Coryneform Bacteria Which Produce Chemical Compounds I

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.

1 . Coryneform bacteria which produce chemical compounds, wherein these have, in addition to at least one copy, present at the natural site (locus), of an open reading frame (ORF), gene or allele which codes for the synthesis of a protein or an RNA, a second, optionally third or fourth copy of the open reading frame (ORF), gene or allele in question at a second, optionally third or fourth site in a form integrated into the chromosome, no nucleotide sequence which is capable of/enables episomal replication, or transposition in microorganisms and no nucleotide sequence(s) which impart(s) resistance to antibiotics being present at the second, optionally third or fourth site, and the second, optionally third or fourth site not relating to open reading frames (ORF), genes or alleles which are essential for the growth of the bacteria and the production of the desired compound. 
     
     
         2 . Coryneform bacteria according to  claim 1  which produce chemical compounds, wherein the corynefo/m bacteria belong to the genus  Corynebacterium.    
     
     
         3 . Coryneform bacteria of the genus  Corynebacterium  according to  claim 2  which produce chemical compounds, wherein these belong to the species  Corynebacterium glutamicum.    
     
     
         4 . Coryneform bacteria according to  claim 1  which produce chemical compounds, wherein the chemical compound is a compound chosen from the group consisting of L-amino acids, vitamins, nucleosides and nucleotides. 
     
     
         5 . Coryneform bacteria according to  claim 1  which produce chemical compounds, wherein the chemical compound is one or more L-amino acids chosen from the group consisting of L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-proline and L-arginine. 
     
     
         6 . Coryneform bacteria according to  claims 1  and  4  which produce chemical compounds, wherein the L-amino acid is L-lysine, and these bacteria have, in addition to at least one copy of an open reading frame (ORF), gene or allele of lysine production present at the natural site (locus), in each case a second, optionally third or fourth copy of the open reading frame (ORF), gene or allele of lysine production in question at in each case a second, optionally third or fourth site in a form integrated into the chromosome. 
     
     
         7 . Coryneform bacteria according to  claim 6  which produce L-lysine, wherein the coryneform bacteria belong to the genus  Corynebacterium.    
     
     
         8 . Coryneform bacteria of the genus  Corynebacterium  according to  claim 7  which produce L-lysine, wherein these belong to the species  Corynebacterium glutamicum.    
     
     
         9 . Coryneform bacteria according to  claim 6  which produce L-lysine, wherein the open reading frame (ORF), gene or allele of lysine production is one or more open reading frame(s), one or more gene(s) or allele(s) chosen from the group consisting of accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysC FBR , lysE, msiK, opcA, oxyR, ppc, ppc FBR , pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigh, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T. 
     
     
         10 . Coryneform bacteria according to  claim 6  which produce L-lysine, wherein the open reading frame, gene or allele of lysine production is one or more gene(s) or allele(s) chosen from the group consisting of dapA, ddh, lysC FBR  and pyc P458S. 
     
     
         11 . Coryneform bacteria according to  claim 6  which produce L-lysine, wherein the open reading frame, gene or allele of lysine production is a lysC FBR  allele which codes for a feed back resistant form of aspartate kinase. 
     
     
         12 . Coryneform bacteria according to  claim 11  which produce L-lysine, wherein the feed back resistant fonts of aspartate kinase coded by the lysC FBR  allele contains an amino acid sequence according to SEQ ID NO:2, SEQ ID NO:2 containing one or more amino acid replacements chosen from the group consisting of A279T, A279V, S301F, T308I, S301Y, G345D, R320G, T311I and S381F. 
     
     
         13 . Coryneform bacteria according to  claim 11  which produce L-lysine, wherein the feed back resistant form of aspartate kinase coded by the lysC FBR  allele includes an amino acid sequence according to SEQ ID NO:4. 
     
     
         14 . Coryneform bacteria according to  claim 11  which produce L-lysine, wherein the coding region of the lysC FBR  allele includes the nucleotide sequence of SEQ ID NO:3. 
     
     
         15 . Coryneform bacteria according to  claim 6  which produce L-lysine, wherein the particular second, optionally third or fourth site is a gene chosen from the group consisting of aecD, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, mqo, pck, pgi and poxB. 
     
