# US 2022/0033862 A1 · Engineered biosynthetic pathways for production of 2-oxoadipate by fermentation

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- Number: US 2022/0033862 A1
- Document ID: US20220033862A1
- Status: Published application
- Kind: A1
- Application number: 17/297,371
- Filed: 2019-11-25
- Published: 2022-02-03
- Earliest claimed: 2018-11-29
- Assignees: Zymergen Inc.
- Applicants: Zymergen Inc.
- Inventors: Anupam Chowdhury; Steven M. Edgar; Alexander Glennon Shearer; Cara Ann Tracewell; Stepan Tymoshenko; Zhihao Wang
- Web page: https://patentgrep.com/patent/US20220033862A1

## CPC

- C12P 7/50: having keto groups, e.g. 2-ketoglutaric acid
- C12N 1/16: Yeasts; Culture media therefor
- C12N 1/20: Bacteria; Culture media therefor
- C12N 9/1025: {Acyltransferases (2.3)}
- C12N 15/77: for Corynebacterium; for Brevibacterium
- C12N 15/81: for yeasts
- C12N 2500/60: Buffer, e.g. pH regulation, osmotic pressure
- C12R 2001/15: Corynebacterium
- C12R 2001/865: Saccharomyces cerevisiae
- C12Y 203/03014: Homocitrate synthase (2.3.3.14)
- 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)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12Y: ENZYMES

## Abstract

The present disclosure describes the engineering of microbial cells for fermentative production of 2-oxoadipate and provides novel engineered microbial cells and cultures, as well as related 2-oxoadipate production methods.

## Claims

### Claim 1

An engineered microbial cell that expresses a heterologous homocitrate synthase, wherein the engineered microbial cell produces 2-oxoadipate.

### Claim 2 (depends on 1)

The engineered microbial cell of claim 1, wherein the engineered microbial cell also expresses a heterologous homoaconitase.

### Claim 3 (depends on 1, 2)

The engineered microbial cell of claim 1 or claim 2, wherein the engineered microbial cell also expresses a heterologous homoisocitrate dehydrogenase.

### Claim 4 (depends on 1, 2, 3)

The engineered microbial cell of any one of claims 1-3, wherein the engineered microbial cell expresses one or more additional enzyme(s) selected from an additional heterologous homocitrate synthase, an additional heterologous homoaconitase, or an additional heterologous homoisocitrate dehydrogenase.

### Claim 5

An engineered microbial cell that expresses a non-native homocitrate synthase, wherein the engineered microbial cell produces 2-oxoadipate.

### Claim 6 (depends on 5)

The engineered microbial cell of claim 5, wherein the engineered microbial cell also expresses a non-native homoaconitase.

### Claim 7 (depends on 5, 6)

The engineered microbial cell of claim 5 or claim 6, wherein the engineered microbial cell also expresses a non-native homoisocitrate dehydrogenase.

### Claim 8 (depends on 5, 6, 7)

The engineered microbial cell of any one of claims 5-7, wherein the engineered microbial cell expresses one or more additional enzyme(s) selected from an additional non-native homocitrate synthase, an additional non-native homoaconitase, or an additional non-native homoisocitrate dehydrogenase.

### Claim 9 (depends on 5, 6, 7)

The engineered microbial cell of 8, wherein the additional enzyme(s) are from a different organism than the corresponding enzyme in claims 5-7.

### Claim 10 (depends on 5, 6, 7, 8, 9)

The engineered microbial cell of any of claims 5-9, wherein the engineered microbial cell comprises increased activity of one or more upstream 2-oxoadipate pathway enzyme(s), said increased activity being increased relative to a control cell.

### Claim 11 (depends on 5, 6, 7, 8, 9, 10)

The engineered microbial cell of any one of claims 5-10, wherein the engineered microbial cell comprises reduced activity of one or more enzyme(s) that consume one or more 2-oxoadipate pathway precursors, said reduced activity being reduced relative to a control cell.

### Claim 12 (depends on 11)

The engineered microbial cell of claim 11, wherein the one or more enzyme(s) that consume one or more 2-oxoadipate pathway precursors comprise alpha-ketoglutarate dehydrogenase or citrate synthase.

