Claims
- 1. A method to inactivate a ketoreductase domain in a modular polyketide synthase, said method comprising changing a conserved amino acid in an SDR active site motif to another amino acid, wherein said SDR active site motif is defined by an amino acid sequence: HX˜6DX˜16-18KX-26SSX˜12YX˜3N, wherein X is any amino acid followed by a subscript indicating a number of amino acids between two conserved residues, and wherein said conserved amino acid that is changed is selected from the group consisting of K, S, S, and Y.
- 2. The method of claim 1 wherein Y is changed to F or X.
- 3. The method of claim 1 wherein S is changed to A or X.
- 4. The method of claim 1, wherein said change is made by altering a coding sequence in a gene encoding said KR.
- 5. The method of claim 1 wherein K is changed to X.
- 6. The method of claim 5 wherein Y is changed to F or X.
- 7. The method of claim 6 wherein one or both S is changed to A or X.
- 8. The method of claim 1, wherein more than one of said conserved amino acids is changed.
- 9. A method to alter a module of a modular polyketide synthase (PKS) such that said module will introduce a cis double bond into a polyketide produced by said PKS, said method comprising, either (A) replacing an entire module for a module with a type 2 KR and DH domains, (B) exchanging a portion of a module between an AT and an ACP of said module for a DH plus a type 2 KR domain of another module, (C) in a module already producing a trans double bond, replacing a KR domain for a type 2 KR domain, (D) in a module containing a type 1 KR domain, changing the KR to a type 2 KR domain by point mutation or replacing the KR with a type 2 KR; and (E) inserting a DH into a module containing a type 2 KR.
- 10. The method of claim 9 wherein said module will introduce a cis double bond by replacing an entire module for a module with a type 2 KR and DH domains.
- 11. The method of claim 9 wherein said module will introduce a cis double bond by exchanging a portion of a module between an AT and an ACP of said module for a DH plus a type 2 KR domain of another module.
- 12. The method of claim 9 wherein said module will introduce a cis double bond by, in a module already producing a trans double bond, replacing a KR domain for a type 2 KR domain.
- 13. The method of claim 9 wherein said module will introduce a cis double bond by in a module containing a type 1 KR domain, changing the KR to a type 2 KR domain by point mutation or replacing the KR with a type 2 KR.
- 14. The method of claim 9 wherein said module will introduce a cis double bond by inserting a DH into a module containing a type 2 KR.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date under 35 USC §119(e) with respect to U.S. Provisional application 60/310,778, filed Aug. 6, 2001. The disclosure of this application is incorporated herein by reference.
Provisional Applications (1)
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Number |
Date |
Country |
|
60310778 |
Aug 2001 |
US |