Coryneform Bacteria Which Produce Chemical Compounds I

Information

  • Patent Application
  • 20070111291
  • Publication Number
    20070111291
  • Date Filed
    December 18, 2006
    17 years ago
  • Date Published
    May 17, 2007
    17 years ago
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
BACKGROUND

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 sequence(s) which impart(s) resistance to antibiotics being present at the second, optionally third or fourth site, and the second, optionally third or fourth site not relating to open reading frames (ORF), genes or alleles which are essential for the growth of the bacteria and the production of the desired compound.


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

    • a) fermentation of coryneform bacteria,
    • 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,
    • b) concentration of the chemical compound(s) in the fermentation broth and/or in the cells of the bacteria,
    • c) isolation of the chemical compound(s), optionally
    • d) with constituents from the fermentation broth and/or the biomass to the extent of >(greater than) 0 to 100 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 compound(s) in the fermentation broth and/or in the cells of the bacteria,
    • c) isolation of the chemical compound(s), optionally
    • d) with constituents from the fermentation broth and/or the biomass to the extent of >(greater than) 0 to 100%.







DETAILED DESCRIPTION OF THE INVENTION

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


Amino acids mean, preferably, L-amino acids, in particular the proteinogenic L-amino acids, chosen from the group consisting of L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-proline and L-arginine and salts thereof, in particular L-lysine, L-methionine and L-threonine. L-Lysine is very particularly preferred.


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


Vitamins mean, in particular, vitamin 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 Kämpfer and Kroppenstedt (Canadian Journal of Microbiology 42, 989-1005 (1996)) and in U.S. Pat. No. 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 U.S. Pat. No. 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).


Other datenbanks e.g. the National Center for Biotechnology Information (NCBI) of the National Library of Medicin (Bethesda, Md., USA) may be also be used.


Further databanks, such as, for example, that of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA) or that of the Swiss Institute of Bioinformatics (Swissport, Geneva, Switzerland) or that of the Protein Information Resource Database (PIR, Washington, D.C., USA) can also be used.


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


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


The invention accordingly also provides a process for the production of coryneform bacteria which produce one or more chemical compounds, which comprises

    • a) isolating the nucleotide sequence of at least one desired ORF, gene or allele, optionally including the expression and/or regulation signals,
    • b) providing the 5′ and the 3′ end of the ORF, gene or allele with nucleotide sequences of the target site,
    • c) preferably incorporating the nucleotide sequence of the desired ORF, gene or allele provided with nucleotide sequences of the target site into a vector which does not replicate or replicates to only a limited extent in coryneform bacteria,
    • d) transferring the nucleotide sequence according to b) or c) into coryneform bacteria, and
    • e) isolating coryneform bacteria in which the nucleotide sequence according to a) is incorporated at the target site, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remaining at the target site.


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


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


Instructions on conventional genetic engineering methods, such as, for example, isolation of chromosomal DNA, plasmid DNA, handling of restriction enzymes etc., are found in Sambrook et al. (Molecular Cloning—A Laboratory Manual (1989) Cold Spring Harbor Laboratory Press). Instructions on transformation and conjugation in coryneform bacteria are found, inter alia, in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), in Schäfer et al. (Journal of Bacteriology 172, 1663-1666 (1990) and Gene 145, 69-73 (1994)) and in Schwarzer and Pühler (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. (U.S. Pat. No. 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, lysCFBR, lysE, msiK, opcA, oxyR, ppc, ppcFBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T. These are summarized and 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 NO: 2, by isoleucine), lysC S301Y (replacement of serine at position 308 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by tyrosine), lysC G345D (replacement of glycine at position 345 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by aspartic acid), lysC R320G (replacement of arginine at position 320 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by glycine), lysC T311I (replacement of threonine at position 311 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by isoleucine), lysC S381F (replacement of serine at position 381 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by phenylalanine).


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


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


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


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


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


Examples of regions of the Corynebacterium glutamicum chromosome representing intergenic regions, prophages, defective phages or phage components are shown in tables 12 and 13. The positions of the DNA regions refer to the genome map of Corynebacterium glutamicum ATCC 13032 as presented in EP-A-1108790 or in the databank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK).

TABLE 1Open reading frames, genes and alleles of lysine productionDescription of the coded enzyme orAccessNameproteinReferenceNumberaccBCAcyl-CoA CarboxylaseJäger et al.U35023EC 6.3.4.14Archives of(acyl-CoA carboxylase)Microbiology(1996) 166: 76-82EP1108790;AX123524WO0100805AX066441accDAAcetyl-CoA CarboxylaseEP1055725EC 6.4.1.2EP1108790AX121013(acetyl-CoA carboxylase)WO0100805AX066443cstACarbon Starvation Protein AEP1108790AX120811(carbon starvation protein A)WO0100804AX066109cysDSulfate AdenylyltransferaseEP1108790AX123177sub-unit IIEC 2.7.7.4(sulfate adenylyltransferase small chain)cysESerine AcetyltransferaseEP1108790AX122902EC 2.3.1.30WO0100843AX063961(serine acetyltransferase)cysH3′-Phosphoadenyl Sulfate ReductaseEP1108790AX123178EC 1.8.99.4WO0100842AX066001(3′-phosphoadenosine 5′-phosphosulfate reductase)cysKCysteine SynthaseEP1108790AX122901EC 4.2.99.8WO0100843AX063963(cysteine synthase)cysNSulfate Adenylyltransferase sub-EP1108790AX123176unit IAX127152EC 2.7.7.4(sulfate adenylyltransferase)cysQTransport Protein CysQEP1108790AX127145(transporter cysQ)WO0100805AX066423dapADihydrodipicolinate SynthaseBonnassie etX53993EC 4.2.1.52al. Nucleic(dihydrodipicolinate synthase)Acids Research18: 6421 (1990)Pisabarro etZ21502al., Journal ofBacteriology175: 2743-2749(1993)EP1108790WO0100805EP0435132EP1067192AX123560EP1067193AX063773dapBDihydrodipicolinate ReductaseEP1108790AX127149EC 1.3.1.26 (dihydrodipicolinate reductase)WO0100843AX063753EP1067192AX137723EP1067193AX137602Pisabarro etX67737al., Journal ofZ21502Bacteriology175: 2743-2749(1993)JP1998215883E16749JP1997322774E14520JP1997070291E12773JP1995075578E08900dapCN-Succinyl AminoketopimelateEP1108790AX127146TransaminaseWO0100843AX064219EC 2.6.1.17EP1136559(N-succinyl diaminopimelatetransaminase)dapDTetrahydrodipicolinate SuccinylaseEP1108790AX127146EC 2.3.1.117WO0100843AX063757(tetrahydrodipicolinateWehrmann et al.AJ004934succinylase)Journal ofBacteriology180: 3159-3165(1998)dapEN-Succinyl DiaminopimelateEP1108790AX127146DesuccinylaseWO0100843AX063749EC 3.5.1.18Wehrmann et al.X81379(N-succinyl diaminopimelateMicrobiologydesuccinylase)140: 3349-3356(1994)dapFDiaminopimelate EpimeraseEP1108790AX127149EC 5.1.1.7WO0100843AX063719(diaminopimelate epimerase)EP1085094AX137620ddhDiaminopimelate DehydrogenaseEP1108790AX127152EC 1.4.1.16WO0100843AX063759(diaminopimelate dehydrogenase)Ishino et al.,Y00151Nucleic AcidsResearch15: 3917-3917(1987)JP1997322774E14511JP1993284970E05776Kim et al.,D87976Journal ofMicrobiologyandBiotechnology5: 250-256(1995)dpsDNA Protection ProteinEP1108790AX127153(protection during starvationprotein)enoEnolaseEP1108790AX127146EC 4.2.1.11WO0100844AX064945(enolase)EP1090998AX136862Hermann et al.,Electrophoresis19: 3217-3221(1998)gapGlyceraldehyde 3-PhosphateEP1108790AX127148DehydrogenaseWO0100844AX064941EC 1.2.1.12Eikmanns etX59403(glyceraldehyde 3-phosphateal., Journal ofdehydrogenase)Bacteriology174: 6076-6086(1992)gap2Glyceraldehyde 3-PhosphateEP1108790AX127146DehydrogenaseWO0100844AX064939EC 1.2.1.12(glyceraldehyde 3-phosphatedehydrogenase 2)gdhGlutamate DehydrogenaseEP1108790AX127150EC 1.4.1.4WO0100844AX063811(glutamate dehydrogenase)Boermann etX59404al., MolecularMicrobiology6: 317-326(1992).Guyonvarch etX72855al., NCBIgnd6-Phosphogluconate DehydrogenaseEP1108790AX127147EC 1.1.1.44AX121689(6-phosphogluconate dehydrogenase)WO0100844AX065125lysCAspartate KinaseEP1108790AX120365EC 2.7.2.4WO0100844AX063743(aspartate kinase)Kalinowski etX57226al., MolecularMicrobiology5: 1197-204(1991)lysCFBRAspartate Kinase feedback resistantsee Table 2(fbr)EC 2.7.2.4(aspartate kinase fbr)lysELysine ExporterEP1108790AX123539(lysine exporter protein)WO0100843AX123539Vrljić et al.,X96471MolecularMicrobiology22: 815-826(1996)msiKSugar ImporterEP1108790AX120892(multiple sugar import protein)opcAGlucose 6-phosphate DehydrogenaseWO0104325AX076272(subunit of glucose 6-phosphatedehydrogenase)oxyRTranscription RegulatorEP1108790AX122198(transcriptional regulator)AX127149ppcFBRPhosphoenol Pyruvate CarboxylaseEP0723011feedback resistantWO0100852EC 4.1.1.31(phosphoenol pyruvate carboxylasefeedback resistant)ppcPhosphoenol Pyruvate CarboxylaseEP1108790AX127148EC 4.1.1.31AX123554(phosphoenol pyruvate carboxylase)O'Reagan etM25819al., Gene77(2): 237-251(1989)pgkPhosphoglycerate KinaseEP1108790AX121838EC 2.7.2.3AX127148(phosphoglycerate kinase)WO0100844AX064943Eikmanns,X59403Journal ofBacteriology174: 6076-6086(1992)pknAProtein Kinase AEP1108790AX120131(protein kinase A)AX120085pknBProtein Kinase BEP1108790AX120130(protein kinase B)AX120085pknDProtein Kinase DEP1108790AX127150(protein kinase D)AX122469AX122468pknGProtein Kinase GEP1108790AX127152(protein kinase G)AX123109ppsAPhosphoenol Pyruvate SynthaseEP1108790AX127144EC 2.7.9.2AX120700(phosphoenol pyruvate synthase)AX122469ptsHPhosphotransferase System Protein HEP1108790AX122210EC 2.7.1.69AX127149(phosphotransferase systemWO0100844AX069154component H)ptsIPhosphotransferase System Enzyme IEP1108790AX122206EC 2.7.3.9AX127149(phosphotransferase systemenzyme I)ptsMGlukose-specific PhosphotransferaseLee et al.,L18874System Enzyme IIFEMSEC 2.7.1.69Microbiology(glucose phosphotransferase systemLetters 119enzyme II)(1-2): 137-145(1994)pycPyruvate CarboxylaseWO9918228A97276EC 6.4.1.1Peters-WendischY09548(pyruvate carboxylase)et al.,Microbiology144: 915-927(1998)pycPyruvate CarboxylaseEP1108790P458SEC 6.4.1.1(pyruvate carboxylase)amino acid exchange P458SsigCSigma Factor CEP1108790AX120368EC 2.7.7.6AX120085(extracytoplasmic functionalternative sigma factor C)sigDRNA Polymerase Sigma Factor DEP1108790AX120753EC 2.7.7.6AX127144(RNA polymerase sigma factor)sigESigma Factor EEP1108790AX127146EC 2.7.7.6AX121325(extracytoplasmic functionalternative sigma factor E)sigHSigma Factor HEP1108790AX127145EC 2.7.7.6AX120939(sigma factor SigH)sigMSigma Factor MEP1108790AX123500EC 2.7.7.6AX127145(sigma factor SigM)talTransaldolaseWO0104325AX076272EC 2.2.1.2(transaldolase)thyAThymidylate SynthaseEP1108790AX121026EC 2.1.1.45AX127145(thymidylate synthase)tktTransketolaseIkeda et al.,AB023377EC 2.2.1.1NCBI(transketolase)tpiTriose Phosphate IsomeraseEikmanns,X59403EC 5.3.1.1Journal of(triose phosphate isomerase)Bacteriology174: 6076-6086(1992)zwa1Cell Growth Factor 1EP1111062AX133781(growth factor 1)zwfGlucose 6-phosphate 1-DehydrogenaseEP1108790AX127148EC 1.1.1.49AX121827(glucose 6-phosphate 1-WO0104325AX076272dehydrogenase)zwfGlucose 6-phosphate 1-DehydrogenaseEP1108790A213TEC 1.1.1.49(glucose 6-phosphate 1-dehydrogenase)amino acid exchange A213T









