Regioselective Hydroxylation of Naringin Dihydrochalcone to Produce Neoeriocitrin Dihydrochalcone by CYP102A1 (BM3) Mutants

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Article Regioselective Hydroxylation of Naringin Dihydrochalcone to Produce Neoeriocitrin Dihydrochalcone by CYP102A1 (BM3) Mutants

1, 1, 1 2 1,3 Thi Huong Ha Nguyen †, Su-Min Woo †, Ngoc Anh Nguyen , Gun-Su Cha , Soo-Jin Yeom , Hyung-Sik Kang 1,3,* and Chul-Ho Yun 1,3,*

1 School of Biological Sciences and Biotechnology, Graduate School, Chonnam National University, Yongbong-ro 77, Gwangju 61186, Korea; [email protected] (T.H.H.N.); [email protected] (S.-M.W.); [email protected] (N.A.N.); [email protected] (S.-J.Y.) 2 Namhae Garlic Research Institute, 2465-8 Namhaedaero, Gyeongsangnamdo 52430, Korea; [email protected] 3 School of Biological Sciences and Technology, Chonnam National University, Yongbong-ro 77, Gwangju 61186, Korea * Correspondence: [email protected] (H.-S.K.); [email protected] (C.-H.Y.); Tel.: +82-62-530-2195 (H.-S.K.); +82-62-530-2194 (C.-H.Y.) These authors equally contributed. † Received: 23 June 2020; Accepted: 21 July 2020; Published: 23 July 2020

Abstract: Naringin dihydrochalcone (DC) is originally derived from the flavonoid naringin, which occurs naturally in citrus fruits, especially in grapefruit. It is used as an artificial sweetener with a strong antioxidant activity with potential applications in food and pharmaceutical fields. At present, enzymatic and chemical methods to make products of naringin DC by hydroxylation reactions have not been developed. Here, an enzymatic strategy for the efficient synthesis of potentially valuable products from naringin DC, a glycoside of phloretin, was developed using Bacillus megaterium CYP102A1 monooxygenase. The major product was identified to be neoeriocitrin DC by NMR and LC-MS analyses. Sixty-seven mutants of CYP102A1 were tested for hydroxylation of naringin DC to produce neoeriocitrin DC. Six mutants with high activity were selected to determine the kinetic 1 parameters and total turnover numbers (TTNs). The kcat value of the most active mutant was 11 min− 1 and its TTN was 315. The productivity of neoeriocitrin DC production increased up to 1.1 mM h− , 1 1 which corresponds to 0.65 g L− h− . In this study, we achieved a regioselective hydroxylation of naringin DC to produce neoeriocitrin DC.

Keywords: CYP102A1; naringin dihydrochalcone; neoeriocitrin dihydrochalcone; regioselective hydroxylation

1. Introduction Dihydrochalcone (DC) is a bicyclic flavonoid family with two aromatic rings and a saturated C3 bridge [1]. DC compounds are mainly found in citrus fruits, grapefruits, and apples, and they play an important role in resisting biotic or abiotic stresses in plant [2,3]. To date, more than 200 DC compounds have been identified from over 30 plant families [4]. As DC compounds show strong antioxidant activities, a large number of studies have researched the potential benefits of DC compounds to human health. They were demonstrated to be effective in preventing different physiopathological processes [3], notably diabetes [5] and bone resorption [6]. In recent years, scientists have more often been attracted by in vitro and in vivo biological activities of DC compounds.

