Methods, reagents and cells for biosynthesizing compounds
AssigneeInvista North America S.A.R.L.
InventorsAlex Van Eck Conradie, Adriana Leonora Botes, Ramdane Haddouche
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Abstract
This document describes biochemical pathways for producing 5-hydroxypentanoate methyl ester and pentanoic acid pentyl 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. 5-hydroxypentanoate methyl esters and pentanoic acid pentyl esters can be enzymatically converted to glutaric acid, 5-aminopentanoate, 5-hydroxypentanoate, cadaverine, or 1,5-pentanediol.
Claims47
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, wherein said polypeptide having fatty acid O-methyltransferase activity is classified under EC 2.1.1.15 and has at least 85% sequence identity to an amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25;
- 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 said polypeptide having monooxygenase activity is classified under EC 1.14.14.- or EC 1.14.15.- and has at least 85% 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,
- the method optionally further comprising enzymatically converting the terminal hydroxyl (C3-8 hydroxyalkyl)-C(═O)OCH3 ester to a terminal hydroxyl C4-9 hydroxyalkanoate.
A method of producing one or more terminal hydroxy-substituted (C4-9alkyl)-OC(═O)—(C3-8alkyl) 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, wherein said polypeptide having alcohol O-acetyltransferase activity is classified under EC 2.3.1.84 and has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 26;
- and
- b) enzymatically converting the (C4-9alkyl)-OC(═O)—(C3-8alkyl) 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,
- the method optionally further comprising enzymatically converting (C4-9 hydroxyalkyl)-OC(═O)—(C3-8 hydroxyalkyl) ester or (C4-9 alkyl)-OC(═O)—(C3-8 hydroxyalkyl) ester to a terminal hydroxyl C4-9 hydroxyalkanoate, wherein the enzymatic conversion uses a polypeptide having esterase activity to convert (C4-9 hydroxyalkyl)-OC(═O)—(C3-8 hydroxyalkyl) ester or (C4-9 hydroxyalkyl)-OC(═O)—(C3-8alkyl) ester to the C4-9 hydroxyalkanoate, wherein said polypeptide having esterase activity is classified under EC 3.1.1.- and has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 27.
A method of enzymatically producing 5-hydroxypentanoate, the method comprising:
- a) enzymatically converting pentanoate to pentanoate methyl ester using a polypeptide having fatty acid O-methyltransferase activity classified under EC 2.1.1.15;
- b) enzymatically converting heptanoate methyl ester to 5-hydroxypentanoate methyl ester using a polypeptide having monooxygenase activity classified under EC 1.14.14.- or EC 1.14.15.-;
- and
- c) enzymatically converting 5-hydroxypentanoate methyl ester to 5-hydroxypentanoate using a polypeptide having demethylase activity classified under EC 2.1.1.- or a polypeptide having esterase activity classified under EC 3.1.1.-.
A method of enzymatically producing 5-hydroxypentanoate, the method comprising:
- a) enzymatically converting pentanoyl-CoA to pentanoic acid pentyl ester using a polypeptide having alcohol O-acetyltransferase activity classified under EC 2.3.1.84;
- b) enzymatically converting pentanoic acid pentyl ester to 5-hydroxypentanoic acid pentyl ester, pentanoic acid 5-hydroxypentyl ester, or 5-hydroxypentanoic acid 5-hydroxypentyl ester using a polypeptide having monooxygenase activity classified under EC 1.14.14.- or EC 1.14.15.-;
- and
- c) enzymatically converting 5-hydroxypentanoic acid pentyl ester, pentanoic acid 5-hydroxypentyl ester, or 5-hydroxypentanoic acid 5-hydroxypentyl ester to 5-hydroxypentanoate using a polypeptide having esterase activity classified under EC 3.1.1.-.
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 is classified under EC 2.1.1.15 and has at least 85% 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 is classified under EC 1.14.14.- or EC 1.14.15.- and has at least 85% 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,
- 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.- and has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 27, said host further producing a terminal hydroxyl C4-9 hydroxyalkanoate.
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 (C4-9alkyl)-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 is classified under EC 2.3.1.84 and has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 26 and said polypeptide having monooxygenase activity is classified under EC 1.14.14.- or EC 1.14.15.- and has at least 85% 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, wherein said (C4-9alkyl)-OC(═O)—(C3-8 hydroxyalkyl) ester is 5-hydroxypentanoic acid pentyl ester, said (C4-9 hydroxyalkyl)-OC(═O)—(C3-8 hydroxyalkyl) ester is 5-hydroxypentanoic acid 5-hydroxypentyl ester, and said (C4-9 hydroxyalkyl)-OC(═O)—(C3-8 alkyl) ester is pentanoic acid 5-hydroxypentyl ester,
- the recombinant host optionally further comprising an exogenous polypeptide having esterase activity and further enzymatically converting pentanoic acid 5-hydroxypentyl ester, 5-hydroxypentanoic acid 5-hydroxypentyl ester, or 5-hydroxypentanoic acid pentyl ester to 5-hydroxypentanoate and/or 1,5-pentanediol, wherein said polypeptide having esterase activity is classified under EC 3.1.1.- and has at least 85% sequence identity to an amino acid sequence set forth in SEQ ID NO:
- 27.
Dependencies are inferred from the claim text.