Assembly-line polyketide synthases (PKSs) are modular multi-enzyme systems with considerable potential for genetic reprogramming. Understanding how they selectively transport biosynthetic intermediates along a defined sequence of active sites could be harnessed to rationally alter PKS product structures. To investigate functional interactions between PKS catalytic and substrate acyl carrier protein (ACP) domains, we employed a bifunctional reagent to crosslink transient domain–domain interfaces of a prototypical assembly line, the 6-deoxyerythronolide B synthase, and resolved their structures by single-particle cryogenic electron microscopy (cryo-EM). Together with statistical per-particle image analysis of cryo-EM data, we uncovered interactions between ketosynthase (KS) and ACP domains that discriminate between intra-modular and inter-modular communication while reinforcing the relevance of conformational asymmetry during the catalytic cycle. Our findings provide a foundation for the structure-based design of hybrid PKSs comprising biosynthetic modules from different naturally occurring assembly lines.

装配线聚酮合酶（PKS）是模块化多酶系统，具有巨大的遗传重编程潜力。了解它们如何沿着确定的活性位点序列选择性地运输生物合成中间体，可以合理地改变 PKS 产品结构。为了研究 PKS 催化域和底物酰基载体蛋白 (ACP) 域之间的功能相互作用，我们采用双功能试剂来交联原型装配线（6-脱氧赤酮内酯 B 合酶）的瞬态域-域界面，并通过单粒子解析其结构低温电子显微镜（cryo-EM）。结合冷冻电镜数据的统计每颗粒图像分析，我们发现了酮合酶 (KS) 和 ACP 结构域之间的相互作用，这些相互作用区分模块内和模块间通信，同时增强催化循环期间构象不对称的相关性。我们的研究结果为基于结构的混合 PKS 设计奠定了基础，该混合 PKS 包含来自不同天然存在的装配线的生物合成模块。