Method for producing L-methionine
The present invention relates to a method for producing L-methionine in which a microorganism is cultured in the presence of L-homoserine and methyl mercaptan, a salt of the same or dimethyl disulfide whereby the L-methionine is accumulated in the culture medium.
1. A method of producing L-methionine, comprising: culturing a microorganism selected from the group consisting of: Escherichia coli, Corynebacterium glutamicum , and Corynebacterium humireducens in a culture medium which, at the start of culturing, comprises L-homoserine and a sulphur source selected from the group consisting of: methyl mercaptan, a methyl mercaptan salt and dimethyl disulfide, wherein said culturing results in the accumulation of said L-methionine in said culture medium, and wherein:
the microorganism has been genetically engineered to increase, compared to the microorganism prior to genetic engineering, the activity of:
i) an enzyme having L-homoserine O-acetyltransferase activity and comprising the amino acid sequence of SEQ ID NO:2; and
ii) an enzyme having O-acetyl-L-homoserine sulfhydrylase activity and comprising the amino acid sequence of SEQ ID NO:4;
by increasing the copy number of nucleotide sequences encoding the enzymes and/or due to nucleotide sequences encoding the enzymes being in functional linkage to a promoter that increases expression of the enzymes; and
during culturing, acetate formed as a result of conversion of O-acetyl-L-homoserine to L-methionine is reused by the microorganism that has been genetically engineered.
2. The method of claim 1 , wherein said L-homoserine O-acetyltransferase activity is encoded by a gene comprising the coding sequence of SEQ ID NO:1.
3. The method of claim 1 , wherein said O-acetyl-L-homoserine sulfhydrylase activity is encoded by a gene comprising the coding sequence of SEQ ID NO:3.
4. The method of claim 1 , wherein the increase in the activity of both the L-homoserine O-acetyltransferase and the O-acetyl-L-homoserine sulfhydrylase is due to an increase in the copy number of nucleotide sequences encoding these enzymes.
5. The method of claim 1 , wherein the increase in the activity of both the L-homoserine O-acetyltransferase and the O-acetyl-L-homoserine sulfhydrylase is due to nucleotide sequences encoding these enzymes being in functional linkage to a promoter that increases expression.
6. The method of claim 5 , wherein said promoter is selected from the group consisting of: a tact promoter (PtacI) comprising the sequence of SEQ ID NO:5; a glucose dependent deo promoter; a tac promoter; a lac promoter; a trp promoter; an Escherichia coli lac operon inducible by lactose or isopropyl ß-D-thiogalactopyranoside; systems using arabinose or rhamnose as inducers; an Escherichia coli cspA promoter; a Lambda PL promoter; and an osmB promoter.
7. The method of claim 1 , wherein at the start of culturing, homoserine is present in the culture medium at about 3.5 g/l to about 6.6 g/l.
8. The method of claim 1 , wherein the amino acid sequence of the enzyme having L-homoserine O-acetyltransferase activity, consists essentially of the amino acid sequence of SEQ ID NO:2.
9. The method of claim 1 , wherein the amino acid sequence of the enzyme having L-homoserine O-acetyltransferase activity, consists of the amino acid sequence of SEQ ID NO:2.
10. The method of claim 1 , wherein the amino acid sequence of the enzyme having O-acetyl-L-homoserine sulfhydrylase activity consists essentially of the amino acid sequence of SEQ ID NO:4.
11. The method of claim 1 , wherein the amino acid sequence of the enzyme having O-acetyl-L-homoserine sulfhydrylase activity consists of the amino acid sequence of SEQ ID NO:4.
12. The method of claim 8 , wherein the amino acid sequence of the enzyme having O-acetyl-L-homoserine sulfhydrylase activity consists essentially of the amino acid sequence of SEQ ID NO:4.
13. The method of claim 9 , wherein the amino acid sequence of the enzyme having O-acetyl-L-homoserine sulfhydrylase activity consists of the amino acid sequence of SEQ ID NO:4.
14. The method of claim 13 , wherein the increase in activity of both the enzyme of i) and the enzyme of ii) is due to an increase in the copy number of nucleotide sequences encoding said enzyme.
15. The method of claim 13 , wherein the increase in activity of both the enzyme of i) and the enzyme of ii) is due to nucleotide sequences encoding said enzyme being in functional linkage to a promoter that increases expression.
16. The method of claim 15 , wherein said promoter is selected from the group consisting of: a tacI promoter (PtacI) comprising the sequence of SEQ ID NO:5; a glucose dependent deo promoter; a tac promoter; a lac promoter; a trp promoter; an Escherichia coli lac operon inducible by lactose or isopropyl ß-D-thiogalactopyranoside; systems using arabinose or rhamnose as inducers; an Escherichia coli cspA promoter; a Lambda PL promoter; and an osmB promoter.