Genetic engineering of natural product biosynthetic gene clusters represents an attractive approach to access new and complex bioactive small molecules. However, due to the large number and size of some genes involved in specialized metabolism, notably those encoding modular polyketide synthase and nonribosomal peptide synthetase megaproteins, it remains difficult to introduce precise genetic mutations to probe domain activity or alter chemical product formation. Here, we report the development and validation of a robust method combining oligonucleotide recombineering and CRISPR/Cas9 targeting for rapid site-directed mutagenesis of cloned pathways, which can be directly transferred to a heterologous host for expression. We rapidly generated 12 point mutations and identified several important determinants of successful mutagenesis, including the protospacer/PAM sequence and presence of regions of local homology. Our approach may be broadly applicable for researchers interested in probing natural product biosynthesis or performing pathway engineering.

中文翻译：

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通过寡核苷酸重组和CRISPR / Cas9靶向进行大型生物合成基因簇的定点诱变。

天然产物生物合成基因簇的遗传工程代表了一种吸引人的方法，可用于获取新的复杂的生物活性小分子。然而，由于参与专门代谢的一些基因的数量和大小特别是编码模块化聚酮化合物合酶和非核糖体肽合成酶大蛋白的基因，因此仍然难以引入精确的基因突变来探测域活性或改变化学产物的形成。在这里，我们报告了结合寡核苷酸重组和CRISPR / Cas9靶向的快速克隆克隆途径的定点诱变的可靠方法的开发和验证，该克隆途径可直接转移至异源宿主进行表达。我们迅速产生了12个点突变，并确定了成功诱变的几个重要因素，包括原间隔物/ PAM序列和局部同源区域的存在。我们的方法可能广泛适用于对探测天然产物生物合成或进行途径工程感兴趣的研究人员。