# US 2018/0002729 A1 · Methods, reagents and cells for biosynthesizing compounds

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2018/0002729 A1
- Document ID: US20180002729A1
- Status: Published application
- Kind: A1
- Application number: 15/642,269
- Filed: 2017-07-05
- Published: 2018-01-04
- Earliest claimed: 2014-06-16
- Assignees: Invista North America S.A R.L.
- Applicants: INVISTA North America S.á r.l.
- Inventors: Alex Van Eck Conradie; Adriana Leonora Botes; Ramdane Haddouche
- Web page: https://patentgrep.com/patent/US20180002729A1

## CPC

- C12P 13/001: {Amines; Imines}
- C07C 47/02: Saturated compounds having —CHO groups bound to acyclic carbon atoms or to hydrogen
- C07C 47/12: containing more than one —CHO group
- C07C 59/01: Saturated compounds having only one carboxyl group and containing hydroxy or O-metal groups
- C07C 59/325: containing —CHO groups
- C07C 223/02: having amino groups bound to acyclic carbon atoms of the carbon skeleton
- C08G 63/06: derived from hydroxycarboxylic acids
- C12N 9/0008: {acting on the aldehyde or oxo group of donors (1.2)}
- C12N 9/0077: {with a reduced iron-sulfur protein as one donor (1.14.15)}
- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- C12N 9/1096: {transferring nitrogenous groups (2.6)}
- C12P 7/24: containing a carbonyl group
- C12P 7/40: containing a carboxyl group {including Peroxycarboxylic acids}
- C12P 7/42: Hydroxy-carboxylic acids
- C12P 7/44: Polycarboxylic acids
- C12P 7/62: Carboxylic acid esters
- C12P 7/6409: Fatty acids
- C12P 7/6436: Fatty acid esters
- C12Y 102/99006: Carboxylate reductase (1.2.99.6)
- C12Y 114/15001: Camphor 5-monooxygenase (1.14.15.1)
- C12Y 114/15003: Alkane 1-monooxygenase (1.14.15.3)
- C12Y 206/01018: Beta-alanine-pyruvate transaminase (2.6.1.18)
- C12Y 206/01019: 4-Aminobutyrate—2-oxoglutarate transaminase (2.6.1.19)
- C12Y 206/01029: Diamine transaminase (2.6.1.29)
- C12Y 206/01048: 5-Aminovalerate transaminase (2.6.1.48)
- C12Y 206/01082: Putrescine aminotransferase (2.6.1.82)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C08G: MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## 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.

## Claims

### Claim at position 1

1-83. (canceled)

### Claim 84

A method of producing 7-hydroxyheptanoate methyl ester in a recombinant host via fermentation, said method comprising:
enzymatically converting heptanoate to heptanoate methyl ester using a polypeptide having fatty acid O-methyltransferase activity; and
enzymatically converting heptanoate methyl ester to 7-hydroxyheptanoate methyl ester using a polypeptide having monooxygenase activity.

### Claim 85 (depends on 84)

The method of claim 84, 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;
said 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, and/or SEQ ID NO: 29.

### Claim 86 (depends on 84)

The method of claim 84, wherein heptanoate is enzymatically produced from heptanoyl-CoA using:
a polypeptide having thioesterase activity; or
a polypeptide having butanal dehydrogenase activity and a polypeptide having aldehyde dehydrogenase activity.

### Claim 87 (depends on 84)

The method of claim 84, wherein heptanoate is enzymatically produced from heptanoyl-CoA using a polypeptide having thioesterase activity, wherein 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: 33, or SEQ ID NO: 34.

### Claim 88 (depends on 86)

The method of claim 86, wherein heptanoyl-CoA is produced from acetyl-CoA and propanoyl-CoA via two cycles of CoA-dependent carbon chain elongation, wherein each of said two cycles of CoA-dependent carbon chain elongation comprises using:
a polypeptide having β-ketothiolase activity or a polypeptide having acetyl-CoA carboxylase activity and a polypeptide having β-ketoacyl-[acp] synthase activity;
a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having 3-oxoacyl-CoA reductase activity;
a polypeptide having enoyl-CoA hydratase activity; and
a polypeptide having trans-2-enoyl-CoA reductase activity.

### Claim 89 (depends on 84)

The method of claim 84, said method further comprising enzymatically converting 7-hydroxyheptanoate methyl ester to 7-hydroxyheptanoate using a polypeptide having demethylase activity or a polypeptide having esterase activity.

### Claim 90 (depends on 89)

The method of claim 89, said method further comprising enzymatically converting 7-hydroxyheptanoate to at least one product chosen from pimelic acid, pimelate semialdehyde, 7-aminoheptanoate, heptamethylenediamine, and 1,7-heptanediol.

### Claim 91 (depends on 89)

The method of claim 89, said method further comprising enzymatically converting 7-hydroxyheptanoate to pimelate semialdehyde using a polypeptide having alcohol dehydrogenase activity, a polypeptide having 6-hydroxyhexanoate dehydrogenase activity, a polypeptide having 5-hydroxypentanoate dehydrogenase activity, a polypeptide having 4-hydroxybutyrate dehydrogenase activity, or a polypeptide having monooxygenase activity.

