A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology
Simon J Moore, Hung-En Lai, Soo-Mei Chee, Ming Toh, Seth Coode, Kameshwari Chengan, Patrick Capel, Christophe Corre, Emmanuel LC de los Santos and Paul S Freemont
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. 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.