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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.
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.
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>
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.
Comparative Study of the Antioxidative Potential of Common Natural Flavonoids and Isoflavonoids
라메쉬,Niranjan Koirala,이주호,이희철,송재경 한국미생물·생명공학회 2013 한국미생물·생명공학회지 Vol.41 No.3
2,2'-diphenylpicrylhydrazyl (DPPH•) assay와 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS+) decolorization assay는 자연상태의 플라보노이드와 이소플라보노이드의 항산화 활성을 확인하는데 사용된다. 억제중간값(half maximal inhibitory concentration(IC50) values)과 트롤록스당량 항산화능값(trolox equivalent antioxidant capacity (TEAC) values)은 DPPH• assay와 ABTS+ assay로 계산되었다. DPPH assay 결과, 쿼세틴(quercetin)은 가장 강한 항산화 능력을 가졌고 뒤이어 피세틴(fisetin), 7,8-디하이드록시플라본(7,8-dihydroxyflavone), 모린(morin), 캠퍼롤(kaempferol) 순이었다. 쿼세틴, 피세틴, 7,8-디하이드록시플라본은 부틸하이드록시 아니솔(butyl hydroxyl anisole)보다 더 높은 항산화 능력을 가졌다. 쿼세틴은 플라보노이드와 이소플로보노이드 중에서 TEAC 값이 가장 높았고 뒤이어 3-하이드록시플로본(3-hydroxyflavone), 피세틴, 7,8-디하이드록시플라본과 모린 순이었다. 다른 나머지 플라보노이드와 이소플라보노이드는 트롤록스 보다 더 약한 ABTS+ 분해능력(scavenging potential)을 가졌다. 테스트된 13개 플라보노이드/이소플라보노이드에서 이소플라보노이드는 플라보노이드보다 매우 약한 항산화 능력을 보였다. The half maximal inhibitory concentration (IC50) values and trolox equivalent antioxidant capacity (TEAC) values were calculated by a 2,2'-diphenylpicrylhydrazyl (DPPH)· assay and a 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS+·)assay, in order to determine the antioxidative activities of the compounds. On the basis of the DPPH assay, quercetin had the strongest antioxidative potential of the flavonoids, followed in order by fisetin, 7,8-dihydroxyflavone, morin and kaempferol. Quercetin, fisetin and 7,8-dihydroxyflavone had higher antioxidant potentials than butyl hydroxyl anisole. Quercetin had the highest TEAC value amongst the flavonoids and isoflavonoids, followed in order by 3-hydroxyflavone, fisetin, 7,8-dihydroxyflavone and morin. Comparatively, isoflavonoids were found to have significantly weaker antioxidative potential than the flavonoids.