Identification, Characterization, and Engineering of a Novel 3-Ketosteroid-Δ ¹ -Dehydrogenase

C1,2-dehydrogenation of sterols by Δ 1 -KstD is critical for synthesizing bioactive steroidal intermediates (e.g., ADD) from agricultural phytosterol byproducts. However, fungal Δ 1 -KstD enzymes remain underexplored. Here, we identified and characterized a novel Fs_KstD via microbial screening, transcriptomics, and heterologous expression in Escherichia coli . Fs_KstD efficiently catalyzed C1,2-dehydrogenation of 4AD, TS, and PG. Using the iCASE computational model, we engineered the T206L mutant, exhibiting a 142.36% activity increase toward 4AD. Molecular dynamics revealed that T206L optimizes active site conformation and strengthens enzyme–substrate interactions. Under optimized conditions with Tween 80, T206L achieved a 95.22% conversion rate. These findings establish fungal Fs_KstD as a promising biocatalyst for valorizing phytosterol feedstocks into high-value steroid intermediates for agricultural and food chemistry.