Methods for altering polyketide synthase genes
OwnerRalph Reidinventor
Life of this patent
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Abstract
Ketoreductase (KR) domains of modular polyketide synthase (PKS) enzymes can be inactivated by one or more point mutations in the domain. Replacement or insertion of a KR domain can be used to introduce a cis or trans double bond into a polyketide by appropriate selection or inactivation of the type of KR domain that codes for a particular stereochemical configuration of a hydroxyl moiety. Inactivation of a DH domain can be used to produce a polyketide having a hydroxyl moiety with a desired stereochemical configuration.
Claims14
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.
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.
Dependencies are inferred from the claim text.