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A streptomyces venezuelae cell-free toolkit for synthetic biology

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Moore, Simon J., Lai, Hung-En, Chee, Soo-Mei, Toh, Ming, Coode, Seth, Chengan, Kameshwari, Capel, Patrick, Corre, Christophe, de los Santos, Emmanuel L. C. and Freemont, Paul S. (2021) A streptomyces venezuelae cell-free toolkit for synthetic biology. ACS Synthetic Biology, 10 (2). pp. 402-411. doi:10.1021/acssynbio.0c00581

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Official URL: http://dx.doi.org/10.1021/acssynbio.0c00581

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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.

Item Type: Journal Article
Subjects: Q Science > QH Natural history
Q Science > QK Botany
Q Science > QR Microbiology
Divisions: Faculty of Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH): Synthetic biology , Streptomyces , Natural products , Prokaryotes
Journal or Publication Title: ACS Synthetic Biology
Publisher: American Chemical Society
ISSN: 2161-5063
Official Date: 26 January 2021
Dates:
DateEvent
26 January 2021Published
17 November 2020Accepted
Volume: 10
Number: 2
Page Range: pp. 402-411
DOI: 10.1021/acssynbio.0c00581
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Open Access
Copyright Holders: © 2021 American Chemical Society
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
EP/K038648/1[EPSRC] Engineering and Physical Sciences Research Councilhttp://dx.doi.org/10.13039/501100000266
ISSF fellowshipWellcome Trusthttp://dx.doi.org/10.13039/100010269
RGS\R1\191186Royal Societyhttp://dx.doi.org/10.13039/501100000288
217528/Z/19/ZWellcome Trusthttp://dx.doi.org/10.13039/100010269

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