Heterologous expression of biosynthetic gene clusters (BGCs) represents an attractive route to the production of new natural products, but is often hampered by poor yields. It is therefore important to develop tools that enable rapid refactoring, gene insertion/deletion, and targeted mutations in BGCs. Ideally, these tools should be highly efficient, affordable, accessible, marker free, and flexible for use with a wide range of BGCs. Here, we present a one-step yeast-based method that enables efficient, cheap, and flexible modifications to BGCs. Using the BGC for the antibiotic bottromycin, we showcase multiple modifications including refactoring, gene deletions and targeted mutations. This facilitated the construction of an inducible, riboswitch-controlled pathway that achieved a 120-fold increase in pathway productivity in a heterologous streptomycete host. Additionally, an unexpected biosynthetic bottleneck resulted in the production of a suite of new bottromycin-related metabolites.

中文翻译：

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快速和稳健的酵母介导途径重构产生多种新的博曲霉素相关代谢物

生物合成基因簇 (BGCs) 的异源表达代表了生产新天然产物的有吸引力的途径，但往往受到低产量的阻碍。因此，开发能够在 BGC 中实现快速重构、基因插入/删除和靶向突变的工具非常重要。理想情况下，这些工具应该是高效的、负担得起的、可访问的、无标记的，并且可以灵活地与各种 BGC 一起使用。在这里，我们提出了一种基于酵母的一步法，可以对 BGC 进行高效、廉价和灵活的修改。我们将 BGC 用于抗生素博特霉素，展示了多种修饰，包括重构、基因缺失和靶向突变。这有助于构建诱导型，核糖开关控制的途径在异源链霉菌宿主中实现了 120 倍的途径生产力增加。此外，一个意想不到的生物合成瓶颈导致了一系列新的博曲霉素相关代谢物的产生。