Tuning architectural organization of eukaryotic P450 system to boost bioproduction in Escherichia coli

• Published in: Nature communications 2024-11, Vol.15 (1), p.10009-12, Article 10009
• Main Authors: Li, Yikui, Li, Jie, Chen, Wei-Kang, Li, Yang, Xu, Sheng, Li, Linwei, Xia, Bing, Wang, Ren
• Format: Article
• Language: English
• Subjects: 38/1; 631/45/603; 631/45/607/1167; 631/61/338/469; 631/61/338/552; 82/16; 82/29; 82/58; 82/80; 82/83; Architecture; Chimeras; Configuration management; Cytochrome; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450; Cytochromes P450; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Human performance; Humanities and Social Sciences; Humans; Membranes; multidisciplinary; N-Terminus; NADPH-ferrihemoprotein reductase; NADPH-Ferrihemoprotein Reductase - genetics; NADPH-Ferrihemoprotein Reductase - metabolism; Natural products; Oxidation-Reduction; Protein Multimerization; Proteins; Reductases; Science; Science (multidisciplinary); Self-assembly
• ISSN: 2041-1723; 2041-1723
• 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. Cytochrome P450 enzymes (P450s) and their redox partner cytochrome P450 reductase (CPR) often perform poorly in bioproduction of natural products by engineered prokaryotic microbes. Here, the authors report spatial architecture-guided P450-CPR assembly for improving the biosynthetic performance of both plant and human P450s in E. coli .