### Claim 13 (depends on 11, 12)

The engineered microbial cell of claim 11 or claim 12, wherein the reduced activity is achieved by replacing a native promoter of a gene for the one or more enzymes that consume one or more 2-oxoadipate pathway precursors with a less active promoter.

### Claim 14

An engineered microbial cell, wherein the engineered microbial cell comprises means for expressing a heterologous homocitrate synthase, wherein the engineered microbial cell produces 2-oxoadipate.

### Claim 15 (depends on 14)

The engineered microbial cell of claim 14, wherein the engineered microbial cell also comprises means for expressing a heterologous homoaconitase.

### Claim 16 (depends on 14, 18)

The engineered microbial cell of claim 14 or claim 18, wherein the engineered microbial cell also comprises means for expressing a non-native homoisocitrate dehydrogenase.

### Claim 17

An engineered microbial cell, wherein the engineered microbial cell comprises means for expressing a non-native homocitrate synthase, wherein the engineered microbial cell produces 2-oxoadipate.

### Claim 18 (depends on 17)

The engineered microbial cell of claim 17, wherein the engineered microbial cell also comprises means for expressing a non-native homoaconitase.

### Claim 19 (depends on 17, 18)

The engineered microbial cell of claim 17 or claim 18, wherein the engineered microbial cell also comprises means for expressing a non-native homoisocitrate dehydrogenase.

### Claim 20 (depends on 14, 15, 16, 17, 18, 19)

The engineered microbial cell of any one of claims 14-19, wherein the engineered microbial cell comprises means for increasing the activity of one or more upstream 2-oxoadipate pathway enzyme(s), said increased activity being increased relative to a control cell.

### Claim 21 (depends on 14, 15, 16, 17, 18, 19, 20)

The engineered microbial cell of any one of claims 14-20, wherein the engineered microbial cell comprises means for reducing the activity of one or more enzyme(s) that consume one or more 2-oxoadipate pathway precursors, said reduced activity being reduced relative to a control cell.

### Claim 22 (depends on 21)

The engineered microbial cell of claim 21, wherein the one or more enzyme(s) that consume one or more 2-oxoadipate pathway precursors comprise alpha-ketoglutarate dehydrogenase or citrate synthase.

### Claim 23 (depends on 21, 22)

The engineered microbial cell of claim 21 or claim 22, wherein the reduced activity is achieved by means for replacing a native promoter of a gene for said one or more enzymes with a less active promoter.

### Claim 24 (depends on 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

The engineered microbial cell of any one of claims 5-23, wherein the engineered microbial cell comprises a fungal cell.

### Claim 25 (depends on 24)

The engineered microbial cell of claim 24, wherein the engineered microbial cell comprises a yeast cell.

### Claim 26 (depends on 25)

The engineered microbial cell of claim 25, wherein the yeast cell is a cell of the genus Saccharomyces.

### Claim 27 (depends on 26)

The engineered microbial cell of claim 26, wherein the yeast cell is a cell of the species cerevisiae.

### Claim 28 (depends on 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)

The engineered microbial cell of any one of claims 5-27, wherein the non-native homocitrate synthase comprises a homocitrate synthase having at least 70% amino acid sequence identity with a homocitrate synthase from Komagataella pastoris or Thermus thermophilus.

### Claim 29 (depends on 28)

The engineered microbial cell of claim 28, wherein the engineered microbial cell comprises a non-native homocitrate synthase having at least 70% amino acid sequence identity with the homocitrate synthase from Komagataella pastoris and a non-native homocitrate synthase having at least 70% amino acid sequence identity with the homocitrate synthase from Thermus thermophilus.

### Claim 30 (depends on 25)

The engineered microbial cell of claim 25, wherein the engineered microbial cell comprises a homocitrate synthase having at least 70 percent amino acid sequence identity to a homocitrate synthase from Schizosaccharomyces pombe (strain 972/ATCC 24843) (Fission yeast) (Uniprot ID No. Q9Y823; SEQ ID NO:90), having amino acid substitution D123N; a homoaconitase having at least 70 percent amino acid sequence identity to a homoaconitase from Saccharomyces cerevisiae (strain ATCC 204508/S288c) (Baker's yeast) (Uniprot ID No. P49367; SEQ ID NO:33); and a homoisocitrate dehydrogenase having at least 70 percent amino acid sequence identity to a homoisocitrate dehydrogenase from Saccharomyces cerevisiae (strain ATCC 204508/S288c) (Baker's yeast) (Uniprot ID No. P40495; SEQ ID NO:11).

