# US 2018/0023102 A1 · Materials and methods utilizing biotin producing mutant hosts for the production of 7-carbon chemicals

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- Number: US 2018/0023102 A1
- Document ID: US20180023102A1
- Status: Published application
- Kind: A1
- Application number: 15/658,958
- Filed: 2017-07-25
- Published: 2018-01-25
- Earliest claimed: 2016-07-25
- Assignees: Invista North America S.A.R.L.
- Applicants: Invista North America S.A.R.L.
- Inventors: Alexander Brett FOSTER; Stephen Thomas CARTMAN; Jonathan KENNEDY
- Web page: https://patentgrep.com/patent/US20180023102A1

## CPC

- C12P 7/44: Polycarboxylic acids
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P 7/24: containing a carbonyl group
- C12P 7/62: Carboxylic acid esters
- C12P 13/001: {Amines; Imines}
- C12P 13/005: {Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids}
- C12Y 102/01: with NAD+ or NADP+ as acceptor (1.2.1)
- C12Y 102/99006: Carboxylate reductase (1.2.99.6)
- C12Y 206/01: Transaminases (2.6.1)
- C12Y 206/01018: Beta-alanine-pyruvate transaminase (2.6.1.18)
- C12Y 301/02: Thioester hydrolases (3.1.2)
- 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)
- C12Y: ENZYMES

## Abstract

Disclosed are methods for regulating biosynthesis of at least one of pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine, 7-aminohelptanol and 1,7-heptanediol (C7 building blocks) using a pathway having a pimeloyl-ACP intermediate, the method including the step of downregulating the activity of BioF. Also disclosed are recombinant hosts by fermentation in which the above methods are performed. Further disclosed are recombinant hosts for producing pimeloyl-ACP, the recombinant host including a deletion of a bioF gene.

## Claims

### Claim 1

A method for regulating biosynthesis of at least one C7 building block using a pathway having a pimeloyl-ACP intermediate, said method comprising downregulating the activity of a BioF enzyme.

### Claim 2

(canceled)

### Claim 3 (depends on 1)

The method of claim 1, wherein the at least one C7 building block is chosen from pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, 7-aminoheptanol, and 1,7-heptanediol.

### Claim 4 (depends on 1)

The method of claim 1, wherein:
the step of downregulating the activity of a BioF enzyme comprises downregulating the expression of a BioF protein;
the step of downregulating the activity of a BioF enzyme comprises downregulating the activity of a BioF protein; and/or
the step of downregulating the activity of a BioF enzyme comprises reducing the activity to zero.

### Claim 5

(canceled)

### Claim 6 (depends on 1)

The method of claim 1, said method further comprising
overexpressing a BioW enzyme and a CoA-specific BioF enzyme.

### Claim 7 (depends on 6)

The method of claim 6, wherein:
said BioW enzyme enzymatically converts pimelic acid to pimeloyl-CoA; or
said CoA-specific BioF enzyme enzymatically converts pimeloyl-CoA to 8-amino-7-oxo-nonanoic acid.

### Claim at position 8

8-10. (canceled)

### Claim 11 (depends on 1)

The method of claim 1, wherein said pathway comprises enzymatically synthesizing a C7 aliphatic backbone from malonyl-ACP via two cycles of methyl-ester shielded carbon chain elongation, and enzymatically forming two terminal functional groups independently chosen from carboxyl, amine, and hydroxyl groups in said backbone, thereby forming the C7 building block.

### Claim 12 (depends on 11)

The method of claim 11, wherein:
a S-adenosyl-L-methionine (SAM)-dependent methyltransferase converts malonyl-ACP to malonyl-ACP methyl ester; and
said two cycles of carbon chain elongation produce pimeloyl-ACP methyl ester from malonyl-ACP methyl ester using a trans-2-enoyl-CoA reductase.

### Claim 13

(canceled)

### Claim 14 (depends on 11)

The method of claim 11, wherein each of said two cycles of carbon chain elongation comprises using (i) a β-ketoacyl-ACP synthase, (ii) a 3-oxoacyl-ACP reductase, (iii) a 3-hydroxyacyl-ACP dehydratase, and (iv) an enoyl-ACP reductase.

### Claim 15

(canceled)

### Claim 16

(canceled)

### Claim 17 (depends on 11)

The method of claim 11, wherein:
said two terminal functional groups are both amine groups;
said two terminal functional groups are both hydroxyl groups;
said at least one C7 building block comprises a terminal amine and a terminal carboxyl group; or
said at least one C7 building block comprises a terminal hydroxyl group and a terminal carboxyl group.

### Claim 18

(canceled)

### Claim at position 17

19-21. (canceled)

### Claim 22 (depends on 11)

The method of claim 11, wherein:
the hydroxyl terminal group is enzymatically formed by a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydratase, or an alcohol dehydrogenase;
the carboxyl terminal group is enzymatically formed by a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a glutaconate CoA-transferase, or a reversible succinyl-CoA ligase; or
the amine terminal group is enzymatically formed by a ω-transaminase or a deacetylase.

### Claim at position 19

23-26. (canceled)

### Claim 27 (depends on 1)

The method of claim 1, wherein an intermediate with a terminal aldehyde group is formed by a carboxylate reductase and enhanced by a phosphopantetheinyl transferase during the biosynthesis of said at least one C7 building block.

### Claim 28

(canceled)

### Claim 29 (depends on 1)

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

### Claim 30 (depends on 29)

The method of claim 29, wherein:
said recombinant host is subjected to a cultivation condition under aerobic, anaerobic, micro-aerobic or mixed oxygen/denitrification cultivation conditions;
said recombinant host is cultured under conditions of nutrient limitation;
said recombinant host is retained using a ceramic hollow fiber membrane to maintain a high cell density during fermentation; or
said recombinant host's tolerance to high concentrations of at least one C7 building block is improved through continuous cultivation in a selective environment.

