Androst-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) are important drug intermediates that can be biosynthesized from phytosterols. However, the C9 hydroxylation of steroids via 3-ketosteroid 9α-hydroxylase (KSH) limits AD and 4-HBC accumulation. Five active KshAs, the oxidation component of KSH, were identified in Mycobacterium fortuitum ATCC 35855 for the first time. The deletion of kshAs indicated that the five KshA genes were jointly responsible for C9 hydroxylation during phytosterol biotransformation. MFKDΔkshA, the five KshAs deficient strain, blocked C9 hydroxylation and produced 5.37 g/L AD and 0.55 g/L 4-HBC. The dual function reductase Opccr knockout and 17β-hydroxysteroid dehydrogenase Hsd4A enhancement reduced 4-HBC content from 8.75 to 1.72% and increased AD content from 84.13 to 91.34%, with 8.24 g/L AD being accumulated from 15 g/L phytosterol. In contrast, hsd4A and thioesterase fadA5 knockout resulted in the accumulation of 5.36 g/L 4-HBC from 10 g/L phytosterol. We constructed efficient AD (MFKDΔkshAΔopccr_hsd4A) and 4-HBC (MFKDΔkshAΔhsd4AΔfadA5) producers and provided insights for further metabolic engineering of the M. fortuitum ATCC 35855 strain for steroid productions. KEY POINTS: • Five active KshAs were first identified in M. fortuitum ATCC 35855. • Deactivation of all five KshAs blocks the steroid C9 hydroxylation reaction. • AD or 4-HBC production was improved by Hsd4A, FadA5, and Opccr modification.

OBJECTIVE: To realize a practical technology for recycling both cyclodextrin and resting-cells at the same time in phytosterol biotransformation using mycobacterial resting cells.
RESULTS: In order to produce 22-hydroxy-23,24-bisnorchol-4-ene-3-one (HBC) efficiently and low-costly, a recycled phytosterols (PS) biotransformation process using mycobacterial resting cells was developed. By optimizing the ratio of hydroxypropyl-β-cyclodextrin (HP-β-CD) and PS to 1:1 (w/w), most products crystallized during the biotransformation process. So, the HBC was easily separated by low-speed (900×g) centrifugation with yield of 92%. The resting cells, HP-β-CD and the residual products and substrates left in the reaction system were reused for another bioconversion cycle after PS supplement. Three continuous cycles were achieved without the supplement of cells and HP-β-CD. In each batch, 80 g L-1 of PS was transformed to HBC with the space-time yield of HBC of 8.9-12.8 g L-1 day-1.
CONCLUSIONS: This strategy reduced the cost of HBC production and simplified the purification process.