Fusion proteins useful for modifying terpenes

Patent metadata

Field	Value
Publication number	US-11939618-B2
Application number	US-202117207097-A
Country	US
Kind code	B2
Filing date	Mar 19, 2021
Priority date	Mar 23, 2020
Publication date	Mar 26, 2024
Grant date	Mar 26, 2024

Abstract

The present invention provides for a fusion protein comprising: (a) a terpene synthase (TS), or a homolog thereof, (b) a peptide linker, and (c) a P450 enzyme, or a homolog thereof.

First claim

We claim:

1. A fusion protein comprising: (a) a terpene synthase (TS) comprising an amino acid sequence having (i) at least 90% identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16, and (ii) the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17) or the amino acid sequence Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18), (b) a peptide linker, and (c) a P450 enzyme comprising (i) an amino acid sequence having at least 90% identity with SEQ ID NO:19 or SEQ ID NO:20, and (ii) a CXG motif or EXXR motif.

2. The fusion protein of claim 1 , wherein the TS comprises an amino acid sequence having at least 95% identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

3. The fusion protein of claim 1 , wherein the TS comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Asp (DDXXD) (SEQ ID NO:17).

4. The fusion protein of claim 1 , wherein the TS comprises the amino acid sequence Asp-Asp-Xaa-Xaa-Glu (DDXXE) (SEQ ID NO:18).

5. The fusion protein of claim 1 , wherein the P450 enzyme comprises an amino acid sequence having at least 95% identity with SEQ ID NO:19 or SEQ ID NO:20.

6. The fusion protein of claim 1 , wherein the P450 enzyme comprises the amino acid sequence FXXGXRXCXG (SEQ ID NO:21).

7. The fusion protein of claim 1 , wherein the peptide linker comprises of 0-1000 amino acid residues.

8. The fusion protein of claim 7 , wherein the peptide linker comprises of 1-5 repeats of the amino acid sequence GSG.

9. A genetically modified host cell capable of producing a modified terpenes, said genetically modified host cell comprising the fusion protein of claim 1 .

10. A nucleic acid comprising a nucleotide sequence encoding the fusion protein of claim 1 operatively linked to a promoter.

11. A vector comprising the nucleic acid of claim 10 .

12. A genetically modified host cell capable of producing a modified terpenes, said genetically modified host cell comprising the nucleic acid of claim 10 or a vector of claim 11 .

13. The genetically modified host cell of claim 12 , wherein the genetically modified host cell is a bacterium.

14. The genetically modified host cell of claim 13 , wherein the bacterium is of the genus Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla , or Paracoccus.

15. The genetically modified host cell of claim 12 , wherein the genetically modified host cell is a eukaryotic cell.

16. The genetically modified host cell of claim 15 , wherein the eukaryotic cell is a yeast.

17. A method for producing a modified terpene comprising: (a) providing the genetically modified host cell of claim 1 , or a culture thereof, (b) culturing or growing the genetically modified host cell to produce the modified terpene, (c) and/or extracting or separating the modified terpene from the culture, and (d) and/or introducing a fuel additive to the extracted or separated the modified terpene.

18. A fuel composition comprising: (a) a modified terpene produced by the fusion protein of claim 1 ; and (b) a fuel additive.

19. The fusion protein of claim 2 , wherein the TS comprises an amino acid sequence having at least 99% identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

20. The fusion protein of claim 19 , wherein the TS comprises an amino acid sequence comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.

21. The fusion protein of claim 5 , wherein the P450 enzyme comprises an amino acid sequence having at least 99% identity with SEQ ID NO:19 or SEQ ID NO:20.

22. The fusion protein of claim 21 , wherein the P450 enzyme comprises an amino acid sequence comprising SEQ ID NO:19 or SEQ ID NO:20.

Assignees

• Univ California

Inventors

• Lee Taek Soon
• Wang Xi

Classifications

• C12P5/007 Primary

  containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00) · CPC title

• C12N9/0073 Primary

  with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13 · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12Y114/13106

  Epi-isozizaene 5-monooxygenase (1.14.13.106) · CPC title