Modular polyketide synthase (PKS) is an ingenious core machine that catalyzes abundant polyketides in nature. Exploring interactions among modules in PKS is very important for understanding the overall biosynthetic process and for engineering PKS assembly-lines. Here, we show that intermodular recognition between the enoylreductase domain ER_1/2 inside module 1/2 and the ketosynthase domain KS₃ inside module 3 is required for the cross-module enoylreduction in azalomycin F (AZL) biosynthesis. We also show that KS₄ of module 4 acts as a gatekeeper facilitating cross-module enoylreduction. Additionally, evidence is provided that module 3 and module 6 in the AZL PKS are evolutionarily homologous, which makes evolution-oriented PKS engineering possible. These results reveal intermodular recognition, furthering understanding of the mechanism of the PKS assembly-line, thus providing different insights into PKS engineering. This also reveals that gene duplication/conversion and subsequent combinations may be a neofunctionalization process in modular PKS assembly-lines, hence providing a different case for supporting the investigation of modular PKS evolution.

模块化聚酮合酶 (PKS) 是一种巧妙的核心机器，可催化自然界中丰富的聚酮化合物。探索 PKS 中模块之间的相互作用对于理解整个生物合成过程和设计 PKS 装配线非常重要。_{在这里，我们表明模块1/2内的烯酰还原酶域 ER 1/2 和模块3}内的酮合成酶域 KS 3之间的模块间识别是偶氮霉素 F (AZL) 生物合成中跨模块烯酰还原所必需的。我们还表明 KS ₄模块 4 充当看门人，促进跨模块烯酰还原。此外，提供的证据表明 AZL PKS 中的模块 3 和模块 6 在进化上是同源的，这使得面向进化的 PKS 工程成为可能。这些结果揭示了模块间识别，进一步了解了 PKS 装配线的机制，从而为 PKS 工程提供了不同的见解。这也表明基因复制/转换和随后的组合可能是模块化 PKS 装配线中的新功能化过程，因此为支持模块化 PKS 进化的研究提供了不同的案例。