Methods and materials for biosynthesizing multifunctional, multivariate molecules via carbon chain modification

Patent metadata

Field	Value
Publication number	US-10801046-B2
Application number	US-201715659505-A
Country	US
Kind code	B2
Filing date	Jul 25, 2017
Priority date	Jul 25, 2016
Publication date	Oct 13, 2020
Grant date	Oct 13, 2020

Abstract

This document describes biochemical pathways for producing a difunctional product having an odd number of carbon atoms in vitro or in a recombinant host, or salts or derivatives thereof, by forming two terminal functional groups selected from carboxyl, amine, formyl, and hydroxyl groups in an aliphatic carbon chain backbone having an odd number of carbon atoms synthesized from (i) acetyl-CoA and propanedioyl-CoA via one or more cycles of methyl ester shielded carbon chain elongation or (ii) propanedioyl-[acp] via one or more cycles of methyl ester shielded carbon chain elongation. The biochemical pathways and metabolic engineering and cultivation strategies described herein rely on enzymes or homologs accepting methyl ester shielded aliphatic carbon chain backbones and maintaining the methyl ester shield for at least one further enzymatic step following one or more cycles of methyl ester shielded carbon chain elongation.

First claim

What is claimed is:

1. A method for biosynthesizing a difunctional product in a recombinant Escherichia host having a BioH deficient background, said method comprising:
enzymatically synthesizing an aliphatic carbon chain backbone having nine, eleven, thirteen, fifteen, seventeen, or nineteen carbon atoms from (i) acetyl-CoA and propanedioyl-CoA via three or more cycles of methyl ester shielded carbon chain elongation or (ii) propanedioyl-[acp] via three or more cycles of methyl ester shielded carbon chain elongation, wherein:
a polypeptide encoded by an exogenous gene and having the activity of a S-adenosyl-L-methionine (SAM)-dependent methyltransferase under EC 2.1.1.197 converts propanedioyl-CoA to propanedioyl-CoA methyl ester or converts propanedioyl-[acp] to propanedioyl-[acp] methyl ester before said three or more cycles of methyl ester shielded carbon chain elongation; and
each of said three or more methyl ester shielded cycles of carbon chain elongation comprises using genes encoding (i) a polypeptide having the activity of a β-ketoacyl-[acp] synthase classified under EC 2.3.1.41, EC 2.3.1.179, or EC 2.3.1.180 or a β-ketothiolase classified under EC 2.3.1.16, (ii) a polypeptide having the activity of a 3-oxoacyl-[acp] reductase classified under EC 1.1.1.100, an acetoacetyl-CoA reductase classified under EC 1.1.1.36, a 3-hydroxyacyl-CoA dehydrogenase classified under EC 1.1.1.35, EC 1.1.1.36, or EC 1.1.1.157, or a 3-hydroxyhutyryl-CoA dehydrogenase classified under EC 1.1.1.157, (iii) an enoyl-CoA hydratase classified under EC 4.2.1.17, EC 4.2.1.119, or EC 4.2.1.150 or a 3-hydroxyacyl-[acp] dehydratase classified under EC 4.2.1.59, and (iv) an enoyl-[acp]reductase classified under EC 1.3.1.9 or EC 1.3.1.10 or a trans-2-enoyl-CoA reductase classified under EC 1.3.1.8, EC 1.3.1.38, or EC 1.3.1.44;
enzymatically forming a first terminal functional group selected from carboxyl, amine, formyl, and hydroxyl groups in said aliphatic carbon chain backbone while maintaining said methyl ester shield for at least one further enzymatic step;
enzymatically forming a second terminal functional group selected from carboxyl, amine, formyl, and hydroxyl groups in said aliphatic carbon chain backbone, thereby forming said difunctional product, wherein:
a carboxyl terminal group is formed by a polypeptide having the activity of a thioesterase classified under EC 3.1.1.2, EC 3.1.1.5, 3.1.2.14, EC 3.1.2.21, or EC 3.1.2.27, an aldehyde dehydrogenase classified under EC 1.2.1.3, a 6-oxohexanoate dehydrogenase classified under EC 1.2.1.63, a CoA-transferase classified under EC 2.8.3.12, or a reversible CoA-ligase classified under EC 6.2.1.5;
an amine terminal group is formed by a polypeptide having the activity of an aminotransferase classified under EC 2.6.1.11, EC 2.6.1.13, EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, EC 2.6.1.82, EC 4.1.1.64, or EC 5.4.3.8 or a deacetylase classified under EC 3.5.1.62;
a formyl terminal group is formed by a polypeptide having the activity of an acetylating aldehyde dehydrogenase classified under EC 1.2.1.10, a succinate semialdehyde dehydrogenase classified under EC 1.2.1.76, or an oxoglutarate dehydrogenase classified under EC 1.2.1.52; or
a hydroxyl terminal group is formed by a polypeptide having the activity of an alcohol dehydrogenase classified under EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, EC 1.1.1.61, EC 1.1.1.184, or EC 1.1.1.258.

