Methods, reagents and cells for biosynthesizing compounds

Patent metadata

Field	Value
Publication number	US-9777302-B2
Application number	US-201514741259-A
Country	US
Kind code	B2
Filing date	Jun 16, 2015
Priority date	Jun 16, 2014
Publication date	Oct 3, 2017
Grant date	Oct 3, 2017

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.

First claim

What is claimed is:

1. A method of producing a terminal hydroxyl (C 3-8 hydroxyalkyl)-C(═O)OCH 3 ester in a recombinant host, said method comprising:
a) enzymatically converting a C 4-9 carboxylic acid to a (C 3-8 alkyl)-C(═O)OCH 3 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 (C 3-8 alkyl)-C(═O)OCH 3 ester to a terminal hydroxyl (C 3-8 hydroxyalkyl)-C(═O)OCH 3 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 (C 3-8 hydroxyalkyl)-C(═O)OCH 3 ester to a terminal hydroxyl C 4-9 hydroxyalkanoate.

2. The method of claim 1 , wherein the C 4-9 carboxylic acid is enzymatically produced from a C 4-9 alkanoyl-CoA using:
a polypeptide having thioesterase activity, wherein said polypeptide having thioesterase activity is classified under EC 3.1.2.- and has at least 85% 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, wherein said polypeptide having butanal dehydrogenase activity is classified under EC 1.2.1.10 or EC 1.2.1.57 and has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30 and said polypeptide having aldehyde dehydrogenase activity is classified under EC 1.2.1.3 or EC 1.2.1.4.

3. The method of claim 1 , wherein a polypeptide having demethylase activity classified under EC 2.1.1.- or a polypeptide having esterase activity classified under EC 3.1.1.- enzymatically converts the terminal hydroxyl (C 3-8 hydroxyalkyl)-C(═O)OCH 3 ester to a terminal hydroxyl C 4-9 hydroxyalkanoate.

4. The method of claim 1 , wherein the C 4-9 carboxylic acid is pentanoate, and is enzymatically converted to pentanoate methyl ester; and the pentanoate methyl ester is enzymatically converted to 5-hydroxypentanoate methyl ester,
the method optionally further comprising 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.-.

5. The method of claim 2 , wherein the C 4-9 alkanoyl-CoA is pentanoyl-CoA and pentanoate is enzymatically produced from pentanoyl-CoA.

6. A method of producing one or more terminal hydroxy-substituted (C 4-9 alkyl)-OC(═O)—(C 3-8 alkyl) esters in a recombinant host, said method comprising:
a) enzymatically converting a C 4-9 alkanoyl-CoA to a (C 4-9 alkyl)-OC(═O)—(C 3-8 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 (C 4-9 alkyl)-OC(═O)—(C 3-8 alkyl) ester to any of (C 4-9 alkyl)-OC(═O)—(C 3-8 hydroxyalkyl) ester, (C 4-9 hydroxyalkyl)-OC(═O)—(C 3-8 hydroxyalkyl) ester, or (C 4-9 hydroxyalkyl)-OC(═O)—(C 3-8 alkyl) ester in said host,
the method optionally further comprising enzymatically converting (C 4-9 hydroxyalkyl)-OC(═O)—(C 3-8 hydroxyalkyl) ester or (C 4-9 alkyl)-OC(═O)—(C 3-8 hydroxyalkyl) ester to a terminal hydroxyl C 4-9 hydroxyalkanoate, wherein the enzymatic conversion uses a polypeptide having esterase activity to convert (C 4-9 hydroxyalkyl)-OC(═O)—(C 3-8 hydroxyalkyl) ester or (C 4-9 hydroxyalkyl)-OC(═O)—(C 3-8 alkyl) ester to the C 4-9 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.

7. The method of claim 6 , wherein (C 4-9 alkyl)-OC(═O)—(C 3-8 alkyl) ester is enzymatically converted to any of (C 4-9 alkyl)-OC(═O)—(C 3-8 hydroxyalkyl) ester, (C 4-9 hydroxyalkyl)-OC(═O)—(C 3-8 hydroxyalkyl) ester, or (C 4-9 hydroxyalkyl)-OC(═O)—(C 3-8 alkyl) 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.

