Patent ID: 7888081

Claim:
A method of making monatin in vitro, the method comprising: a) providing D-tryptophan and pyruvate to a reactor, forming a first composition which includes monatin produced via a multi-step equilibrium pathway in which D-tryptophan is converted to indole-3-pyruvate, indole-3-pyruvate is converted to 2-hydroxy 2-(indol-3-ylmethyl)-4-keto glutarie (MP), and MP is converted to R,R-monatin; b) providing to the reactor a first-step polypeptide capable of facilitating the conversion of D-tryptophan to indole-3-pyruvate, wherein the first-step polypeptide is chosen from 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), D-amino acid (D-alanine) aminotransferase (EC 2.6.1.21), or a combination thereof; c) providing to the reactor a second-step polypeptide capable of facilitating the conversion of indole-3-pyruvate to MP, wherein the second-step polypeptide is chosen from 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); d) providing to the reactor a third-step polypeptide capable of facilitating the conversion of MP to R—R-monatin, wherein the third-step polypeptide is chosen from tryptophan aminotransferase (2.6.1.27), tyrosine (aromatic) aminotransferase (2.6.1.5), tryptophan dehydrogenase (1.4.1.19), tryptophan-phenylpyruvate transaminase (EC 2.6.1.28), aspartate aminotransferase (EC 2.6.1.1), glutamate dehydrogenase (1.4.1.2-4), phenylalanine dehydrogenase (EC 1.4.1.20), D-amino acid dehydrogenase (1.4.99.1), D-amino acid (D-alanine) aminotransferase (EC 2.6.1.21), D-methionine-pyruvate aminotransferase (2.6.1.41), and combinations thereof; e) removing, inhibiting, inactivating or degrading the second-step polypeptide to form a second composition; and f) providing alanine to the second composition to drive the conversion of MP to R,R-monatin.