A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology.

Simon J. Moore,Kameshwari Chengan,Soo-Mei Chee,Ming Toh,Paul S. Freemont,Hung-En Lai,Patrick Capel,Seth Coode,Emmanuel LC de los Santos,Christophe Corre

doi:10.1021/acssynbio.0c00581

Simon J. Moore, Kameshwari Chengan + Show 8 more

Open Access

https://doi.org/10.1021/acssynbio.0c00581

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Abstract

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.

Highlights

Streptomyces bacteria are environmental specialists that synthesize rich repertoires of natural products such as antibiotics
We focus on upgrading our S. venezuelae system to elevate protein synthesis
We demonstrate its broader potential for cell-free synthetic biology, namely, for characterizing DNA parts and activating some model biosynthetic pathways

Summary

Introduction

Streptomyces bacteria are environmental specialists (e.g., soil, marine, desert) that synthesize rich repertoires of natural products such as antibiotics. Much of this genetic information is locked up and cryptically regulated within biosynthetic gene clusters; regions of genomic DNA that harbor enzymes and other proteins (e.g., transporters, resistance markers). The key limitation in awakening these clusters for natural product discovery is silent gene expression and recalcitrant genetics. Traditional strategies to overcome this include genetic modification of the host organism to bypass native regulatory elements, and the “capture” of the cluster and expression in a heterologous host.[1] But this can take several weeks to months to complete with varying levels of success: some cryptic clusters remain dormant due to obscure native regulation. Fundamental tools that aid these efforts are of major interest to the natural product community

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