Polypeptides and biosynthetic pathways for the production of monatin and its precursors

Abstract

Methods and compositions that can be used to make monatin from glucose, tryptophan, indole-3-lactic acid, indole-3-pyruvate, and 2-hydroxy 2-(indol-3-ylmethyl)-4-keto glutaric acid, are provided. Methods are also disclosed for producing the indole-3-pyruvate and 2-hydroxy2-(indol-3-ylmethyl)-4-keto glutaric acid intermediates. Compositions provided include nucleic acid molecules, polypeptides, chemical structures, and cells. Methods include in vitro and in vivo processes, and the in vitro methods include chemical reactions.

1 . A method for producing monatin or salt thereof comprising:
 (a) contacting a cell with tryptophan, wherein the cell expresses   (i) a first polypeptide that facilitates the conversion of tryptophan to indole-3-pyruvate;   (ii) a second polypeptide that facilitates the conversion of indole-3-pyruvate to 2-hydroxy2-(indol-3-ylmethyl)-4-keto glutaric acid, wherein the second polypeptide is an aldolase; and   (iii) a third polypeptide that facilitates the conversion of 2-hydroxy2-(indol-3-ylmethyl)-4-keto glutaric acid to monatin or salt thereof;   wherein at least one of the first and third polypeptides are selected from the group consisting of HEXAspC aminotransferase (SEQ ID NO:76) and YfdZ (SEQ ID NO:92);   (b) culturing the cell under conditions whereby monatin or salt thereof is produced; and   (c) harvesting monatin or salt thereof.   
     
     
         2 . The method of  claim 1 , wherein the first polypeptide and the third polypeptide are the same. 
     
     
         3 . The method of  claim 1 , wherein the aldolase is selected from the group consisting of 4-hydroxy-2-oxoglutarate aldolase (KHG) (EC 4.1.3.16) and 4-hydroxy-4-methyl-2-oxoglutarate aldolase (ProA) (EC 4.1.3.17). 
     
     
         4 . The method of  claim 3 , wherein the aldolase is derived from  Z. mobilis, C. testosteroni, S. meliloti, B. japonicum, S. paucimobilis, Y. pestis, R. metallidurans, Y. pseudotuberculosis, R. leguminosarum, N. aromaticivorans, P. putida, M magnetotacticum, R. palustris, X. campestris, X. axonopodis , or  S. avermitilis.    
     
     
         5 . The method of  claim 1 , wherein the cell is an  E. coli  cell. 
     
     
         6 . The method of  claim 1 , wherein one or more of the first, second, and third polypeptides are encoded by an exogenous nucleic acid. 
     
     
         7 . The method of  claim 1 , wherein the tryptophan is D-tryptophan.