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

Area of Science:

• Biotechnology
• Synthetic Biology
• Biochemical Engineering

Background:

• Testosterone production traditionally involves complex multi-step chemical or biocatalytic processes from 4-androstenedione.
• Phytosterols are abundant and renewable precursors for steroid synthesis.
• There is a need for more sustainable and cost-effective methods for testosterone manufacturing.

Purpose of the Study:

• To develop a streamlined and sustainable two-stage bioconversion strategy for direct testosterone production from phytosterols.
• To engineer a microbial strain for efficient accumulation of 4-androstenedione.
• To enhance the catalytic efficiency of ketoreductase for improved testosterone yield.

Main Methods:

• Metabolic engineering of Mycobacterium neoaurum NRRL B-3805 by gene deletion (kstd1, kshB1) and overexpression (hsd4A) to maximize 4-androstenedione production.
• Structure-guided redesign of ketoreductase through molecular docking and dynamics analysis, followed by site-directed mutagenesis (G141A substitution).
• Integrated two-stage bioconversion in a single bioreactor, eliminating intermediate purification steps.

Main Results:

• Engineered strain MnB^{Δkstd1/ΔkshB1/p261-hsd4A} achieved 12.3 g/L of 4-androstenedione in 6 days ( >88% molar yield).
• Redesigned ketoreductase exhibited a 5.1-fold increase in catalytic efficiency, converting 4-androstenedione to testosterone.
• The integrated process yielded 12.05 g/L testosterone (86% overall molar yield from phytosterol) with record productivity (0.6 g/L/h).

Conclusions:

• The developed two-stage bioconversion strategy offers a cost-effective and environmentally sustainable route for industrial testosterone production.
• Synergistic application of metabolic engineering and structure-guided enzyme design accelerates steroid biomanufacturing.
• This approach demonstrates a significant advancement in sustainable biomanufacturing of steroid-based pharmaceuticals.