Nucleotide sequences which code for the cysD, cysN, cysK, cysE and cysH genes

Abstract

Nucleotide sequences from coryneform bacteria which code for the cysD, cysN, cysK, cysE and cysH genes and a process for the fermentative preparation of amino acids using bacteria in which the genes mentioned are enhanced, a process for the fermentative preparation of L-amino acids using coryneform bacteria in which at least the cysD gene, the cysN gene, the cysK gene, the cysE gene and/or the cysH gene is present in enhanced form, and the use of polynucleotides which contain the sequences according to the invention as hybridization probes and a process for the preparation of an L-methionine-containing animal feedstuffs additive from fermentation broths.

1 - 20 . (canceled)  
     
     
         21 . A modified coryneform bacterium in which at least one of the cysD gene, cysN gene, cysK gene, cysE gene and the cysH gene are enhanced relative to an unmodified coryneform bacterium.  
     
     
         22 . The modified coryneform bacterium of  claim 21 , wherein at least one of cysD gene, cysN gene, cysK gene, cysE gene and the cysH gene are over-expressed.  
     
     
         23 . Microorganism DSM 14308 deposited at the Deutsche Sammlung für Mikroorganismen und Zellkulturen.  
     
     
         24 .  Escherichia coli  strain DH5αmcr/pEC-XK99EcysKa1ex deposited as DSM 14310 deposited at the Deutsche Sammlung für Mikroorganismen und Zellkulturen.  
     
     
         25 .  Escherichia coli  strain DH5αmcr/pEC-XK99EcysDa1ex deposited as DSM 14311 deposited at the Deutsche Sammlung für Mikroorganismen und Zelikulturen.  
     
     
         26 .  Escherichia coli  strain DH5αmcr/pEC-XK99EcysHa1ex as DSM 14315 deposited at the Deutsche Sammlung für Mikroorganismen und Zellkulturen.  
     
     
         27 . A process for the fermentative preparation of an L-amino acid comprising: 
 (a) fermenting the modified coryneform bacterium of  claim 21  in a medium wherein the modified coryneform bacterium produce the L-amino acid    (b) concentrating the L-amino acid in the medium or in the cells of the bacteria, and    (c) isolating the L-amino acid.    
     
     
         28 . The process of  claim 27 , wherein the amino acid is L-lysine.  
     
     
         29 . The process of  claim 27 , wherein the L-amino acid is L-cysteine or L-methionine.  
     
     
         30 - 31 . (canceled)  
     
     
         32 . The process of  claim 27 , wherein the bacteria are transformed with a plasmid vector, and the plasmid vector comprises at least one polynucleotide which encodes a gene product selected from the group consisting of cysD, cysN, cysK, cysE and cysH.  
     
     
         33 - 35 . (canceled)  
     
     
         36 . The process of  claim 27 , wherein the modified coryneform bacterium further comprises at least one gene whose expression is enhanced, wherein the at least one gene is selected from the group consisting of 
 the dapA gene which codes for dihydrodipicolinate synthase,    the gap gene which codes for glyceraldehyde 3-phosphate dehydrogenase,    the tpi gene which codes for triose phosphate isomerase,    the pgk gene which codes for 3-phosphoglycerate kinase,    the zwf gene which codes for glucose 6-phosphate dehydrogenase,    the pyc gene which codes for pyruvate carboxylase,    the mqo gene which codes for malate-quinone oxidoreductase,    the lysC gene which codes for a feed-back resistant aspartate kinase,    the lysE gene which codes for lysine export,    the hom gene which codes for homoserine dehydrogenase,    the ilvA gene which codes for threonine dehydratase,    the ilvA(Fbr) allele which codes for a feed back resistant threonine dehydratase,    the ilvBN gene which codes for acetohydroxy-acid synthase,    the ilvD gene which codes for dihydroxy-acid dehydratase, and    the zwa1 gene which codes for the Zwa1 protein.    
     