### Claim 92 (depends on 89)

The method of claim 89, said method further comprising:
enzymatically converting 7-hydroxyheptanoate to 1,7-heptanediol using a polypeptide having carboxylate reductase activity and a polypeptide having alcohol dehydrogenase activity, wherein said polypeptide having carboxylate reductase is optionally enhanced by gene product of sfp; and
optionally further comprising enzymatically converting 1,7-heptanediol to heptamethylenediamine using a polypeptide having alcohol dehydrogenase activity and a polypeptide having ω-transaminase activity.

### Claim 93 (depends on 91)

The method of claim 91, said method further comprising:
enzymatically converting pimelate semialdehyde to pimelic acid using a polypeptide having 5-oxopentanoate dehydrogenase activity, a polypeptide having 6-oxohexanoate dehydrogenase activity, a polypeptide having 7-oxoheptanoate dehydrogenase activity, a polypeptide having aldehyde dehydrogenase activity, or a polypeptide having monooxygenase activity;
enzymatically converting pimelate semialdehyde to 7-aminoheptanoate using a polypeptide having ω-transaminase activity; or
enzymatically converting pimelate semialdehyde to heptamethylenediamine using at least one polypeptide chosen from a polypeptide having carboxylate reductase activity and a polypeptide having ω-transaminase activity, wherein said polypeptide having carboxylate reductase is optionally enhanced by gene product of sfp.

### Claim 94 (depends on 93)

The method of claim 93, said method comprising enzymatically converting pimelate semialdehyde to 7-aminoheptanoate using a polypeptide having ω-transaminase activity, and further comprising enzymatically converting 7-aminoheptanoate to heptamethylenediamine using:
at least one polypeptide chosen from a polypeptide having carboxylate reductase activity and a polypeptide having ω-transaminase activity, wherein the polypeptide having carboxylate reductase is optionally enhanced by gene product of sfp; and
optionally a polypeptide having deacetylase activity or a polypeptide having acetylputrescine deacetylase activity.

### Claim 95 (depends on 84)

The method of claim 84, wherein said recombinant host is:
subjected to a cultivation strategy under aerobic, anaerobic or, micro-aerobic cultivation conditions;
cultured under conditions of nutrient limitation; and/or
retained using a ceramic membrane to maintain a high cell density during fermentation.

### Claim 96 (depends on 84)

The method of claim 84, wherein the principal carbon source fed to the recombinant host is a biological feedstock or non-biological feedstock.

### Claim 97 (depends on 84)

The method of claim 84, wherein:
said biological feedstock is or derives from monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste; or
said non-biological feedstock is or derives from natural gas, syngas, CO2/H2, methanol, ethanol, benzoate, non-volatile residue (NVR) caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.

### Claim 98 (depends on 84)

The method of claim 84, wherein said recombinant host is:
a prokaryote chosen from the genera Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus, and Rhodococcus; or
a eukaryote chosen from the genera Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula, and Kluyveromyces.

### Claim 99 (depends on 84)

The method of claim 84, wherein said recombinant host is:
a prokaryote chosen from Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtilis, Lactobacillus delbrueckii, Lactococcus lactis, and Rhodococcus equi; or
a eukaryote chosen from Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans, and Kluyveromyces lactis.

### Claim 100 (depends on 84)

The method of claim 84, wherein said recombinant host:
comprises at least one attenuated enzyme chosen from:
a polypeptide having polyhydroxyalkanoate synthase activity;
a polypeptide having acetyl-CoA thioesterase activity;
a polypeptide having propanoyl-CoA thioesterase activity;
a polypeptide having phosphotransacetylase forming acetate activity;
a polypeptide having acetate kinase activity;
a polypeptide having lactate dehydrogenase activity;
a polypeptide having menaquinol-fumarate oxidoreductase activity;
a polypeptide having 2-oxoacid decarboxylase activity producing isobutanol;
a polypeptide having alcohol dehydrogenase activity forming ethanol;
a polypeptide having triose phosphate isomerase activity;
a polypeptide having pyruvate decarboxylase activity;
a polypeptide having glucose-6-phosphate isomerase activity;
a polypeptide having transhydrogenase activity dissipating the cofactor gradient;
a polypeptide having glutamate dehydrogenase activity specific for the cofactor used to create a gradient;
a NADH/NADPH-utilizing polypeptide having glutamate dehydrogenase activity;
a polypeptide having pimeloyl-CoA dehydrogenase activity;
a polypeptide having acyl-CoA dehydrogenase activity accepting C7 building blocks and central precursors as substrates;
a polypeptide having glutaryl-CoA dehydrogenase activity; and
a polypeptide having pimeloyl-CoA synthetase activity; and/or
overexpresses at least one gene encoding a polypeptide chosen from:
a polypeptide having acetyl-CoA synthetase activity;
a polypeptide having propionate-CoA synthetase activity;
a polypeptide having 6-phosphogluconate dehydrogenase activity;
a polypeptide having transketolase activity;
a polypeptide having puridine nucleotide transhydrogenase activity;
a polypeptide having formate dehydrogenase activity;
a polypeptide having glyceraldehyde-3P-dehydrogenase activity;
a polypeptide having malic enzyme activity;
a polypeptide having glucose-6-phosphate dehydrogenase activity;
a polypeptide having fructose 1,6 diphosphatase activity;
a polypeptide having L-alanine dehydrogenase activity;
a polypeptide having L-glutamate dehydrogenase activity;
a polypeptide having L-glutamine synthetase activity;
a polypeptide having lysine transporter activity;
a polypeptide having dicarboxylate transporter activity; and
a polypeptide having multidrug transporter activity.

