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^*

^*Corresponding author for this work

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

5 Citations (Scopus)

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	Methods in Enzymology
Subtitle of host publication	Metabolons and Supramolecular Enzyme Assemblies
Publisher	Elsevier Inc.
Chapter	4
Pages	63-82
Number of pages	20
Volume	617
ISBN (Electronic)	978-0-12-817074-8
DOIs	https://doi.org/10.1016/bs.mie.2018.12.018
Publication status	E-pub ahead of print - 4 Feb 2019

Publication series

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

Research Groups and Themes

BrisSynBio
Bristol BioDesign Institute
BCS and TECS CDTs

Keywords

Polyketide synthase
protein expression
enzymology
protein structure
natural products
Synthetic biology

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BrisSynBio: Bristol Centre for Synthetic Biology
Woolfson, D. N. (Principal Investigator)
31/07/14 → 31/03/22
Project: Research

Structural and Functional Interrogation of the Abyssomicin C Biosynthetic Pathway
Maschio, L. (Author), Race, P. (Supervisor) & Willis, C. (Supervisor), 29 Sept 2020
Student thesis: Doctoral Thesis › Doctor of Philosophy (PhD)

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Maschio, L., Parnell, A., Lees, N. R., Willis, C., Berger-Schaffitzel, C., Stach, J. E. M., & Race, P. (2019). Cloning, Expression and Purification of Intact Polyketide Synthase Modules. In Methods in Enzymology: Metabolons and Supramolecular Enzyme Assemblies (Vol. 617, pp. 63-82). (Methods in Enzymology). Elsevier Inc.. Advance online publication. https://doi.org/10.1016/bs.mie.2018.12.018

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keywords = "Polyketide synthase, protein expression, enzymology, protein structure, natural products, Synthetic biology",

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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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

PY - 2019/2/4

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N2 - 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.

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

U2 - 10.1016/bs.mie.2018.12.018

DO - 10.1016/bs.mie.2018.12.018

M3 - Chapter in a book

C2 - 30784415

VL - 617

T3 - Methods in Enzymology

SP - 63

EP - 82

BT - Methods in Enzymology

PB - Elsevier Inc.

ER -

Cloning, Expression and Purification of Intact Polyketide Synthase Modules

Abstract

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Research Groups and Themes

Keywords

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Projects

BrisSynBio: Bristol Centre for Synthetic Biology

Student theses

Structural and Functional Interrogation of the Abyssomicin C Biosynthetic Pathway

Cite this