Organisms for the production of cyclohexanone
A non-naturally occurring microbial organism has cyclohexanone pathways that include at least one exogenous nucleic acid encoding a cyclohexanone pathway enzyme. A pathway includes a 2-ketocyclohexane-1-carboxyl-CoA hydrolase (acting on C—C bond), a 2-ketocyclohexane-1-carboxylate decarboxylase and an enzyme selected from a 2-ketocyclohexane-1-carboxyl-CoA hydrolase (acting on thioester), a 2-ketocyclohexane-1-carboxyl-CoA transferase, and a 2-ketocyclohexane-1-carboxyl-CoA synthetase. A pathway includes an enzyme selected from a 6-ketocyclohex-1-ene-1-carboxyl-CoA hydrolase (acting on C—C bond), a 6-ketocyclohex-1-ene-1-carboxyl-CoA synthetase, a 6-ketocyclohex-1-ene-1-carboxyl-CoA hydrolase (acting on thioester), a 6-ketocyclohex-1-ene-1-carboxyl-CoA transferase, a 6-ketocyclohex-1-ene-1-carboxyl-CoA reductase, a 6-ketocyclohex-1-ene-1-carboxylate decarboxylase, a 6-ketocyclohex-1-ene-1-carboxylate reductase, a 2-ketocyclohexane-1-carboxyl-CoA synthetase, a 2-ketocyclohexane-1-carboxyl-CoA transferase, a 2-ketocyclohexane-1-carboxyl-CoA hydrolase (acting on thioester), a 2-ketocyclohexane-1-carboxylate decarboxylase, and a cyclohexanone dehydrogenase. A pathway includes an adipate semialdehyde dehydratase, a cyclohexane-1,2-diol dehydrogenase, and a cyclohexane-1,2-diol dehydratase. A pathway includes a 3-oxopimelate decarboxylase, a 4-acetylbutyrate dehydratase, a 3-hydroxycyclohexanone dehydrogenase, a 2-cyclohexenone hydratase, a cyclohexanone dehydrogenase and an enzyme selected from a 3-oxopimeloyl-CoA synthetase, a 3-oxopimeloyl-CoA hydrolase (acting on thioester), and a 3-oxopimeloyl-coA transferase. Each these pathways can include a PEP carboxykinase. A method for producing cyclohexanone includes culturing these non-naturally occurring microbial organisms.
1. A non-naturally occurring microbial organism, wherein said microbial organism comprises exogenous nucleic acids encoding each of the following cyclohexanone pathway enzymes:
(1) a PEP carboxykinase,
(2) a 2-ketocyclohexane-1-carboxyl-CoA hydrolase (acting on C—C bond),
(3) a 2-ketocyclohexane-1-carboxylate decarboxylase, and
(4) an enzyme selected from the group consisting of
(i) a 2-ketocyclohexane-1-carboxyl-CoA hydrolase (acting on thioester),
(ii) a 2-ketocyclohexane-1-carboxyl-CoA transferase, and
(iii) a 2-ketocyclohexane-1-carboxyl-CoA synthetase,
wherein said cyclohexanone pathway enzymes are expressed in a sufficient amount to produce cyclohexanone.
2. The non-naturally occurring microbial organism of claim 1 , wherein said exogenous nucleic acids are heterologous nucleic acids.
3. The non-naturally occurring microbial organism of claim 1 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
4. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism has a native pimeloyl-CoA pathway.
5. A method for producing cyclohexanone, comprising culturing a non-naturally occurring microbial organism a, wherein said microbial organism comprises nucleic acids encoding each of the following cyclohexanone pathway enzymes:
(1) a PEP carboxykinase,
(2) a 2-ketocyclohexane-1-carboxyl-CoA hydrolase (acting on C—C bond),
(3) a 2-ketocyclohexane-1-carboxylate decarboxylase, and
(4) an enzyme selected from the group consisting of
(i) a 2-ketocyclohexane-1-carboxyl-CoA hydrolase (acting on thioester),
(ii) a 2-ketocyclohexane-1-carboxyl-CoA transferase, and
(iii) a 2-ketocyclohexane-1-carboxyl-CoA synthetase,
wherein said cyclohexanone pathway enzymes are expressed in a sufficient amount to produce cyclohexanone, under conditions and for a sufficient period of time to produce cyclohexanone.
6. The method of claim 5 , wherein said exogenous nucleic acids are heterologous nucleic acids.
7. The method of claim 5 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
8. The method of claim 5 , wherein said microbial organism has a native pimeloyl-CoA pathway.
9. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism further comprises at least one exogenous nucleic acid encoding an enzyme selected from the group consisting of an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a glutaryl-CoA dehydrogenase, a oxopimeloyl-CoA:glutaryl-CoA acyltransferase, a 3-hydroxypimeloyl-CoA dehydrogenase, a 3-hydroxypimeloyl-CoA dehydratase, and a pimeloyl-CoA dehydrogenase.
10. The method of claim 5 , wherein said microbial organism further comprises at least one exogenous nucleic acid encoding an enzyme selected from the group consisting of an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a glutaryl-CoA dehydrogenase, a oxopimeloyl-CoA:glutaryl-CoA acyltransferase, a 3-hydroxypimeloyl-CoA dehydrogenase, a 3-hydroxypimeloyl-CoA dehydratase, or a pimeloyl-CoA dehydrogenase.