Engineering a Highly meta -Selective Biphenyl Monooxygenase for the Biosynthesis of High-Value Polymer Precursors

Abstract

The meta-selective C–H functionalization of aromatic substrates lacking meta-directing electron-withdrawing groups remains challenging. To engineer the regioselective enzymatic conversion of biphenyl (BP) to 3,3′-dihydroxybiphenyl (33DHBP), an industrial precursor to high-performance polymers, substrate positioning within the active site must be precisely controlled. Here, we report the regioselective catalytic promiscuity engineering of toluene/o-xylene monooxygenase (ToMO) to achieve sequential, meta-selective hydroxylation of the non-natural biaryl BP, where distinct ToMO variants were engineered to catalyze meta-selective hydroxylation of BP, 3-hydroxybiphenyl (3HBP), or both. Variants to catalyze the meta-selective hydroxylation of BP were designed using an automated docking workflow, with predicted binding poses consistent with the observed regioselectivity; this resulted in the development of the I100V-E103V-F205G variant that hydroxylated BP with 100% apparent meta-selectivity. Additional substitutions, especially L268A and L402A, were introduced to widen the long hydrophobic active-site access channel, further improving meta-selective BP hydroxylation in Pseudomonas putida. Variants to catalyze the meta-selective hydroxylation of 3HBP were selected through a structure-based residue scan of 437 active-site substitutions, resulting in the identification of the I100V-E103V-F176H variant that hydroxylated 3HBP with over 90% apparent meta-selectivity and no activity toward BP. In addition, the combined I100V-E103V-I162Y-F205G variant hydroxylated 3HBP with over 90% apparent meta-selectivity and improved hydroxylation of BP. This study demonstrates structural and computational monooxygenase engineering for the regioselective hydroxylation of non-natural biaryls, enabling the production of 33DHBP as a valuable precursor to specialized polymers.