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

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 D_3. 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.