7 with aqueous ammonia and autoclaved. The sterile subs trate and vitamin solutions, as well as the CaC03 autoclaved 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 33sC and 80o 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 winch 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.

~a'hl r~ 1 fl Strain OD Lysine HC1 (660 nm) gll DSM13994 12.0 19.1 DSM13994g1u::lysC 9.9 20.0 DSM12866 12.5 14.9 DSM15039 11.4 16.2 DSM12866pck::lysC 12.6 16.5 DSM12866aecD::lysC ~ 12.0 15.9 DSM12866g1u::ddh ~ 11.0 15.5 DSM12856aecD::dapA ~ 11.1 16.2 DSM12866pck::pyc = 10.9 16.9 i Brief Description of the Figures:
5 The base pair numbers stated are approximate values obtained in the context of reproducibility of measurements.
Figure 1: I~iap of the plasmid pKl8mobsacBglul 1.
The abbreviations and designations used have the following meaning:
KanR: Kanamycin resistance gene HindIII: Cleavage site of the restriction enzyme HindIII

BamHI: Cleavage site of the restriction enzyme BamHI

lysC: lySCFBR 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 pKl8mobsacBaecD1 1.

The abbreviations following and designations used have the meaning:

KanR: Kanamycin resistance gene SalI: Cleavage site of the restriction enzyme SalI
lysC: lysCFBR 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 pKl8mobsacBpckl-1.

The abbreviations following and designations used have the meaning:

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

lysC: lysCFBR 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 .

The abbreviations following and designations used have the meaning:

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

EcoRI Cleavage site of the restriction enzyme ECORI

BamHI: Cleavage site of the restriction enzyme BamHI

ddh: ddh gene gluA gluA gene gluB': 5' terminal fragment of the gluB gene 'gluB: 3' terminal fragment of the gluB gene gluC gluC gene gluD': 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 Map of the plasmid pKl8mobsacBaecD2_1.
5:

The abbreviations and designations following used have the meaning:

KanR: I~anamycin resistance gene EcoRI Cl.eavag~ site of the restriction enzyme ECORI

Sall: Cleavage site of the restriction enzyme SalI

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 pKl8mobsacBpckl 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

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identified at the bottom of this page I. IDENTIFICATION OF THE MICROORGANISM
I

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pKlBmobsacBglul 1 DSM 14243 II. SCIENTIFIC DESCRIPTION AND/OR
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III. RECEB~T AND ACCEPTANCE

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The microorganism identified under I above wes received by this International Depository 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).

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Form DSMZ-Bpl4 (sole page) OI96 BUDAPEST TREATY ON THE INTERNATIONAL

FOR THE PURPOSES OF PATENT PROCEDURE
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issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY
identified at the bottom of this page I. DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM

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III. VIABIL1TY STATEMENT

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On that date, the said microorganism was (X)' viable ( )' no longer viable IV. CONDITIONS UNDER WHICH TfIE
VIABIISTY TEST HAS BEEN PERFORMED

V. INTERNATIONAL DEPOSTfARY AUTHORITY

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' Address: Mascheroder Weg 1b /~
~
~

D-38124 Braunschweig ~
_.~s~~ I

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 1 D.2(a) (ii) and (iii), refer to the most recent viability test.
' Mark with a cross the applicable box.
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Form DSMZ-BP19 (sale page) 0196 SEøLIENCE LISTING
<110> Degussa AG