     
         16 . Coryneform bacteria according to  claim 6  which produce L-lysine, wherein the particular second, optionally third or fourth site is a site chosen from the group consisting of intergenic regions of the chromosome, prophages contained in the chromosome and defective phages contained in the chromosome. 
     
     
         17 . Coryneform bacteria according to  claim 15  which produce L-lysine, wherein the particular second, optionally third or fourth site is the aecD gene site. 
     
     
         18 . Coryneform bacteria according to  claim 15  which produce L-lysine, wherein the particular second, optionally third or fourth site is the gluB gene site. 
     
     
         19 . Coryneform bacteria according to  claim 15  which produce L-lysine, wherein the particular second, optionally third or fourth site is the pck gene site. 
     
     
         20 . Process for the preparation of chemical compounds by fermentation of coryneform bacteria, in which the following steps are carried out: 
       a) fermentation of coryneform bacteria, which 
       a1) which have, in addition to at least one copy, present at the natural site (locus), of an open reading frame (ORF), gene or allele which codes for the synthesis of a protein or an RNA, a second, optionally third or fourth copy of this open reading frame (ORF), gene or allele at a second, optionally third or fourth site in a form integrated into the chromosome, no nucleotide sequence which is capable of/enables episomal replication or transposition in microorganisms and no nucleotide sequence(s) which impart(s) resistance to antibiotics being present at the second, optionally third or fourth site, and the second, optionally third or fourth site not relating to open reading frames (ORF), genes or alleles which are essential for the growth of the bacteria and the production of the desired compound, and 
       a2) in which the intracellular activity of the corresponding protein is increased, in particular the nucleotide sequence which codes for this protein is over-expressed, 
       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. %. 
     
     
         21 . Process according to  claim 20 , wherein the coryneform bacteria belong to the genus  Corynebacterium.    
     
     
         22 . Process according to  claim 20 , wherein the coryneform bacteria of the genus  Corynebacterium  belong to the species  Corynebacterium glutamicum.    
     
     
         23 . Process according to  claim 20 , wherein the chemical compound is a compound chosen from the group consisting of L-amino acids, vitamins, nucleosides and nucleotides. 
     
     
         24 . Process according to  claim 20 , wherein the chemical compound is one or more L-amino acids chosen from the group consisting of L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-proline and L-arginine. 
     
     
         25 . Process according to  claim 24 , wherein the chemical compound is L-lysine. 
     
     
         26 . Process for the preparation of L-lysine, which comprises the following steps: 
       a) fermentation of coryneform bacteria which have, in addition to at least one copy of an open reading frame (ORF), gene or allele of lysine production present at the natural site (locus), in each case a second, optionally third or fourth copy of the open reading frame (ORF), gene or allele of lysine production in question at in each case a second, optionally third or fourth site in a form integrated into the chromosome
 under conditions which allow expression of the said open reading frames (ORF), genes or alleles mentioned. 
 
     
     
         27 . Process for the preparation of L-lysine according to  claim 26 , wherein the open reading frame (ORF), gene or allele of lysine production is an open reading frame, a gene or allele chosen from the group consisting of accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lysC FBR , lysE, msiK, opcA, oxyR, ppc, ppc FBR , pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T. 
     
     
         28 . Process for the preparation of L-lysine according to  claim 26 , wherein the open reading frame (ORF), gene or allele of lysine production is a gene or allele chosen from the group consisting of dapA, ddh, lysC FBR  and pyc P458S. 
     
     
         29 . Process for the preparation of L-lysine according to  claim 26 , wherein the open reading frame (ORF), gene or allele of lysine production is a lysC FBR  allele which codes for a feed back resistant form of aspartate kinase. 
     
     
         30 . Process for the preparation of L-lysine according to  claim 29 , wherein the feed back resistant form of aspartate kinase coded by the lysC FBR  allele contains an amino acid sequence according to SEQ ID NO:2, SEQ ID NO:2 containing one or more amino acid replacements chosen from the group consisting of A279T, A279V, S301F, T308I, S301Y, G345D, R320G, T311I and S381F. 
     
