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 6-hydroxyhexanoate methyl ester and hexanoic acid hexyl 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 enzymes. 6-hydroxyhexanoate methyl esters and hexanoic acid hexyl ester can be enzymatically converted to adipic acid, adipate semialdehyde, 6-aminohexanoate, 6-hydroxyhexanoate, hexamethylenediamine, and 1,6-hexanediol.
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 and having at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24;
- 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, said polypeptide having monooxygenase activity classified under EC 1.14.14.- or EC 1.14.15.- and having at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO:
- 13-15, SEQ ID NO:27 or SEQ ID NO:28,
- 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 has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:
- 32, or SEQ ID NO:33, or
- a polypeptide having butanal dehydrogenase activity and a polypeptide having aldehyde dehydrogenase activity.
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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 using a polypeptide having alcohol O-acetyltransferase activity, said polypeptide having alcohol O-acetyltransferase activity and having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 25;
- 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 using a polypeptide having monooxygenase activity, said polypeptide having monooxygenase activity classified under EC 1.14.14.- or EC 1.14.15.- and having at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO:
- 13-15, SEQ ID NO:27 or SEQ ID NO:28.
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A method of enzymatically producing 6-hydroxyhexanoate, the method comprising:
- a) enzymatically converting hexanoate to hexanoate methyl ester using a polypeptide having fatty acid O-methyltransferase activity;
- b) enzymatically converting hexanoate methyl ester to 6-hydroxyhexanoate methyl ester using a polypeptide having monooxygenase activity;
- and
- c) enzymatically converting 6-hydroxyhexanoate methyl ester to 6-hydroxyhexanoate using a polypeptide having demethylase activity or a polypeptide having esterase activity.
A method of enzymatically producing 6-hydroxyhexanoate, the method comprising:
- a) enzymatically converting hexanoyl-CoA to hexanoic acid hexyl ester using a polypeptide having alcohol O-acetyltransferase activity;
- b) enzymatically converting hexanoic acid hexyl ester to 6-hydroxyhexanoic acid hexyl ester, hexanoic acid 6-hydroxyhexyl ester or 6-hydroxyhexanoic acid 6-hydroxyhexyl ester using a polypeptide having monooxygenase activity;
- and
- c) enzymatically converting 6-hydroxyhexanoic acid hexyl ester, hexanoic acid 6-hydroxyhexyl ester or 6-hydroxyhexanoic acid 6-hydroxyhexyl ester to 6-hydroxyhexanoate 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 polypeptide having monooxygenase activity has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO:
- 13-15, SEQ ID NO:27 and/or, SEQ ID NO:28,
- the recombinant host optionally further comprising 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:
- 25, and said polypeptide having monooxygenase activity has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO:
- 13-15, SEQ ID NO:27 and/or, SEQ ID NO:28,
- the recombinant host optionally further comprising one or more of the following exogenous polypeptides:
- (i) a polypeptide having β-ketothiolase activity or a polypeptide having acetyl-CoA carboxylase activity and a polypeptide having O-ketoacyl-[acp] synthase activity, (ii) a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having 3-oxoacyl-CoA reductase activity, (iii) a polypeptide having enoyl-CoA hydratase activity, and (iv) a polypeptide having trans-2-enoyl-CoA reductase activity.
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Dependencies are inferred from the claim text.