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Biotechnological Production of Dimethoxyflavonoids Using a Fusion Flavonoid O‑Methyltransferase Possessing Both 3′- and 7‑O‑Methyltransferase Activities Danbi Lee,†,§ Hye Lin Park,†,§ Sang-Won Lee,†,‡ Seong Hee Bhoo,*,† and Man-Ho Cho*,† †

Graduate School of Biotechnology and ‡Crop Biotech Institute, Kyung Hee University, Yongin 17104, Republic of Korea S Supporting Information *

ABSTRACT: Although they are less abundant in nature, methoxyflavonoids have distinct physicochemical and pharmacological properties compared to common nonmethylated flavonoids. Thus, enzymatic conversion and biotransformation using genetically engineered microorganisms of flavonoids have been attempted for the efficient production of methoxyflavonoids. Because of their regiospecificity, more than two flavonoid O-methyltransferases (FOMTs) and enzyme reactions are required to biosynthesize di(or poly)methoxyflavonoids. For the one-step biotechnological production of bioactive di-O-methylflavonoids, we generated a multifunctional FOMT fusing a 3′-OMT (SlOMT3) and a 7-OMT (OsNOMT). The SlOMT3/OsNOMT fusion enzyme possessed both 3′- and 7-OMT activities to diverse flavonoid substrates, which were comparable to those of individual SlOMT3 and OsNOMT. The SlOMT3/OsNOMT enzyme also showed 3′- and 7-OMT activity for 7- or 3′-O-methylflavonoids, respectively, suggesting that the fusion enzyme can sequentially methylate flavonoids into di-O-methylflavonoids. The biotransformation of the flavonoids quercetin, luteolin, eriodictyol, and taxifolin using SlOMT3/OsNOMT-transformed Escherichia coli generated corresponding di-O-methylflavonoids, rhamnazin, velutin, 3′,7-di-O-methyleriodictyol, and 3′,7-di-Omethyltaxifolin, respectively. These results indicate that dimethoxyflavonoids may be efficiently produced from nonmethylated flavonoid precursors through a one-step biotransformation using the engineered E. coli harboring the SlOMT3/OsNOMT fusion gene.

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activity against bacteria such as Escherichia coli, Staphylococcus aureus, and Bacillus pumilus as well as the fungus Sacchromyces cerevisiae.14 Rhamnazin was also reported to be a potential anticancer drug because it inhibits the expression of vascular endothelial growth factor receptor 2, which is important in angiogenesis and tumor cell growth.15 3′,7-Di-O-methylluteolin (velutin) is a potent anti-inflammatory agent that more strongly inhibits nuclear factor-kappa B activation than luteolin does.16 The polymethoxyflavonoids nobiletin, tangeretin, and quercetagetin showed a reversal effect in multidrug resistance to anticancer drugs.17 Nobiletin was also reported to have a protective effect on UV radiation-induced skin photodamage.18 The O-methylation of flavonoids is catalyzed by FOMTs.6,19−21 Many studies have shown that FOMTs are highly regiospecific. Naringenin OMT (OsNOMT) from rice introduces a methyl group to the 7-OH of flavonoid substrates.20 An OMT (SlOMT3) isolated from tomatoes methylates the 3′- and 5′-OHs of flavonoids.21 CrOMT6 from Catharanthus roseus transfers a methyl group to the 4′−OH of flavonoids.19 In contrast to their regiospecificity, FOMTs have been reported to use a wide range of flavonoids as substrates.8,20−24 Because of their strict regiospecificity and broad substrate preference, FOMTs have been considered

lavonoids are a class of natural phenolic compounds with a wide range of health-beneficial and pharmacological activities including antioxidant, anti-inflammatory, antimicrobial, and anticancer activities.1−4 Because of their useful biological activities, flavonoids have been widely used as functional ingredients in the food, cosmetic, and pharmaceutical industries. A wide range of the biological activities of flavonoids are based on their structural diversity, which is due to the degree of oxidation of the carbon skeleton, different hydroxylation patterns, and other modifications.4−6 Methylation of the hydroxy groups of flavonoids is a common modification and contributes to their structural diversity, which can alter and/or improve their biological activities and pharmacological properties.6−8 It was reported that di-O-methylchrysin showed markedly improved intestinal anti-inflammatory properties compared with chrysin.9 Methylation of the flavones chrysin and apigenin led to a drastic increase of their hepatic metabolic stability and intestinal absorption.7 Several studies also demonstrated that methylation is important for the antimicrobial activities of flavonoids. A previous study reported that 4′-O-methylnaringenin (ponciretin) and 7-O-methylnaringenin (sakuranetin) possessed antiHelicobacter pylori activity.10,11 Sakuranetin is a well-known phytoalexin in rice possessing antimicrobial activity against fungal and bacterial phytopathogens.12,13 3′,7-Di-O-methylquercetin (rhamnazin) showed a broad spectrum antimicrobial © 2017 American Chemical Society and American Society of Pharmacognosy

Received: December 19, 2016 Published: April 21, 2017 1467

DOI: 10.1021/acs.jnatprod.6b01164 J. Nat. Prod. 2017, 80, 1467−1474

Journal of Natural Products

Article

Figure 1. Chemical structure of the flavonoids used in the present study.

