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

1α,25-dihydroxyvitamin D3 (1α,25(OH)2VD3), the biologically active form of Vitamin D3 (VD3), plays a crucial role in a wide range of physiological processes. However, the efficient biosynthesis of 1α,25(OH)2VD3 is hindered by the low activity of natural enzymes, poor solubility of substrate VD3, 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 VD3 solubility and resting cells eliminated metabolic loss. This synergistic strategy achieved a record-high 1α,25(OH)2VD3 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)2VD3 production and a general strategy for the biohydroxylation of hydrophobic compounds.