Generation of acyl amino acids

Abstract

Engineered polypeptides useful in synthesizing acyl amino acids are provided. Also provided are methods of making acyl amino acids using engineered polypeptides. In certain embodiments, an acyl amino acid produced using compositions and/or methods of the present invention comprises cocoyl glutamate.

1 . A method of producing an acyl amino acid, comprising steps of:
 providing an engineered polypeptide comprising a fatty acid linkage domain, a peptide synthetase domain, and a thioesterase domain;   which fatty acid linkage domain, peptide synthetase domain and thioesterase domain are covalently linked;   providing fatty acid recognized by the fatty acid linkage domain;   providing an amino acid recognized by the peptide synthetase domain;   incubating the engineered polypeptide, fatty acid, and amino acid under conditions and for a time sufficient for the fatty acid linkage domain to link the fatty acid to the amino acid to generate an acyl amino acid;   incubating the engineered polypeptide and the acyl amino acid under conditions and for a time sufficient for the thioesterase domain to catalyze release of the acyl amino acid from the engineered polypeptide.   
     
     
         2 . A method of producing an acyl amino acid, comprising steps of:
 providing an engineered polypeptide comprising a fatty acid linkage domain, a peptide synthetase domain, and a reductase domain;   which fatty acid linkage domain, peptide synthetase domain and reductase domain are covalently linked;   providing a fatty acid recognized by the fatty acid linkage domain;   providing an amino acid recognized by the peptide synthetase domain;   incubating the engineered polypeptide, fatty acid, and amino acid under conditions and for a time sufficient for the fatty acid linkage domain to link the fatty acid to the amino acid to generate an acyl amino acid;   incubating the engineered polypeptide and the acyl amino acid under conditions and for a time sufficient for the reductase domain to catalyze release of the acyl amino acid from the engineered polypeptide.   
     
     
         3 . The method of  claim 2 , wherein fatty acid linkage domain is a beta-hydroxy fatty acid linkage domain. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 3 , wherein the beta-hydroxy fatty acid is beta-hydroxy myristic acid. 
     
     
         6 . The method of  claim 2 , wherein the fatty acid linkage domain is at least 70% identical to the surfactin synthetase complex fatty acid linkage domain. 
     
     
         7 .- 9 . (canceled) 
     
     
         10 . The method of  claim 2 , wherein the amino acid is glutamate. 
     
     
         11 . The method of  claim 2 , wherein the peptide synthetase domain is at least 70% identical to the first peptide synthetase domain of the surfactin synthetase complex SrfA-A polypeptide subunit. 
     
     
         12 .- 14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein the thioesterase domain is at least 70% identical to the surfactin synthetase complex thioesterase domain. 
     
     
         16 .- 18 . (canceled) 
     
     
         19 . The method of  claim 15 , wherein the thioesterase domain comprises SEQ ID NO: 2. 
     
     
         20 . The method of  claim 2 , wherein the reductase domain is at least 70% identical to the linear gramicidin synthetase complex reductase domain. 
     
     
         21 .- 23 . (canceled) 
     
     
         24 . The method of  20 , wherein the reductase domain comprises SEQ ID NO: 1. 
     
     
         25 . The method of  claim 2 , wherein:
 the amino acid is glutamate;   the fatty acid is beta-hydroxy myristic acid;   and the acyl amino acid is cocoyl glutamate.   
     
     
         26 . The method of  claim 2 , wherein the engineered polypeptide is introduced into a host cell. 
     
     
         27 . The method of  claim 26 , wherein the host cell is  Bacillus subtilis.    
     
     
         28 . The method of  claim 26 , wherein the host cell is a plant cell. 
     
     
         29 . A transgenic plant comprising an engineered polypeptide comprising:
 (A) a fatty acid linkage domain, a peptide synthetase domain, and a thioesterase domain;   which fatty acid linkage domain, peptide synthetase domain and thioesterase domain are covalently linked; or   (B) a fatty acid linkage domain, a peptide synthetase domain, and a reductase domain;   which fatty acid linkage domain, peptide synthetase domain and reductase domain are covalently linked.   
     
     
         30 . (canceled) 
     
     
         31 . The transgenic plant of  claim 29 , wherein the fatty acid linkage domain is a beta-hydroxy fatty acid linkage domain.