Functional characterization of two reductase KshBs in Mycobacterium fortuitum and their applications to C9 non-hydroxylated steroid production

Abstract

3-ketosteroid-9α-hydroxylase (KSH), a two-component monooxygenase consisting of a Rieske oxygenase KshA and a ferredoxin reductase KshB, is a crucial enzyme involved in C-9 hydroxylation and is indispensable in the microbial catabolism of sterols. However, the in vivo function of KshB remains unclear. In this study, two reductase subunits, KshB1 and KshB2 from Mycobacterium fortuitum ATCC 35855, were first characterized and then engineered in the strain MFΔkstD, which produces 9-OHAD (9α-hydroxy-4-androstene-3,17-dione), to construct the strains producing AD (4-androstene-3,17-dione). The transcriptional level and specific enzyme activity of KshB1 under phytosterols induction was significantly higher compared to KshB2. After knocking out kshB1, AD peaked at 1.77 g/L at 48 h, subsequently being fully converted to 9-OHAD. Furthermore, we developed the stable AD-producer MFΔkstDΔkshB by null mutation both kshB1 and kshB2, although no AD was detected by single knocking out kshB2. Through gene complementation and the bioconversion of phytosterols, KshB1 emerged as the primary reductase in the KSH system, with KshB2 serving a complementary role. The stable AD-producer MFΔkstDΔkshB could produce 5.18 g/L, 6.91 g/L, and 9.03 g/L AD from the transformation of 10 g/L, 15 g/L, and 20 g/L phytosterols, respectively. In conclusion, these findings highlight a new strategy for the metabolic engineering of Mycobacterium fortuitum, involving the inactivation of the reductase KshB to facilitate the production of C9 non-hydroxylated steroidal intermediates.