# US 11,078,499 B2 · Host cells and methods for producing toluene biochemically

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- Number: US 11,078,499 B2
- Document ID: US11078499B2
- Status: Granted
- Kind: B2
- Application number: 16/286,411
- Filed: 2019-02-26
- Published: 2020-08-27
- Granted: 2021-08-03
- Earliest claimed: 2018-02-27
- Assignees: The Regents of the University of California
- Applicants: Harry R. Beller; Andria V. Rodrigues; Kamrun Zargar
- Inventors: Harry R. Beller; Andria V. Rodrigues; Kamrun Zargar
- Web page: https://patentgrep.com/patent/US11078499B2

## CPC

- C12P 5/005: {aromatic (naphthacene C12P29/00)}
- C12N 1/38: Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound (C12N1/34 takes precedence)
- C12N 9/14: Hydrolases (3)
- C12N 9/88: Lyases (4.)
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N 15/79: Vectors or expression systems specially adapted for eukaryotic hosts
- C12P 17/10: Nitrogen as only ring hetero atom
- C12Y 401/01: Carboxy-lyases (4.1.1)
- 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

The present invention provides for a genetically modified host cell comprising a first polypeptide comprising a sequence having at least 70% amino acid sequence identity with a phenylacetate decarboxylase, and having an enzymatic activity to decarboxylate a phenylacetic acid into a toluene and a carbon dioxide, and a second polypeptide comprising a sequence having at least 70% amino acid sequence identity with a phenylacetate decarboxylase activating enzyme, and having an enzymatic activity to cleave a S-adenosylmethionine (SAM) to form a methionine and a 5′-deoxyadenosyl radical.

## Claims

### Claim 1

A genetically modified host cell comprising a first polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 and a conserved qlycyl radical motif comprising R at position 812, V at position 813, G at position 815, F at position 816, L at position 823, Q at position 828, I at position 831, and R at position 834, and a conserved C at position 482, of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, and having an enzymatic activity to decarboxylate a phenylacetic acid into a toluene and a carbon dioxide, and a second polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4 and conserved amino acid residues: C at position 33, C at position 37, and C at position 40 of the amino acid sequence of SEQ ID NO:3, or C at position 39, C at position 43, and C at position 46 of the amino acid sequence of SEQ ID NO:4, and having an enzymatic activity to cleave a S-adenosylmethionine (SAM) to form a methionine and a 5′-deoxyadenosyl radical, wherein said 5′-deoxyadenosyl radical can activate the first polypeptide; wherein the first polypeptide or the second polypeptide is heterologous to the genetically modified host cell.

### Claim 2 (depends on 1)

The genetically modified host cell of claim 1, wherein the conserved glycyl radical motif of the first polypeptide comprises the amino acid sequence RVXGX12QX5R (SEQ ID NO:5), RVAGFX6LX4QX2IX2R (SEQ ID NO:6), or RVAGFSAYFITLCPEVQXEIVSR (SEQ ID NO:7).

### Claim 3 (depends on 1)

The genetically modified host cell of claim 1, wherein the first polypeptide comprises the amino acid sequence GCVXSG (SEQ ID NO: 9) or GCVQQSIIGG (SEQ ID NO: 10).

### Claim 4 (depends on 1)

The genetically modified host cell of claim 1, wherein the second polypeptide comprises the amino acid sequence CXXXCXXC (SEQ ID NO: 10), CXXXCXXCXN (SEQ ID NO:11), CPLRCLWC (SEQ ID NO:12), GXRX3FX2GCX3CX2CXN (SEQ ID NO: 13), or FLKGCPLRCLWCSNPE (SEQ ID NO:14).

### Claim 5

A genetically modified host cell comprising a first nucleic acid encoding a first polypeptide operatively linked a first promoter, wherein the first polypeptide comprises an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 and a conserved qlycyl radical motif comprising R at position 812, V at position 813, G at position 815, F at position 816, L at position 823, Q at position 828, 1 at position 831, and R at position 834, and a conserved C at position 482, of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, and having an enzymatic activity to decarboxylate a phenylacetic acid into a toluene and a carbon dioxide; and the first nucleic acid, or a second nucleic acid, encoding a second polypeptide operatively linked to the first promoter or a second promoter, wherein the second polypeptide comprises an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4 and conserved amino acid residues: C at position 33, C at position 37, and C at position 40 of the amino acid sequence of SEQ ID NO:3, or C at position 39, C at position 43, and C at position 46 of the amino acid sequence of SEQ ID NO:4, and having an enzymatic activity to cleave a S-adenosylmethionine (SAM) to form a methionine and a 5′-deoxyadenosyl radical; wherein the genetically modified host cell is capable of expressing the first and the second polypeptide and the first polypeptide or the second polypeptide is heterologous to (i) the genetically modified host cell, or (ii) the first promoter or the second promoter.

### Claim 6 (depends on 1)

A method of producing a substituted or unsubstituted toluene or 2-methyl-1H-indole in a genetically modified host cell, the method comprising culturing the genetically modified host cell of claim 1 in a medium under a suitable condition such that the culturing results in the genetically modified host cell producing the substituted or unsubstituted toluene or 2-methyl-1H-indole.

### Claim 7 (depends on 6)

The method of claim 6, wherein the medium comprises S-adenosylmethionine (SAM) and the genetically modified host cell uptakes or absorbs SAM and/or an unsubstituted or substituted phenylacetic acid from the medium.

### Claim 8 (depends on 6)

The method of claim 6, wherein the genetically modified host cell is capable of endogenously synthesizing SAM and/or an unsubstituted or substituted phenylacetic acid from a carbon source.

### Claim 9 (depends on 6)

The method of claim 6, further comprising introducing a first and/or second nucleic acids encoding the first and/or second polypeptide into the genetically modified host cell, wherein the introducing step is prior to the culturing step.

### Claim 10 (depends on 6)

The method of claim 6, wherein the method further comprises separating the substituted or unsubstituted toluene or 2-methyl-1H-indole from the genetically modified host cell and/or the medium, wherein the separating step is subsequent, concurrent or partially concurrent with the culturing step.

### Claim 11 (depends on 1)

The genetically modified host cell of claim 1, wherein the first polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the second polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

### Claim 12 (depends on 1)

The genetically modified host cell of claim 1, wherein the first polypeptide comprises an amino acid sequence having at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the second polypeptide comprises an amino acid sequence having at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

### Claim 13 (depends on 12)

The genetically modified host cell of claim 12, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.