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scientists have more often been attracted by in vitro and in vivo biological activities of DC Catalystscompounds.2020, 10, 823 2 of 14 Naringin DC (3,5-dihydroxy-4[3-(4-hydroxyphenyl)propanoyl]phenyl 2-O-(6-deoxy-α-L- mannopyranosyl)-Naringin DCβ-L (3,5-dihydroxy-4[3-(4-hydroxyphenyl)propanoyl]phenyl-glucopyranoside) (Figure 1) is known as a widely used artificial 2-O-(6-deoxy- sweetenerα-l -mannopyranosyl)-[7,8]. Naringin DCβ-l -glucopyranoside)is produced when (Figure naringin1) is is known treated as with a widely a strong used artificialbase, such sweetener as potassium [ 7,8]. Naringinhydroxide, DC and is produced then catalytically when naringin hydrogenated. is treated Naringin with a strong is a flavanone-7- base, such asO potassium-glycoside hydroxide,between the andflavanone then catalytically naringenin hydrogenated. and the disaccharide Naringin isneoh a flavanone-7-esperidose.O -glycosideNaringin DC between has thea sweet flavanone value naringeninapproximately and the300 disaccharidetimes higher neohesperidose.than that of sucrose Naringin [9]. Naringin DC has DC a sweethas high value antioxidant approximately activity, 300which times performs higher than better that free-radical of sucrose [9 ].scavenging Naringin DCthan has its high corresponding antioxidant activity,flavanone which naringin performs [10]. betterBesides, free-radical naringin scavenging DC is a glycoside than its correspondingof phloretin that flavanone shows an naringin inhibitory [10]. effect Besides, on active naringin transport DC is a of glycosideglucose ofinto phloretin cells by thatSGLT1 shows and an SGLT2 inhibitory [8]. Nari effectngin on activeDC was transport suggested of glucose as a promising into cells therapeutic by SGLT1 andagent SGLT2 for [Alzheimer’s8]. Naringin DCdisease was suggestedtreatment asagainst a promising multiple therapeutic effects that agent reduce for Alzheimer’s Aβ levels, suppress disease treatmentneuroinflammation, against multiple and eenhanceffects that neurogenesis reduce Aβ levels, [8]. The suppress antioxidant neuroinflammation, and noncalorie and sweetener enhance neurogenesisabilities of naringin [8]. The DC antioxidant can make and it be noncalorie a potential sweetener compound abilities for applications of naringin in DC food, can beverages, make it be and a potentialpharmaceuticals compound [11]. for applications in food, beverages, and pharmaceuticals [11]. CytochromeCytochrome P450 P450 (CYP (CYP or or P450) P450) is is known known as as one one of of the the largest largest enzyme enzyme families families found found in in all all organisms.organisms. P450s P450s catalyze catalyze the the oxidation oxidation of variousof variou endogenouss endogenous and and xenobiotic xenobiotic compounds compounds [12]. [12]. Due Due to theirto their diversity diversity of substrates, of substrates, P450s P450s are are attractive attractive as biocatalystsas biocatalysts for for producing producing chemicals, chemicals, including including bioactivebioactive compounds compounds and and pharmaceuticals pharmaceuticals [13 ,[13,14].14]. CYP102A1 CYP102A1 (P450 (P450 BM3) BM3) from fromBacillus Bacillus megaterium megateriumis a self-suis a ffiself-sufficientcient monooxygenase monooxygenase enzyme, whichenzyme, is naturally which fusedis naturally to its redox fused partner, to its a mammalian-likeredox partner, a diflavinmammalian-like reductase. diflavin Engineered reductase. CYP102A1 Engineered mutants CYP102A1 have been mutants extensively have obtained been extensively through rationalobtained designthrough and rational directed evolutiondesign and to catalyzedirected the evolution oxidation to of catalyze several non-natural the oxidation substrates, of several environmental non-natural chemicals,substrates, and environmental pharmaceuticals chemical [15–19s,]. and It was pharmaceuticals also suggested [15–19]. that the engineeredIt was also CYP102A1suggested canthat bethe developedengineered as aCYP102A1 potential biocatalystcan be developed for biotechnology as a potent applicationsial biocatalyst [20 ,21for]. biotechnology applications [20,21].In this study, we have tried to find an enzymatic strategy for the production of products from naringinIn this DC. study, A large we set have of CYP102A1 tried to find mutants an enzymatic were used strategy for the for e ffithecient production synthesis ofof products potentially from valuablenaringin products DC. A large from set naringin of CYP102A1 DC. To themutants best of were our knowledge,used for the the efficient enzymatic synthesis hydroxylation of potentially of naringinvaluable DC products has not from previously naringin been DC. reported. To the best This of work our isknowledge, the first report the enzymatic on enzymatic hydroxylation synthesis of of neoeriocitrinnaringin DC DC, has a not major previously product ofbeen naringin reported. DC This (Figure work1). is the first report on enzymatic synthesis of neoeriocitrin DC, a major product of naringin DC (Figure 1).

Figure 1. Chemical structures of naringin DC and neoeriocitrin DC. The conversion of the substrate, Figure 1. Chemical structures of naringin DC and neoeriocitrin DC. The conversion of the substrate, naringin DC, to its corresponding product, neoeriocitrin DC, is catalyzed by CYP102A1 in the presence naringin DC, to its corresponding product, neoeriocitrin DC, is catalyzed by CYP102A1 in the of NADPH. An enzymatic reaction site on naringin DC is marked by a star. presence of NADPH. An enzymatic reaction site on naringin DC is marked by a star. 2. Results and Discussion 2. Results and Discussion 2.1. Hydroxylation of Naringin DC by CYP102A1 Mutants 2.1. Hydroxylation of Naringin DC by CYP102A1 Mutants First, to determine the ability of CYP102A1 to hydroxylate naringin DC, the catalytic activity of the wild typeFirst, (WT) to determine and its 60 mutantsthe ability [12 of,14 CYP102A1,19,22–25] towardto hydroxylate naringin naringin DC were DC, tested the at catalytic 200 µM substrateactivity of forthe 30 wild min at type 37 ◦ C(WT) (Figure and2 ).its The 60 60mutants mutants [12,14,19,22–25] used for ﬁrst screening toward werenaringin selected DC basedwere ontested our previousat 200 µM workssubstrate showing for 30 their min improved at 37 °C catalytic(Figure activities2). The 60 on mu a numbertants used of non-naturalfor first screening substrates, were such selected as natural based productson our andprevious pharmaceuticals works showing (each mutanttheir improved bears amino cata acidlyticsubstitutions activities on relative a number to WT of CYP102A1,non-natural assubstrates, summarized such in Supplementaryas natural products Table S1). and To obtainpharmaceuticals more highly (each active mutant mutants, bears the randomized amino acid DNA library obtained from the M16V2 library (see Materials and Methods) was screened using a

