Cloning, Expression and Purification of Intact Polyketide Synthase Modules
Citation
Maschio, L, Parnell, A, Lees, NR, Willis, C, Berger-Schaffitzel, C, Stach, JEM & Race, P, 2018, ‘Cloning, Expression and Purification of Intact Polyketide Synthase Modules’. in: [forthcoming Methods in Enzymology volume]. Elsevier Inc., pp. 63-82
Abstract
Polyketides are a structurally and functionally diverse family of bioactive natural products that have proven to be a rich source of pharmaceutical and agrochemical lead compounds. Many polyketides are biosynthesized by large multifunctional megaenzymes termed type 1 modular polyketide synthases (PKSs). These systems possess a distinctive assembly line-like architecture, comprising a series of linearly arranged, multi-domain extension modules, housed in sequence within giant polypeptide chains. Due to their inherently modular structures, PKSs represent attractive targets for re-engineering, enabling access to functionally optimized ‘non-natural’ natural products. In this chapter we describe methods for the molecular cloning, recombinant over-expression, and purification of intact PKS modules and multi-modular PKS polypeptides. The usefulness of these methods is demonstrated by applying them to the study of the abyssomicin C PKS, a >1 MDa multi-modular synthase responsible for the biosynthesis of a polyketide antimicrobial lead compound.
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