# US 2014/0193864 A1 · Methods Of Producing 7-Carbon Chemicals Via Pyruvate And Succinate Semialdehyde Aldol Condensation

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- Number: US 2014/0193864 A1
- Document ID: US20140193864A1
- Status: Published application
- Kind: A1
- Application number: 14/138,992
- Filed: 2013-12-23
- Published: 2014-07-10
- Earliest claimed: 2012-12-31
- Assignees: Invista North America S.A R.L.
- Applicants: INVISTA North America S.á r.l.
- Inventors: Adriana Leonora Botes; Alex Van Eck Conradie; Changlin Chen; Paul S. Pearlman
- Web page: https://patentgrep.com/patent/US20140193864A1

## CPC

- C12P 7/40: containing a carboxyl group {including Peroxycarboxylic acids}
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P 7/18: polyhydric
- C12P 7/24: containing a carbonyl group
- C12P 7/42: Hydroxy-carboxylic acids
- C12P 7/44: Polycarboxylic acids
- C12P 13/001: {Amines; Imines}
- C12P 13/002: {Nitriles (-CN)}
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

This document describes biochemical pathways for producing one or more of pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine and 1,7-heptanediol by forming one or two terminal functional groups, comprised of carboxyl, amine or hydroxyl groups, in a C7 aliphatic backbone substrate produced from succinate semialdehyde or pyruvate. These pathways, metabolic engineering and cultivation strategies described herein rely on the aldol condensation of succinate semialdehyde and pyruvate.

## Claims

### Claim 1

A method for biosynthesizing a product selected from the group consisting of pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine and 1,7-heptanediol, said method comprising enzymatically synthesizing a seven carbon chain aliphatic backbone from succinate semialdehyde and pyruvate via aldol condensation 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-CoA or pimelate semialdehyde.

### Claim 3 (depends on 1)

The method of claim 1, wherein the product of the aldol condensation, 2,4-dihydroxyhept-2-enedioate or 4-hydroxy-2-oxo-pimelate, is converted to a) pimeloyl-CoA using one or more of (i) a 2-hydroxyhepta-2,4-dienedioate hydratase, (ii) an enoate reductase, (iii) a 2-hydroxyglutarate dehydrogenase, (iv) a glutaconate CoA-transferase, (v) a 2-hydroxyglutaryl-CoA dehydratase, or (vi) an enoyl-CoA reductase or an enoyl-[acp] reductase; or b) pimelate semialdehyde using one or more of (vii) a 2-hydroxyhepta-2,4-dienedioate hydratase, (viii) an enoate reductase, (ix) a 2-hydroxyglutarate dehydrogenase, (x) a glutaconate CoA-transferase, (xi) a 2-hydroxyglutaryl-CoA dehydratase, or (xii) a carboxylate reductase.

### Claim 4

(canceled)

### Claim 5 (depends on 3)

The method of claim 3, wherein said enoate reductase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16 and SEQ ID NO:17.

### Claim 6 (depends on 1)

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

### Claim 7

(canceled)

### Claim 8 (depends on 6)

The method of claim 6, wherein said product comprises a terminal amine and a terminal carboxyl group; or a terminal hydroxyl group and a terminal carboxyl group.

### Claim 9

(canceled)

### Claim 10 (depends on 6)

The method of claim 6, wherein said two terminal functional groups are amine or said two terminal functional groups are hydroxyl groups.

### Claim 11

(canceled)

### Claim 12 (depends on 10)

The method of claim 10, wherein a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydratase, or an alcohol dehydrogenase enzymatically forms the two hydroxyl groups.

### Claim 13 (depends on 1)

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

### Claim 14 (depends on 13)

The method of claim 13, wherein said thioesterase has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

### Claim 15 (depends on 8)

The method of claim 8, wherein a ω-transaminase or a deacetylase enzymatically forms the amine group.

