Improvement of 9α-hydroxyandrost-4-ene-3,17-dione production in Mycolicibacterium neoaurum by regulation of cell wall formation and transcriptional regulator PadR.

The biotransformation of phytosterol into high value steroid intermediates such as 9α-hydroxyandrost-4-ene-3,17-dione (9-OHAD) in Mycolicibacterium is the cornerstone of the steroid pharmaceuticals. However, the limited permeability of the dense mycobacterial cell wall severely hinders the efficient transportation of phytosterol and their bioconversion to 9-OHAD. In this study, we disrupted the genetic pathways involved in trehalose biosynthesis, trehalose recycle and by-product formation, leading to alterations in cell wall formation, cell permeability and 9-OHAD productivity. This manipulation led to an increase of 63.7% in the yield of 9-OHAD, reaching 10.8 g/L at a phytosterol concentration of 20 g/L in shake flask. The enhancement of cell permeability and 9-OHAD production were achieved through the deletion of genes TreS, TreY, OtsA, LpqY, and SugC, as well as the inactivation of regulator PadR. Notably, it was found that the increase in TMM content of cell wall components via TLC analysis directly affected the distribution of 9-OHAD within and outside the cell, ultimately leading to an increase in extracellular production of 9-OHAD from 12% to 32.1%. Therefore, this study provides with an effective strategy for enhancing 9-OHAD production by increasing cell permeability while minimizing by-product 4-AD formation.