<120> Coryneform bacteria which produce chemical compounds I

<130> 010301 BT

<160> 22 <170> PatentIn version 3.1 <210> 1 <211> 1263 <212> DNA

<213> Corynebacterium glutamicum <220>

<221> CDS

<222> (1)..(1263) <223> lysC wild-type gene <400>

gtg gccctg gtcgtacagaaa tatggcggttcctcgctt gagagtgcg 48 Met AlaLeu ValValGlnLys TyrGlyGlySerSerLeu GluSerAla gaa cgcatt agaaacgtcget gaacggatcgttgccacc aagaagget 96 Glu ArgIle ArgAsnValAla GluArgIleValAlaThr LysLysAla 3 gga aatgat gtcgtggttgtc tgctccgcaatgggagac accacggat 144 Gly AsnAsp ValValValVal CysSerAlaMetGlyAsp ThrThrAsp gaa cttcta gaacttgcagcg gcagtgaatcccgttccg ccagetcgt 192 4 Glu LeuLeu GluLeuAlaAla AlaValAsnProValPro ProAlaArg gaa atggat atgctcctgact getggtgagcgtatttct aacgetctc 240 Glu MetAsp MetLeuLeuThr AlaGlyGluArgIleSer AsnAlaLeu gtc gccatg getattgagtcc cttggcgcagaagcccaa tctttcacg 288 Val AlaMet AlaIleGluSer LeuGlyAlaGluAlaGln SerPheThr ggc tctcag getggtgtgctc accaccgagcgccacgga aacgcacgc 336 G1y SerGln AlaGlyValLeu ThrThrGluArgHisGly AsnAlaArg 55 att gttgat gtcactccaggt cgtgtgcgtgaagcactc gatgagggc 384 Ile ValAsp ValThrProGly ArgValArgGluAlaLeu AspG1uGly aag atctgc attgttgetggt ttccagggtgttaataaa gaaacccgc 432 Lys I1eCys IleValAlaGly PheGlnGlyValAsnLys GluThrArg gat gtcaccacg ttgggtcgtggtggttct gacaccact gcagttgcg 480 Asp Va1ThrThr LeuGlyArgGlyGlySer AspThrThr AlaValAla ttg gcagetget ttgaacgct~gatgtgtgt gagatttac tcggacgtt 528 Leu AlaAlaAla LeuAsnAlaAspValCys GluIleTyr SerAspVal gac ggtgtgtat accgetgacccgcgcatc gttcctaat gcacagaag 576 1 Asp GlyValTyr ThrAlaAspProArgIle ValProAsn AlaGlnLys ctg gaaaagctc agcttcgaagaaatgctg gaacttget getgttggc 624 Leu GluLysLeu SerPheGluGluMetLeu GluLeuAla AlaValGly tcc aagattttg gtgctgcgcagtgttgaa tacgetcgt gcattcaat 672 Ser LysIleLeu ValLeuArgSerValGlu TyrAlaArg AlaPheAsn gtg ccactt cgcgtacgctcgtcttat agtaatgatccc ggcactttg 720 Val ProLeu ArgValArgSerSerTyr SerAsnAspPro GlyThrLeu 2 att gccggc tctatggaggatattcct gtggaagaagca gtccttacc 768 Ile AlaGly SerMetGluAspI1ePro ValGluGluAla ValLeuThr ggt gtcgca accgacaagtccgaagcc aaagtaaccgtt ctgggtatt 816 3 Gly ValAla ThrAspLysSerG1uAla LysValThrVal LeuGlyIle tcc gataag ccaggcgaggetgcgaag gttttccgtgcg ttggetgat 864 Ser AspLys ProGlyG1uAlaAlaLys ValPheArgAla LeuAlaAsp gca gaaatc aacattgacatggttctg cagaacgtctct tctgtagaa 912 Ala GluIle AsnIleAspMetValLeu GlnAsnValSer SerValGlu 40 , gac ggcacc accgacatcaccttcacc tgccctcgttcc gacggccgc 960 Asp GlyThr ThrAspIleThrPheThr CysProArgSer AspGlyArg 45 cgc gcgatg gagatcttgaagaagctt caggttcagggc aactggacc 1008 Arg AlaMet GluIleLeuLysLysLeu GlnValG1nGly AsnTrpThr aat gtgctt tacgacgaccaggtcggc aaagtctccctc gtgggtget 1056 50 Asn Va1Leu TyrAspAspGlnValGly LysValSerLeu ValG1yAla ggc atgaag tctcacccaggtgttacc gcagagttcatg gaagetctg 1104 Gly MetLys SerHisProGlyValThr AlaGluPheMet GluAlaLeu cgc gatgtc aacgtgaacatcgaattg atttccacctct gagattcgt 1152 Arg AspVal AsnValAsnIleGluLeu IleSerThrSer GluIleArg att tcc gtg ctg atc cgt gaa gat gat ctg gat get get gca cgt gca 1200 Ile Ser Va1 Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala ttg cat gag cag ttc cag ctg ggc ggc gaa gac gaa gcc gtc gtt tat 1248 Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp G1u Ala Val Val Tyr gca ggc acc gga cgc 1263 1 0 Ala Gly Thr G1y Arg <210> 2 <211> 421 15 <212> PRT
<213> Corynebacterium glutamicum <400> 2 2 0 Met Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Va1 Ala Thr Lys Lys Ala ~5 Gly Asn Asp Val Val Val Val Cys Ser A1a Met Gly Asp Thr Thr Asp Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Va1 Pro Pro A1a Arg Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn A1a Leu 3 5 Val A1a Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala 5 0 Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val Asp Gly Val Tyr Thr Ala Asp Pro Arg .Ile Val Pro Asn Ala Gln Lys Leu Glu Lys Leu Ser Phe Glu G1u Met Leu Glu Leu Ala Ala Val Gly Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn $0 210 215 220 Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro G1y Thr Leu Ile Ala Gly Ser Met G1u Asp Ile Pro Val Glu Glu Ala Val Leu Thr Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly I1e 1 Ser AspLysProGly Glu'AlaAlaLysVal PheArgAlaLeuAlaAsp Ala GluIleAsnIle AspMetValLeuGln AsnVa1SerSerValGlu Asp GlyThrThrAsp IleThrPheThrCys ProArgSerAspGlyArg Arg AlaMetG1uIle LeuLysLysLeuGln ValGlnGlyAsnTrpThr Asn ValLeuTyrAsp AspGlnValG1yLys ValSerLeuValGlyAla 2 Gly MetLysSerHis ProGlyValThrAla GluPheMetGluA1aLeu Arg AspValAsnVal AsnIleGluLeuIle SerThrSerGluIleArg Ile SerValLeuIle ArgGluAspAspLeu AspAlaAlaA1aArgAla Leu HisGluGlnPhe GlnLeuGlyGlyGlu AspGluAlaValValTyr Ala GlyThrGlyArg 40 <210> 3 <211> 1263 <212> DNA

