Claims
- 1. A method to produce a desired polyketide which method comprises:
- (a) providing a functional modular polyketide synthase (PKS) comprising at least a first and second module wherein said PKS has been modified to prevent its utilization of a native starter unit for said modular PKS by inactivation of the catalytic domain of the ketosynthase of said first module, but wherein said PKS is able to incorporate a diketide substrate into at least a triketide;
- (b) adding to said modified PKS a diketide that is a substrate for the modified PKS;
- (c) incubating the modified PKS and said diketide under conditions wherein said polyketide is synthesized; and
- (d) optionally recovering the polyketide.
- 2. The method of claim 1 wherein one amino acid in the catalytic domain of said first module ketosynthase has been altered.
- 3. The method of claim 2 wherein said amino acid is cysteine.
- 4. The method of claim 3 wherein said cysteine is replaced by alanine.
- 5. The method of claim 1 wherein said modified PKS is contained in host cells.
- 6. The method of claim 5 wherein said cells are heterologous to said modified PKS.
- 7. The method of claim 5 wherein said cells are Streptomyces.
- 8. The method of claim 5 wherein said cells are permeable to said diketide.
- 9. The method of claim 5 wherein said cells have been modified to delete a native PKS contained in said cells.
- 10. The method of claim 9 wherein said cells are S. coelicolor CH999.
- 11. The method of claim 5 wherein the diketide is that obtained by the coupling of a starter unit which is acetyl CoA, malonamyl Co-A, propionyl Co-A, butyryl Co-A, isobutyryl Co-A, isovaleryl Co-A, benzoyl Co-A, aminobenzoyl Co-A, aminohydroxybenzoyl Co-A, or thiophene carboxyl Co-A, with an extender unit which is malonyl Co-A, methylmalonyl Co-A or ethylmalonyl Co-A.
- 12. The method of claim 1 wherein said diketide is in the form of an N-acetyl cysteamine (NAc) thioester.
- 13. The method of claim 12 wherein said diketide is (2S,3R)-2-methyl-3-hydroxypentanoyl-NAc thioester.
- 14. The method of claim 1 wherein the diketide is that obtained by the coupling of a starter unit which is acetyl CoA, malonamyl Co-A, propionyl Co-A, butyryl Co-A, isobutyryl Co-A, isovaleryl Co-A, benzoyl Co-A, aminobenzoyl Co-A, aminohydroxybenzoyl Co-A, or thiophene carboxyl Co-A, with an extender unit which is malonyl Co-A, methylmalonyl Co-A or ethylmalonyl Co-A.
CROSS-REFERENCE TO RELATED APPLICATION
This application is related to provisional patent application Ser. No. 60/003,338, filed Jul. 6, 1995, from which priority is claimed under 35 U.S.C. .sctn.119(e) (1) and which is incorporated herein by reference in its entirety.
REFERENCE TO GOVERNMENT CONTRACT
This invention was made with United States Government support in the form of a grant from the National Institutes of Health (GM22172 and CA 66736-01).
US Referenced Citations (1)
Number |
Name |
Date |
Kind |
5824513 |
Katz |
Oct 1998 |
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Foreign Referenced Citations (1)
Number |
Date |
Country |
WO 9313663 |
Jul 1993 |
WOX |
Non-Patent Literature Citations (4)
Entry |
Donadio S, et al, (1993) An erythromycin analog produced by reprogramming of polyketide synthesis. Proc.Natl.Acad.Sci.U.S.A. 90: 7119-7123. |
Donadio S, et al, (1992) Biosynthesis of the erythromycin macrolactone and a rational approach for producing hydrid macrolides. Gene 115: 97-103. |
Omura, et al., "Inhibition of the Biosynthesis of Leucomycin, a Macrolide Antibiotic, by Cerulenin," J. Biochem. 75: 193-195 (1974). |
Rudd, et al., "Genetics of Actinorhodin Biosynthesis by Streptomyces Coelicolor A3(2)," J. Gen. Microbiol. 114:35-43 (1979). |