Optimization And Application Of UDP-rhamnose Glycosyltransferase From Ginseng

Ginseng contains more than 200 kinds of ginsenosides,which are the main active ingredients of ginseng.The main aglycogens of ginsenosides are protopanoxadiol and protopanaxatriol,and glycosylation plays a key role in ginsenoside structural and functional diversity.Glycosylation modification includes direct glycosylation of saponins and sugar chain extension.The direct glycosylation of saponins is usually modified by glucose moiety,and the extension of sugar chain includes rhamnose,xylose and arabinose besides glucose.The extension of these different sugar gives ginsenoside different activities and physiological functions.For example,ginsenoside Rg2 which has rhamnose-extended sugar chain at C-6possess significant therapeutic effects on cardiovascular diseases.In our preliminary study,we systematically identified the functions of glycosyltransferases from UGT94 Q family of Panax ginseng and Panax notoginseng,and obtained glycosyltransferases that catalyze glucose extension at saponins C-3,C-6 and C-20,and found some of them also had catalytic activity of rhamnose extension.However,the catalytic activity was low.We optimized one of the glycosyltransferases,UGT29-24,to improve its catalytic efficiency from ginsenoside Rh1 to ginsenoside Rg2.First,we compared the PSPG box between UGT29-24 and reported UDP-rhamnose glycosyltransferase,then we mutated the UGT29-24 and obtained The mutant UGT29-24m2(S343G,A359P)(hereafter referred as UGT29-24m2).The results indicated that compared with UGT29-24,when we just supply UDP-rhamnose,the conversion rate of ginsenoside Rg2 from ginsenoside Rh1 catalyzed by UGT29-24m2 increased from 0.4% to 11.6%.Then,we performed homologous modeling of UGT29-24 and finished molecular docking analysis about UGT29-24 with sugar donor UDP-rhamnose and sugar receptor ginsenoside Rh1.According to the molecular docking results,we predicted the key amino acid sites,which may be particularly important for stabilizing methyl groups of UDP-rhamnose.So We performed iterative mutations obtained mutant UGT29-24m4(S343G,A359 P,F15W,F362Y)(hereafter referred as UGT29-24m4).The results showed that compared with UGT29-24m2,when we just supply UDP-rhamnose,the conversion rate of ginsenoside Rg2 from ginsenoside Rh1 catalyzed by UGT29-24m4 increased from 11.6% to 53%.Next,we performed Homology modeling of UGT29-24m4 and molecular docking analysis about UGT29-24m4 with sugar donor UDP-rhamnose and sugar receptor ginsenoside Rh1,and the results showed that the binding energy between UGT29-24m4 and UDP-rhamnose significantly decrease,compared with UGT29-24 and UDP-rhamnose,which lead to the better combination between UDP-rhamnose glycosyltransferase and sugar donor UDP-rhamnose.Then,We mutated the key amino acid site I54,which maybe affect the size of substrate combine pocket,and the results showed that compared with UGT29-24m4,when we just supply UDP-rhamnose,the conversion rate of ginsenoside Rg2 from ginsenoside Rh1 catalyzed by UGT29-24m5 increased from 53% to 93.5%.We have modified the UDP-rhamnose glycosyltransferase,and obtained the iterative mutant UGT29-24m5,the conversion rate of ginsenoside Rg2 from ginsenoside Rh1 catalyzed by UGT29-24m5 is 232 times of UGT29-24.Finally we texted the protein activities between UGT29-24 and UGT29-24m5.HPLC results showed that the conversion rate of UGT29-24 catalyzed ginsenoside Rh1 form ginsenoside Rg2 was 0.4% but UGT29-24m5 was 93.5%,when only supply UDP-rhamnose.When UDP-rhamnose and UDP-glucose were supplied in equal quantities,UGT29-24 catalyzed ginsenoside Rh1 form ginsenoside Rg2 with a conversion rate of 0,but UGT29-24m5 was 13.8%.To complete the construction of ginsenoside Rg2 yeast cell factory,we attempted to construct the UDP-rhamnose synthesis pathway in ginsenoside Rh1 yeast chassis cells.Rhamnose-synthase VVRHM-NRS,which can synthesize UDP-rhamnose-using NADH,was integrated into the genome of ginsenoside Rh1 yeast chassis cells,and then UDP-rhamnose glycosyltransferase UGT29-24m5 was integrated into the single copy site of the yeast genome to complete the construction of ginsenoside Rg2 yeast cell factory.The yield of ginsenoside Rg2 reached 1 mg/L by shaking flask fermentationIn this study,we optimized the UDP-rhamnose glycosyltransferase UGT29-24 from ginseng by protein engineering method,and its catalytic activity of UDP-rhamnose was greatly improved,which provided a new approach and method for the optimization and modification of UDP-rhamnose glycosyltransferase.Meanwhile,in this study,the optimized mutant UGT29-24m5 and rhamnose-synthesis pathway were successfully integrated into ginsenoside Rh1 yeast chassis cells,and achieved the heterologous synthesis of ginsenoside Rg2.