Boldenone and Testosterone Production from Phytosterol via One-Pot Cascade Biotransformations

Area of Science:

• Biotechnology and Industrial Microbiology
• Steroid Biotransformation
• Enzyme Engineering

Background:

• Testosterone (TS) and boldenone (BD) are crucial steroids with wide applications in medicine and veterinary science.
• Current production methods often involve complex chemical synthesis or less efficient enzymatic routes.
• Green and sustainable production of steroids from readily available precursors like phytosterols (PS) is highly desirable.

Purpose of the Study:

• To develop a novel cascade biotransformation process for the green production of testosterone (TS) and boldenone (BD) from phytosterols (PS).
• To investigate the enzymatic capabilities of *Curvularia* sp. VKM F-3040 for steroid conversion.
• To explore the potential for synthesizing 7-hydroxylated steroids through biotransformation.

Main Methods:

• Utilized *Curvularia* sp. VKM F-3040 for the conversion of androstadienedione (ADD) to boldenone (BD).
• Developed a two-stage cascade biotransformation: Stage 1 involved *Mycolicibacterium neoaurum* converting PS to androstenedione (AD) or ADD; Stage 2 used *Curvularia* sp. to reduce AD/ADD to TS/BD.
• Employed transcriptome analysis to identify candidate genes encoding 17β-hydroxysteroid dehydrogenases (17β-HSDs) and P450 7-monooxygenase. Investigated the effect of cycloheximide (CHX) on hydroxylation.

Main Results:

• *Curvularia* sp. efficiently converted ADD to BD (up to 97% yield).
• The developed cascade biotransformation achieved over 90% efficiency in converting AD/ADD to TS/BD from PS.
• Identified inducible P450_cur 7-monooxygenase activity leading to significant 7α-hydroxytestosterone (7α-OH-TS) production (up to 83%) from AD, inhibited by cycloheximide.

Conclusions:

• The *Curvularia* sp. strain exhibits significant biotechnological potential for steroid synthesis.
• Cascade biotransformation offers a promising green route for producing testosterone, boldenone, and novel 7-hydroxylated steroids.
• The findings open prospects for the sustainable industrial synthesis of valuable steroid compounds.