Adipate (ester or thioester) synthesis

Abstract

The present invention relates to a method for preparing an adipate ester or thioester. The invention further relates to a method for preparing adipic acid from said ester or thioester. Further the invention provides a number of methods for preparing an intermediate for said ester or thioester. Further the invention relates to a method for preparing 6-amino caproic acid (6-ACA), a method for preparing 5-formyl valeric acid (5-FVA), and a method for preparing caprolactam. Further, the invention relates to a host cell for use in a method according to the invention.

1 - 31 . (canceled) 
     
     
         32 . A non-naturally occurring microbial organism, comprising a microbial organism having an adipate thioester pathway comprising at least one exogenous nucleic acid encoding a set of adipate thioester pathway enzymes expressed in a sufficient amount to produce an adipate thioester, wherein said set of adipate thioester pathway enzymes convert a succinate thioester and an acetate thioester to a 3-oxoadipate thioester, a 3-oxoadipate thioester to a 3-hydroxyadipate thioester, a 3-hydroxyadipate thioester to a 5-carboxy-2-pentenoate thioester and a 5-carboxy-2-pentenoate thioester to an adipate thioester. 
     
     
         33 . The non-naturally occurring microbial organism of  claim 32 , wherein said adipate thioester pathway is an adipyl-CoA pathway, said set of adipate thioester pathway enzymes are a set of adipyl-CoA pathway enzymes, said adipyl-CoA pathway enzymes are expressed in a sufficient amount to produce adipyl-CoA, and said set of adipyl-CoA pathway enzymes convert a succinyl-CoA and an acetyl-CoA to a 3-oxoadipyl-CoA, a 3-oxoadipyl-CoA to a 3-hydroxyadipyl-CoA, a 3-hydroxyadipyl-CoA to a 5-carboxy-2-pentenoyl-CoA and a 5-carboxy-2-pentenoyl-CoA to an adipyl-CoA. 
     
     
         34 . The non-naturally occurring microbial organism of  claim 33 , wherein said set adipyl-CoA pathway enzymes comprise a succinyl-CoA:acetyl-CoA acyl transferase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-hydroxyadipyl-CoA dehydratase and a 5-carboxy-2-pentenoyl-CoA reductase. 
     
     
         35 . The non-naturally occurring microbial organism of  claim 34 , further comprising an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, wherein said adipate pathway enzyme is selected from the group consisting of an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl-CoA:acetyl-CoA transferase and an adipyl-CoA hydrolase. 
     
     
         36 . The non-naturally occurring microbial organism of  claim 34 , wherein said at least one exogenous nucleic acid encodes a set of adipate pathway enzymes expressed in a sufficient amount to produce adipate, said set of adipate pathway enzymes comprising an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl-CoA:acetyl-CoA transferase and an adipyl-CoA hydrolase. 
     
     
         37 . The non-naturally occurring microbial organism of  claim 32 , further comprising an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, wherein said adipate pathway enzyme is selected from the group consisting of a succinyl-CoA:acetyl-CoA acyl transferase, a 3-oxoadipyl-CoA transferase, a 3-oxoadipate reductase, a 3-hydroxyadipate dehydratase, and a 2-enoate reductase. 
     
     
         38 . The non-naturally occurring microbial organism of  claim 32 , wherein said at least one exogenous nucleic acid encodes a set of adipate pathway enzymes expressed in a sufficient amount to produce adipate, said set of adipate pathway enzymes comprising a succinyl-CoA:acetyl-CoA acyl transferase, a 3-oxoadipyl-CoA transferase, a 3-oxoadipate reductase, a 3-hydroxyadipate dehydratase, and a 2-enoate reductase. 
     
     
         39 . The non-naturally occurring microbial organism of  claim 32 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         40 . The non-naturally occurring microbial organism of  claim 32 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         41 . A method for producing an adipate thioester, comprising culturing the non-naturally occurring microorganism of  claim 32  under conditions and for a sufficient period of time to produce an adipate thioester. 
     
     
         42 . The method for producing an adipate thioester of  claim 41 , wherein said adipate thioester is adipyl-CoA. 
     
     
         43 . A method for producing adipate, comprising producing an adipate thioester according to the method of  claim 41 , wherein the non-naturally occurring microorganism further comprises an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, wherein said adipate pathway enzyme is selected from the group consisting of an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl-CoA:acetyl-CoA transferase and an adipyl-CoA hydrolase, and culturing the non-naturally occurring microorganism under conditions and for a sufficient period of time to produce adipate. 
     
     
         44 . The method of  claim 41 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         45 . The method of  claim 41 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         46 . A method for producing adipyl-CoA, comprising culturing a host microbial organism in which at least one exogenous nucleic acid is introduced under conditions and for a sufficient period of time to produce adipyl-CoA, wherein said host microbial organism comprises at least one exogenous nucleic acid encoding a set of adipyl-CoA pathway enzymes that are expressed in a sufficient amount to produce adipyl-CoA, wherein said set of adipyl-CoA pathway enzymes convert succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA, 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA and 5-carboxy-2-pentenoyl-CoA to adipyl-CoA. 
     
     
         47 . The method of  claim 46 , wherein said host microbial organism is in a substantially anaerobic culture medium. 
     
     
         48 . The method of  claim 46 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         49 . The method of  claim 46 , wherein said set of adipyl-CoA pathway enzymes comprise a succinyl-CoA:acetyl-CoA acyl transferase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-hydroxyadipyl-CoA dehydratase and a 5-carboxy-2-pentenoyl-CoA reductase. 
     
     
         50 . A method for producing adipate, comprising culturing a host microbial organism in which at least one exogenous nucleic acid is introduced under conditions and for a sufficient period of time to produce adipate, wherein said host microbial organism comprises at least one exogenous nucleic acid encoding a set of adipyl-CoA pathway enzymes that are expressed in a sufficient amount to produce adipyl-CoA, wherein said set of adipyl-CoA pathway enzymes convert succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA, 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA and 5-carboxy-2-pentenoyl-CoA to adipyl-CoA, and wherein said host microbial organism comprises at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, wherein said adipate pathway enzyme is selected from the group consisting of an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl-CoA:acetyl-CoA transferase and an adipyl-CoA hydrolase. 
     
     
         51 . The method of  claim 50 , wherein said host microbial organism is in a substantially anaerobic culture medium. 
     
     
         52 . The method of  claim 50 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         53 . The method of  claim 50 , wherein said at least one exogenous nucleic acid encoding an adipate pathway enzyme encodes a set of adipate pathway enzymes that are expressed in a sufficient amount to produce adipate, said set of adipate pathway enzymes comprising an adipyl-CoA synthetase, a phosphotransadipylase/adipate kinase, an adipyl-CoA:acetyl-CoA transferase and an adipyl-CoA hydrolase. 
     
     
         54 . A method for producing adipate, comprising culturing a host microbial organism in which at least one exogenous nucleic acid is introduced under conditions and for a sufficient period of time to produce adipate, wherein said host microbial organism comprises at least one exogenous nucleic acid encoding a set of adipyl-CoA pathway enzymes that are expressed in a sufficient amount to produce adipyl-CoA, wherein said set of adipyl-CoA pathway enzymes convert succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA, 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA and 5-carboxy-2-pentenoyl-CoA to adipyl-CoA, and wherein said host microbial organism comprises at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, wherein said adipate pathway enzyme is selected from the group consisting of a succinyl-CoA:acetyl-CoA acyl transferase, a 3-oxoadipyl-CoA transferase, a 3-oxoadipate reductase, a 3-hydroxyadipate dehydratase, and a 2-enoate reductase. 
     
     
         55 . The method of  claim 54 , wherein said host microbial organism is in a substantially anaerobic culture medium. 
     
     
         56 . The method of  claim 54 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         57 . The method of  claim 54 , wherein said at least one exogenous nucleic acid encoding an adipate pathway enzyme encodes a set of adipate pathway enzymes that are expressed in a sufficient amount to produce adipate, said set of adipate pathway enzymes comprising a succinyl-CoA:acetyl-CoA acyl transferase, a 3-oxoadipyl-CoA transferase, a 3-oxoadipate reductase, a 3-hydroxyadipate dehydratase, and a 2-enoate reductase. 
     
     
         58 . A non-naturally occurring microbial organism, comprising a microbial organism having an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, said adipate pathway comprising 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthetase, phosphotranshydroxyadipylase/2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. 
     
     
         59 . The non-naturally occurring microbial organism of  claim 58 , wherein said microbial organism comprises two exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         60 . The non-naturally occurring microbial organism of  claim 58 , wherein said microbial organism comprises three exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         61 . The non-naturally occurring microbial organism of  claim 58 , wherein said microbial organism comprises four exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         62 . The non-naturally occurring microbial organism of  claim 58 , wherein said microbial organism comprises five exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         63 . The non-naturally occurring microbial organism of  claim 62 , wherein said five exogenous nucleic acids encode 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthetase, phosphotranshydroxyadipylase/2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA: acetyl-CoA transferase or adipyl-CoA hydrolase. 
     
     
         64 . The non-naturally occurring microbial organism of  claim 58 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         65 . The non-naturally occurring microbial organism of  claim 58 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         66 . A method for producing adipate, comprising culturing a non-naturally occurring microbial organism having an adipate pathway, said pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, under conditions and for a sufficient period of time to produce adipate, said adipate pathway comprising 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthetase, phosphotranshydroxyadipylase/2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. 
     
