Materials and Methods for Producing 6-Carbon Monomers

Abstract

This document describes materials and methods for, for example, producing 6-hydroxyhexanoic acid using a β-ketothiolase or synthase and an alcohol O-acetyltransferase to form a 6-acetyloxy-3-oxohexanoyl-CoA intermediate. This document describes biochemical pathways for producing 6-hydroxyhexanoic acid using a β-ketothiolase or synthase and an alcohol O-acetyltransferase to form a 6-acetyloxy-3-oxohexanoyl-CoA intermediate. 6-hydroxyhexanoic acid can be enzymatically converted to adipic acid, caprolactam, 6-aminohexanoic acid, hexamethylenediamine or 1,6-hexanediol. This document also describes recombinant hosts producing 6-hydroxyhexanoic acid as well as adipic acid, caprolactam, 6-aminohexanoic acid, hexamethylenediamine and 1,6-hexanediol.

1 .- 44 . (canceled) 
     
     
         45 . A recombinant host comprising at least one exogenous nucleic acid sequence encoding (i) a polypeptide having the activity of an alcohol O-acetyltransferase classified under EC 2.3.1.84, (ii) a polypeptide having the activity of a β-ketothiolase or synthase classified under EC 2.3.1.16, EC 2.3.1.41, EC 2.3.1.174, EC 2.3.1.179, or EC 2.3.1.180, (iii) a polypeptide having the activity of a thioesterase classified under EC 3.1.2.- or a CoA transferase classified under EC 2.8.3-, (iv) a polypeptide having the activity of an esterase classified under EC 3.1.1.1, EC 3.1.1.6, or EC 3.1.1.85, and one or more exogenous nucleic acid sequences encoding (v) a polypeptide having the activity of a 3-hydroxyacyl-CoA dehydrogenase classified under EC 1.1.1.35, EC 1.1.1.36, or EC 1.1.1.157 or a 3-oxoacyl-CoA reductase classified under EC 1.1.1.100, (vi) a polypeptide having the activity of an enoyl-CoA hydratase classified under EC 4.2.1.17 or EC 4.2.1.119, and/or (vii) a polypeptide having the activity of a trans-2-enoyl-CoA reductase classified under EC 1.3.1.38, EC 1.3.1.44, or EC 1.3.1.8, said recombinant host producing 6-hydroxyhexanoate. 
     
     
         46 . The recombinant host of  claim 45 , further comprising one or more exogenous nucleic acid sequences encoding: a polypeptide having the activity of a monooxygenase in the cytochrome P450 family, a polypeptide having the activity of an alcohol dehydrogenase classified under EC 1.1.1.-, a polypeptide having the activity of a 4-hydroxybutanoate dehydrogenase classified under EC 1.1.1.-, a polypeptide having the activity of a 5-hydroxyvalerate dehydrogenase classified under EC 1.1.1.-, a polypeptide having the activity of a 6-hydroxyhexanoate dehydrogenase classified under EC 1.1.1.258, a polypeptide having the activity of a 7-oxoheptanoate dehydrogenase classified under EC 1.2.1.-, a polypeptide having the activity of a 6-oxohexanoate dehydrogenase classified under EC 1.2.1.-, a polypeptide having the activity of a 5-oxovalerate dehydrogenase classified under EC 1.2.1.-, and/or a polypeptide having the activity of an aldehyde dehydrogenase classified under EC 1.2.1.3, said recombinant host further producing adipic acid. 
     
     
         47 . The recombinant host of  claim 45 , further comprising one or more exogenous nucleic acid sequences encoding: a polypeptide having the activity of a monooxygenase in the cytochrome P450 family, a polypeptide having the activity of a ω-transaminase classified under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82, a polypeptide having the activity of a 6-hydroxyhexanoate dehydrogenase classified under EC 1.1.1.258, a polypeptide having the activity of a 5-hydroxypentanoate dehydrogenase classified under EC 1.1.1.-, a polypeptide having the activity of a 4-hydroxybutyrate dehydrogenase classified under EC 1.1.1.-, and/or a polypeptide having the activity of an alcohol dehydrogenase classified under EC 1.1.1.2, said recombinant host further producing 6-aminohexanoate. 
     
