Biochemical characterization of a regiospecific flavonoid 3’-O -methyltransferase from orange

Citrus plants have diverse methoxyflavonoids including, chrysoeriol, isosakuranetin, and nobiletin. In plants, O ‑methyltransferases (OMTs) participate in the methylation of a vast array of secondary metabolites, including flavonoids, phenylpropanoids, and alkaloids. To identify functional OMTs involved in the formation of methoxyflavonoids, orange ( Citrus sinensis ) OMT (CsOMT) genes were retrieved from the Citrus Genome Database. The phylogenetic relationships with functional OMTs suggested that three CsOMTs, CsOMT15, CsOMT16, and CsOMT30, are possible candidates for flavonoid OMTs (FOMTs). These CsOMTs were heterologously expressed in Escherichia coli , and their OMT activity was examined with flavonoid substrates. Of the examined CsOMTs, CsOMT16 catalyzed the regiospecific 3’‑ O ‑methylation of flavonoids to the respective 3’‑methoxyflavonoids. A kinetic study demonstrated that CsOMT16 accepts diverse flavonoids as a substrate with a comparable preference. The flavonoids eriodictyol, luteolin, and quercetin were efficiently converted to homoeriodictyol, chrysoeriol, and isorhamnetin by CsOMT16‑ transformed E. coli cells, respectively. These findings suggest that CsOMT16 contributes to the methoxyflavonoid formation in orange and is applicable to the biotechnological production of 3’‑methoxyflavonoids.


Introduction
Flavonoids are a structurally diverse class of plant secondary metabolites, which are known to have healthbeneficial activities [1][2][3][4][5].The structural diversity of flavonoids comes from the different oxidation statuses of the carbon skeletons and substitutions in their hydroxyl groups.Flavonoids are divided into several subclasses, such as flavanones, flavones, and flavonols, based on the oxidation status of the middle ring [6].Flavonoid backbones can be substituted by a variety of functional groups, including methyl, isoprenyl, and glycosyl moieties [7].The hydroxyl groups of flavonoids are open-modified with one or more methyl groups [7][8][9].O-Methylation affects the physiochemical properties and biological activities of flavonoids [7,[10][11][12].
In plants, O-methyltransferases (OMTs) are responsible for the methylation of a wide array of secondary metabolites.OMT transfers a methyl group from S-adenosyl L-methionine (SAM) to the hydroxyl group of an acceptor molecule [8,9].Two classes of SAM-dependent OMTs are involved in the methylation of phenolic compounds.Class I OMT methylates hydroxycinnamoyl-CoAs and is involved in the formation of monolignols [9].Class II OMT participates in the methylation of an array of phenolic compounds, including flavonoids and isoflavonoids [8,9,13].Flavonoid OMTs (FOMTs) accept diverse flavonoid substrates, whereas they typically transfer a methyl group to a specific position of flavonoids [8,[14][15][16].Due to their broad substrate selectivity and strict regiospecificity, FOMTs are considered a useful tool for the tailored production of methoxyflavonoids [17,18].
Citrus plants are a rich source of methoxyflavonoids.Citrus fruit and juices are reported to have diverse methoxyflavonoids, such as hesperetin, isosakuranetin, chrysoeriol, and nobiletin, and their respective glycosides [19][20][21].To identify functional FOMTs, orange (C.sinensis) OMT genes (CsOMTs) were retrieved from the Citrus Genome Database.Based on the phylogenetic relationships with functional OMTs, three CsOMTs were selected as FOMT candidates and were heterologously expressed in E. coli as a His-tag fusion protein.The OMT activity and kinetic properties of the recombinant CsOMT proteins toward flavonoids were examined in the present study.Biotransformation of flavonoids using the E. coli transformants harboring CsOMT was also performed for the production of methoxyflavonoids.
First-strand cDNA was synthesized from the total RNA extracted from orange peel by the procedures described previously [16].The CsOMT16 (accession number; OR786463) gene was amplified by polymerase chain reaction (PCR) from the first-strand cDNA.The primers used were 5′-GGCAT ATG GGT TCA ACC AGT TCA GAA -3′ and 5′-CCGGA TCC TCA AGC ACT CTT GAG AAA TTCC-3′ (NdeI and BamHI sites are underlined, respectively).The PCR product was cloned into the pJET1.2/bluntvector.After sequence confirmation, the CsOMT16 gene was inserted into the pET-28a vector.Even though it was attempted several times, the CsOMT15 and CsOMT30 genes could not amplify from the first-strand cDNA of orange.Therefore, the synthetic CsOMT15 and CsOMT30 genes individually cloned in the pET-28b vector were obtained from Bionics (Seoul, Korea).For the heterologous expression of CsOMTs, the resulting constructs were individually transformed into E. coli BL21 (DE3) and Rosetta2 (DE3) cells.