useful tools for the biotechnological production of bioactive Omethylflavonoids.8 Ponciretin was produced from naringenin by biotransformation using the engineered E. coli expressing soybean 4′-FOMT.22 The recombinant rice ROMT-9 heterologously expressed in Yarrowia lioplytica was used in the enzymatic production of the bitter-masking flavonoid homoeriodictyol.25 Some bioactive O-methylflavonoids such as rhamnazin, velutin, and nobiletin contain more than two methyl groups in different aromatic rings and locations in flavonoid backbones.14−18 Because of their regiospecificity, more than two FOMTs and methylation reactions are required for the biosynthesis of polymethoxyflavonoids. In the present study, for the one-step biotechnological production of polymethoxyflavonoids, we generated a fusion FOMT by combining the previously identified 3′/5′-OMT (SlOMT3) and 7-OMT (OsNOMT) and expressed it in E. coli. The fusion FOMT showed both original 3′/5′- and 7-OMT activities to diverse nonmethylated and methylated flavonoids. We attempted the biotechnological production of bioactive di-O-methylflavonoids using E. coli harboring the fusion FOMT gene and demonstrated that the nonmethylated flavonoids quercetin, luteolin, eriodictyol, and taxifolin are successfully converted to the corresponding di-O-methyl products, rhamnazin, velutin, 3′,7-di-O-methyleriodictyol, and 3′,7-di-O-methyltaxifolin, respectively.

When a recombinant fusion protein is generated by direct fusion of two proteins, it is frequently misfolded and loses its biological activities.29−31 To avoid these unwanted effects, we inserted an EAAAK peptide linker between two OMT proteins in the generation of the SlOMT3/OsNOMT fusion protein (Figure 2A). (EAAAK)n linkers are helical-forming rigid

Figure 2. Generation of the SlOMT3/OsNOMT fusion protein. (A) Construction of the expression vector for the His-tagged SlOMT3/ OsNOMT fusion protein linked with an EAAAK peptide linker. The SlOMT3 and OsNOMT genes were combined by overlap extension PCR. The fusion gene was cloned into the His-tag vector pET-28a(+). L, EAAAK peptide linker; MCS, multicloning site. (B) Purification of the SlOMT3/OsNOMT fusion protein from E. coli transformants. M, size marker; 1, crude extract from uninduced E. coli transformant; 2, crude extract from IPTG-induced E. coli transformant; 3, proteins after ammonium sulfate fractionation; and 4, affinity-purified SlOMT3/ OsNOMT fusion protein.

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RESULTS AND DISCUSSION Generation of the SlOMT3/OsNOMT Fusion Enzyme. Flavonoids are often present as (poly)-methylated analogues in nature, which have a wide range of valuable biological activities.15,17,26,27 Although it is possible to methylate most hydroxy groups in flavonoids, 7- and 3′/5′−OHs are predominant flavonoid O-methylation locations (Figure 1).6 Bioactive di-O-methylflavonoids such as velutin and rhamnazin are methylated at both the 7-OH and 3′-OH positions. The trimethoxyflavonoid 3′,5′,7-tri-O-methyltricetin has anticancer activity by inhibiting DNA topoisomerase I.28 For the biosynthesis of polymethoxyflavonoids, more than two OMT reactions are required because of the strict regiospecificity of FOMTs. In the present study, we generated a multifunctional FOMT possessing both 3′/5′- and 7-OMT activities by genetically fusing SlOMT3 and OsNOMT and applied it to the biotransformation of flavonoids for the one-step production of polymethoxyflavonoids.

linkers, which have been applied to the construction of many recombinant fusion proteins to maintain their independent bioactivities.31 The SlOMT3 and OsNOMT genes were combined by overlap extension PCR, and the linker was introduced between two genes by use of the overlapping primer set containing the linker sequence. The resulting fusion gene was cloned into the His-tag vector (Figure 2A). The N-terminal His-tagged fusion protein with the linker (SlOMT3/ OsNOMT) was heterologously expressed in E. coli by 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at the 1468



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Figure 3. FOMT activity assay of the SlOMT3/OsNOMT fusion protein and individual SlOMT3 and OsNOMT proteins. (A) SlOMT3 and SlOMT3/OsNOMT showed the 3′-OMT activity toward rhamnetin (S) yielding rhamnazin (P). (B) OsNOMT and SlOMT3/OsNOMT showed 7-OMT activity toward naringenin (S) yielding sakuranetin (P). S, substrate; P, reaction product; C, authentic methylated compounds (rhamnazin, C for panel A; and sakuranetin, C for panel B).

