Improvement of the 25-hydroxyvitamin D3 production in a CYP109A2-expressing Bacillus megaterium system

• Published in: Journal of biotechnology Vol. 325; pp. 355 - 359
• Main Authors: Abdulmughni, Ammar, Erichsen, Björn, Hensel, Jürgen, Hannemann, Frank, Bernhardt, Rita
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 10-01-2021
• Subjects: Bacillus megaterium; Calcifediol; Cytochrome P450; Putative redox-partner binding site; Site-directed mutagenesis; Putative redox-partner binding site; Calcifediol; Cytochrome P450; Site-directed mutagenesis; Bacillus megaterium
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2020.09.027

•Demonstrating the importance of the region between amino acids T103-A106 of CYP109A2 for the hydroxylation of vitamin D3.•Increasing the productivity of CYP109A2-based whole-cell system by optimizing the reaction conditions.•Using the mutant T103A under optimized conditions resulted in a 5-fold higher amount of 25(OH)VD3 compared to the wild type.
Calcifediol (25(OH)VD3) is a physiologically very important vitamin D3 metabolite and of high pharmaceutical importance, due to its potential for treating not only vitamin D3 deficiencies but also coronary diseases and cancer.
Previously, we established a whole-cell Bacillus megaterium-based system using the cytochrome P450 CYP109A2 for the biotransformation of vitamin D3 into its metabolite 25-hydroxyvitamin D3. In this study, we demonstrate the importance of the region between amino acids T103 and A106 for the catalytic activity of CYP109A2 towards vitamin D3 as a substrate. In order to increase the productivity of the system, reaction conditions (xylose, vitamin D3, saponin, 2-hydroxypropyl-β-cyclodextrin) were optimized for the in vivo production of 25-hydroxyvitamin D3.
With cells producing the T103A mutant, a productivity of 282.7 mg/L/48 h was achieved under the optimized conditions. This value is two times higher than that obtained in the control reaction with the wild-type enzyme in this study and five times higher than that obtained in a previous study.