Abstract

The sustainable bioproduction of high-value aromatic compounds is a significant challenge in synthetic biology, often constrained by the complexity of multi-step metabolic pathways. To address this, we developed a cytochrome P450-driven plug-and-play system that integrates directed enzyme evolution with systems-level metabolic engineering. Using a structure-guided approach, we engineered the fatty acid hydroxylase P450BM3 to create variants capable of catalyzing regio-divergent hydroxylation of 2-phenylethanol to generate either (R)-1-phenylethanediol ((R)-PED) or tyrosol with exceptional regioselectivity (>99%) and enantioselectivtiy (100% ee). Implementation of these optimized enzymes in a tailored Yarrowia lipolytica chassis, engineered for high-level 2-phenylethanol synthesis, enabled the de novo production of (R)-PED and tyrosol. Scale-up in a 5-L bioreactor yielded 12.4 g/L of (R)-PED, the highest titer reported to date. Furthermore, this platform facilitated the first biosynthesis of salidroside in this yeast and serendipitously uncovered promiscuous glycosyltransferase activity, leading to record-level production of 2-phenylethyl-β-D-glucopyranoside (13.0 g/L). This study establishes a scalable and adaptable framework for plug-and-play oxidative biocatalysis, significantly expanding the scope of sustainable biomanufacturing.