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Catalysts 2020, 10, x FOR PEER REVIEW 3 of 14 substitutions relative to WT CYP102A1, as summarized in Supplementary Table S1). To obtain more highly active mutants, the randomized DNA library obtained from the M16V2 library (see Materials Catalystssubstitutions2020, 10, relative 823 to WT CYP102A1, as summarized in Supplementary Table S1). To obtain more3 of 14 highlyand Methods) active mutants, was screened the random usingized a colorimetric DNA library (blue) obtained colony-based from the M16V2method library and HPLC (see Materials analysis and(Figure Methods) 3). Finally, was seven screened mutants using were a colorimetric selected from (blue) approximately colony-based 500 method blue colonies and HPLC (from analysisM524 to colorimetric(FigureM850 in 3). Supplementary Finally, (blue) colony-basedseven mutantsTable S1). method were The selected selection and HPLC fr omwas analysis approximately based (Figureon the 3500mutants’). Finally, blue colonies expression seven mutants(from levels M524 were and to −1 selectedM850catalytic in from Supplementaryactivity approximately of naringin Table DC 500 S1). hydroxylation blue The colonies selection (Figure (from was based M524 2). The on to mutants M850the mutants’ in M601 Supplementary expression(7.1 min ), levelsM620 Table and(8.2 S1). −1 −1 −1 −1 Thecatalyticmin selection), M788 activity (5.3 was ofmin based naringin), and on M850 theDC mutants’hydroxylation (8.0 min expression) showed (Figure higher levels 2). The catalytic and mutants catalytic activity M601 activity than (7.1 M16V2 min of− naringin1), (3.4 M620 min (8.2 DC). 1 1 1 hydroxylationmin−1In), M788the HPLC (5.3 (Figure min chromatogram,2−1).), Theand mutantsM850 one (8.0 M601 minor min− (7.11 )and showed min one− major ),higher M620 product catalytic (8.2 min were −activity), observed M788 than (5.3 M16V2(Figure min− ), (3.43). and Among min M850−1). 1 1 (8.0all mintestedIn −the) mutants, showedHPLC chromatogram, higher26 mutants catalytic showed one activity minor apparent than and one M16V2 but major very (3.4 product minlow −activity). were observed toward naringin(Figure 3). DC Among (<0.5 −1 allmin testedIn). the CYP102A1 HPLCmutants, chromatogram, WT 26 mutantsdid not show oneshowed minor any apparent apparent and one major butactivities. very product low Meanwhile, wereactivity observed towardseven (Figure mutants naringin3). (G1, Among DC M179, (<0.5 all −11 testedminM601,−1) mutants,. M620,CYP102A1 M221, 26mutants WT M788, did showednot and show M850) apparent any showed apparent but veryhigh activities. lowcatalytic activity Meanwhile, activity toward for seven naringin naringin mutants DC DC (< (G1,0.5(>5 minM179,min− ).) −1 −1 CYP102A1M601,(Figure M620, 2). WTMutants M221, did not G1M788, show (10.2 and anymin M850) apparent) and showed M221 activities. (9.8 high min Meanwhile,catalytic) showed activity sevenapproximately for mutants naringin (G1,three-fold DC M179, (>5 highermin M601,−1) 1 M620,(Figurecatalytic M221, 2). activity Mutants M788, toward and G1 M850)(10.2 naringin min showed −DC1) and than high M221 that catalytic of(9.8 M16V2. min activity−1) showed for naringin approximately DC (>5 minthree-fold− ) (Figure higher2). 1 1 MutantscatalyticSix G1mutantsactivity (10.2 towardwere min− selected) naringin and M221 for DC further (9.8 than min experiment that− )of showed M16V2.s to approximatelydetermine the kinetic three-fold parameters higher and catalytic total activityturnoverSix toward mutants numbers naringin were (TTNs). selected DC M16V2 than for that furtherwas of selected M16V2. experiment as it wass to determineused a template the kinetic to make parameters the DNA and library. total turnoverM179Six showed mutants numbers a medium were (TTNs). selected activity M16V2 for and was further the selected other experiments fouras it mutantswas used to determine (G1, a template M221, the M620, tokinetic make and the parametersM850) DNA had library. high and totalM179activities. turnover showed numbersa medium (TTNs). activity M16V2and the wasother selected four mutants as it was (G1, used M221, a template M620, and to M850) make thehad DNAhigh library.activities. M179 showed a medium activity and the other four mutants (G1, M221, M620, and M850) had high activities.

Figure 2. Catalytic activity of naringin DC hydroxylation by CYP102A1 mutants. The reactions contained 200 µM naringin DC as a substrate in 100 mM potassium phosphate buffer (pH 7.4) and FigureFigure 2.2. CatalyticCatalytic activityactivity of naringinnaringin DCDC hydroxylationhydroxylation by CYP102A1CYP102A1 mutants. The reactions 0.20 µM CYP102A1. NADPH-generating systems were added to initiate the reaction, and the reaction containedcontained 200200µ µMM naringinnaringin DCDC asas aa substratesubstrate inin 100100 mMmM potassiumpotassium phosphatephosphate bubufferﬀer (pH 7.4) and mixtures were incubated for 30 min at 37 °C. 0.200.20µ µMM CYP102A1. CYP102A1. NADPH-generatingNADPH-generating systemssystems werewere addedadded toto initiate the reaction, and the reaction mixturesmixtures were were incubated incubated for for 30 30 min min atat 3737◦ °C.C.

Figure 3. HPLC chromatogram of naringin DC and its products formed by CYP102A1 mutant M221. The peaks of reaction mixtures of HPLC chromatograms were identiﬁed by comparing their retention

times with those of neoeriocitrin DC (tR = 20.8 min) and naringin DC (tR = 23.4 min). The retention time of M1 was 18.4 min. Catalysts 2020, 10, x FOR PEER REVIEW 4 of 14

Figure 3. HPLC chromatogram of naringin DC and its products formed by CYP102A1 mutant M221. The peaks of reaction mixtures of HPLC chromatograms were identified by comparing their retention Catalyststimes2020 with, 10, 823 those of neoeriocitrin DC (tR = 20.8 min) and naringin DC (tR = 23.4 min). The retention4 of 14 time of M1 was 18.4 min.

2.2.2.2. Optimal Expression ofof CYP102A1CYP102A1 M221M221 MutantMutant ToTo findfind the the best bestEscherichia Escherichia coli colistrain strain for for protein protein expression expression of the of M221the M221 mutant, mutant, the plasmid the plasmid M221 (inM221 pCW (in vector)pCW vector) was transformed was transformed to a set to ofa set competent of competentE. coli E.cells coli cells (DH5 (DH5α-F’IQ,α-F’IQ, BL21, BL21, SHu SHuffleffle T7, Rosetta,T7, Rosetta, MG1655, MG1655, and and JM109). JM109). The The P450 P450 expression expression levels levels at diatff differenterent induction induction periods periods (12 (12 to 28to 28 h) andh) and incubation incubation temperatures temperatures (20 and (20 25 and◦C) 25 were °C) analyzed. were analyzed. The MG1655 The MG1655 strain had strain a higher had capability a higher ofcapability producing of producing recombinant recombinant M221 protein M221 than protein other than strains. other Thus, strains. the Thus, MG1655 the strainMG1655 was strain selected was forselected the next for experiments.the next experiments. The protein The expressionprotein expr levelession of level the M221 of the enzyme M221 enzyme was determined was determined by the 2+ 2+ evaluationby the evaluation of CO di offference CO difference spectrum, spectrum, a typical Fea typicalCO versus Fe2+·CO Fe versusspectrum Fe2+ ofspectrum the heme of group the heme [26], andgroup obtained [26], and after obtained 12 to 28after h culture12 to 28 at h 20 culture and 25 at ◦20C. and OD 60025 and°C. OD the600 protein and the expression protein expression level are proportionallevel are proportional from 12 to 20from h (Figure 12 to 420A). hAfter (Figure that, 4A the). cellAfter growth that, ratethe cell reached growth the stationaryrate reached phase, the butstationary the P450 phase, expression but the levelP450 expression showed di fflevelerence showed rates difference between 20 rates and between 25 ◦C. At20 and 20 ◦ C,25 the°C. At P450 20 expression°C, the P450 achieved expression stability achieved up to stability 28 h. Meanwhile, up to 28 h. theMeanwhile, P450 expression the P450 increased expression up increased to 24 h and up reachedto 24 h and the reached highest levelthe highest of approximately level of approximately 0.5 nmol of M2210.5 nmol/mL of (24 M221/mL h point time)(24 h atpoint 25 ◦ time)C. At at 28 25 h culture°C. At 28 at 25h culture◦C, the at P450 25 level°C, the decreased. P450 level The decreased. MG1655 strainThe MG1655 showed strain the capability showed ofthe producing capability the of highestproducing expression the highest level expression of M221 among level of tested M221 strains among (Figure tested4 stB).rains (Figure 4B).

FigureFigure 4.4. Optimal expression ofof CYP102A1 M221M221 mutantmutant inin thethe E.E. colicoli strainstrain MG1655.MG1655. ((A)) TheThe culturescultures

werewere growngrown atat 2020 oror 2525◦ °CC upup toto 2828 h.h. OD600600 andand P450 P450 concentration concentration were were measured measured at indicated time. ((BB)) CO-diCO-differenceﬀerence spectrumspectrum ofofM221 M221at at24 24h h culture culture time time at at 25 25◦ °C.C.

2.3.2.3. Characterizing a Major Product ofof NaringinNaringin DCDC byby CYP102A1CYP102A1 MutantsMutants ProductsProducts andand thethe substratesubstrate were were characterized characterized by by results results of of the the HPLC HPLC (Figure (Figure3), 3), LC-MS LC-MS (Figures (Figures5 and5 and6), and6), and NMR NMR spectroscopy spectroscopy (Figure (Figure7). Naringin 7). Naringin DC’s minor DC’s and minor major and products major made products by CYP102A1 made by mutantsCYP102A1 were mutants M1 and neoeriocitrinwere M1 DC,and respectively. neoeriocitrin The formationDC, respectively. of a monohydroxylated The formation product of as aa majormonohydroxylated product was conﬁrmed product byas LC-MSa major (Figure product5). The was minor confirmed product by (M1) LC-MS has m(Figure/z 596, which 5). The indicates minor twoproduct protons (M1) were has deletedm/z 596, from which the indicates monohydroxylated two protons product were deleted (m/z 598). from However, the monohydroxylated the M1 product 1 1 formationproduct (m/z rate 598). is too However, low (1.5 the min M1− )product compared formation to that rate of the is too major low product (1.5 min (8.4−1) compared min− ) (Figure to that3 of). Here,the major we mainly product focus (8.4 on min the−1 production) (Figure 3). of Here, the major we mainly catechol focus product. on the production of the major catecholThe product. major product was prepared by preparative HPLC (Figure6), and its chemical structure was identiﬁed by NMR (Figure7). The chemical shifts and splitting patterns of the major product’s 1H and 13C NMR spectra are shown (see also Supplementary Figure S1–S3 for NMR spectra).

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Figure 5. LC-MS analysis of naringin DC and its products produced by CYP102A1 M221 mutant. (A) LC-MS chromatogram of naringin DC and its products; (B) Naringin DC shows m/z 582; (C) The minor product (M1) shows m/z 596; (D) The major product (M2) of m/z 598 was found to be monohydroxylated.

FigureThe major 5. LC-MS product analysis was prepared of naringin by DCpreparative and its products HPLC (Figure produced 6), by and CYP102A1 its chemical M221 structure mutant. was Figure 5. LC-MS analysis of naringin DC and its products produced by CYP102A1 M221 mutant. (A) 1 identifiedA by NMR (Figure 7). The chemical shifts and splittingB patterns of mthez majorC product’s H LC-MS( ) LC-MS chromatogram chromatogram of ofnaring naringinin DC DC and and its products;products; ( (B) Naringin) Naringin DC DC shows shows/ 582;m/z (582;) The (C minor) The and 13productC NMR (M1) spectra shows arem/z shown596; (D) (see The majoralso Supplementary product (M2) of m Figure/z 598 was S1–S3 found for to NMR be monohydroxylated. spectra). minor product (M1) shows m/z 596; (D) The major product (M2) of m/z 598 was found to be monohydroxylated.

The major product was prepared by preparative HPLC (Figure 6), and its chemical structure was identified by NMR (Figure 7). The chemical shifts and splitting patterns of the major product’s 1H and 13C NMR spectra are shown (see also Supplementary Figure S1–S3 for NMR spectra).

Figure 6. Figure 6. HPLC and LC-MS analyses of naringin DC’s major product produced by CYP102A1 M221 mutant. (A) preparative-HPLC chromatogram of the major product of naringin DC (C18 column, mutant. (A) preparative-HPLC chromatogram of the major product of naringin DC (C18 column, 10 10 150 mm, gradient from 10% to 100% methanol, 3 mL/min). (B) LC-MS analyses of the major × 150× mm, gradient from 10% to 100% methanol, 3 mL/min). (B) LC-MS analyses of the major product product DC [M+H]+ 599 (m/z 598). DC [M+H]+ 599 (m/z 598).

Figure 6. HPLC and LC-MS analyses of naringin DC’s major product produced by CYP102A1 M221 mutant. (A) preparative-HPLC chromatogram of the major product of naringin DC (C18 column, 10 × 150 mm, gradient from 10% to 100% methanol, 3 mL/min). (B) LC-MS analyses of the major product DC [M+H]+ 599 (m/z 598).

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FigureFigure 7. 7. (A()A Chemical) Chemical structure structure of ofthe the major major product product (neoeriocitrin (neoeriocitrin DC) DC) from from naringin naringin DC. DC.Red: Red:1H chemical1H chemical shift shift values values (multiplicity (multiplicity and and coupling coupling constants constants [Hz] [Hz] in in parenthesis); parenthesis); Black: Black: 1313CC chemical chemical shiftshift values values which which were were assigned assigned from from 2D 2D HMBC HMBC NMR NMR spectra. spectra. (B (B) )2D 2D NMR NMR spectra spectra of of the the major major product,product, neoeriocitrin neoeriocitrin DC. DC. (S (Seeee also also Supplementary Supplementary Figu Figurere S1-S3 S1–S3 for for NMR NMR spectra.) spectra.)

2.4.2.4. Kinetic Kinetic Parameters Parameters and and TTNs TTNs of of Naringin Naringin DC DC Hydroxylation Hydroxylation by by CYP102A1 CYP102A1 Mutants Mutants SixSix mutants mutants (M16V2, (M16V2, G1, G1, M179, M179, M221, M221, M620, M620, and and M850) M850) that that showed showed high high rates rates of of naringin naringin DC DC productproduct formation formation among among tested mutants were selectedselected andand usedusedto to measure measure the the kinetic kinetic parameters parameters of ofnaringin naringin DC DC hydroxylation hydroxylation (Table (Tab1le). 1). WT WT CYP102A1 CYP102A1 did did not not exhibit exhibi appreciablet appreciable activity activity by by which which to todetermine determine reliable reliable kinetic kinetic parameters. parameters. M16V2 M16V2 was was used used as a controlas a control because because it was usedit was as used a template as a for the DNA library. The k values of mutants G1 (11 min 1) and M221 (10.8 min 1) increased template for the DNA library.cat The kcat values of mutants G1 (11 min− −1) and M221 (10.8 min−−1) increased compared to M16V2 by 41% and 38%, respectively. The K values of G1, M179, and M221 decreased compared to M16V2 by 41% and 38%, respectively. The Kmm values of G1, M179, and M221 decreased to half, and mutants M620 and M850 showed 1.5–2.8-fold increases in K values when compared to to half, and mutants M620 and M850 showed 1.5–2.8-fold increases in Kmm values when compared to that of M16V2. The catalytic efficiencies (k /K ) of neoeriocitrin DC formation by mutants G1, M179, that of M16V2. The catalytic efficiencies (kcatcat/Kmm) of neoeriocitrin DC formation by mutants G1, M179, 1µ 1 andand M221 M221 were were 0.151, 0.151, 0.070, 0.070, and and 0.137 0.137 (min (min−−1µMM−1−), ),which which were were more more efficient efficient than than that that of of M16V2 M16V2 by by 3.1-,3.1-, 1.4-, 1.4-, and and 2.8-fold, 2.8-fold, respectively. respectively. M620 M620 and and M850 M850 showed showed decreased decreased catalytic catalytic efficiencies efficiencies due due to to increased K values. increased Kmm values.

Table 1. Kinetic parameters of naringin DC hydroxylation by CYP102A1. The chimeric mutant M16V2 Table 1. Kinetic parameters of naringin DC hydroxylation by CYP102A1. The chimeric mutant M16V2 and selected mutants (G1, M179, M221, M620, and M850). and selected mutants (G1, M179, M221, M620, and M850). 1 1 1 Enzymes kcat (min−−1) Km (µM) kcat/Km−1(min−1 µM− ) Enzymes k (min ) Km (μM) k /K (min μM ) M16V2cat 7.8 0.5 160 25cat m 0.049 0.008 ± ± ± G1M16V2 11.0 7.8 ± 0.50.3 160 73± 25 5 0.049 ± 0.151 0.008 0.011 ± ± ± M179G1 11.0 5.3 ±0.2 0.3 73 76± 5 9 0.151 ± 0.070 0.011 0.008 ± ± ± M221 10.8 0.2 79 6 0.137 0.011 M179 5.3 ± 0.2 76 ± 9± 0.070 ± 0.008± M620 5.7 0.7 441 101 0.013 0.003 M221 10.8± ± 0.2 79 ± 6± 0.137 ± 0.011± M850 4.5 0.4 243 43 0.019 0.004 M620 5.7 ±± 0.7 441 ± 101± 0.013 ± 0.003± M850 4.5 ± 0.4 243 ± 43 0.019 ± 0.004

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FourFour mutants mutants (M16V2, (M16V2, G1, G1, M221, M221, and and M850) M850) were were selected selected and and used used to to measure measure the the TTNs TTNs of of naringinnaringin DC DC hydroxylation. hydroxylation. When When the the assays assays were were carried carried out out at at the the reaction reaction times times of of 20 20 min, min, 30 30 min, min, 11 h, h, 2 2 h, h, and and 4 4 h, h, overall overall product product formation formation was was in in the the range range of of 105 105 to to 315 315 TTNs TTNs (Figure (Figure8). 8). All All mutants mutants showedshowed increased increased neoeriocitrin neoeriocitrin DC DC formation formation rate, rate, which which was was 1.1–1.8-fold 1.1–1.8-fold higher higher than than M16V2 M16V2 during during indicatedindicated reaction reaction time. time. In In addition, addition, the the results results showed showed that that neoeriocitrin neoeriocitrin DC DC is is stable stable at at least least up up to to 22 h h and and then then the the product product seems seems to to degrade. degrade. For For a a 1 1 h h reaction reaction with with M221 M221 or or G1, G1, the the productivity productivity was was 1 1 1 1.11.1 mM mM h −h−1,, whichwhich correspondscorresponds to to 0.65 0.65 g g L L−−1 h−1. .

