Cloning, expression, and purification of intact polyketide synthase modules
Laurence Maschio1, Alice E Parnell1, Nicholas R Lees2
1School of Biochemistry, Biomedical Sciences Building, University Walk, University of Bristol, Bristol, United Kingdom; BrisSynBio Synthetic Biology Research Centre, Life Sciences Building, University of Bristol, Bristol, United Kingdom.
Methods in Enzymology
|February 21, 2019
Summary
Researchers developed methods to study large enzyme complexes called polyketide synthases (PKSs). These methods enable the engineering of PKSs for creating novel pharmaceutical compounds.
Area of Science:
- Biochemistry
- Natural Product Synthesis
- Enzymology
Background:
- Polyketides are diverse bioactive natural products crucial for pharmaceuticals and agrochemicals.
- Type I modular polyketide synthases (PKSs) are large enzymes responsible for polyketide biosynthesis.
- PKSs feature a modular assembly line architecture, making them targets for reengineering.
Purpose of the Study:
- To describe methods for molecular cloning, recombinant over-expression, and purification of PKS modules and polypeptides.
- To demonstrate the utility of these methods using the abyssomicin C PKS.
Main Methods:
- Molecular cloning of PKS genes.
- Recombinant over-expression of PKS modules and polypeptides.
- Purification of large PKS enzymes.
Main Results:
- Successful cloning, expression, and purification of intact PKS modules and multimodular PKS polypeptides.
- Demonstrated application of these methods to the >1MDa abyssomicin C PKS.
Conclusions:
- The described methods are effective for studying complex PKS systems.
- These techniques facilitate the investigation and potential reengineering of PKSs for novel natural product discovery.
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