Systematic engineering of plant cytochrome P450 system identifies a comprehensive strategy for expression of highly functional P450 enzymes in Escherichia coli
Cytochrome P450s catalyse diverse and unique chemical reactions, which makes them invaluable enzymes in nature and industry. Metabolic engineers leverage these unique catalytic properties when refactoring plant biosynthetic pathways into microbial cell factories. However, due to their hydrophobic anchor, microbial expression of membrane-bound cytochrome P450s is challenging. An arsenal of protein engineering strategies was developed to improve their expression in Escherichia coli, but extensive screening is often necessary to tailor the engineering approach to an individual enzyme. Here, we propose a universal strategy that allows the expression of highly active cytochrome P450s in E. coli by systematically evaluating six common N-terminal modifications and their effect on in vivo activity of enzymes from the CYP79 and CYP83 families. We identified transmembrane domain truncation as the only strategy that had a significantly positive effect on all seven tested enzymes, increasing product titres between 2- to 170-fold. When comparing the changes in protein titre and product generation, we show that higher expression does not always translate to higher in vivo activity, thus making protein titre an unreliable screening target. Our results demonstrate that transmembrane domain truncation improves in vivo activity across a broad range of cytochrome P450s with diverse N-terminal sequences and could be applied as the modification-of-choice to avoid the time-consuming screening process and accelerate the future design of E. coli cell factories.