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Title: Metabolic engineering of Eescherichia colibl21 (de3) for the production of methylated/glycosylated flavonoids and their biological activities
Authors: Koirala, Niranjan
Keywords: Metabolic engineering
Biological activity
Issue Date: 9-Nov-2017
Abstract: Flavonoids (polyphenolic natural products) are ubiquitous plant secondary metabolites and have been recognized to be potent pharmaceutical agents by several research groups. Hydroxyl groups on flavonoids are both important for the antioxidizing capacity and key points for further modification resulting in O -methylation, -glycosylation, - sulfation, or -acylation. Methylation of the flavonoids via theirs free hydroxyl gro ups or carbon atom dramatically increases their metabolic stability and enhance s the membrane transport, leading to facilitated absorption and greatly increased oral bioavailability. Glycosylation usually improves the solubility, absorption, dis tribution, metabolism, and excretion (SAD ME properties) of the drugs. The effects of O -glycosylation and acylation have been most explored in vitro concerning antioxidizing properties as well as stability and solubility. After knowing the thorough insights in glycosylation and methylation, we also knew that individually each modifications were having some demerits. Only methylations will increase the metabolic stability and biological activities but the drugs solubility will decrease due to liphophilic (methyl) group attached to it. Similarly only glycosylation will just increase the solubility without having a remarkable biological activity enhancement to the original compounds (not in all cases) or sometimes decreasing the original activity as well. This led us to hypothesize the combined effects of methylation and subsequent glycosylation of flavonoids. Firstly we hypothesized that the methylation of these flavonoids will significantly increase their metabolic stability and biological activities, and then secondly, theirs subsequently glycosylated products will have enhanced solubility and better drug transport capability, making it more significant for formulation of pharmaceutical applications. To serve this purpose, genetically engineered Escherichia coli was reconstructed by harboring E. coli K12-derived S -adenosylmethionine (SAM) synthase ( metK; accession number: K02129) for enhancement of SAM as precursor and a Streptomyces avermitilis -originated O -methyltransferase ( SaOMT2 ; accession number: NP_823558) for methylation of naringenin, apigenin, quercetin and isoflavonoids (genistein and daidzein) as preferred substrates. The formation of desired products via biotransformation including sakuranetin, genkwanin, rhamnetin, 7-O-methyl-genistein and 7-O-methyl-daidzein were confirmed individually by chromatography data such as HPLC, LC-TOF-MS and NMR ( H and C) as well. Furthermore, substrates 1 13 concentration, culture time and media parameters were optimized using fed batch fermentor scaled up to 3 L to obtain the maximum yield of the products. Combination of optimum culture conditions, the maximum yield of sakuranetin, genkwanin, rhamnetin, 7- O -methyl genistein and 7- O -methyl daidzein was 194 µM, 170 µM, 196 µM, 164 µM 382 µM when 200 µM of naringenin, apigenin, quercetin and genistein and 400 µM of daidzein were supplemented in the separate cultures. Furthermore, sakuranetin was purified in large scale and used as a substrate for in vitro glycosylation by YjiC to produce glucose and galactose derivatives of sakuranetin. Similarly purified rhamnetin was used for the in vivo rhamnosylation and xylosylation in engineered E. a a coli to produce rhamnetin-3- O - -L-rhamnoside and rhamnetin-3- O - -L-xyloside. Alongsides in this present study, an O -methyltransferase SpOMT2884 , originating from Streptomyces peucetius ATCC 27952, was cloned, expressed, and applied for the production of target metabolite from Escherichia coli . SpOMT2884 catalyzed O - methylation of different classes of flavonoids such as flavones (7,8-dihydroxyflavone (7,8-DHF), luteolin), flavonols (quercetin, rutin), flavanone (naringenin), and isoflavonoids (daidzein, formononetin). Biotransformation of 7,8-DHF, a preferred substrate of SpOMT2884, in a grown-induced culture of E. coli BL21 (DE3) harboring the recombinant pET-28a-SpOMT2884 stoichiometically converted 7,8-DHF into 7- hydroxy-8-methoxyflavone, which was confirmed by liquid chromatography, mass spectrometry and various nuclear magnetic resonance (NMR) spectroscopy analyses. The maximum yield of 7-hydroxy-8-methoxyflavone was 192 µM (52.57 mg/L), representing