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An in vitro platform for engineering and harnessing modular polyketide synthases
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An in vitro platform for engineering and harnessing modular polyketide synthases
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Journal Article
An in vitro platform for engineering and harnessing modular polyketide synthases
2020
Overview
To harness the synthetic power of modular polyketide synthases (PKSs), many aspects of their biochemistry must be elucidated. A robust platform to study these megadalton assembly lines has not yet been described. Here, we in vitro reconstitute the venemycin PKS, a short assembly line that generates an aromatic product. Incubating its polypeptides, VemG and VemH, with 3,5-dihydroxybenzoic acid, ATP, malonate, coenzyme A, and the malonyl-CoA ligase MatB, venemycin production can be monitored by HPLC and NMR. Multi-milligram quantities of venemycin are isolable from dialysis-based reactors without chromatography, and the enzymes can be recycled. Assembly line engineering is performed using pikromycin modules, with synthases designed using the updated module boundaries outperforming those using the traditional module boundaries by over an order of magnitude. Using combinations of VemG, VemH, and their engineered derivatives, as well as the alternate starter unit 3-hydroxybenzoic acid, a combinatorial library of six polyketide products is readily accessed.
A robust platform to study modular polyketide synthases (PKSs) in vitro is still unavailable. Here, the authors report the reconstitution of the venemycin PKS, engineer hybrid venemycin/pikromycin PKSs, and obtain much improved yields through employing the updated module boundaries.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ 82/16
/ 82/80
/ 82/83
/ Bacterial Proteins - chemistry
/ Bacterial Proteins - genetics
/ Bacterial Proteins - metabolism
/ Dialysis
/ High-performance liquid chromatography
/ Humanities and Social Sciences
/ Modules
/ NMR
/ Polyketide Synthases - chemistry
/ Polyketide Synthases - genetics
/ Polyketide Synthases - metabolism
/ Science