TABLE 2










lysCFBR alleles which code for feed back resistant aspartate


kinases










Name of the
Further

Access


allele
information
Reference
Number





lysCFBR-E05108

JP 1993184366-A
E05108




(sequence 1)


lysCFBR-E06825
lysC A279T
JP 1994062866-A
E06825




(sequence 1)


lysCFBR-E06826
lysC A279T
JP 1994062866-A
E06826




(sequence 2)


lysCFBR-E06827

JP 1994062866-A
E06827




(sequence 3)


lysCFBR-E08177

JP 1994261766-A
E08177




(sequence 1)


lysCFBR-E08178
lysC A279T
JP 1994261766-A
E08178




(sequence 2)


lysCFBR-E08179
lysC A279V
JP 1994261766-A
E08179




(sequence 3)


lysCFBR-E08180
lysC S301F
JP 1994261766-A
E08180




(sequence 4)


lysCFBR-E08181
lysC T308I
JP 1994261766-A
E08181




(sequence 5)


lysCFBR-E08182

JP 1994261766-A
E08182




(sequence 6)


lysCFBR-E12770

JP 1997070291-A
E12770




(sequence 13)


lysCFBR-E14514

JP 1997322774-A
E14514




(sequence 9)


lysCFBR-E16352

JP 1998165180-A
E16352




(sequence 3)


lysCFBR-E16745

JP 1998215883-A
E16745




(sequence 3)


lysCFBR-E16746

JP 1998215883-A
E16746




(sequence 4)


lysCFBR-I74588

US 5688671-A
I74588




(sequence 1)


lysCFBR-I74589
lysC A279T
US 5688671-A
I74589




(sequence 2)


lysCFBR-I74590

US 5688671-A
I74590




(sequence 7)


lysCFBR-I74591
lyaC A279T
US 5688671-A
I74591




(sequence 8)


lysCFBR-I74592

US 5688671-A
I74592




(sequence 9)


lysCFBR-I74593
lysC A279T
US 5688671-A
I74593




(sequence 10)


lysCFBR-I74594

US 5688671-A
I74594




(sequence 11)


lysCFBR-I74595
lysC A279T
US 5688671-A
I74595




(sequence 12)


lysCFBR-I74596

US 5688671-A
174596




(sequence 13)


lysCFBR-I74597
lysC A279T
US 5688671-A
I74597




(sequence 14)


lysCFBR-X57226
lysC S301Y
EP0387527
X57226




Kalinowski et al.,




Molecular and




General Genetics




224: 317-324 (1990)


lysCFBR-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
WO0063388




(sequence 17)


lysCFBR
lysC S301F
US3732144


lysCFBR
lysC S381F


lysCFBR

JP6261766




(sequence 1)


lysCFBR
lysC A279T
JP6261766




(sequence 2)


lysCFBR
lysC A279V
JP6261766




(sequence 3)


lysCFBR
lysC S301F
JP6261766




(sequence 4)


lysCFBR
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 coded

Access


name
enzyme or protein
Reference
Number





aecD
beta C-S Lyase
Rossol et al., Journal
M89931



EC 2.6.1.1
of Bacteriology



(beta C-S lyase)
174(9): 2968-77 (1992)


ccpA1
Catabolite Control
WO0100844
AX065267



Protein
EP1108790
AX127147



(catabolite control



protein A1)


ccpA2
Catabolite Control
WO0100844
AX065267



Protein
EP1108790
AX121594



(catabolite control



protein A2)


citA
Sensor Kinase CitA
EP1108790
AX120161



(sensor kinase CitA)


citB
Transcription Regulator
EP1108790
AX120163



CitB



(transcription regulator



CitB)


citE
Citrate Lyase
WO0100844
AX065421



EC 4.1.3.6
EP1108790
AX127146



(citrate lyase)


fda
Fructose Bisphosphate
von der Osten et al., Molecular
X17313



Aldolase
Microbiology



EC 4.1.2.13
3(11): 1625-37 (1989)



(fructose 1,6-



bisphosphate aldolase)


gluA
Glutamate Transport ATP-
Kronemeyer et al.,
X81191



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): 1252-8 (1995)



system permease)


luxR
Transcription Regulator
WO0100842
AX065953



LuxR
EP2108790
AX123320



(transcription regulator



LuxR)


luxS
Histidine Kinase LuxS
EP1108790
AX123323



(histidine kinase LuxS)

AX127145


lysR1
Transcription Regulator
EP1108790
AX064673



LysR1

AX127144



(transcription regulator



LysR1)


lysR2
Transcription Activator
EP1108790
AX123312



LysR2



(transcription regulator



LysR2)


lysR3
Transcription Regulator
WO0100842
AX065957



LysR3
EP1108790
AX127150



(transcription regulator



LysR3)


menE
O-Succinylbenzoic Acid
WO0100843
AX064599



CoA Ligase
EP1108790
AX064193



EC 6.2.1.26

AX127144



(O-succinylbenzoate CoA



ligase)


mqo
Malate-Quinone
Molenaar et al., Eur.
AJ224946



Oxidoreductase
Journal of Biochemistry



(malate-quinone-oxidoreductase)
1; 254(2): 395-403 (1998)






pck
Phosphoenol Pyruvate
WO0100844
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
WO0100844
AX064959



EC 1.2.3.3
EP1096013
AX137665



(pyruvate oxidase)


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-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) 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.


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


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

  • a) fermentation of coryneform bacteria, in particular Corynebacterium glutamicum, characterized in that these have, in addition to at least one of the copy of an open reading frame (ORF), gene or allele of methionine production or threonine production present at the natural site (locus), in each case a second, optionally third or fourth copy of the open reading frame (ORF), gene or allele in question at in each case a second, optionally third or fourth site in integrated form, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics being present at the particular second, optionally third or fourth site,
    • 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, cysK, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd, glyA, hom, homFBR, lysC, lysCFBR, metA, metB, metE, metH, metY, msiK, opcA, oxyR, ppc, ppcFBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T. These are summarized and 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 homeFBR alleles which code for a “feed back” resistant homoserine dehydrogenase.


The second, optionally third or fourth copy of the open reading frame (ORF), gene or allele of 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, ccpA1, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, metD, metK, pck, pgi, poxB and zwa2. These are summarized and 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.


Examples of regions of the Corynebacterium glutamicum chromosome representing intergenic regions, prophages, defective phages or phage components are shown in tables 12 and 13. The positions of the DNA regions refer to the genome map of Corynebacterium glutamicum ATCC 13032 as presented in EP-A-1108790 or in the databank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK).

TABLE 4Open reading frames, genes and alleles of methionineproductionDescription of the coded enzyme orAccessNameproteinReferenceNumberAccBCAcyl-COA CarboxylaseJäger et al.U35023EC 6.3.4.14Archives of(acyl-COA carboxylase)Microbiology(1996) 166: 76-82EP1108790;AX123524WO0100805AX066441AccDAAcetyl-COA CarboxylaseEP1055725EC 6.4.1.2EP1108790AX121013(acetyl-CoA carboxylase)WO0100805AX066443AecDCystathionine beta-LyaseRossol et al.,M89931EC 4.4.1.8Journal of(cystathionine beta-lyase)Bacteriology174: 2968-2977(1992)CstACarbon Starvation Protein AEP1108790AX120811(carbon starvation protein A)WO0100804AX066109CysDSulfate AdenylyltransferaseEP1108790AX123177sub-unit IIEC 2.7.7.4(sulfate adenylyltransferase smallchainCysESerine AcetyltransferaseEP1108790AX122902EC 2.3.1.30WO0100843AX063961(serine acetyltransferase)CysH3′-Phosphoadenyl Sulfate ReductaseEP1108790AX123178EC 1.8.99.4WO0100842AX066001(3′-phosphoadenosine 5′-phosphosulfate reductase)CysKCysteine SynthaseEP1108790AX122901EC 4.2.99.8WO0100843AX063963(cysteine synthase)CysNSulfate Adenylyltransferase sub-EP1108790AK123176unit IAX127152EC 2.7.7.4(sulfate adenylyltransferase)CysQTransport protein CysQEP1108790AX127145(transporter cysQ)Wo0100805AX066423DpsDNA Protection ProteinEP1108790AX127153(protection during starvationprotein)EnoEnolaseEP1108790AX127146EC 4.2.1.11WO0100844AX064945(enolase)EP1090998AX136862Hermann et al.,Electrophoresis19: 3217-3221(1998)FdaFructose Bisphosphate Aldolasevan der Osten etX17313EC 4.1.2.13al., Molecular(fructose bisphosphate aldolase)Microbiology3: 1625-1637(1989)GapGlyceraldehyde 3-PhosphateEP1108790AX127148DehydrogenaseWO0100844AX064941EC 1.2.1.12Eikmanns et al.,X59403(glyceraldehyde 3-phosphateJournal ofdehydrogenase)Bacteriology174: 6076-6086(1992)gap2Glyceraldehyde 3-PhosphateEP1108790AX127146DehydrogenseWO0100844AX064939EC 1.2.1.12(glyceraldehyde 3-phosphatedehydrogenase 2)GdhGlutamate DehydrogenaseEP1108790AX127150EC 1.4.1.4WO0100844AX063811(glutamate dehydrogenase)Boermann et al.,X59404MolecularMicrobiology6: 317-326(1992);Guyonvarch etX72855al., NCBIGlyAGlycine/SerineEP1108790AX127146HydroxymethyltransferaseAX121194EC 2.1.2.1(glycine/serinehydroxymethyltransferase)Gnd6-Phosphogluconate DehydrogenaseEP1108790AX127147EC 1.1.1.44AX121689(6-phosphogluconate dehydrogenase)WO0100844AX065125HomHomoserine DehydrogenasePeoples et al.,Y00546EC 1.1.1.3Molecular(homoserine dehydrogenase)Microbiology2: 63-72 (1988)homFBRHomoserine Dehydrogenase feedbackReinscheid etresistant (fbr)al., Journal ofEC 1.1.1.3Bacteriology(homoserine dehydrogenase fbr)173: 3228-30(1991)LysCAspartate KinaseEP1108790AX120365EC 2.7.2.4WO0100844AX063743(aspartate kinase)Kalinowski etX57226al., MolecularMicrobiology5: 1197-204(1991)lysCFBRAspartate Kinase feedback resistantsee Table 2(fbr)EC 2.7.2.4(aspartate kinase fbr)MetAHomoserine AcetyltransferasePark et al.,AF052652EC 2.3.1.31Molecular Cells(homoserine acetyltransferase)8: 286-94 (1998)MetBCystathionine γ-LyaseHwang et al.,AF126953EC 4.4.1.1Molecular Cells(cystathionine gamma-synthase)9: 300-308 (1999)MetEHomocysteine MethyltransferaseEP1108790AX127146EC 2.1.1.14AX121345(homocysteine methyltransferase)MetHHomocysteine MethyltransferaseEP1108790AX127148(Vitamin B12-dependent)AX121747EC 2.1.1.14(homocysteine methyltransferase)MetYAcetylhomoserine SulfhydrolaseEP1108790AX120810(acetylhomoserine sulfhydrolase)AX127145MsiKSugar ImporterEP1108790AX120892(multiple sugar import protein)OpcAGlucose 6-phosphate DehydrogenaseWO0104325AX076272(subunit of glucose 6-phosphatedehydrogenase)OxyRTranscription RegulatorEP1108790AX122198(transcriptional regulator)AX127149ppcFBRPhosphoenol Pyruvate CarboxylaseEP0723011feedback resistentWO0100852EC 4.1.1.31(phosphoenol pyruvate carboxylasefeedback resistant)PpcPhosphoenol Pyruvate CarboxylaseEP1108790AX127148EC 4.1.1.31AX123554(phosphoenol pyruvate carboxylase)O'Reagan et al.,M25819Gene 77(2): 237-251(1989)PgkPhosphoglycerate KinaseEP1108790AX121838EC 2.7.2.3AX127148(phosphoglycerate kinase)WO0100844AX064943Eikmanns,X59403Journal ofBacteriology174: 6076-6086(1992)PknAProtein Kinase AEP1108790AX120131(protein kinase A)AX120085PknBProtein Kinase BEP1108790AX120130(protein kinase B)AX120085PknDProtein Kinase DEP1108790AX127150(protein Kinase D)AX122469AX122468PknGProtein Kinase GEP1108790AX127152(protein Kinase G)AX123109PpsAPhosphoenol Pyruvate SynthaseEP1108790AX127144EC 2.7.9.2AX120700(phosphoenol pyruvate synthase)AX122469PtsHPhosphotransferase System Protein HEP1108790AX122210EC 2.7.1.69AX127149(phosphotransferase systemWO0100844AX069154component H)PtsIPhosphotransferase System Enzyme IEP1108790AX122206EC 2.7.3.9AX127149(phosphotransferase systemenzyme I)PtsMGlucose-specific PhosphotransferaseLee et al., FEMSL18874System Enzyme IIMicrobiologyEC 2.7.1.69Letters 119(glucose phosphotransferase system(1-2): 137-145enzyme II)(1994)PycPyruvate CarboxylaseWO9918228A97276EC 6.4.1.1Peters-WendischY09548(pyruvate carboxylase)et al.,Microbiology144: 915-927(1998)PycPyruvate CarboxylaseEP1108790P458sEC 6.4.1.1(pyruvate carboxylase)amino acid exchange P458SSigCSigma Factor CEP1108790AX120368EC 2.7.7.6AX120085(extracytoplasmic functionalternative sigma factor C)SigDRNA Polymerase Sigma Factor DEP1108790AX120753EC 2.7.7.6AX127144(RNA polymerase sigma factor)SigESigma Factor EEP1108790AX127146EC 2.7.7.6AX121325(extracytoplasmic functionalternative sigma factor E)SigHSigma Factor HEP1108790AX127145EC 2.7.7.6AX120939(sigma factor SigH)SigMSigma Factor MEP1108790AX123500EC 2.7.7.6AX127153(sigma factor SigM)TalTransaldolaseWO0104325AX076272EC 2.2.1.2(transaldolase)ThyAThymidylate SynthaseEP1108790AX121026EC 2.1.1.45AX127145(thymidylate synthase)TktTransketolaseIkeda et al.,AB023377EC 2.2.1.1NCBI(transktolase)TpiTriose Phosphate IsomeraseEikmanns,X59403EC 5.3.1.1Journal of(triose phosphate isomerase)Bacteriology174: 6076-6086(1992)zwalCell Growth Factor 1EP1111062AX133781(growth factor 1)ZwfGlucose 6-phosphate 1-DehydrogenaseEP1108790AX127148EC 1.1.1.49AX121827(glucose 6-phosphate 1-WO0104325AX076272dehydrogenase)ZwfGlucose 6-phosphate 1-DehydrogenaseEP1108790A213TEC 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











