Substrate tunnel redesign of short-chain dehydrogenase enabled efficient biocatalytic production of the TRPV1 antagonist trans-4-tert-butylcyclohexanol

Abstract

trans-4-tert-Butylcyclohexanol (trans-1b), an antagonist for the transient receptor potential channel vanilloid subfamily member 1 (TRPV1) used in sensitive skin cosmetics, faces sustainability challenges in conventional chemical synthesis. While biocatalytic approaches employing carbonyl reductases offer eco-friendly alternatives, existing enzymes lack sufficient activity and trans-selectivity for this substrate. In this study, an NADH-dependent short-chain dehydrogenase from Escherichia coli K12, UCPA, was identified to exhibit excellent diastereoselectivity (de) and moderate catalytic activity for trans-1b production. Structure-guided redesign of the substrate tunnel generated a small library of 12 mutants. The top variant, Y187A, achieved a 15.6-fold activity enhancement. When integrated with enzyme-coupled NADH regeneration, this mutant enabled complete conversion of 1 M 4-tert-butylcyclohexanone (1a) to trans-1b within 10 h, showing both excellent trans-selectivity (>99.9%) and yield (>99.0%). This biocatalyst also demonstrated broad applicability, efficiently reducing five additional para-alky-substituted cyclohexanones to the corresponding trans-alcohols. Molecular dynamics simulations revealed that the activity improvements arose from a widened substrate tunnel, an optimized hydrophobic binding microenvironment, and increased flexibility in the critical loop region. This work establishes a rational tunnel engineering strategy for short-chain dehydrogenases (SDRs) and delivers a robust biocatalyst for sustainable trans-1b synthesis.