SlOMT3 methylated all examined subclasses of flavonoids containing 3′- or 3′/5′-OH groups. OsNOMT was identified as the sakuranetin biosynthesis enzyme in rice by catalyzing the 7O-methylation of naringenin.20 Other than naringenin, OsNOMT was reported to methylate diverse flavonoids, such as apigenin, luteolin, kaempferol, and quercetin.20 In addition, evaluation of the OsNOMT activity toward other flavonoids not examined previously showed that it catalyzed the methylation of eriodictyol, taxifolin, and 3′-O-methylflavonoids (Table 1). The SlOMT3/OsNOMT fusion protein showed comparable 3′/5′- and 7-OMT activities to individual SlOMT3 and OsNOMT (Table 1). These findings indicate that SlOMT3 and OsNOMT methylate a broad range of flavonoids regardless of the subclasses, and the SlOMT3/OsNOMT fusion enzyme can be applied to produce (poly)-methoxyflavonoids. Kinetic analyses of SlOMT3/OsNOMT using single O-methylation substrates (7-O-methyleriodictyol and rhamnetin for 3′-OMT reactions; and naringenin, homoeriodictyol, and isorhamnetin for 7-OMT reactions) were also performed (Table 2). The kinetic properties are in agreement with the relative activities of SlOMT3/OsNOMT to flavonoid substrates. For the one-step production of 3′,7- or 3′,5′,7-Omethylflavonoids, SlOMT3/OsNOMT should use 7-O-methylflavonoids and 3′ (or 3′/5′)-O-methylflavonoids as substrates (Figure 4). The 3′-OMT activity of SlOMT3/OsNOMT and SlOMT3 toward the 7-O-methylflavonoids 7-O-methyleriodictyol, 7-O-methyltaxifolin, 7-O-methylluteolin, and rhamnetin was also examined (Figure 1). SlOMT3/OsNOMT consumed rhamnetin, 7-O-methyleriodictyol, and 7-O-methylluteolin as substrates with strong 3′-OMT activity toward rhamnetin (Table 1). The relative activity of SlOMT3/OsNOMT toward 7-methylated flavonoids was similar to that of SlOMT3, indicating that it has comparable enzymatic properties to the original SlOMT3 enzyme. The 7-OMT activities of SlOMT3/ OsNOMT and OsNOMT were evaluated with the 3′- or 3′/5′O-methylflavonoids homoeriodictyol (3′-O-methyleriodictyol), 3′-O-methyltaxifolin, chrysoeriol (3′-O-methylluteolin), isorhamnetin (3′-O-methylquercetin), tricin (3′/5′-di-O-methyltricetin), and syringetin (3′/5′-di-O-methylmyricetin) (Figure 1). SlOMT3/OsNOMT used homoeriodictyol, 3′-O-methyl-

induction temperature of 18 °C. SDS-PAGE analysis of the soluble and insoluble protein fractions from the IPTG-induced E. coli transformants showed that the expressed proteins were detected in both soluble and insoluble fractions, indicating that a part of the SlOMT3/OsNOMT fusion proteins are expressed as a soluble form (Figure S1, Supporting Information). The recombinant SlOMT3/OsNOMT protein was purified by (NH4)2SO4 fractionation and Ni2+ affinity chromatography (Figure 2B). The molecular mass of the purified SlOMT3/ OsNOMT protein was determined to be 82 kDa by SDSPAGE, which agrees well with the predicted size of the fusion protein with His-tag. It was previously reported that the recombinant alfalfa ChOMT and IOMT are homodimers.32 To elucidate the native conformation of the recombinant SlOMT3/OsNOMT protein, gel filtration chromatography was performed with the purified proteins. Gel filtration chromatography of the recombinant SlOMT3/OsNOMT showed two protein peaks with the estimated molecular masses of 157.7 and 74.4 kDa, respectively, indicating that SlOMT3/ OsNOMT exist in both dimeric and monomeric forms (Figure S2, Supporting Information). This result suggested that dimerization of recombinant SlOMT3/OsNOMT is partially hindered by the fusion of two OMTs. SlOMT3/OsNOMT Has Both 3′/5′- and 7-OMT Activities toward Diverse Flavonoids. To confirm whether the SlOMT3/OsNOMT fusion protein has both original flavonoid 3′/5′- and 7-OMT activities, enzyme reactions were performed using rhamnetin (7-O-methylquercetin) and naringenin as the flavonoid substrates for 3′- and 7-OMT activities, respectively. HPLC analysis showed that SlOMT3/OsNOMT consumed rhamnetin and naringenin, and produced 3′-O-methylrhamnetin (rhamnazin) and sakuranetin, respectively (Figure 3). These results indicate that the recombinant SlOMT3/OsNOMT is a multifunctional FOMT possessing both flavonoid 3′- and 7OMT activities. In a previous study, the OMT activity of SlOMT3 was examined with a wide array of flavonoids and isoflavonoids.21 It was found that SlOMT3 methylates flavonoids, whereas it showed no OMT activity to isoflavonoids. Although flavonols such as quercetin and laricitrin were its preferable substrates, 1469



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Table 1. Substrate Preference of Recombinant SlOMT3, OsNOMT, and SlOMT3/OsNOMT Enzymes Expressed in E. coli relative activity (%)a SlOMT3 substrate

(3′-OMT)

SlOMT3/OsNOMT (3′-OMT)

OsNOMT

(7-OMT)

(7-OMT)

100 + 92

100 41 69

+ 57

10 56

78 + 78

73 198 96

− −

− −

80 +

87 83

111 -

139 -

Flavanone naringenin eriodictyol homoeriodictyol 7-O-methyleriodictyol taxifolin 3′-O-methyltaxifolin 7-O-methyltaxifolin

24b

+c

21 21 Dihydroflavonol △d △ −e

− Flavone

apigenin luteolin chrysoeriol 7-O-methylluteolin tricetin tricin

93b

+

20 +

28 + Flavonol

kaempferol quercetin rhamnetin isorhamnetin syringetin

222b 100

+ 100

Figure 4. Reaction scheme of the biotechnological production of dimethoxyflavonoids from nonmethylated flavonoids using SlOMT3/ OsNOMT-transformed E. coli. The SlOMT3/OsNOMT fusion enzyme catalyzes the 3′- and 7-O-methylations of flavonoid substrates, and vice versa. Regiospecific OMT activities for the corresponding reaction steps are in the parentheses.