<213> Corynebacterium glutamicum 45 <220>

<221> CDS

<222> (1)..(1263) <223> lysC-fbrallele T311I
lysC

5fl <400> 3 gtg gcc ctg gta cagaaatatggc ggttcctcgcttgagagt gcg 48 gtc Met A1a Leu Val GlnLysTyrGly G1ySerSerLeuGluSer A1a Val 5 gaa cgc att aac gtcgetgaacgg atcgttgccaCCaagaag get 96 5 aga Glu Arg 21e Asn ValAlaGluArg IleValAlaThrLysLys Ala Arg gga aat gat gtg gttgtctgctcc gcaatgggagacaccacg gat 1.44 gtc 6 Gly Asn Asp Val Va1ValCysSer AlaMetGlyAspThrThr Asp 0 Val gaa ctt cta gaa ctt gca gcg gca gtg aat ccc gtt ccg c.ca get cgt 192 Glu Leu Leu Glu Leu Ala Ala A1a Val Asn Pro Val Pro Pro Ala Arg gaa atg gat atg ctc ctg act get ggt gag cgt att tct aac get ctc 240 Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu gtc gcc atg get att gag tcc .ctt ggc gca gaa gcc caa tct ttc acg 288 Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr ggc tct cag get ggt gtg ctc acc acc gag cgc cac gga aac gca cgc 336 Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg att gtt gat gtc act cca ggt cgt gtg cgt gaa gca ctc gat gag ggc 384 Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly aag atc tgc att gtt get ggt ttc cag ggt gtt aat aaa gaa acc cgc 432 Lys Ile Cys Ile Val Ala Gly Phe G1n Gly Val Asn Lys Glu Thr Arg 2 5 gat gtc acc acg ttg ggt cgt ggt ggt tct gac acc act gca gtt gcg 480 Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala ttg gca get get ttg aac get gat gtg tgt gag att tac tcg gac gtt 528 3 0 Leu Ala A1a Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val gac ggt gtg tat acc get gac ccg cgc atc gtt cct aat gca cag aag 576 Asp Gly Va1 Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn A1a Gln Lys ctg gaa aag ctc agc ttc gaa gaa atg ctg gaa ctt get get gtt ggc 624 Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala A1a Val Gly tcc aag att ttg gtg ctg cgc agt gtt gaa tac get cgt gca ttc aat 672 Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn gtg cca ctt cgc gta cgc tcg tct tat agt aat gat ccc ggc act ttg 720 Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu att gcc ggc tct atg gag gat att cct gtg gaa gaa gca gtc ctt acc 768 5 0 Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr ggt gtc gca acc gac aag tcc gaa gcc aaa gta acc gtt ctg ggt att 816 Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Va1 Thr Val Leu G1y Ile 2g0 265 270 tcc gat aag cca ggc gag get gcg aag gtt ttc cgt gcg ttg get gat 864 Ser Asp Lys Pro Gly G1u Ala Ala Lys Val Phe Arg Ala Leu A1a Asp gca gaaatcaac attgacatggtt ctgcagaacgtctcttct gtagaa 912 Ala GluIleAsn I1eAspMetVal LeuGlnAsnValSerSer ValGlu gac ggcaccacc gacatcatcttc acctgccctcgttccgac ggccgc 960 Asp GlyThrThr AspIleIlePhe ThrCysProArgSerAsp GlyArg cgc gcgatggag atcttgaagaag cttcaggttcagggcaac tggacc 1008 1,.0Arg AlaMetGlu IleLeuLysLys LeuGlnValGlnGlyAsn TrpThr aat gtgctttac gacgaccaggtc ggcaaagtctccctcgtg ggtget 1056 Asn ValLeuTyr AspAspGlnVal GlyLysValSerLeuVal GlyAla ggc atgaagtct cacccaggtgtt accgcagagttcatggaa getctg 1104 Gly MetLysSer HisProGlyVal ThrAlaGluPheMetGlu AlaLeu cgc gatgtcaac gtgaacatcgaa ttgatttccacctctgag attcgt 1152 Arg AspValAsn ValAsnIleGlu LeuIleSerThrSerGlu I1eArg 2 att tccgtgctg atccgtgaagat gatctggatgetgetgca cgtgca 1200 Ile SerValLeu IleArgGluAsp AspLeuAspAlaAlaAla ArgAla ttg catgagcag ttccagctgggc ggcgaagacgaagccgtc gtttat 1248 3flLeu HisGluGln PheG1nLeuGly GlyGluAspGluA1aVal ValTyr 405. 410 415 gca ggcaccgga cgc 1263 Ala GlyThrGly Arg <210> 4 <211> 421 <212> PRT