### Claim 16 (depends on 15)

The method of claim 15, 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 17 (depends on 1)

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

### Claim 18 (depends on 17)

The method of claim 17, 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 19 (depends on 1)

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

### Claim 20

(canceled)

### Claim 21

(canceled)

### Claim 22

(canceled)

### Claim 23

(canceled)

### Claim 24 (depends on 19)

The method of claim 19, wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks.

### Claim 25 (depends on 24)

The method of claim 24, 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.

### Claim 26 (depends on 24)

The method of claim 24, 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 27 (depends on 19)

The method of claim 19, wherein the host is a prokaryote.

### Claim 28 (depends on 27)

The method of claim 27, 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 29 (depends on 19)

The method of claim 19, wherein the host is a eukaryote.

### Claim 30 (depends on 29)

The method of claim 29, 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 31 (depends on 19)

The method of any one of claims 19, wherein the host's tolerance to high concentrations of a C7 building block is improved through continuous cultivation in a selective environment.

### Claim 32 (depends on 19)

The method of claim 19, wherein said host comprises one or more of the following attenuated enzymes: a lactate dehydrogenase, a menaquinol-fumarate oxidoreductase, an alcohol dehydrogenase producing ethanol, a pyruvate decarboxylase, a 2-oxoacid decarboxylase generating isobutanol, a polymer synthase, a NADH-specific L-glutamate dehydrogenase, a NADH-consuming transhydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase that degrades C7 building blocks and their precursors; a glutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase.

### Claim 33 (depends on 19)

The method of claim 19, wherein said host overexpresses one or more genes encoding: a PEP carboxykinase, a PEP carboxylase, a pyruvate carboxylase, a PEP synthase, a formate dehydrogenase, a L-alanine dehydrogenase; a NADPH-specific L-glutamate dehydrogenase; a diamine transporter; a dicarboxylate transporter; and/or a multidrug transporter.

### Claim 34

A recombinant host comprising at least one exogenous nucleic acid encoding an 4-hydroxy-2-oxopimelate aldolase, a 2-hydroxyhepta-2,4-dienedioate hydratase, an enoate reductase, a 2-hydroxyglutarate dehydrogenase, a glutaconate CoA-transferase, a 2-hydroxyglutaryl-CoA dehydratase, and an enoyl-CoA reductase or an enoyl-[acp] reductase, said host producing pimeloyl-CoA.

### Claim 35

A recombinant host comprising at least one exogenous nucleic acid encoding an 4-hydroxy-2-oxopimelate aldolase, a 2-hydroxyhepta-2,4-dienedioate hydratase, an enoate reductase, a 2-hydroxyglutarate dehydrogenase, a glutaconate CoA-transferase, a 2-hydroxyglutaryl-CoA dehydratase, and a carboxylate reductase, said host producing pimelate semialdehyde.

### Claim 36 (depends on 34)

The recombinant host of claim 34, said host comprising at least one exogenous nucleic acid encoding one or more of an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a CoA-transferase, a reversible CoA-ligase, an acetylating aldehyde dehydrogenase, or a carboxylate reductase, said host producing pimelic acid or pimelate semialdehyde.

### Claim 37 (depends on 36)

The recombinant host of claim 36, said host further comprising a) at least one exogenous nucleic acid encoding a ω-transaminase, said host producing 7-aminoheptanoate; b) at least one exogenous nucleic acid encoding a 4-hydroxybutyrate dehydrogenase, a 5-hydroxypentanoate dehydrogenase or a 6-hydroxyhexanoate dehydrogenase, said host producing 7-hydroxyheptanoic acid; or c) at least one exogenous nucleic acid encoding a ω-transaminase, a deacetylase, an N-acetyl transferase oran alcohol dehydrogenase, said host producing heptamethylenediamine.

### Claim 38

(canceled)

### Claim 39

(canceled)

### Claim 40 (depends on 37)

The recombinant host of claim 37, said host further comprising at least one exogenous nucleic acid encoding a (i) carboxylate reductase or (ii) an alcohol dehydrogenase, said host producing 1,7-heptanediol.