To determine the relative activity, 50 μM of each substrate and 1 μg of purified enzymes were used for the reaction. The 100% relative activities of SlOMT3/OsNOMT for rhamnetin and naringenin are 34.01 nmol min−1mg−1 and 79.78 nmol min−1mg−1, respectively. The 100% relative activities of SlOMT3 for rhamnetin and OsNOMT for naringenin are 95.06 nmol min−1mg−1 and 165.15 nmol min−1mg−1, respectively. bThese values were recalculated from the previously reported data by Cho et al.21 cThe plus signs indicate that the tested flavonoids were used as substrates. The relative activities toward the substrates were not determined. dThe triangles indicated the weak OMT activities of the recombinant enzymes toward flavonoid substrates. eThe minus signs indicate that the recombinant enzymes had no detectable OMT activities toward flavonoid substrates. a

Biotransformation Kinetics of the Di-O-methylation of Flavonoids by SlOMT3/OsNOMT-transformed E. coli. Owing to their regiospecificity, FOMTs are thought to be a useful tool for the production of O-methylflavonoids.8 Several studies have reported the in vitro synthesis of O-methylflavonoids using purified FOMTs.19,25,33 Recently, biotechnological approaches using genetically engineered microorganisms harboring an FOMT gene have been attempted in order to produce O-methylflavonoids because biotransformation has advantages such as no cofactor requirement, decreased formation of byproducts, and ease of scale-up.8 To date, many studies have demonstrated the production of mono-Omethylflavonoids by biotransformation using microorganisms containing an FOMT gene.21−24,34−39 Willits et al.40 reported the biotechnological production of poly-O-methylflavonoids using mixed-cultures of E. coli transformants expressing a single OMT from Mentha x piperita. In the present study, the biotechnological production of di-O-methylflavonoids from

taxifolin, chrysoeriol, and isorhamnetin with different preferences, but showed no detectable OMT activity toward tricin or syringetin (Table 1). OsNOMT showed a similar substrate preference to 3′- or 3′/5′-O-methylflavonoid substrates to that of SlOMT3/OsNOMT (Table 1). The wide substrate preferences of SlOMT3/OsNOMT toward 7- and 3′-Omethylflavonoids suggest that it is able to be applied to produce di-O-methylflavonoids, and its preferable nonmethylated flavonoid substrates are eriodictyol, luteolin, and quercetin.

Table 2. Kinetic Parameters of Recombinant SlOMT3/OsNOMTa substrate 7-O-methyleriodictyol rhamnetin naringenin homoeriodictyol isorhamnetin a

Km (μM) 4.32 1.33 1.16 2.95 1.24

± ± ± ± ±

0.20 0.11 0.07 0.25 0.23

Vmax (nmol min−1 mg−1)

kcat (s−1)

± ± ± ± ±

0.046 0.035 0.116 0.063 0.163

33.53 25.18 83.96 45.35 118.0

4.28 4.62 5.19 1.38 7.21

kcat/Km (μM−1 s−1) 1.07 2.61 9.97 2.12 13.1

× × × × ×

10−2 10−2 10−2 10−2 10−2

The results are the means and ± SD of two independent experiments. 1470



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of final products obtained within 4 h of biotransformation at the examined substrate concentrations compared to eriodictyol and taxifolin (Figure 5). Under the experimental conditions, the biotransformation of eriodictyol into the di-O-methyl product required 18−24 h (Figure 5A). Biotransformation of taxifolin was much slower than that of the other substrates. SlOMT3/ OsNOMT showed weak OMT activity to taxifolin, particularly its 3′-OMT activity (Table 1); consequently, the bioconversion rate of taxifolin into the mono-O-methyl intermediates was slow (Figure 5B). Although a substantial amount of 7-O-methyl intermediate was formed, it was hardly consumed by SlOMT3/ OsNOMT as a substrate for the di-O-methyl product because the fusion OMT has no detectable activity toward 7-Omethyltaxifolin. These results demonstrate that SlOMT3/ OsNOMT-transformed E. coli cells are useful to produce diO-methylflavonoids from nonmethylated flavonoids, such as eriodictyol, luteolin, and quercetin. Because SlOMT3/ OsNOMT showed weak or no detectable OMT activities toward the flavonoids tricetin, tricin, syringetin, and 7-Omethyltaxifolin, other FOMTs with strong OMT activity toward these flavonoids need to be identified for the efficient biotechnological production of diverse di-O-methylflavonoids. Production of Di-O-methylflavonoids by Biotransformation Using SlOMT3/OsNOMT-Transformed E. coli. The bioconversion efficiency of flavonoids toward their methylated products varies depending on the sort of substrate, reaction conditions, FOMTs, and microorganisms used.21−24,38,39 At a substrate concentration of 70 μM, E. coli containing POMT-7, a 7-FOMT from poplar, bioconverted more than 80% of apigenin, quercetin, luteolin, and kaempferol into the corresponding O-methyl analogues.23 E. coli harboring SaOMT-2 from Streptomyces avermitilis mostly converted naringenin into sakuranetin at a substrate concentration of 60 μM.24 SaOMT-2-transformed E. coli was also applied to methylate genistein and daidzen. In the case of genistein, the bioconversion yields were about 65 and 3% at substrate concentrations of 100 and 1000 μM, respectively.39 In the present study, different concentrations (10, 50, and 250 μM) of each flavonoid substrate were added to the SlOMT3/OsNOMT-transformed E. coli culture, and their biotransformation into di-O-methylflavonoid products was monitored over a 2−24 h time period (Figure S4, Supporting Information). Except for the case of taxifolin, SlOMT3/ OsNOMT-transformed E. coli showed higher bioconversion yields at lower concentrations of nonmethylated flavonoid substrates (Figure 6). Under the experimental conditions, the bioconversion yields of eriodictyol and luteolin into the di-Omethyl products were 86.7 and 88.1%, respectively, at a substrate concentration of 10 μM (Figure 6A,C). Although SlOMT3/OsNOMT-transformed E. coli effected the dimethylation of flavonoids, the conversion yields of eriodictyol and luteolin were comparable to those of the reported highly efficient bioconversions of flavonoids into the mono-Omethylflavonoids.22−24 At a substrate concentration of 10 μM, the bioconversion yield of quercetin was 48.8%. The biotransformations of eriodictyol and luteolin at a substrate concentration of 50 μM resulted in conversion yields of 57.1 and 37.1%, respectively (Figure 6). At the same substrate concentration, the bioconversion yield of quercetin was 21.0% (Figure 6). These results indicate that SlOMT3/OsNOMTtransformed E. coli efficiently bioconverts eriodictyol and luteolin compared to quercetin. This is consistent with a previous result where the bioconversion yields of SlOMT3-

