A Novel Enzyme-mediated Synthesis Of Acylated Steroidal Glycosides

Glycosylation of natural products is widely exist in plants,animals and microorganisms.Regulate the activity of organisms by glycosylation or the futher steps by acylation and methylation of macromolecules living matters and small molecules of nonliving materials.The glycosylation process of living organisms is mainly accomplished by different types and functions of glycosyltransferase(GTs),Meanwhile,a variety of natural products with important medicinal value are produced.The glycosylation modification andfuther derivativeof natural products plays an important role in improvingpharmacological activity,bioavailability and reducing toxicity.Steroid substances are ubiquitous in organisms.In plants,mainly exists in the form of steroidal glycosides and acylated steroidal glycosides.A variety of steroids exist because of the differences in steroid parent nucleus types,saccharide units,acyl types and the location of acetylation.Medicinal plant Ornithogalum saundersiae containing a variety of significant anticancer activity compound-Steroidal saponins,such as OSW-1.Because of its low content,difficult extractionand the tedious synthesis steps,which has seriously hindered further development.In this study,19 Glycosyltransferase related genes glycosyltransferases were cloned,steroidal glycation products were synthesized by a steroid specific glycosyl transferase.Further,a acyltransferase which can catalyze the acetylation of steroidal glycosides was found in Escherichia coli.A novel method for the biosynthesis of acetylated steroidal glycosides was designed.8 acylated steroidal glycosides were successfully obtained and their pharmacological activities were screened by MTT.The results showed that there is an obvious inhanced antitumor activity compared with steroidal glycosides which were not acylated(esterified).1,Cloning and expression of OsGTsTotal RNA was extracted from sterile bulb tissue of O.saundersiae,and been use as a template for reverse transcription to obtain the cDNA.19 GTs genes were cloned using nested PCR.And then,according to the method of In-Fusion,primers were designed for expression vector construction of target genes.And the prokaryotic expression plasmids were constructed successfully.The expression was induced by the way of chaperone co expression,and the soluble expression was achieved.2,Functional identification of glycosyltransferase and enzymatic propertiesThe substrates of the glycosyltransferase include the sugar donor and the receptor,formation of the three element complex with glycosyl transferase.Bioinformatics analysis was carried out on the obtained OsGTs gene.Determine the types of major sugar donor and receptor.Glycosylation was performed using different types of small molecules as sugar receptors.UDP-Glc,UDP-Xyl,UDP-GlcA,UDP-Gal,UDP-GalA,UDP-Ara and UDP-GlcNAc were selected as the sugar donors.The glycosylated products were identified by HPCL,mass spectrometry and NMR.OsSGT1 were identified as a steroid specific glycosyltransferase.The optimum reaction temperature-50?,the optimum pH-9.0-11.0 and the effects of the heavy metal ions on the catalytic activity of OsSGT1 were also investigated.3,Explore and identification of steroidal glycoside acetyltransferase from E.coliGlycosyltransferase crude enzyme obtained from Escherichia coli lysate,the corresponding acetylation products of steroid glycosides were also found in the glycosylation products of the sterols.Bioinformatic analysis of the genome of E.coli BL21(DE3),three sequence of O-acetyltransferase genes were obtained.Primers were designed to obtain 3 O-acetyltransferases:EOAT1/EOAT2 and EOAT3.Construction of expression vector,expression and functional identification of EOAT2 and then the function of steroidal glycosides acetylation were determined.4,Biosynthesis and application of acetylated steroidal glycosidesSteroidal compounds testosterone and estradiol as substrates,corresponding glycosylations was performed by OsSGT1,and then,with the endogenous or exogenous Acetyl-CoA to form testosterone 17?-O-?-D-glucoside-O-acetyl and estradiol 17?-O-?-D-glucoside-O-acetyl,respectively.Utilize this pathway the acetylation of steroidal glycoside were obtained with different loci of glycoside by biosynthesis.These acetylated steroidal glycosides were screened for their pharmacological activity,and were found to have enhanced anti human lung cancer cell(NCI-H460)activity.5,Studies on squalene synthase(OcSQSl)Analysis the transcriptome sequencing data of Ornithogalum caudatum,obtained the full-length 1230 bp of squalene synthase unigene,ORF analysis showed that the open reading frame of 1044 bp,named OcSQS1,bioinformatics analysis showed that the isoelectric point is 5.01.Phylogenetic analysis showed that it belongs to the SQS family.The expression vector was constructed and expressed with SDS-PAGE electrophoresis andWestern-blot assay.In vitro enzymatic reaction detected by GC-MS,The result shows that the truncation mutants of C-terminal membrane binding region can catalyze the formation of squalene FPP.