Cloning, Expression and Purification of Intact Polyketide Synthase Modules

Laurence Maschio; Alice Parnell; Nick R Lees; Chris Willis; Christiane Berger-Schaffitzel; James E M Stach; Paul Race

doi:10.1016/bs.mie.2018.12.018

Cloning, Expression and Purification of Intact Polyketide Synthase Modules

Laurence Maschio, Alice Parnell, Nick R Lees, Chris Willis, Christiane Berger-Schaffitzel, James E M Stach, Paul Race

Research output: Chapter in Book/Report/Conference proceeding › Chapter in a book

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.

Original language	English
Title of host publication	[forthcoming Methods in Enzymology volume]
Publisher	Elsevier Inc.
Chapter	4
Pages	63-82
Number of pages	20
Volume	617
DOIs	https://doi.org/10.1016/bs.mie.2018.12.018
Publication status	Accepted/In press - 31 Oct 2018

Publication series

Name	Methods in Enzymology
Publisher	Academic Press
ISSN (Print)	0076-6879

Structured keywords

BrisSynBio
Bristol BioDesign Institute
BCS and TECS CDTs

Keywords

Polyketide synthase
protein expression
enzymology
protein structure
natural products
Synthetic biology

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Maschio, L., Parnell, A., Lees, N. R., Willis, C., Berger-Schaffitzel, C., Stach, J. E. M., & Race, P. (Accepted/In press). Cloning, Expression and Purification of Intact Polyketide Synthase Modules. In [forthcoming Methods in Enzymology volume] (Vol. 617, pp. 63-82). (Methods in Enzymology). Elsevier Inc.. https://doi.org/10.1016/bs.mie.2018.12.018

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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 {\textquoteleft}non-natural{\textquoteright} 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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author = "Laurence Maschio and Alice Parnell and Lees, {Nick R} and Chris Willis and Christiane Berger-Schaffitzel and Stach, {James E M} and Paul Race",

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Cloning, Expression and Purification of Intact Polyketide Synthase Modules. / Maschio, Laurence; Parnell, Alice; Lees, Nick R; Willis, Chris ; Berger-Schaffitzel, Christiane; Stach, James E M; Race, Paul.

[forthcoming Methods in Enzymology volume]. Vol. 617 Elsevier Inc., 2018. p. 63-82 (Methods in Enzymology).

Research output: Chapter in Book/Report/Conference proceeding › Chapter in a book

TY - CHAP

T1 - Cloning, Expression and Purification of Intact Polyketide Synthase Modules

AU - Maschio, Laurence

AU - Parnell, Alice

AU - Lees, Nick R

AU - Willis, Chris

AU - Berger-Schaffitzel, Christiane

AU - Stach, James E M

AU - Race, Paul

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AB - 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.

KW - Polyketide synthase

KW - protein expression

KW - enzymology

KW - protein structure

KW - natural products

KW - Synthetic biology

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DO - 10.1016/bs.mie.2018.12.018

M3 - Chapter in a book

C2 - 30784415

VL - 617

T3 - Methods in Enzymology

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BT - [forthcoming Methods in Enzymology volume]

PB - Elsevier Inc.

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