### Claim 31 (depends on 30)

The engineered microbial cell of claim 30, wherein the engineered microbial cell is a Saccharomyces cerevisiae cell or a Yarrowia lipolytica cell.

### Claim 32 (depends on 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

The engineered microbial cell of any one of claims 7-23, wherein the engineered microbial cell is a bacterial cell.

### Claim 33 (depends on 32)

The engineered microbial cell of claim 32, wherein the bacterial cell is a cell of the genus Corynebacterium.

### Claim 34 (depends on 33)

The engineered microbial cell of claim 33, wherein the bacterial cell is a cell of the species glutamicum.

### Claim 35 (depends on 34)

The engineered microbial cell of claim 34, wherein the non-native homocitrate synthase comprises a homocitrate synthase having at least 70% amino acid sequence identity with a homocitrate synthase selected from the group consisting of Thermus thermophilus, Saccharomyces cerevisiae, Candida dubliniensis, Ustilaginoidea virens, Schizosaccharomyces cryophilus, and Komagataella pastoris.

### Claim 36 (depends on 35)

The engineered microbial cell of claim 35, wherein the non-native homocitrate synthase comprises a homocitrate synthase having at least 70% amino acid sequence identity with a homocitrate synthase from Thermus thermophilus or Saccharomyces cerevisiae.

### Claim 37 (depends on 36)

The engineered microbial cell of claim 36, wherein the engineered microbial cell comprises a non-native homocitrate synthase having at least 70% amino acid sequence identity with the homocitrate synthase from Thermus thermophilus and a non-native homocitrate synthase having at least 70% amino acid sequence identity with the homocitrate synthase from Saccharomyces cerevisiae.

### Claim 38 (depends on 34, 35, 36, 37)

The engineered microbial cell of any one of claims 34-37, wherein the engineered microbial cell also expresses a non-native homoaconitase having at least 70% amino acid sequence identity with a homoaconitase selected from the group consisting of Ogataea parapolymorpha, Komagataella pastoris, Ustilaginoidea virens, Ceratocystis fimbriata f. sp. Platani, and Gibberella moniliformis.

### Claim 39 (depends on 38)

The engineered microbial cell of claim 38, wherein the non-native homoaconitase comprises a homoaconitase having at least 70% amino acid sequence identity with a homoaconitase from Ogataea parapolymorpha.

### Claim 40 (depends on 34, 35, 36, 37, 38, 39)

The engineered microbial cell of any one of claims 34-39, wherein the wherein the engineered microbial cell also expresses a non-native homoisocitrate dehydrogenase having at least 70% amino acid sequence identity with a homoisocitrate dehydrogenase selected from the group consisting of Ogataea parapolymorpha, Candida dubliniensis, and Saccharomyces cerevisiae.

### Claim 41 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)

The engineered microbial cell of any one of claims 1-40, wherein the wherein the engineered microbial cell also expresses a non-native homoisocitrate dehydrogenase having at least 70% amino acid sequence identity with a homoisocitrate dehydrogenase from Ogataea parapolymorpha.

### Claim 42 (depends on 34)

The engineered microbial cell of claim 34, wherein the engineered microbial cell comprises a homocitrate synthase having at least 70 percent amino acid sequence identity to a homocitrate synthase from Schizosaccharomyces pombe (strain 972/ATCC 24843) (Fission yeast) (Uniprot ID No. Q9Y823; SEQ ID NO:90), having amino acid substitution D123N; a homoaconitase having at least 70 percent amino acid sequence identity to a homoaconitase from Saccharomyces cerevisiae (strain ATCC 204508/S288c) (Baker's yeast) (Uniprot ID No. P49367; SEQ ID NO:33); and a homoisocitrated dehydrogenase having at least 70 percent amino acid sequence identity to a homoisocitrate dehydrogenase from Saccharomyces cerevisiae (strain ATCC 204508/S288c) (Baker's yeast) (Uniprot ID No. P40495; SEQ ID NO:11).

