Methods, reagents and cells for biosynthesizing compounds
AssigneeInvista North America S.A R.L.
InventorsAdriana Leonora Botes, Alex Van Eck Conradie, Ramdane Haddouche
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Abstract
This document describes biochemical pathways for producing 7-hydroxyheptanoate methyl ester and heptanoic acid heptyl ester using one or more of a fatty acid O-methyltransferase, an alcohol O-acetyltransferase, and a monooxygenase, as well as recombinant hosts expressing one or more of such exogenous enzymes. 7-hydroxyheptanoate methyl esters and heptanoic acid heptyl esters can be enzymatically converted to pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, or 1,7-heptanediol.
Claims83
A method of producing a terminal hydroxyl (C3-8 hydroxyalkyl)-C(═O)OCH3 ester in a recombinant host, said method comprising:
- a) enzymatically converting a C4-9 carboxylic acid to a (C3-8 alkyl)-C(═O)OCH3 ester using a polypeptide having fatty acid O-methyltransferase activity, said polypeptide having fatty acid O-methyltransferase activity having at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25, wherein the C4-9 carboxylic acid is enzymatically produced from a C4-9 alkanoyl-CoA using:
- a polypeptide having thioesterase activity, said polypeptide having thioesterase activity having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:
- 33, or SEQ ID NO:34, or
- a polypeptide having butanal dehydrogenase activity and a polypeptide having aldehyde dehydrogenase activity;
- and
- b) enzymatically converting the (C3-8 alkyl)-C(═O)OCH3 ester to a terminal hydroxyl (C3-8 hydroxyalkyl)-C(═O)OCH3 ester using a polypeptide having monooxygenase activity, wherein the monooxygenase is classified under EC 1.14.14.- or EC 1.14.15.-, and the polypeptide having monooxygenase activity has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO:
- 14, SEQ ID NO:15, SEQ ID NO:
- 16, SEQ ID NO:28 or SEQ ID NO:29.
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A method of producing one or more terminal hydroxy-substituted (C4-9 alkyl)-OC(═O)—(C3-8 alkyl) esters in a recombinant host, said method comprising:
- a) enzymatically converting a C4-9 alkanoyl-CoA to a (C4-9 alkyl)-OC(═O)—(C3-8 alkyl) ester in said host;
- and
- b) enzymatically converting the (C4-9 alkyl)-OC(═O)—(C3-8 alkyl) ester to any of (C4-9 alkyl)-OC(═O)—(C3-8 hydroxyalkyl) ester, (C4-9 hydroxyalkyl)-OC(═O)—(C3-8 hydroxyalkyl) ester, or (C4-9 hydroxyalkyl)-OC(═O)—(C3-8 alkyl) ester in said host, wherein the C4-9 alkanoyl-CoA is enzymatically converted to the (C4-9 alkyl)-OC(═O)—(C3-8 alkyl) ester using a polypeptide having alcohol O-acetyltransferase activity, said polypeptide having alcohol O-acetyltransferase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 26,
- the method optionally further comprising enzymatically converting (C4-9 hydroxyalkyl)-OC(═O)—(C3-8 hydroxyalkyl) ester or (C4-9 hydroxyalkyl)-OC(═O)—(C3-8 alkyl) ester to a terminal hydroxyl C4-9 hydroxyalkanoate.
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A method of enzymatically producing 7-hydroxyheptanoate, the method comprising:
- a) enzymatically converting heptanoate to heptanoate methyl ester using a polypeptide having fatty acid O-methyltransferase activity;
- b) enzymatically converting heptanoate methyl ester to 7-hydroxyheptanoate methyl ester using a polypeptide having monooxygenase activity;
- and
- c) enzymatically converting 7-hydroxyheptanoate methyl ester to 7-hydroxyheptanoate using a polypeptide having demethylase activity or a polypeptide having esterase activity.
A method of enzymatically producing 7-hydroxyheptanoate, the method comprising:
- a) enzymatically converting heptanoyl-CoA to heptanoic acid heptyl ester using a polypeptide having alcohol O-acetyltransferase activity;
- b) enzymatically converting heptanoic acid heptyl ester to 7-hydroxyheptanoic acid heptyl ester, heptanoic acid 7-hydroxyheptyl ester or 7-hydroxyheptanoic acid 7-hydroxyheptyl ester using a polypeptide having monooxygenase activity;
- and
- c) enzymatically converting 7-hydroxyheptanoic acid heptyl ester, heptanoic acid 7-hydroxyheptyl ester or 7-hydroxyheptanoic acid 7-hydroxyheptyl ester to 7-hydroxyheptanoate using a polypeptide having esterase activity.
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A recombinant host comprising at least one exogenous nucleic acid encoding (i) a polypeptide having fatty acid O-methyltransferase activity and (ii) a polypeptide having monooxygenase activity, said recombinant host producing a terminal hydroxyl (C3-8 hydroxyalkyl)-C(═O)OCH3 ester, wherein said polypeptide having fatty acid O-methyltransferase activity has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25 and said monooxygenase has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO:
- 14, SEQ ID NO:15, SEQ ID NO:
- 16, SEQ ID NO:28 and/or SEQ ID NO:29,
- wherein said recombinant host optionally further comprises an exogenous polypeptide having demethylase activity classified under EC 2.1.1.- or a polypeptide having esterase activity classified under EC 3.1.1.-, said host further producing a terminal hydroxyl C4-9 hydroxyalkanoate.
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A recombinant host comprising at least one exogenous nucleic acid encoding (i) a polypeptide having alcohol O-acetyltransferase activity and (ii) a polypeptide having monooxygenase activity, and producing any of a (C4-9 alkyl)-OC(═O)—(C3-8 hydroxyalkyl) ester, a (C4-9 hydroxyalkyl)-OC(═O)—(C3-8 hydroxyalkyl) ester, or a (C4-9 hydroxyalkyl)-OC(═O)—(C3-8 alkyl) ester, wherein said polypeptide having alcohol O-acetyltransferase activity has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 26, and said monooxygenase has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO:
- 14, SEQ ID NO:15, SEQ ID NO:
- 16, SEQ ID NO:28 and/or SEQ ID NO:29, wherein said (C4-9 alkyl)-OC(═O)—(C3-8 hydroxyalkyl) ester is heptanoic acid 7-hydroxyheptyl ester, said (C4-9 hydroxyalkyl)-OC(═O)—(C3-8 hydroxyalkyl) ester is 7-hydroxyheptanoic acid 7-hydroxyheptyl ester, and said (C4-9 hydroxyalkyl)-OC(═O)—(C3-8 alkyl) ester is 7-hydroxyheptanoic acid heptyl ester,
- the recombinant host cell optionally further comprising an exogenous polypeptide having esterase activity and further enzymatically converting heptanoic acid 7-hydroxyheptyl ester, 7-hydroxyheptanoic acid 7-hydroxyheptyl ester, and 7-hydroxyheptanoic acid heptyl ester to one or more of 7-hydroxyheptanoate and 1,7-heptanediol, wherein said polypeptide having esterase activity has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO:
- 27.
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Dependencies are inferred from the claim text.