Expression and purification of recombinant CsOMTs
The E. coli transformants harboring each CsOMT/ pET-28 construct were inoculated into LB medium supplemented with kanamycin (25 µg/mL) and grown at 37 °C until the cell population reached an OD 600 of ~ 0.8.Different concentrations of IPTG were added to the cultures, and the cells were then grown at various temperatures to induce recombinant CsOMT expression.After overnight induction, the cells were harvested by centrifugation and stored in a freezer until use.The cells were resuspended in phosphate-buffered saline (10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , 137 mM NaCl, and 2.7 mM KCl) supplemented with lysozyme (1 mg/mL) and PMSF (1 mM), and the resulting cell suspensions were then sonicated to break cells.After the removal of cell debris, the crude protein extract was applied to a Ni-NTA agarose column.The recombinant CsOMT proteins were eluted with different concentrations of imidazole in 50 mM Tris-HCl buffer (pH 8.0, 300 mM NaCl).Protein purification was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) using a 12% polyacrylamide gel.

FOMT assay and kinetic analysis
FOMT reactions of recombinant CsOMTs were carried out in 20 mM Tris-HCl buffer (pH 7.5) containing 50 µM flavonoid substrate and 100 µM SAM.The reaction was initiated by the individual addition of recombinant CsOMT protein.After incubation of the reaction mixtures at 30 °C for 30 min, a one-tenth volume of 5 N HCl was added to stop the reaction.The reaction mixtures were extracted twice with ethyl acetate, and the resulting extracts were then dried in vacuo.The residues were redissolved in dimethyl sulfoxide and analyzed by reversed-phase HPLC equipped with a Sunfire C 18 column (Waters, Milford, MA, USA) using a linear gradient of 25-60% acetonitrile in 3% acetic acid-water for 25 min with detection at 280 nm.Liquid chromatography-mass spectrometry (LC-MS) analysis of CsOMT reaction mixtures and authentic 3'-methoxyflavonoids was carried out with an Agilent 6410 Triple Quadrupole LC-MS System (Agilent Technologies, Santa Clara, CA) to identify the reaction products.Mass spectra were obtained with the ion source of negative-mode electrospray ionization.
Kinetic analysis of CsOMT reactions was performed with different concentrations of flavonoids.Eriodictyol and luteolin concentrations for kinetic analysis were 0.5-10 µM.The concentrations of quercetin and rhamnetin were 0.5-50 µM.

Biotransformation of flavonoids using CsOMT16-transformed E. coli
The E. coli cells bearing the CsOMT16/pET-28a construct were grown in LB medium supplemented with kanamycin (25 µg/mL) by the above-described method.The E. coli culture was added with 0.1 mM IPTG and followed by 4 h incubation at 25 °C to induce CsOMT16 expression.After the induction, the culture was centrifuged, and the medium was discarded.The cell pellet was resuspended in the same volume of fresh LB medium supplemented with kanamycin (25 µg/mL).Each flavonoid substrate was added to the cell suspension at a concentration of 50 μM and further incubated at 25 °C.An aliquot of the culture was harvested at the selected time points and centrifuged to remove the cells.Each cell-free medium was extracted twice with ethyl acetate.The resulting extracts were dried in vacuo.The residues were individually redissolved in a small volume of dimethyl sulfoxide and analyzed by reversed-phase HPLC using the method described above.

Phylogenetic analysis and molecular characterization of CsOMTs
Plant genomes contain several dozens of class II OMT genes, of which a few encode biochemically functional OMTs [14,26,27].More than a hundred CsOMTs were retrieved from the Citrus Genome Database (https:// www.citru sgeno medb.org/) by searching for the gene ontology term O-methyltransferase activity.Class II OMTs are known to have an O-methyltransferase domain (InterPro ID: IPR001077) and a dimerisation domain of plant methyltransferase (InterPro ID: IPR012967) [28][29][30].A domain search of CsOMTs using the InterPro search tool (https:// www.ebi.ac.uk/ inter pro/) revealed that 32 members harbor both O-methyltransferase and dimerisation domains, designating them as CsOMT1-CsOMT32 [31].

