Direct Conversion of Phytosterol to Testosterone via an Integrated Microbial-Enzymatic Strategy

Testosterone is an essential androgenic steroid traditionally produced from 4-androstenedione through multistep chemical or biocatalytic processes. Here, we report a streamlined and sustainable "two-stage" bioconversion strategy for direct testosterone production from phytosterols. In the first stage, Mycobacterium neoaurum NRRL B-3805 was metabolically reprogrammed to maximize 4-androstenedione accumulation by deleting kstd1 and kshB1 and overexpressing hsd4A, generating strain MnB^{Δkstd1/ΔkshB1/p261-hsd4A}. This engineered strain produced 12.3 g L^{- 1} of 4-androstenedione within 6 days, corresponding to a molar yield exceeding 88%. In the second stage, structure-guided redesign of ketoreductase markedly enhanced its catalytic efficiency toward 4-androstenedione. Molecular docking and dynamics analyses identified residues G141 and M215 as critical contributors to substrate recognition. The G141A substitution optimized the local binding environment by modulating hydrophobic and polar interactions, leading to a 5.1-fold increase in catalytic efficiency. Under optimized reaction conditions, the engineered enzyme achieved complete conversion of in situ-generated 4-androstenedione to testosterone in a single bioreactor, yielding 12.05 g/L of testosterone with an overall molar yield of 86% from phytosterol and delivering a record spatiotemporal productivity of 0.6 g L^{- 1} h^{- 1}. By eliminating intermediate 4-androstenedione extraction and purification, this integrated strategy establishes a cost-effective and environmentally sustainable route for industrial testosterone manufacture. More broadly, our work illustrates how synergistic metabolic engineering and structure-guided enzyme design can be combined to accelerate steroid biomanufacturing.