Preliminary Study On Epothilone Glycosyltransferases

Epothilone,frst reported in 1993,is a class of 16-membered polyketide natural products only from Sorangium cellulosum,which shows excellent inhibitory activities against multi-drug-resistant tumors,especialiiy paclitaxel-resistant tumor cells.Epothilone can bind to the ?-subunit of the ?,?-tubulin heterodimer,promoting the polymerization and inhibiting the depolymerization,which cause the proliferating cells death.In 2008,a semi-synthetic analogue of epothilone B,Ixabepilone,was approved by the FDA for the treatment of breast cancer in patients.However,since normal cells and tumors have similar properties,epothilone B analogues also have significant cytotoxicity on normal proliferating cells,limiting the further clinical use.According to the physical and biological properties of glycosylated macrolide compounds,we hypothesis that epothilone glycosylation will significantly decrease the cytotoxicity,and provide broader prospects for the application of epothilones.Glycosylated epothilone A was detected in the fermentation broth of Sorangium cellulosum So0157-2 in the early stage,which was the first time to find glycosylated epothilone in Sorangium cellulosum.We hypothesized that the potential glycosyltransferases catalyze the glycosylation reaction,and our work focused on the discovery of predicted glycosyltransferases for glycosylation of epothilone.According to the analysis of Cazy website,it was confirmed that Sorangium cellulosum So0157-2 has 64 predicted glycosyltransferases,and we discovered the predicted glycosyltransferase near the epothilone gene cluster.Hence,the glycosyltransferase were cloned,vectors construction and protein expression.However,the glycosylated epothilones were not detected in vitro enzymatic activities.Therefore,we tried to explore the genes encoding the potential glycosyltransferases in the whole genome and search for the exogenous glycosyltransferases for epothilones glycosylation.Bacillus is the rich source of glycosyltransferases,large numbers of glycosyltransferases from Bacillus are highly adaptable to different substrates,the glycosyltransferases are regiospecific rather than highly substrate specific.Therefore,these glycosyltransferases are usually applied to enzymatic synthesize glycosides derivatives and have been reported for numerous compounds glycosylation.Glycosyltransferase YjiC,derived from the Bacillus licheniformis glycosyltransferase 1 family,has been reported the activities for epothilone A glycosylation.What's more,the glycosyltransferase YjiC can not only has the activities against epothilone A,but also has the abilities for epothilone B glycosylation.In this study,the experiments were carried out through two aspects:On one hand,glycosyltransferase YjiC was tested to verify the glycosylation functions by gene cloning,heterologous expression and enzyme activities in vitro;on the other hand,glycosyltransferase YjiC was purified for the enzymatic properties exploration to determine the optimum temperature and optimum pH.