| 摘要： |
Biosynthesis of simvastatin, the active pharmaceutical ingredient of cholesterol-lowering drug Zocor, has drawn increasing global attention in recent years. Although single-step in vivo production of monacolin J, the intermediate biosynthetic precursor of simvastatin, has been realized by utilizing lovastatin hydrolase (PcEST) in our previous study, about 5% of residual lovastatin is still a problem for industrial production and quality control. In order to improve conversion efficiency and reduce lovastatin residues, modification of PcEST is carried out through directed evolution and a novel two-step high-throughput screening method. The mutant Q140L shows 18-fold improved whole-cell activity as compared to the wild-type, and one fold enhanced catalytic efficiency and 3 degrees C increased T-50(10) over the wild-type are observed by characterizing the purified protein. Finally, the engineered A. terreus strain overexpressing Q140L mutant exhibited the increased conversion efficiency and the reduced lovastatin residues by comparing with A. terreus strain overexpressing the wild-type PcEST, where almost 100% of the produced lovastatin is hydrolyzed to monacolin J. Therefore, this improved microbial cell factory can realize single-step bioproduction of monacolin J in a more efficient way, providing an attractive and eco-friendly substitute over the existing chemical synthetic routes of monacolin J and promoting complete bioproduction of simvastatin at industrial scale. |