     
         31 . Process for the preparation of L-lysine according to  claim 29 , wherein the feed back resistant form of aspartate kinase coded by the lysC FBR  allele includes an amino acid sequence according to SEQ ID NO:4. 
     
     
         32 . Process for the preparation of L-lysine according to  claim 29 , wherein the coding region of the lysC FBR  allele includes the nucleotide sequence of SEQ ID NO:3. 
     
     
         33 . Process for the preparation of L-lysine according to  claim 26 , wherein the particular second, optionally third or fourth site is a site chosen from the group consisting of aecD, ccpA1, ccpA2, citA, citB, citE, fda, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, mqo, pck, pgi and poxB. 
     
     
         34 . Process for the preparation of L-lysine according to  claim 26 , wherein the second, optionally third or fourth site is the aecD gene site. 
     
     
         35 . Process for the preparation of L-lysine according to  claim 26 , wherein the second, optionally third or fourth site is the gluB gene site. 
     
     
         36 . Process for the preparation of L-lysine according to  claim 26 , wherein the second, optionally third or fourth site is the pck gene site. 
     
     
         37 . Process for the production of coryneform bacteria which produce one or more chemical compounds, which comprises 
       a) isolating the nucleotide sequence of at least one desired ORF, gene or allele which codes for a protein or an RNA, optionally including the expression and/or regulation signals, preferably from coryneform bacteria, 
       b) providing the 5′ and the 3′ end of the ORF, gene or allele with nucleotide sequences of the target site, 
       c) preferably incorporating the nucleotide sequence of the desired ORF, gene or allele provided with nucleotide sequences of the target site into a vector which does not replicate or replicates to only a limited extent in coryneform bacteria, 
       d) transferring the nucleotide sequences according to b) or c) into coryneform bacteria, and 
       e) isolating coryneform bacteria in which the nucleotide sequence(s) according to a) is incorporated at the target site, no nucleotide sequence(s) which is(are) capable of/enable(s) episomal replication or transposition in microorganisms, and no nucleotide sequence(s) which impart(s) resistance to antibiotics remaining at the target site. 
     
     
         38 . Plasmid pK18mobsacBglu1 — 1 shown in  FIG. 1  and deposited in the form of a pure culture of the strain  E. coli  DH5αmcr/pK18mobsacBglu1 — 1 (=DH5alpha mcr/pK18mobsacBglu1 — 1) under number DSM14243. 
     
     
         39 . Plasmid pK18mobsacBaecD1 — 1 shown in  FIG. 2  and deposited in the form of a pure culture of the strain  E. coli  DH5αmcr/pK18mobsacBaecD1 — 1 (=DH5alphamcr/pK18mobsacBaecD1 — 1) under number DSM15040. 
     
     
         40 .  Corynebacterium glutamicum  strain DSM12866glu::lysC deposited in the form of a pure culture under number DSM15039. 
     
     
         41 . Coryneform bacteria according to  claim 1 , wherein the second, optionally third or fourth site is a site chosen from the group consisting of intergenic regions of the chromosome, prophages contained in the chromosome and defective phages contained in the chromosome. 
     
     
         42 . Coryneform bacteria according to  claim 41 , wherein the intergenic region is selected from table 12. 
     
     
         43 . Coryneform bacteria according to  claim 41 , wherein the prophages contained in the chromosome and defective phages contained in the chromosome are selected from table 13. 
     
     
         44 . Process according to  claim 20 , wherein said second, third or fourth site is selected from the group consisting of intergenic regions of the chromosome, prophages contained in the chromosome and defective phages contained in the chromosome. 
     
     
         45 . Process according to  claim 44 , wherein the intergenic regions are selected from table 12. 
     
     
         46 . Process according to  claim 44 , wherein the prophages contained in the chromosome and defective phages contained in the chromosome are selected from table 13. 
     
     
         47 . Process according to  claim 37 , wherein said nucleotide sequence site is selected from the group consisting of intergenic regions of the chromosome, prophagbes contained in the chromosome and defective phages contained in the chromosome. 
     
     
         48 . Process according to  claim 47 , wherein the intergenic regions are selected from table 12.