nonmethylated flavonoids using E. coli harboring the SlOMT3/ OsNOMT fusion gene was attempted. To confirm the possible application of SlOMT3/OsNOMTtransformed E. coli in the biotechnological production of di-Omethylflavonoids from nonmethylated flavonoids, biotransformation was performed with eriodictyol, taxifolin, luteolin, and quercetin, which contain hydroxyl groups at both C-3′ and C-7. Each flavonoid substrate (50 μM) was added to the E. coli culture, and the formation of mono- and di-O-methylflavonoids was monitored (Figure 5 and Figure S3, Supporting

Figure 5. Biotransformation kinetics of nonmethylated flavonoids using E. coli expressing SlOMT3/OsNOMT. The E. coli transformant expressing SlOMT3/OsNOMT converted nonmethylated flavonoids (―○―), eriodictyol (A), taxifolin (B), luteolin (C), and quercetin (D), into the corresponding 3′,7-di-O-methyl products (―●―), 3′,7-di-O-methyleriodictyol, 3′,7- di-O-methyltaxifolin, 3′,7-di-O-methylluteolin, and 3′,7-di-O-methylquercetin, respectively. Fresh cultures of the IPTG-induced E. coli transformants were supplemented with 50 μM of each nonmethylated flavonoid substrate, and the production of the di-O-methylflavonoid was analyzed over time. 3′- O-Mono- (---■---) or 7-O-mono- (---□---) methylated intermediates were also monitored. The results represent the mean ± SD of three independent experiments.

Information). Since SlOMT3/OsNOMT possesses both 3′and 7-OMT activities, during biotransformation, nonmethylated substrates are expected to be first converted into either 3′or 7-O-methylflavonoids and sequentially transformed into 3′,7di-O-methylflavonoids (Figure 4). Indeed, 3′- or 7-mono-Omethylflavonoid intermediates were observed immediately after starting the biotransformation (Figure 5). With decreasing mono-O-methyl intermediates, di-O-methylflavonoid products were produced in the culture medium (Figure 5). This result demonstrates that di-O-methylflavonoids are able to be produced from nonmethylated flavonoids by biotransformation using SlOMT3/OsNOMT-transformed E. coli. In parallel to the relative activity of SlOMT3/OsNOMT to flavonoid substrates, luteolin and quercetin were rapidly bioconverted to di-O-methylflavonoids, with the highest levels 1471



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SpOMT2884 in Terrific broth was 1.3- and 1.6-fold higher than those obtained in LB and M9 minimal media, respectively. In addition to the optimized biotransformation conditions, enhanced internal AdoMet production led to the increased production of 7-O-methylisoflavonoids, such as 7-O-methylgenistein and 7-O-methyldaidzein by engineered E. coli harboring SaOMT-2 and metK encoding AdoMet synthase.39 Therefore, for more efficient production of di-O-methylflavonoids, the reaction conditions including substrate concentration and culture conditions such as type of media, cell population, and reaction time need to be further optimized.

