Identification of SpySystem-linked CYP73A5-ATR2 by LC-MSMS

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, we here 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; the N-termini-bridged architecture is validated to improve the biosynthesis of both plant and human P450 systems. Furthermore, the architectural configuration of protein assemblies has an inherent effect on the biosynthesis 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 biosynthesis of protein complexes, particularly those related to eukaryotic membranes, in prokaryotic and even eukaryotic hosts.