Rational design of 17β-hydroxysteroid dehydrogenase type3 for improving testosterone production with an engineered Pichia pastoris

Abstract Testosterone (TS) is a critical androgenic steroid that regulates human metabolism and maintains secondary sexual characteristics. The biotransformation from 4-androstene-3,17-done (4-AD) to TS is limited by the poor catalytic activity of 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3). Herein, we explored the structural characteristics and catalytic mechanism of 17β-HSD3 and adopted the rational design strategy to improve its catalytic activity. Molecular docking and molecular dynamics simulations revealed the substrate-binding pocket and the binding mode of 4-AD to 17β-HSD3. We located the pivotal residues and regulated their hydrophobicity and polarity. The obtained G186R/Y195W variant formed additional electrostatic interaction and hydrogen bond with 4-AD, increasing the binding affinity between the variant and 4-AD. Therefore, the G186R/Y195W variant produced 3.98 g/L of TS, which increased to 297%. The combination of structural and mechanism resolution drives the implementation of the rational design strategy, which provides guidance for bioproduction of TS catalyzed by 17β-HSD3.