WO2003040373A2 - Bacteries coryneformes generatrices de composes chimiques - Google Patents

Bacteries coryneformes generatrices de composes chimiques Download PDF

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WO2003040373A2
WO2003040373A2 PCT/EP2002/008464 EP0208464W WO03040373A2 WO 2003040373 A2 WO2003040373 A2 WO 2003040373A2 EP 0208464 W EP0208464 W EP 0208464W WO 03040373 A2 WO03040373 A2 WO 03040373A2
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gene
site
lysine
allele
coryneform bacteria
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PCT/EP2002/008464
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English (en)
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WO2003040373A3 (fr
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Brigitte Bathe
Caroline Reynen
Bettina Möckel
Georg Thierbach
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Degussa Ag
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Priority to EP02760293A priority Critical patent/EP1414970A2/fr
Priority to CA002455878A priority patent/CA2455878A1/fr
Priority to BR0211723-1A priority patent/BR0211723A/pt
Priority to AU2002325923A priority patent/AU2002325923A1/en
Priority to US10/358,405 priority patent/US7160711B2/en
Publication of WO2003040373A2 publication Critical patent/WO2003040373A2/fr
Publication of WO2003040373A3 publication Critical patent/WO2003040373A3/fr
Priority to US11/612,208 priority patent/US20070111291A1/en
Priority to US12/565,533 priority patent/US20100159523A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium

Definitions

  • 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.
  • 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.
  • the plasmid including the cloned biosynthesis gene, is integrated into the chromosomal biosynthesis gene of the microorganism (Reinscheid et al . , Applied and Environmental Microbiology 60(1), 126-132 (1994); Jetten et al . , Applied Microbiology and Biotechnology 43(l):76-82 (1995)).
  • a disadvantage of this method is that the nucleotide sequences of the plasmid and of the antibiotic resistance gene necessary for the selection remain in the microorganism. This is a disadvantage, for example, for the disposal and utilization of the biomass.
  • 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.
  • the inventors had the object of providing new measures for improved fermentative preparation chemical compounds using coryneform bacteria.
  • 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.
  • ORF open reading frame
  • the invention also provides processes for the preparation of one or more chemical compounds, in which the following steps are carried out:
  • nucleotide sequence which is capable of/enables episomal replication or transposition in microorganisms no nucleotide sequence (s) which impart (s) resistance to antibiotics being present at the second, optionally third or fourth site, and the second, optionally third or fourth site not relating to open reading frames (ORF) , genes or alleles which are essential for the growth of the bacteria and the production of the desired compound, and a2) in which the intracellular activity of the corresponding protein is increased, in particular the nucleotide sequence which codes for this protein is over-expressed, b) concentration of the chemical compoun (s) in the fermentation broth and/or in the cells of the bacteria,
  • the invention also pr.ovides processes for the preparation of one or more chemical compounds, which comprise the following steps:
  • 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,
  • ORF open reading frames
  • Chemical compounds are to be understood, in particular, as meaning amino acids, vitamins, nucleosides and nucleotides.
  • the biosynthesis pathways of these compounds are known and are available in the prior art.
  • Amino acids mean, preferably, L-amino acids, in particular the proteinogenic L-amino acids, chosen from the group consisting of L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L- leucine, L-tyrosine, L-phenylalanine, L-histidine, L- lysine, L-tryptophan, L-proline and L-arginine and salts thereof, in particular L-lysine, L-methionine and L- threonine.
  • L-Lysine is very particularly preferred.
  • Proteinogenic amino acids are understood- as meaning the amino acids which occur in natural proteins, that is to say in proteins of microorganisms, plants, animals and humans.
  • Vitamins mean, in particular, vitamin Bl (thiamine) , vitamin B2 (riboflavin) , vitamin B5 (pantothenic acid) , vitamin B6 (pyridoxines) , vitamin B12 (cyanocobalamin) , nicotinic acid/nicotinamide, vitamin M (folic acid) and vitamin E (tocopherol) and salts thereof, pantothenic acid being preferred.
  • Nucleosides and nucleotides mean, inter alia, S-adenosyl- methionine, inosine-5 ' -monophosphoric acid and guanosine- 5 ' -monophosphoric acid and salts thereof.
  • the coryneform bacteria are, in particular, those of the genus Corynebacterium. of the genus Corynebacterium, the species Corynebacterium glutamicum, Corynebacterium ammoniagenes and Corynebacterium thermoaminogenes are preferred. Information on the taxonomic classification of strains of this group of bacteria is to be found, inter alia, in Kampfer and Kroppenstedt (Canadian Journal of Microbiology 42, 989-1005 (1996)) and in US-A-5,250, 434.
  • Suitable strains of the species Corynebacterium glutamicum are, in particular, the known wild-type strains
  • Suitable strains of the species Corynebacterium ammoniagenes are, in particular, the known wild-type strains
  • thermoaminogenes are, in particular, the known wild-type strains
  • Strains with the designation "ATCC” can be obtained from the American Type Culture Collection (Manassas, VA, USA) . Strains with the designation “FERM” can be obtained from the National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan) . The strains of Corynebacterium thermoaminogenes mentioned (FERM BP-1539, FERM BP-1540, FERM BP-1541 and FERM BP-1542) are described in US-A 5,250,434.
  • Open reading frame 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.
  • Alleles are in general understood as meaning alternative forms of a given gene.
  • the forms are distinguished by differences in the nucleotide sequence.
  • 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.
  • the natural site of the lysC gene or of an lysC FBR allele, which codes for a "feed back" resistant aspartate kinase from Corynebacterium glutamicum is the lysC site or lysC locus or lysC gene site with the directly adjacent genes or open reading frames orfX and leuA on one flank and the asd gene on the other flank.
  • “Feed back” resistant aspartate kinase is understood as meaning aspartate kinases which, compared with the wild- type form, have a lower sensitivity to inhibition by mixtures of lysine and threonine or mixtures of AEC (aminoethylcysteine) and threonine or lysine by itself or AEC by itself. Strains which produce L-lysine typically contain such "feed back" resistant or desensitized aspartate kinases .
  • 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) .
  • 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 .
  • the nucleotide sequence of the desired ORF, gene or allele 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,
  • 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
  • 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.
  • 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.
  • 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%.
  • 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.
