Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, California 92037, United States.
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92037, United States.
ACS Synth Biol. 2020 Jul 17;9(7):1917-1922. doi: 10.1021/acssynbio.0c00265. Epub 2020 Jul 6.
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.
天然产物生物合成基因簇的遗传工程代表了一种有吸引力的方法，可以获得新的和复杂的生物活性小分子。然而，由于一些参与特殊代谢的基因数量和大小很大，特别是那些编码模块化聚酮合酶和非核糖体肽合酶的大型蛋白，因此仍然难以引入精确的遗传突变来探测结构域活性或改变化学产物的形成。在这里，我们报告了一种结合寡核苷酸重组和 CRISPR/Cas9 靶向的强大方法的开发和验证，该方法可用于快速定点突变克隆途径，该途径可直接转移到异源宿主进行表达。我们迅速产生了 12 个点突变，并确定了几个成功突变的重要决定因素，包括原间隔区/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天然产物生物合成进行探索或进行途径工程的研究人员。
