Improving the biotransformation efficiency of soybean phytosterols in Mycolicibacterium neoaurum by the combined deletion of fbpC3 and embC in cell envelope synthesis

Biotransformation of soybean phytosterols into 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) by mycobacteria is the core step in the synthesis of adrenocortical hormone. However, the low permeability of the dense cell envelope largely inhibits the overall conversion efficiency of phytosterols. The antigen 85 (Ag85) complex encoded by fbpA, fbpB, and fbpC was proposed as the key factor in the combined catalysis of mycoloyl for producing mycolyl-arabinogalactan (m-AG) and trehalose dimycolate (TDM) in mycobacterial cell envelope. Herein, we confirmed that fbpC3 was essential for the biotransformation of trehalose monomycolate (TMM) to TDM in Mycolicibacterium neoaurum. The deficiency of this gene raised the cell permeability, thereby enhancing the steroid uptake and utilization. The 9-OHAD yield in the fbpC3-deficient 9-OHAD-producing strain was increased by 21.3%. Moreover, the combined deletion of fbpC3 and embC further increased the 9-OHAD yield compared to the single deletion of fbpC3. Finally, after 96 h of bioconversion in industrial resting cells, the 9-OHAD yield of 11.2 g/L was achieved from 20 g/L phytosterols and the productivity reached 0.116 g/L/h. In summary, this study suggested the critical role of the fbpC3 gene in the synthesis of TDM in M. neoaurum and verified the feasibility of improving the bioconversion efficiency of phytosterols through the cell envelope engineering strategy.