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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Anaerobic degradation of 2-chloro-4-nitroaniline by Geobacter sp. KT7 and Thauera aromatica KT9.

2-chloro-4-nitroaniline is a nitroaromatic compound widely used in industrial and agricultural sectors, causing serious environmental problems. This compound and some of its analogs were utilized by two Fe3+-reducing microbial strains Geobacter sp. KT7 and Thauera aromatica KT9 isolated from contaminated sediment as sole carbon and nitrogen sources under anaerobic conditions. The anaerobic degradation of 2-chloro-4-nitroaniline by the mixed species was increased approximately by 45% compared to that of individual strains. The two isolates' crossfeeding, nutrient sharing and cooperation in the mixed culture accounted for the increase in degradation rates. The determination of degradation pathways showed that Geobacter sp. KT7 transformed the nitro group in 2-chloro-4-nitroaniline to the amino group following by the dechlorination process, while T. aromatica KT9 dechlorinated the compound before removing the nitro group and further transformed it to aniline. This study provided an intricate network of 2-chloro-4-nitroaniline degradation in the bacterial mixture and revealed two parallel routes for the substrate catabolism.