Compositions and methods for producing podophyllotoxin derivatives

Patent metadata

Field	Value
Publication number	US-10472688-B2
Application number	US-201615242525-A
Country	US
Kind code	B2
Filing date	Aug 20, 2016
Priority date	Aug 21, 2015
Publication date	Nov 12, 2019
Grant date	Nov 12, 2019

Abstract

The present invention provides compositions and methods for biosynthetically producing podophyllotoxin intermediates and derivatives including enzymes and their equivalents involved in the biosynthetic production of podophyllotoxin intermediates and derivatives.

First claim

What is claimed is:

1. A genetically engineered host cell for use in biosynthesis of a podophyllotoxin intermediate or podophyllotoxin derivative, comprising:
operably linked to a promoter, at least one nucleic acid molecules having at least 70% sequence identity to a sequence selected from SEQ ID NOS: 4-8 and encoding at least one polypeptide involved in the biosynthesis of podophyllotoxin intermediates and derivatives,
wherein the biosynthesis is initiated with a starting material suitable to produce the podophyllotoxin intermediate or podophyllotoxin derivative, and
wherein the biosynthesis is completed with the recovery of the podophyllotoxin intermediate or podophyllotoxin derivative from the host cell.

2. The host cell in accordance to claim 1 , wherein the at least one nucleic acid molecule encoding at least one polypeptide involved in biosynthetically producing podophyllotoxin intermediates or podophyllotoxin derivatives has a sequence selected from SEQ ID NOS: 4-8.

3. The host cell in accordance to claim 1 , wherein the at least one polypeptide is selected from the group consisting of O-methyltransferase 3 (OMT3), CYP71CU1, O-methyltransferase 1 (OMT1), 2-oxoglutarate/Fe(II)-dependent dioxygenases (2-ODD), and CYP71BE54.

4. The host cell in accordance to claim 1 , wherein the podophyllotoxin intermediate or podophyllotoxin derivative is 5′-desmethoxy-yatein, 5′-desmethyl-yatein, yatein, deoxypodophyllotoxin, or 4′-desmethyl-deoxypodophyllotoxin.

5. The host cell in accordance to claim 3 , wherein the at least one polypeptide is OMT3 and is encoded by a nucleic acid molecule having at least 70% sequence identity to SEQ ID No: 4.

6. The host cell in accordance to claim 5 , wherein said polypeptide has at least 90% sequence identity to the amino acid sequence for OMT3 set forth in SEQ ID No: 11.

7. The host cell in accordance to claim 3 , wherein the at least one polypeptide is CYP71CU1 and is encoded by a nucleic acid molecule having at least 70% sequence identity to SEQ ID No: 5.

8. The host cell in accordance to claim 7 , wherein said polypeptide has at least 90% sequence identity to the amino acid sequence for CYP71CU1 set forth in SEQ ID No: 12.

9. The host cell in accordance to claim 3 , wherein the at least one polypeptide is OMT1 and is encoded by a nucleic acid molecule having at least 70% sequence identity to SEQ ID No: 6.

10. The host cell in accordance to claim 9 , wherein said polypeptide has at least 90% sequence identity to the amino acid sequence for OMT1 set forth in SEQ ID No: 13.

11. The host cell in accordance to claim 3 , wherein the at least one polypeptide is 2-ODD and is encoded by a nucleic acid molecule having at least 70% sequence identity to SEQ ID No: 7.

12. The host cell in accordance to claim 11 , wherein said polypeptide has at least 90% sequence identity to the amino acid sequence for 2-ODD set forth in SEQ ID No: 14.

13. The host cell in accordance to claim 3 , wherein the at least one polypeptide is CYP71BE54 and is encoded by a nucleic acid molecule having at least 70% sequence identity to SEQ ID No: 8.

14. The host cell in accordance to claim 13 , wherein said polypeptide has at least 90% sequence identity to the amino acid sequence for CYP71BE54 set forth in SEQ ID No: 15.

15. The host cell in accordance to claim 1 , comprising OMT3 or, operably linked to a promoter, a nucleic acid molecule having at least 70% sequence identity to SEQ ID NO: 4, and wherein the podophyllotoxin intermediate or derivative is 5′-desmethoxy-yatein.

16. The host cell in accordance to claim 1 , comprising CYP71CU1 or, operably linked to a promoter, a nucleic acid molecule having at least 70% sequence identity to SEQ ID NO: 5, and wherein the podophyllotoxin intermediate or derivative is 5′-desmethyl-yatein.

17. The host cell in accordance to claim 1 , comprising OMT1 or, operably linked to a promoter, a nucleic acid molecule having at least 70% sequence identity to SEQ ID NO: 6, and wherein the podophyllotoxin intermediate or derivative is yatein.

18. The host cell in accordance to claim 1 , comprising 2-ODD or, operably linked to a promoter, a nucleic acid molecule having at least 70% sequence identity to SEQ ID NO: 7, and wherein the podophyllotoxin intermediate or derivative is deoxypodophyllotoxin.

19. The host cell in accordance to claim 1 , comprising CYP71BE54 or, operably linked to a promoter, a nucleic acid molecule having at least 70% sequence identity to SEQ ID NO: 8, and wherein the podophyllotoxin intermediate or derivative is 4′-desmethyl-deoxypodophyllotoxin.

Assignees

• Univ Leland Stanford Junior

Inventors

• Lau Warren
• Sattely Elizabeth

Classifications

• C12P17/181

  Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12Y114/21 Primary

  with NADH or NADPH as one donor, and the other dehydrogenated (1.14.21) · CPC title

• C12N9/0008

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12N9/1007 Primary

  Methyltransferases (general) (2.1.1.) · CPC title