Semi-rational engineering of 3-Ketosteroid-Δ1-dehydrogenase boosts catalytic efficiency and robustness for steroid bioconversion

Steroid drugs play a pivotal role in clinical therapeutics, with androsta-1,4-diene-3,17-dione (ADD) serving as a critical intermediate whose efficient biosynthesis relies on the catalytic activity of 3-ketosteroid-Δ1-dehydrogenase (KstD). Building upon the previously engineered KstD₂^ep variant, this study employed semi-rational design strategies to enhance KstD₂’s high-substrate-loading adaptability. Key residues (V332, L334, G534) were systematically identified through homology modeling and molecular docking, followed by constructing a combinatorial mutant library. Through alanine scanning and iterative screening of single/multiple-site mutations, the optimal mutant KstD₂^ep (V332E/L334T/G534V) demonstrated a 1.1-fold enhancement in catalytic efficiency compared to the KstD₂^ep. Molecular dynamics simulations confirmed significantly enhanced structural stability in the mutant. Whole-cell catalytic optimization revealed expanded operational tolerance to temperature fluctuations, pH variations, and co-solvent exposure. Implementing high-density fermentation coupled with fed-batch substrate supplementation, the process achieved a 1.5-fold increase in substrate conversion efficiency, yielding 117.75 g/L ADD. These advancements position the engineered variant as a high-potential candidate for scalable steroid biotransformation, addressing key barriers to enzymatic stability and process efficiency for enzyme-driven biosynthesis of steroidal pharmaceutical intermediates.