  • plasmid vectors which, as a function of the conditions under which the host or carrier is cultured, replicate or do not replicate.
  • a temperature-sensitive plasmid for coryneform bacteria which can replicate only at temperatures below 31 S C has been described by Nakamura et al. (US-A-6, 303, 383) .
  • the invention furthermore provides coryneform bacteria, in particular of the genus Corynebacterium, which produce L- lysine, characterized in that these have, in addition to at least one of the copy of an open reading frame (ORF) , gene or allele of lysine production present at the natural site (locus) , in each case a second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele in question at in each case a second, optionally third or fourth site in integrated form, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics being present at the particular second, optionally third or fourth site.
  • ORF open reading frame
  • the invention also furthermore provides a process for the preparation of L-lysine, which comprises the following steps:
  • 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
  • ORF open reading frame
  • 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,
  • ORF open reading frames
  • 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.
  • genes or alleles include, inter alia, the following open reading frames, genes or alleles: accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysC FBR , lysE, msiK, opcA, oxyR, ppc, ppc FBR , pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsl, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM
  • lysC FBR alleles which code for a "feed back" resistant aspartate kinase.
  • Various lysC FBR alleles are summarized and explained in Table 2.
  • lysC FBR alleles are preferred: lysC A279T (replacement of alanine at position 279 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by threonine) , lysC A279V (replacement of alanine at position 279 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by valine), lysC S301F (replacement of serine at position 301 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by phenylalanine), lysC T308I (replacement of threonine at position 308 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by isoleucine) , lysC S301Y (replacement of serine at position 308 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by is
  • the lysC FBR allele lysC T311I replacement of threonine at position 311 of the aspartate kinase protein coded, according to SEQ ID NO: 2, by isoleucine
  • the nucleotide sequence of which is shown as SEQ ID NO: 3 is particularly preferred; the amino acid sequence of the aspartate kinase protein coded is shown as SEQ ID NO : 4.
  • the second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele of lysine production in question can be integrated at in each case a second, optionally third or fourth site.
  • the following open reading frames, genes or nucleotide sequences, inter alia, can be used for this: aecD, ccpAl, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysRl, lysR2, lysR3 , menE, mqo, pck, pgi, poxB and zwa2, in particular the genes aecD, gluA, gluB, gluC, gluD and pck. These are summarized and explained in Table 3.
  • the sites mentioned include, of course, not only the coding regions of the open reading frames or genes mentioned, but also the regions or nucleotide sequences lying upstream which are responsible for expression and regulation, such as, for example, ribosome binding sites, promoters, binding sites for regulatory proteins, binding sites for regulatory ribonucleic acids and attenuators. These regions in general lie in a range of 1-800, 1-600, 1-400, 1-200, 1-100 or 1-50 nucleotides upstream of the coding region. In the same way, regions lying downstream, such as, for example, transcription terminators, are also included. These regions in general lie in a range of 1-400, 1-200, 1-100, 1-50 or 1-25 nucleotides downstream of the coding region.
  • Intergenic regions in the chromosome that is to say nucleotide sequences without a coding function, can furthermore be used.
  • prophages or defective phages contained in the chromosome can be used for this .
  • a prophage is understood as meaning a bacteriophage, in particular the genome thereof, where this is replicated together with the genome of the host and the formation of infectious particles does not take place.
  • a defective phage is understood as meaning a prophage, in particular the genome thereof, which, as a result of various mutations, has lost the ability to form so-called infectious particles.
  • Defective phages are also called cryptic. Prophages and defective phages are often present in integrated form in the chromosome of their host. Further details exist in the prior art, for example in the textbook by Edward A. Birge (Bacterial and Bacteriophage Genetics, 3 rd ed., Springer-Verlag, New York, USA, 1994) or in the textbook by S. Klaus et al . (Bakterienviren, Gustav Fischer Verlag, Jena, Germany, 1992) .
  • the invention accordingly also provides a process for the production of coryneform bacteria which produce L-lysine, which comprises
  • 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
  • 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.
  • ORF open reading frame
  • the invention also furthermore provides a process for the preparation of L-methionine and/or L-threonine, which comprises the following steps:
  • 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,
  • ORF open reading frames
  • 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.
  • genes or alleles include, inter alia, the following open reading frames, genes or alleles: accBC, accDA, aecD, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd, glyA, horn, hom FBR , lysC, lysC FBR , metA, metB, metE, metH, etY, msiK, opcA, oxyR, ppc, ppc FBR , pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsl, ptsM, pyc, pyc P458S, sigC, sigD, sigE
  • the second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele of methionine production in question can be integrated at in each case a second, optionally third or fourth site.
  • the following open reading frames, genes or nucleotide sequences, inter alia, can be used for this: brnE, brnF, brnQ, ccpAl, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, luxR, luxS, lysRl, lysR2, lysR3, menE, etD, metK, pck, pgi, poxB and zwa2.
  • 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.
  • prophages or defective phages contained in the chromosome can be used for this .
  • 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.
  • genes or alleles include, inter alia, the following open reading frames, genes or alleles: accBC, accDA, cstA, cysD, cysE, cysH, cysl, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd, horn, hom FBR , lysC, lysC FBR , msiK, opcA, oxyR, ppc, ppc FBR , pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsl, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi,
  • the second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele of threonine production in question can be integrated at in each case a second, optionally third or fourth site.
  • the following open reading frames, genes or nucleotide sequences, inter alia, can be used for this: ccpAl, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, glyA, ilvA, ilvBN, ilvC, ilvD, luxR, luxS, lysRl, lysR2, lysR3 , mdh, menE, metA, metD, pck, poxB, sigB and zwa2.
  • 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.
  • prophages or defective phages contained in the chromosome can be used for this.
  • the invention accordingly also provides a process for the production of coryneform bacteria which produce L- methionine and/or -threonine, which comprises
  • 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
  • e) isolating coryneform bacteria in which the nucleotide sequence according to a) is incorporated at the target site, no nucleotide sequence which is capable, of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics remaining at the target site.
  • the invention furthermore provides coryneform bacteria, in particular of the genus Corynebacterium, which produce - valine, wherein these have, in addition to at least one of the copy of an open reading frame (ORF) , gene or allele of valine production present at the natural site (locus) , in each case a second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele in question at in each case a second, optionally third or fourth site in integrated form, no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of/enables transposition and no nucleotide sequence which imparts resistance to antibiotics being present at the particular second, optionally third or fourth site.
