Study On The Biotransformation Of Dammarane-type Triterpene Saponins

Dammarane-type triterpene saponins are the main pharmacological active components distributing in the genus of Panax.There are nearly 300 saponins reported from Panax species.Some ginsenosides,which are saponins with one to three monosaccharides,are proved to have better anti-tumour activity such as ginsenoside CK and Rh2.However,the content of these active saponins,named as rare saponins,is very low in the original medicinal materials,which restricts further development and utilization of these compounds.Therefore,it is very significant to look for new ways to produce such rare saponins for medicinal application.The biosynthesis of saponins begins at the cyclization of 2,3-oxidosqualene followed by hydroxylation and glycosidation.Glycosyltransferase is involved in the last and key modification step occurring in the process with glycosylation on the aglycones.It is possible to obtain rare ginsenosides using appropriate glycosyltransferase on the aglycone(PPD or PPT)in vitro.In this study,candidate glycosyltransferase genes selected from plant and microbe were used to obtain rare ginsenosides and the main research results are as follows:1 Glycosyltransferase selection and bioinformatic analysisThe nucleotide sequence of UGTPg1 was analyzed with tBLASTx and five other glycosyltransferase genes namely UGT71G1,UGT73K1,UGT74M1,PnUGT82 and Pn UGT95 were chosen according to the known information.Protparam,SignalP-4.1,Prosite,SOPMA and SWISS-MODEL online analysis tools were used for the bioinformatic analysis.2 Cloning and expression of the candidate glycosyltransferase genesSix candidate genes were respectively constructed into pETM6 expression vector and introduced into E.coli BL21starTM(DE3)host cells.Total proteins were extracted from individual recombinant E.coli cells after ultrasonic disruption and analyzed by SDS-PAGE electrophoresis.SDS-PAGE results showed that all the six genes were expressed as proteins with expected molecular weight.3 Catalytic property of the candidate glycosyltransferasesCatalytic property of the candidate glycosyltransferases were investigated with recombinant strains.As monitored by UPLC-ESI-MS,strains carrying GT1syn and GT95syn respectively could glycosylate the C20-OH of PPD to produce CK among the six strains.While the converted products of the remaining four strains and the control group were not detected.The catalytic activity of GT95syn was discovered as a new glycosyltransferase involved in the biosynthesis of ginsenosides.In addition,strain BL21*-GT95syn could also glycosylate the C20-OH of PPT to produce F1 and then glycosylate the C6-OH of F1 to produce Rgl.Moreover,the substrates could be both 20(S)and 20(R)-type ginsenosides.Crude enzyme of GT95syn was also investigated in vivo,and the results showed that GT95syn had a better catalytic efficiency than strain BL21*-GT95syn.Moreover,the pH value,temperature,reaction time,solvent,the concentration of glucose,and other parameters in the catalytic systems were also optimized to obtain an optimal condition.4 Cloning and expression of glycosyltransferase genes from Bacillus subtilisTwo glycosyltransferase genes GTK1 and GTC1 were successfully cloned from Bacillus subtilis by PCR,and then constructed into pETM6 prokaryotic expression vector and introduced into E.coli BL21starTM(DE3)host cells,respectively.Total proteins were extracted from individual recombinant E.coli cells with ultrasonic disruption and analyzed by SDS-PAGE electrophoresis.SDS-PAGE results showed that both GTK1 and GTC1 were expressed successfully.As monitored by UPLC-ESI-MS,both GTK1-and GTC1-modified E.coli glycosylated the C3-OH of PPD to form Rh2.5 Construction of a two-step glycosylation pathwayp2GT95synK1,an ePathBrick expression vector harboring two copies of GT95syn and one copy of GTK1 genes,was contructed with the aim to recreate a two-step glycosylation pathway from PPD to CK then to F2,or from PPD to Rh2 then to F2.As monitored by UPLC-ESI-MS,E.coli containing p2GT95synK1 implemented the successive glycosylation at C20-OH and C3-OH of PPD and yield the expected F2 in the biotransformation broth.6 Application of Fungi in the biosynthesis of saponinsThe biotransformation ability of twenty strains of fungi on protopanaxatriol(PPT)were explored.Five of them,strains 17,29,X-6-2,X-2-2 and X-D3-2,could use PPT as substrate.Strain 29,Trametes hirsuta as identified with 18S rDNA,was selected for scale up experiments and the products were isolated and identified as 26-hydroxyl-20(S)-protopanaxtriol(1),27-hydroxyl-20(S)-protopanaxtriol(2)and 26-hydroxyl-3-oxo-20(S)-protopanaxatriol(3)by NMR and HR-ESI-MS.Among them,compound 3 was a new compound.In summary Panax notoginseng GT95syn and Bacillus subtilis GTK1 and GTC1 selectively hydroxylazed the C20-OH and C3-OH of PPD and PPT to produce rare ginsenosides CK,Rh2,F1,and F2.Besides,E.coli engineered with a two-step glycosylation route containing GT95 and GTK1 successive glycosylated C20-OH and C3-OH of 20(R)-PPD and yield 20(R)-F2.This study demonstrated that rare ginsenosides could be produced through E.coli engineered with UTG genes.Moreover,some new PPT derivatives with low polarity were obtained through oxidative modification of PPT by Trametes hirsuta,which increased the diversity of saponins.