Natural products are important because of their significant pharmaceutical properties such as antiviral, antimicrobial, and anticancer activity. Recent breakthroughs in DNA sequencing reveal that a great number of cryptic natural product biosynthetic gene clusters are encoded in microbial genomes, for example, those of Streptomyces species. However, it is still challenging to access compounds from these clusters because many source organisms are uncultivable or the genes are silent during laboratory cultivation. To address this challenge, we develop an efficient cell-free platform for the rapid, in vitro total biosynthesis of the nonribosomal peptide valinomycin as a model. We achieve this goal in two ways. First, we used a cell-free protein synthesis (CFPS) system to express the entire valinomycin biosynthetic gene cluster (>19 kb) in a single-pot reaction, giving rise to approximately 37 μg/L of valinomycin after optimization. Second, we coupled CFPS with cell-free metabolic engineering system by mixing two enzyme-enriched cell lysates to perform a two-stage biosynthesis. This strategy improved valinomycin production ~5000-fold to nearly 30 mg/L. We expect that cell-free biosynthetic systems will provide a new avenue to express, discover, and characterize natural product gene clusters of interest in vitro.

天然产物之所以重要，是因为它们具有显着的药物特性，例如抗病毒，抗微生物和抗癌活性。DNA测序的最新突破表明，微生物基因组（例如链霉菌属物种）的微生物基因组中编码了大量隐秘的天然产物生物合成基因簇。但是，从这些簇中获取化合物仍然具有挑战性，因为在实验室培养过程中许多来源生物无法培养或基因沉默。为了应对这一挑战，我们开发了一个高效的无细胞平台，用于快速，体外总生物合成非核糖体肽缬霉素的模型。我们通过两种方式实现这一目标。首先，我们使用无细胞蛋白质合成（CFPS）系统在单罐反应中表达整个缬霉素生物合成基因簇（> 19 kb），经过优化后产生了约37μg/ L的缬霉素。第二，我们通过混合两种富含酶的细胞裂解液，将CFPS与无细胞代谢工程系统结合起来，进行两阶段的生物合成。该策略将缬氨霉素的产量提高了约5000倍，达到近30 mg / L。我们期望无细胞的生物合成系统将为体外表达，发现和表征感兴趣的天然产物基因簇提供一条新途径。