Method for the preparation of L-amino acids from D-amino acids
The invention relates to recombinant microorganisms which, in comparison to the starting organism, have a higher concentration or activity of a D-amino acid oxidase, of an L-amino acid dehydrogenase, of an NADH cosubstrate regenerating enzyme and, if necessary, of a catalase. The invention also includes methods for preparing L-amino acids from D-amino acids using of these microorganisms.
1. A method for manufacturing an L-amino acid from a D-amino acid comprising:
a) reacting a recombinant microorganism with said D-amino acid, wherein:
i) said recombinant microorganism has been made by transforming a starting microorganism with one or more vectors comprising polynucleotides encoding the enzymes: D-amino acid oxidase, wherein said D-amino acid oxidase is the D-amino acid oxidase of A. protophormiae; L-amino acid dehydrogenase, wherein said L-amino acid dehydrogenase is the L-amino acid dehydrogenase of Bacillus cereus ; and an enzyme that regenerates NADH, wherein said enzyme that regenerates NADH is the malate dehydrogenase from A. protophormiae;
ii) relative to said starting microorganism, said recombinant microorganism overexpresses each of said enzymes;
iii) the reaction is carried out under conditions in which each of said enzymes is active; and
b) isolating said L-amino acid.
2. The method of claim 1 , wherein relative to said starting microorganism, said recombinant microorganism has at least one additional copy of:
a) a polynucleotide encoding said D-amino acid oxidase;
b) a polynucleotide encoding said L-amino acid dehydrogenase; and
c) a polynucleotide encoding said enzyme that regenerates NADH.
3. The method of either claim 1 , wherein, in comparison to the starting organism, said recombinant microorganism has a higher concentration of E. coli catalase.
4. The method of either claim 1 , wherein said recombinant microorganism is from a genus selected from the group consisting of: Bacillus; Staphylococcus; Streptomyces ; and Escherichia.
5. The method of either claim 1 , wherein said recombinant microorganism is of the species Escherichia coli.
6. The method of claim 1 , wherein the cell wall of said recombinant microorganism is permeabilized by chemical or physical measures to allow for the absorption of a D-amino acid from the solution.
7. The method of either claim 1 , wherein said D-amino acid is selected from the group consisting of: lysine, arginine, phenylalanine, valine, omithine, leucine, histidine, norleucine, tyrosine, alanine, glutamate, and methionine.
8. The method of claim 1 , wherein L-malate or L-malic acid is present when the polynucleotide encoding malate dehydrogenase is overexpressed.
9. The method of claim 1 , wherein:
a) said D-amino acid oxidase is selected from the group consisting of: the D-amino acid oxidase of A. protophormiae; the D-amino acid oxidase of Trigonopsis variabilis ; the D-aspartate oxidase of Bos taurus ; the D-amino acid oxidase of Rhodosporidium ; and the D-amino acid oxidase of Rhototorula gracillis;
b) said L-amino acid dehydrogenase is selected from the group consisting of: the L-leucine dehydrogenase of Bacillus cereus ; the L-phenylalanine dehydrogenase of Rhodococcus ; the L-lysine dehydrogenase of Home sapiens ; the L-alanine dehydrogenase of Bacillus subtilis ; and the glutamate dehydrogenase of Bos taurus;
c) said polynucleotide encoding an enzyme that regenerates NADH is selected from the group consisting of: a formate dehydrogenase; a malate dehydrogenase; and an alcohol dehydrogenase, each from A. protophormiae, T. variabilis and E. coli.
10. The method of claim 9 , wherein, in comparison to the starting organism said recombinant microorganism has a higher concentration of E. coli catalase.
11. The recombinant organism of claim 9 , wherein said recombinant microorganism is from a genus selected from the group consisting of: Bacillus; Staphylococcus; Streptomyces ; and Escherichia.
12. The recombinant microorganism of claim 9 , wherein said recombinant microorganism is of the species Escherichia coli.
13. The method of claim 9 , wherein the cell wall of said recombinant microorganism is permeabilized by chemical or physical measures to allow for the absorption of a D-amino acid from the solution.
14. The method of claims 9 , wherein said D-amino acid is selected from the group consisting of: lysine, arginine, phenylalanine, valine, ornithine, leucine, histidine, norleucine, tyrosine, alanine, glutamate, cephalosporine, and methionine.
15. The method of claim 1 , wherein L-malate or L-malic acid is present when the polynucleotide encoding malate dehydrogenase is overexpressed.