Directed evolution of a cytochrome P450 monooxygenase for improved perillyl alcohol biosynthesis

Perillyl alcohol is a naturally occurring terpene with promising anticancer properties. However, like other oxidized terpenes, its limited biosynthetic availability hinders its development as a therapeutic lead. Cytochrome P450 monooxygenase CYP153A6 catalyzes the key hydroxylation of limonene to yield perillyl alcohol in Escherichia coli, yet the enzyme is the rate-limiting step in this pathway and has not previously been improved by directed evolution. Here, we report the development of a genetically encoded biosensor based on the cymene repressor CymR from Pseudomonas putida F1, engineered to selectively detect perillyl alcohol over its non-hydroxylated precursor limonene, and its application to the directed evolution of CYP153A6. A single substitution (S77L) in the CymR effector-binding pocket dramatically increased biosensor sensitivity and dynamic range toward perillyl alcohol while maintaining discrimination against limonene. The tailored CymR-S77L biosensor was coupled to a CYP153A6 random mutant library to enable high-throughput fluorescence-based screening of variants. Ultimately, seven CYP153A6 variants, some with multiple mutations, were identified to have improved perillyl alcohol titers. Deconvolution of beneficial mutations revealed that a single substitution, A287T, located near the predicted CYP153A6:ferredoxin reductase interface, was sufficient to increase perillyl alcohol productivity 4.2-fold relative to wild-type without compromising regio- or chemoselectivity. These results demonstrate that biosensor-guided directed evolution is an effective and selective strategy for engineering P450-dependent terpene hydroxylation pathways and establish a generalizable platform for developing monoterpene-responsive biosensors and applying them to the directed evolution of terpene-modifying enzymes.