Journal of Biotechnology 184 (2014) 128–137 Contents lists available at ScienceDirect Journal of Biotechnology j ourna l ho me pa ge: www.elsevier.com/locate/jbiotec Methylation and subsequent glycosylation of 7,8-dihydroxyflavone Niranjan Koirala 1 , Ramesh Prasad Pandey 1 , Prakash Parajuli, Hye Jin Jung, Jae Kyung Sohng ∗ Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, Asansi, Chungnam 336-708, Republic of Korea a r t i c l e i n f o Article history: Received 10 March 2014 Received in revised form 12 May 2014 Accepted 13 May 2014 Available online 22 May 2014 Keywords: Methylation Glycosylation 7,8-Dihydroxyflavone Streptomyces peucetius Cytoprotective a b s t r a c t An O-methyltransferase SpOMT2884, originating from Streptomyces peucetius ATCC 27952, was cloned, expressed, and applied for the production of target metabolite from Escherichia coli. Biochemical charac- terization of the 25 kDa recombinant protein by in vitro and in vivo experiments showed that SpOMT2884 was an S-adenosyl-l-methionine-dependent O-methyltransferase. 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). Biotransforma- tion 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 stoichiometrically 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. In order to improve the biotransformation substrate, time and media parameters were optimized and the production was scaled up using a 3-L fermentor. The maximum yield of 7-hydroxy-8-methoxyflavone was 192 M (52.57 mg/L), representing almost 96% bioconversion within 12 h, when 200 M of 7,8-DHF was supplemented in the culture. Fur- ther, 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 7th hydroxyl position of 7-hydroxy-8-methoxyflavone. Biological activity showed that 7-hydroxy-8-methoxyflavone had long term cytoprotective and antioxidant effects compared to 7,8-DHF suggesting that methylation enhances the stability of substrate and glycosylation has proved to increase the water solubility. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Flavones are widely distributed in celery and parsley, among a number of other vegetables and herbs (Hertog et al., 1993; Peterson and Dwyer, 1998). Since, they are present in many daily consumed foods, vegetables, beverages, and fruits; they are the active ingre- dients of human diet. But, the pharmacological application of these compounds is limited because of the low water solubility and instability. Moreover, the free hydroxyl groups are susceptible to glucoronidation, sulfation and oxidation reactions in the intestine and liver that avoids them to pass intact into the systemic circula- tion (Otake et al., 2002). Methylation of the free hydroxyl groups in the flavones dramatically increases their metabolic stability and enhances the membrane transport, leading to facilitated absorp- tion and greatly increased oral bioavailability (Walle, 2009). Some of the methylated flavones show remarkable inhibitory effects on ∗ Corresponding author. Tel.: +82 41 530 2246; fax: +82 41 544 2919. E-mail address: sohng@sunmoon.ac.kr (J.K. Sohng). 1 These authors are equally contributed to this work. carcinogen activating enzymes (Wen and Walle, 2006), effects on multidrug resistance proteins (MRPs), and fungicidal properties (Bernini et al., 2011). 7,8-Dihydroxyflavone (7,8-DHF) is a flavonoid that is abun- dantly present in fruits and vegetables (Harborne and Williams, 2002). It can cross the blood–brain barrier (Andero et al., 2011) has the potential to act as a mammalian neurotrophin, brain-derived neurotrophic factor (BDNF) ligand for TrkB (Mantilla and Ermilov, 2012), and is capable of inducing angiogenesis (Williams, 2011). 7,8-DHF has been identified as a potent TrkB agonist (Jang et al., 2010). It also prevents aging-induced morphological changes, and plasticity of neurons. In addition, it can raise the cellular glutathione levels, which ultimately reduces the reactive oxygen species (ROS) production caused by glutamate, reducing the chances of neurode- generative diseases induced by ROS (Chen et al., 2011). Findings from Huai group have suggested that neuro-protective 7,8-DHF has also a vasorelaxing and antihypertensive properties (Huai et al., 2014) suggesting its use in treatment of cardiovascular diseases. S-adenosyl-l-methionine (AdoMet) dependent O- methyltransferase (OMT) functions in transferring methyl moiety in the biosynthesis of several natural products (Wils et al., 2013) http://dx.doi.org/10.1016/j.jbiotec.2014.05.005 0168-1656/© 2014 Elsevier B.V. All rights reserved.