almost 96 % bioconversion within 12 h, when 200 µM of 7,8-DHF was supplemented in the culture during fermentation. Further, the 7-hydroxy-8- methoxyflavone was purified in large scale and was used as a substrate separately for in vitro glycosylation to produce glucose, galactose and 2-deoxyglucose conjugated at 7 hydroxyl position of 7-hydroxy-8-methoxyflavone. Finally, three novel compounds t h viz 7- O-ß- D-galactoxy-8-methoxyflavone, 7- O-ß- D-glucoxy-8-methoxyflavone and 7- O-ß- D-2-deoxyglucoxy-8-methoxyflavone were produced using 7-hydroxy-8- methoxyflavone as a substrate. Biological activity showed that 7-hydroxy-8- methoxyflavone had long term cytoprotective and antioxidant effects compared to 7,8- DHF suggesting methylation enhances the stability of substrate and glycosylation has proved to increase the water solubility. We also carried out glycosylation and subsequent malonylation of isoflavonoids in this research. In order to increase the availability of malonyl-CoA, a critical precursor of a ß malonyltransferases (GmIF7MAT), genes for the acyl-CoA carboxylase and subunits ( nfa 9890 and nfa 9940), biotin ligase ( nfa 9950), and acetyl-CoA synthetase ( nfa 3550) 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. Engineered E. coli produced 175.7 µM (75.90 mg/L) of genistin and 14.2 µM (7.37 mg/L) genistin 6''- O -malonate. Similar conditions produced 162.2 µM (67.65 mg/L) daidzin and 12.4 µM (6.23 mg/L) daidzin 6''- O -malonate when 200 µM 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. In the present study, we have carried out extensive preliminary biological activity assays to all the compounds produced in our study. To uncover the pharmaceutical significances of the produced compounds, we investigated the inhibitory effect of sakuranetin, genkwanin and rhamnetin on the growth of several cancer cell lines that have not been previously reported. Sakuranetin exhibited a stronger inhibitory activity than naringenin on the growth of cancer (AGS, B16F10, and U87MG) cells, demonstrating that sakuranetin may possess greater potential as an anti-cancer agent than naringenin. Furthermore, our data reports for the first time on the anti-melanogenic property of sakuranetin. Therefore, sakuranetin could be utilized as a new therapeutic agent with anti-carcinogenic as well as anti-melanogenic properties. We also demonstrated that, in comparison with quercetin, rhamnetin has better suppressive ability on cancer cell growth as well as melanogenesis. Our results support that rhamnetin can be considered as an attractive therapeutic agent with improved pharmacological property in both cancer therapy and skin medication. We also evaluated the pharmaceutical potential of genkwanin as a chemotherapeutic agent for cancer treatment. Genkwanin showed a more increased inhibition activity, compared to apigenin, on the growth of cancer cells (B16F10, HepG2, and U87MG) as well as angiogenesis of HUVECs stimulated by VEGF. These results indicate that genkwanin may have more enhanced potential as an anti-cancer and anti-angiogenic agent than apigenin. Notably, our report on the inhibitory potential of genkwanin on angiogenesis suggests that the compound can be developed as a new angiogenesis inhibitor for the treatment of angiogenesis-associated diseases. Furthermore, potent inhibitory activities of the isoflavonoid methoxides against the growth of cancer line (B16F10, AGS and HepG2) and endothelical (HUVEC) cells were investigated and demonstrated. 7- O -methyl genistein and 7- O -methyl daidzein significantly inhibited TNF- -induced invasion of HUVECs at 20 µM, a concentration at which no cytotoxicity was observed. Notably, 7- O -methyl daidzein the most effectively suppressed the invasion of HUVECs induced by TNF- amongst four compounds tested. Taken together this research work described the metabolic engineering and biotransformation processes for the optimal production of methylated and glycosylated flavonoids and isoflavonoids. Improvement of production, purification of products, and characterization of produced compounds and preliminary in vitro biological activities of these derivatives being manufactured were studied in detail. Key words : Metabolic engineering, Biotransformation, Flavonoids, Methylation, Glycosylation, Spectrometry, Fermentation, Biological activity. v
Description: Dissertation submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in the Department of Life Sciences and Biochemical Engineering, Sun Moon University, 2015.
Appears in Collections:500 Natural sciences and mathematics

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