Improving the bioconversion of phytosterols to 9α-hydroxy-4-androstene-3,17-dione by disruption of acyltransferase SucT and TmaT associated with the mycobacterial cell wall synthesis

The bioconversion of low value-added phytosterols into high value-added 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) in Mycolicibacterium neoaurum is a representative step in the steroid pharmaceutical industry. However, the complex mycobacterial cell walls with extremely low permeability and flowability greatly decrease the overall conversion efficiency. Herein, we preliminarily identified two key acyltransferases encoded by Mn_TmaT and Mn_SucT required for the proper synthesis of cell wall in mycobacteria and achieved a significant increase in cell permeability by disrupting them without affecting the cell wall structural stability. At length, the destruction of Mn_TmaT and Mn_SucT alone increased the conversion rate of 9-OHAD from 45.3% (6.67 ± 0.39 g/L) to 62.4% (9.19 ± 0.58 g/L) and 67.9% (10.02 ± 0.62 g/L) while the continuous destruction of Mn_TmaT and Mn_SucT did not further improve the conversion efficiency of 9-OHAD. Notably, it was investigated that the continuous destruction of Mn_TmaT and Mn_SucT led to alterations in both the covalent and non-covalent binding layers of the cell wall, resulting in excessive changes in cell morphology and structure, which ultimately decreased 9-OHAD production. Therefore, this study deciphered a pivotal biosynthetic path of cell wall and provided an efficient and feasible construction strategy of 9-OHAD synthesis in mycobacteria.