### Claim 31

(canceled)

### Claim 32

(canceled)

### Claim 33 (depends on 29)

The method of claim 29, wherein the principal carbon source fed to the fermentation derives from biological or non-biological feedstocks.

### Claim 34 (depends on 33)

The method of claim 33, wherein:
the biological feedstock is, or derives from, at least one feedstock chosen from monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, and municipal waste; or
the non-biological feedstock is, or derives from, at least one feedstock chosen from natural gas, syngas, CO2/H2, methanol, ethanol, benzoate, non-volatile residue (NVR) caustic wash waste stream from cyclohexane oxidation processes, and terephthalic acid/isophthalic acid mixture waste streams.

### Claim 35

(canceled)

### Claim 36

(canceled)

### Claim 37 (depends on 29)

The method of claim 29, wherein:
said recombinant host is a prokaryote is chosen from the genii Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia acidovorans, Bacillus, Lactobacillus, Lactococcus, and Rhodococcus; or
said recombinant chosen is a eukaryote chosen from the genii Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula, and Kluyveromyces.

### Claim 38 (depends on 37)

The method of claim 37, wherein:
said prokaryote is Escherichia coli, Clostridium liunadahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum Cupriavidus necator, Cupriavidus metallidurans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Bacillus subtillis, Lactobacillus delbrueckii, Lactococcus lactis, or Lactococcus lactis; or
said eukaryote is Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans, or Kluyveromyces lactis.

### Claim at position 32

39-57. (canceled)

### Claim 58 (depends on 29)

The method of claim 29, wherein:
said recombinant host comprises one or more of attenuated enzymes chosen from—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, a transhydrogenase dissipating the NADH or NADPH imbalance, an glutamate dehydrogenase dissipating the NADH or NADPH imbalance, 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; and a pimeloyl-CoA synthetase; or
said recombinant host overexpresses an acetyl-CoA synthetase, a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a formate dehydrogenase: a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a fructose 1,6 diphosphatase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase specific to the NADH or NADPH used to generate a co-factor imbalance; a methanol dehydrogenase, a formaldehyde dehydrogenase, a diamine transporter a dicarboxylate transporter an S-adenosylmethionine synthetase and/or a multidrug transporter.

### Claim 59

(canceled)

### Claim 60

A recombinant host for producing pimeloyl-ACP, wherein:
said recombinant host comprising a deletion of a bioF gene;
said recombinant host expresses a BioF at a level below that of a wild type host;
said recombinant host does not express BioF activity; or
said recombinant host comprises at least one exogenous nucleic acid encoding bioW and a CoA-specific bioF.

### Claim at position 36

61-64. (canceled)

### Claim 65

A nucleic acid construct or expression vector comprising:
a polynucleotide encoding a polypeptide having 8-amino-7-oxononanoate synthase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having 8-amino-7-oxononanoate synthase activity is a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 23;
a polypeptide having thioesterase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having thioesterase activity is chosen from: (a) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 1; (b) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 21; and (c) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 22;
a polynucleotide encoding a polypeptide having carboxylate reductase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having carboxylate reductase activity is chosen from: (a) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 2: (b) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 3; (c) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 4; (d) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 5; (e) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 6; and (f) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 7;
a polynucleotide encoding a polypeptide having ω-transaminase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having ω-transaminase activity is chosen from: (a) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 8: (b) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 9; (c) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 10 (d) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 11: (e) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 12; and (f) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 13;
a polynucleotide encoding a polypeptide having phosphopantetheinyl transferase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having phosphopantetheinyl transferase activity is chosen from: (a) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 14 and (b) a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 15;
a polynucleotide encoding a polypeptide having pimeloyl-ACP methyl ester esterase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having pimeloyl-ACP methyl ester esterase activity is a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 17;
a polynucleotide encoding a polypeptide having 6-carboxyhexanoate-CoA ligase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having 6-carboxyhexanoate-CoA ligase activity is a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 18;
a polypeptide having 8-amino-7-oxononanoate synthase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having 8-amino-7-oxononanoate synthase activity is a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 19; or
a polynucleotide encoding a polypeptide having malonyl-ACP O-methyltransferase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having malonyl-ACP O-methyltransferase activity is a polypeptide having at least 70% sequence identity or homology to the polypeptide of SEQ ID NO: 20.

### Claim at position 38

66-75. (canceled)

### Claim 76 (depends on 1)

A bio-derived, bio-based, or fermentation-derived product, wherein said product comprises:
i. a composition comprising at least one bio-derived, bio-based, or fermentation-derived compound biosynthesized according to claim 1, or any combination thereof;
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 bio-derived, bio-based, or fermentation-derived 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 obtained by molding the bio-derived, bio-based, or fermentation-derived polymer of ii. or the bio-derived, bio-based, or fermentation-derived resin 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 of i., bio-derived, bio-based, or fermentation-derived compound of i., bio-derived, bio-based, or fermentation-derived polymer of ii., bio-derived, bio-based, or fermentation-derived resin of iii., or 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 composition of i., bio-derived, bio-based, or fermentation-derived compound of i., bio-derived, bio-based, or fermentation-derived polymer of ii., bio-derived, bio-based, or fermentation-derived resin of iii., bio-derived, bio-based, or fermentation-derived formulation of v., or bio-derived, bio-based, or fermentation-derived molded substance of iv., or any combination thereof.