Rational design of 17β-hydroxysteroid dehydrogenase type3 for improving testosterone production with an engineered Pichia pastoris. (December 2021)

• Record Type: Journal Article
• Authors: Ding, Juanjuan; You, Shengping; Zhang, Jiaxing; Zhang, Hongtao; Wang, Hui; Zhang, Wei; Qi, Wei; Su, Rongxin; He, Zhimin
• Is Part Of: Bioresource technology. Volume 341(2021)
• Publication Date: 2021-12
• Subjects: Testosterone -- 17β-hydroxysteroid dehydrogenase type 3 -- Homology modeling -- Molecular dynamics simulation -- Rational design; Biomass -- Periodicals; Biomass energy -- Periodicals; Bioremediation -- Periodicals; Agricultural wastes -- Periodicals; Factory and trade waste -- Periodicals; Organic wastes -- Periodicals; Bioénergie -- Périodiques; Déchets agricoles -- Périodiques; Déchets industriels -- Périodiques; Déchets organiques -- Périodiques; Déchets (Combustible) -- Périodiques; 662.88
• DOI: 10.1016/j.biortech.2021.125833
• Languages: English
• ISSNs: 0960-8524

Abstract

Graphical abstract: Highlights: This report resolves the substrate-binding pocket and binding mode of 17β-HSD3. Rational design reconstructs the substrate-binding pocket of 17β-HSD3. G186R/Y195W variant has strengthened the binding affinity of 4-AD to 17β-HSD3. The TS yield of engineered Pichia pastoris was 3.98 g/L, which increased to 297%. 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.