Method for producing l-glutamine by fermentation and l-glutamine producing bacterium

Abstract

L-Glutamine is produced by culturing a coryneform bacterium which has L-glutamine producing ability and has been modified so that its intracellular glutamine synthetase activity should be enhanced, preferably which has been further modified so that its intracellular glutamate dehydrogenase activity should be enhanced, in a medium to produce and accumulate L-glutamine in the medium and collecting the L-glutamine.

1 . An isolated coryneform bacterium comprising a modification such that said glutamine synthetase activity is enhanced compared to an unmodified coryneform bacterium, said modification comprising increasing expression amount of a polynucleotide encoding a protein having glutamine synthetase activity by increasing the copy number of said polynucleotide or modifying an expression control sequence of said polynucleotide so that expression of said polynucleotide is enhanced.  
     
     
         2 . The bacterium according to  claim 1 , wherein said polynucleotide encodes a protein having 90% homology to the amino acid sequence of SEQ ID NO: 25.  
     
     
         3 . The bacterium according to  claim 2 , wherein said polynucleotide is obtainable from a coryneform bacterium by PCR using primers of SEQ ID NO: 4 and SEQ ID NO: 5 under stringent conditions entailing 1×SSC, 0.1% SDS at 60° C., and the DNA is obtainable from a coryneform bacterium.  
     
     
         4 . The bacterium according to  claim 2 , wherein said polynucleotide encodes a protein having the amino acid sequence of SEQ ID NO: 25  
     
     
         5 . The bacterium according to  claim 1 , wherein the bacterium has been further modified so that intracellular glutamate dehydrogenase activity is enhanced compared to an unmodified coryneform bacterium.  
     
     
         6 . The bacterium according to  claim 5 , wherein the glutamate dehydrogenase activity is enhanced by increasing expression amount of a polynucleotide encoding a glutamate dehydrogenase.  
     
     
         7 . The bacterium according to  claim 6 , wherein the expression amount of said polynucleotide encoding a glutamate dehydrogenase is increased by increasing the copy number of said polynucleotide encoding a glutamate dehydrogenase or modifying an expression control sequence of said polynucleotide encoding a glutamate dehydrogenase so that expression of said polynucleotide encoding a glutamate dehydrogenase is increased.  
     
     
         8 . The bacterium of  claim 1 , wherein said bacterium belongs to the genus  Brevibacterium  or  Corynebacterium.    
     
     
         9 . The bacterium of  claim 1 , which is selected from the group consisting of  Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium alkanolyticum, Corynebacterium callunae, Corynebacterium glutamicum, Corynebacterium lilium, Corynebacterium melassecola, Corynebacterium thermoaminogenes, Corynebacterium herculis, Brevibacterium divaricatum, Brevibacterium flavum, Brevibacterium immariophilum, Brevibacterium lactofermentum, Brevibacterium roseum, Brevibacterium saccharolyticum, Brevibacterium thiogenitalis, Brevibacterium ammoniagenes, Brevibacterium album, Brevibacterium cerium,  and  Microbacterium ammoniaphilum.    
     
     
         10 . The bacterium of  claim 1 , wherein said modification comprises replacing the native expression control promoter of said polynucleotide encoding a protein having glutamine synthetase activity with a strong promoter sequence.  
     
     
         11 . The bacterium of  claim 10 , wherein said strong promoter sequence is selected from the group consisting of a lac promoter, a trp promoter, and a trc promoter.  
     
     
         12 . A method for producing L-glutamine, comprising cultivating the bacterium of  claim 1  in a culture medium for a time sufficient to produce L-glutamine; and collecting the L-glutamine produced