Eukaryotic cytochrome P450 enzymes, generally colocalizing with their redox partner cytochrome P450 reductase (CPR) on the cytoplasmic surface of organelle membranes, often perform poorly in prokaryotic cells, whether expressed with CPR as a tandem chimera or free-floating individuals, causing a low titer of heterologous chemicals. To improve their biosynthetic performance in Escherichia coli, here, we architecturally design self-assembled alternatives of eukaryotic P450 system using reconstructed P450 and CPR, and create a set of N-termini-bridged P450-CPR heterodimers as the counterparts of eukaryotic P450 system with N-terminus-guided colocalization. The covalent counterparts show superior and robust biosynthetic performance, and the N-termini-bridged architecture is validated to improve the biosynthetic performance of both plant and human P450 systems. Furthermore, the architectural configuration of protein assemblies has an inherent effect on the biosynthetic performance of N-termini-bridged P450-CPR heterodimers. The results suggest that spatial architecture-guided protein assembly could serve as an efficient strategy for improving the biosynthetic performance of protein complexes, particularly those related to eukaryotic membranes, in prokaryotic and even eukaryotic hosts.

真核细胞色素 P450 酶通常与其氧化还原伴侣细胞色素 P450 还原酶 （CPR） 共定位在细胞器膜的细胞质表面，在原核细胞中通常表现不佳，无论是用 CPR 表达为串联嵌合体还是自由漂浮的个体，导致异源化学物质的滴度低。为了提高它们在大肠杆菌中的生物合成性能，在这里，我们使用重建的 P450 和 CPR 在架构上设计了真核 P450 系统的自组装替代品，并创建了一组 N 端桥接的 P450-CPR 异二聚体作为具有 N 端引导共定位的真核 P450 系统的对应物。共价对应物显示出卓越而稳健的生物合成性能，并且 N 端桥结构经过验证，可改善植物和人类 P450 系统的生物合成性能。此外，蛋白质组装体的结构构型对 N 端桥式 P450-CPR 异二聚体的生物合成性能具有固有影响。结果表明，空间结构引导的蛋白质组装可以作为一种有效的策略，以改善原核甚至真核宿主中蛋白质复合物的生物合成性能，特别是与真核膜相关的蛋白质复合物。