     
         67 . The method of  claim 66 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         68 . The method of  claim 66 , wherein said microbial organism comprises two exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         69 . The method of  claim 66 , wherein said microbial organism comprises three exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         70 . The method of  claim 66 , wherein said microbial organism comprises four exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         71 . The method of  claim 66 , wherein said microbial organism comprises five exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         72 . The method of  claim 71 , wherein said five exogenous nucleic acids encode 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthetase, phosphotranshydroxyadipylase/2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. 
     
     
         73 . The method of  claim 66 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         74 . A host microbial organism in which at least one exogenous nucleic acid is introduced, wherein said host microbial organism has, an adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, said adipate pathway comprising 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthetase, phosphotranshydroxyadipylase/2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. 
     
     
         75 . The host microbial organism of  claim 74 , wherein said host microbial organism comprises two exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         76 . The host microbial organism of  claim 74 , wherein said host microbial organism comprises three exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         77 . The host microbial organism of  claim 74 , wherein said host microbial organism comprises four exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         78 . The host microbial organism of  claim 74 , wherein said host microbial organism comprises five exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         79 . The host microbial organism of  claim 78 , wherein said five exogenous nucleic acids encode 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthetase, phosphotranshydroxyadipylase/2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. 
     
     
         80 . The host microbial organism of  claim 74 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         81 . The host microbial organism of  claim 74 , wherein said host microbial organism is in a substantially anaerobic culture medium. 
     
     
         82 . A method for producing adipate, comprising culturing a host microbial organism in which at least one exogenous nucleic acid is introduced, wherein said culturing is under conditions and for a sufficient period of time to produce adipate, wherein said host microbial organism has an adipate pathway, said adipate pathway comprising at least one exogenous nucleic acid encoding an adipate pathway enzyme expressed in a sufficient amount to produce adipate, said adipate pathway comprising 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthetase, phosphotranshydroxyadipylase/2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. 
     
     
         83 . The method of  claim 82 , wherein said host microbial organism is in a substantially anaerobic culture medium. 
     
     
         84 . The method of  claim 82 , wherein said host microbial organism comprises two exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         85 . The method of  claim 82 , wherein said host microbial organism comprises three exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         86 . The method of  claim 82 , wherein said host microbial organism comprises four exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         87 . The method of  claim 82 , wherein said host microbial organism comprises five exogenous nucleic acids each encoding an adipate pathway enzyme. 
     
     
         88 . The method of  claim 87 , wherein said five exogenous nucleic acids encode 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoA synthetase, phosphotranshydroxyadipylase/2-hydroxyadipate kinase or 2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. 
     
     
         89 . The method of  claim 82 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         90 . A recombinant microorganism comprising at least one exogenous nucleic acid encoding enzymes that catalyze reactions that result in the production of an adipate thioester, wherein said enzymes convert a succinate thioester and an acetate thioester to a 3-oxoadipate thioester, a 3-oxoadipate thioester to a 3-hydroxyadipate thioester, a 3-hydroxyadipate thioester to a 5-carboxy-2-pentenoate thioester and a 5-carboxy-2-pentenoate thioester to an adipate thioester. 
     
     
         91 . The microorganism of  claim 90 , wherein said adipate thioester is adipyl-CoA and said enzymes convert succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA, 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA and 5-carboxy-2-pentenoyl-CoA to adipyl-CoA. 
     
     
         92 . The microorganism of  claim 91 , wherein said enzymes comprise a succinyl-CoA:acetyl-CoA acyl transferase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-hydroxyadipyl-CoA dehydratase and a 5-carboxy-2-pentenoyl-CoA reductase. 
     
     
         93 . The microorganism of  claim 92 , wherein said microorganism further comprises at least one exogenous nucleic acid encoding at least one enzyme that catalyzes the conversion of adipyl CoA to adipate. 
     
     
         94 . The microorganism of  claim 90 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         95 . The microorganism of  claim 90 , wherein said microorganism is in oxygen limited or anaerobic conditions. 
     
     
         96 . A method for producing an adipate thioester, comprising incubating the microorganism of  claim 90  under conditions such that said adipate thioester is produced. 
     
     
         97 . The method of  claim 96 , wherein said adipate thioester is adipyl-CoA. 
     
     
         98 . A method for producing adipate, comprising incubating the microorganism of  claim 93  under conditions such that adipate is produced. 
     
     
         99 . The method of  claim 96 , wherein said microorganism is in oxygen limited or anaerobic conditions. 
     
     
         100 . The method of  claim 96 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.