3.1 KshA1 promotes the 9-position hydroxylation of the steroid nucleus
The engineered strains Mycobacterium neoaurum NwIB-HK86 W and HK86-K are 9-OH-AD production strains in the resting cell-cyclodextrin system. Under substrate feeding of 50 g/L and the mass ratio of PS and HP-β-CD (1:4, w/w), the 9-OH-AD yields of the two strains peaked at 22.18 g/L and 25.10 g/L, respectively, on the 4th day of reaction (Fig. 1). The yield of 9-OH-AD begins to decline after 4 days because it was degraded in the later stages of transformation and the production rate was less than degradation rate. The composition of the transformation products of the two strains is shown in Table 2, and the purity of the main product 9-OH-AD in broth was 77% and 78% respectively. Overexpression of kshA1 in mutant strains promoted the level of 9-position hydroxylation. The production of 9-OH-AD was increased in HK86-K, while large amounts of incomplete degradation by-products such as 9,22-dihydroxy-23,24-bisnorchol-4-en-3-one (DHBC) and 9,24-dihydroxychol-4-en-3-one (DHC) were also accumulated at the end of biotransformation. The excessive by-products will hamper the downstream process and reduce the yield of biotransformation.