Description




Gene
of the coded

Access


name
enzyme or protein
Reference
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., Archives
M89931



branched-chain amino
of Microbiology
AX066841



acids
169(4): 303-12 (1998)
AX127150



(branched-chain amino
WO0100805



acid transport system
EP1108790



carrier protein)


ccpA1
Catabolite Control
WO0100844
AX065267



Protein
EP1108790
AX127147



(catabolite control



protein A1)


ccpA2
Catabolite Control
WO0100844
AX065267



Protein
EP1108790
AX121594



(catabolite control



protein A2)


citA
Sensor Kinase CitA
EP1108790
AX120161



(sensor kinase CitA)


citB
Transcription
EP1108790
AX120163



Regulator CitB



(transcription



regulator CitB)


citE
Citrate Lyase
WO0100844
AX065421



EC 4.1.3.6
EP1108790
AX127146



(citrate lyase)


ddh
Diaminopimelate
Ishino et al., Nucleic
S07384



Dehydrogenase
Acids Research 15: 3917
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 transport
177(5): 1152-8 (1995)



system permease)


luxR
Transcription
WO0100842
AX065953



Regulator LuxR
EP1108790
AX123320



(transcription



regulator LuxR)


luxS
Histidine Kinase LuxS
EP1108790
AX123323



(histidine kinase LuxS)

AX127145


lysR1
Transcription
EP1108790
AX064673



Regulator LysR1

AX127144



(transcription



regulator LysR1)


lysR2
Transcription
EP1108790
AX123312



Activator LysR2



(transcription



regulator LysR2)


lysR3
Transcription
WO0100842
AX065957



Regulator LysR3
EP1108790
AX127150



(transcription



regulator LysR3)


menE
O-Succinylbenzoic
WO0100843
AX064599



Acid CoA Ligase
EP1108790
AX064193



EC 6.2.1.26

AX127144



(O-succinylbenzoate



CoA ligase)


metD
Transcription
EP1108790
AX123327



Regulator MetD

AX127153



(transcription



regulator MetD)


metK
Methionine Adenosyl
WO0100843
AX063959



Transferase
EP1108790
AX127148



EC 2.5.1.6



(S-adenosylmethionine



synthetase)


pck
Phosphoenol Pyruvate
WO0100844
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
WO0100844
AX064959



EC 1.2.3.3
EP1096013
AX137665



(pyruvate oxidase)


zwa2
Cell Growth Factor 2
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, sigM, tal, thyA, tkt, tpi, thrB, thrC, thrE, zwa1, 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 homFBR alleles which code for a “feed back” resistant homoserine dehydrogenase.


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


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


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


Examples of regions of the Corynebacterium glutamicum chromosome representing intergenic regions, prophages, defective phages or phage components are shown in tables 12 and 13. The positions of the DNA regions refer to the genome map of Corynebacterium glutamicum ATCC 13032 as presented in EP-A-1108790 or in the databank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK).

TABLE 6Open reading frames, genes and alleles of threonine productionDescription of the coded enzyme orAccessNameproteinReferenceNumberaccBCAcyl-CoA CarboxylaseJäger et al.U35023EC 6.3.4.14Archives of(acyl-CoA carboxylase)Microbiology166: 76-82 (1996)EP1108790AX123524WO0100805AX066441accDAAcetyl-CoA CarboxylaseEP1055725EC 6.4.1.2EP1108790AX121013(acetyl-CoA carboxylase)WO0100805AX066443cstACarbon Starvation Protein AEP1108790AX120811(carbon starvation protein A)WO0100804AX066109cysDSulfate AdenylyltransferaseEP1108790AX123177sub-unit IIEC 2.7.7.4(sulfate adenylyltransferase smallchain)cysESerine AcetyltransferaseEP1108790AX122902EC 2.3.1.30WO0100843AX063961(serine acetyltransferase)cysH3′-Phosphoadenyl Sulfate ReductaseEP1108790AX123178EC 1.8.99.4WO0100842AX066001(3′-phosphoadenosine 5′-phosphosulfate reductase)cysKCysteine SynthaseEP1108790AX122901EC 4.2.99.8WO0100843AX063963(cysteine synthase)cysNSulfate Adenylyltransferase sub-EP1108790AX123176unit IAX127152EC 2.7.7.4(sulfate adenylyltransferase)cysQTransport protein CysQEP1108790AX127145(transporter cysQ)WO0100805AX066423dpsDNA Protection ProteinEP1108790AX127153(protection during starvationprotein)enoEnolaseEP1108790AX127146EC 4.2.1.11WO0100844AX064945(enolase)EP1090998AX136862Hermann et al.,Electrophoresis19: 3217-3221(1998)fdaFructose Bisphosphate Aldolasevan der Osten etX17313EC 4.1.2.13al., Molecular(fructose bisphosphate aldolase)Microbiology3: 1625-1637(1989)gapGlyceraldehyde 3-PhosphateEP1108790AX127148DehydrogenaseWO0100844AX064941EC 1.2.1.12Eikmanns et al.,X59403(glyceraldehyde 3-phosphateJournal ofdehydrogenase)Bacteriology174: 6076-6086(1992)gap2Glyceraldehyde 3-PhosphateEP1108790AX127146DehydrogenaseWO0100844AX064939EC 1.2.1.12(glyceraldehyde 3-phosphatedehydrogenase 2)gdhGlutamate DehydrogenaseEP1108790AX127150EC 1.4.1.4WO0100844AX063811(glutamate dehydrogenase)Boermann et al.,X59404MolecularMicrobiology6: 317-326(1992);Guyonvarch etX72855al., NCBIgnd6-Phosphogluconate DehydrogenaseEP1108790AX127147EC 1.1.1.44AX121689(6-phosphogluconate dehydrogenase)WO0100844AX065125homHomoserine DehydrogenasePeoples et al.,Y00546EC 1.1.1.3Molecular(homoserine dehydrogenase)Microbiology2: 63-72 (1988)homFBRHomoserine Dehydrogenase feedbackReinscheid etresistant (fbr)al., Journal ofEC 1.1.1.3Bacteriology(homoserine dehydrogenase fbr)173: 3228-30(1991)lysCAspartate KinaseEP1108790AX120365EC 2.7.2.4WO0100844AX063743(aspartate kinase)Kalinowaki etX57226al., MolecularMicrobiology5: 1197-204(1991)lysCFBRAspartate Kinase feedback resistentsee Table 2(fbr)EC 2.7.2.4(aspartate kinase fbr)msiKSugar ImporterEP1108790AX120892(multiple sugar import protein)opcAGlucose 6-Phosphate DehydrogenaseWO0104325AX076272(subunit of glucose 6-phosphatedehydrogenase)oxyRTranscription RegulatorEP1108790AX122198(transcriptional regulator)AX127149ppcFBRPhosphoenol Pyruvate CarboxylaseEP0723011feedback resistentWO0100852EC 4.1.1.31(phosphoenol pyruvate carboxylasefeedback resistant)ppcPhosphoenol Pyruvate CarboxylaseEP1108790AX127148EC 4.1.1.31AX123554(phosphoenol pyruvate carboxylase)O'Reagan et al.,M25819Gene 77(2): 237-251(1989)pgkPhosphoglycerate KinaseEP1108790AX121838EC 2.7.2.3AX127148(phosphoglycerate kinase)WO0100844AX064943Eikmanns,X59403Journal ofBacteriology174: 6076-6086(1992)pknAProtein Kinase AEP1108790AX120131(protein kinase A)AX120085pknBProtein Kinase BEP1108790AX120130(protein kinase B)AX120085pknDProtein Kinase DEP1108790AX127150(protein kinase D)AX122469AX122468pknGProtein Kinase GEP1108790AX127152(protein kinase G)AX123109ppsAPhosphoenol Pyruvate SynthaseEP1108790AX127144EC 2.7.9.2AX120700(phosphoenol pyruvate synthase)AX122469ptsHPhosphotransferase System Protein HEP1108790AX122210EC 2.7.1.69AX127149(phosphotransferase systemWO0100844AX069154component H)ptsIPhosphotransferase System Enzyme IEP1108790AX122206EC 2.7.3.9AX127149(phosphotransferase systemenzyme I)ptsMGlukose-specific PhosphotransferaseLee et al., FEMSL18874System Enzyme IIMicrobiologyEC 2.7.1.69Letters 119(glucose phosphotransferase-system(1-2): 137-145enzyme II)(1994)pycPyruvate CarboxylaseWO9918228A97276EC 6.4.1.1Peters-WendischY09548(pyruvate carboxylase)et al.,Microbiology144: 915-927(1998)pycPyruvate CarboxylaseEP1108790P458SEC 6.4.1.1(pyruvate carboxylase)amino acid exchange P458SsigCSigma Factor CEP1108790AX120368EC 2.7.7.6AX120085(extracytoplasmic functionalternative sigma factor C)sigDRNA Polymerase Sigma Factor DEP1108790AX120753EC 2.7.7.6AX127144(RNA polymerase sigma factor)sigESigma Factor EEP1108790AX127146EC 2.7.7.6AX121325(extracytoplasmic functionalternative sigma factor B)sigHSigma Factor HEP1108790AX127145EC 2.7.7.6AX120939(sigma factor SigH)sigMSigma Factor MEP1108790AX123500EC 2.7.7.6AX127153(sigma factor SigM)talTransaldolaseWO0104325AX076272EC 2.2.1.2(transaldolase)thrBHomoserine KinasePeoples et al.,Y00546EC 2.7.1.39Molecular(homoserine kinase)Microbiology2: 63-72 (1988)thrCThreonine SynthaseHan et al.,X56037EC 4.2.99.2Molecular(threonine synthase)Microbiology4: 1693-1702(1990)thrEThreonine ExporterEP1085091AX137526(threonine export carrier)thyAThymidylate SynthaseEP1108790AX121026EC 2.1.1.45AX127145(thymidylate synthase)tktTransketolaseIkeda et al.,AB023377EC 2.2.1.1NCBI(transketolase)tpiTriose phosphate IsomeraseEikmanns,X59403EC 5.3.1.1Journal of(triose phosphate isomerase)Bacteriology174: 6076-6086(1992)zwalCell Growth Factor 1EP1111062AX133781(growth factor 1)zwfGlucose 6-Phosphate 1-DehydrogenaseEP1108790AX127148EC 1.1.1.49AX121827(glucose 6-phosphate 1-WO0104325AX076272dehydrogenase)zwfGlucose 6-Phosphate 1-DehydrogenaseEP1108790A213TEC 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 coded

