Biosynthesis of paclitaxel intermediate

1. A method comprising:

reacting a substrate represented by formula I with a tyrocidine synthetase (TycA) to produce an aminopropanoyl-CoA of formula II,

wherein:

X is hydrogen; Y is hydrogen or OH; and Ring is an unsubstituted or substituted aryl, heteroaryl, (C4-C10) cycloalkyl, or (C4-C9) heterocycloakyl;

mixing said aminopropanoyl-CoA of formula II with Baccatin III of the formula:

to form a mixture; and

reacting said mixture with a baccatin III O-phenylpropanoyltransferase (BAPT) to produce N-debenzoyl-2′-deoxypaclitaxel of the formula:

wherein each of X, Y, and Ring has the same meaning as defined above.

2. The method of claim 1 , wherein said substrate of formula I comprises a structure represented by formula III or formula IV:

wherein:

X is hydrogen; Y is hydrogen or OH; Z is CH, CH 2 , oxygen (O) or nitrogen (NH or NH 2 ); and R 1 is selected from the group consisting of hydrogen, alkyl, amino, hydroxyl, cyano, carboxy, nitro, thio, alkoxy, and halogen.

3. The method of claim 1 , wherein said aminopropanoyl-CoA of formula II comprises a structure of formula V or VI:

wherein:

X is hydrogen; Y is hydrogen or OH; Z is CH, CH 2 , oxygen (O) or nitrogen (NH or NH 2 ); and R 1 is selected from the group consisting of hydrogen, alkyl, amino, hydroxyl, cyano, carboxy, nitro, thio, alkoxy, and halogen.

4. The method of claim 1 , wherein said Tyrocidine synthetase A is selected from the group consisting of:

a. a polypeptide comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO: 5, or SEQ ID NO: 6;

b. a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with the exception of a substitution of a serine at an amino acid position corresponding to position 563 of the amino acid sequence of SEQ ID NO: 1, wherein said substitution does not have a hydroxy in its side chain;

c. a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with the exception of a substitution of a serine at an amino acid position corresponding to position 563 of the amino acid sequence of SEQ ID NO: 1, wherein said substitution is an alanine, valine, isovaline, leucine, isoleucine, proline, glycine, arginine, lysine, histidine, tryptophan, phenylalanine, methionine or cysteine;

d. a polypeptide comprising an amino acid sequence that shares at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6;

e. a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7;

f. a polypeptide encoded by a nucleotide sequence that shares at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 7; and

g. a combination thereof.

5. The method of claim 1 , wherein said reacting of said substrate of formula I comprises a mixture of said Tyrocidine synthetase A, said substrate of formula I, ATP and a divalent cation.

6. The method of claim 1 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

7. The method of claim 1 , wherein the method further comprises reacting said N-debenzoyl-2′-deoxypaclitaxel with N-debenzoyl-2′-deoxypaclitaxel:N-benzoyltransferase (NDTBT) to produce paclitaxel of the formula:

wherein:

each of X, Y, and Ring are defined as in claim 1 .

8. The method of claim 7 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

9. The method of claim 1 , wherein said method further comprises reacting a 10-deactyl Baccatin Ill of the formula:

with a 10-deacetylbaccatin III acetyltransferase (DBAT) to produce said Baccatin III.

10. The method of claim 9 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

11. The method of claim 9 , wherein said method further comprises reacting a 2-debenzoyltaxane of the formula:

with a taxane 2α-O-benzoyltransferase (TBT) to produce said 10-deacetyl Baccatin III.

12. The method of claim 11 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

13. The method of claim 11 , wherein said method further comprises reacting a taxa-4(5),11(12)-diene-5α-ol of the formula:

with a taxadienol 5α-O-acetyl-transferase (TAT) to produce said 2-debenzoyltaxane.

14. The method of claim 13 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

15. The method of claim 13 , wherein said method further comprises reacting a taxa-4(5),11(12)-diene of the formula:

with a taxadiene 5α-hydroxylase to produce said taxa-4(5),11(12)-diene-5α-ol.

16. The method of claim 15 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

17. The method of claim 15 , wherein said method further comprises reacting a geranylgeranyl diphosphate of the formula:

with a taxadiene synthase to produce said taxa-4(5),11(12)-diene.

18. The method of claim 17 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.

19. The method of claim 17 , wherein method further comprises reacting a isopentenyl diphosphate of the formula:

and diemethylallyl diphosphate of the formula:

with a geranylgeranyl diphosphate synthetase to produce said geranylgeranyl diphosphate.

20. The method of claim 19 , wherein said method is performed in vitro, in a cell-free reaction, in a cultured cell, or in a cultured cell during a plant cell fermentation process.