Heterologous expression and purification of CsOMTs
The cDNAs of CsOMT15, CsOMT16, and CsOMT30 were cloned into either the pET-28a or pET-28b vectors.The resulting constructs were individually transformed into E. coli BL21 and Rosetta2 cells for heterologous expression of CsOMT15, CsOMT16, and CsOMT30 as a His-tag fusion protein.CsOMT16 and CsOMT30 were successfully expressed in E. coli BL21 cells at 25 °C of growth temperature by 0.1 mM and 0.5 mM IPTG, respectively.Although the expression was attempted under various induction conditions, CsOMT15 was not expressed as a soluble form in both E. coli BL21 and Rosetta2 cells.Recombinant CsOMT16 and CsOMT30 proteins were purified by Ni 2+ affinity chromatography to examine their biochemical properties (Fig. 3).An SDS-PAGE analysis showed that the molecular sizes of the purified CsOMTs were consistent with the theoretical molecular masses of the His-tagged CsOMT16 and CsOMT30 proteins (Fig. 3).

FOMT activity and regiospecificity of CsOMTs
To identify biochemically functional CsOMTs, SAMdependent FOMT activity of recombinant CsOMT16 and CsOMT30 proteins was examined with various subclasses of flavonoid substrates, including flavanones, flavones, and flavonols (Fig. 4).An HPLC analysis of the CsOMT reactions showed that CsOMT16 consumed Fig. 1 Phylogenetic relationships between the selected CsOMTs and other functional OMTs.The evolutionary history of CsOMTs and other OMTs was analyzed using the Neighbor-joining method.The scale bar represents the evolutionary distances computed using the JTT matrix-based method.A phylogenetic tree was drawn in MEGA X (https:// www.megas oftwa re.net/).The OMT sequences used to draw phylogenetic tree were CaFOMT  diverse flavonoids, such as eriodictyol, luteolin, and quercetin, and yielded the reaction products (Fig. 5).
As expected, the products of the CsOMT16 reactions showed 14 daltons higher mass than their substrates in the mass spectra, indicating that CsOMT16 catalyzes the SAM-dependent methylation of flavonoids (Additional file 1: Fig. S2).Unlike the CsOMT16 reactions, no product peak was detected in the CsOMT30 reactions with flavonoid substrates.This result indicates that CsOMT16 is a biochemically functional FOMT and CsOMT30 has no FOMT activity.CsOMT16 accepted diverse substrates of different flavonoid backbones, although its FOMT activity varied considerably depending on flavonoids (Table 2).This property is well consistent with the broad substrate specificity of other FOMTs [14-18, 26, 27].It has been known that FOMTs catalyze the regiospecific O-methylation of flavonoids [14][15][16][17][18]27]. CsOMT16 used most flavonoids examined as a substrate.However, no product was detected in CsOMT16 reactions with naringenin, apigenin, and Fig. 2 The conserved signature motifs of class II OMT in CsOMTs, SlOMT3, AtCOMT, and ObFOMT5.The amino acid sequences of CsOMTs were aligned with those of class II OMT SlOMT3, AtCOMT, and ObFOMT5 using Clustal-W.The SAM binding motifs and the COMT motifs were indicated under the aligned sequences with their consensus sequences (X indicates any amino acid).Red triangles indicate the catalytic residues of class II OMT.Identical and similar amino acids are shaded in black and grey, respectively kaempferol (Table 2).These flavonoids commonly have no 3'-OH in their backbones (Fig. 4), suggesting that CsOMT16 requires an OH group at the 3'-position.Indeed, the reaction products from eriodictyol, luteolin, and quercetin showed identical retention times with the authentic 3'-methoxyflavonoids, homoeriodictyol, chrysoeriol, and isorhamnetin, respectively (Fig. 5).A LC-MS analysis also showed that the fragmentation pattern of the products of the CsOMT16 reactions was almost identical with that of the corresponding 3'-methoxyflavonoids (Additional file 1: Fig. S2), indicating that CsOMT16 Even though there are a few exceptions, flavonoid 3′-OMTs commonly catalyze methylation at the 3′and 5′-positions of substrates because the 3′-and 5′-positions of flavonoids are chemically equivalent [14,38,39].Similarly, CsOMT16 catalyzed both 3'and 5'-O-methylation of flavonoids, which contain 3'and 5'-OH groups.Myricetin and tricetin harboring 3'-and 5'-OH groups were used by CsOMT16 and yielded the dimethylated products (Table 2).A LC-MS analysis of the CsOMT16 reaction with myricetin and laricitrin (3'-methylmyricetin) revealed that the fragmentation pattern of the methylated products of these substrates was almost identical with that of syringetin (3' ,5'-dimethylmyricetin), indicating that CsOMT16 can catalyze an additional 5'-O-methylation of flavonoids when the 5'-OH is available.These results demonstrate that CsOMT16 is a FOMT catalyzing the 3'-O-methylation of flavonoids and likely contributes to orange having diverse methoxyflavonoids.

