A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology

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 mu g/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 mu g/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.

Automated summary

TX-TL systems show promise in studying natural product biosynthetic pathways with reduced experimental time and controlled conditions. Developing specialized cell-free systems is crucial, and the updated Streptomyces TX-TL protocol improves protein yield, demonstrating combined transcription, translation, and biosynthesis of Streptomyces metabolic pathways in a single reaction pot.