<213> Corynebacterium glutamicum <400> 4 Met Ala Leu Val Lys Tyr Gly Gly Ser GluSer Val Gln Ser Leu Ala Glu Arg Ile Asn Ala G1u Arg Ile Ala LysLys Arg Val Val Thr Ala 5 Gly Asn Asp Val Val Cys Ser Ala Gly ThrThr ~ Val Val Met Asp Asp Glu Leu Leu Leu Ala Ala Val Asn Val ProAla Glu Ala Pro Pro Arg Glu Met Asp Leu Thr Ala Gly Glu Ile AsnAla Met Leu Arg Ser Leu Val Ala Met Ile Ser Leu Gly Ala Ala SerPhe Ala Glu Glu Gln Thr Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg 1 0 Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala Leu Ala Ala Ala Leu Asn Ala Asp Va1 Cys Glu Ile T'yr Ser Asp Val Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly Ser Lys Ile Leu Val Leu Arg Ser Va1 Glu 'I'yr Ala Arg Ala the Asn ~ 5 Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu A1a Val Leu Thr Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg A1a Leu Ala Asp Ala G1u Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu 4 0 Asp G1y Thr Thr Asp Ile Ile Phe Thr Cys Pro Arg Ser Asp Gly Arg Arg Ala Met Glu Ile Leu Lys Lys Leu G1n Val Gln Gly Asn Trp Thr Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu Arg Asp Val Asn Val Asn I1e Glu Leu Ile Ser Thr Ser Glu Ile Arg 5 5 Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala 385 390 395 ' 400 Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu A1a Val Val Tyr b0 Ala Gly Thr Gly Arg <210> 5 <211> 28 <212> DNA

<213> Artificial sequence <220>

<221> misc feature _ <222> (1). (28) 1 <223> Primer lysClbeg <400> 5 taggatcctc cggtgtctga ccacggtg 28 <210> 6 <211> 29 <212> DNA

<213> Artificial sequence <220>

<221> misc feature _ <222> (1). (29) <223> Primer lysC2end <~oo>

acggatccgc tgggaaattg cgctcttcc 29 <210> 7 <211> 28 <212> DNA

<213> Artificial sequence <220>

<221> misc feature 3 _ 5 <222> (1). (28) <223> Primer gluBgll <400> 7 taagatctgt gttggacgtc atggcaag 28 <210> 8 <211> 28 <212> DNA

