Compositions and methods for producing podophyllotoxin derivatives

Patent metadata

Field	Value
Publication number	US-2017088872-A1
Application number	US-201615242525-A
Country	US
Kind code	A1
Filing date	Aug 20, 2016
Priority date	Aug 21, 2015
Publication date	Mar 30, 2017
Grant date	—

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 . An isolated nucleic acid molecule encoding an enzyme involved in synthesizing podophyllotoxin intermediates and derivatives and having at least 90% sequence identity to nucleic acid SEQ ID NO:1, and conservative variants thereof.

2 . An isolated nucleic acid molecule encoding an enzyme involved in synthesizing podophyllotoxin intermediates and derivatives and having at least 90% sequence identity to nucleic acid SEQ ID NO:2, and conservative variants thereof.

3 . An isolated nucleic acid molecule encoding an enzyme involved in synthesizing podophyllotoxin intermediates and derivatives and having at least 90% sequence identity to nucleic acid SEQ ID NO:3, and conservative variants thereof.

4 . An isolated nucleic acid molecule encoding an enzyme involved in synthesizing podophyllotoxin intermediates and derivatives and having at least 90% sequence identity to nucleic acid SEQ ID NO:4, and conservative variants thereof.

5 . An isolated nucleic acid molecule encoding an enzyme involved in synthesizing podophyllotoxin intermediates and derivatives and having at least 90% sequence identity to nucleic acid SEQ ID NO:5, and conservative variants thereof.

6 . An isolated nucleic acid molecule encoding an enzyme involved in synthesizing podophyllotoxin intermediates and derivatives and having at least 90% sequence identity to nucleic acid SEQ ID NO:6, and conservative variants thereof. An isolated nucleic acid molecule encoding an enzyme involved in synthesizing podophyllotoxin intermediates and derivatives and having at least 90% sequence identity to nucleic acid SEQ ID NO:7, and conservative variants thereof.

8 . An isolated nucleic acid molecule encoding an enzyme involved in synthesizing podophyllotoxin intermediates and derivatives and having at least 90% sequence identity to nucleic acid SEQ ID NO:8, and conservative variants thereof.

9 . An isolated nucleic acid molecule encoding an enzyme involved in synthesizing podophyllotoxin intermediates and derivatives and having at least 90% sequence identity to nucleic acid SEQ ID NO:9, and conservative variants thereof.

10 . An isolated nucleic acid molecule encoding an enzyme involved in synthesizing podophyllotoxin intermediates and derivatives and having at least 90% sequence identity to nucleic acid SEQ ID NO:10, and conservative variants thereof.

11 . A vector comprising one or more of the nucleic acid molecules according to any of claims 1 - 10 , wherein said one or more nucleic acid molecule is operably linked to a nucleic acid molecule comprising a promoter sequence, conferring expression of one or more enzymes involved in synthesizing podophyllotoxin intermediates and derivatives.

12 . A host cell comprising one or more of the nucleic acid molecules according to any of claims 1 - 10 for use in expressing enzymes involved in synthesizing podophyllotoxin intermediates and derivatives.

13 . A host cell for producing 4′-desmethylepipodophyllotoxin, comprising nucleic acid SEQ ID NO:1 for converting pinoresinol to secoisolariciresinol; nucleic acid SEQ ID NO:2 for converting secoisolariciresinol to matairesinol; nucleic acid SEQ ID NO:3 for converting matairesinol to pluviatolide; nucleic acid SEQ ID NO:4 for converting pluviatolide to 5′-desmethoxy-yatein; nucleic acid SEQ ID NO:5 for converting 5′-desmethoxy-yatein into 5′-desmethyl-yatein; nucleic acid SEQ ID NO:6 for converting 5′-desmethyl-yatein into yatein; nucleic acid SEQ ID NO:7 for converting yatein into deoxypodophyllotoxin; nucleic acid SEQ ID NO:8 for converting deoxypodophyllotoxin into 4′-desmethyl-deoxypodophyllotoxin; and nucleic acid SEQ ID NO:9 for converting 4′-desmethyl-deoxypodophyllotoxin into 4′-desmethyl-epipodophyllotoxin.

14 . The host cell of claim 12 , wherein the host cell is a plant, yeast, bacterial, insect or mammalian cell.

15 . The host cell of claim 13 , wherein the host cell is a plant, yeast, bacterial, insect or mammalian cell.

16 . The host cell of claim 13 , furthermore comprising nucleic acid SEQ ID NO:10 or a conservative variant thereof.

17 . The host cell of claim 14 , wherein the plant cell is Nicotiana benthamiana.

18 . The host cell of claim 15 , wherein the plant cell is Nicotiana benthamiana.

19 . A method for producing a podophyllotoxin intermediate and derivative in a host cell, comprising culturing and transforming the host cell with one or more of the nucleic acid molecules according to any of claims 1 - 10 , adding starting material suitable to produce the podophyllotoxin intermediate and derivative to culture medium; and
recovering the podophyllotin intermediate and derivative from culture medium.

20 . The method of claim 19 , wherein the host cell is a plant, yeast, bacterial, insect or mammalian cell.

21 . A method for producing 4′-desmethyl-epipodophyllotoxin in a host cell, comprising culturing and transforming the host cell with nucleic acid SEQ ID NO:1 for converting pinoresinol to secoisolariciresinol; nucleic acid SEQ ID NO:2 for converting secoisolariciresinol to matairesinol; nucleic acid SEQ ID NO:3 for converting matairesinol to pluviatolide; nucleic acid SEQ ID NO:4 for converting pluviatolide to 5′-desmethoxy-yatein; nucleic acid SEQ ID NO:5 for converting 5′-desmethoxy-yatein into 5′-desmethyl-yatein; nucleic acid SEQ ID NO:6 for converting 5′-desmethyl-yatein into yatein; nucleic acid SEQ ID NO:7 for converting yatein into deoxypodophyllotoxin; nucleic acid SEQ ID NO:8 for converting deoxypodophyllotoxin into 4′-desmethyl-deoxypodophyllotoxin; and nucleic acid SEQ ID NO:9 for converting 4′-desmethyl-deoxypodophyllotoxin into 4′-desmethyl-epipodophyllotoxin; adding pinoresinol as starting material to culture medium; and recovering 4′-desmethylepipodophyllotoxin from culture medium.

22 . The method of claim 21 , wherein the host cell is a plant, yeast, bacterial, insect or mammalian cell.

23 . The method of claim 21 , wherein the host cell furthermore comprises nucleic acid SEQ ID NO:10 or a conservative variant thereof.

Assignees

• Univ Leland Stanford Junior

Inventors

• Lau Warren
• Sattely Elizabeth

Classifications

• C12Y201/01

  Methyltransferases (2.1.1) · CPC title

• C12P17/181

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

• C12N9/001

  acting on the CH-CH group of donors (1.3) · CPC title

• C12Y201/01042

  Flavone 3'-O-methyltransferase (2.1.1.42) · CPC title

• C12N9/0073

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