Access


name
enzyme or portein
Reference
Number





ccpA1
Catabolite Control
WO0100844
AX065267



Protein
EP1108790
AX127147



(catabolite control protein A1)


ccpA2
Catabolite Control
WO0100844
AX065267



Protein
EP1108790
AX121594



(catabolite control



protein A2)


citA
Sensor Kinase CitA
EP1108790
AX120161



(sensor kinase CitA)


citB
Transcription Regulator
EP1108790
AX120163



CitB



(transcription regulator



CitB)


citE
Citrate Lyase
WO0100844
AX065421



EC 4.1.3.6
EP1108790
AX127146



(citrate lyase)


ddh
Diaminopimelate
Ishino et al., Nucleic Acids
S07384



Dehydrogenase
Research 15: 3917
AX127152



EC 1.4.1.16
(1987)



(diaminopimelate
EP1108790



dehydrogenase)


gluA
Glutamate Transport ATP-binding
Kronemeyer et al., Journal
X81191



Protein
of Bacteriology



(glutamate transport ATP-
177(5): 1152-8 (1995)



binding protein)


gluB
Glutamate-binding Protein
Kronemeyer 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.,
X81191



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
Möckel et al., Journal
A47044



EC 4.2.1.16
of Bacteriology 174
L01508



(threonine dehydratase)
(24), 8065-8072 (1992)
AX127150




EP1108790


ilvBN
Acetolactate Synthase
Keilhauer et al.,
L09232



EC 4.1.3.18
Journal of Bacteriology



(acetolactate synthase)
175(17): 5595-603 (1993)




EP1108790
AX127147


ilvC
Reductoisomerase
Keilhauer et al.,
C48648



EC 1.1.1.86
Journal of Bacteriology
AX127147



(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 Regulator
WO0100842
AX065953



LysR
EP1108790
AX123320



(transcription regulator



LuxR)


luxS
Histidine Kinase LuxS
EP1108790
AX123323



(histidine kinase LuxS)

AX127153


lysR1
Transcription Regulator
EP1108790
AX064673



LysR1

AX127144



(transcription regulator



LysR1)


lysR2
Transcription Activator
EP1108790
AX123312



LysR2



(transcription regulator



LysR2)


lysR3
Transcription Regulator
WO0100842
AX065957



LysR3
EP1108790
AX127150



(transcription regulator



LysR3)


mdh
Malate Dehydrogenase
WO0100844
AX064895



EC 1.1.1.37



(malate dehydrogenase)


menE
O-Succinylbenzoic Acid
WO0100843
AX064599



CoA Ligase
EP1108790
AX064193



EC 6.2.1.26

AX127144



(O-succinylbenzoate CoA



ligase)


metA
Homoserine O-
Park et al., Molecular
AX063895



Acetyltransferase
Cells 30; 8(3): 286-94
AX127145



EC 2.3.1.31
(1998)



(homoserine O-
WO0100843



acetyltransferase)
EP1108790


metD
Transcription Regulator
EP1108790
AX123327



MetD

AX127153



(transcription regulator



MetD)


pck
Phosphoenol Pyruvate
WO0100844
AJ269506



Carboxykinase

AX065053



(phosphoenol pyruvate



carboxykinase)


poxB
Pyruvate Oxidase
WO0100844
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 allele with nucleotide sequences of the target site,
  • c) preferably incorporating the nucleotide sequence of the desired ORF, gene or allele provided with nucleotide sequences of the target site into a vector which does not replicate or replicates to only a limited extent in coryneform bacteria,
  • d) transferring the nucleotide sequence according to b) or c) into coryneform bacteria, and
  • e) isolating coryneform bacteria in which the nucleotide sequence according to a) is incorporated at the target site, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remaining at the target site.


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, ptsi, ptsM, sigc, sigD, sigE, sigH, sigM, tpi, zwa1. These are summarized and explained in Table 8. These include in particular the ilvBN alleles which code for a valine-resistant acetolactate synthase.


The second, optionally third or fourth copy of the open reading frame (ORF), gene or allele of valine 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, ccpA1, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, glyA, ilvA, luxR, lysR1, lysR2, lysR3, panB, panC, poxB and zwa2. These are summarized and 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 nucleotide sequences without a coding function, can furthermore be used. Finally, prophages or defective phages contained in the chromosome can be used for this.


Examples of regions of the Corynebacterium glutamicum chromosome representing intergenic regions, prophages, defective phages or phage components are shown in tables 12 and 13. The positions of the DNA regions refer to the genome map of Corynebacterium glutamicum ATCC 13032 as presented in EP-A-1108790 or in the databank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK).

TABLE 8Open reading frames, genes and alleles of valine productionDescription of the coded enzyme orAccessNameproteinReferenceNumberbrnEFExport of branched-chain amino acidsEP1096010(branched chain amino acid export)Kennerknecht etAF454053al., NCBIcstACarbon Starvation Protein AEP1108790AX120811(carbon starvation protein A)WO0100804AX066109dpsDNA Protection ProteinEP1108790AX127153(protection during starvationprotein)enoEnolaseEP1108790AX127146EC 4.2.1.11WO0100844AX064945(enolase)EP1090998AX136862Hermann et al.,Electrophoresis19: 3217-3221(1998)fdaFructose Bisphosphate Aldolasevan der Osten et al.,X17313EC 4.1.2.13 (fructose bisphosphate aldolase)MolecularMicrobiology3: 1625-1637(1989)gapGlyceraldehyde 3-PhosphateEP1108790AX127148DehydrogenaseWO0100844AX064941EC 1.2.1.12Eikmanns et al.,X59403(glyceraldehyde 3-phosphateJournal ofdehydrogenase)Bacteriology174: 6076-6086(1992)gap2Glyceraldehyde 3-PhosphateEP1108790AX127146DehydrogenaseWO0100844AX064939EC 1.2.1.12(glyceraldehyde 3-phosphatedehydrogenase 2)gdhGlutamate DehydrogenaseEP1108790AX127150EC 1.4.1.4WO0100844AX063811(glutamate dehydrogenase)Boermann et al.,X59404MolecularMicrobiology6: 317-326(1992);Guyonvarch etX72855al., NCBIilvBNAcetolactate SynthaseKeilhauer etL09232EC 4.1.3.18al., Journal of(acetolactate synthase)Bacteriology175(17): 5595-603(1993)EP1108790AX127147ilvCIsomeroreductaseKeilhauer etC48648EC 1.1.1.86al., Journal ofAX127147(acetohydroxy acidBacteriologyisomeroreductase)175(17): 5595-603(1993)EP1108790ilvDDihydroxy-acid DehydrataseEP1006189AX136925EC 4.2.1.9(dihydroxy acid dehydratase)ilvETransaminase BEP1108790AX127150EC 2.6.1.42AX122498(transaminase B)msiKSugar ImporterEP1108790AX120892(multiple sugar import protein)pgkPhosphoglycerate KinaseEP1108790AX121838EC 2.7.2.3AX127148(phosphoglycerate kinase)WO0100844AX064943Eikmanns,X59403Journal ofBacteriology174: 6076-6086(1992)ptsHPhosphotransferase System Protein HEP1108790AX122210EC 2.7.1.69AX127149(phosphotransferase systemWO0100844AX069154component H)ptsIPhosphotransferase System Enzyme IEP1108790AX122206EC 2.7.3.9AX127149(phosphotransferase systemenzyme I)ptsMGlucose-specific PhosphotransferaseLee et al., FEMSL18874System Enzyme IIMicrobiologyEC 2.7.1.69Letters 119(glucose phosphotransferase-system(1-2): 137-145enzyme II)(1994)sigCSigma Factor CEP1108790AX120368EC 2.7.7.6AX120085(extracytoplasmic functionalternative sigma factor C)sigDRNA Polymerase Sigma Factor DEP1108790AX120753EC 2.7.7.6AX127144(RNA polymerase sigma factor)sigESigma Factor EEP1108790AX127146EC 2.7.7.6AX121325(extracytoplasmic functionalternative sigma factor E)sigHSigma Factor HEP1108790AX127145EC 2.7.7.6AX120939(sigma factor SigH)sigMSigma Factor MEP1108790AX123500EC 2.7.7.6AX127153(sigma factor SigM)tpiTriose Phosphate IsomeraseEikmanns,X59403EC 5.3.1.1Journal of(triose phosphate isomerase)Bacteriology174: 6076-6086(1992)zwalCell Growth Factor 1EP1111062AX133781(growth factor 1)









TABLE 9










Target sites for integration of open reading frames, genes


and alleles of valine production










Gene
Description of the coded

Access


name
enzyme or protein
Reference
Number





aecD
beta C-S Lyase
Rossol et al., Journal
M89931



EC 2.6.1.1
of Bacteriology



(beta C-S lyase)
174(9): 2968-77 (1992)


ccpA1
Catabolite Control
WO0100844
AX065267



Protein
EP1108790
AX127147



(catabolite control



protein A1)


ccpA2
Catabolite Control
WO0100844
AX065267



Protein
EP1108790
AX121594



(catabolite control



protein A2)


citA
Sensor Kinase CitA
EP1108790
AX120161



(sensor kinase CitA)


citB
Transcription Regulator
EP1108790
AX120163



CitB



(transcription regulator



CitB)


citE
Citrate Lyase
WO0100844
AX065421



EC 4.1.3.6
EP1108790
AX127146



(citrate lyase)


ddh
Diaminopimelate
Ishino et al., Nucleic Acids
S07384



Dehydrogenase
Research 15: 3917
AX127152



EC 1.4.1.16
(1987)



(diaminopimelate dehydrogenase)
EP1108790


gluA
Glutamate Transport ATP-
Kronemeyer et al.,
X81191



binding Protein
Journal of Bacteriology



(glutamate transport ATP-
177(5): 1152-8 (1995)



binding protein)


gluB
Glutamate-binding Protein
Kronemeyer 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.,
X81191



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
Möckel et al., Journal
A47044



EC 4.2.1.16
of Bacteriology 174
L01508



(threonine dehydratase)
(24), 8065-8072 (1992)
AX127150




EP1108790


luxR
Transcription Regulator
WO0100842
AX065953



LuxR
EP1108790
AX123320



(transcription regulator



LuXR)


lysR1
Transcription Regulator
EP1108790
AX064673



LysR1

AX127144



(transcription regulator



LysR1)


lysR2
Transcription Activator
EP1108790
AX123312



LysR2



(transcription regulator



LysR2)


lysR3
Transcription Regulator
WO0100842
AX065957



LysR3
EP1108790
AX127150



(transcription regulator



LysR3)


panB
Ketopantoate
US6177264
X96580



Hydroxymethyltransferase



EC 2.1.2.11



(ketopantoate



hydroxymethyltransferase)


panC
pantothenate Synthetase
US6177264
X96580



EC 6.3.2.1



(pantothenate synthetase)


poxB
Pyruvate Oxidase
WO0100844
AX064959



EC 1.2.3.3
EP1096013
AX137665



(pyruvate oxidase)


zwa2
Cell Growth Factor 2
EP1106693
AX113822



(growth factor 2)
EP1108790
AX127146









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

  • a) isolating the nucleotide sequence of at least one desired ORF, gene or allele of valine production, optionally including the expression and/or regulation signals,
  • b) providing the 5′ and the 3′ end of the ORF, gene or allele with nucleotide sequences of the target site,
  • c) preferably incorporating the nucleotide sequence of the desired ORF, gene or allele provided with nucleotide sequences of the target site into a vector which does not replicate or replicates to only a limited extent in coryneform bacteria,
  • d) transferring the nucleotide sequence according to b) or c) into coryneform bacteria, and
  • e) isolating coryneform bacteria in which the nucleotide sequence according to a) is incorporated at the target site, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remaining at the target site.


