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

Abstract 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 (chole...

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Main Authors: Dennis Kosian, Max Willistein, Ralf Weßbecher, Constantin Eggers, Oliver May, Matthias Boll
Format: Article
Language:English
Published: BMC 2024-01-01
Series:Microbial Cell Factories
Subjects:
Online Access:https://doi.org/10.1186/s12934-024-02303-6
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author Dennis Kosian
Max Willistein
Ralf Weßbecher
Constantin Eggers
Oliver May
Matthias Boll
author_facet Dennis Kosian
Max Willistein
Ralf Weßbecher
Constantin Eggers
Oliver May
Matthias Boll
author_sort Dennis Kosian
collection DOAJ
description Abstract 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.
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spelling doaj.art-91dc032b60b34ee89ed3e979c15785ac2024-01-21T12:40:30ZengBMCMicrobial Cell Factories1475-28592024-01-0123111310.1186/s12934-024-02303-6Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recyclingDennis Kosian0Max Willistein1Ralf Weßbecher2Constantin Eggers3Oliver May4Matthias Boll5Faculty of Biology – Microbiology, University of FreiburgFaculty of Biology – Microbiology, University of FreiburgFaculty of Biology – Microbiology, University of FreiburgFaculty of Biology – Microbiology, University of FreiburgDSM Nutritional Products, Koninklijke DSM N.V.Faculty of Biology – Microbiology, University of FreiburgAbstract 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.https://doi.org/10.1186/s12934-024-02303-6Vitamin D325-hydroxyvitamin D3CalcifediolCalcitriolWhole-cell biocatalysisThauera aromatica
spellingShingle Dennis Kosian
Max Willistein
Ralf Weßbecher
Constantin Eggers
Oliver May
Matthias Boll
Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling
Microbial Cell Factories
Vitamin D3
25-hydroxyvitamin D3
Calcifediol
Calcitriol
Whole-cell biocatalysis
Thauera aromatica
title Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling
title_full Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling
title_fullStr Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling
title_full_unstemmed Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling
title_short Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling
title_sort highly selective whole cell 25 hydroxyvitamin d3 synthesis using molybdenum dependent c25 steroid dehydrogenase and cyclodextrin recycling
topic Vitamin D3
25-hydroxyvitamin D3
Calcifediol
Calcitriol
Whole-cell biocatalysis
Thauera aromatica
url https://doi.org/10.1186/s12934-024-02303-6
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