The Inhibitory Effect of Cyclodextrin on Oxygen Bioavailability Is a Key Factor for the Metabolic Flux Redistribution Toward Steroid Alcohols in Phytosterol Resting Cells Bioconversion.

In the present work, we tried to identify the mechanism why by which the steroid alcohols accumulated when hydroxypropyl-β-cyclodextrin (HP-β-CD) was present to enhance the sterol conversion rate. Compared with the bioconversion system without HP-β-CD, the reaction rate was greatly improved in presence of HP-β-CD, but the steroid alcohols largely accumulated concurrently. In a reaction system with an enhanced reaction rate, the higher intracellular NADH/NAD level was detected, and the production of steroid alcohols increased also. Mycobacterium neoaurum mutants with higher KshA activity (3-ketosteroid 9α-hydrolase, a monooxygenase hydroxylating the nucleus at C-9 at the expense of NAD(P)H consumption) reduced the steroid alcohol production, and in the meantime, the NADH/NAD level was decreased consequently. Further research found that oxygen availability was seriously inhibited by the cyclodextrin in a reaction system. These results indicated that NADH formed in the bioconversion was not properly regenerated via the respiratory chain because of the poor oxygen bioavailability. The inhibitory effect of cyclodextrin on oxygen bioavailability is a key factor for the metabolic flux redistribution toward steroid alcohols in phytosterol resting cells bioconversion.