Engineered metabolic pathways in microbial cell factories often have no natural organization and have challenging flux imbalances, leading to low biocatalytic efficiency. Modular polyketide synthases (PKSs) are multienzyme complexes that synthesize polyketide products via an assembly line thiotemplate mechanism. Here, we develop a strategy named mimic PKS enzyme assembly line (mPKSeal) that assembles key cascade enzymes to enhance biocatalytic efficiency and increase target production by recruiting cascade enzymes tagged with docking domains from type I cis-AT PKS. We apply this strategy to the astaxanthin biosynthetic pathway in engineered Escherichia coli for multienzyme assembly to increase astaxanthin production by 2.4-fold. The docking pairs, from the same PKSs or those from different cis-AT PKSs evidently belonging to distinct classes, are effective enzyme assembly tools for increasing astaxanthin production. This study addresses the challenge of cascade catalytic efficiency and highlights the potential for engineering enzyme assembly.

微生物细胞工厂中的工程代谢途径通常没有自然组织，并且具有挑战性的通量不平衡，导致生物催化效率低下。模块化聚酮合酶 (PKS) 是多酶复合物，可通过流水线硫代模板机制合成聚酮产物。在这里，我们开发了一种名为模拟 PKS 酶组装线 (mPKSeal) 的策略，该策略组装关键级联酶以提高生物催化效率并通过从 I 型顺式-AT PKS 中招募标记有对接域的级联酶来提高目标产量。我们将这一策略应用于工程化大肠杆菌中的虾青素生物合成途径用于多酶组装，可将虾青素产量提高 2.4 倍。来自相同 PKS 或来自明显属于不同类别的不同顺式-AT PKS 的对接对是增加虾青素产量的有效酶组装工具。这项研究解决了级联催化效率的挑战，并强调了工程酶组装的潜力。