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In vitro glycosylation of isoflavonoids using YjiC from Bacillus licheniformis DSM 13
Prakash Parajuli,Ramesh Prasad Pandey,Jae KyungSohng 한국당과학회 2013 한국당과학회 학술대회 Vol.2013 No.1
Isoflavonoids are the plant secondary metabolites biogenetically derived from 2-phenylchroman skeleton of flavonoids. They are particularly prevalent in subfamily of Leguminosae; Papilonoidae. The remarkable biological properties of isoflavonoids described as antimicrobial, antioxidant, anti-inflammatory, estrogenic and cancer chemoprotectant. To enhance the bioavailability and biological properties of isoflavonoids, their diversification via acetylation, malonylation, hydroxylation, prenylation, glycosylation are under study. Glycosylation is one of the important tools to diversify and extend such biological functions in natural products and secondary metabolites. The process is catalyzed by UDP-glycosyltransferases in the formation of glycosidic linkages by transferring sugar moiety from a donor substrate to an acceptor. In present study, we have analyzed the in vitro enzymatic reactions of isoflavonoids using YjiC, a glycosyltransferase from Bacillus licheniformis DSM 13. UDP-D-glucose was considered as a sugar donor and isoflavonoids genestein, diadzein, formononetin and biochanin A as acceptor substrates. Reaction products were analyzed by HPLC and high resolution LC-QTOF-ESI/MS which revealed the detection of two mono-glucosides and one di-glucoside in genestein and daidzein where as single mono glucoside with formononetin and biochanin A. Glycosylation at the probable positions of 4’ and 7 hydroxyl groups of the genestein and daidzein was suspected while biochanin A - 7-hydroxyl position might have been prominent. In case of formononetin having a single hydroxyl at 7th position, high resolution LC-QTOF-ESI/MS analysis conforms the exact configuration and positioning of glucose attachment. Although glycosides of the rests of isoflavonoids were detected from high resolution LC-QTOF-ESI/MS analysis, the exact configuration of sugar attachment is yet to be identified.
Enzymatic and Microbial Biosynthesis of Natural Products Glycosides
Ramesh Prasad Pandey,PrakashParajuli,AnilShrestha,JaeKyungSohng 한국당과학회 2018 한국당과학회 학술대회 Vol.2018 No.07
Post-biosynthesis modifications of natural products (NPs) provides opportunity to bring chemical diversity to the parent molecule. Such modifications usually play vital roles in executing biological activities of the molecules. Thus, engineering of molecules by diverse post-modifications is increasingly becoming a tool to design or produce novel biologically potent biologics. To develop the rapid and sustainable system for the production of different flavonoid glycosides, central metabolic pathway for the production of pool of UDP-0-glucose, UDP-0-xylose, TDP-L -rhamnose, TDP-0-viosamine, TOP 4-amino 4,6-dideoxy-D-galactose, and TOP 3-amino 3,6-dideoxy-D-galactose was engineered in E. coli BL21 (DE3) cells. Different glycosyltransferases were engaged to transfer sugar moieties to aglycones. Several flavonoids and isoflavonoid glycosides including natural and non-natural o- and C- glycosides were produced by microbial cell fermentation. In a different approach of enzymatic biosynthesis, a number of glycodiversified flavonoids were generated using several NDP-sugars and GTs in vitro. As a result, glycodiversified resveratrol, flavonol, epothilone A, mupirocin conjugated with glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugars were produced. Multiple glycosides of other flavonoids, isoflavonoids, chalcones, stilbenes, xanthonoids, anthraquinones, anthracyclines, and terpenoids were also generated with significantly high yield. Some of the selected glycosides exhibited promising anticancer, antibacterial, immunomodulatory, anti-inflammatory, antioxidant, and anti-asthmatic activities in in vitro and in vivo mouse models. This approach of microbial and enzymatic synthesis of novel glycosides derivatives of NPs using highly flexible and promiscuous enzymes from diverse sources opened up possibility of development of new molecules with better stability, bioavailability, and novel biological activity.
Glucosylation of Isoflavonoids in Engineered Escherichia coli
Ramesh Prasad Pandey,PRAKASHPARAJULI,Niranjan Koirala,이주호,박용일,송재경 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.2
A glycosyltransferase, YjiC, from Bacillus licheniformis has been used for the modification of the commercially available isoflavonoids genistein, daidzein, biochanin A and formononetin. The in vitro glycosylation reaction, using UDP-α-D-glucose as a donor for the glucose moiety and aforementioned four acceptor molecules, showed the prominent glycosylation at 4′ and 7 hydroxyl groups, but not at the 5th hydroxyl group of the A-ring, resulting in the production of genistein 4′-O-β-D-glucoside, genistein 7-O- β-D-glucoside (genistin), genistein 4′,7-O-β-D-diglucoside, biochanin A-7-O-β-D-glucoside (sissotrin), daidzein 4′-O-β- D-glucoside, daidzein 7-O-β-D-glucoside (daidzin), daidzein 4′, 7-O-β-D-diglucoside, and formononetin 7-O-β-D-glucoside (ononin). The structures of all the products were elucidated using high performance liquid chromatographyphoto diode array and high resolution quadrupole time-offlight electrospray ionization mass spectrometry (HR QTOFESI/ MS) analysis, and were compared with commercially available standard compounds. Significantly higher bioconversion rates of all four isoflavonoids was observed in both in vitro as well as in vivo bioconversion reactions. The in vivo fermentation of the isoflavonoids by applying engineered E. coli BL21(DE3)/ΔpgiΔzwfΔushA overexpressing phosphoglucomutase (pgm) and glucose 1-phosphate uridyltransferase (galU), along with YjiC, found more than 60% average conversion of 200 μM of supplemented isoflavonoids, without any additional UDP-α-D-glucose added in fermentation medium, which could be very beneficial to large scale industrial production of isoflavonoid glucosides.
Biosynthesis of a novel fistein glycoside from engineered Escherichia coli
Biplav Shrestha,Ramesh Prasad Pandey,PrakashParajuli,SumangalaDarsandhari,Jae Kyung Sohng 한국당과학회 2018 한국당과학회 학술대회 Vol.2018 No.01
Fisetin (3,7,3’,4’-tetrahydroxyflavone) belongs to the flavonol subgroup of flavonoids and is found in several fruits and vegetables. Fisetin has high medicinal value and is useful natural agent against cancer and evaluated for its potential inhibitory role against cancer on cells and animal models. To efficiently diversify the therapeutic uses of fisetin, Escherichia coli was harnessed as a production factory and E. coli BL21(DE3)/ΔpgiΔzwfΔgalU mutant was engineered by overexpressing thymidine diphosphate (dTDP)-D-glucose synthase (tgs), dTDP-D-glucose 4,6-dehydratase (dh), and a sugar aminotransferase (wecE) from different sources to produce a pool of dTDP-4-amino-4,6-dideoxy-D-gal actose in the cell cytosol. To this recombinant mutant, two Arabidopsis thaliana glycosyltransferases (ArGT-3 and ArGT-4) were overexpressed to generate two glycosylation platforms (E. coli BL21(DE3)/ΔpgiΔzwfΔgalUTDW-3 and E. coli BL21(DE3)/Δ pgiΔzwfΔgalUTDW-4), which were accessed for the glycosylation of fisetin. As a result, one of the two systems, E. coli BL21(DE3)/ΔpgiΔzwfΔgalUTDW-3, was able to conjugate 4-amino-4,6-dideoxy-D-galactose sugar at the 3-OH position of fisetin, producing an unnatural fisetin 3-O-4-amino-4,6-dideoxy-D-galactoside.