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-vivo Glycosylation of genistein in E. coli using GmIF7GT from Glycine max

Niranjan Koirala,Ramesh Prasad Pandey,Na-Rae Lee,Jae Kyung Sohng 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

Isoflavonoids, polyphenols are biologically active natural products which are abundantly present in soyabean seeds during development. Clinical studies have suggested positive effects of isoflavonoids in human health and nutrition, such as reduction in the risks of hormonally dependent cancers, menopausal symptoms, osteoporosis, and cardiovascular disease. However, the insolubility and instability of those polyphenols prevent the wide applications of these medicinally important compounds. Therefore, the generation of isoflavonoid derivatives by glycosylation gained much attention and interest. In this study, we used E.coli BL21 (DE3) as an expression host for gmIF7Gt, a Glycine max derived glycosyl transferase, cell biocatalyst for the production of glycosylated derivatives of genistein. The supplementation of 0.2mM of genistein in the growing induced culture of E. coli BL21 (DE3) harboring PET15b-GmIF7GT resulted novel spots in TLC. These spots were further analyzed by HPLC and LC-TOF ESI/MS. The exact molecular mass analysis confirmed the production of Genistein glucoside. Further, structural elucidation and product enhancement is necessary for large scale production of this compound. However, this study reveals a method that might be useful for the biosynthesis of glycosylated isoflavonoids and related compounds by in-vivo glycosylation.

Tuning the Production of Malonylated-Glycosylated-Isoflavonoids in Engineered E. coli

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

Towards the Production of Rhamnetin in Escherichia coli

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

Generation of Novel Compound by Enzymatic Modification of 7,8-Dihydroxyflavone by Methylation and Glycosylation

Niranjan Koirala,Ramesh Prasad Pandey,Jae Kyung Sohng 한국당과학회 2013 한국당과학회 학술대회 Vol.2013 No.1

Methylation and glycosylation are two different but vital modification processes in plants to generate diversified secondary metabolites. Methylation increases the lipophilicity of compounds whereas glycosylation enhances the solubility. Most of the methylated and glycosylated natural compounds are significantly active against pathogens and cancers. In this study, an O-methyltransferase gene, designated as SpOMT-2884, was identified from Streptomyces peucetius ATCC 27952. To find the exact substrates for SpOMT-2884, we tested several compounds in-vitro. As a result SpOMT-2884 catalyzed O-methylation of flavonoids such a 7,8-dihydroxyflavone (7,8-DHF), quercetin, luteolin, fisetin and rutin. 7,8-DHF was found to be the best substrate. We further proceeded for in vivo biotransformation of 7,8-DHF where we used E. coli BL21 (DE3) expressing SpOMT-2884 cell, as a biocatalyst for the production of methylated derivative of 7,8-DHF. The supplementation of 0.2 mM of 7, 8-DHF in the growing induced culture of E.coli BL21 (DE3) harboring pET28-SpOMT-2884 recombinant resulted in the production of 7-methyl-8-hydroxyflavone which was confirmed by HPLC (Rt: 17 min), high resolution LC-QTOF-ESI/MS (m/z+ 269.08) and NMR spectroscopy. Further, this enzymatically synthesized methylated derivative of 7, 8-DHF was used as a substrate in vitro for glycosylation by Yjic, a glycosyltransferase from Bacillus licheniformis DSM13. This in-vitro reaction mixture analysis revealed the presence of glycosylated product which was confirmed by HPLC and LC-QTOF-ESI/MS (m/z+ 431.13). The glycosylation of the target was further supported by our findings from in silico docking analysis. Inparticular, this study demonstrated the potential for enzymatic biosynthesis of novel methylated cum glycosylated 7,8-DHF.

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

Production of novel derivative of 7,8-dihydroxyf1avone in E. coli and in vitro glycosylation of the metabolite

Niranjan Koirala,Ramesh Prasad Pandey,Jae Kyung Sohng 한국당과학회 2014 한국당과학회 학술대회 Vol.2014 No.1

Enzymatic Modification of 7,8-Dihydroxyflavone by Methylation and Glycosylation

Niranjan Koirala,Ramesh Prasad Pandey,Jae Kyung Sohng 한국당과학회 2013 한국당과학회 학술대회 Vol.2013 No.1

Methylation and glycosylation are two different but vital modification processes in plants to generate diversified secondary metabolites. Methylation increases the lipophilicity of compounds whereas glycosylation enhances the solubility. Most of the methylated and prenylated natural compounds are significantly active against cancers. 7, 8-dihydroxyflavone is a member of a flavonoid family of chemicals, which are abundant in fruits and vegetables. The compound’s selective effects suggest that it could be the founder of a new class of brain-protecting drugs as it can cross the blood-brain barrier by triggering tyrosine kinase B (TrkB) proving it could be a powerful anti-oxidant and neuroprotectant drug. In this study, we used E. coli BL21 (DE3) expressing SPOMT 2884, a Streptomyces peucetius ATCC 27952 derived O-methyltransferase, cell as a biocatalyst for the production of methylated derivative of 7, 8-Dihydroxy flavone. The supplementation of 0.2 mM of 7, 8-dihydroxyflavone in the growing induced culture of E. coli BL21 (DE3) harboring pET28-SPOMT2884 recombinant resulted in the production of a mono methylated compound which was confirmed by HPLC (Rt:17 min) and high resolution LC-QTOF-ESI/MS (m/z+ 269.08). Further, this enzymatically synthesized methylated derivative of 7, 8-DHF was purified in large amount and used as a substrate for in-vitro glycosylation by Yjic, a glycosyltransferase from Bacillus licheniformis DSM13. This reaction mixture analysis revealed the presence of glycosylated product which was analyzed by TLC and HPLC. Further structural elucidation is necessary for the confirmation of methylation and glycosylation positions. However, this study reveals a method that might be useful for the enzymatic biosynthesis of the methylated compound and subsequent modification of the same by glycosylation.

Glycosylation and subsequent malonylation of isoflavonoids in E. coli: strain development, production and insights into future metabolic perspectives.

Koirala, Niranjan,Pandey, Ramesh Prasad,Thang, Duong Van,Jung, Hye Jin,Sohng, Jae Kyung Published by Stockton Press on behalf of the Socie 2014 Journal of industrial microbiology & biotechnology Vol.41 No.11

<P>Genistin and daidzein exhibit a protective effect on DNA damage and inhibit cell proliferation. Glycosylation and malonylation of the compounds increase water solubility and stability. Constructed pET15b-GmIF7GT and pET28a-GmIF7MAT were used for the transformation of Escherichia coli and bioconversion of genistein and daidzein. To increase the availability of malonyl-CoA, a critical precursor of GmIF7MAT, genes for the acyl-CoA carboxylase α and β subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) from Nocardia farcinia were also introduced. Thus, the isoflavonoids were glycosylated at position 7 by 7-O-glycosyltranferase and were further malonylated at position 6() of glucose by malonyl-CoA: isoflavone 7-O-glucoside-6()-O-malonyltransferase both from Glycine max. Engineered E. coli produced 175.7 ?M (75.90 mg/L) of genistin and 14.2 ?M (7.37 mg/L) genistin 6''-O-malonate. Similar conditions produced 162.2 ?M (67.65 mg/L) daidzin and 12.4 ?M (6.23 mg/L) daidzin 6''-O-malonate when 200 ?M of each substrate was supplemented in the culture. Based on our findings, we speculate that isoflavonoids and their glycosides may prove useful as anticancer drugs with added advantage of increased solubility, stability and bioavailability.</P>

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.