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