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EXPERIMENTAL SECTION

General Experimental Procedures. All flavonoids used were purchased from Indofine Chemical Company (Hillsborough, NJ, U.S.A.) and ChromaDex (Irvine, CA, U.S.A.) except for several 3′- or 7-O-methylflavonoids. Homoeriodictyol (3′-O-methyleriodictyol) and 3′-O-methyltaxifolin were synthesized by biotransformation using previously generated SlOMT3-transformed E. coli.21 7-O-Methyleriodictyol, 7-O-methylluteolin, and 7-O-methyltaxifolin were produced by biotransformation using the engineered E. coli expressing OsNOMT. Each O-methylflavonoid was purified by preparative TLC. HPLC-grade water, MeCN, and HOAc were purchased from Avantor Performance Materials (Center Valley, PA, U.S.A.). S-Adenosyl methionine (AdoMet) and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Generation of the SlOMT3/OsNOMT Fusion Gene by Overlap Extension PCR. For cloning of OsNOMT, total RNA was extracted from UV-treated leaves of rice (Oryza sativa cv. Dongjin) using TRIzol reagent (Invitrogen, Waltham, MA, U.S.A.). cDNA was synthesized from the total RNAs of rice by RT-PCR using a firststrand cDNA synthesis kit (Roche Diagnostics, Indianapolis, IN, U.S.A.). The OsNOMT gene was amplified by PCR from first-strand cDNA. The primer sets used were 5′-GGCATATGGGAGACATGGTGAGCCCG-3′ and 5′-CCGAATTCTTACTTTGTGAACTCGAGAGC-3′, which contain NdeI and EcoRI restriction sites (underlined), respectively. 20 The resulting PCR product was subcloned into the pJET1.2 vector (Thermo Scientific, Waltham, MA, U.S.A.). After sequence confirmation, the OsNOMT gene was cut with the appropriate restriction enzymes and inserted into the pET28a(+) vector (Novagen, Madison, WI, U.S.A.). The resulting construct was transformed into E. coli BL21 (DE3) cells. SlOMT3 from the miniature tomato cultivar was previously cloned into the pET-28a(+) vector and transformed into E. coli cells.21 To fuse SlOMT3 and OsNOMT with a linker between the genes, the two genes were amplified and combined by overlap extension PCR using an In-fusion HD cloning kit (Clontech, Mountain View, CA, U.S.A.). The primer sets used were 5′- ATGGGTCGCGGATCCATGGGTTCAACAAGCCTA-3′ and 5′-TTTCGCCGCCGCTTCCTTGGTGAATTCCATAAT-3′ for SlOMT3, and 5′GAAGCGGCGGCGAAAGGAGACATGGTGAGCCCGGTGG-3′ and 5′-CTCGAGTGCGGCCGCTTACTTTGTGAACTCGAG-3′ for OsNOMT (the linker sequences are underlined). The sequences to insert the linker were contained in the reverse primer for SlOMT3 and forward primer for OsNOMT, respectively. After sequence confirmation, the SlOMT3/OsNOMT fusion gene was inserted into the pET-28a(+) vector. The resulting constructs were transformed into E. coli BL21 (DE3) cells to express the fusion enzyme. Expression and Purification of SlOMT3/OsNOMT and OsNOMT. E. coli transformant harboring SlOMT3/OsNOMT was grown in Luria−Bertani (LB) medium supplemented with kanamycin (25 μg/mL) at 37 °C until the cells reached an OD600 of ∼0.6, at which point 0.1 mM IPTG was added to induce the production of the SlOMT3/OsNOMT fusion protein. The culture was incubated at 18 °C for 16 h, and the cells were harvested by centrifugation (4800g for 15 min). The cell pellets were resuspended in phosphate-buffered saline (PBS) supplemented with lysozyme (1 mg/mL) and phenylmethylsulfonyl fluoride (1 mM), and followed by sonication to lyse the

Figure 6. Production of di-O-methylflavonoids by one-step biotransformation of nonmethylated flavonoids using E. coli expressing SlOMT3/OsNOMT. The highest bioconversion yields and the maximum amounts (μM) of the 3′,7-di-O-methyl products from different concentrations (10, 50, and 250 μM) of the nonmethylated substrates eriodictyol (A), taxifolin (B), luteolin (C), and quercetin (D) by E. coli expressing SlOMT3/OsNOMT were determined. Bioconversion yield is defined as 3′,7-di-O-methyl product concentration/substrate concentration × 100. The results represent the mean ± SD of three independent experiments.

transformed E. coli for eriodictyol and luteolin were higher than that for quercetin.21 It has been reported that the biotransformation conditions for the highest bioconversion yields are different from those producing the highest amounts of methylated products in the culture media.38,39 In the case of the mono-O-methylation of 7,8-dihydroxyflavone (DHF) by E. coli harboring SpOMT2884 from Streptomyces peucetius, the highest bioconversion yield of >90% was obtained at a substrate concentration of 100 μM, whereas the maximum content (152 μM) of the O-methyl product was produced in the culture supplemented with 200 μM of substrate.38 Both the bioconversion yields and product contents decreased when substrate concentrations higher than 200 μM of 7,8-DHF were used in the biotransformation. Our results also showed that the maximum contents of the di-Omethyl products were 80.1, 32.9, and 36.7 μM when 250 μM of eriodictyol, taxifolin, and luteolin were supplemented, respectively, although the bioconversion yields were the highest at a substrate concentration of 10 μM (Figure 6A−C and Figures S4A−C, Supporting Information). The biotransformation of quercetin showed a maximum di-O-methyl product concentration of 9.7 μM at 50 μM substrate concentration (Figure 6D and Figure S4D, Supporting Information). In the present study, it was demonstrated that SlOMT3/ OsNOMT-transformed E. coli is applicable for the one-step biotechnological production of valuable di-O-methylflavonoids such as velutin, 3′,7-di-O-methyleriodictyol, and rhamnazin from nonmethylated flavonoid precursors. The bioconversion efficiency of flavonoids using microorganisms harboring an FOMT gene could be increased by optimization of the biotransformation conditions. Koirala et al.37 showed that the biotransformation efficiency of 7,8-DHF by E. coli harboring 1472