Substrate preferences of CsOMT16
Like other FOMTs, CsOMT16 methylates diverse flavonoids to different extents.Under saturated conditions of substrates, CsOMT16 showed the strongest OMT activity toward quercetin among the examined flavonoids (Table 2).In the same condition, the activity of CsOMT16 for rhamnetin was about 15-fold lower than that for quercetin.CsOMT16 exhibited moderate OMT activity for eriodictyol and luteolin relative to those for quercetin and rhamnetin (Table 2).
To further elucidate the biochemical properties of CsOMT16, the kinetic parameters of recombinant CsOMT16 toward the selected flavonoid substrates, eriodictyol, luteolin, quercetin, and rhamnetin, were determined.In agreement with its relative activity, CsOMT16 has the highest V max and k cat values of 89.33 pmol min −1 ug −1 and 3.76 min −1 for quercetin, respectively (Table 3).The V max value for rhamnetin was the lowest among those for the examined substrates.The V max values of CsOMT16 for eriodictyol and luteolin were 32.25 pmol min −1 ug −1 and 60.62 pmol min −1 ug −1 , respectively.CsOMT16 showed comparable binding affinity to eriodictyol, luteolin, and quercetin, with K m values of 2.62, 1.17, and 4.52 µM, respectively (Table 3).CsOMT16 had the lowest K m value of 0.25 µM for rhamnetin.Although its V max and K m values were considerably different, CsOMT16 exhibited comparable k cat /K m values to the flavonoids examined, ranging from 8.66 × 10 3 to 3.65 × 10 4 M −1 s −1 (Table 3).The kinetic analysis demonstrates that CsOMT16 accepts a broad range of flavonoid substrates with a minor difference in catalytic activity.
To evaluate the application of CsOMT16 in the biotechnological production of 3'-methoxyflavonoids,  After the induction of CsOMT16, the flavonoids (50 μM) were individually added to the culture, and then the production of the respective 3'-methoxyflavonoids was monitored.Eriodictyol was almost consumed by the E. coli cells within 2 h and was converted to an equivalent amount of homoeriodictyol (Fig. 6a).In parallel with the substrate preference of CsOMT16, the bioconversion of luteolin and quercetin by the CsOMT16-transformed E. coli was faster than that of eriodictyol.The bioconversion yield of eriodictyol homoeriodictyol in the first 30 min of biotransformation was 46.8%, and that of luteolin and quercetin was 56.3 and 56.6%, respectively.The production of the respective 3'-O-methylated products from luteolin and quercetin was reached at a maximum level after 1 h of biotransformation and was then declined (Fig. 6b, c).The maximum biotransformation yield of chrysoeriol and isorhamnetin from luteolin and quercetin was 90.5 and 71.3%, respectively, at the substrate concentration of 50 μM.Methoxyflavonoids have valuable biological activities and have thus been considered functional agents in the food and pharmaceutical industries [11,12,[45][46][47][48]. 3'-Methoxyflavonoids such as homoeriodictyol, chrysoeriol, and isorhamnetin have been reported to have anti-cancer, anti-inflammatory, and anti-oxidation activities [4,5,46,49,50].Homoeriodictyol has bitter masking effects and is thus applicable in food and pharmaceuticals as a taste modifier [51].Homoeriodictyol and chrysoeriol exhibit anti-microbial activities against bacteria and fungi [5,45].For the application of FOMTs in the biotechnological production of valuable methoxyflavonoids, their biochemical characteristics, including substrate preferences, regiospecificity, and kinetic properties, need to be elucidated.In this study, CsOMT16 was isolated from orange and identified as a flavonoid 3'-OMT.Biotransformation using E. coli cells harboring CsOMT16 also showed the successful production of homoeriodictyol, chrysoeriol, and isorhamnetin from eriodictyol, luteolin, and quercetin, respectively.These results indicate that whole-cell microorganisms bearing CsOMT16 are applicable for the biotechnological production of 3'-methoxyflavonoids.

Table 2
Substrate preference of recombinant expressed in E. coli a The OMT activity toward flavonoids respective to that toward quercetin at a substrate concentration of 50 µM b Minus signs indicate that no product was detected in the HPLC analysis c Plus signs represent that the flavonoids tested were used as a substrate that yielded a product peak in the HPLC analysis.The relative activities of the substrates were not determined

Table 3
Kinetic parameters of recombinant CsOMT16 a