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

Metabolic Engineering of E. coli for Regiospecific Modifications of Naringenin to produce Astragalin

Ramesh Prasad Pandey,Sailesh Malla,Byung-Gee Kim,Kyung Sohng 한국당과학회 2013 한국당과학회 학술대회 Vol.2013 No.1

We successfully produced astragalin (AST) from regiospecific modifications of naringenin (NRN) in Escherichia coli BL21(DE3). The exogenously supplied NRN was converted into dihydrokaempferol (DHK) and then kaempferol (KMF) in the presence of flavanone-3-hydroxylase (f3h) and flavonone synthase (fls1) from Arabidopsis thaliana, respectively. KMF was further modified to produce AST by 3-O-glucosylation utilizing the endogeneous UDP-glucose in presence of UGT78K1 from Glycine max. To increase the intracellular UDP-glucose concentration by channeling the carbon flux toward UDP-glucose at the branch point of glucose-6-phosphate (G6P), the chromosomal glucose phosphate isomerase (pgi) and D-glucose-6-phosphate dehydrogenase (zwf) were knocked-out in E. coli BL21(DE3). The two enzymes directly involved in the synthesis of UDP-glucose from G6P, phosphoglucomutase (nfa44530) from Nocardia farcinia and glucose-1-phosphate uridylyltransferase (galU) from E. coli K12 were overexpressed, which successfully diverted the carbon flow from glycolysis to the synthesis of UDP-glucose. Furthermore, to prevent the dissociation of UDP-glucose into UDP and glucose, the UDP-glucose hydrolase (ushA) was deleted. The E. coliΔpgiΔzwfΔushA mutant harboring the UDP-glucose biosynthetic pathway and the aforementioned genes for the regiospecific glucosylation produced 109.3 mg/L (244μM) of AST representing 48.8% conversion from 500 μM of NRN in 60 h without any supplementation of extracellular UDP-glucose.

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.

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.

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.

Glycosylation of small molecules: generation of novel pharmaceutical compounds for future drug use

Ramesh Prasad Pandey,Rit Bahadur Gurung,Prakash Parajuli,Jae Kyung Sohng 한국당과학회 2013 한국당과학회 학술대회 Vol.2013 No.1

The biological activity of many natural products and pharmaceutically used drugs are attributed by the glycosidic residues attached to it. Generally, the glycosylation determines the competence of the most of the molecules as a drug. Therefore, engineering the sugar moiety in the natural products is one of the most efficient ways of generating novel compounds with diverse pharmacological properties. The screening and engineering of flexible glycosyltransferases from different sources and applying those enzymes for the biosynthesis of novel small molecule glycosides is still significantly demanding. In this context, we have identified a novel glycosyltransferase (YjiC) from Bacillus licheniformis and applied to glycosylate small molecule natural products like, flavonoids, isoflavonoids, chalcones, stillbenes, curcuminoids etc. The substrate flexibility of thus identified glycosyltaransferase is found to have broad, which resulted to generate a number of natural product glycosides including novel compounds. The enzyme glycosylates non-regiospecifically at suitable hydroxyl groups to produce O-glucosides of compounds. Moreover, this glycosyltransferase has shown flexibility towards NDP-sugar donors. This property of the enzyme would help to engineer the sugar moiety of most of the small molecules which might exhibit potentially different biological activities. The exchange of sugar moiety might help to modulate the biological application of the parent bioactive compounds. Plenty of biologically active aglycones and their glycoside derivatives are characterized to have diverse biological activities against wide-range of diseases. The production of such pharmaceutically important glycoside compounds and their glyco-engineering to alter biological function would be noteworthy in scientific world to develop a novel drug for future use.

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.

In Vitro Type I Modular Polyketide Synthase Catalysis for New Antibiotics

Ramesh Prasad Pandey,송재경 대한화학회 2018 Bulletin of the Korean Chemical Society Vol.39 No.4

Bioconversion of Tetracycline Antibiotics to Novel Glucoside Derivatives by Single-Vessel Multienzymatic Glycosylation

( Ramesh Prasad Pandey ),( Luan Luong Chu ),( Tae-su Kim ),( Jae Kyung Sohng ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.2

The single-vessel multienzyme UDP-α-D-glucose recycling system was coupled with a forward glucosylation reaction to produce novel glucose moiety-conjugated derivatives of different tetracycline antibiotic analogs. Among five tetracycline analogs used for the reaction, four molecules (chlorotetracycline, doxytetracycline, meclotetracycline, and minotetracycline) were accepted by a glycosyltransferase enzyme, YjiC, from Bacillus licheniformis to produce glucoside derivatives. However, the enzyme was unable to conjugate sugar units to rolitetracycline. All glucosides of tetracycline derivatives were characterized by ultraviolet absorbance maxima, ultra-pressure liquid chromatography coupled with photodiode array, and high-resolution quadruple time-of-flight electrospray mass spectrometry analyses. These synthesized glucosides are novel tetracycline derivatives.