Role Identification and Application of SigD in the Transformation of Soybean Phytosterol to 9α-Hydroxy-4-androstene-3,17-dione in Mycobacterium neoaurum.

9α-Hydroxy-4-androstene-3,17-dione (9-OHAD) is a valuable steroid pharmaceutical intermediate which can be produced by the conversion of soybean phytosterols in mycobacteria. However, the unsatisfactory productivity and conversion efficiency of engineered mycobacterial strains hinder their industrial applications. Here, a sigma factor D (sigD) was investigated due to its dramatic downregulation during the conversion of phytosterols to 9-OHAD. It was determined as a negative regulator in the metabolism of phytosterols, and the deletion of sigD in a 9-OHAD-producing strain significantly enhanced the titer of 9-OHAD by 18.9%. Furthermore, a high yielding strain was constructed by the combined modifications of sigD and choM2, a key gene in the phytosterol metabolism pathway. After the modifications, the productivity of 9-OHAD reached 0.071 g/L/h (10.27 g/L from 20 g/L phytosterol), which was 22.5% higher than the original productivity of 0.058 g/L/h (8.37 g/L from 20 g/L phytosterol) in the industrial resting cell biotransformation system.