### Claim 101

A recombinant host comprising at least one exogenous nucleic acid encoding a polypeptide having monooxygenase activity and at least one nucleic acid encoding a polypeptide having fatty acid O-methyltransferase activity, wherein:
said 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; and
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 recombinant host produces 7-hydroxyheptanoate methyl ester.

### Claim 102 (depends on 101)

The recombinant host of claim 101, said recombinant host 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 said host further produces 7-hydroxyheptanoate.

### Claim 103 (depends on 102)

The recombinant host of claim 102, said recombinant host further comprising at least one exogenous polypeptide chosen from:
a polypeptide having 4-hydroxybutyrate dehydrogenase activity;
a polypeptide having 5-hydroxypentanoate dehydrogenase activity;
a polypeptide having 5-oxopentanoate dehydrogenase activity;
a polypeptide having 6-hydroxyhexanoate dehydrogenase activity;
a polypeptide having 6-oxohexanoate dehydrogenase activity;
a polypeptide having 7-oxoheptanoate dehydrogenase activity;
a polypeptide having acetylputrescine deacetylase activity;
a polypeptide having alcohol dehydrogenase activity;
a polypeptide having aldehyde dehydrogenase activity;
a polypeptide having carboxylate reductase activity;
a polypeptide having deacetylase activity; and
a polypeptide having ω-transaminase activity.

### Claim 104 (depends on 102)

The recombinant host of claim 102, said recombinant host further comprising:
an exogenous polypeptide having β-ketothiolase activity or an exogenous polypeptide having acetyl-CoA carboxylase activity and an exogenous polypeptide having β-ketoacyl-[acp] synthase activity;
an exogenous polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity or an exogenous polypeptide having 3-oxoacyl-CoA reductase activity;
an exogenous polypeptide having enoyl-CoA hydratase activity; and
an exogenous polypeptide having trans-2-enoyl-CoA reductase activity.

### Claim 105 (depends on 102)

The recombinant host of claim 102, said recombinant host further comprising:
an exogenous polypeptide having thioesterase activity; or
an exogenous polypeptide having butanal dehydrogenase activity and an exogenous polypeptide having aldehyde dehydrogenase activity.

### Claim 106 (depends on 90)

A method of producing a bio-based polymer or resin comprising at least one bio-derived seven-carbon compound chosen from pimelic acid, pimelate semialdehyde, 7-aminoheptanoate acid, 7-hydroxyheptanoate, heptamethylenediamine, and 1,7-heptanediol, said method comprising:
producing said at least one bio-derived seven-carbon compound using the method of claim 90; and
chemically reacting said at least one bio-derived seven-carbon compound with itself or another compound in a polymer-producing reaction or resin-producing reaction.

### Claim 107 (depends on 106)

A method of producing a molded product comprising producing a polymer or resin using the method of claim 106 and forming said polymer or resin into a molded product.

### Claim 108 (depends on 90)

A bio-derived product, bio-based product, or fermentation-derived product, wherein said product comprises:
(i) at least one bio-derived, bio-based, or fermentation-derived compound produced using the method of claim 90 or a composition comprising said at least one bio-derived, bio-based, or fermentation-derived compound;
(ii) a bio-derived, bio-based, or fermentation-derived polymer comprising the bio-derived, bio-based, or fermentation-derived composition or compound of (i), or any combination thereof;
(iii) a bio-derived, bio-based, or fermentation-derived resin comprising the bio-derived, bio-based, or fermentation-derived compound or composition of (i) or any combination thereof, or the bio-derived, bio-based, or fermentation-derived polymer of (ii), or any combination thereof;
(iv) a molded substance obtainable by molding the bio-derived, bio-based, or fermentation-derived polymer of (ii) or the bio-derived, bio-based, or fermentation-derived of (iii), or any combination thereof;
(v) a bio-derived, bio-based, or fermentation-derived formulation comprising the bio-derived, bio-based, or fermentation-derived composition or compound of (i), the bio-derived, bio-based, or fermentation-derived polymer of (ii), the bio-derived, bio-based, or fermentation-derived resin of (iii), the bio-derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof; or
(vi) a bio-derived, bio-based, or fermentation-derived semi-solid or a non-semi-solid stream, comprising the bio-derived, bio-based, or fermentation-derived compound or composition of (i), the bio-derived, bio-based, or fermentation-derived polymer of (ii), the bio-derived, bio-based, or fermentation-derived resin of (iii), the bio-derived, bio-based, or fermentation-derived formulation of (v), the bio-derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof.