Microbial Biosynthesis of Antibacterial Chrysoeriol in Recombinant Escherichia Coli and Bioactivity Assessment

catalysts

Article Microbial Biosynthesis of Antibacterial Chrysoeriol in Recombinant Escherichia coli and Bioactivity Assessment

Puspalata Bashyal 1,†, Prakash Parajuli 1,† , Ramesh Prasad Pandey 1,2,* and Jae Kyung Sohng 1,2,* 1 Department of Life Science and Biochemical Engineering, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 31460, Korea; [email protected] (P.B.); [email protected] (P.P.) 2 Department of Pharmaceutical Engineering and Biotechnology, Sun Moon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 31460, Korea * Correspondence: [email protected] (R.P.P.); [email protected] (J.K.S.); Tel.: +82-(41)-530-2246 (J.K.S.); Fax: +82-(41)-544-2919 (J.K.S.) † These authors contributed equally to this work.

Received: 18 December 2018; Accepted: 22 January 2019; Published: 24 January 2019

Abstract: Various flavonoid derivatives including methoxylated flavones display remarkable biological activities. Chrysoeriol is a methoxylated flavone of great scientific interest because of its promising anti-microbial activities against various Gram-negative and Gram-positive bacteria. Sustainable production of such compounds is therefore of pronounced interest to biotechnologists in the pharmaceutical and nutraceutical industries. Here, we used a sugar O-methyltransferase enzyme from a spinosyn biosynthesis gene cluster of Saccharopolyspora spinosa to regioselectively produce chrysoeriol (15% conversion of luteolin; 30 µM) in a microbial host. The biosynthesized chrysoeriol was structurally characterized using high-resolution mass spectrometry and various nuclear magnetic resonance analyses. Moreover, the molecule was investigated against 17 superbugs, including thirteen Gram-positive and four Gram-negative pathogens, for anti-microbial effects. Chrysoeriol exhibited antimicrobial activity against nine pathogens in a disc diffusion assay at the concentration of 40 µg per disc. It has minimum inhibitory concentration (MIC) values of 1.25 µg/mL against a methicillin-resistant Staphylococcus aureus 3640 (MRSA) for which the parent luteolin has an MIC value of sixteen-fold higher concentration (i.e., 20 µg/mL). Similarly, chrysoeriol showed better anti-microbial activity (~1.7-fold lower MIC value) than luteolin against Proteus hauseri, a Gram-negative pathogen. In contrast, a luteolin 40-O-methylated derivative, diosmetin, did not exhibit any anti-microbial activities against any tested pathogen.

Keywords: biotransformation; Chrysoeriol; anti-bacterial agent; regioselective

1. Introduction Chrysoeriol is a 30-O-methoxy flavone, chemically a derivative of luteolin, which belongs to the flavone group of compounds and is abundantly present in many types of plants, vegetables, and fruits, including medicinal herbs [1]. It has been studied for various biological functions, such as neuroprotective [2], cardio protection [3], anti-inflammation, anti-cancer [4], and antimicrobial effects [5]. Chrysoeriol is well known for its anti-CK2 activity, which inhibits different molecular forms of CK2 at a lower concentration than other flavonoids [6]. Structurally, luteolin has a catechol group in its B ring, and these catechol groups tend to be modified by catechol-O-methyltransferases, which are widely distributed in human organs, such as the liver, intestines, lungs, and brain [7]. One of the

Catalysts 2019, 9, 112; doi:10.3390/catal9020112 www.mdpi.com/journal/catalysts Catalysts 2019, 9, 112 2 of 15 derivatives, diosmetin, which has a 40-methoxy group (para-position) in luteolin, is considered to be more common than chrysoeriol, which has 30-methoxy (meta-position) luteolin. However, both of these compounds are reported to be natural prodrugs in cancer prevention [8]. In particular, chrysoeriol has been naturally isolated from Artemisia, which is a large genus of plants, and contains an artemisinin derivative, which is now used worldwide to treat malaria, which is caused by Plasmodium falciparum [9]. Hence, it might be possible that chrysoeriol could have anti-microbial activities. Although flavonoids from natural products have been showing antibacterial actions against various pathogenic organisms, including a well-known methicillin-resistant Staphylococcus aureus (MRSA), and synergistic antimicrobial effects [10,11]. However, a core flavonoid compound, luteolin, has anti-MRSA activity that was previously reported to act on the bacterial cell wall, as was further proved mechanistically by disrupting the MRSA cytoplasmic membrane [12,13]. Chrysoeriol has also been found to have anti-microbial effects against different bacteria [14]. These are interesting experimental facts that suggest that such flavonoids and their derivatives might have pharmaceutical importance and industrial applications. Besides Artemisia, chrysoeriol was also isolated from other plant sources for various reasons: Digitalis purpurea, for the induction of nitric-acid synthase by blocking nuclear factor activator protein (κB AP-1) [15]; Artemisia copa, vasorelaxing and hypotensive effects study through Ca2+ inhibition influx [16]; Capsicum frutescens, to study and compare the antioxidant and antimicrobial activities of phenolic compounds [14]; and Tecoma stans, lipase inhibitory activity for type 2 diabetes mellitus [17]. Most of these important compounds are either extracted from plant biomass or chemically synthesized. Neither of these techniques is environmentally friendly or economical. Recently, the application of a genetic engineering tool to design a microbial platform has been widely used to synthesize such compounds by expression of a specific plant or microbial genetic materials. Microbes, especially actinomycetes, are a diverse source of simple and complex natural products that cover most of the antibiotics in use today [18,19]. The biosynthetic pathway of these natural products involves post-secondary metabolite modification enzymes, which have recently been mimicked by researchers to use for flavonoid modifications in microbial hosts, such as Escherichia coli. Luteolin is one of the abundant flavones found in plants. It occurs as both O- and C-glycosides. A recent review shows that the abundance of apigenin and luteolin in free and glycosides form in different citrus, teas, fruits, vegetables, olive oil, honey, and dry herbal plants [20]. However, the occurrence of chrysoeriol is limited in plants, and it is not easily available from commercial vendors. In contrast, luteolin is easily obtainable from diverse suppliers at a relatively cheaper price. In this context, the biosynthesis of chrysoeriol using luteolin as starting precursor would be a more economical, eco-friendly, and sustainable approach. This study highlights the microbial production of an important antimicrobial agent, chrysoeriol, by the use of a post-modifying enzyme, sugar O-methyltransferase (SpnK) from Saccharopolyspora spinose (Figure1). This is the first report of using microbial sugar (rhamnose) O-methyltransferase from a spinosyn biosynthesis gene cluster to catalyze plant metabolite luteolin methylation at the 30-OH position. Recently, we observed SpnK as a promiscuous enzyme with broad substrate flexibility towards plant polyphenols [21,22]. Thus, here, we employed SpnK to catalyze an O-methylation reaction over luteolin to synthesize chysoeriol. The microbially produced chrysoeriol was compared to luteolin and diosmetin for anti-bacterial activity against various Gram-positive and Gram-negative bacteria. Chrysoeriol exhibited strong anti-microbial activity in a smaller dose than that needed for luteolin or diosmetin. Catalysts 2019, 9, 112 3 of 15 Catalysts 2018, 8, x FOR PEER REVIEW 3 of 15

Figure 1. Schematic representation of the biosynthesis of chrysoeriol from Escherichia coli and its Figure 1. Schematic representation of the biosynthesis of chrysoeriol from Escherichia coli and its antibacterial test against human pathogens. antibacterial test against human pathogens. 2. Results 2. Results 2.1. Biosynthesis of Luteolin O-methoxide 2.1. Biosynthesis of Luteolin O-methoxide The recombinant strain of E. coli BL21 (DE3) harboring pET32a + SpnK was cultured and prepared for biotransformationThe recombinant as strain mentioned of E. coli in SectionBL21 (DE3)4. Luteolin harboring as substrate pET32a + was SpnK exogenously was cultured fed, and samplesprepared were for biotransformation prepared for the as product mentioned analyses. in Section Sample 4. Luteolin analysis as substrate by high was performance exogenously liquid fed, chromatographyand samples were photo-diode prepared for array the product (HPLC-PDA) analyses. showed Sample a productanalysis peakby high at aperformance retention time liquid (tR ) ofchromat 22.4 minography after the photo substrate-diode peak array at (tHPLCR 20.2- minPDA as) showed compared a product to the control peak at reaction, a retention which time showed (tR) of a single22.4 min peak after at t theR 20.2 substrate min, which peak resemblesat tR 20.2 min to the as compared only substrate-injected to the control chromatogramreaction, which (Figure showed2A). a Luteolinsingle peak was at converted tR 20.2 min, to awhich methoxide resembles derivative to the withonly ansubstrate approximately-injected 15%chromatogram (30 µM) conversion (Figure 2A rate). withoutLuteolin any was biotransformation converted to a methoxide optimization derivative and metabolic with an engineering approximate ofly the 15% host (30 strain. µM) Theconversion reaction mixturerate without was furtherany biotransformation analyzed by high-resolution optimization and quadrupole metabolic time-of-ﬂight engineering electrosprayof the host strain. ionization The massreaction spectrometry mixture was (HRQTOF-ESI-MS). further analyzed The by producthigh-resolution mass fragment quadrupole [M + H] timem/z-of301.0720-flight electrospray matched the calculatedionization mass mass of spectrometry luteolin O-methoxide (HRQTOF [M-ESI +- H]MSm/z). The301.0712 product (Figure mass 2fragmentB). This conﬁrmed [M + H] m/z the 301.0720 product tomatche be O-methylatedd the calculated luteolin, mass but of the luteolin position O of-methoxide the methylation [M + wasH] m/z unknown. 301.0712 (Figure 2B). This confirmed the product to be O-methylated luteolin, but the position of the methylation was unknown.

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Figure 2. (A) HPLC-PDA chromatogram analyses of (i) the biotransformation reaction sample Figurecompared 2. (A to) (iiHPLC) the- controlPDA chromatogram reaction sample, analyses (iii) luteolin of (i) the standard, biotransformation and (iv) the purifiedreaction product. sample compared(B) HRQTOF-ESI/MS to (ii) the control confirmation reaction ofsample, methoxylated (iii) luteolin luteolin standard and its, and UV–vis (iv) the analysis. purified product. (B) HRQTOF-ESI/MS confirmation of methoxylated luteolin and its UV–vis analysis. 2.2. Purification and Structural Elucidation of the Metabolite 2.2. PurificationThe biotransformation and Structural wasElucidation carried of outthe M inetabolite largevolume to collect luteolin-O-methoxide for structuralThe biotransformation elucidation and further was carried biological out activity in large tests. volume The to biotransformation collect luteolin-O reaction-methoxide mixture for structuralwas extracted elucidation with a and double further volume biological of ethyl activity acetate, tests. and The the biotransformation crude extracted reaction was subjected mixture to wasprep-HPLC extracted for with purification. a double volume The luteolinof ethylO -methoxideacetate, and derivativethe crude extracted was purified was tosubjected the level to ofprep 98%- HPLCpurity for(Figure purification.2A (iv)) .The The luteolin purified-O product-methoxide was derivative dried by lyophilization,was purified to dissolvedthe level of in 98% deuterated purity (Fig. 2A (iv)). The purified product was dried by lyophilization, dissolved in deuterated dimethyl

Catalysts 2019, 9, 112 5 of 15 dimethyl sulfoxide, and analyzed by nuclear magnetic resonance (NMR) spectroscopy (900 MHz) for structural elucidation. The 1H NMR analysis of the purified product showed multiple peaks between 3.0 ppm and 13.0 ppm. When compared to the 1H-NMR with the standard luteolin, a distinct singlet peak at δ 3.90 ppm was found in the product, which is absent in luteolin. Most of the other peaks were at the corresponding places of luteolin except a singlet peak for 30-OH, which was missing (Figure3A and Table1). The peak appeared at δ 3.90 ppm, singlet, corresponding to the chemical shift position of the methoxy protons showing an integration value of ~3.0, as expected for the methoxy functional group present in the sample. Moreover, the intact 5-OH, 7-OH, and 40-OH singlet proton peaks at δ 12.96, 10.85, and 9.98 ppm, respectively, but missing 30-OH (which usually appears at δ 9.50 ppm in luteolin) gave a shred of solid evidence that the luteolin biotransformed metabolite could be 30-O-methoxy luteolin (chrysoeriol). The same sample was further analyzed by 13C-NMR. The total peaks of the compound displayed 16 carbons, one carbon more than that of luteolin. A distinct carbon peak at the upfield region at δ 56.4 ppm resembled the suspected peak for methoxy carbon (Figure3B). The other peaks for the carbon of the product were similar to the exogenously fed luteolin. The assignment of 1H and 13C-NMR was also compared with the previously published reports, which showed exactly similar spectral values to chrysoeriol [23,24]. Furthermore, two-dimensional (2D)-NMR analyses, such as heteronuclear single quantum coherence spectroscopy (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments were performed to find the exact position of methylation. HSQC showed a cross peak illustrating a correlation between the protons (δ 3.90 ppm) and the carbon of the methoxy group (56.43 ppm). Moreover, HMBC showed a cross peak depicting the correlations between C-30 (δ 148.50 ppm) and the protons of the methoxy group (δ 3.90 ppm) (Figure4). There was no cross peak showing a correlation of C-40 (δ 151.20 ppm) with the methoxy protons in both the HSQC and HMBC analyses (Figures S1 and S2). Along with these pieces of evidence from various NMR analyses, the structure of the compound was confirmed to be chrysoeriol.

1 13 Table 1. H and C-NMR of chrysoeriol (DMSO-d6, 900 MHz).

1H-NMR 13C-NMR Position of Protons Position of Carbons 5-OH 12.96 1 - 7-OH 10.85 2 164.15 40-OH 9.98 3 103.68 60 7.57 (d, J = 2.19 Hz) 4 182.28 20 7.56 (s) 5 161.90 50-H 6.94 (d, J = 8.33 Hz) 6 99.29 3-H 6.90 (s) 7 164.60 8-H 6.52 (d, J = 2.1 Hz) 8 94.52 6-H 6.21 (d, J = 2.15 Hz) 9 157.80 10 104.14 10 121.99 20 110.66 30 148.50 30-OCH 3.90 (s) 3 40 151.20 50 116.24 60 120.83 O-CH3 56.43 Catalysts 2019, 9, 112 6 of 15 Catalysts 2018, 8, x FOR PEER REVIEW 6 of 15

FigureFigure 3.3. NMRNMR analysis.analysis. ( (AA)) 11H-NMRH-NMR and and (B (B) )1313C-NMRC-NMR spectra spectra of ofchrysoeriol chrysoeriol isolated isolated after after the the biotransformationbiotransformation ofof luteolinluteolin from recombinant E.E. coli coli harboringharboring the the PET28a+SpnK PET28a+SpnK plasmid. plasmid.

2.3.2.3. Antimicrobial Antimicrobial Activityactivity of of Lut Luteolin,eolin, Chrysoeriol, Chrysoeriol, and and Diosmetin Diosmetin ChrysoeriolChrysoeriol waswas collected in a a sufficient sufﬁcient amount amount from from a amicrobial microbial biotransformation biotransformation reaction reaction andand puriﬁed,purified, and a a powder powder form form of of this this compound compound was was used used for the for biological the biological tests. tests.Firstly, Firstly,the thecompound compound was was tested tested for for antibacterial antibacterial activity activity against against several several Gram-positive Gram-positive and and Gram-negative Gram-negative humanhuman pathogens, pathogens, as as described described in in Table Table2. The2. The antibacterial antibacterial activity activity of chrysoeriolof chrysoeriol was was compared compared with itswith close its derivatives,close derivatives, such assuch luteolin as luteolin and diosmetin.and diosmetin.

Table 2. Antibacterial activities test against Gram-positive and Gram-negative bacteria via disc diffusion assay. The zone of inhibition (diameter) due to luteolin, diosmetin, and chrysoeriol against different pathogens is noted in mm.

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Zone of Inhibition (mm) Luteolin Chrysoeriol Diosmetin

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GramTable-P 2.ositiveAntibacterial Strains activities test against Gram-positive andGram-negative bacteria via disc CatalystsdiffusionCatalystsCatalysts 2018 2018, 8 assay. ,2018 x, 8FOR, ,x 8 FOR,The PEERx FOR PEER zone REVIEW PEER REVIEW of REVIEW inhibition (diameter) due to luteolin, diosmetin, and chrysoeriol7 againstof7 15of7 15of 15 S. aureus CCARM 3640 (MRSA) 9 ± 0.2 15 ± 0.3 ND different pathogens is noted in mm. S. aureus CCARM 3089 (MRSA) 9 ± 0.12 ZoneZone Zoneof Inhibitionof14 ofInhibition ± Inhibition 0.35 (mm) (mm) (mm) ND Zone of Inhibition (mm) S. aureus CCARM 33591(MRSA) LuteolinLuteolin9Luteolin ± Luteolin0.10 ChrysoeriolChrysoeriolChrysoeriolChrysoeriolND DiosmetinDiosmetinDiosmetinDiosmetinND S. aureus CCARM 0205 (MSSA) 9 ± 0.2 9 ± 0.2 ND - - - - S. aureus CCARM 0204 (MSSA) 10 ± 0.2 9 ± 0.12 ND S. aureus CCARM 0027 (MSSA) ND ND ND Gram-PositiveGramGramGram-Positive-Positive-P Strainsositive Strains Strains Strains S. aureusaureusCCARM CCARM 3640 3090 (MRSA) (MRSA) 912± ±0.2 0.3 15 15 ±± 0.210.3 ND ND S. aureusS. aureusS. aureus CCARM CCARM CCARM 3640 3640 (MRSA)3640 (MRSA) (MRSA) 9 ± 90.2 ±9 0.2 ± 0.2 15 ±15 0.3 15± 0.3 ± 0.3 NDND ND S. aureusaureusCCARM CCARM 3089 3634 (MRSA) (MRSA) 9 ±9 ±0.12 0.1 14 14 ± 0.250.35 ND ND S.S. aureus aureusS. aureusS.CCARM aureus CCARM CCARM CCARM 33591(MRSA) 3089 3089 (MRSA)3089 (MRSA) (MRSA) 9 ± 90.120.10 ±9 0.12 ± 0.12 14 ±14 0.35 14 ND± 0.35 ± 0.35 NDND ND S. aureusaureusCCARM CCARM 0205 3635 (MSSA) (MRSA) 911± ±0.2 0.11 10 9 ± 0.150.2 ND ND S.S. aureus aureusS. aureusS.CCARM aureus CCARM CCARM CCARM 0204 33591(MRSA) (MSSA) 33591(MRSA) 33591(MRSA) 9 10 ±± 90.10 ±0.29 0.10 ± 0.10 ND 9 ±ND 0.12ND NDND ND Bacillus subtilis ATCC 6633 8 ± 0.1 9 ± 0.2 ND S.S. aureus aureusS. aureusS.CCARM aureus CCARM CCARM CCARM 0027 0205 (MSSA) 0205 (MSSA)0205 (MSSA) (MSSA) 9 ND± 90.2 ±9 0.2 ± 0.2 9 ± 90.2 ND±9 0.2 ± 0.2 NDND ND EnterococcusS. aureus CCARM faecalis 3090 (MRSA)19433 12 ±ND0.3 15ND± 0.21 ND ND S.S. aureus aureusS. aureusS.CCARM aureus CCARM CCARM CCARM 3634 0204 (MRSA) 0204 (MSSA)0204 (MSSA) (MSSA) 10 9 ± ±10 0.10.2 10± 0.2 ± 0.2 9 ± 14 90.12 ±±9 0.12 ±0.25 0.12 NDND ND Enterococcus faecalis 19434 ND ND ND S.S. aureus aureusS. aureusS.CCARM aureus CCARM CCARM CCARM 3635 0027 (MRSA) 0027 (MSSA)0027 (MSSA) (MSSA) 11 ND± 0.11ND ND ND 10 ND± 0.15ND NDND ND ± ± KocuriaBacillus subtilis rhizophillaATCC NBRC 6633 12708 88 ±0.1 0.2 9ND0.2 ND ND EnterococcusS. aureusS. aureusS. aureus CCARM faecalis CCARM CCARM19433 3090 3090 (MRSA)3090 (MRSA) (MRSA) 12 ND±12 0.3 12± 0.3 ± 0.3 15 ±15 0.21 15 ND± 0.21 ± 0.21 NDND ND GramEnterococcusS. aureusS.- aureusS.N egativeaureus CCARM faecalis CCARM CCARM Strains19434 3634 3634 (MRSA)3634 (MRSA) (MRSA) 9 ND± 90.1 ±9 0.1 ± 0.1 14 ±14 0.25 14 ND± 0.25 ± 0.25 NDND ND Kocuria rhizophilla NBRC 12708 8 ± 0.2 ND ND SalmonellaGram-NegativeS. aureusS. aureusS. aureus CCARM enterica CCARM Strains CCARM ATCC3635 3635 (MRSA)3635 14028 (MRSA) (MRSA) 11 8±11 ±0.11 11±0.2 0.11 ± 0.11 10 ±10 0.15ND 10± 0.15 ± 0.15 NDND NDND Salmonella enterica ATCC 14028 8 ± 0.2 ND ND BacillusBacillusBacillus subtilis subtilis subtilis ATCC ATCC ATCC 6633 6633 6633 8 ± 80.1 ±8 0.1 ± 0.1 9 ± 90.2 ±9 0.2 ± 0.2 NDND ND Klebsiella pneumonia pneumoniaATCC ATCC 10031 10031 NDND ND ND ND ND EscherichiaEnterococcusEnterococcusEnterococcus coli coliATCC faecalis ATCC faecalis 25922faecalis 19433 25922 19433 19433 ND NDND ND NDNDND ND NDND NDND Proteus hauseri NBRC 3851 ND 14 ± 0.2 ND ProteusEnterococcusEnterococcusEnterococcus hauseri faecalis NBRC faecalis faecalis 19434 3851 19434 19434 NDNDND ND ND14ND ± 0.2ND NDND NDND ND: not detected. KocuriaKocuriaKocuria rhizophilla rhizophilla rhizophilla NBRC NBRC NBRC 12708 12708 12708 ND:8 ± 80.2 not±8 0.2 ± detected. 0.2 NDND ND NDND ND GramGramGram-Negative-N-egativeNegative Strains Strains Strains SalmonellaSalmonellaSalmonella enterica enterica enterica ATCC ATCC ATCC 14028 14028 14028 8 ± 80.2 ±8 0.2 ± 0.2 NDND ND NDND ND KlebsiellaKlebsiellaKlebsiella pneumonia pneumonia pneumonia ATCC ATCC ATCC 10031 10031 10031 NDND ND NDND ND NDND ND EscherichiaEscherichiaEscherichia coli coli ATCC coli ATCC ATCC 25922 25922 25922 NDND ND NDND ND NDND ND ProteusProteusProteus hauseri hauseri hauseri NBRC NBRC NBRC 3851 3851 3851 NDND ND 14 ±14 0.2 14± 0.2 ± 0.2 NDND ND ND:ND: notND: notdetected. not detected. detected.

FigureFigure 4.4. ZoomedZoomed-in-in view view of of (A ()A HMBC) HMBC and and (B) HSQC (B) HSQC-DEPT-DEPT analyses analyses of chrysoeriol of chrysoeriol.. The cross The peak cross peakshows shows the correlation the correlation of the of proton the proton of methoxy of methoxy-carbon-carbon with C with-3′ of C-3the0 benzeneof the benzene ring in ringHMBC. in HMBC.The Thecross cross peak peak in HSQC in HSQC shows shows the correlation the correlation between between the proton the proton of theof methoxy the methoxy-proton-proton group groupand the and thecarbon carbon of the of themethyl methyl group group attached attached at C3’ at- C3’-OH.OH.

2.3.1.2.3.1. Disc DiscDiffusion Diffusion AssayAssay ForForFigure theFiguretheFigure 4. antibacterialantibacterial Zoomed 4. Zoomed 4. Zoomed-in view-in test, -viewin of view ( aAof ) paper-discpaper (ofHMBCA )( AHMBC) -HMBC discand and (Bdiffusion diffusionand) (HSQCB )( BHSQC) HSQC-DEPT assay- assayDEPT- DEPTanalyses wasanalyses was analyses perfoof performed. chrysoeriol of chrysoeriolrmed.of chrysoeriol .All The All. The threecross. The three cross peak crosscompounds, peak compounds, peak luteolin,luteolin,shows chrysoeriol, chrysoeriolshowsshows the thecorrelation the correlation ,correlation andand of aa similartheof theofproton the proton 4′ 4proton 0-of-OO methoxy- -methoxyofmethoxy methoxyof methoxy-carbon derivative-carbon derivative-carbon with with C with- of3′ C ofof -luteolin 3′C luteolinthe -of3′ theofbenzene the benzene (diosmetin), benzene (diosmetin), ring ring in ring HMBC. in were HMBC.in wereHMBC. The prepared The prepared The at at cross peakcross in peak HSQC in HSQC shows shows the correlation the correlation between between the proton the proton of the ofmethoxy the methoxy-proton-proton group groupand the and the the concentrationcross peak of in 10 HSQC mg/mL. shows Allthe thecorrelation compounds between at the the proton ﬁnal of concentration the methoxy-proton of 40 groupµg/disc and the (4 µL) were carboncarboncarbon of the of theofmethyl the methyl methyl group group groupattached attached attached at C3’ at -C3’atOH. C3’-OH. -OH. added to each disc, which was placed over Mueller-Hinton agar (MHA) plates spread with bacterial strains.2.3.1.2.3.1. 2.3.1.Disc The Disc D diameterDisciffusion Diffusion Diffusion A ofssay A thessay A ssay zone of inhibition was measured along with the paper disc after a clear zone of inhibition was observed. The comparative study showed that the diameter of the zone of ForFor theFor the antibacterial the antibacterial antibacterial test, test, atest, paper a paper a paper-disc-disc -diffusiondisc diffusion diffusion assay assay assaywas was perfowas perfo rmed.performed.rmed. All All three All three threecompounds, compounds, compounds, inhibition was greater for chrysoeriol than for luteolin against all the Gram-positive bacteria except luteolin,luteolin,luteolin, chrysoeriol chrysoeriol chrysoeriol, and, and a, andsimilar a similar a similar 4′-O 4′--methoxy O4′-Omethoxy-methoxy derivative derivative derivative of luteolinof ofluteolin luteolin (diosmetin), (diosmetin), (diosmetin), were were preparedwere prepared prepared at at at S. aureus CCARM 0027 (MSSA), Enterococcus faecalis 19433, and Enterococcus faecalis 19434 (Table2).

Catalysts 2018, 8, x FOR PEER REVIEW 8 of 15 the concentration of 10 mg/mL. All the compounds at the final concentration of 40 µg/disc (4 µL) were added to each disc, which was placed over Mueller-Hinton agar (MHA) plates spread with bacterial strains. The diameter of the zone of inhibition was measured along with the paper disc after aCatalysts clear zone2019, of9, 112inhibition was observed. The comparative study showed that the diameter of the zone8 of 15 of inhibition was greater for chrysoeriol than for luteolin against all the Gram-positive bacteria except S. aureus CCARM 0027 (MSSA), Enterococcus faecalis 19433, and Enterococcus faecalis 19434 (Table 2). Interestingly, luteolin inhibited Gram-negative bacterial strains, such as Salmonella enterica ATCC 14028 Interestingly, luteolin inhibited Gram-negative bacterial strains, such as Salmonella enterica ATCC and Kocuria rhizophila NBRC 12708, but chrysoeriol did not show any activity against them. Chrysoeriol 14028 and Kocuria rhizophila NBRC 12708, but chrysoeriol did not show any activity against them. showed much better activity against Proteus hauseria NBRC 3851, but luteolin and diosmetin both were Chrysoeriol showed much better activity against Proteus hauseria NBRC 3851, but luteolin and negative (Table2). diosmetin both were negative (Table 2). 2.3.2. Measurements of Colony-Forming Units 2.3.2. Measurements of Colony-Forming Units The growth-inhibition activity of luteolin and chrysoeriol was investigated in liquid by measuring The growth-inhibition activity of luteolin and chrysoeriol was investigated in liquid by the number of colony-forming units (log CFU/mL) in each compound-treated sample of the measuring the number of colony-forming units (log CFU/mL) in each compound-treated sample of two pathogens S. aureus 3640 (MRSA), a Gram-positive strain, and Proteus hauseri NBRC 3851, the two pathogens S. aureus 3640 (MRSA), a Gram-positive strain, and Proteus hauseri NBRC 3851, a a Gram-negative pathogen. These two pathogens were selected based on the largest zone of inhibition Gram-negative pathogen. These two pathogens were selected based on the largest zone of inhibition observed from the disc diffusion assay for the abovementioned pathogens (Table2). As depicted in observed from the disc diffusion assay for the abovementioned pathogens (Table 2). As depicted in Figures5 and6, the CFUs were measured at 2, 4, and 6 h. The number of CFUs remained almost Figure 5 and Figure 6, the CFUs were measured at 2, 4, and 6 h. The number of CFUs remained almost constant in the control samples until 6 h (Figure5). However, the decrease in the number of CFUs was constant in the control samples until 6 h (Figure 5). However, the decrease in the number of CFUs seen in both the luteolin- and chrysoeriol-treated bacterial samples. For the luteolin-treated S. aureus was seen in both the luteolin- and chrysoeriol-treated bacterial samples. For the luteolin-treated S. (Figures5b and6A), the number of bacteria slightly decreased over time. However, it was nil when aureus (Figure 5b and Figure 6A), the number of bacteria slightly decreased over time. However, it the same number of bacteria was treated with chrysoeriol within 2 h. A similar result was observed was nil when the same number of bacteria was treated with chrysoeriol within 2 h. A similar result when the same set of compounds were used for the treatment of P. hauseri (Figures5a and6B). Luteolin was observed when the same set of compounds were used for the treatment of P. hauseri (Figure 5a killed Gram-positive S. aureus more than Gram-negative P. hauseri. However, chrysoeriol killed both and Figure 6B). Luteolin killed Gram-positive S. aureus more than Gram-negative P. hauseri. Gram-positive S. aureus and Gram-negative P. hauseri within 2 h. However, chrysoeriol killed both Gram-positive S. aureus and Gram-negative P. hauseri within 2 h.

(a)

Figure 5. Cont.

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(b)

(b) Figure 5. Colony-forming unit (CFU) count of chrysoeriol- and luteolin-treated microbes. (a) Proteus Figurehauseri 5.. ControlColony-forming (A) 0 h, (B unit) 2 h (CFU), (C) 4 count h, and of ( chrysoeriol-D) 6 h. Luteolin and-treated luteolin-treated (E) 2 h, ( microbes.F) 4 h, and ( a()GProteus) 6 h. hauseriChrysoeriolFigure. Control 5. Colony-treated (A-)forming ( 0H h,) 2 ( Bh), unit( 2I) h, 4 (CFU) (hC, )and 4 h, count(J and) 6 h of (. D( bchrysoeriol)) 6Staphylococcus h. Luteolin-treated- and aureusluteolin 3640 (E-treated) 2 (MRSA) h, (F microbes.) 4. h,Control and (a ( G)( AProteus)) 6 0 h. Chrysoeriol-treatedh,hauseri (B) 2 h,. Control (C) 4 h, (andA (H) 0)( D 2h) h,, 6(B (h.I)) 2Luteolin 4 h h,, and(C) 4- (treatedJ h), 6 and h. ( b( E()D)Staphylococcus 2) 6h, h (.F )Luteolin 4 h, and aureus- treated(G) 63640 h. ( ChrysoeriolE) (MRSA). 2 h, (F) Control4- treatedh, and ( A((HG)) 0) 26 h, h. (Bh,Chrysoeriol) ( 2I) h, 4 (h,C )and 4 h,- (treatedJ and) 6 h. (D ()H 6) h.2 h Luteolin-treated, (I) 4 h, and (J) (6E h). 2 ( h,b) (StaphylococcusF) 4 h, and (G )aureus 6 h. Chrysoeriol-treated 3640 (MRSA). Control (H) 2(A h,) 0 (I)h, 4 ( h,B) and 2 h, ( J()C 6) 4 h. h, and (D) 6 h. Luteolin-treated (E) 2 h, (F) 4 h, and (G) 6 h. Chrysoeriol-treated (H) 2 h, (I) 4 h, and (J) 6 h.

Figure 6. The antibacterial activity of luteolin and chrysoeriol against (A) Staphylococcus aureus and Figure 6. The antibacterial activity of luteolin and chrysoeriol against (A) Staphylococcus aureus and (B) Proteus hauseri. The number of colonies is expressed in terms of log CFU/mL. (B) Proteus hauseri. The number of colonies is expressed in terms of log CFU/mL. 2.3.3. MeasurementFigure 6. The antibacterial of MIC Values activity of luteolin and chrysoeriol against (A) Staphylococcus aureus and 2.3.3. Measurement(B) Proteus hauseri of .MIC The numbervalues of colonies is expressed in terms of log CFU/mL. The minimum inhibitory concentration (MIC) values of both luteolin and chrysoeriol were measuredThe minimum for two selected inhibitory pathogens, concentrationS. aureus (MIC)3640 values(MRSA) of and bothP. hauseriluteolinNBRC and chrysoeriol 3851. The resultswere 2.3.3. Measurement of MIC values showedmeasured MIC for values two selected of 20 µ pathogens,g/mL and 1.25S. aureusµg/mL 3640 for (MRSA) luteolin and and P. chrysoeriol, hauseri NBRC respectively, 3851. The results against S.showed aureusThe MIC3640 minimum values (MRSA). of inhibitory 20 Similarly, µg/mL and concentration the 1.25 MIC µg/mL values (MIC) for were luteolin values 50 µ andg/mL of chrysoeriol both and luteolin 30 µ,g/mL respectively, and against chrysoeriol againstP. hauseri wereS. NBRCaureusmeasured 38513640 for(MRSA).for luteolintwo selected Similarly, and chrysoeriol, pathogens, the MIC values respectivelyS. aureus were 3640 50 (Figure µg/mL(MRSA) S3). and and The 30 P.µg/mL results hauseri against showed NBRC P. a 3851. hauseri 16 times The NBRC lowerresults MIC3851showed valuefor luteolin MIC of chrysoeriol values and chrysoeriolof 20 than µg/mL that, respectively and of parent 1.25 µg/mL luteolin (Figure for against Sluteolin3). TheS. resultand aureus. chrysoeriols showedIn contrast, a, respectively,16 times the MIC lower valueagainst MIC of S. chrysoeriolvalueaureus of 3640 chrysoeriol was (MRSA). only 1.7than Similarly, times that lower of the parent MIC for P. values luteolin hauseri were. against 50 µg/mL S. aur andeus. 30In µg/mL contrast, against the MICP. hauseri value NBRC of chrysoeriol3851 for luteolin was only and 1.7 chrysoeriol times lower, respectively for P. hauseri (Figure. S3). The results showed a 16 times lower MIC 3.value Discussion of chrysoeriol than that of parent luteolin against S. aureus. In contrast, the MIC value of 3. Discussion chrysoeriolNumerous was pathogens only 1.7 times including lower both for Gram-negativeP. hauseri. and Gram-positive bacteria are resistant to multipleNumerous antibiotics. pathogens Pathogens including always both tend Gram to ﬁnd-negative ways toand resist Gram the-positive treatment. bacteria According are resistant to the to list 3. Discussion ofmultiple threatening antibiotics. pathogens Pathogens given always by the Worldtend toHealth find ways Organization to resist the (WHO), treatment.Proteus According, E. coli, to Klebsiella the list , andof threateningPseudomonasNumerous pathogens fallpathogens under given theincluding deadlyby the Worldboth infectious Gram Health- pathogens,negative Organization and and Gram methicillin-resistant(WHO),-positive Proteus bacteria, E. coli,S. are aureusKlebsiella resistantfalls, to underandmultiple Pseudomonas high antibiotics. risk, thus fall suggestingunderPathogens the deadly always a need infectious totend develop to find pathogens, new ways antibiotics to andresist methicillin the for treatment. their- controlresistant According ( www.who.intS. aureus to fallsthe list ). Thus,underof threatening there high isrisk an ,pathogens urgentthus suggesting need given to develop aby need the toWorld antibiotics develop Health new that Organization antibiotics are effective for (WHO), against their control multipleProteus (www.who.int, E. drug-resistant coli, Klebsiella). , and Pseudomonas fall under the deadly infectious pathogens, and methicillin-resistant S. aureus falls under high risk, thus suggesting a need to develop new antibiotics for their control (www.who.int).

Catalysts 2019, 9, 112 10 of 15 pathogens. Recent reports show increasing infections by the aforementioned pathogens in hospitals. In this context, the development and discovery of new antibacterial agents effective against a broad spectrum of multi-drug-resistant Gram-positive and Gram-negative pathogens is of prime importance. The search for new antibiotics takes a significantly long time and requires several approval and clinical trial steps. Thus, many researchers develop compounds based on structure-activity relationships (SAR) and optimize them for better activities against particular target pathogen based on the previously known structure and their activities [25,26]. Yet, the development of molecules by post-modifications and other derivatization is another widely accepted approach for developing novel molecules with potent activities [27–29]. Recently, diverse plant and microbial secondary metabolites have been modified by enzymatic and microbial biotransformation with the aim of developing novel agents with stronger activities against diverse pathogens, cancers, and other communicable and non-communicable diseases. When compared with chemical synthesis, enzymatic synthesis is the most advantageous. Chemical methods are long, health hazardous, and result in more toxic intermediates. However, whole-cell biotransformation uses the fully grown cells, which contain all the needed enzymes and cofactors for the chemical reaction to occur within the cell. The adequate concentration of co-factors and enzymes provides the favorable conditions that modulate the activity of multi-enzymatic complexes, resulting in the increase of reaction conversion rates [30]. The production of antibiotics via microbial biotransformation not only involves supply of purified precursor molecules but may also encompass complex substrates that can be biotransformed into compounds rich in active substances [28]. In this study, a simple approach of regiospecific modification of luteolin, a plant metabolite, was applied to modify it into a methyl-group-conjugated chrysoeriol derivative. A sugar O-methyltransferase gene spnK from actinomycetes S. spinosa was cloned into an E. coli BL21 (DE3) host for selective biotransformation of luteolin. As a result, we successfully achieved chrysoeriol, a 30-O-methylated derivative in the culture broth. A similar derivative, but methyl-group conjugated at the 40-O-position of luteolin, diosmetin, is abundantly present in plants as diosmin. However, the occurrence of chrysoeriol is limited. Thus, biosynthesis of this molecule is of significant interest among biochemists. Moreover, the molecule is active against a wide range of Gram-negative and Gram-positive pathogens, including WHO-listed critical and high-risk pathogens such as MRSA and Proteus. A study by Teffo et. al. [31] showed four methyl-ether derivatives of kaempferol, such as 3, 5, 7-trihydroxy-40-methoxyflavone, 5, 7, 40-trihydroxy-3, 6-dimethoxyflavone, 5, 7-dihydroxy-3, 6, 40-trimethoxyflavone (santin), and 5-hydroxy -3, 7, 40-trimethoxyflavone, from the leaf powder of Dodonaea viscosa Jacq. var. angustifolia. These molecules showed MIC values ranging from 16 µg/mL to above 250 µg/mL against S. aureus, Enterococcus faecalis, E. coli, and Pseudomonas aeruginosa. Similarly, Choi et. al. [32] recently developed a bio-renovation technique to produce diverse derivatives of genistein using whole cells of Bacillus amyloliquefaciens KCTC 13588. Among the isolated four derivatives, 40-O-isopropyl genistein exhibited antibacterial activity against MRSA and MSSA strains with better activities (MIC 8 µg/mL or above) than genistein. However, the 30-O-methylated luteolin derivative, also called chrysoeriol, exhibited a significantly lower MIC value of 1.25 µg/mL against S. aureus CCARM 3640 (MRSA), which is 16 times lower than that of its parent compound and only 2.5 times higher than ampicillin [33]. It is not only active against MRSA, but it is also active against seven other Gram-positive strains (MRSA and MSSA) and a Gram-negative P. hauseri strain (Table2). Among all the tested organisms, chrysoeriol exhibited better activity than luteolin in the disc diffusion assay when a 40 µg/disc compound dissolved in DMSO was loaded over a sterile disc. We also compared the activity using diosmetin under identical conditions and concentrations. However, it did not show any activity against the tested pathogens. The evidence revealed a significance of modification of luteolin at the 30-OH, particularly by the alkyl group. However, a previous report showed the significance of free 30, 40, 50-trihydroxy groups in the B-ring and of the free 3-OH group for antibacterial activity [31]. Flavonoids inhibited DNA synthesis in Gram-negative P. vulgaris and Catalysts 2019, 9, 112 11 of 15

RNA synthesis in Gram-positive S. aureus [34]. Other potential targets of polyphenols have long been studied [34,35]. The reports also provide evidence that the lowest MIC value reported against S. aureus is (0.06–2.0) µg/mL by panduratin A [36]. Similarly, other bioactive polyphenols include epigallocatechin gallate, which killed 56 clinical isolates of Helicobacter pylori, causative agents of ulcers and gastric cancers, at MIC values of 100 µg/mL [37]. All such evidence shows the anti-microbial potential of diverse polyphenols isolated from plants. However, selective production of these molecules from native plants suffers from several difficulties, such as the high consumption of both time and plant biomass, low yield, and the difficulty of extraction and purification. Thus, microbial production of such bioactive molecules by simple biotransformation using engineered microbial cells is always superlative. In this study, we produced bioactive chrysoeriol from luteolin by a simple biotransformation approach using an E. coli BL21 (DE3) host harboring the spnK gene. The metabolite showed significantly improved antibacterial activity against a wide range of Gram-positive MRSA and MSSA and Gram-negative P. hauseri pathogens. Therefore, chrysoeriol has great potential as an antibiotic against superbugs.

4. Materials and Methods

4.1. General Procedures Luteolin and diosmetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was purchased from GeneChem, Inc. (Daejeon, Korea). All the other chemicals and reagents used in this study were of the highest chemical grade. E. coli cells were grown in Luria-Bertani (LB) broth or an agar medium supplemented with ampicillin 100 µg/mL. Previously constructed plasmid pET32a+ SpnK [19] was used for transformation into E. coli BL21 (DE) for biotransformation in LB medium. Pathogenic strains such as Staphylococcus. aureus CCARM 3640 (MRSA), S. aureus CCARM 3089 (MRSA), S. aureus CCARM 33591 (MRSA), S. aureus CCARM 0205 (MSSA), S. aureus CCARM 0204 (MSSA), S. aureus CCARM 0027 (MSSA), S. aureus CCARM 3090 (MRSA), S. aureus CCARM 3634 (MRSA), and S. aureus CCARM 3635 (MRSA), Salmonella enterica ATCC 14028, Kocuria rhizophilla NBRC 12708, Bacillus subtilis ATCC 6633, Proteus hauseri NBRC 3851, Enterococcus faecalis 19433, E. faecalis 19434, Klebsiella pneumonia ATCC 10031, and E. coli ATCC 25922 were obtained from Professor Seung-Young Kim (Sun Moon University, Korea) [32].

4.2. Whole-Cell Biotransformation and Validation A recombinant strain of E. coli BL21 (DE3) harboring pET32a+ SpnK was inoculated in 5 mL of LB broth medium and incubated at 37 ◦C supplementing ampicillin antibiotic (100 mg/mL) for the selection and maintenance of the recombinant strain. Out of the 5 mL culture, 500 µL as seed culture was transferred to the same medium (50 mL culture volume) in two different shake flasks and incubated at 37 ◦C. One flask culture was induced with 0.5 mM IPTG after the optical density ◦ at 600 nm (OD600nm) reached 0.7; the other was not induced, but they were both incubated at 20 C for 20 h in a similar condition. The culture without IPTG induction was a control for the experiment. Both cultures were supplemented with 200 µM of luteolin as substrate dissolved in dimethylsulfoxide (DMSO) and were incubated at 20 ◦C for 48 h at 200 rpm for the bioconversion experiment. Later, a culture broth of 500 µL from each flask was transferred to micro-centrifuge tubes, and an equal volume of chilled high-grade methanol was added, mixed vigorously by vortex, and centrifuged at 13,475× g for 15 min. The supernatant separated from the cell debris was analyzed by a high-performance liquid chromatography coupled with a photodiode array (HPLC-PDA) detector, followed by confirmation via high-resolution quantitative time-of-flight electrospray-ionization mass spectrometry (HRQTOF-ESI-MS). For collecting a sample to characterize structurally, the biotransformation experiment was done using two 2-L shake flasks, each containing 500 mL of culture. The pure fraction of the compound was collected via preparative HPLC. Catalysts 2019, 9, 112 12 of 15

4.3. Analytical Methods The reverse-phase high-performance liquid-chromatography photo-diode array (HPLC-PDA) analysis was performed with a C18 column (Mightysil RP-18 GP (4.6 mm × 250 mm, 5 µm) connected to a PDA (290 nm) using binary conditions of H2O (0.1% trifluoroacetic acid buffer) and 100% acetonitrile (ACN) at a flow rate of 1 mL/min for 35 min. At this condition, the ACN concentration began from 20% (0–5 min), 70% (5–20 min), 100% (20–30 min), 50% (30–33 min), and 20% (33–35 min). The purification of the compounds was carried out by preparative HPLC with a C18 column (YMC-Pack ODS-AQ, 250 mm × 20 mm ID., 10 µm) connected to a UV detector (290 nm) using a 36-min binary program with ACN of 20% (0–5 min), 50% (5–12 min), 70% (12–20 min), 90% (20–28 min), 50% (28–33 min), and 20% (33–36 min) at a flow rate of 10 mL/min. The HRQTOF-ESI-MS was performed in positive ion mode using an Acquity mass spectrometer (Waters, Milford, MA, USA), which was coupled with a Synapt G2-S system (Waters). However, for nuclear magnetic resonance (NMR) studies, a purified molecule was dissolved in hexadeuterodimethyl-sulfoxide (DMSO-d6) from Sigma-Aldrich. The compound was characterized by a 900-MHz NMR spectrometer equipped with the TCI CryoProbe (5 mm). In addition to 1H and 13C, two-dimensional NMRs, such as heteronuclear single-quantum correlation [HSQC] and heteronuclear multiple-bond correlation [HMBC], were performed for structural confirmation.

4.4. Biological Activities

4.4.1. Disc Diffusion Assay The antibacterial activity of the newly synthesized compound was ﬁrst examined by disc diffusion assay using the aforementioned microbial strains cultured in a Mueller Hinton agar (MHA) plate (Difco, Baltimore, MD, USA). Paper discs of 7-mm diameter were prepared and autoclaved. The discs were placed in each plate in a marked region after spreading the pathogen uniformly. Then, 4 microliters of 10 µg/mL of luteolin, diosmetin, and chrysoeriol were added to each disc, and the plates were kept for bacterial growth in a 37 ◦C incubator. The zone of inhibition was observed after the pathogen growth was visible. The diameter of the zone of inhibition was noted for each pathogen. Dimethyl sulfoxide (DMSO) was used as a control for the zone of inhibition, as all the compounds were dissolved in DMSO.

4.4.2. Measurement of the Colony-Forming Unit (CFU) The colony-forming unit was observed for two pathogens, S. aureus 3640 (MRSA) and Proteus hauseri NBRC 3851. The pathogens were cultured until the O.D600 reached around 0.5. The cells were taken by centrifugation at 3000 rpm for 10 min, and the aliquot was removed. The cells were suspended in 5 mL of phosphate buffer (pH 7.0). Then, 1 mL of cells in phosphate buffer was taken into separate polypropylene tubes, in which 40 µg/mL of each compound was added separately. The result was compared with standard luteolin and the control (no compound added), which were also prepared under the identical conditions. The experimental cells were incubated at 37 ◦C in an incubator under shaking condition. Samples were taken at 0 min, 2 h, 4 h, and 6 h for each of the luteolin, chrysoeriol, and control. The sample was appropriately diluted and plated on MHA and incubated at 37 ◦C in a plate incubator for overnight growth. The colony-forming units (CFU) were counted the following day.

4.4.3. Measurement of Minimum Inhibitory Concentration (MIC) Minimum inhibitory concentration (MIC) was also calculated for two strains, S. aureus 3640 (MRSA), which Gram-positive, and Proteus hauseri NBRC 3851, which is a Gram-negative pathogen. The pathogens were cultured until they reached O.D600 of 0.6. LB broth (1 mL/tube) was dispensed into the 15-mL polypropylene tube. Different concentrations (80 µg/mL, 60 µg/mL, 50 µg/mL, 40 µg/mL, 30 µg/mL, 20 µg/mL, 10 µg/mL, 5 µg/mL, 2.5 µg/mL, 1.25 µg/mL, 625 ng/mL, 312.5 ng/mL, 156 ng/mL, and 78 ng/mL) of the compounds (chrysoeriol and luteolin) were added into each tube, Catalysts 2019, 9, 112 13 of 15

in which 20 µL of inoculum were inoculated from the culture of O.D600 of 0.6. The tubes were kept for growth at 37 ◦C. After 16 h of incubation, growth (turbidity) was monitored by the unaided eye, and the MIC was noted. The growth was compared to sterile MH broth and culture without antibacterial compounds as controls. The test was repeated three times to conﬁrm the MIC value.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/9/2/112/s1, Figure S1. HMBC analysis of chrysoeriol (DMSO-d6,900 MHz); Figure S2. HSQC-DEPT analysis of chrysoeriol (DMSO-d6,900 MHz); and Figure S3. Culture tubes showing inhibition at different concentration: (A) Staphylococcus aureus 3640 (MRSA) (i) chrysoeriol and (ii) luteolin; (B) Proteus hauseri NBRC 3851 (i) chrysoeriol and (ii) luteolin. Author Contributions: P.P. and P.B. prepared the compound. P.B. performed the antimicrobial test. P.B., P.P., and R.P.P. prepared the manuscript. R.P.P., P.P., and P.B. analyzed data. R.P.P. and J.K.S. supervised the work. Funding: This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant no: PJ013137), Rural Development Administration, Republic of Korea. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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