Method of producing L-amino acid
Provided is a method of producing an L-amino acid such as L-glutamic acid and the like. An L-amino-acid is produced by cultivating a coryneform bacterium having L-amino acid-producing ability in a culture medium, which has been modified so as to have one or more of the following modifications: (A) a modification for increasing activity of acetate kinase, (B) a modification for increasing activity of fructose-1,6-bisphosphatase, (C) a modification for decreasing activity of pyruvate dehydrogenase, (D) a modification for decreasing activity of aspartate transaminase, and (E) a modification for decreasing activity of malic enzyme; and collecting the L-amino acid from the culture medium and/or the bacterial cells.
1 . A method of producing an L-amino acid, comprising:
cultivating a coryneform bacterium having L-amino acid-producing ability in a culture medium to accumulate an L-amino acid in the culture medium and/or in cells of the bacterium; and
collecting the L-amino acid from the culture medium and/or the cells;
wherein the L-amino acid is L-glutamic acid family, and
wherein the bacterium has a modification for increasing activity of acetate kinase, and optionally an additional a modification for increasing activity of fructose-1,6-bisphosphatase, combinations thereof;
wherein expression of the gene encoding acetate kinase is increased by increasing the copy number of the gene and/or by modifying an expression regulatory sequence of the gene;
wherein the acetate kinase is selected from the group consisting of:
(i) a protein comprising the amino acid sequence of SEQ ID NO: 8 or 10,
(ii) a protein comprising the amino acid sequence of SEQ ID NO: 8 or 10 except that said amino acid sequence includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and has acetate kinase activity, and
(iii) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8 or 10, and has acetate kinase activity;
wherein the fructose-1,6-bisphosphatase is selected from the group consisting of:
(i) a protein comprising the amino acid sequence of SEQ ID NO: 12 or 14,
(ii) a protein comprising the amino acid sequence of SEQ ID NO: 12 or 14 except that said amino acid sequence includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and has fructose-1,6-bisphosphatase activity, and
(iii) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 12 or 14, and has fructose-1,6-bisphosphatase activity;
wherein the bacterium has been further modified so that the activity of phosphoketolase is increased as compared to an unmodified bacterium; and
wherein the bacterium is of the genus Corynebacterium.
2 . The method according to claim 1 ,
wherein the activity of the acetate kinase is increased by increasing expression of a gene encoding acetate kinase, and
wherein the activity of the fructose-1,6-bisphosphatase is increased by increasing expression of a gene encoding fructose-1,6-bisphosphatase.
3 . The method according to claim 2 , wherein expression of the gene encoding fructose-1,6-bisphosphatase is increased by increasing the copy number of the gene and/or by modifying an expression regulatory sequence of the gene.
4 . The method according to claim 1 , wherein the phosphoketolase is D-xylulose-5-phosphate phosphoketolase and/or fructose 6-phosphate phosphoketolase.
5 . The method according to claim 1 , wherein the bacterium is Corynebacterium glutamicum.
6 . The method according to claim 1 , wherein the L-glutamic acid is ammonium L-glutamate or sodium L-glutamate.