A Streptomyces venezuelae cell-free toolkit for synthetic biology
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Author(s)
Type
Journal Article
Abstract
Prokaryotic cell-free coupled transcription–translation (TX-TL) systems are emerging as a powerful tool to examine natural product biosynthetic pathways in a test tube. The key advantages of this approach are the reduced experimental time scales and controlled reaction conditions. To realize this potential, it is essential to develop specialized cell-free systems in organisms enriched for biosynthetic gene clusters. This requires strong protein production and well-characterized synthetic biology tools. The Streptomyces genus is a major source of natural products. To study enzymes and pathways from Streptomyces, we originally developed a homologous Streptomyces cell-free system to provide a native protein folding environment, a high G+C (%) tRNA pool, and an active background metabolism. However, our initial yields were low (36 μg/mL) and showed a high level of batch-to-batch variation. Here, we present an updated high-yield and robust Streptomyces TX-TL protocol, reaching up to yields of 266 μg/mL of expressed recombinant protein. To complement this, we rapidly characterize a range of DNA parts with different reporters, express high G+C (%) biosynthetic genes, and demonstrate an initial proof of concept for combined transcription, translation, and biosynthesis of Streptomyces metabolic pathways in a single “one-pot” reaction.
Date Issued
2021-02-19
Date Acceptance
2020-11-17
Citation
ACS Synthetic Biology, 2021, 10 (2), pp.402-411
ISSN
2161-5063
Publisher
American Chemical Society
Start Page
402
End Page
411
Journal / Book Title
ACS Synthetic Biology
Volume
10
Issue
2
Copyright Statement
© 2021 The Authors. Published by American Chemical Society. This publication is licensed under CC-BY. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
Identifier
https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000621601500019&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=a2bf6146997ec60c407a63945d4e92bb
Subjects
Biochemical Research Methods
Biochemistry & Molecular Biology
cell-free protein synthesis
cell-free synthetic biology
in vitro transcription-translation
Life Sciences & Biomedicine
natural products
Science & Technology
Streptomyces
Publication Status
Published
Coverage Spatial
United States
Date Publish Online
2021-01-26