# US 2014/0193865 A1 · Methods Of Producing 7-Carbon Chemicals From Long Chain Fatty Acids Via Oxidative Cleavage

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- Number: US 2014/0193865 A1
- Document ID: US20140193865A1
- Status: Published application
- Kind: A1
- Application number: 14/139,142
- Filed: 2013-12-23
- Published: 2014-07-10
- Earliest claimed: 2012-12-31
- Assignees: INVISTA North America S.a r.I.
- Applicants: INVISTA North America S.á r.I.
- Inventors: Adriana Leonora Botes; Alex Van Eck Conradie; Changlin Chen; Paul S. Pearlman
- Web page: https://patentgrep.com/patent/US20140193865A1

## CPC

- C12P 7/46: Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
- C12P 7/18: polyhydric
- C12P 7/42: Hydroxy-carboxylic acids
- C12P 7/44: Polycarboxylic acids
- C12P 13/001: {Amines; Imines}
- C12P 13/005: {Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids}
- 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)}

## Abstract

This document describes biochemical pathways for producing pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or 1,7-heptanediol by forming two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in a C7 aliphatic backbone substrate. These pathways, metabolic engineering and cultivation strategies described herein rely on the fatty acid synthesis pathway and oxidative cleavage of long chain acyl-[acp] intermediates by a monooxgenase (e.g., cytochrome P450) such as that encoded by BioI from microorganisms such as Bacillus subtillis.

## Claims

### Claim 1

A method for biosynthesizing a product selected from the group consisting of pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine and 1,7-heptanediol, said method comprising enzymatically synthesizing two seven carbon chain aliphatic backbones from a long chain acyl-[acp] intermediate and enzymatically forming two terminal functional groups selected from the group consisting of carboxyl, amine, and hydroxyl groups in said backbone, thereby forming the product.

### Claim 2 (depends on 1)

The method of claim 1, wherein the seven carbon chain aliphatic backbone is pimeloyl-[acp] and heptanoate.

### Claim 3 (depends on 2)

The method of claim 2, wherein pimeloyl-[acp] and heptanoate are enzymatically synthesized from acetyl-CoA and malonyl-CoA via fatty acid synthesis and the oxidative cleavage of a long chain acyl-[acp] intermediate by a monooxgenase encoded by BioI.

### Claim 4 (depends on 1)

The method of claim 1, wherein said two terminal functional groups are the same.

### Claim 5 (depends on 1)

The method of claim 1, wherein said two terminal functional groups are different.

### Claim 6 (depends on 5)

The method of claim 5, wherein said product comprises a terminal amine and a terminal carboxyl group or a terminal hydroxyl group and a terminal carboxyl group.

### Claim 7

(canceled)

### Claim 8 (depends on 4)

The method of claim 4, wherein said two terminal functional groups are amine or said two terminal functional groups are hydroxyl groups.

### Claim 9

(canceled)

### Claim 10 (depends on 8)

The method of claim 8, wherein (i) a monooxygenase, an oxidoreductase and ferredoxin or (ii) a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase or a 4-hydroxybutyrate dehydrogenase or (iii) an alcohol dehydrogenase enzymatically forms the two hydroxyl groups.

### Claim 11 (depends on 10)

The method of claim 10, wherein said monooxygenase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO: 14-16.

### Claim 12 (depends on 1)

The method of claim 1, wherein a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, or a 6-oxohexanoate dehydrogenase, enzymatically forms a terminal carboxyl group.

### Claim 13 (depends on 12)

The method of claim 12, wherein said thioesterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:23, or SEQ ID NO:24.

### Claim 14 (depends on 1)

The method of claim 1, wherein a ω-transaminase or a deacetylase enzymatically forms an amine group.

### Claim 15 (depends on 14)

The method of claim 14, wherein said ω-transaminase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO. 8-13.

### Claim 16 (depends on 1)

The method of claim 1, wherein a carboxylate reductase and a phosphopantetheinyl transferase form a terminal aldehyde group as an intermediate in forming the product.

### Claim 17 (depends on 16)

The method of claim 16, wherein said carboxylate reductase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NO. 2-7.

### Claim 18 (depends on 1)

The method of claim 1, wherein said method is performed in a recombinant host by fermentation.

### Claim 19

(canceled)

### Claim 20

(canceled)

### Claim 21

(canceled)

### Claim 22 (depends on 18)

The method of claim 18, wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks.

### Claim 23 (depends on 22)

The method of claim 22, wherein the 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; and wherein the 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 24

(canceled)

### Claim 25 (depends on 18)

The method of claim 18, wherein the host is a prokaryote.

### Claim 26 (depends on 25)

The method of claim 25, wherein said prokaryote is from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; from the genus Corynebacteria such as Corynebacterium glutamicum; from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans; from the genus Delftia acidovorans, from the genus Bacillus such as Bacillus subtillis; from the genes Lactobacillus such as Lactobacillus delbrueckii; from the genus Lactococcus such as Lactococcus lactis or from the genus Rhodococcus such as Rhodococcus equi.

### Claim 27 (depends on 18)

The method of claim 18, wherein the host is a eukaryote.

### Claim 28 (depends on 27)

The method of claim 27, wherein said eukaryote is from the genus Aspergillus such as Aspergillus niger; from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; from the genus Yarrowia such as Yarrowia lipolytica, from the genus Issatchenkia such as Issathenkia orientalis, from the genus Debaryomyces such as Debaryomyces hansenii, from the genus Arxula such as Arxula adenoinivorans, or from the genus Kluyveromyces such as Kluyveromyces lactis.

### Claim 29

(canceled)

### Claim 30 (depends on 18)

The method of claim 18, wherein said host comprises one or more of the following attenuated enzymes: polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, an acetyl-CoA specific β-ketothiolase, a phosphotransacetylase forming acetate, an acetate kinase, a lactate dehydrogenase, a menaquinol-fumarate oxidoreductase, a 2-oxoacid decarboxylase producing isobutanol, an alcohol dehydrogenase forming ethanol, a triose phosphate isomerase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase, NADPH-consuming transhydrogenase, an NADPH-specific glutamate dehydrogenase, a NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase accepting C7 building blocks and central precursors as substrates; a glutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase.

### Claim 31 (depends on 18)

The method of claim 18, wherein said host overexpresses one or more genes encoding: an acetyl-CoA carboxylase, an acetyl-CoA synthetase, a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a fructose 1,6 diphosphatase; a L-alanine dehydrogenase; a NADPH L-glutamate dehydrogenase; a diamine transporter; a dicarboxylate transporter; and/or a multidrug transporter.

### Claim 32

A recombinant host comprising at least one exogenous nucleic acid encoding a monooxygenase and an aldehyde dehydrogenase, said host producing pimeloyl-[acp] and heptanoate.

### Claim 33 (depends on 32)

The recombinant host of claim 32, said host further comprising one or more of a monooxygenase, a thioesterase, an aldehyde dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, or a carboxylate reductase, said host producing pimelic acid, pimelate semialdehyde, or 7-hydroxyheptanoate.

### Claim 34 (depends on 33)

The recombinant host of claim 33, said host further comprising a) a ω-transaminase, said host producing 7-aminoheptanoate and/or b) one or more of a carboxylate reductase, a ω-transaminase, a deacetylase, an N-acetyl transferase, or an alcohol dehydrogenase, said host producing heptamethylenediamine.

### Claim 35

(canceled)

### Claim 36 (depends on 33)

The recombinant host of claim 33, said host further comprising a carboxylate reductase or an alcohol dehydrogenase, said host producing 1,7-heptanediol.