8. The method of claim 6 , wherein the C 4-9 alkanoyl-CoA is pentanoyl-CoA and is enzymatically converted to pentanoic acid pentyl ester; and pentanoic acid pentyl ester is enzymatically converted to any of 5-hydroxypentanoic acid pentyl ester, 5-hydroxypentanoic acid 5-hydroxypentyl ester, or pentanoic acid 5-hydroxypentyl ester using a polypeptide having monooxygenase activity classified under EC 1.14.14.- or EC 1.14.15.-,
the method optionally further comprising enzymatically converting 5-hydroxypentanoic acid 5-hydroxypentyl ester or 5-hydroxypentanoic acid pentyl ester to 5-hydroxypentanoate, said enzymatic conversion of 5-hydroxypentanoic acid 5-hydroxypentyl ester or 5-hydroxypentanoic acid pentyl ester to 5-hydroxypentanoate using a polypeptide having esterase activity classified under EC 3.1.1.-.

9. The method of claim 8 , said method further comprising enzymatically converting 5-hydroxypentanoic acid 5-hydroxypentyl ester or pentanoic acid 5-hydroxypentyl ester to 1,5-pentanediol, said enzymatic conversion of 5-hydroxypentanoic acid 5-hydroxypentyl ester or pentanoic acid 5-hydroxypentyl ester to 1,5-pentanediol using a polypeptide having esterase activity classified under EC 3.1.1.-,
the method optionally further comprising enzymatically converting 1,5-pentanediol to 5-hydroxypentanoate using a polypeptide having alcohol dehydrogenase activity classified under EC 1.1.1- and a polypeptide having aldehyde dehydrogenase activity classified under EC 1.2.1.3 or EC 1.2.1.4.

10. The method of claim 8 , wherein the method further comprises enzymatically converting 5-hydroxypentanoic acid 5-hydroxypentyl ester or 5-hydroxypentanoic acid pentyl ester to 5-hydroxypentanoate, said enzymatic conversion of 5-hydroxypentanoic acid 5-hydroxypentyl ester or 5-hydroxypentanoic acid pentyl ester to 5-hydroxypentanoate using a polypeptide having esterase activity classified under EC 3.1.1.-,
said method further comprising enzymatically converting 5-hydroxypentanoate to glutarate semialdehyde, said enzymatic conversion of 5-hydroxypentanoate to glutarate semialdehyde using:
a polypeptide having alcohol dehydrogenase activity classified under EC 1.1.1.-,
a polypeptide having 6-hydroxyhexanoate dehydrogenase activity classified under EC 1.1.1.258,
or
a polypeptide having cytochrome P450 monooxygenase activity classified under EC 1.14.14.-,
the method optionally further comprising enzymatically converting glutarate semialdehyde to glutaric acid using a polypeptide having 7-oxoheptanoate dehydrogenase activity classified under EC 1.2.1.-, a polypeptide having 6-oxohexanoate dehydrogenase activity classified under EC 1.2.1.63, a polypeptide having 5-oxopentanoate dehydrogenase activity classified under EC 1.2.1.20, a polypeptide having aldehyde dehydrogenase activity classified under EC 1.2.1.3, or a polypeptide having

Assignees

• Invista North America S Á R L
• Invista North America Sarl

Inventors

• Conradie Alex Van Eck
• Botes Adriana Leonora
• Haddouche Ramdane

Classifications

• C07C47/12

  containing more than one —CHO group · CPC title

• C12P7/44

  Polycarboxylic acids · CPC title

• C12Y102/99006

  Carboxylate reductase (1.2.99.6) · CPC title

• C12P7/6409

  Fatty acids · CPC title

• C12Y206/01019

  4-Aminobutyrate—2-oxoglutarate transaminase (2.6.1.19) · CPC title