In vitro Cas9-assisted editing of modular polyketide synthase genes to produce desired natural product derivatives
In vitro Cas9-assisted editing of modular polyketide synthase genes to produce desired natural product derivatives
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DOI：
10.1038/s41467-020-17769-2
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发表时间：
2020-08-11
影响因子：
16.6
通讯作者：
Shin-ya, Kazuo
中科院分区：
文献类型：
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作者：
Kudo, Kei;Hashimoto, Takuya;Shin-ya, Kazuo
天然产物药物开发的一个主要瓶颈是衍生化，这是微调先导化合物的关键。一个有希望的解决方案是改进大环内酯类等中间分子的生物合成机制。尽管大量的研究已经为I型模块化聚酮合成酶(PKSS)的蛋白质工程建立了各种方法，但由于PKS基因模块之间的高度序列相似性，精确定位PKS基因的期望区域仍然是一个挑战。在这里，我们报告了一种创新的技术，它采用体外Cas9反应和Gibson组装来编辑I型模块化PKS基因的靶区。以雷帕霉素PKS为模板的概念验证实验表明，编辑后的生物合成基因簇的异源表达几乎产生了所有所需的衍生物。我们的结果与模块化PKS的混杂一致，因此，我们的技术将为未来的药物开发提供一个合理设计的天然产物衍生物的平台。已经报道了几种不同的遗传策略来修饰聚酮合成酶，但高度重复的模块结构使这一点变得困难。在这里，作者报告了一种改进的Cas9反应和Gibson组装，以在体外编辑聚酮合成酶基因的靶区。
One major bottleneck in natural product drug development is derivatization, which is pivotal for fine tuning lead compounds. A promising solution is modifying the biosynthetic machineries of middle molecules such as macrolides. Although intense studies have established various methodologies for protein engineering of type I modular polyketide synthase(s) (PKSs), the accurate targeting of desired regions in the PKS gene is still challenging due to the high sequence similarity between its modules. Here, we report an innovative technique that adapts in vitro Cas9 reaction and Gibson assembly to edit a target region of the type I modular PKS gene. Proof-of-concept experiments using rapamycin PKS as a template show that heterologous expression of edited biosynthetic gene clusters produced almost all the desired derivatives. Our results are consistent with the promiscuity of modular PKS and thus, our technique will provide a platform to generate rationally designed natural product derivatives for future drug development. Several different genetic strategies have been reported for the modification of polyketide synthases but the highly repetitive modular structure makes this difficult. Here the authors report on an adapted Cas9 reaction and Gibson assembly to edit a target region of the polyketide synthases gene in vitro.