Host cells and methods for producing toluene biochemically

Patent metadata

Field	Value
Publication number	US-11078499-B2
Application number	US-201916286411-A
Country	US
Kind code	B2
Filing date	Feb 26, 2019
Priority date	Feb 27, 2018
Publication date	Aug 3, 2021
Grant date	Aug 3, 2021

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.

First claim

What is claimed is:

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.

2. The genetically modified host cell of claim 1 , wherein the conserved glycyl radical motif of the first polypeptide comprises the amino acid sequence RVXGX 12 QX 5 R (SEQ ID NO:5), RVAGFX 6 LX 4 QX 2 IX 2 R (SEQ ID NO:6), or RVAGFSAYFITLCPEVQXEIVSR (SEQ ID NO:7).

3. 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).

4. 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).

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.

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

Assignees

• Beller Harry R
• Rodrigues Andria V
• Zargar Kamrun
• Univ California

Inventors

• Beller Harry R
• Rodrigues Andria V
• Zargar Kamrun

Classifications

• C12N1/38

  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) · CPC title

• C12P17/10

  Nitrogen as only ring hetero atom · CPC title

• C12N9/14 Primary

  Hydrolases (3) · CPC title

• C12Y401/01

  Carboxy-lyases (4.1.1) · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title