The invention also furthermore provides a process for the preparation of L-tryptophane, 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 tryptophane 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 tryptophane in the fermentation broth,
  • c) isolation of the tryptophane 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 tryptophane 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 tryptophane production.


These include, inter alia, the following open reading frames, genes or alleles: aroA, aroB, aroC, aroD, aroE, aroG, aroK, cstA, eno, gap, gap2, gnd, ppsA, rpe, serA, serB, serC, tal, thyA, tkt, tpi, trpA, trpB, trpC, trpD optionally comprising at least one of the amino acid exchanges selected from the group consisting of A21ST (exchange of alanine at position 215 against threonine), D138A (exchange of aspartic acid at position 138 against alanine), S149F (exchange of serine at position 149 against phenylalanine) and A162E (exchange of alanine at position 162 against glutamic acid), trpE, trpEFBR comprising e.g. the amino acid exchange S38R (exchange of serine at position 38 against arginine), trpG, trpL optionally comprising the mutation W14*, zwa1, zwf optionally comprising the amino acid exchange A213T (exchange of alanine at position 213 against threonine). These are summarized and explained in Table 10. These include in particular the tryptophane operon comprising trpE, trpG, trpD, trpC and trpA and optionally trpL. Furthermore these include in particular a trpEFBR allele which codes for a tryptophane-resistant anthranilate synthase.


The second, optionally third or fourth copy of the open reading frame (ORF), gene or allele of tryptophane 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: ccpA1, ccpA2, citA, citB, citE, cysE, gluA, gluB, gluC, gluD, glyA, luxR, luxS, lysR1, lysR2, lysR3, menE, pgi, pheA, poxB and zwa2. These are summarized and explained in Table 11.


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.


Examples of regions of the Corynebacterium glutamicum chromosome representing intergenic regions, prophages, defective phages or phage components are shown in tables 12 and 13. The positions of the DNA regions refer to the genome map of Corynebacterium glutamicum ATCC 13032 as presented in EP-A-1108790 or in the databank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK).

TABLE 10Open reading frames, genes and alleles of tryptophaneproductionDescription of the coded enzyme orAccess-Gene nameproteinReferenceNumberaroAEnolpyruvylsahikimate PhosphateO'Donohue etAF114233Synthaseal., NCBIEC 2.5.1.19(enolpyruvylshikimate 3-phosphatesynthase)aroBDehydroquinate SynthetaseBurke et al.,AF124600EC 4.6.1.3NCBI(dehydroquinate synthetase)aroCChorismate SynthaseBurke et al.,AF124600EC 4.6.1.4NCBI(chorismate synthase)aroDDehydroquinate DehydrataseJoy et al.,AF124518EC 4.2.1.10NCBI(dehydroquinate dehydratase)aroEShikimate DehydrogenaseJoy et al.,AF124518EC 1.1.1.25NCBI(shikimate dehydrogenase)aroGDehydro-3-DeoxyphosphoheptonateChen et al.,L07603AldolaseFEMSEC4.1.2.15Microbioliology(dehydro-3-deoxyphosphoheptonateLettersaldolase)107: 223-230(1993).aroKShikimate KinaseBurke et al.,AF124600EC 2.7.1.71NCBI(shikimate kinase)cstACarbon Starvation Protein AEP1108790AX120811(carbon starvation protein A)WO0100804AX066109enoEnolaseEP1108790AX127146EC 4.2.1.11WO0100844AX064945(enolase)EP1090998AX136862Hermann et al.,Electrophoresis19: 3217-3221(1998)gapGlyceraldehyde-3-PhosphateEP1108790AX127148DehydrogenaseWO0100844AX064941EC 1.2.1.12Eikmanns etX59403(glyceraldehyde-3-phosphateal., Journal ofdehydrogenase)Bacteriology174: 6076-6086(1992)gap2Glyceraldehyde-3-PhosphateEP1108790AX127146DehydrogenaseWO0100844AX064939EC 1.2.1.12(glyceraldehyde-3-phosphatedehydrogenase 2)gnd6-Phosphogluconate DehydrogenaseEP1108790AX127147EC 1.1.1.44AX121689(6-phosphogluconate dehydrogenase)WO0100844AX065125ppsAPhosphoenolpyruvate SynthetaseEP1108790AX127144Ec 2.7.9.2AX120700(phosphoenolpyruvate-synthase)rpeRibulose-Phosphate EpimeraseEP1108790AX127148EC 5.1.3.1AX121852(ribulose-phosphate-epimerase)serAPhosphoglycerate DehydrogenaseEP1108790AX127147EC1.1.1.95AX121499(phosphoglycerate-dehydrogenase)serBPhosphoserine PhosphataseEP1108790AX127144EC 3.1.3.3AX120551(phosphoserine phosphatase)serCPhosphoserine AminotransferaseEP1108790AX127145EC 2.6.1.52AX121012(phosphoserine aminotransferase)talTransaldolaseWO0104325AX076272EC 2.2.1.2(transaldolase)thyAThymidylate SynthaseEP1108790AX121026EC 2.1.1.45AX127145(thymidylate synthase)tktTransketolaseIkeda et al.,AB023377EC 2.2.1.1NCBI(transketolase)tpiTriose-phosphate IsomeraseEikmanns,X59403EC 5.3.1.1Journal of(triose-phosphate isomerase)Bacteriology174: 6076-6086(1992)trpATryptophane Synthase (alpha Kette)Matsui et al.,X04960EC 4.2.1.20Nucleic Acids(tryptophan synthase (alpha chain))Research14: 10113-10114(1986)trpBTryptophane Synthase (beta Kette)Matsui et al.,X04960EC 4.2.1.20Nucleic Acids(tryptophan synthase (beta chain))Research14: 10113-10114(1986)trpCPhosphoribosylanthranilateMatsui et al.,X04960IsomeraseNucleic AcidsEC 5.3.1.24Research(phosphoribosylanthranilate14: 10113-10114isomerase)(1986)trpDAnthranilateMatsui et al.,X04960PhosphoribosyltransferaseNucleic AcidsEC 2.4.2.18Research(anthranilate14: 10113-10114phosphoribosyltransferase)(1986)trpDAnthranilateO'Gara et al.,A125T,PhosphoribosyltransferaseApplied andD138A,EC 2.4.2.18EnvironmentalS149F,anthranilateMicrobiologyA162E(phosphoribosyltransferase)61: 4477-4479amino acid exchanges A125T, D138A,(1995)S149F, A162EtrpEAnthranilate Synthase Komponente IMatsui et al.,X04960EC 4.1.3.27Nucleic Acids(anthranilate synthase component I)Research14: 10113-10114(1986)trpEAnthranilat Synthase Component IMatsui et al.,fbrfeedback resistentJournal ofEC 4.1.3.27Bacteriology(anthranilate synthase component I169: 5330-5332feedback resistant)(1987)trpGAnthranilate Synthase Komponente IIMatsui et al,X04960EC 4.1.3.24Nucleic Acids(anthranilate synthase componentResearchII)14: 10113-10114(1986)trpLTrp Operon Leader PeptideMatsui et al.,X04960(trp operon leader peptide)Nucleic AcidsResearch14: 10113-10114(1986)trpLTrp Operon LeaderpeptidHerry et al.,W14*(trp operon leader peptideApplied andmutation W14*)EnvironmentalMicrobiology59: 791-799(1993)zwalCell Growth Factor 1EP1111062AX133781(growth factor 1)zwfGlucose-6-phosphat1-1-DehydrogenaseEP1108790AX127148EC 1.1.1.49AX121827(glucose-6-phosphate-1-WO0104325AX076272dehydrogenase)zwfGlucose-6-phosphate-1-DehydrogenaseEP1108790A213TEC 1.1.1.49(glucose-6-phosphate-1-dehydrogenase)amino acid exchange A213T









TABLE 11










Target sites for integration of open reading frames, genes


and alleles of tryptophane production











Description




Gene
of the coded

Access


name
enzyme or protein
Reference
Number





ccpA1
Catabolite Control
WO0100844
AX065267



Protein
EP1108790
AX127147



(catabolite control



protein A1)


ccpA2
Catabolite Control
WO0100844
AX065267



Protein
EP1108790
AX121594



(catabolite control



protein A2)


citA
Sensor-Kinase CitA
EP1108790
AX120161



(sensor kinase CitA)


citB
Transcription
EP1108790
AX120163



Regulator CitB



(transcription regulator



CitB)


citE
Citrate-Lyase
WO0100844
AX065421



EC 4.1.3.6
EP1108790
AX127146



(citrate lyase)


cysE
Serine O-
EP1108790
AX122902



Acetyltransferase



EC 2.3.1.30



(serine O-



acetyltransferase)


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 (glutamate
Journal of Bacteriology



binding 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.,
X81191



Permease
journal of Bacteriology



(glutamate transport
177(5): 1152-8 (1995)



system permease)


glyA
glycine
JP1997028391
E12594



hydroxymethyl-



transferase EC 2.1.2.1



(glycine



hydroxymethyl-



transferase)


luxR
Transkription
WO0100842
AX065953



Regulator LuxR
EP1108790
AX123320



(transcription regulator



LuxR)


luxS
Histidine Kinase LuxS
EP1108790
AX123323



(histidine kinase LuxS)

AX127153


lysR1
Transkription
EP1108790
AX064673



Regulator LysR1

AX127144



(transcription regulator



LysR1)


lysR2
Transkription
EP1108790
AX123312



Activator LysR2



(transcription regulator



LysR2)


lysR3
Transkription
WO0100842
AX065957



Regulator LysR3
EP1108790
AX127150



(transcription regulator



LysR3)


menE
O-Succinylbenzoic
WO0100843
AX064599



acid-CoA-Ligase
EP1108790
AX064193



EC 6.2.1.26

AX127144



(O-succinylbenzoate-



CoA ligase)


pgi
Glucose-6-Phosphate-
EP1087015
AX136015



Isomerase
EP1108790
AX127146



EC 5.3.1.9



(glucose-6-phosphate



isomerase)


pheA
Prephenate
Follettie et al.,
M13774



Dehydratase
Journal of Bacteriology



EC 4.2.1.51
167: 695-702 (1986)



(prephenate



dehydratase)


poxB
pyruvate-Oxidase
WO0100844
AX064959



EC 1.2.3.3
EP1096013
AX137665



(pyruvate oxidase)


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-valine, which comprises

  • a) isolating the nucleotide sequence of at least one desired ORF, gene or allele of valine production, optionally including the expression and/or regulation signals,
  • b) providing the 5′ and the 3′ end of the ORF, gene or allele with nucleotide sequences of the target site,
  • c) preferably incorporating the nucleotide sequence of the desired ORF, gene or allele provided with nucleotide sequences of the target site into a vector which does not replicate or replicates to only a limited extent in coryneform bacteria,
  • d) transferring the nucleotide sequence according to b) or c) into coryneform bacteria, and


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

TABLE 12Intergenic regions as target sites for integration of openreading frames, genes and allelesPosition ofPosition ofAccesssequencesequenceReferencenumberstartendEP1108790AX120085192176194501EP1108790AX127145235840237311EP1108790AX127145236096237311EP1108790AX127148322628330877EP1108790AX127148334045336467EP1108790AX127148289565291841EP1108790AX127149154823161111EP1108790AX127149190088193497EP1108790AX1271492739828707EP1108790AX1271496147862944EP1108790AX127149116234117561EP1108790AX127149140847144605EP1108790AX127150113274114324EP1108790AX127152244281246403









TABLE 13










Target sites coding for phages or phage components suitable


for integration of open reading frames, genes and alleles














Position of
Position of




Access
sequence
Sequence



Reference
number
start
end
















EP1108790
AX127149
50474
51049



EP1108790
AX127149
67886
68587



EP1108790
AX127151
72893
73480



EP1108790
AX127149
88231
89445



EP1108790
AX127148
139781
140155



EP1108790
AX127148
140546
141001



EP1108790
AX127149
194608
195294



EP1108790
AX127147
200185
200940



EP1108790
AX127147
208157
208450



EP1108790
AX127149
269616
269948



EP1108790
AX127148
336468
338324



EP1108790
AX127148
342235
342681



EP1108790
AX127148
343518
345356



EP1108790
AX127148
345872
346207










During work on the present invention, it was possible to incorporate a second copy of an lysCFBR allele into the gluB gene of Corynebacterium glutamicum such that no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remained at the gluB gene site. This strain, which is called DSM13994glu::lysC, carries the lysCFBR allele lysC T311I at its natural lysC site and a second copy of the lysCFBR allele lysC T311I at a second site (target site), namely the gluB gene. A plasmid with the aid of which the incorporation of the lysC R allele into the gluB gene can be achieved is shown in FIG. 1. It carries the name pK18mobsacBglu11.