  • ORF open reading frame
  • the invention also furthermore provides a process for the preparation of L-valine, which comprises the following steps :
  • 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,
  • ORF open reading frames
  • 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.
  • genes or alleles include, inter alia, the following open reading frames, genes or alleles: brnE, brnF, brnEF, cstA, cysD, dps, eno, fda, gap, gap2, gdh, ilvB, ilvN, ilvBN, ilvC, ilvD, ilvE siK, pgk, ptsH, ptsl, ptsM, sigC, sigD, sigE, sigH, sigM, tpi, zwal .
  • Table 8 include in particular the acetolactate synthase which codes for a valine-resistant .
  • the second, optionally third or fourth copy of the open reading frame (ORF) , gene or allele of threonine production in question can be integrated at in each case a second, optionally third or fourth site.
  • the following open reading frames, genes or nucleotide sequences, inter alia, can be used for this: aecD, ccpAl, ccpA2 , citA, citB, citE, ddh, gluA, gluB, gluC, gluD, glyA, ilvA, luxR, lysRl, lysR2, lysR3, panB, panC, poxB and zwa2.
  • 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.
  • prophages or defective phages contained in the chromosome can be used for this.
  • the invention accordingly also provides a process for the production of coryneform bacteria which produce L-valine, which comprises
  • 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
  • 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.
  • This strain which is called DSMl3994glu: :lysC, carries the lysC FBR allele lysC T311I at its natural lysC site and a second copy of the lysC FBR allele lysC T311I at a second site (target site) , namely the gluB gene.
  • a plasmid with the aid of which the incorporation of the lysC FBR allele into the gluB gene can be achieved is shown in Figure 1. It carries the name pK18mobsacBglul_l .
  • This strain which is called DSMl2866glu: :lysC, carries the wild-type form of the lysC gene at its natural lysC site and a second copy of the lysC gene in the form of the lysC FBR allele lysC T311I at a second site (target site) , namely the gluB gene. It has been deposited under number DSM15039 at the Deutsche Sammlung f ⁇ r Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) . A plasmid with the aid of which the incorporation of the lys ' C FBR allele into the gluB gene can be achieved is shown in Figure 1. It carries the name pKl ⁇ mobsacBglul_l .
  • This strain which is called DSM12866aecD: :lysC, carries the wild-type form of the lysC gene at its natural lysC site and a second copy of the lysC gene in the form of the lysC FBR allele lysC T311I at a second site (target site) , namely the aecD gene.
  • a plasmid with the aid of which the incorporation of the lysC FBR allele into the aecD gene can be achieved is shown in Figure 2. It carries the name pKl8mobsacBaecDl_l .
  • This strain which is called DSMl2866pck: :lysC, carries the wild-type form of the lysC gene at its natural lysC site and a second copy of the lysC gene in the form of the lysC FBR allele lysC T311I at a second site (target site) , namely the pck gene.
  • a plasmid with the aid of which the incorporation into the pck gene can be achieved is shown in Figure 3. It carries the name pKl8mobsacBpckl_l .
  • This strain which is called DSMl2866pck: :pyc, carries a copy of the wild-type form of the pyc gene at its natural pyc site and a second copy of the pyc gene in the form of the pyc allele pyc P458S at a second site (target site) , namely the pck gene.
  • a plasmid with the aid of which the incorporation of the pyc allele into the pck gene can be achieved is shown in Figure 6. It carries the name pK18mobsacBpckl_3.
  • the coryneform bacteria produced according to the invention can be cultured continuously or discontinuously in the batch process (batch culture) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of production of chemical compounds .
  • batch culture batch culture
  • feed process fed batch
  • repetitive feed process repeated fed batch process
  • 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
  • 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.
  • 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.
  • oxygen or oxygen-containing gas mixtures such as e.g. air, are introduced into the culture.
  • the temperature of the culture is usually 20 a C to 45 a C, and preferably 25 S C to
  • 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 .
  • DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
  • the Corynebacterium glutamicum strain DSM13994 was produced by multiple, non-directed mutagenesis, selection and mutant selection from C. glutamicum ATCC13032.
  • the strain is resistant to the lysine analogue S- (2-aminoethyl) -L- cysteine and has a feed back-resistant aspartate kinase which is insensitive to inhibition by a mixture of lysine and threonine (in each case 25 mM) .
  • the nucleotide sequence of the lysC FBR allele of this strain is shown as SEQ ID NO: 3. It is also called lysC T311I in the following.
  • the amino acid sequence of the aspartate kinase protein coded is shown as SEQ ID NO:4.
  • the strain DSM12866 was produced from C. glutamicum ATCC13032 by non-directed mutagenesis and selection of the mutants with the best L-lysine accumulation. It is methionine-sensitive. Growth on minimal medium comprising L-methionine can be re-established by addition of threonine.
  • This strain has the wild-type form of the lysC gene shown as SEQ ID N0:1. The corresponding amino acid sequence of the wild-type aspartate kinase protein is shown as SEQ ID NO: 2.
  • a pure culture of this strain was deposited on 10th June 1999 at the Deutsche Sammlung f ⁇ r 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
  • 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:
  • lysC2end (SEQ ID NO: 6) : 5 AC(G GAT CC)G CTG GGA AAT TGC GCT CTT CC 3
  • the primers shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al . (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) .
  • the primers allow amplification of a DNA section of approx. 1.7 kb in length, which carries the lysC gene or allele.
  • the primers moreover contain the sequence for a cleavage site of the restriction endonuclease BamHI, which is marked by parentheses in the nucleotide sequence shown above.
  • the amplified DNA fragment of approx. 1.7 kb in length which carries the lysC allele of the strain DSM13994 is identified by electrophoresis in a 0.8% agarose gel, isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .
  • Ligation of the fragment is then carried out by means of the Topo TA Cloning Kit (Invitrogen, Leek, The Netherlands, Cat. Number K4600-01) in the vector pCRII-TOPO.
  • the ligation batch is transformed in the E. coli strain TOP10 (Invitrogen, Leek, The Netherlands) .
  • Selection of plasmid- carrying cells is made by plating out the transformation batch on kanamycin (50 mg/1) -containing LB agar with X-Gal (5-bromo-4-chloro-3-indolyl ⁇ -D-galactopyranoside, 64 mg/1) .
