Engineering of isopropanol-tolerant carbonyl reductase for cofactor-free efficient testosterone synthesis.

Testosterone (TS), a vital androgen and key precursor of hormonal pharmaceuticals, plays a critical role in reproductive health. Although the enzymatic method is regarded as a green TS preparation process, its low productivity and dependence on exogenous cofactors still limit its scalability. In the present study, we addressed these challenges by engineering AxSDR, an isopropanol-tolerant carbonyl reductase from Algoriella xinjiangensis, to enable cofactor-free TS synthesis from 4-androstenedione (4AD). The wild-type AxSDR failed to catalyze 4AD due of the presence of a bottleneck with large steric hindrance that obstructed the positioning of the 17-keto group of 4AD. Through iterative protein engineering, a five-point mutant (G94N/H145G/Y188A/K209S/Y247I, Mu5) was constructed with a spatially unhindered catalytic center, which facilitated the spontaneous binding of the 17-keto group of 4AD. Focusing on the imbalance between reduction of 4AD and oxidation of isopropanol that exists in Mu5, a synergistic dual-enzyme system of Mu5 and its wild type (Mu5-WT) was constructed and achieved the full conversion of 150 mM 4AD in 12 h, yielding a record space-time yield of 3.6 g/L/h. This study establishes a cofactor-independent biocatalytic platform for TS production, offering a cost-effective and sustainable alternative to conventional chemical methods of TS production.