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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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WRAP-Streptomyces-venezuelae-cell-free-toolkit-synthetic-biology-2021.pdf - Published Version - Requires a PDF viewer. Available under License Creative Commons Attribution 4.0. Download (4Mb) | Preview |
Official URL: http://dx.doi.org/10.1021/acssynbio.0c00581
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 | |||||||||||||||
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| Subjects: | Q Science > QH Natural history Q Science > QK Botany Q Science > QR Microbiology |
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| 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: |
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| 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: |
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