A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding because of the lack of specific functions or substrates in the host cell, the cell’s sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in vitro in a minimal complex PURE system to directly run multistep reactions. Specifically, we synthesize indigoidine and rhabdopeptides with the in vitro produced multidomain nonribosomal peptide synthetases BpsA and KJ12ABC from the organisms Streptomyces lavendulae and Xenorhabdus KJ12.1, respectively. These in vitro produced proteins are analyzed in yield, post-translational modification and in their ability to synthesize the natural compounds, and compared to recombinantly produced proteins. Our study highlights cell-free PURE system as suitable setting for the characterization of biosynthetic gene clusters that can potentially be harnessed for the rapid engineering of biosynthetic pathways.

中文翻译：

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从生物合成基因簇的基因组 DNA 中无细胞合成天然化合物。

通过优化和工程化的生物合成途径，可以在活宿主细胞中产生多种化学物质。尽管取得了成功，但由于宿主细胞中缺乏特定功能或底物、细胞在重要生理过程中对异源成分的敏感性或跨膜传质受限，通路工程仍然要求很高。在这项研究中，我们展示了参与天然化合物生物合成的复杂多域蛋白可以通过在最小复杂 PURE 系统中体外编码 DNA来直接运行多步反应。具体来说，我们用体外产生的多域非核糖体肽合成酶 BpsA 和 KJ12ABC 从生物体合成靛蓝和弹状肽分别为Streptomyces lavendulae和Xenorhabdus KJ12.1。分析这些体外产生的蛋白质的产量、翻译后修饰及其合成天然化合物的能力，并与重组产生的蛋白质进行比较。我们的研究强调无细胞 PURE 系统是表征生物合成基因簇的合适环境，可潜在地用于生物合成途径的快速工程。