Methods, reagents and cells for biosynthesizing compounds
AssigneeInvista North America S.A.R.L.
InventorsAdriana Leonora Botes, Alex Van Eck Conradie
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Abstract
This document describes biochemical pathways for producing 2,4-pentadienoyl-CoA by forming one or two terminal functional groups, comprised of carboxyl or hydroxyl group, in a C5 backbone substrate such as glutaryl-CoA, glutaryl-[acp] or glutarate methyl ester. 2,4-pentadienoyl-CoA can be enzymatically converted to 1,3-butadiene.
Claims62
A method of biosynthesizing glutarate methyl ester in a recombinant host, the method comprising enzymatically converting at least one of malonyl-[acp] and malonyl-CoA to glutarate methyl ester in said host using a polypeptide having malonyl-CoA O-methyltransferase activity, a polypeptide having thioesterase activity, or a combination thereof, wherein said polypeptide having malonyl-CoA O-methyltransferase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 21,
- wherein malonyl-CoA is enzymatically converted to malonyl-CoA methyl ester using said at least one polypeptide having malonyl-CoA O-methyltransferase activity;
- or
- wherein malonyl-[acp] is enzymatically converted to malonyl-[acp] methyl ester using said at least one polypeptide having malonyl-CoA O-methyltransferase activity,
- the method optionally further comprising enzymatically converting glutarate methyl ester to glutarate semialdehyde methyl ester in said host using at least one polypeptide having carboxylate reductase activity.
- wherein said enzymatic conversion occurs in the host using a polypeptide having malonyl-CoA O-methyltransferase activity, a polypeptide having thioesterase activity, or a combination thereof.
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A method of making glutarate, said method comprising (i) enzymatically converting glutaryl-[acp] methyl ester to glutaryl-[acp] or glutaryl-CoA methyl ester to glutaryl-CoA using a polypeptide having pimeloyl-[acp] methyl ester methylesterase activity, and (ii) enzymatically converting glutaryl-[acp] or glutaryl-CoA to glutarate using at least one polypeptide having thioesterase activity, reversible CoA-ligase activity, a CoA-transferase activity, an acylating dehydrogenase activity, an aldehyde dehydrogenase activity, a glutarate semialdehyde dehydrogenase activity, or a succinate-semialdehyde dehydrogenase activity, wherein said polypeptide having pimeloyl-[acp] methyl ester methylesterase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 1, wherein:
- glutaryl-[acp] or glutaryl-CoA is enzymatically converted to glutaric acid using a polypeptide having thioesterase activity;
- glutaryl-[acp] or glutaryl-CoA is enzymatically converted to glutaric acid using a polypeptide having reversible CoA-ligase activity or a CoA-transferase activity;
- or
- wherein glutaryl-[acp] or glutaryl-CoA is enzymatically converted to glutaric acid using a polypeptide having an acylating dehydrogenase activity, an aldehyde dehydrogenase activity glutarate semialdehyde dehydrogenase activity, or a succinate-semialdehyde dehydrogenase activity.
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A recombinant host cell comprising at least one exogenous nucleic acid encoding a polypeptide having malonyl-CoA O-methyltransferase activity;
- and a polypeptide having thioesterase activity, the host producing glutarate methyl ester, wherein the polypeptide having malonyl-CoA O-methyltransferase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 21, wherein the polypeptide having thioesterase activity has at least 70% sequence identity to any one of the amino acid sequences set forth in any one of SEQ ID NOs:
- 17-18,
- the recombinant host optionally further comprising an exogenous polypeptide having carboxylate reductase activity, wherein the polypeptide having carboxylate reductase activity has at least 70% sequence identity to any one of the amino acid sequences set forth in any one of SEQ ID NOs:
- 2-7, said host further producing glutarate semialdehyde methyl ester.
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A recombinant host comprising at least one exogenous nucleic acid encoding a polypeptide having pimeloyl-[acp] methyl ester methylesterase activity, and at least one polypeptide having an activity selected from the group consisting of thioesterase activity, reversible CoA-ligase activity, a CoA-transferase activity, an acylating dehydrogenase activity, an aldehyde dehydrogenase activity, a glutarate semialdehyde dehydrogenase activity, and a succinate-semialdehyde dehydrogenase activity, wherein said polypeptide having pimeloyl-[acp] methyl ester methylesterase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 1.
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A method of increasing the activity of a polypeptide having carboxylate reductase activity on a substituted or unsubstituted C4-C8 dicarboxylic acid, the method comprising enzymatically converting said C4-C8 dicarboxylic acid to a HOC(═O)(C2-C6 alkyl)-C(═O)OCH3 ester using a polypeptide having malonyl-CoA methyltransferase activity before enzymatically converting the HOC(═O)(C2-C6 alkyl)-C(═O)OCH3 ester to a HC(═O)(C2-C6 alkyl)-C(═O)OCH3 using a polypeptide having carboxylate reductase activity, the method optionally further comprising converting said HC(═O)(C2-C6 alkyl)-C(═O)OCH3 to a HOCH2(C2-C6 alkyl)-C(═O)OCH3 using a polypeptide having dehydrogenase activity.
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Dependencies are inferred from the claim text.