     
         37 . The process of  claim 27 , wherein the modified coryneform bacterium further comprises at least one gene whose expression is attenuated, wherein the at least one gene is selected from the group consisting of 
 the pck gene which codes for phosphoenol pyruvate carboxykinase,    the pgi gene which codes for glucose 6-phosphate isomerase,    the poxB gene which codes for pyruvate oxidase, and    the zwa2 gene which codes for the Zwa2 protein.    
     
     
         38 . The process of  claim 27 , wherein the modified coryneform bacterium further comprises at least one gene whose expression is attenuated, wherein the at least one gene is selected from the group consisting of 
 the aecD gene which codes for cystathionine β-lyase, and    the metB gene which codes for cystathionine synthase.    
     
     
         39 . The process of  claim 27 , wherein the modified coryneform bacterium is  Corynebacterium glutamicum.    
     
     
         40 . The process of  claim 39 , wherein the  Corynebacterium glutamicum  is strain DSM5715/pEC-XK99EcysDa1ex.  
     
     
         41 . The process of  claim 39 , wherein the  Corynebacterium glutamicum  is strain DSM5715/pEC-XK99EcysKa1ex.  
     
     
         42 . The process of  claim 39 , wherein the  Corynebacterium glutamicum  is strain DSM5715/pEC-XK99EcysEb1ex.  
     
     
         43 . The process of  claim 39 , wherein the  Corynebacterium glutamicum  is strain DSM5715/pEC-XK99EcysHa1ex.  
     
     
         44 . (canceled)  
     
     
         45 . A process of the preparation of an L-methionine-containing animal feedstuffs additive from a fermentation broth, comprising: 
 (a) culturing and fermenting an L-methionine-producing microorganism in a fermentation medium;    (b) removing water from the L-methionine-containing fermentation broth;    (c) removing an amount of from 0 to 100 wt. % of the biomass formed during the fermentation; and    (d) drying the fermentation broth obtained according to (b) and/or (c) to obtain the animal feedstuffs additive in powder or granule form.    
     
     
         46 . The process of  claim 45 , wherein genes in the biosynthesis pathway of L-methionine are enhanced.  
     
     
         47 . The process of  claim 45 , wherein the metabolic pathways which reduce the formation of L-methionine are at least partly eliminated in the microorganism.  
     
     
         48 . The process of  claim 45 , wherein the expression of the polynucleotides which code for cysD, cysN, cysK, cysE or cysH is enhanced in the microorganism.  
     
     
         49 . The process of  claim 45 , wherein the expression of the polynucleotides which code for cysD, cysN, cysK, cysE or cysH is over-expressed in the microorganism.  
     
     
         50 . The process of  claim 45 , wherein microorganism is  Corynebacterium glutamicum.    
     
     
         51 . The process of  claim 50 , wherein the  Corynebacterium glutamicum  strain is DSM5715/pEC-XK99EcysDa1ex.  
     
     
         52 . The process of  claim 50 , wherein the  Corynebacterium glutamicum  is strain DSM5 715/pEC-XK99EcysKa1ex.  
     
     
         53 . The process of  claim 50 , wherein the  Corynebacterium glutamicum  is strain DSM5715/pEC-XK99EcysEb1ex.  
     
     
         54 . The process according to  claim 50 , wherein the  Corynebacterium glutamicum  strain is DSM5715/pEC-XK99EcysHa1ex.  
     
     
         55 . The process of  claim 45 , further comprising one or more of the following steps: 
 e) adding one or more organic substances to the products obtained according to b), c) and/or d);    f) adding auxiliary substances selected from the group consisting of silicas, silicates, stearates, grits, and bran to the substances obtained according to (b) to (e) for stabilization and to increase the storability; or    g) converting the substances obtained according to (b) to (f) into a form which is stable in an animal stomach by coating with film-forming agents.    
     
     
         56 . The process of  claim 55 , which comprises step (e) and wherein the said organic substances are selected from the group consisting of L-methionine, D-methionine, a racemic mixture D,L-methionine, and combinations thereof.  
     
     
         57 . The process of  claim 45 , wherein some of the biomass is removed.  
     