2. The method of claim 1 , wherein said aliphatic carbon chain backbone is i) nonanedioyl-[acp] methyl ester or nonanedioyl-CoA methyl ester, ii) undecanedioyl-[acp] methyl ester or undecanedioyl-CoA methyl ester, iii) tridecanedioyl-[acp]methyl ester or tridecanedioyl-CoA methyl ester, iv) pentadecanedioyl-[acp] methyl ester or pentadecanedioyl-CoA methyl ester, v) heptadecanedioyl-[acp] methyl ester or heptadecanedioyl-CoA methyl ester, or vi) nonadecanedioyl-[acp] methyl ester or nonadecanedioyl-CoA methyl ester.

3. The method of claim 1 , wherein:
said at least one further enzymatic step comprises the enzymatic conversion of said aliphatic carbon chain backbone to a monomethyl carboxylate using a polypeptide having the activity of a thioesterase classified under EC 3.1.1.2, EC 3.1.1.5, EC 3.1.2.14, EC 3.1.2.21, or EC 3.1.2.27; and
said at least one further enzymatic step also produces holo-ACP or holo-CoA.

4. The method of claim 3 , wherein:
said at least one further enzymatic step comprises the enzymatic conversion of nonanedioyl-[acp] methyl ester to monomethyl nonanedioate or nonanedioyl-CoA methyl ester to monomethyl nonanedioate;
said at least one further enzymatic step comprises the enzymatic conversion of undecanedioyl-[acp] methyl ester to monomethyl undecanedioate or undecanedioyl-CoA methyl ester to monomethyl undecanedioate;
said at least one further enzymatic step comprises the enzymatic conversion of tridecanedioyl-[acp] methyl ester to monomethyl tridecanedioate or tridecanedioyl-CoA methyl ester to monomethyl tridecanedioate;
said at least one further enzymatic step comprises the enzymatic conversion of pentadecanedioyl-[acp] methyl ester to monomethyl pentadecanedioate or pentadecanedioyl-CoA methyl ester to monomethyl pentadecanedioate;
said at least one further enzymatic step comprises the enzymatic conversion of heptadecanedioyl-[acp] methyl ester to monomethyl heptadecanedioate or heptadecanedioyl-CoA methyl ester to monomethyl heptadecanedioate; or
said at least one further enzymatic step comprises the enzymatic conversion of nonadecanedioyl-[acp] methyl ester to monomethyl nonadecanedioate or nonadecanedioyl-CoA methyl ester to monomethyl nonadecanedioate.

5. The method of claim 1 , wherein said at least one further enzymatic step further comprises the enzymatic conversion of said monomethyl carboxylate to a monomethyl carboxylate semialdehyde using a polypeptide having the activity of a carboxylate reductase classified under EC 1.2.99.6.

6. The method of claim 5 , wherein:
said at least one further enzymatic step comprises the enzymatic conversion of monomethyl nonanedioate to methyl 9-oxononanoate;
said at least one further enzymatic step comprises the enzymatic conversion of monomethyl undecanedioate to methyl 11-oxoundecanoate;
said at least one further enzymatic step comprises the enzymatic conversion of monomethyl tridecanedioate to methyl 13-oxotridecanoate;
said at least one further enzymatic step comprises the enzymatic conversion of monomethyl pentadecanedioate to methyl 15-oxopentadecanoate;
said at least one further enzymatic step comprises the enzymatic conversion of monomethyl heptadecanedioate to methyl 17-oxoheptadecanoate; or
said at least one further enzymatic step comprises the enzymatic conversion of monomethyl nonadecanedioate to methyl 19-oxononadecanoate.

7. The method of claim 5 , wherein said at least one further enzymatic step further comprises the enzymatic conversion of said monomethyl carboxylate semialdehyde to a monomethyl aminocarboxylate using a polypeptide having the activity of an aminotransferase classified under EC 2.6.1.11, EC 2.6.1.13, EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, EC 2.6.1.82, EC 4.1.1.64, or EC 5.4.3.8.

8. The method of claim 7 , wherein:
said at least one further enzymatic step comprises the enzymatic conversion of methyl 9-oxononanoate to monomethyl 9-aminononanoate;
said at least one further enzymatic step comprises the enzymatic conversion of methyl 11-oxoundecanoate to monomethyl 11-aminoundecanoate;
said at least one further enzymatic step comprises the enzymatic conversion of methyl 13-oxotridecanoate to monomethyl 13-aminotridecanoate;
said at least one further enzymatic step comprises the enzymatic conversion of methyl 15-oxopentadecanoate to monomethyl 15-aminopentadecanoate;

Assignees

• Invista North America Sarl

Inventors

• Foster Alexander Brett
• Cartman Stephen Thomas
• Kennedy Jonathan
• Simmons William Joseph

Classifications

• C12N15/09 Primary

  Recombinant DNA-technology · CPC title

• C12Y101/01035

  3-Hydroxyacyl-CoA dehydrogenase (1.1.1.35) · CPC title

• C12P7/44

  Polycarboxylic acids · CPC title

• C12P13/005

  Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids · CPC title

• C12Y101/01258

  6-Hydroxyhexanoate dehydrogenase (1.1.1.258) · CPC title