High efficiency biosynthesis of 1α,25-Dihydroxyvitamin D3 via engineered dihydroxylase and deep eutectic solvent based resting cell bioprocess

1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2 VD 3 ), the biologically active form of Vitamin D 3 (VD 3 ), plays a crucial role in a wide range of physiological processes. However, the efficient biosynthesis of 1α,25(OH) 2 VD 3 is hindered by the low activity of natural enzymes, poor solubility of substrate VD 3 , and its intracellular metabolic loss. Herein, we developed an integrated bioprocess by synergizing engineered dihydroxylase (CYP109A2-H) with a deep eutectic solvent (DES) based resting cell system to overcome these limitations. A semi-rational design strategy was employed to enhance the performance of the enzyme, which yielded several beneficial mutants including E81G that increased the catalytic titer by 43.6%. Concurrently, a DES resting cell system was constructed, wherein DES dramatically increased VD 3 solubility and resting cells eliminated metabolic loss. This synergistic strategy achieved a record-high 1α,25(OH) 2 VD 3 space–time yield of 33.15 mg·L −1 ·d −1 , establishing the most efficient single-batch bioconversion reported to date. This work provides a sustainable and efficient platform for 1α,25(OH) 2 VD 3 production and a general strategy for the biohydroxylation of hydrophobic compounds.