FigureFigure 8. 8.TTNs TTNs of of naringin naringin DC DC hydroxylation hydroxylation by by CYP102A1 CYP102A1 mutants. mutants. The The reactions reactions contained contained 500 500µ µMM µ naringinnaringin DC DC substrate substrate and and 0.40 0.40 µMM of of M16V2, M16V2, G1, G1, M221, M221, or or M850 M850 in in a a 100-mM 100-mM potassium potassium phosphate phosphate bubufferffer (pH (pH 7.4). 7.4). NADPH-generatingNADPH-generating systemssystems werewere addedadded toto initiate initiate the the reaction, reaction, and and the the reaction reaction mixturesmixtures were were incubated incubated for for 20 20 min, min, 30 30 min, min, 1 1 h, h, 2 2 h, h, and and 4 4 h h at at 37 37◦C. °C. 2.5. Spectral Titration of Naringin DC toward CYP102A1 Mutants 2.5. Spectral Titration of Naringin DC toward CYP102A1 Mutants The differences of binding affinity between M16V2 and selected mutants toward the substrate The differences of binding affinity between M16V2 and selected mutants toward the substrate naringin DC were analyzed by spectral binding titration (Figure9). Binding of naringin DC to CYP102A1 naringin DC were analyzed by spectral binding titration (Figure 9). Binding of naringin DC to G1, M179, and M221 produced a typical Type II spectral shift, with an increase at 420 nm and a CYP102A1 G1, M179, and M221 produced a typical Type II spectral shift, with an increase at 420 nm decrease at 390 nm, indicating an increase in the low-spin fraction of the enzyme. The spectrally and a decrease at 390 nm, indicating an increase in the low-spin fraction of the enzyme. The spectrally determined dissociation constants (Kd) of naringin DC to G1, M179, and M221 were 1.6, 2.2, and 1.4 µM, determined dissociation constants (Kd) of naringin DC to G1, M179, and M221 were 1.6, 2.2, and 1.4 respectively (Figure9). This result indicates that naringin DC can bind to the active sites of the mutants µM, respectively (Figure 9). This result indicates that naringin DC can bind to the active sites of the with a high affinity (Kd of 1–2 µM). However, M16V2 did not show an apparent spectral change. mutants with a high affinity (Kd of 1–2 µM). However, M16V2 did not show an apparent spectral It was suggested that the catechol moieties of polyphenol compounds are important for their change. biological antiadipogenesis, antiobesity, and anticancer functions [27]. The biological activities of resveratrol and its catechol product, piceatannol, were reported to have antioxidation, antiobesity, anti-inflammatory and anticancer abilities [28–30]. Piceatannol was shown to be more potent than resveratrol in inhibitory effects on adipogenesis, obesity, and carcinogenesis [31]. Polydatin, a glycoside of resveratrol, was reported to have many biomedical properties related to antioxidation, antiplatelet aggregation, cardioprotective activity, and anti-inflammatory and immune-regulating functions [32]. Astringin, a catechol product of polydatin, was found to have a more potential antioxidative activity than polydatin [33] and a potential cancer chemopreventive activity [34]. Moreover, 7,3040-trihydroxyisoflavone (7,3040-THIF) (a catechol product of daidzein), but not daidzein itself, inhibited UVB-induced skin tumor in hairless mice. Thus, 7,3040-THIF is considered a new candidate chemoprotective agent [35]. Recently, we found that 3-OH phloretin, a catechol product of phloretin, shows an inhibitory effect on adipocyte differentiation [36]. All of the catechol products mentioned above can be efficiently produced using bacterial P450s [19,22,36,37].

Catalysts 2020, 10, 823 8 of 14 Catalysts 2020, 10, x FOR PEER REVIEW 8 of 14

Figure 9. Binding titration of naringin DC to CYP102A1.CYP102A1. The assay contained 0–20 µµMM naringin DC substrate in 100 mM potassium phosphate buffer buﬀer (7.4) and 1.0 µMµM of M16V2 ( A), G1 ( B), M179 ( C),), and M221M221 ((DD).). The The inset inset of of each each panel panel (B (–BD–)D shows) shows a plot a plot of inducedof induced Soret Soret absorbance absorbance change change (∆A390–420 (ΔA390-) versus420) versus the relevantthe relevant concentration concentration of naringin of naringin DC. SpectrallyDC. Spectrally determined determined dissociation dissociation constants constants (Kd)

were(Kd) were also shown.also shown.