  • the plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel.
  • the resulting plasmid is called pCRIITOPOlysC .
  • the nucleotide sequence of the amplified DNA fragment or PCR product is determined by the dideoxy chain termination method of Sanger et al . (Proceedings of the National Academy of Sciences USA, 74:5463-5467 (1977)) using the "ABI Prism 377" sequencing apparatus of PE Applied Biosysterns (Weiterstadt, Germany) .
  • the sequence of the coding region of the PCR product is shown in SEQ ID No : 3.
  • the amino acid sequence of the associated aspartate kinase protein is shown in SEQ ID NO : 4.
  • the base thymine is found at position 932 of the nucleotide sequence of the coding region of the lysC FBR allele of strain DSM13994 (SEQ ID NO: 3) .
  • the base cytosine is found at the corresponding position of the wild-type gene (SEQ ID NO:l) .
  • the amino acid isoleucine is found at position 311 of the amino acid sequence of the aspartate kinase protein of strain DSM13994 (SEQ ID No: 4) .
  • the amino acid threonine is found at the corresponding position of the wild-type protein (SEQ ID No:2) .
  • the lysC allele which contains the base thymine at position 932 of the coding region and accordingly codes for an aspartate kinase protein which contains the amino acid isoleucine at position 311 of the amino acid sequence, is called the lysC FBR allele or lysC T311I in the following.
  • the plasmid pCRIITOPOlysC which carries the lysC FBR allele lysC T311I, was deposited in the form of a pure culture of the strain E. coli TOP 10/pCRIITOPOlysC under number DSM14242 on 20th April 2001 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.
  • the Corynebacterium glutamicum strain ATCC13032 is used as the donor for the chromosomal DNA. From the strain ATCC13032, chromosomal DNA is isolated using the conventional methods (Eikmanns et al . , Microbiology 140: 1817 - 1828 (1994)). With the aid of the polymerase chain reaction, a DNA fragment which carries the gluB gene and surrounding regions is amplified. On the basis of the sequence of the gluABCD gene cluster known for C. glutamicum (Kronemeyer et al . , Journal of Bacteriology, 177: 1152 - 1158 (1995)) (Accession Number X81191) , the following primer oligonucleotides are chosen for the PCR:
  • gluBgll (SEQ ID NO: 7) :
  • gluBgl2 (SEQ ID NO: 8) :
  • the primers shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) .
  • the primers allow amplification of a DNA fragment of approx 1.7 kb in size, which carries the gluB gene and surrounding regions .
  • the surrounding regions are a sequence section approx. 0.33 kb in length upstream of the gluB gene, which represents the 3' end of the gluA gene, and a sequence section approx. 0.44 kb in length downstream of the gluB gene, which represents the 5' end of the gluC gene.
  • the primers moreover contain the sequence for the cleavage site of the restriction endonuclease Bglll, which is marked by parentheses in the nucleotide sequence shown above.
  • the amplified DNA fragment of approx. 1.7 kb in length which carries the gluB gene and surrounding regions is identified by means of electrophoresis in a 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .
  • Ligation of the fragment is then carried out by means of the TOPO TA Cloning Kit (Invitrogen, Leek, The Netherlands, Cat. Number K4600-01) in the vector pCRII-TOPO.
  • the ligation batch is transformed in the E. coli strain TOP10 (Invitrogen, Leek, The Netherlands) .
  • Selection of plasmid- carrying cells is made by plating out the transformation batch on kanamycin (50 mg/1) -containing LB agar with X-Gal (5-bromo-4-chloro-3-indolyl ⁇ -D-galactopyranoside, 64 mg/1) .
  • the plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel.
  • the resulting plasmid is called pCRII-TOPOglu.
  • the plasmid pCRII-TOPOglu is cleaved with the restriction enzyme Bglll (Amersham-Pharmacia, Freiburg, Germany) and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the gluB fragment of approx. 1.7 kb is isolated from the agarose gel and employed for ligation with the obilizable cloning vector pKl ⁇ mobsacB described by Schafer et al. (Gene 14: 69-73 (1994)).
  • plasmid- carrying cells are made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold Spring Harbor, New York, 1989) , which is supplemented with 50 mg/1 kanamycin.
  • Plasmid DNA is isolated from a transfor ant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis.
  • the plasmid is called pK18mobsacBglul.
  • Plasmid DNA was isolated from the strain DSM14242 (see Example 1.1), which carries the plasmid pCRIITOPOlysC, and cleaved with the restriction enzyme BamHI (Amersham- Pharmacia, Freiburg, Germany) , and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the lysC FBR - containing DNA fragment of approx. 1.7 kb in length was isolated from the agarose gel and employed for ligation with the vector pKl ⁇ mobsacBglul described above.
  • the E. coli strain DH5 ⁇ mcr (Life Technologies GmbH, Düsseldorf, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol.
  • plasmid-carrying cells are made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold Spring Harbor, New York, 1989) , which was supplemented with 50 mg/1 kanamycin.
  • Plasmid DNA is isolated from a transformant with the aid of the QlAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis.
  • the plasmid is called pKl8mobsacBglul_l.
  • a map of the plasmid is shown in Figure 1.
  • DH5alphamcr/pKl8mobsacBglul_l under number DSM14243 on 20.04.2001 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty.
  • the vector pKl8mobsacBglul_l described in Example 1.2 is transferred by the protocol of Schafer et al. (Journal of Microbiology 172: 1663-1666 (1990)) into the C. glutamicum strain DSM13994 by conjugation.
  • the vector cannot replicate independently in DSM13994 and is retained in the cell only if it has integrated into the chromosome.
  • Selection of clones or transconjugants with integrated pKl8mobsacBglul_l is made by plating out the conjugation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual.
  • Kanamycin-resistant transconjugants are plated out on LB agar plates with 25 mg/1 kanamycin and incubated for 48 hours at 33°C.
  • the clones are cultured for 20 hours in LB liquid medium and then plated out on LB agar with 10% sucrose and incubated for 48 hours .
  • the plasmid pKl8mobsacBglul_l contains, in addition to the kanamycin resistance gene, a copy of the sacB gene which codes for levan sucrase from Bacillus subtilis.
