A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology.

Autor: Moore SJ; Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.; Department Section of Structural and Synthetic Biology, Department of Infectious Disease; Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.; School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, U.K., Lai HE; Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.; Department Section of Structural and Synthetic Biology, Department of Infectious Disease; Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K., Chee SM; Department Section of Structural and Synthetic Biology, Department of Infectious Disease; Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.; The London Biofoundry, Imperial College Translation & Innovation Hub, White City Campus, 80 Wood Lane, London W12 0BZ, U.K., Toh M; Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.; Department Section of Structural and Synthetic Biology, Department of Infectious Disease; Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K., Coode S; School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, U.K., Chengan K; School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, U.K., Capel P; Warwick Integrative Synthetic Biology Centre, School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, U.K., Corre C; Warwick Integrative Synthetic Biology Centre, School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, U.K., de Los Santos EL; Warwick Integrative Synthetic Biology Centre, School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, U.K., Freemont PS; Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.; Department Section of Structural and Synthetic Biology, Department of Infectious Disease; Imperial College London, South Kensington Campus, Exhibition Road, London, SW7 2AZ, U.K.; The London Biofoundry, Imperial College Translation & Innovation Hub, White City Campus, 80 Wood Lane, London W12 0BZ, U.K.; UK Dementia Research Institute Care Research and Technology Centre, Imperial College London, Hammersmith Campus, Du Cane Road, London, W12 0N, U.K.
Jazyk: angličtina
Zdroj: ACS synthetic biology [ACS Synth Biol] 2021 Feb 19; Vol. 10 (2), pp. 402-411. Date of Electronic Publication: 2021 Jan 26.
DOI: 10.1021/acssynbio.0c00581
Abstrakt: 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.
Databáze: MEDLINE
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