A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology

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.