  • the expression which can be induced by sucrose leads to the formation of levan sucrase, which catalyses the synthesis of the product" levan, which is toxic to C. glutamicum.
  • Only those clones in which the integrated pKl8mobsacBglul_l has excised as the consequence of a second recombination event therefore grow on LB agar.
  • the second copy of the lysC FBR allele manifests itself in the chromosome at the gluB locus, or the original gluB locus of the host remains.
  • gluBgll (SEQ ID NO: 7) :
  • gluBgl2 (SEQ ID NO: 8) :
  • the primers allow amplification of a DNA fragment approx. 1.7 kb in size in control clones with the original gluB locus. In clones with a second copy of the lysC FBR allele in the chromosome at the gluB locus, DNA fragments with a size of approx. 3.4 kb are amplified.
  • the amplified DNA fragments are identified by means of electrophoresis in a 0.8% agarose gel.
  • the plasmid pKl8mobsacBglul_l is transferred into the C. glutamicum strain DSM12866 by conjugation.
  • a clone which, in addition to the copy of the wild-type gene present at the lysC locus, has a second copy of the lysC gene in the form of the lysC BR allele lysC T311I at the gluB locus in the chromosome was identified in the manner described in 1.3. This clone was called strain DSMl2866glu: :lysC.
  • Corynebacterium glutamicum strain according to the invention which carries a second copy of an lysC FBR allele in the gluB gene was deposited in the form of a pure culture of the strain Corynebacterium glutamicum DSMl2866glu: : lysC on 5th June 2002 under number DSM15039 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty. 1.5 'Construction of the replacement vector pKl8mobsacBpckl_l
  • the Corynebacterium glutamicum strain ATCC13032 is used as the donor for the chromosomal DNA. From the strain ATCC13032, chromosomal DNA is isolated using the conventional methods (Eikmanns et al., Microbiology 140: 1817 - 1828 (1994)). With the aid of the polymerase chain reaction, a DNA fragment which carries the pck gene and surrounding regions is amplified. On the basis of the sequence of the pck gene known for C. glutamicum (EP1094111 and Riedel et al . , Journal of Molecular and Microbiological Biotechnology 3:573-583 (2001)) (Accession Number AJ269506) , the following primer oligonucleotides are chosen for the PCR:
  • pck_end (SEQ ID NO: 10) :
  • the primers shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) .
  • the primers allow amplification of a DNA fragment of approx.2.9 kb in size, which carries the pck gene and adjacent regions.
  • the primers moreover contain the sequence for the cleavage site of the restriction endonuclease Bglll, which is marked by parentheses in the nucleotide sequence shown above.
  • the amplified DNA fragment of approx. 2.9 kb in length which carries the pck gene and surrounding regions is identified by means of electrophoresis in a 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) . Ligation of the fragment is then carried out by means of the TOPO TA Cloning Kit (Invitrogen, Leek, The Netherlands, Cat. Number K4600-01) in the vector pCRII-TOPO. The ligation batch is transformed in the E. coli strain TOP10 (Invitrogen, Leek, The Netherlands) . Selection of plasmid- carrying cells is made by plating out the transformation batch on kanamycin (50 mg/1) -containing LB agar with X-Gal (64 mg/1) .
  • the plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel.
  • the resulting plasmid is called pCRII-TOPOpck.
  • the plasmid pCRII-TOPOpck is cleaved with the restriction enzyme Bglll (Amersham-Pharmacia, Freiburg, Germany) and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the pck fragment of approx. 2.9 kb is isolated from the agarose gel and employed for ligation with the mobilizable cloning vector pKl ⁇ mobsacB described by Schafer et al . (Gene 14: 69-73 (1994)).
  • the E. coli Strain DH5 ⁇ (Grant et al.; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989) Selection of plasmid- carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold Spring Harbor, New York, 1989), which is supplemented with 50 mg/1 kanamycin.
  • Plasmid DNA is isolated from a 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 pKl ⁇ mobsacBpckl.
  • Example 1.1 which carries the plasmid pCRIITOPOlysC, and cleaved with the restriction enzyme BamHI (Amersham- Pharmacia, Freiburg, Germany) , and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the lysC FBR - containing DNA fragment approx. 1.7 kb long was isolated from the agarose gel and employed for ligation with the vector pKl ⁇ mobsacBpckl described above. This is cleaved beforehand with the restriction enzyme BamHI, dephosphorylated with alkaline phosphatase (Alkaline
  • the E. coli strain DH5cxmcr (Life Technologies GmbH,
  • 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 pKl ⁇ mobdsacBpckl_l .
  • a map of the plasmid is shown in Figure 3.
  • Example 1.3 the plasmid pKl ⁇ mobsacBpckl_l. described in Example 1.5 is transferred into the C. glutamicum strain DSM12866 by conjugation. Selection is made for targeted recombination events in the chromosome of C. glutamicum DSMl2 ⁇ 66 as described in Example 1.3. Depending on the position of the second recombination event, after the excision the second copy of the lysC FBR allele manifests itself in the chromosome at the pck locus, or the original pck locus of the host remains.
  • pck_end (SEQ ID NO: 10):
  • the primers allow amplification of a DNA fragment approx. 2.9 kb in size in control clones with the original pck locus.
  • DNA fragments with a size of approx. 4.6 kb are amplified.
  • the amplified DNA fragments are identified by means of electrophoresis in a 0.6% agarose gel.
  • a clone which, in addition to the copy of the wild-type gene present at the lysC locus, has a second copy of the lysC gene in the form of the lysC FBR allele lysC T311I at the pck locus in the chromosome was identified in this manner.
  • This clone was called strain DSMl2 ⁇ 66pck: :lysC.
  • 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 - l ⁇ 28 (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_end (SEQ ID NO: 12) :
  • 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. ⁇ % 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 pUCl ⁇ then takes place (Norrander et al . , Gene 26:101-106 (1983)).
  • This is cleaved beforehand with the restriction enzymes Bglll and Smal, dephosphorylated, mixed with the aecD-carrying fragment of approx. 1.5 kb, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany) .
  • the ligation batch is transformed in the E. coli strain TOP10 (Invitrogen, Leek, The Netherlands) .
  • Selection of plasmid-carrying cells is made by plating out the transformation batch on kanamycin (50 mg/1) -containing LB agar with X-Gal (64 mg/1).
