L-Amino Acid-Producing Bacterium and Method for Producing L-Amino Acid

Abstract

An L-amino acid is produced by culturing a Methylophilus bacterium which can grow by using methanol as the main carbon source and has L-amino acid-producing ability, for example, a Methylophilus bacterium in which dihydrodipicolinate synthase activity and aspartokinase activity are enhanced by transformation of cells with a DNA coding for dihydrodipicolinate synthase that is desensitized to feedback inhibition by L-lysine and a DNA coding for aspartokinase that is desensitized to feedback inhibition by L-lysine, or a Methylophilus bacterium which is casamino acid auxotrophic, in a medium containing methanol as a main carbon source, to produce and accumulate an L-amino acid in culture, and collecting the L-amino acid from the culture.

1 . A method for producing cells of a  Methylophilus methylotrophus  strain with an increased content of an L-amino acid comprising culturing a  Methylophilus methylotrophus  strain which is able to produce the L-amino acid in a medium.  
     
     
         2 . The method according to  claim 1 , wherein the L-amino acid is selected from the group consisting of L-lysine, L-valine, L-leucine, L-isoleucine, and L-threonine.  
     
     
         3 . The method according to  claim 1 , wherein said strain is able to produce L-lysine, and is modified to enhance dihydrodipicolinate synthase activity as compared to a wild-type  Methylophilus methylotrophus  strain, and wherein said dihydrodipicolinate synthase is selected from the group consisting of: 
 (a) a protein encoded by a DNA comprising nucleotides 1268 to 2155 of SEQ ID NO: 9; and    (b) a protein having dihydrodipicolinate synthase activity and encoded by a DNA comprising nucleotides 1268 to 2155 of SEQ ID NO: 9, except that substitution, deletion, or addition of one to 10 amino acids is present in the amino acid sequence of said protein,    and wherein said activity is enhanced by a method selected from the group consisting of:    i) increasing the copy number of said DNA in said strain,    ii) placing multiple copies of said DNA on the chromosome of said strain, and    iii) replacing a native promoter with a stronger promoter upstream of said DNA.    
     
     
         4 . The method according to  claim 3 , wherein said strain is further modified to enhance an activity or activities of one, two or three enzymes selected from the group consisting of aspartic acid semialdehyde dehydrogenase, dihydrodipicolinate reductase, and diaminopimelate decarboxylase as compared to a wild-type  Methylophilus methylotrophus  strain by a method selected from the group consisting of: 
 i) increasing the copy number(s) of a DNA(s) encoding said one, two, or three enzyme(s) in said strain,    ii) placing multiple copies of said DNA(s) on the chromosome of said strain, and    iii) replacing a native promoter with a stronger promoter upstream of said DNA(s).    
     
     
         5 . The method according to  claim 1 , wherein said strain has L-lysine-producing ability, and is modified to enhance dihydrodipicolinate synthase activity and aspartokinase activity as compared to a wild-type  Methylophilus methylotrophus  strain, and wherein said dihydrodipicolinate synthase is selected from the group consisting of: 
 (a) a protein encoded by a DNA comprising nucleotides 1268 to 2155 of SEQ ID NO: 9; and    (b) a protein having dihydrodipicolinate synthase activity and encoded by a DNA comprising nucleotides 1268 to 2155 of SEQ ID NO: 9, except that substitution, deletion, or addition of one to 10 amino acids is present in the amino acid sequence of said protein,    and wherein said aspartokinase is selected from the group consisting of:    (a) a protein encoded by a DNA comprising nucleotides 510 to 1736 of SEQ ID NO: 5; and    (b) a protein having aspartokinase activity and encoded by a DNA comprising nucleotides 510 to 1736 of SEQ ID NO: 5, except that substitution, deletion, or addition of one to 10 amino acids is present in the amino acid sequence of said protein,    and wherein said activities are enhanced by a method selected from the group consisting of:    i) increasing the copy number of said DNAs in said strain,    ii) placing multiple copies of said DNAs on the chromosome of said strain, and    iii) replacing a native promoter with a stronger promoter upstream of said DNAs.    
     
     
         6 . The method according to  claim 5 , wherein said strain is further modified to enhance an activity or activities of one, two, or three enzymes selected from the group consisting of aspartic acid semialdehyde dehydrogenase, dihydrodipicolinate reductase and diaminopimelate decarboxylase as compared to a wild-type  Methylophilus methylotrophus  strain by a method selected from the group consisting of: 
 i) increasing the copy number(s) of a DNA(s) encoding said one, two, or three enzyme(s) in said strain,    ii) placing multiple copies of said DNA(s) on the chromosome of said strain, and    iii) replacing a native promoter with a stronger promoter upstream of said DNA(s).    
     
     
         7 . The method according to  claim 5 , wherein the dihydrodipicolinate synthase activity and the aspartokinase activity are enhanced as compared to a wild-type  Methylophilus methylotrophus  strain by transformation with a DNA coding for said dihydrodipicolinate synthase and a DNA coding for said aspartokinase.  
     
     
         8 . The method according to  claim 1 , wherein said strain is modified to enhance aspartokinase activity as compared to a wild-type  Methylophilus methylotrophus  strain, and wherein said aspartokinase is selected from the group consisting of: 
 (a) a protein encoded by a DNA comprising nucleotides 510 to 1736 of SEQ ID NO: 5; and    (b) a protein having aspartokinase activity and encoded by a DNA comprising nucleotide numbers 510 to 1736 of SEQ ID NO: 5, except that substitution, deletion, or addition of one to 10 amino acids is present in the amino acid sequence of said protein,    and wherein said activity is enhanced by a method selected from the group consisting of:    i) increasing the copy number of said DNA in said strain,    ii) placing multiple copies of said DNA on the chromosome of said strain, and    iii) replacing a native promoter with a stronger promoter upstream of said DNA.    
     
     
         9 . The isolated strain according to  claim 8 , wherein said strain is further modified to enhance activities of homoserine dehydrogenase, homoserine kinase and threonine synthase as compared to a wild-type  Methylophilus methylotrophus  strain by a method selected from the group consisting of: 
 i) increasing the copy numbers of DNAs encoding homoserine dehydrogenase, homoserine kinase and threonine synthase in said strain,    ii) placing multiple copies of said DNAs on the chromosome of said strain, and    iii) replacing a native promoter with a stronger promoter upstream of said DNAs, and wherein said strain has L-threonine-producing ability.    
     
     
         10 . The method according to  claim 8 , wherein the L-amino acid is L-lysine.  
     
     
         11 . The method according to  claim 10 , wherein said strain is further modified to enhance an activity or activities of one, two or three enzymes selected from the group consisting of aspartic acid semialdehyde dehydrogenase, dihydrodipicolinate reductase and diaminopimelate decarboxylase as compared to a wild-type  Methylophilus methylotrophus  strain by a method selected from the group consisting of: 
 i) increasing the copy number(s) of a DNA(s) encoding said one, two, or three enzyme(s) in said strain,    ii) placing multiple copies of said DNA(s) on the chromosome of said strain, and    iii) replacing a native promoter with a stronger promoter upstream of said DNA(s).