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

The global prevalence of vitamin D (VitD) deficiency associated with numerous acute and chronic diseases has led to strategies to improve the VitD status through dietary intake of VitD-fortified foods and VitD supplementation. In this context, the circulating form of VitD₃ (cholecalciferol) in the human body, 25-hydroxy-VitD₃ (calcifediol, 25OHVitD₃), has a much higher efficacy in improving the VitD status, which has motivated researchers to develop methods for its effective and sustainable synthesis. Conventional monooxygenase-/peroxygenase-based biocatalytic platforms for the conversion of VitD₃ to value-added 25OHVitD₃ are generally limited by a low selectivity and yield, costly reliance on cyclodextrins and electron donor systems, or by the use of toxic co-substrates.

In this study, we used a whole-cell approach for biocatalytic 25OHVitD₃ synthesis, in which a molybdenum-dependent steroid C25 dehydrogenase was produced in the denitrifying bacterium Thauera aromatica under semi-aerobic conditions, where the activity of the enzyme remained stable. This enzyme uses water as a highly selective VitD₃ hydroxylating agent and is independent of an electron donor system. High density suspensions of resting cells producing steroid C25 dehydrogenase catalysed the conversion of VitD₃ to 25OHVitD₃ using either O₂ via the endogenous respiratory chain or externally added ferricyanide as low cost electron acceptor. The maximum 25OHVitD₃ titer achieved was 1.85 g L^-1 within 50 h with a yield of 99%, which is 2.2 times higher than the highest reported value obtained with previous biocatalytic systems. In addition, we developed a simple method for the recycling of the costly VitD₃ solubiliser cyclodextrin, which could be reused for 10 reaction cycles without a significant loss of quality or quantity.

The established steroid C25 dehydrogenase-based whole-cell system for the value-adding conversion of VitD₃ to 25OHVitD₃ offers a number of advantages in comparison to conventional oxygenase-/peroxygenase-based systems including its high selectivity, independence from an electron donor system, and the higher product titer and yield. Together with the established cyclodextrin recycling procedure, the established system provides an attractive platform for large-scale 25OHVitD₃ synthesis.