  • the plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel.
  • the resulting plasmid is called pUCl ⁇ aecD.
  • 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. ⁇ %) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the lysC FBR -containing DNA fragment approx. 1.7 kb in length is isolated from the agarose gel and employed for ligation with the vector pUCl ⁇ aecD described above.
  • plasmid-carrying cells are made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold Spring Harbor, New York, 1989), which was supplemented with 50 mg/1 kanamycin.
  • Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis.
  • the plasmid is called pUCl ⁇ aecDl.
  • the plasmid pUCl ⁇ aecDl is cleaved with the restriction enzyme Kpnl and then treated with Klenow polymerase.
  • the plasmid is then cleaved with the restriction enzyme Sail (Amersham-Pharmacia, Freiburg, Germany) and after separation in an agarose gel (0. ⁇ %) 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 pKl ⁇ mobsacB described by Schafer et al. (Gene 14: 69-73 (1994)).
  • This is cleaved beforehand with the restriction enzymes Smal and Sail and dephosphorylated with alkaline phosphatase (Alkaline
  • the E. coli strain DH5 ⁇ (Grant et al.; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989) . Selection of plas id- carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al . , Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold Spring Harbor, New York, 19 ⁇ 9) , which is supplemented with 50 mg/1 kanamycin.
  • Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis.
  • the plasmid is called pKl ⁇ mobsacBaecDl_l.
  • a map of the plasmid is shown in Figure 2.
  • Example 1.3 the plasmid pKl ⁇ mobsacBaecDl_l described in Example 1.4 is transferred into the C. glutamicum strain DSM12866 by conjugation. Selection is made for targeted recombination events in the chromosome of C. glutamicum DSM12866 as described in Example 1.3. Depending on the position of the second recombination event, after the excision the second copy of the lysC FBR allele manifests itself in the chromosome at the aecD locus, or the original aecD locus of the host remains.
  • aecD_end (SEQ ID NO: 12) : 5 s AGC ACC ACA ATC AAC GTG AG 3
  • the primers allow amplification of a DNA fragment approx. 2.1 kb in size in control clones with the original aecD locus.
  • 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 lysC FBR allele lysC T311I at the aecD locus in the chromosome was identified in this manner.
  • This clone was called strain DSMl2 ⁇ 66aecD: :lysC.
  • 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 - l ⁇ 2 ⁇ (1994)). With the aid of the polymerase chain reaction, a DNA fragment which carries the gluB gene and surrounding regions is amplified. On the basis of the sequence of the gluABCD gene cluster known for C. glutamicum (Kronemeyer et al . , Journal of Bacteriology, 177: 1152 - 1156 (1995); EP1106790) (Accession Number X81191 and AX127149) , the following primer oligonucleotides are chosen for the PCR:
  • gluA_beg (SEQ ID NO: 13) : 5 CAC GGT TGC TCA TTG TAT CC 3
  • gluD_end (SEQ ID NO: 14) :
  • 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
  • the primers allow amplification of a DNA fragment of approx 4.4 kb in size, which carries the gluB gene and surrounding regions.
  • the amplified DNA fragment is identified by means of electrophoresis in a 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .
  • Ligation of the fragment is then carried out by means of the TOPO TA Cloning Kit (Invitrogen, Leek, The Netherlands, Cat. Number K4600-01) in the vector pCRII-TOPO.
  • the ligation batch is transformed in the E. coli strain TOP10 (Invitrogen, Leek, The Netherlands) .
  • Selection of plasmid- carrying cells is made by plating out the transformation batch on kanamycin (50 mg/1) -containing LB agar with X-Gal (64 mg/1) .
  • the plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel .
  • the resulting plasmid is called pCRII- T0P0glu2.
  • the plasmid pCR.Il-TOPOglu2 is cleaved with the restriction enzymes EcoRI and Sail (Amersham-Pharmacia, Freiburg, Germany) and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the gluB fragment of approx. 3.7 kb is isolated from the agarose gel and employed for ligation with the mobilizable cloning vector pKl ⁇ mobsacB described by Schafer et al. (Gene 14, 69-73 (1994)).
  • the E. coli Strain DH5 ⁇ (Grant et al . ; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989) . Selection of plasmid- carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al . , Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold Spring Harbor, New York, 19 ⁇ 9) , which is supplemented with 50 mg/1 kanamycin.
  • Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis.
  • the plasmid is called pKl ⁇ mobsacBglu2.
  • a DNA fragment which carries the ddh gene and surrounding regions is also amplified with the aid of the polymerase chain reaction.
  • the following primer oligonucleotides are chosen for the PCR:
  • 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) .
  • 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.
  • 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) .
  • the E. coli strain DH5oancr (Life Technologies GmbH, Düsseldorf, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989). Selection of plasmid-carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold
  • Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis.
  • the plasmid is called pKl8mobsacBglu2_l.
  • a map of the plasmid is shown in Figure 4.
  • Example 1.3 the plasmid pKl8mobsacBglu2_l described in Example 2.1 is transferred into the C. glutamicum strain DSM12866 by conjugation. Selection is made for targeted recombination events in the chromosome of C. glutamicum DSM12866 as described in Example 1.3. Depending on the position of the second recombination event, after the excision the second copy of the ddh gene manifests itself in the chromosome at the gluB locus, or the original gluB locus of the host remains.
  • gluD_end (SEQ ID NO: 14) :
  • the primers allow amplification of a DNA fragment approx. 4.4 kb in size in control clones with the original glu 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 .
  • 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:
  • the primers shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al . (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) .
  • the primers allow amplification of a DNA fragment of approx 2.1 kb in size, which carries the aecD gene and adjacent regions.
  • the amplified DNA fragment of approx. 2.1 kb in length is identified by means of electrophoresis in a 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .
  • the DNA fragment purified is cleaved with the restriction enzyme Bglll and EcoRV (Amersham Pharmacia, Freiburg, Germany) .
  • the ligation of the fragment in the vector pUCl ⁇ then takes place (Norrander et al . , Gene 26:101-106
  • 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.
  • a further DNA fragment which carries the dapA gene and surrounding regions is amplified.
  • the following primer oligonucleotides are chosen for the PCR:
  • dapA_beg (SEQ ID NO: 17) :
  • dapA_end (SEQ ID NO: 18) :
  • 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) .
