A Designer Ternary Deep Eutectic Solvent Enhancing the Efficiency of the Sequential Two-Step Biohydroxylation of Progesterone

The core hydroxylation of steroids plays a pivotal role in steroid drug synthesis. However, direct hydroxylation of sp3 C–H bonds by chemical catalysis is challenging due to poor selectivity and substantial environmental pollution. Fungal biotransformation may be a solution to these issues. In this study, Mortierella sp. CICC 40250 was identified as being capable of hydroxylating progesterone through a two-step process, yielding 6β,11α-dihydroxyprogesterone. The dihydroxy metabolite was evaluated using Swiss ADME, and the results suggested that it might exhibit bioavailability and bioactivity. To address the limitation associated with the low solubility of the substrate in the reaction system, 36 types of deep eutectic solvents (DESs) were constructed for the sequential two-step biohydroxylation of progesterone. The newly designed ternary DES (Pro/EG/Glc at a molar ratio of 2:1:1) exhibited excellent biocompatibility and enhanced the cell membrane permeability. Compared with the original system, the ternary DES system achieved a higher progesterone conversion rate of 80.5% and increased the time-space yield of 6β,11α-diOH-PG by 3.3-fold in the 5-L fermenter. DESs were inherently natural and nontoxic. Consequently, they are environmentally friendly and well-suited for the green synthesis of steroid drugs. The results demonstrated the significant potential of solvent engineering in the development of efficient and sustainable biocatalytic solutions.