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 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 DSM12866glu::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 für Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures). A plasmid with the aid of which the incorporation of the lysCFBR allele into the gluB gene can be achieved is shown in FIG. 1. It carries the name pK18mobsacBglu11.


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 T3111 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 FIG. 2. It carries the name pK18mobsacBaecD11.


During work on the present invention, it was furthermore possible to incorporate a copy of an lysCFBR 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 DSM12866 pck::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 pck gene. A plasmid with the aid of which the incorporation into the pck gene can be achieved is shown in FIG. 3. It carries the name pK18mobsacBpck11.


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 DSM12866glu::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 FIG. 4. It carries the name pK18mobsacBgluB21.


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 FIG. 5. It carries the name pK18mobsacBaecD21.


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 DSM12866 pck::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 FIG. 6. It carries the name pK18mobsacBpck13.


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


The culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).


Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e.g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and organic acids, such as e.g. acetic acid or lactic acid, can be used as the source of carbon. These substances can be used individually or as a mixture.


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


Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus.


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


Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH of the culture. Antifoams, such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, such as e.g. antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as e.g. air, are introduced into the culture. The temperature of the culture is usually 20° C. to 45° C., and preferably 25° C. 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 the invention, in particular the coryneform bacteria which produce L-lysine, have an unexpectedly high stability. They were stable for at least 10-20, 20-30, 30-40, 40-50, preferably at least 50-60, 60-70, 70-80 and 80-90 generations or cell division cycles.


The following microorganisms have been deposited:


The strain Corynebacterium glutamicum DSM12866glu::lysC was deposited in the form of a pure culture on 5 Jun. 2002 under number DSM15039 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ=German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty.


The plasmid pK18mobsacBglu11 was deposited in the form of a pure culture of the strain E. coli DH5αmcr/pK18mobsacBglu11 (=DH5alphamcr/pK18mobsacBglu11) on 20 Apr. 2001 under number DSM14243 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.


The plasmid pK18mobsacBaecD11 was deposited in the form of a pure culture of the strain E. coli DH5αmcr/pK18mobsacBaecD11 (=DH5alphamcr/pK18mobsacBaecD11) on 5 Jun. 2002 under number DSM15040 at the Deutsche Sammlung für Mikroorganismen und 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 DSM12866


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


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


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


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

lysC1beg (SEQ ID No: 5):5′ TA(G GAT CC)T CCG GTG TCT GAC CAC GGT G 3′lysC2end: (SEQ ID NO: 6):5′ AC(G GAT CC)G CTG GGA AAT TGC GCT CTT CC 3′


The primers 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/l)-containing LB agar with X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside, 64 mg/l).


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 NO:4.


The base thymine is found at position 932 of the nucleotide sequence of the coding region of the lysC5R allele of strain DSM13994 (SEQ ID NO:3). The base cytosine is found at the corresponding position of the wild-type gene (SEQ ID NO: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 20 Apr. 2001 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.


1.2 Construction of the Replacement Vector pK18mobsacBglu11


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

gluBgl1 (SEQ ID NO: 7):5′ TA(A GAT CT)G TGT TGG ACG TCA TGG CAA G 3′gluBgl2 (SEQ ID NO: 8):5′ AC(A GAT CT)T GAA GCC AAG TAC GGC CAA G 3′


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


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


Ligation of the fragment is then carried out by means of the TOPO TA Cloning Kit (Invitrcgen, 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/l)-containing LB agar with X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside, 64 mg/l).


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


The plasmid pCRII-TOPOglu is cleaved with the restriction enzyme 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 gluB fragment of approx. 1.7 kb is isolated from the agarose gel and employed for ligation with the mobilizable cloning vector pK18mobsacB described by Schäfer et al. (Gene 14: 69-73 (1994)). This is cleaved beforehand with the restriction enzyme BamHI and dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim), mixed with the gluB fragment of approx. 1.7 kb, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany).


The E. coli strain DH5α (Grant et al.; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, N.Y., 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, N.Y., 1989), which is supplemented with 50 mg/l 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 pK18mobsacBglu1.


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 lysCFBR-containing DNA fragment of approx. 1.7 kb in length was isolated from the agarose gel and employed for ligation with the vector pK18mobsacBglu1 described above. This is cleaved beforehand with the restriction enzyme BamHI, 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 Ligase (Amersham-Pharmacia, Freiburg, Germany).


The E. coli strain DH5αmcr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, N.Y., 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, N.Y., 1989), which was supplemented with 50 mg/l 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 pK18mobsacBglu11. A map of the plasmid is shown in FIG. 1.


The plasmid pK18mobsacBglu11 was deposited in the form of a pure culture of the strain E. coli DH5αmcr/pK18mobsacBglu11 (=DH5alphamcr/pK18mobsacBglu11) under number DSM14243 on 20.04.2001 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.


1.3 Incorporation of a Second Copy of the lysCFBR Allele lysC T311I into the Chromosome (Target Site: gluB Gene) of the Strain DSM13994 by Means of the Replacement Vector pK18mobsacBglu11


The vector pK18mobsacBglu11 described in Example 1.2 is transferred by the protocol of Schäfer 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 pK18mobsacBglu11 is made by plating out the conjugation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor, N.Y., 1989), which is supplemented with 15 mg/l kanamycin and 50 mg/l nalidixic acid. Kanamycin-resistant transconjugants are plated out on LB agar plates with 25 mg/l 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 pK18mobsacBglu11, like the starting plasmid pK18mobsacB, 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 pK18mobsacBglu11 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.

gluBgl1 (SEQ ID NO: 7):5′ TA(A GAT CT)G TGT TGG ACG TCA TGG CAA G 3′gluBgl2 (SEQ ID NO: 8):5′ AC(A GAT CT)T GAA GCC AAG TAC GGC CAA G 3′


The primers 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 0.8% agarose gel.


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


1.4 Incorporation of a Second Copy of the lysC Gene in the Form of the lysCFBR Allele lysC T311I into the Chromosome (Target Site: gluB Gene) of the Strain DSM12866 by Means of the Replacement Vector pK18mobsacBglu11


As described in Example 1.3, the plasmid pK18mobsacBglu11 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 DSM12866glu::lysC.


The Corynebacterium glutamicum strain according to the invention which carries a second copy of an lysCFBR allele in the gluB gene was deposited in the form of a pure culture of the strain Corynebacterium glutamicum DSM12866glu::lysC on 5 Jun. 2002 under number DSM15039 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.


1.5 Construction of the Replacement Vector pK18mobsacBpck11


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

pck_beg (SEQ ID NO: 9):5′ TA(A GAT CT)G CCG GCA TGA CTT CAG TTT 3′pck_end (SEQ ID NO: 10):5′ AC(A GAT CT)G GTG GGA GCC TTT CTT GTT ATT3′


The primers shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press). The primers allow amplification of a DNA fragment of approx 2.9 kb in size, which carries the pck gene and adjacent regions. The primers moreover contain the sequence for the cleavage site of the restriction endonuclease 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 identified by means of electrophoresis in a 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden).


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


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 pK18mobsacB described by Schäfer et al. (Gene 14: 69-73 (1994)). This is cleaved beforehand with the restriction enzyme BamHI and dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim), mixed with the pck fragment of approx. 2.9 kb, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany).


The E. coli Strain DH5α (Grant et al.; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, N.Y., 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, N.Y., 1989), which is supplemented with 50 mg/l 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 pK18mobsacBpck1.


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 lysCFBR-containing DNA fragment approx. 1.7 kb long was isolated from the agarose gel and employed for ligation with the vector pK18mobsacBpck1 described above. This is cleaved beforehand with the restriction enzyme BamHI, 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 Ligase (Amersham-Pharmacia, Freiburg, Germany).


The E. coli strain DH5αmcr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, N.Y., 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, N.Y., 1989), which was supplemented with 50 mg/l 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 pK18mobdsacBpck11. A map of the plasmid is shown in FIG. 3.


1.6 Incorporation of a Second Copy of the lysC Gene in the Form of the lysCFBR Allele lysC T311I into the Chromosome (Target Site: pck Gene) of the Strain DSM12866 by Means of the Replacement Vector pK18mobsacBpck11


As described in Example 1.3, the plasmid pK18mobsacBpck11 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 NO: 9):5′ TA(A GAT CT)G CCG GCA TGA CTT CAG TTT 3′pck_end (SEQ ID NO: 10):5′ AC(A GAT CT)G GTG GGA GCC TTT CTT GTT ATT3′


The primers 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 lysCFBR allele lysC T311I at the pck locus in the chromosome was identified in this manner. This clone was called strain DSM12866 pck::lysC.


1.7 Construction Of the Replacement Vector pK18mobsacBaecD11


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 al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press). The primers allow amplification of a DNA fragment of approx 2.1 kb in size, which carries the aecD gene and adjacent regions.


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


The DNA fragment purified is cleaved with the restriction enzyme BamHI and EcoRV (Amersham Pharmacia, Freiburg, Germany). The ligation of the fragment in the vector pUC18 then takes place (Norrander et al., Gene 26:101-106 (1983)). This is cleaved beforehand with the restriction enzymes BglII and SmaI, 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/l)-containing LB agar with X-Gal (64 mg/l).


The plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel. The resulting plasmid is called pUC18aecD.


Plasmid DNA was isolated from the strain DSM14242 (see Example 1.1) which carries the plasmid pCRIITOPOlysC and cleaved with the restriction enzyme BamHI (Amersham-Pharmacia, Freiburg, Germany) and then treated with Klenow polymerase. After separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the lysCFBR-Containing DNA fragment approx. 1.7 kb in length is isolated from the agarose gel and employed for ligation with the vector pUC18aecD described above. This is 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 Ligase (Amersham-Pharmacia, Freiburg, Germany).


The E. coli strain DH5αmcr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, N.Y., 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, N.Y., 1989), which was supplemented with 50 mg/l 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 pUC18aecD1.


The plasmid pUC18aecD1 is cleaved with the restriction enzyme KpnI and then treated with Klenow polymerase. The plasmid is then cleaved with the restriction enzyme SalI (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 pK18mobsacB described by Schäfer et al. (Gene 14: 69-73 (1994)). This is cleaved beforehand with the restriction enzymes SmaI and SalI and dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim), mixed with the fragment of approx. 3.2 kb which carries aecD and lysC, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany).


The E. coli strain DH5α (Grant et al.; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, N.Y., 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, N.Y., 1989), which is supplemented with 50 mg/l 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 pK18mobsacBaecD11. A map of the plasmid is shown in FIG. 2.


The plasmid pK18mobsacBaecD11 was deposited in the form of a pure culture of the strain E. coli DH5αmcr/pK18mobsacBaecD11 (=DH5alphamcr/pK18mobsacBaecD11) on 5 Jun. 2002 under number DSM15040 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.


1.8 INCORPORATION of a Second Copy of the lysC Gene as the lysCFBR Allele into the Chromosome (Target Site: aecD Gene) of the Strain DSM12866 by Means of the Replacement Vector pK18mobsacBaecD11


As described in Example 1.3, the plasmid pK18mobsacBaecD11 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 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 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 lysCFBR 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 0.8% 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 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 pK18mobsacBglu21


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 Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press). The primers allow amplification of a DNA fragment of approx 4.4 kb in size, which carries the gluB gene and surrounding regions.


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


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


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 pCRII-TOPOglu2 is cleaved with the restriction enzymes EcoRI and SalI (Amersham-Pharmacia, Freiburg, Germany) and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the gluB fragment of approx. 3.7 kb is isolated from the agarose gel and employed for ligation with the mobilizable cloning vector pK18mobsacB described by Schafer 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 T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany).


