Highly selective whole-cell 25-hydroxyvitamin D₃ synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling

Vitamin D (VitD) deficiency, which is associated with several acute and chronic diseases, is prevalent in populations worldwide and has inspired researchers to develop strategies to increase serum VitD levels via VitD-fortified food and supplementation.¹ In this context, the physiologically active form in the human body, 25-OH-VitD₃, has been shown to be significantly more effective as a supplement.² Enzymatic systems to produce this circulating form of VitD₃ typically use oxygen-dependent P450 monooxygenases, but these enzymes often produce undesired by-products and require costly NAD(P)H.³

As an alternative, we have developed a VitD₃-hydroxylating whole-cell system using a water-dependent steroid C25 dehydrogenase (S25DH) from denitrifying Sterolibacterium denitrificans in the related Thauera aromatica K172 as heterologous production host. Using resting cells of the S25DH-producing whole-cell biocatalyst at high densities, potassium ferricyanide as electron acceptor, 2-hydroxypropyl-β-cyclodextrin (HPCD) as solubiliser and ethanol as co-solvent, we demonstrated the formation of up to 1.85 g L^-1 within 50 h at a 100 mL scale, making this the highest published 25-OH-VitD₃ titer achieved to date. In addition, no common by-products such as the di-hydroxlated 1α,25-di-OH-VitD₃ were formed. We also showed that the VitD₃-solubilising HPCD can be recycled and used for up to 10 consecutive conversion cycles without a significant loss of quality or quantity, making the entire process economically feasible.⁴

1 Holick, M. et al., Rev. Endocr. Metab. Disord. 18, 153-165 (2017)

2 Bouillon, R. and Quesada-Gomez, J., J. Steroid Biochem. Mol. Biol. 228, 106248 (2023)

3 Wang, Z. et al., Biotechnol. Biofuels 15, 109 (2022)

4 Kosian, D. et al., Microb. Cell Fact. 23, 30 (2024)