Integrated Metabolic Engineering and Substrate Pretreatment for Sustainable Androstenedione Production from Phytosterols

Androstenedione (AD) is a key steroidal intermediate widely used in the synthesis of hormonal steroid drugs. Efficient bioconversion of phytosterols into AD not only enhances their industrial value but also promotes the development of a sustainable bioeconomy. In this study, we employed two strategies to enhance AD production in Mycolicibacterium neoaurum (MNR): (i) optimizing metabolic flux by deleting the transcription factor FdmR to upregulate the phytosterol side-chain degradation pathway and overexpressing icl1 and sdhD to prevent the accumulation of metabolic intermediates and (ii) enhancing the bioavailability of phytosterols using high-pressure homogenization (HPH) in a waste cooking oil/HP-β-cyclodextrin (WCO/HP-β-CD) cosolvent system. Under the optimized conditions, engineered strain QCM3-ΔFdmR-icl1-sdhD produced 9.29 g/L AD in 72 h, representing a 1.5-fold increase over the control strain QCM3. In addition, the use of repetitive batch fermentation reduced the production cycle by 46.7%, further improving the efficiency and process sustainability. This work provides novel strategies for sustainable AD biosynthesis that can be extended to the production of other high-value steroid intermediates.