A streptomyces venezuelae cell-free toolkit for synthetic biology
| Autor: | Patrick Capel, Christophe Corre, Ming Toh, Kameshwari Chengan, Simon J. Moore, Seth Coode, Soo-Mei Chee, Emmanuel L. C. de los Santos, Hung-En Lai, Paul S. Freemont |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
Streptomyces venezuelae Biomedical Engineering Q1 01 natural sciences Biochemistry Genetics and Molecular Biology (miscellaneous) Streptomyces 03 medical and health sciences Synthetic biology chemistry.chemical_compound Biosynthesis 010608 biotechnology Protein biosynthesis Gene 030304 developmental biology 2. Zero hunger 0303 health sciences Cell-free protein synthesis biology Chemistry QH QK General Medicine biology.organism_classification QR Metabolic pathway Biochemistry |
| ISSN: | 2161-5063 |
| Popis: | 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.\ud \ud |
| Databáze: | OpenAIRE |
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