  • the dapA-containing DNA fragment approx. 1.4 kb in length is employed for ligation with the vector pUCl ⁇ aecD described above. This is cleaved beforehand with the restriction enzyme Stul, mixed with the DNA fragment of approx. 1.4 kb, and the mixture is treated with T4 DNA Ligase (Amersham-Pharmacia, Freiburg, Germany) .
  • the E. coli strain DH5otmcr (Life Technologies GmbH, Düsseldorf, Germany) is then transformed with the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989). Selection of plasmid-carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold Spring Harbor, New York, 1989), which was supplemented with 50 mg/1 kanamycin.
  • Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis.
  • the plasmid is called pUC18aecD2.
  • the plasmid pUC18aecD2 is cleaved with the restriction enzyme Sail and partly with EcoRI (Amersham-Pharmacia, Freiburg, Germany) and after separation in an agarose gel (0.8%) with the aid of the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) the fragment of approx. 2.7 kb which carries aecD and dapA is isolated from the agarose gel and employed for ligation with the mobilizable cloning vector pKl ⁇ mobsacB described by Schafer et al . (Gene 14: 69-73 (1994).).
  • the E. coli strain DH5 ⁇ (Grant et al . ; Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) is then transformed with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989) . Selection of plasmid- carrying cells is made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold Spring Harbor, New York, 1989), which is supplemented with 50 mg/1 kanamycin.
  • Plasmid DNA is isolated from a transformant with the aid of the QIAprep.Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis.
  • the plasmid is called pKl8mobsacBaecD2_l.
  • a map of the plasmid is shown in Figure 5.
  • Example 1.3 the plasmid pKl ⁇ mobsacBaecD2_l described in Example 3.1 is transferred into the C. glutamicum strain DSM12666 by conjugation. Selection is made for targeted recombination events in the chromosome of C. glutamicum DSMl2 ⁇ 66 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.
  • 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 .
  • 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. ⁇ % agarose gel.
  • Example 1.5 is used as the base vector for insertion of the pyc allele.
  • a DNA fragment which carries the pyc gene and surrounding regions is also amplified with the aid of the polymerase chain reaction.
  • the following primer oligonucleotides are chosen for the PCR:
  • the primers shown are synthesized by MWG Biotech and the PCR reaction is carried out by the standard PCR method of Innis et al . (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) .
  • the primers allow amplification of a DNA fragment of approx 3.6 kb in size, which carries the pyc gene.
  • the primers moreover contain the sequence for the cleavage site of the restriction endonuclease Mlul, which is marked by parentheses in the nucleotide sequence shown above.
  • the amplified DNA fragment of approx. 3.6 kb in length, which carries the pyc gene, is cleaved with the restriction endonuclease Mlul, identified by means of electrophoresis in a 0.8% agarose gel and isolated from the gel and purified by conventional methods (QIAquick Gel Extraction Kit, Qiagen, Hilden) .
  • the fragment carrying the pyc gene is employed for ligation in the vector pKl ⁇ mobsacBpckl 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 DH ⁇ mcr (Life Technologies GmbH, Düsseldorf, Germany) is then transformed with the ligation batch (Hanahan ; In: DNA Cloning. A Practical Approach. Vol. 1, ILR-Press, Cold Spring Harbor, New York, 1989) .
  • plasmid-carrying cells are made by plating out the transformation batch on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold Spring Harbor, New York, 1989), which was supplemented with 50 mg/1 kanamycin.
  • Plasmid DNA is isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction cleavage and subsequent agarose gel electrophoresis.
  • the plasmid is called pKl8mobsacBpckl_2.
  • EP-A-1108790 describes a point mutation in the pyc gene for C. glutamicum which allows improved L-lysine production.
  • the allele is called pyc P458S.
  • the following primer oligonucleotides are chosen for the linear amplification:
  • P458S-1 (SEQ ID NO: 21) : 5' GGATTCATTGCCGATCAC (TCG) CACCTCCTTCAGGCTCCA 3'
  • the primers shown are synthesized by MWG Biotech.
  • the codon for serine, which is to replace the proline at position 45 ⁇ , is marked by parentheses in the nucleotide sequence shown above.
  • the plasmid pKl ⁇ mobsacBpckl_2 described in Example 4.1 is employed with the two primers, which are each complementary to a strand of the plasmid, for linear amplification by means of Pfu Turbo DNA polymerase.
  • Pfu Turbo DNA polymerase Pfu Turbo DNA polymerase.
  • the newly synthesized broken, mutated vector DNA is transformed in the E. coli strain XLl Blue (Bullock, Fernandez and Short, BioTechniques (5) 376-379 (1987)). After the transformation, the XLl Blue cells repair the breaks in the mutated plasmids . Selection of the transformants was carried out on LB medium with kanamycin 50 mg/1. The plasmid obtained is checked by means of restriction cleavage, after isolation of the DNA, and identified in agarose gel. The DNA sequence of the mutated DNA fragment ⁇ _. checked by sequencing. The sequence of the PCR product coincides with the sequence described Ohnishi et al. (2002). The resulting plasmid is called pKl8mobsacBpckl_3. A map of the plasmid is shown in Figure 6.
  • the plasmid pKl ⁇ mobsacBpckl_3 described in Example 4.2 is transferred as described in Example 1.3 into the C. glutamicum strain DSM12866 by conjugation. Selection is made for targeted recombination events in the chromosome of C. glutamicum DSM12866 as described in Example 1.3. Depending on the position of the second recombination event, after de excision the second copy of the pyc allele manifests itself in the chromosome at the pck locus, or the original pck locus of the host remains .
  • the primers allow amplification of a DNA fragment approx. 2.9 kb in size in control clones with the original 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 DSMl2866pck: :pyc.
  • the cultures are first incubated on a brain-heart agar plate (Merck, Darmstadt, Germany) for 24 hours at
  • 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 a 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.
  • Glucose (autociaved separately) 50 g/1
  • the CSL corn steep liquor
  • MOPS morpholinopropanesulfonic acid
  • the salt solution are brought to pH 7 with aqueous ammonia and autociaved.
  • Culturing is carried out in a 10 ml volume in a 100 ml conical flask with baffles. Culturing is carried out at 33 a 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, Kunststoff) . The amount of lysine formed is determined wich an amino acid analyzer from Eppendorf- BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivation with ninhydrin detection.
