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 Unnatural Flavonoid Glyconjugates in Escherichia coli by Expressing Arabidopsis thaliana Glycosyltransferase

Ramesh Prasad Pandey,Hei Chan Lee,Jae Kyung Sohng 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1

Glycosylation of small molecule based therapeutics and natural products influences biological activities by altering molecular and cellular specificities. E. coli has been engineered by expressing unnatural TDP-4-amino-4, 6-dideoxy-D-galactose biosynthetic pathway genes to produce pools of activated TDP-aminodeoxysugar, and glycosyltransferase gene from A. thaliana. Quercetin and Kaempferol Deoxyaminosugar conjugates were produced by whole cell biocatalysis and the products were analysed by TLC and Proton NMR. This strategy of in vivo glycosylation offers vast combinatorial biosynthesis potential to produce glycorandomized natural products of pharmaceutical importance by simple fermentation.

Biosynthesis of quercetin glycosides in Escherichia coli by whole cell biotransformation using Arabidopsis thaliana Glycosyltransferase

Ramesh Prasad Pandey,Hei Chan Lee,Jae Kyung Sohng 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

Diverse Epothilone A Glycosides : NoveI Anticancer Compounds

Ramesh Prasad Pandey,Prakash Parajuli,Jae Kyung Sohng 한국당과학회 2014 한국당과학회 학술대회 Vol.2014 No.1

In vivo glycorandomization in E. coli for natural product diversification

Ramesh Prasad Pandey,Dinesh Simkhada,Jae Kyung Sohng 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1

Stabilization, detoxification, and solubilization of small molecules based therapeutics and natural products can be altered by glycosylation. In vitro glycosylation of small molecules is difficult to scale up as well as costly to implement in industrial level. To overcome these hurdles, Escherichia coli has been engineered by heterologous overexpression of TDP-4-amino-4, 6-dideoxy-D-galactose biosynthetic gene clusters, and glycosyltransferase gene to produce a range of small molecule glycosides. By applying this metabolic engineering approach, flavonoids, the polyphenolic secondary plant metabolites- Quercetin and Kaempferol were glycosylated to produce Quercetin glycoside and Kaempferol glycoside by whole cell biotransformation. This strategy of in vivo glycosylation offers vast combinatorial biosynthesis potential to produce glycosylated natural products by simple fermentation. Key Words: glycosylation, heterologous overexpression, biotransformation, combinatorial biosynthesis potential.

Probing 3-Hydroxyflavone for in vitro Glycorandomization of Flavonols

Ramesh Prasad PANDEY,Prakash PARAJULI,Niranjan KOIRALA,Rit Bahadur GURUNG,Joo-Ho LEE,Hye Jin JUNG,Jae Kyung SOHNG 한국생물공학회 2014 한국생물공학회 학술대회 Vol.2014 No.4

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.

Biosynthesis of 3-O-xylosyl quercetin in Escherichia coli

Ramesh Prasad Pandey,Jun-Ho Cho,Jae Kyung Sohng 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

To diversify the therapeutic uses of quercetin, Escherichia coli was exploited as a production factory, by assembly of various bacterial and plant UDP-xylose sugar biosynthetic genes and glycosyltransferase. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were over-expressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21 (DE3)/Δpgi, E. coli BL21 (DE3)/Δzwf, E. coli BL21 (DE3)/ΔpgiΔzwf, and E. coli BL21 (DE3)/ΔpgiΔzwfΔushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase, and galU integrated E. coli BL21 (DE3)/Δpgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. The highest production of 3-O-xylosyl quercetin was achieved with E. coli BL21 (DE3)/ ΔpgiΔzwfΔushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM) representing 54.75 % bioconversion, which was ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5 ml culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98% when the reaction was scaled up in a 3 L fermentor at 36 h.

In vitro and in vivo production of phloretin glucosides by using Bacillus licheniformis glycosyltransferase

Ramesh Prasad Pandey,Le Tai Feng,Mi Kyoung Kim,Jae Kyung Sohng 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

A novel GT1 family glycosyltransferase, YjiC from Bacillus licheniformis ATCC 14580 has been PCR amplified, cloned, and expressed in E. coli BL21 (DE3) expression host. The expression of 396 amino acid long gene generated ~45 kDa N-terminal his-tag protein. The protein has been purified and used for the in vitro glycosylation of phloretin. Moreover, the enzyme was also applied for the in vivo glycosylation of the same phloretin molecule. The in vitro and in vivo study found that YjiC can glycosylate at different positions of hydroxyl groups of phloretin molecule very efficiently with conversion rate of ~98%. Four different products has been identified from the in vitro as well as in vivo reaction as phloretin-2’-glucoside, phloretin-4’-glucoside, and two different diglucosides of phloretin. All the products have been confirmed by TLC, HPLC, LC-MS, and former two products has been further confirmed by NMR analysis.

Glucosylation of Isoflavonoids in Engineered Escherichia coli

Pandey, Ramesh Prasad,Parajuli, Prakash,Koirala, Niranjan,Lee, Joo Ho,Park, Yong Il,Sohng, Jae Kyung Korean Society for Molecular and Cellular Biology 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-${\alpha}$-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 $5^{th}$ hydroxyl group of the A-ring, resulting in the production of genistein 4'-O-${\beta}$-D-glucoside, genistein 7-O-${\beta}$-D-glucoside (genistin), genistein 4',7-O-${\beta}$-D-diglucoside, biochanin A-7-O-${\beta}$-D-glucoside (sissotrin), daidzein 4'-O-${\beta}$-D-glucoside, daidzein 7-O-${\beta}$-D-glucoside (daidzin), daidzein 4', 7-O-${\beta}$-D-diglucoside, and formononetin 7-O-${\beta}$-D-glucoside (ononin). The structures of all the products were elucidated using high performance liquid chromatography-photo diode array and high resolution quadrupole time-of-flight electrospray ionization mass spectrometry (HR QTOF-ESI/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 $BL21(DE3)/{\Delta}pgi{\Delta}zwf{\Delta}ushA$ overexpressing phosphoglucomutase (pgm) and glucose 1-phosphate uridyltransferase (galU), along with YjiC, found more than 60% average conversion of $200{\mu}M$ of supplemented isoflavonoids, without any additional UDP-${\alpha}$-D-glucose added in fermentation medium, which could be very beneficial to large scale industrial production of isoflavonoid glucosides.