4 CONCLUSION
Mycobacterium could degrade the sidechain of PS to produce the steroidal intermediates 9-OH-AD. In this study, the production of 9-OH-AD from PS biotransformation was significantly improved by modulating the key gene expression of kshA1 and hsd4A in mycobacterial mutants. The augment of gene hsd4A could effectively promote the side-chain degradation of PS and decrease the incomplete degradation products, DHBC, but increase the complete degradation product of 4-androstene-3,17-dione (AD) due to lack of 3-Ketosteroid 9α-Hydroxylase KshA1 activities. Therefore, co-expression of kshA1 and hsd4A in an appropriate pattern in mycobacterial could optimize their expression level and achieve a better balance of the Hsd4A and KshA1 activity. Further decreasing the PS conversion rate could be best for the action of the Hsd4A and KshA1 activities to transfer the metabolic flux to the target product of 9-OH-AD and decrease the byproducts formation. Finally, the yield and purity of 9-OH-AD in PS transformation by HK86 K-A mutant were increased from 22.18 g/L and 77.13% to 28.27 g/L and 87.84%, respectively, with a molar yield of 78.32%.