Nucleotide sequences which code for the rpoB gene

Abstract

The present invention relates to polynucleotides corresponding to the rpoB gene and which encode the β-subunit of RNA polymerase B, methods of producing L-amino acids, and methods of screening for polynucleotides which encode proteins having activity of the β-subunit of RNA polymerase B.

1 - 87 . (canceled)  
     
     
         88 . An isolated or purified polynucleotide which encodes a variant of the polypeptide of SEQ ID NO: 2, 
 wherein said polypeptide variant comprises the amino acid sequence of SEQ ID NO: 2, except for one or more amino acid substitutions between positions 1-10, positions 190-200 or positions 420-450, and    wherein lysine production is improved by the expression of said isolated or purified polynucleotide in a  coryneform  bacterium compared to the expression of the polypeptide of SEQ ID NO: 2 in the same type of  coryneform  bacterium.    
     
     
         89 . The polynucleotide of  claim 88 , which encodes a polypeptide that comprises one or more amino acid substitutions between positions 1-10 of SEQ ID NO: 2.  
     
     
         90 . The polynucleotide of  claim 88 , which encodes a polypeptide in which L-proline at position 5 of SEQ ID NO: 2 is substituted by L-leucine, L-isoleucine or L-valine.  
     
     
         91 . The polynucleotide of  claim 88 , which encodes a polypeptide comprising one or more amino acid substitutions between positions 190-200 of SEQ ID NO: 2.  
     
     
         92 . The polynucleotide of  claim 88 , which encodes a polypeptide in which L-serine at position 196 of SEQ ID NO: 2 is substituted by L-phenylalanine or L-tyrosine.  
     
     
         93 . The polynucleotide of  claim 88 , which encodes a polypeptide that comprises one or more amino acid substitutions between positions 420-450 of SEQ ID NO: 2.  
     
     
         94 . The polynucleotide of  claim 88 , which encodes a polypeptide in which L-leucine at position 424 of SEQ ID NO: 2 is substituted by L-proline or L-arginine.  
     
     
         95 . The polynucleotide of  claim 88 , which encodes a polypeptide in which L-serine at position 425 of SEQ ID NO: 2 is substituted by L-threonine or L-alanine.  
     
     
         96 . The polynucleotide of  claim 88 , which encodes a polypeptide in which L-glutamine at position 426 of SEQ ID NO: 2 is substituted by L-leucine or L-lysine.  
     
     
         97 . The polynucleotide of  claim 88 , which encodes a polypeptide in which L-aspartic acid at position 429 of SEQ ID NO: 2 is substituted by L-isoleucine, L-valine or L-leucine.  
     
     
         98 . The polynucleotide of  claim 88 , which encodes a polypeptide in which L-histidine at position 439 of SEQ ID NO: 2 is substituted by a proteogenic amino acid other than L-histidine.  
     
     
         99 . The polynucleotide of  claim 88 , which encodes a polypeptide in which L-serine at position 444 of SEQ ID NO: 2 is substituted by L-leucine, L-tyrosine or L-tryptophan.  
     
     
         100 . The polynucleotide of  claim 88 , which encodes a polypeptide in which L-leucine at position 446 of SEQ ID NO: 2 is substituted by L-proline or L-isoleucine.  
     
     
         101 . The polynucleotide of  claim 88 , which encodes a polypeptide comprising SEQ ID NO: 2, except for one or more of the following substitutions: L-proline at position 5 substituted by L-leucine, L-serine at position 196 substituted by L-phenylalanine, L-aspartate at position 429 substituted by L-valine, or L-histidine at position 439 substituted by L-tyrosine.  
     
     
         102 . The polynucleotide of  claim 88  which encodes the polypeptide of SEQ ID NO: 4.  
     
     
         103 . The polynucleotide of  claim 88 , which comprises SEQ ID NO: 3.  
     
     
         104 . The polynucleotide of  claim 88  encoding the polypeptide of SEQ ID NO: 6.  
     
     
         105 . The polynucleotide of  claim 88 , which comprises SEQ ID NO: 5.  
     
     
         106 . The full complement of the polynucleotide of  claim 88 .  
     
     
         107 . A vector comprising the polynucleotide of  claim 88 .  
     
     
         108 . A shuttle vector or plasmid vector comprising the polynucleotide of  claim 88 .  
     
     
         109 . A host cell comprising the polynucleotide of  claim 88 .  
     
     
         110 . The host cell of  claim 109 , wherein the polynucleotide is integrated in the chromosome.  
     
     
         111 . The host cell of  claim 109 , wherein the polynucleotide is present on a plasmid.  
     
     
         112 . The host cell of  claim 109 , which is a  coryneform  bacterium.  
     
     
         113 . The host cell of  claim 109 , which is of the genus  Corynebacterium.    
     
     
         114 . The host cell of  claim 109 , which is selected from the group consisting of  Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium thermoaminogenes, Corynebacterium melassecola, Brevibacterium flavum, Brevibacterium lactofermentum , and  Brevibacterium divaricatum.    
     
     
         115 . A method for making an amino acid comprising: 
 culturing the host cell of  claim 109  for a time and under conditions suitable for the production of said amino acid, and    recovering or isolating said amino acid.    
     
     
         116 . The method of  claim 115 , wherein said amino acid is L-lysine.  
     
     
         117 . The method of  claim 115 , wherein said amino acid is L-glutamate.  
     
     
         118 . A method for making a variant of the polypeptide of SEQ ID NO:  2  comprising: 
 culturing the host cell of  claim 109  for a time and under conditions suitable for expression of said polypeptide, and    recovering or isolating said polypeptide.    
     
     
         119 . A variant of the polypeptide of SEQ ID NO: 2, 
 wherein said polypeptide variant comprises the amino acid sequence of SEQ ID NO: 2, except for one or more amino acid substitutions between positions 1-10, positions 190-200 or positions 420-450, and    wherein lysine production is improved by expression of said isolated or purified polynucleotide in a  coryneform  bacterium compared to expression of the polypeptide of SEQ ID NO: 2 in the same type of  coryneform  bacterium.