Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, 210014, China.
Jiangsu Key Laboratory for the Research and Utilization of Plant Resources, Nanjing, 210014, China.
Nat Commun. 2024 Nov 19;15(1):10009. doi: 10.1038/s41467-024-54259-1.
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.
真核细胞细胞色素 P450 酶通常与它们的氧化还原伴侣细胞色素 P450 还原酶 (CPR) 一起定位于细胞器膜的细胞质表面，无论是与 CPR 作为串联嵌合体表达还是自由漂浮的个体，在原核细胞中表现都很差，导致异源化学品的产量低。为了提高真核 P450 系统在大肠杆菌中的生物合成性能，在这里，我们使用重构的 P450 和 CPR 对真核 P450 系统进行了结构设计，构建了一组 N 端桥接的 P450-CPR 异源二聚体作为具有 N 端引导共定位的真核 P450 系统的对应物。共价对应物显示出优越和强大的生物合成性能，并且 N 端桥接结构已被验证可提高植物和人 P450 系统的生物合成性能。此外，蛋白质组装的结构配置对 N 端桥接的 P450-CPR 异源二聚体的生物合成性能具有内在影响。结果表明，空间架构引导的蛋白质组装可以作为提高蛋白质复合物生物合成性能的有效策略，特别是那些与真核膜相关的蛋白质复合物，无论是在原核生物还是真核生物宿主中。