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cells. After sonication, the cell debris was removed by centrifugation (15 900g for 20 min), and the supernatant was obtained. The Histagged SlOMT3/OsNOMT protein was purified by salt fractionation and affinity chromatography. The crude extract was mixed with (NH4)2SO4 (60% saturation) and the mixture was gently agitated at 4 °C for 30 min. After centrifugation at 15 900g for 20 min, the precipitate was resuspended with 33% saturated (NH4)2SO4 in PBS for back extraction. The suspension was agitated at 4 °C for 10 min and centrifuged at 15 900g for 20 min. The supernatant from the salt fractionation was mixed with Ni-NTA agarose beads (Qiagen, Hilden, Germany) and incubated at 4 °C for 2 h with shaking. The mixture was packed into a chromatography column and washed with a fivecolumn volume of 20 mM imidazole in Tris buffer (50 mM Tris, pH 8.0, 300 mM NaCl). The SlOMT3/OsNOMT protein was eluted with 50 mM imidazole in the same buffer. The expression of OsNOMT in the E. coli cells was induced with 0.1 mM IPTG at 25 °C for 6 h. The recombinant OsNOMT protein was purified by Ni2+-affinity chromatography as described above. FOMT Assay. The reaction mixtures consisted of 50 μM flavonoid substrates and 100 μM AdoMet in 20 mM Tris-HCl (pH 7.5) to a final volume of 500 μL. The purified enzymes were added into the reaction mixtures to initiate the enzyme reaction, and the mixtures were incubated at 30 °C for 30 min. The reactions were stopped by adding 50 μL of 5 N HCl, and the reaction products were extracted twice with 500 μL of EtOAc. The EtOAc extracts were dried in vacuo and redissolved in 100 μL of MeOH. Each reaction product was analyzed by reverse-phase HPLC equipped with a Sunfire C18 column (Waters, Milford, MA, U.S.A.) using a linear gradient of 25−60% MeCN in 3% HOAc-water for 25 min with detection at 280 nm. In the kinetic analyses of SlOMT3/OsNOMT, different concentrations (0.5−10 μM) of each flavonoid substrate and different amounts (0.5−2.5 μg) of the purified proteins were contained in the reaction mixtures. After 10 min incubation at 30 °C, the reactions were stopped and analyzed by reverse-phase HPLC according to abovedescribed procedure. Biotransformation of Flavonoids Using SlOMT3/OsNOMTTransformed E. coli. The E. coli transformants harboring SlOMT3/ OsNOMT were grown and induced with IPTG as described above. After induction for 6 h, the cells were harvested and resuspended in the same volume of fresh LB medium containing kanamycin (25 μg/ mL). Different concentrations (10, 50, and 250 μM) of each flavonoid substrate were added to a fresh culture and incubated at 25 °C. The flavonoid substrates used in the biotransformation were eriodictyol, taxifolin, luteolin, and quercetin. An aliquot of the culture was harvested at a selected time point, and cell-free medium was obtained by centrifugation. The medium was extracted twice with 500 μL of EtOAc, and the extracts were analyzed by reverse-phase HPLC with the previously described method.

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ORCID

Man-Ho Cho: 0000-0002-4064-5271 Author Contributions §

D.L. and H.L.P. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Next-Generation BioGreen 21 Program (Project No: PJ01107501) funded by the Rural Development Administration, and the Midcareer Researcher Program (NRF-2016R1A2B4014276) through NRF grant funded by the Ministry of Education, Science and Technology, Republic of Korea.