<213> Artificial sequence <220>

<221> misc feature _ <222> (1). (28) <223> Primer gluBgl2 <400> 8 acagatcttg aagccaagta cggccaag 28 <210> 9 <211> 27 <212 > DNA

<213> Artificial sequence <220>

$fl<221> misc feature <222> (1). (27) <223> Primer pck beg <400> 9 taagatctgc cggcatgact tcagttt 27 <210> 10 <211> 30 <212> DNA

<213> Artificial sequence <220>

<221> misc feature _ <222> (1). {30) <223> Primer pck_end <400> so acagatctgg tgggagcctt tcttgttatt 30 <210> 11 <211> 20 <212> DNA

<213> Corynebacterium glutamicum <220>

<221> misc feature 2 _ 5 <222> (1). (20) <223> Primer aecD beg <400> 11 gaacttacgc caagctgttc 20 <210> 12 <211> 20 <212> DNA

<213> Corynebacterium glutamicum <220>

<221> misc feature _ <222> (1). (20) <223> Primer aecD_end <400> 12 agcaccacaa tCaacgtgag 20 <210> 13 <211> 20 <212> DNA

<213> Corynebacterium glutamicum <220>

<221> misc feature _ <222> (1). (20) <223> Primer gluA_beg <400> 13 cacggttgct cattgtatcc 20 <210> 14 <211> 20 <212> DNA

<213> Corynebacterium glutamicum <220>

<221> misc_feature <222> (1)..(20) <223> Primer gluD_end <400> 14 cgaggcgaat cagacttctt 2p <210> 15 <211> 20 <212> DNA

<213> Corynebacterium glutamicum <220>

1 <221> misc_feature <222> (1)..(20) <223> Primer ddh_beg <400> 15 2 ctgaatcaaa ggcggacatg 20 <210> 16 <211> 20 <212> DNA

2 <2l3> Corynebacterium glutamicum <220>

<221> misc feature <222> (1)..(20) 3 <223> Primer ddh end <400> 16 tcgagctaaa ttagacgtcg 20 3 <210> 17 <211> 20 <212> DNA

<213> Corynebacterium glutamicum 40 <220>

<221> misc feature _ <222> (1). (20) <223> Primer dapA beg 45 <400> 17 cgagccagtg aacatgcaga 20 <210> 18 <211> 20 50 <212> DNA

<213> Corynebacterium glutamicum <220>

<221> misc feature 5 _ 5 <222> (1). (20) <223> Primer dapA_end <400> 18 cttgagcacc ttgcgcagca 20 <210> 19 <211> 28 <212> DNA
<213> Artificial sequence <220>
<221> misc_feature <222> (1). (28) <223> Primer pyc beg <400> 19 tcacgcgtct tgaagtcgtg caggtcag 28 <210> 20 <211> 28 <212> DNA
<213> Artificial sequence <220>
2 0 <221> misc_feature <222> (1). (28) <223> Primer pyc_end <400> 20 ~ 5 tcacgcgtcg cctcctccat gaggaaga 28 <210> 21 <211> 39 <212> DNA
3 0 <213> Corynebacterium glutamicum <220>
<221> misc_feature <222> (1). (39) 3 5 <223> Primer P458S-1 <400> 21 ggattcattg ccgatcactc gcacctcctt caggctcca 39 40 <210> 22 <211> 39 <212> DNA
<213> Corynebacterium glutamicum 45 <220>
<221> misc_feature <222> (1)..(39) <223> Primer P458S-2 <400> 22 gtggaggaag tccgaggtcg agtgatcggc aatgaatcc 39

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, msiK, opcA, oxyR, ppc, ppc FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, 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, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, 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 a1) 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, ppc FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T.

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, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, 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, 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 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 pK18mobsacBglu1_1 shown in Figure 1 and deposited in the form of a pure culture of the strain E. coli DH5.alpha.mcr/pK18mobsacBglu1_1 (= DH5alpha mcr/pK18mobsacBglu1_1) under number DSM14243.

39. Plasmid pK18mobsacBaecD1_1 shown in Figure 2 and deposited in the form of a pure culture of the strain E. coli DH5.alpha.mcr/pK18mobsacBaecD1_1 (=
DH5alphamcr/pK18mobsacBaecD1_1) under number DSM15040.

40. Corynebacterium glutamicum strain DSM12866glu::lysC
deposited in the form of a pure culture under number DSM15039.