The E. coli Strain DH5α (Grant et al.; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, N.Y., 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, N.Y., 1989), which is supplemented with 50 mg/l 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 pK18mobsacBglu2.


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

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 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden).


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


The E. coli strain DH5αmcr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, N.Y., 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, N.Y., 1989), which was supplemented with 50 mg/l 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 pK18mobsacBglu21. A map of the plasmid is shown in FIG. 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 pK18mobsacBglu21


As described in Example 1.3, the plasmid pK18mobsacBglu21 described in Example 2.1 is transferred into the C. glutamicum strain DSM12866 by conjugation. Selection is made for targeted recombination events in the chromosome of C. glutamicum DSM12866 as described in Example 1.3. Depending on the position of the second recombination event, after the excision the second copy of the ddh gene manifests itself in the chromosome at the gluB locus, or the original gluB locus of the host remains.


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

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


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


A clone which, in addition to the copy present at the ddh locus, has a second copy of the ddh gene at the gluB locus in the chromosome was identified in this manner. This clone was called strain DSM12866glu::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 pK18mobsacBaecD21


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 al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press). The primers allow amplification of a DNA fragment of approx 2.1 kb in size, which carries the aecD gene and adjacent regions.


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


The DNA fragment purified is cleaved with the restriction enzyme BglII and EcoRV (Amersham Pharmacia, Freiburg, Germany). The ligation of the fragment in the vector pUC18 then takes place (Norrander et al., Gene 26:101-106 (1983)). This is cleaved beforehand with the restriction enzymes BamHI and SmaI 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/l)-containing LB agar with X-Gal (64 mg/l).


The plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel. The resulting plasmid is called pUC18aecD.


With the aid of the polymerase chain reaction, a further DNA fragment which carries the dapA gene and surrounding regions is amplified. On the basis of the sequence of the dapA gene known for C. glutamicum (Bonassi et al., Nucleic Acids Research 18:6421 (1990)) (Accession Number X53993 and AX127149), the following primer oligonucleotides are chosen for the PCR:

dapA_beg (SEQ ID NO: 17):5′ CGA GCC AGT GAA CAT GCA GA 3′dapA_end (SEQ ID NO: 18):5′ CTT GAG CAC CTT GCG CAG CA 3′


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


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


After purification, the dapA-containing DNA fragment approx. 1.4 kb in length is employed for ligation with the vector pUC18aecD described above. This is cleaved beforehand with the restriction enzyme StuI, mixed with the DNA fragment of approx. 1.4 kb, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany).


The E. coli strain DH5αmcr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, N.Y., 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, N.Y., 1989), which was supplemented with 50 mg/l kanamycin.


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


The plasmid pUC18aecD2 is cleaved with the restriction enzyme SalI and partly with EcoRI (Amersham-Pharmacia, Freiburg, Germany) and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the fragment of approx. 2.7 kb which carries aecD and dapA is isolated from the agarose gel and employed for ligation with the mobilizable cloning vector pK18mobsacB described by Schäfer 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 Ligase (Amersham-Pharmacia, Freiburg, Germany).


The E. coli strain DH5α (Grant et al.; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, N.Y., 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, N.Y., 1989), which is supplemented with 50 mg/l 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 pK18mobsacBaecD21. A map of the plasmid is shown in FIG. 5.


3.2 Incorporation of a Second Copy of the dapA Gene into the Chromosome (Target Site: aecD Gene) of the Strain DSM12866 by Means of the Replacement Vector pK18mobsacBaecD21


As described in Example 1.3, the plasmid pK18mobsacBaecD21 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 aecD locus of the host remains.


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

aecD_beg (SEQ ID NO: 11):5′ GAA CTT ACG CCA AGC TGT TC 3′aecD_end (SEQ ID NO: 12):5′ AGC ACC ACA ATC AAC GTG AG 3′


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


The amplified DNA fragments are identified by means of electrophoresis in a 0.8% 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 pK18mobsacBpck13


The replacement vector pK18mobsacBpck1 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 primer oligonucleotides are chosen for the PCR:

pyc_beg (SEQ ID NO: 19):5′ TC(A CGC GT)C TTG AAG TCG TGC AGG TCA G 3′pyc_end (SEQ ID NO: 20):5′ TC(A CGC GT)C GCC TCC TCC ATG AGG AAG A 3′


The primers shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press). The primers allow amplification of a DNA fragment of approx 3.6 kb in size, which carries the pyc gene. The primers moreover contain the sequence for the cleavage site of the restriction endonuclease MluI, which is marked by parentheses in the nucleotide sequence shown above.


The amplified DNA fragment of approx. 3.6 kb in length, which carries the pyc gene, is cleaved with the restriction endonuclease 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, Hilden).


After purification, the fragment carrying the pyc gene is employed for ligation in the vector pK18mobsacBpck1 described. This is cleaved beforehand with the restriction enzyme BssHII, dephosphorylated with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany), mixed with the DNA fragment of approx. 3.6 kb which carries the pyc gene, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany).


The E. coli strain DH5αmcr (Life Technologies GmbH, Karlsruhe, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, N.Y., 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, N.Y., 1989), which was supplemented with 50 mg/l 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 pK18mobsacBpck12.


4.2 Construction of the pyc Allele pyc P458S by Means of Site-Specific Mutagenesis of the Wild-Type pyc Gene


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

P458S-1 (SEQ ID NO: 21):5′ GGATTCATTGCCGATCAC (TCG) CACCTCCTTCAGGCTCCA 3′P458S-2 (SEQ ID NO: 22):5′ GTGGAGGAAGTCCGAGGT (CGA) GTGATCGGCAATGAATCC 3′


The primers shown are synthesized by MWG Biotech. The codon for serine, which is to replace the proline at position 458, is marked by parentheses in the nucleotide sequence shown above. The plasmid pK18mobsacBpck12 described in Example 4.1 is employed with the two primers, which are each complementary to a strand of the plasmid, for linear amplification by means of Pfu Turbo DNA polymerase. By this lengthening of the primers, a mutated plasmid with broken circular strands is formed. The product of the linear amplification is treated with DpnI—this endonuclease cleaves the methylated and half-methylated template DNA specifically. The newly synthesized broken, mutated vector DNA is transformed in the E. coli strain 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/l. 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 is checked by sequencing. The sequence of the PCR product coincides with the sequence described Ohnishi et al. (2002). The resulting plasmid is called pK18mobsacBpck13. A map of the plasmid is shown in FIG. 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 pK18mobsacBpck13


The plasmid pK18mobsacBpck13 described in Example 4.2 is transferred as described in Example 1.3 into the C. glutamicum strain DSM12866 by conjugation. Selection is made for targeted recombination events in the chromosome of C. glutamicum DSM12866 as described in Example 1.3. Depending on the position of the second recombination event, after 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 in Example 1.5 for the construction of the replacement plasmid are chosen for the PCR.

pck_beg (SEQ ID NO: 9):5′ TA(A GAT CT)G CCG GCA TGA CTT CAG TTT 3′pck_end (SEQ ID NO: 10):5′ AC(A GAT CT)G GTG GGA GCC TTT CTT GTT ATT3′


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


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 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 DSM12866 pck::pyc.


EXAMPLE 5

Preparation of Lysine


The C. glutamicum strains DSM13994glu::lysC, DSM12866glu::lysC, DSM12866 pck::lysC, DSM12866aecD::lysC, DSM1286 glu::ddh, DSM12866aecD::dapA and DSM12866 pck::pyc obtained in Example 1, 2, 3 and 4 are cultured in a nutrient medium 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 33° C. Starting from this agar plate culture, a preculture is seeded (10 ml medium in a 100 ml conical flask). The medium MM is used as the medium for the preculture. The preculture is incubated for 24 hours at 33° 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 MMCSL5g/lMOPS20g/lGlucose (autoclaved separately)50g/lSalts:(NH4)2SO425g/lKH2PO40.1g/lMgSO4*7 H2O1.0g/lCaCl2*2 H2O10mg/lFeSO4*7 H2O10mg/lMnSO4*H2O5.0mg/lBiotin (sterile-filtered)0.3mg/lThiamine * HCl (sterile-filtered)0.2mg/lCaCO325g/l


The CSL (corn steep liquor), MOPS (morpholinopropanesulfonic acid) and the salt solution are brought to pH 7 with aqueous ammonia and autoclaved. The sterile substrate and vitamin solutions, as well as the CaCO3 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 33° C. and 80% atmospheric humidity.


After 48 hours, the OD is determined at a measurement wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of lysine formed is determined with 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 14.

TABLE 14ODLysine HClStrain(660 nm)g/lDSM1399412.019.1DSM13994glu::lysC9.920.0DSM1286612.514.9DSM1503911.416.2DSM12866pck::lysC12.616.5DSM12866aecD::lysC12.015.9DSM12866glu::ddh11.015.5DSM12866aecD::dapA11.116.2DSM12866pck::pyc10.916.9


EXAMPLE 6

Integration of a Copy of the lysC_T311I Allele into the Intergenic Area ncode1 of the Chromosome of the Strain DSM13992


6.1 Construction of the Exchange Vector pK18mobsacBnc1::lysC


The Corynebacterium glutamicum strain DSM13994 (see example 1) is used as a donor for the chromosomal DNA. Chromosomal DNA is isolated from the strain DSM13994 with the customary methods (Eikmanns et al., Microbiology 140: 1817-1828 (1994). With the help of the polymerase chain reaction (PCR), a DNA fragment which encompasses an intergenic area of the chromosome labelled as “ncode1” (SEQ ID NO: 23) is amplified. This area lies within the positions 27398 to 28707 of the sequence of the Corynebacterium glutamicum genome, which is accessible under the access code AX127149 (see table 12). Due to the known sequence of this area, the following primer oligonucleotides are selected for the PCR:

Primer ncode_1 (SEQ ID NO: 24):5′ GA(A GAT CT)A AGC TCT ATT GTC CCC TAC G 3′Primer ncode_2 (SEQ ID NO: 25):5′ GAT CCT TTT AAA AGC CAG TAA CAA G 3′Primer ncode_3 (SEQ ID NO: 26):5′ CTT GTT ACT GGC TTT TAA AAG GAT CCT ATT AAA GAACAC TCC CCT AT 3′Primer ncode_4 (SEQ ID NO: 27):5′ GA(A GAT CT)C GAC TCT GGC TAA TTG CTA C 3′


The primers shown are synthesized by the company MWG Biotech and the PCR reaction is carried out using the standard PCR method of Innis et al. (PCR protocols. A guide to methods and applications, 1990, Academic Press), in which first two products are amplified with the primer combinations ncode1 and ncode2 or ncode3 and ncode4, which then serve as a template for the primer combination ncode1 and ncode4 together in a second PCR. In this manner, the selected primers enable the amplification of an approx. 1.2 kb sized DNA fragment which bears an artificially created interface of the restriction endonuclease BamHI in the center of the intergenic area (emphasized by underlining, (SEQ ID NO: 28)). Furthermore, the primers contain the sequence for the interface of the restriction endonuclease BglII, which is marked with brackets in the nucleotide series shown above.


The amplified DNA fragment of a length of approx. 1.2 kb which bears the intergenic area ncode1 is identified by means of electrophoresis in an 0.8% agarose gel and is isolated from the gel and cleaned using the customary methods (QIAquick Gel Extraction Kit, Qiagen, Hilden).


Next, the ligation of the fragment using the Topo TA Cloning Kit (Invitrogen, Leek, Netherlands, Cat. Number K4600-01) into the vector pCRII-TOPO takes place. The ligation culture is transformed into the E. coli strain TOP10 (Invitrogen, Leek, Netherlands). The selection of plasmide-bearing cells takes place through plating of the transformation culture onto Kanamycin (50 mg/l)-containing LB agar with X-Gal (64 mg/l).


Following isolation of the DNA, the plasmide thus obtained is verified using restriction splitting and identified in the agarose gel. The plasmide obtained is called pCRII-TOPOnc.


The plasmide pCRII-TOPOnc is cut with the restriction enzyme BglII (Amersham-Pharmarcia, Freiburg, Germany), and following splitting in an agarose gel (0.8%), the approx. 1.2 kb fragment is isolated out of the agarose gel with the aid of the Qiagenquick Gel Extraction Kit (Qiagen, Hilden, Germany) and used for ligation with the mobilizable cloning vector pK18mobsacB described in Schäfer et al. (Gene 14, 69-73 (1994)). This is first split with the restriction enzyme BamHI and dephosphorylized with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim), mixed with the approx. 1.2 kb fragment and the culture is treated with T4-DNA ligase (Amersham-Pharmacia, Freiburg, Germany).


