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A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology

Moore, Simon J., Lai, Hung-En, Chee, Soo-Mei, Toh, Ming, Coode, Seth, Chengan, Kameshwari, Capel, Patrick, Corre, Christophe, de los Santos, Emmanuel LC, Freemont, Paul S. and others. (2021) A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology. ACS Synthetic Biology, . ISSN 2161-5063. E-ISSN 2161-5063. (doi:10.1021/acssynbio.0c00581) (KAR id:86561)


Prokaryotic cell-free coupled transcription-translation (TX-TL) systems are emergingas a powerful tool to examine natural product biosynthetic pathways in a test-tube.The key advantages of this approach are the reduced experimental timescales andcontrolled reaction conditions. To realise this potential, it is essential to developspecialised cell-free systems in organisms enriched for biosynthetic gene clusters.This requires strong protein production and well-characterised synthetic biology tools.The Streptomyces genus is a major source of natural products. To study enzymes andpathways from Streptomyces, we originally developed a homologous Streptomycescell-free system to provide a native protein folding environment, a high G+C (%) tRNApool and an active background metabolism. However, our initial yields were low (36μg/mL) and showed a high level of batch-to-batch variation. Here, we present anupdated high-yield and robust Streptomyces TX-TL protocol, reaching up to yields of266 μg/mL of expressed recombinant protein. To complement this, we rapidlycharacterise a range of DNA parts with different reporters, express high G+C (%)biosynthetic genes and demonstrate an initial proof of concept for combinedtranscription, translation, and biosynthesis of Streptomyces metabolic pathways in asingle ‘one-pot’ reaction.

Item Type: Article
DOI/Identification number: 10.1021/acssynbio.0c00581
Uncontrolled keywords: Cell-free synthetic biology, Streptomyces, natural products, in vitro transcriptiontranslation, cell-free protein synthesis
Subjects: Q Science > Q Science (General)
Divisions: Divisions > Division of Natural Sciences > Biosciences
Depositing User: S.J. Moore
Date Deposited: 11 Feb 2021 12:48 UTC
Last Modified: 26 Jan 2022 00:00 UTC
Resource URI: (The current URI for this page, for reference purposes)

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