     
         58 . The process of  claim 55 , wherein some of the biomass is removed.  
     
     
         59 . The process of  claim 45 , wherein up to 100% of the biomass is removed.  
     
     
         60 . The process of  claim 55 , wherein up to 100% of the biomass is removed.  
     
     
         61 . The process of  claim 45 , wherein the water content is up to 5 wt. %.  
     
     
         62 . The process of  claim 55 , wherein the water content is up to 5 wt. %.  
     
     
         63 . The process of  claim 50 , wherein the water content is less than 2 wt. %.  
     
     
         64 . The process of  claim 45 , wherein the water content is less than 2 wt. %.  
     
     
         65 . The process of  claim 55 , wherein the water content is less than 2 wt. %.  
     
     
         66 . The process of  claim 55 , wherein the film-forming agents are metal carbonates, silicas, silicates, alginates, stearates, starches, gums or cellulose ethers.  
     
     
         67 . An animal feedstuff additive prepared according to  claim 45 .  
     
     
         68 . The animal feedstuff additive of  claim 67 , which comprises 1 wt. % to 80 wt. % L-methionine, D-methionine, D,L-methionine or a mixture thereof, based on the dry weight of the animal feedstuffs additive.  
     
     
         69 - 70 . (canceled)  
     
     
         71 . The modified coryneform bacterium of  claim 21 , wherein the cysD gene is enhanced and wherein the cysD gene codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 2, the cysD gene comprises nucleotides 232 to 1143 of SEQ ID No. 1, or at least one sequence which hybridizes with the sequence fully complementary to sequence nucleotides 232 to 1143 of SEQ ID No. 1, wherein the hybridization conditions comprise washing in 2×SSC at a temperature of from 50 to 68° C. and which encodes a polypeptide with sulfate adenylyltransferase activity.  
     
     
         72 . The modified coryneform bacterium of  claim 21 , wherein the cysN gene is enhanced and wherein the cysN gene codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 3, the cysN gene comprises nucleotides 1146 to 2444 of SEQ ID No. 1, or at least one sequence which hybridizes with the sequence fully complementary to sequence nucleotides 1146 to 2444 of SEQ ID No. 1, wherein the hybridization conditions comprise washing in 2×SSC at a temperature of from 50 to 68° C. and which encodes a polypeptide with sulfate adenylyltransferase activity.  
     
     
         73 . The modified coryneform bacterium of  claim 21 , wherein the cysK gene is enhanced and wherein the cysK gene codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 5, the cysK gene comprises nucleotides 271 to 1203 of SEQ ID No. 4, or at least one sequence which hybridizes with the sequence fully complementary to sequence nucleotides 271 to 1203 of SEQ ID No. 4, wherein the hybridization conditions comprise washing in 2×SSC at a temperature of from 50 to 68° C. and which encodes a polypeptide with cysteine synthase A activity.  
     
     
         74 . The modified coryneform bacterium of  claim 21 , wherein the cysE gene is enhanced and wherein the cysE gene codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 6, the cysE gene comprises nucleotides 1392 to 1955 of SEQ ID No.4, or at least one sequence which hybridizes with the sequence fully complementary to sequence nucleotides 1392 to 1955 of SEQ ID No.4, wherein the hybridization conditions comprise washing in 2×SSC at a temperature of from 50 to 68° C. and which encodes a polypeptide with serine acetyltransferase activity.  
     
     
         75 . The modified coryneform bacterium of  claim 21 , wherein the cysH gene is enhanced and wherein the cysH gene codes for a polypeptide which comprises the amino acid sequence of SEQ ID No. 8, the cysH gene comprises nucleotides 250 to 1032 of SEQ ID No. 7, or at least one sequence which hybridizes with the sequence fully complementary to sequence nucleotides 250 to 1032 of SEQ ID No. 7, wherein the hybridization conditions comprise washing in 2×SSC at a temperature of from 50 to 68° C. and which encodes a polypeptide with 3′-phopshoadenylyl sulfate reductase activity.