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(1) Tapas, A. R.; Sakarkar, D. M.; Kakde, R. B. Trop. J. Pharm. Res. 2008, 7, 1089−1099. (2) Agrawal, A. D. Int. J. Pharm. Sci. Nanotechnol. 2011, 4, 1394− 1398. (3) Romagnolo, D. F.; Selmin, O. I. J. Nutr. Gerontol. Geriatr. 2012, 31, 206−238. (4) Kumar, S.; Pandey, A. K. Sci. World J. 2013, 2013, 1. (5) Heim, K. E.; Tagliaferro, A. R.; Bobilya, D. J. J. Nutr. Biochem. 2002, 13, 572−584. (6) Kim, B. G.; Sung, S. H.; Chong, Y.; Lim, Y.; Ahn, J. H. J. Plant Biol. 2010, 53, 321−329. (7) Walle, T. Mol. Pharmaceutics 2007, 4, 826−832. (8) Koirala, N.; Thuan, N. H.; Ghimire, G. P.; Thang, D. V.; Sohng, J. K. Enzyme Microb. Technol. 2016, 86, 103−116. (9) During, A.; Larondelle, Y. Biochem. Pharmacol. 2013, 86, 1739− 1746. (10) Kim, D. H.; Bae, E. A.; Han, M. J. Biol. Pharm. Bull. 1999, 22, 422−424. (11) Zhang, L.; Kong, Y.; Wu, D.; Zhang, H.; Wu, J.; Chen, J.; Ding, J.; Hu, L.; Jiang, H.; Shen, X. Protein Sci. 2008, 17, 1971−1978. (12) Kodama, O.; Miyakawa, J.; Akatsuka, T.; Kiyosawa, S. Phytochemistry 1992, 31, 3807−3809. (13) Park, H. L.; Yoo, Y.; Hahn, T. R.; Bhoo, S. H.; Lee, S. W.; Cho, M. H. Molecules 2014, 19, 18139−18151. (14) Omosa, L. K.; Amugune, B.; Ndunda, B.; Milugo, T. K.; Heydenreich, M.; Yenesew, A.; Midiwo, J. O. S. Afr. J. Bot. 2014, 91, 58−62. (15) Yu, Y.; Cai, W.; Pei, C.; Shao, Y. Biochem. Biophys. Res. Commun. 2015, 458, 913−919. (16) Kang, J.; Xie, C.; Li, Z.; Nagarajan, S.; Schauss, A. G.; Wu, T.; Wu, X. Food Chem. 2011, 128, 152−157. (17) Ohtani, H.; Ikegawa, T.; Honda, Y.; Kohyama, N.; Morimoto, S.; Shoyama, Y.; Juichi, M.; Naito, M.; Tsuruo, T.; Sawada, Y. Pharm. Res. 2007, 24, 1936−1943. (18) Tanaka, S.; Sato, T.; Akimoto, N.; Yano, M.; Ito, A. Biochem. Pharmacol. 2004, 68, 433−439. (19) Schröder, G.; Wehinger, E.; Lukačin, R.; Wellmann, F.; Seefelder, W.; Schwab, W.; Schröder, J. Phytochemistry 2004, 65, 1085−1094. (20) Shimizu, T.; Lin, F.; Hasegawa, M.; Okada, K.; Nojiri, H.; Yamane, H. J. Biol. Chem. 2012, 287, 19315−19325. (21) Cho, M. H.; Park, H. L.; Park, J. H.; Lee, S. W.; Bhoo, S. H.; Hahn, T. R. J. Korean Soc. Appl. Biol. Chem. 2012, 55, 749−755. (22) Kim, D. H.; Kim, B. G.; Lee, Y.; Ryu, J. Y.; Lim, Y.; Hur, H. G.; Ahn, J. H. J. Biotechnol. 2005, 119, 155−162. (23) Kim, B. G.; Kim, H.; Hur, H. G.; Lim, Y.; Ahn, J. H. J. Biotechnol. 2006, 126, 241−247.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01164. SDS-PAGE of the soluble and insoluble protein fractions from E. coli transformants expressing SlOMT3/OsNOMT; estimation of the native molecular weight of the SlOMT3/OsNOMT protein by gel filtration; biotransformation of nonmethylated flavonoids using E. coli transformants harboring an empty pET28a vector; and biotransformation of nonmethylated flavonoids using E. coli expressing SlOMT3/OsNOMT (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +82 31 201 2632. Fax: +82 31 203 8127. 1473



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(24) Kim, B. G.; Jung, B. R.; Lee, Y.; Hur, H. G.; Lim, Y.; Ahn, J. H. J. Agric. Food Chem. 2006, 54, 823−828. (25) Liu, Q.; Liu, L.; Zhou, J.; Shin, H. D.; Chen, R. R.; Madzak, C.; Li, J.; Du, G.; Chen, J. J. Biotechnol. 2013, 167, 472−478. (26) Cho, J. G.; Song, N. Y.; Nam, T. G.; Shrestha, S.; Park, H. J.; Lyu, H. N.; Kim, D. O.; Lee, G.; Woo, Y. M.; Jeong, T. S.; Baek, N. I. J. Agric. Food Chem. 2013, 61, 10354−10359. (27) Yahagi, T.; Yakura, N.; Matsuzaki, K.; Kitanaka, S. J. Nat. Med. 2014, 68, 414−420. (28) Zahir, A.; Jossang, A.; Bodo, B.; Provost, J.; Cosson, J.-P.; Sevenet, T. J. Nat. Prod. 1996, 59, 701−703. (29) Bai, Y.; Ann, D. K.; Shen, W.-C. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 7292−7296. (30) Zhao, H. L.; Yao, X. Q.; Xue, C.; Wang, Y.; Xiong, X. H.; Liu, Z. M. Protein Expression Purif. 2009, 61, 73−77. (31) Chen, X.; Zaro, J. L.; Shen, W.-C. Adv. Drug Delivery Rev. 2013, 65, 1357−1369. (32) Zubieta, C.; He, X.-Z.; Dixon, R. A.; Noel, J. P. Nat. Struct. Biol. 2001, 8, 271−279. (33) De Luca, V.; Ibrahim, R. K. Arch. Biochem. Biophys. 1985, 238, 596−605. (34) Hosny, M.; Rosazza, J. P. N. J. Nat. Prod. 1999, 62, 1609−1612. (35) Jeon, Y. M.; Kim, B. G.; Ahn, J. H. J. Microbiol. Biotechnol. 2009, 19, 491−494. (36) Malla, S.; Koffas, M. A. G.; Kazlauskas, R. J.; Kim, B. G. Appl. Environ. Microbiol. 2012, 78, 684−694. (37) Kim, M. J.; Kim, B. G.; Ahn, J. H. Appl. Microbiol. Biotechnol. 2013, 97, 7195−7204. (38) Koirala, N.; Pandey, R. P.; Parajuli, P.; Jung, H. J.; Sohng, J. K. J. Biotechnol. 2014, 184, 128−137. (39) Koirala, N.; Thuan, N. H.; Ghimire, G. P.; Jung, H. J.; Oh, T. J.; Sohng, J. K. Biotechnol. Appl. Biochem. DOI: 10.1002/bab.1452. (40) Willits, M. G.; Giovanni, M.; Prata, R. T. N.; Kramer, C. M.; De Luca, V.; Steffens, J. C.; Graser, G. Phytochemistry 2004, 65, 31−41.

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