Following this, the E. coli strain DH5α (Grant et al.; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is transformed with the ligation culture (Hanahan, In. DNA cloning. A practical approach. Vol. 1.


ILR-Press, Cold Spring Harbor, N.Y., 1989). The selection of the plasmide-bearing cells takes place through plating of the transformation cultures onto LB agar (Sambrock et al., Molecular Cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor, N.Y., 1989) which is supplemented with 25 mg/l Kanamycin.


Plasmide DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from the company Qiagen and verified using restriction splitting with the enzyme XbaI and following agarose gel electrophoresis. The plasmide is called pK18mobsacBnc.


The plasmide pCRIITOPOlysC described in example 1 is cut using the restriction enzyme BamHI (Amersham-Pharmarcia, Freiburg, Germany). Following splitting in an agarose gel (0.8%) with the aid of the Qiagenquick Gel Extraction Kit (Qiagen, Hilden, Germany), the approx. 1.7 kb long, lysC_T311I containing DNA fragment is isolated out of the agarose gel and used for ligation with the vector pK18mobsacBnc described above. This is first split with the restriction enzyme BamHI, dephosphorylized with alkaline phosphatase (Alkaline Phosphatase, Boehringer Mannheim, Germany), mixed with the approx. 1.7 kb DNA fragment and the culture is treated with T4-DNA ligase (Amersham-Pharmacia, Freiburg, Germany).


Following this, the E. coli Strain DH5αmcr (Life Technologies GmbH, Karlsruhe, Germany) is transformed using the ligation culture (Hanahan, In. DNA cloning. A practical approach. Vol. 1. ILR-Press, Cold Spring Harbor, N.Y., 1989). The selection of the plasmide-bearing cells takes place through plating of the transformation culture onto LB agar (Sambrock et al., Molecular Cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor, N.Y., 1989), which was supplemented with 25 mg/l Kanamycin.


Plasmide DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from the company Qiagen isolated and verified through restriction splitting with the enzymes HindIII and XbaI and following agarose gel electrophoresis. The plasmide is called pK18mobsacBnc::lysC. A card of the plasmide is shown in FIG. 7.


6.2 Integration of a Second Copy of the lysC Gene in the Form of the lysCFBRm Allele lysC T311I into the Chromosome (Target Site: the Intergenic Area ncode1) of the Strain DSM13992, Using the Exchange Vector pK18mobsacBnc::lysC


The vector pK18mobsacBnc::lysC named in example 6.1 is transferred into the C. glutamicum strain DSM13992 according to a modified protocol of Schäfer et al. (1990 Journal of Microbiology 172: 1663-1666).


The Corynebacterium glutamicum strain DSM13992 was manufactured by repeated, undirected mutagenesis, selection and mutant selection from C. glutamicum ATCC13032. The strain is resistant to the antibiotic Streptomycin and phenotypically resistant to the lysine analogon S-(2-Aminoethyl)-L-Cystein. However, the strain has a wild-type aspartate kinase which is sensitive to inhibition by a mixture of lysine and therein (25 mM each). A pure culture of this strain was filed on Jan. 16, 2001 with the Deutsche Sammlung für Mikroorganismen und Zellkulturen [=German Collection of Microorganisms and Cell Cultures] (DSMZ, Braunschweig, Germany) in accordance with the Budapest Convention.


The vector pK18mobsacBnc::lysC cannot replicate independently in DSM13992 and only remains in the cell if it has integrated into the chromosome through recombination.


The selection of clones with integrated pK18mobsacBnc::lysC takes place through plating of the conjugation culture onto LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor, N.Y., 1989) which was supplemented with 15 mg/l Kanamycin and 50 mg/l nalidixin acid. Clones that have begun to grow are plated onto LB agar plates with 25 mg/l Kanamycin and incubated for 16 hours at 33° C. To bring about the excision of the plasmide through a second recombination event, the clones are started with 10% sucrose after the 16-hour incubation in the LB liquid medium. The plasmide pK18mobsacB contains a copy of the sacB gene, which changes sucrose into levan, which in turn is toxic to C. glutamicum.


Consequently, only clones in which the integrated pK18mobsacBnc::lysC has excised again grow on LB agar with sucrose. In dependency from the position of the second recombination event, during excision, either the copy of the lysC with the surrounding intergenic area ncode1 will excise together with the plasmide, or only the intergenic area ncode1 will excise.


In order to prove that the copy of lysC has remained in the intergenic area ncode1 of the chromosome, approximately 20 colonies which show the phenotype “Growth in the Presence of Sucrose” and “Non-Growth in the Presence of Kanamycin” according to the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) are researched with the aid of the polymerase chain reaction. In this, a DNA fragment from the chromosomal colonies of the DNA which carries the lysC gene as well as the surrounding areas is amplified. The following primer oligonucleotides are selected for the PCR.

3371V (SEQ ID NO: 29):5′ TAT CAT GCG GTG AGC TGT GA 3′3372N (SEQ ID NO: 30):5′ TAG GGG TGA TGT GCT ACT GT 3′


In control clones with the original ncode1 location, the primers enable the amplification of an approx. 1.3 kb sized DNA fragment. In clones with a copy of the lysC gene in the intergenic area ncode1 of the chromosome, DNA fragments with a size of approx. 3.0 kb are amplified.


The amplified DNA fragments are identified using electrophoresis in an 0.8% agarose gel.


In this manner, a clone was identified which in addition to the copy existing in the lysC location, has a second copy of the lysCFBR allele lysC T311I in the intergenic area ncode1 in the chromosome. This clone was named as the strain DSM13992nc::lysC.


BRIEF DESCRIPTION OF THE FIGURES

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



FIG. 1: Map of the plasmid pK18mobsacBglu11.


The abbreviations and designations used have the following meaning:

  • KanR: Kanamycin resistance gene
  • HindIII: Cleavage site of the restriction enzyme HindIII
  • BanHI: 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



FIG. 2: Map of the plasmid pK18mobsacBaecD11.


The abbreviations and designations used have the following 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



FIG. 3: Map of the plasmid pK18mobsacBpck11.


The abbreviations and designations used have the following 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



FIG. 4: Map of the plasmid pK18mobsacBgluB21.


The abbreviations and designations used have the following 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′: 51 terminal fragment of the gluB gene
  • ′gluB: 31 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



FIG. 5: Map of the plasmid pK18mobsacBaecD21.


The abbreviations and designations used have the following meaning:

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



FIG. 6: Map of the plasmid pK18mobsacBpck13.


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



FIG. 7: Map of the plasmid pK18mobsacBnc::lysC


The abbreviations and designations used have the following meaning:

  • KanR: Kanamycin resistance gene
  • BamHI: Cleavage site of the restriction enzyme BamHI
  • HindIII Cleavage site of the restriction enzyme HindIII
  • XbaI Cleavage site of the restriction enzyme XbaI
  • lysC: lysCFBR allele, lysC T311I
  • sacB: sacB gene
  • RP4mob: mob region with the replication origin for the transfer (oriT)
  • oriV: Replication origin V

Claims
  • 1-48. (canceled)
  • 49. A process for producing L-amino acids, which comprises fermentation of coryneform bacterium comprising two or more copies of a coryneform gene selected from the group consisting of accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysCFBR, lysE, msiK, opcA, oxyR, ppc, ppcFBR, 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 within the bacterium's chromosome, wherein said coryneform gene contains no nucleotide sequence encoding antibiotic resistance, wherein said coryneform bacterium produces enhanced levels of L-amino acid over a coryneform bacterium having one copy of said coryneform gene, under conditions which allow expression of said genes.
  • 50. The process of claim 49, comprising fermentation of a coryneform bacterium wherein at least one coryneform gene selected from the group consisting of accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysCFBR, lysE, msiK, opcA, oxyR, ppc, ppcFBR, 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 is inserted into one or more coryneform chromosomal sites selected from the group consisting of ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, mqo, pck, pgi, and poxB.
  • 51. The process of claim 49, wherein the L-amino acid produced is selected 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.
  • 52. The process of claim 51, wherein the L-amino acid produced is L-lysine.
  • 53. The process of claim 51, comprising fermentation of a coryneform bacterium, wherein two or more copies of a lysCFBR variant gene are within the bacterium chromosome, wherein said lysCFBR variant gene is encoded by one or more amino acid substitutions selected from the group consisting of A279T, A279V, S301F, T3081, S301Y, G345D, R320G, T311I, and S381F of an amino acid sequence as set forth in SEQ ID NO: 2, wherein said coryneform bacterium produces enhanced levels of L-amino acid over a coryneform bacterium having one copy of said lysCFBR variant gene.
  • 54. The process of claim 49, wherein the coryneform bacterium is a member of the genus of Corynebacterium.
  • 55. The process of claim 54, wherein the Corynebacterium is a Corynebacterium glutamicum.
  • 56. The process of claim 49, comprising fermentation of a coryneform bacterium wherein no nucleotide sequences of said genes is capable of episomal replication in the coryneform bacterium.
  • 57. The process of claim 49, comprising fermentation of coryneform bacterium wherein no nucleotide sequence of said genes is capable of transposition in the coryneform bacterium.
  • 58. The process of claim 49, comprising fermentation of coryneform bacterium wherein no nucleotide sequence imparts resistance to antibiotics within the chromosome of said bacterium.
  • 59. The process of claim 49, comprising fermentation of coryneform bacterium wherein at least one copy of said gene is inserted in an intergenic region of the chromosome, or a target site for a prophage nucleotide sequence contained within the chromosome, or a target site for a defective phage nucleotide sequence contained within the chromosome.
  • 60. The process of claim 59, comprising fermentation of coryneform bacterium wherein the intergenic region of the C. glutamicum chromosome is selected from the group consisting of nucleotide positions 192,176 to 194,501; 235,840 to 237,311; 236,096 to 237,311; 322,628 to 330,877; 334,045 to 336,467; 289,565 to 291,841; 154,823 to 161,111; 190,088 to 193,497; 27,398 to 28,707; 61,478 to 62,944; 116,234 to 117,561; 140,847 to 144,605; 113,274 to 114,324; and 244,281 to 246,403.
  • 61. The process of claim 59, comprising fermentation of coryneform bacterium wherein the target site of prophage and defective prophages suitable for integration of open reading frames, genes or alleles within a C. glutamicum chromosome are selected from the group consisting of nucleotide positions 50,474 to 51,049; 67,886 to 68,587; 72,893 to 73,480; 88,231 to 89,445; 139,781 to 140,155; 140,546 to 141,001; 194,608 to 195,294; 200,185 to 200,940; 208,157 to 208,450; 269,616 to 269,948; 336,468 to 338,324; 342,235 to 342,681; 343,518 to 345,356; and 345,872 to 346,207.
  • 62. The process of claim 49, which comprises: a) fermentation of a coryneform bacterium comprising two or more copies of a coryneform gene selected from the group consisting of accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysCFBR, lysE, msiK, opcA, oxyR, ppc, ppcFBR, 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 within the bacterium's chromosome, wherein said coryneform gene contains no nucleotide sequence encoding antibiotic resistance, wherein said coryneform bacterium produces enhanced levels of L-amino acid over a coryneform bacterium having one copy of said coryneform gene, under conditions which allow expression of said genes, b) concentration of the L-amino acid in the fermentation broth and/or in the cells of the bacterium, and c) isolation of said L-amino acid.
  • 63. The process of claim 62, wherein step (c) results in production of a composition in which the isolated L-amino acid is present at a weight percent (wt. %) in the range of from greater than 0 to 100 wt. % relative to constituents from the fermentation broth and/or the biomass.
Parent Case Info

This is a continuation-in-part application of International Patent Appl. No. PCT/EP02/08464 filed on Jul. 30, 2002 which claims priority to U.S. Prov. Appl. No. 60/309,878, filed Aug. 6, 2001.

Provisional Applications (1)
Number Date Country
60309878 Aug 2001 US
Divisions (1)
Number Date Country
Parent 10358405 Feb 2003 US
Child 11612208 Dec 2006 US
Continuation in Parts (1)
Number Date Country
Parent PCT/EP02/08464 Jul 2002 US
Child 10358405 Feb 2003 US