Nucleic acid molecules encoding modified polyketide synthases containing polylinkers and uses thereof
AssigneesBiotica Technology Limited, Pfizer Inc.
InventorsJohannes Kellenberger, Peter Francis Leadlay, James Staunton, Kim Jonelle Stutzman-Engwall, Hamish Alastair Irvine McArthur
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Abstract
Nucleic acid molecules encoding at least part of a Type I polyketide synthase, and having a polylinker with multiple restriction enzyme sites in place of one or more PKS genes encoding enzymes associated with reduction, optionally further including nucleic acid incorporated into the polylinker, the further nucleic acid encoding one or more reductive enzymes; plasmids incorporating such nucleic acids; host cells transfected with such plasmids; methods relating thereto.
Claims24
An isolated recombinant nucleic acid molecule encoding a Type I polyketide synthase (PKS) wherein said nucleic acid molecule is produced by:
- a) providing a polynucleotide encoding at least an extension module of a PKS wherein said extension module contains at least one reductive domain;
- b) deleting a portion of the polynucleotide which corresponds to said at least one reductive domain of said PKS;
- and
- c) replacing said deleted portion with a polylinker region having multiple restriction enzyme sites;
- wherein said deleted portion corresponding to said at least one reductive domain is at least one domain selected from the group consisting of:
- a β-ketoreductase (KR) domain, a dehydratase (DH) domain and an enoyl reductase (ER) domain;
- wherein said polylinker is an in-frame addition to the PKS;
- and wherein said polylinker encodes a polypeptide.
Dependencies are inferred from the claim text.