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.