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 timescales and controlled reaction conditions. In order to realise this potential, specialised cell-free systems in organisms enriched for biosynthetic gene clusters, with strong protein production and well-characterised synthetic biology tools, is essential. 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 characterise 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.

中文翻译：

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委内瑞拉链霉菌无细胞合成生物学工具包

无原核无细胞偶联转录翻译（TX-TL）系统正在成为一种强大的工具，可以在试管中检查天然产物的生物合成途径。这种方法的主要优点是减少了实验时间，并控制了反应条件。为了实现这一潜力，必不可少的是在富含生物合成基因簇的生物体中建立专门的无细胞系统，并具有强大的蛋白质生产能力和特征明确的合成生物学工具。链霉菌属是天然产物的主要来源。为了研究链霉菌的酶和途径，我们最初开发了一种同源链霉菌无细胞系统，以提供天然蛋白质折叠环境，高G + C（％）tRNA库和活跃的背景代谢。然而，我们的初始产量较低（36μg/ mL），批次间差异很大。在这里，我们提出了一个更新的高产且健壮的链霉菌TX-TL协议，达到表达的重组蛋白的266μg/ mL的产量。为了补充这一点，我们用不同的报道分子快速鉴定了一系列DNA片段，表达了高G + C（％）生物合成基因，并证明了在一个单一罐中链霉菌代谢途径的组合转录，翻译和生物合成的初步概念证明反应。