Patent ID: 8153405

Claim:
A method of producing monatin by converting tryptophan to indole-3-pyruvate using a first aminotransferase, converting indole-3-pyruvate to 2-hydroxy 2-(indole-3-ylmethyl)-4-keto glutaric acid (“monatin precursor”) using an aldolase, and converting monatin precursor to monatin using either the first aminotransferase or a second aminotransferase, the method comprising: a) genetically engineering a Gram-negative bacteria to express: i) a nucleic acid that encodes the first aminotransferase, wherein the first aminotransferase is selected from the group consisting of tryptophan aminotransferase (EC 2.6.1.27), tyrosine (aromatic) aminotransferase (EC 2.6.1.5), tryptophan dehydrogenase (EC 1.4.1.19), tryptophan-phenylpyruvate transaminase (EC 2.6.1.28), aspartate aminotransferase (EC 2.6.1.1.), L-amino acid oxidase (EC 1.4.3.2), D-amino acid dehydrogenase (EC 1.4.99.1), D-amino acid oxidase (EC 1.4.3.3), and D-amino acid (D-alanine) aminotransferase (EC 2.6.1.21); ii) where a second aminotransferase is present, a nucleic acid that encodes the second aminotransferase, wherein the second aminotransferase is selected from the group consisting of tryptophan aminotransferase (EC 2.6.1.27), tyrosine (aromatic) aminotransferase (EC 2.6.1.5), tryptophan dehydrogenase (EC 1.4.1.19), aspartate aminotransferase (EC 2.6.1.1.), glutamate dehydrogenase (EC 1.4.1.2-4), phenylalanine dehydrogenase (EC 1.4.1.20), D-amino acid dehydrogenase (EC 1.4.99.1), and D-amino acid (D-alanine) aminotransferase (EC 2.6.1.21); iii) a nucleic acid that encodes the aldolase, wherein the aldolase is selected from the group consisting of 4-hydroxy-2-oxoglutarate glyoxylate-lyase (EC 4.1.3.16) and 4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase (EC 4.1.3.17); and iv) a nucleic acid that encodes one or more bacterial TolC polypeptides capable of secreting monatin; b) culturing the Gram-negative bacteria, wherein the Gram-negative bacteria expresses nucleic acids sufficient for the intracellular synthesis of monatin; c) secreting monatin using the one or more TolC polypeptides; and d) collecting at least a portion of the secreted monatin.