Mutants for the preparation of D-amino acids
View Patent ↗Escherichia. coli mutants which can be used for the synthesis of D-amino acids, and to such a process. The mutants are distinguished by deficiencies in particular enzymes which break down D-amino acids and include those which produce D-amino acids via the carbamoylase/hydantoinase route.
1. An isolated Escherichia coli strain
having its dadA gene mutated or deleted so that said gene does not express a functional D-amino acid oxidase; and
having its dsdA gene mutated or deleted so that said gene does not express a functional D-serine dehydratase;
wherein said strain has been transformed with and expresses a D-carbamoylase gene and a D-hydantoinase gene,
wherein the strain is Escherichia coli strain DSM15182 (ET4), and
wherein the isolated strain exhibits reduced breakdown of produced D-amino acids when cultured in a suitable medium.
2. A process for preparing a D-amino acid comprising:
culturing in a suitable medium the isolated Escherichia coli strain of claim 1 , and
recovering a D-amino acid,
wherein the isolated strain exhibits reduced breakdown of produced D-amino acids when cultured.
3. The process of claim 2 , wherein the D-amino acid that is recovered is D-serine.
4. The process of claim 2 , wherein the D-amino acid that is recovered is D-methionine.
5. The process of claim 2 , wherein the D-amino acid that is recovered is D-tryptophan.
6. The process of claim 2 , wherein the D-amino acid that is recovered is D-phenylalanine.
7. The process of claim 2 , wherein the D-amino acid that is recovered is D-aminobutyric acid.
8. A method for producing a D-amino acid via the carbamoylase/hydantoinase route comprising culturing an isolated microorganism that has been transformed with and expresses a D-carbamoylase gene and a D-hydantoinase gene;
wherein said microorganism lacks at least one gene that expresses a functional D-amino oxidase and D-amino acid dehydratase;
wherein said microorganism is selected from the group consisting of Escherichia coli DCM15181 (ET3) and Escherichia coli DSM15182 (ET4), and
wherein the isolated strain exhibits reduced breakdown of produced D-amino acids when cultured.
9. The isolated Escherichia coli strain of claim 1 , wherein the isolated strain exhibits reduced breakdown of produced D-amino acids to less than 10% when cultured for at least 10 hours.
10. The process of claim 2 , wherein the isolated strain exhibits reduced breakdown of produced D-amino acids to less than 10% when cultured for at least 10 hours.
11. The process of claim 8 , wherein the isolated strain exhibits reduced breakdown of produced D-amino acids to less than 10% when cultured for at least 10 hours.
12. The isolated Escherichia coli strain of claim 1 , wherein the amino acid breakdown provided by the isolated strain is less than an amino acid breakdown provided by a strain which has unaltered dadA and dsdA genes.
13. The process of claim 2 , wherein the amino acid breakdown provided by the isolated strain is less than an amino acid breakdown provided by a strain which has unaltered dadA and dsdA genes.
14. The method of claim 8 , wherein the amino acid breakdown provided by the isolated strain is less than an amino acid breakdown provided by a strain which has unaltered dadA and dsdA genes.
15. An isolated Escherichia coli strain that has been transformed with and expresses a D-carbamoylase gene and a D-hydantoinase gene;
wherein said strain is Escherichia coli DCM15181 (ET3) that lacks a dadA gene that expresses a functional D-amino oxidase.