### Claim 43 (depends on 32)

The engineered microbial cell of claim 32, wherein the bacterial cell is a Bacillus subtilis cell.

### Claim 44 (depends on 43)

The engineered microbial cell of claim 43, wherein the engineered microbial cell comprises a homocitrate synthase having at least 70 percent amino acid sequence identity to a homocitrate synthase from Saccharomyces cerevisiae (strain ATCC 204508/S288c) (Baker's yeast) (Uniprot ID No. P48570; SEQ ID NO:35); a homoaconitase having at least 70 percent amino acid sequence identity to a homoaconitase from Neosartorya fumigata (strain ATCC MYA-4609/Af293/CBS 101355/FGSC A1100) (Aspergillus fumigatus) (Uniprot ID No. Q4WUL6; SEQ ID NO:83), which includes a deletion of amino acid residues 2-41 and 721-777, relative to the full-length sequence; and a homoisocitrate dehydrogenase having at least 70 percent amino acid sequence identity to a homoisocitrate dehydrogenase from Saccharomyces cerevisiae (strain ATCC 204508/S288c) (Baker's yeast) (Uniprot ID No. P40495; SEQ ID NO:11).

### Claim 45 (depends on 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)

The engineered microbial cell of any one of claims 5-41, wherein, when cultured, the engineered microbial cell produces 2-oxoadipate at a level at least 100 μg/L of culture medium.

### Claim 46 (depends on 45)

The engineered microbial cell of claim 45, wherein, when cultured, the engineered microbial cell produces 2-oxoadipate at a level at least 20 mg/L of culture medium.

### Claim 47 (depends on 46)

The engineered microbial cell of claim 46, wherein, when cultured, the engineered microbial cell produces 2-oxoadipate at a level at least 75 mg/L of culture medium.

### Claim 48 (depends on 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)

A culture of engineered microbial cells according to any one of claims 5-47.

### Claim 49 (depends on 48)

The culture of claim 48, wherein the substrate comprises a carbon source and a nitrogen source selected from the group consisting of urea, an ammonium salt, ammonia, and any combination thereof.

### Claim 50 (depends on 48, 49)

The culture of claim 48 or claim 49, wherein the engineered microbial cells are present in a concentration such that the culture has an optical density at 600 nm of 10-500.

### Claim 51 (depends on 48, 49, 50)

The culture of any one of claims 48-50, wherein the culture comprises 2-oxoadipate.

### Claim 52 (depends on 48, 49, 50, 51)

The culture of any one of claims 48-51, wherein the culture comprises 2-oxoadipate at a level at least 100 μg/L of culture medium.

### Claim 53 (depends on 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)

A method of culturing engineered microbial cells according to any one of claims 5-46, the method comprising culturing the cells under conditions suitable for producing 2-oxoadipate.

### Claim 54 (depends on 53)

The method of claim 53, wherein the method comprises fed-batch culture, with an initial glucose level in the range of 1-100 g/L, followed controlled sugar feeding.

### Claim 55 (depends on 53, 54)

The method of claim 53 or claim 54, wherein the fermentation substrate comprises glucose and a nitrogen source selected from the group consisting of urea, an ammonium salt, ammonia, and any combination thereof.

### Claim 56 (depends on 53, 54, 55)

The method of any one of claims 53-55, wherein the culture is pH-controlled during culturing.

### Claim 57 (depends on 53, 54, 55, 56)

The method of any one of claims 53-56, wherein the culture is aerated during culturing.

### Claim 58 (depends on 53, 54, 55, 56, 57)

The method of any one of claims 53-57, wherein the engineered microbial cells produce 2-oxoadipate at a level at least 100 μg/L of culture medium.

### Claim 59 (depends on 53, 54, 55, 56, 57, 58)

The method of any one of claims 53-58, wherein the method additionally comprises recovering 2-oxoadipate from the culture.

### Claim 60

A method for preparing 2-oxoadipate using microbial cells engineered to produce 2-oxoadipate, the method comprising:
(a) expressing a non-native homocitrate synthase in microbial cells;
(b) cultivating the microbial cells in a suitable culture medium under conditions that permit the microbial cells to produce 2-oxoadipate, wherein the 2-oxoadipate is released into the culture medium; and
(c) isolating 2-oxoadipate from the culture medium.