  • the base pair numbers stated are approximate values obtained in the context of reproducibility of measurements.
  • Figure 1 Map of the plasmid pKl8mobsacBglul_l .
  • KanR Kanamycin resistance gene
  • HindiII Cleavage site of the restriction enzyme
  • lysC lysC FBR allele, lysC T311I
  • gluB ' 5 ' 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)
  • Figure 2 Map of the plasmid pKl ⁇ mobsacBaecDl_l.
  • KanR Kanamycin resistance gene
  • lysC lysC FBR allele, lysC T311I
  • sacB sacB gene
  • RP4mob mob region with the replication origin for the transfer (oriT) oriV: Replication origin V
  • Figure 3 Map of the plasmid pKl ⁇ mobsacBpckl_l.
  • KanR Kanamycin resistance gene
  • lysC lysC* BK allele, lysC T311I
  • sacB sacB gene
  • RP4mob mob region with the replication origin for the transfer (oriT)
  • Figure 4 Map of the plasmid pKl8mobsacBgluB2_l.
  • KanR Kanamycin resistance gene
  • ddh ddh gene gluA gluA gene
  • gluB 1 5 ' terminal fragment of the gluB gene
  • gluD 1 5 ' terminal fragment of the gluD gene
  • sacB sacB gene
  • RP4mob mob region with the replication origin for the transfer (oriT)
  • Figure 5 Map of the plasmid pKl ⁇ mobsacBaecD2_l.
  • KanR Kanamycin resistance gene
  • dapA dapA gene
  • sacB sacB gene
  • RP4mob mob region with the replication origin for the transfer (oriT)
  • KanR Kanamycin resistance gene
  • sacB sacB gene
  • RP4mob mob region with the replication origin for the transfer (oriT)
  • the microorganism identified under I. above was accompanied by:
  • This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2002-06-05 (Date of the original deposit) 1 .
  • microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).
  • the microorganism identified under I. above was accompanied by:
  • microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).
  • the microorganism identified under I. above was accompanied by:
  • This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2001 -04 - 20 (Date of the original deposit) 1 .
  • microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).

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Abstract

L'invention porte sur des bactéries coryneformes qui, en plus d'une copie au moins, présente au site naturel (locus) d'un cadre de lecture ouvert (ORF), d'un gène ou d'un allèle, codant pour la synthèse d'une protéine ou d'un ARN, produisent, dans chaque cas une deuxième copie et éventuellement une troisième ou une quatrième copie présentes dans chaque cas dans un deuxième site ou éventuellement dans un troisième ou une quatrième site, sous une forme intégrée au chromosome. L'invention porte également sur un procédé de préparation de composés chimiques par fermentation de ces bactéries.
PCT/EP2002/008464 2001-08-06 2002-07-30 Bacteries coryneformes generatrices de composes chimiques WO2003040373A2 (fr)

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EP02760293A EP1414970A2 (fr) 2001-08-06 2002-07-30 Bacteries coryneformes generatrices de composes chimiques
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BR0211723-1A BR0211723A (pt) 2001-08-06 2002-07-30 Bactérias corineformes que produzem compostos quìmicos i
AU2002325923A AU2002325923A1 (en) 2001-08-06 2002-07-30 Production of l-lysine by genetically modified corynebacterium glutamicum strains
US10/358,405 US7160711B2 (en) 2001-08-06 2003-02-05 Coryneform bacteria which produce chemical compounds I
US11/612,208 US20070111291A1 (en) 2001-08-06 2006-12-18 Coryneform Bacteria Which Produce Chemical Compounds I
US12/565,533 US20100159523A1 (en) 2001-08-06 2009-09-23 Coryneform Bacteria Which Produce Chemical Compounds I

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WO2004069996A2 (fr) * 2003-02-05 2004-08-19 Degussa Ag Bacteries et procede de production de composes chimiques au moyen desdites bacteries i
WO2006008103A1 (fr) * 2004-07-20 2006-01-26 Basf Aktiengesellschaft Unites d'expression de p1-35
WO2006100211A1 (fr) 2005-03-24 2006-09-28 Degussa Gmbh Alleles mutes du gene zwf (g6pdh) tire de corynebacteries pour la production accrue de lysine
US7135313B2 (en) 2001-10-16 2006-11-14 Degussa Ag Method for producing L-lysine or L-lysine containing feed additives with a cornebacteria containing a mutated lysC
WO2006125714A2 (fr) 2005-05-24 2006-11-30 Evonik Degussa Gmbh Alleles du gene opca provenant de bacteries coryneformes
WO2007141111A2 (fr) * 2006-06-02 2007-12-13 Evonik Degussa Gmbh Procédé de production d'un additif alimentaire pour animaux contenant de la l-lysine
DE102007005072A1 (de) 2007-02-01 2008-08-07 Evonik Degussa Gmbh Verfahren zur fermentativen Herstellung von Cadaverin
WO2009088049A1 (fr) 2008-01-10 2009-07-16 Ajinomoto Co., Inc. Procédé de fabrication d'une substance désirée par un procédé de fermentation
DE102008001874A1 (de) 2008-05-20 2009-11-26 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren
US7785779B2 (en) 2003-12-18 2010-08-31 Paik Kwang Industrial Co., Ltd. P EF-TU expression units
WO2010149574A1 (fr) 2009-06-25 2010-12-29 Evonik Degussa Gmbh Procédé pour préparer des acides aminés l par fermentation
EP2354235A1 (fr) 2005-10-05 2011-08-10 Evonik Degussa GmbH Méthode de production d'acides aminés L par fermentation au moyen de bactéries corynéformes
WO2011124477A2 (fr) 2010-03-30 2011-10-13 Evonik Degussa Gmbh Procédé de production par fermentation de l-ornithine
DE102010025124A1 (de) 2010-06-25 2011-12-29 Forschungszentrum Jülich GmbH Verfahren zur Herstellung von D-Aminosäuren, Mikroorganismus, sowie Vektor
EP2479279A1 (fr) 2011-01-20 2012-07-25 Evonik Degussa GmbH Procédé de fabrication fermentative d'acides aminés contenant du soufre
WO2012114256A1 (fr) 2011-02-22 2012-08-30 Basf Se Procédés et microorganismes recombinants pour la production de cadavérine
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