Efficient 9α-hydroxy-4-androstene-3,17-dione production by engineered Bacillus subtilis co-expressing Mycobacterium neoaurum 3-ketosteroid 9α-hydroxylase and B. subtilis glucose 1-dehydrogenase with NADH regeneration

3-Ketosteroid 9α-hydroxylase (KSH, consisting of KshA and KshB), a key enzyme in steroid metabolism, can catalyze the transformation of 4-androstene-3,17-dione (AD) to 9α-hydroxy-4-androstene-3,17-dione (9OHAD) with NADH as coenzyme. In this work, KSH from Mycobacterium neoaurum JC-12 was successfully cloned and overexpressed in Bacillus subtilis 168. The expression and purification of KSH was analyzed by SDS-PAGE and KSH activity assay. Preliminary characterization of KSH was performed using purified KshA and KshB. The results showed that KSH was very unstable, and its activity was inhibited by most metal ions, especially Zn(2+). The whole-cells of recombinant B. subtilis, co-expression of KSH and glucose 1-dehydrogenase (GDH), were used as biocatalyst to convert AD to 9OHAD. The biocatalyst, in which the intracellular NADH was regenerated, efficiently catalyzed the bioconversion of AD to 9OHAD with a conversion rate of 90.4 % and productivity of 0.45 g (L h)(-1), respectively. This work proposed a strategy for efficiently producing 9OHAD by using B. subtilis as a promising whole-cell biocatalyst host and co-expressing KSH and GDH to construct a NADH regeneration system.