     
         48 . The recombinant host of  claim 47 , further comprising an exogenous nucleic acid sequence encoding a polypeptide having the activity of an amidohydrolase classified under EC 3.5.2.-, said recombinant host further producing caprolactam. 
     
     
         49 . The recombinant host of  claim 45 , further comprising one or more exogenous nucleic acid sequences encoding: a polypeptide having the activity of a carboxylate reductase classified under EC 1.2.99.6, a polypeptide having the activity of a ω-transaminase classified under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82, a polypeptide having the activity of an acyl lysine deacylase classified under EC 3.5.1.17, a polypeptide having the activity of a N-acetyl transferase classified under EC 2.3.1.32, and/or a polypeptide having the activity of an alcohol dehydrogenase classified under EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184, said recombinant host further producing hexamethylenediamine. 
     
     
         50 . The recombinant host of  claim 45 , further comprising an exogenous nucleic acid sequence encoding a polypeptide having the activity of a carboxylate reductase classified under EC 1.2.99.6 and an exogenous nucleic acid sequence encoding a polypeptide having the activity of an alcohol dehydrogenase classified under EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184, said recombinant host further producing 1,6-hexanediol. 
     
     
         51 . The recombinant host of  claim 45 , further comprising one or more exogenous nucleic acid sequences encoding: a polypeptide having the activity of a glutamate synthase classified under EC 1.4.1.13, a polypeptide having the activity of a 2-oxoglutarate decarboxylase classified under EC 4.1.1.71, a polypeptide having the activity of a branch-chain decarboxylase classified under EC 4.1.1.72, a polypeptide having the activity of a glutamate decarboxylase classified under EC 4.1.1.15 or EC 4.1.1.18, a polypeptide having the activity of a ω-transaminase classified under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, or EC 2.6.1.96, a polypeptide having the activity of a CoA-ligase classified under EC 6.2.1.-, a polypeptide having the activity of a CoA-transferase classified under EC 2.8.3.-, and/or a polypeptide having the activity of an alcohol dehydrogenase classified under EC 1.1.1.61. 
     
     
         52 . The recombinant host of  claim 45 , wherein said polypeptide having the activity of a β-ketothiolase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 16. 
     
     
         53 . The recombinant host of  claim 45 , wherein said polypeptide having the activity of an enoyl-CoA reductase has at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 23-28. 
     
     
         54 . The recombinant host  claim 45 , wherein said polypeptide having the activity of an alcohol O-acetyltransferase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. 
     
     
         55 . The recombinant host  claim 45 , wherein said polypeptide having the activity of a carboxylate reductase has at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 2-6. 
     
     
         56 . The recombinant host of  claim 45 , wherein said polypeptide having the activity of a ω-transaminase has at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 7-12. 
     
     
         57 . The recombinant host of, wherein said polypeptide having the activity of an esterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 18. 
     
     
         58 .- 71 . (canceled) 
     
     
         72 . A nucleic acid construct or expression vector comprising
 (a) a polynucleotide encoding a polypeptide having the activity of a β-ketothiolase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a β-ketothiolase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 16; or   (b) a polynucleotide encoding a polypeptide having the activity of a trans-2-enoyl-CoA reductase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a trans-2-enoyl-CoA reductase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28; or   (c) a polynucleotide encoding a polypeptide having the activity of ω-transaminase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of ω-transaminase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; or   (d) a polynucleotide encoding a polypeptide having the activity of a phosphopantetheinyl transferase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having phosphopantetheinyl transferase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 13 or 14;   (e) a polynucleotide encoding a polypeptide having the activity of an alcohol-O-acetyltransferase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of an alcohol-O-acetyltransferase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 17;   (f) a polynucleotide encoding a polypeptide having the activity of a carboxylate reductase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a carboxylate reductase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6; or   (g) a polynucleotide encoding a polypeptide having the activity of a esterase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a esterase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 15;   (h) a polynucleotide encoding a polypeptide having the activity of a pimeloyl-[acp] methyl ester esterase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a pimeloyl-[acp] methyl ester esterase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 18; or   (i) a polynucleotide encoding a polypeptide having the activity of a CoA-transferase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a CoA-transferase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 19; or   (j) a polynucleotide encoding a polypeptide having the activity of a decarboxylase, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having the activity of a decarboxylase is selected from the group consisting of a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.   
     
     
         73 . A composition comprising the nucleic acid construct or expression vector of  claim 72 .