It iswas known suggested that glycosylation that the catechol is an essentialmoieties mechanismof polyphenol for compounds diverse biological are important functions for and their the structurebiological ofantiadipogenesis, natural flavonoids antiobesity, in plants [and38,39 antica]. Glycosylationncer functions of flavonoids[27]. The bi canological modify activities color and of tasteresveratrol properties and [its39 ,catechol40]. Furthermore, product, piceatannol, it leads to their were strong reported solubility to have and antioxidation, stability in water antiobesity, [41–43], whichanti-inflammatory helps to improve and anticancer physiological abilities and [28–30]. pharmacological Piceatannol propertieswas shown that to be increase more potent compound than bioavailabilityresveratrol in [43inhibitory,44]. Naringin effects DC on is aadipogenesis, glycoside of phloretin obesity, thatand shows carcinogenesis several beneficial [31]. Polydatin, antioxidant, a anticancerglycoside of [45 resveratrol,,46], and antiobesity was reported [47] e fftoects. have Phloretin many biomedical is widely used properties as a cosmeceutical related to antioxidation, ingredient for UVantiplatelet protection aggregation, [48]. However, cardioprotective the biological functionsactivity, ofand a catecholanti-inflammatory product of naringin and immune-regulating DC, neoeriocitrin DC,functions have not[32]. been Astringin, reported a untilcatechol now. product Therefore, of thepolydatin, production was offound neoeriocitrin to have DCa more from potential naringin DCantioxidative by CYP102A1 activity reported than herepolydatin should [33] be aand good a potential strategy tocancer obtain chemopreventive it. Furthermore, toxicologicalactivity [34]. researchMoreover, is needed7,3′4′-trihydroxyisoflavone if neoeriocitrin DC is(7,3 applied′4′-THIF) as a(a sweetener. catechol product of daidzein), but not daidzein itself,In inhibited this study, UVB-induced we found that skin neoeriocitrin tumor in DC, hairless can be mice. produced Thus, by 7,3 an′4 enzymatic′-THIF is considered biotransformation a new usingcandidate CYP102A1. chemoprotective It is now agent possible [35]. Recently, to study neoeriocitrinwe found that DC’s 3-OH biological phloretin, functions, a catechol suchproduct as itsof antiobesity,phloretin, shows anti-inflammatory, an inhibitory andeffect anticancer on adipocyte abilities. differentiation Further investigation [36]. All of is the necessary catechol to products improve thementioned production above of neoeriocitrincan be efficiently DC to produc meet theed minimumusing bacterial space-time P450s yield [19,22,36,37]. and a minimum final product concentrationIt is known for that industrial glycosylation application is an [essential49]. Although mechanism we tried for whole-celldiverse biological biocatalysis functions experiments and the withstructureE. coli ofexpressing natural flavonoids CYP102A1 in genesplants for[38,39]. improved Glycosylation production of flavonoids of neoeriocitrin can modify DC, no color products and oftaste naringin properties DC were[39,40]. obtained Furthermore, (results it not leads shown). to their Surface strong displaysolubility [50 and] and stability export in of water CYP102A1 [41–43], to thewhich periplasmic helps to spaceimprove [51 ]physiological of E. coli might and be goodpharmacological whole cell systems properties for thethat industrial increase application.compound Thisbioavailability result indicates [43,44]. that Naringin the carbohydrate DC is a moietyglycoside of naringin of phloretin DC, may that inhibit shows its several transport beneficial into the E.antioxidant, coli cells because anticancer phloretin, [45,46], the aglyconeand antiobesity of naringin [47] DC, caneffects. enter Phloretin into the cells is andwidely be hydroxylated used as a tocosmeceutical 3-OH product ingredient [36]. for UV protection [48]. However, the biological functions of a catechol product of naringin DC, neoeriocitrin DC, have not been reported until now. Therefore, the production of neoeriocitrin DC from naringin DC by CYP102A1 reported here should be a good strategy to obtain it. Furthermore, toxicological research is needed if neoeriocitrin DC is applied as a sweetener. In this study, we found that neoeriocitrin DC, can be produced by an enzymatic biotransformation using CYP102A1. It is now possible to study neoeriocitrin DC’s biological functions, such as its antiobesity, anti-inflammatory, and anticancer abilities. Further investigation is

Catalysts 2020, 10, 823 9 of 14

3. Materials and Methods

3.1. Materials Glucose-6-phosphate, glucose-6-phosphate dehydrogenase from baker’s yeast, naringin DC, and β-nicotinamide adenine dinucleotide phosphate (NADP+) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals and solvents with the highest grade were obtained from commercial suppliers

3.2. Optimal Expression of CYP102A1 M221 Mutant The plasmid of M221 (in pCW vector) was transformed to a set of competent E. coli cells (DH5α-F’IQ, BL21, SHuffle T7, Rosetta, MG1655, and JM109) and spread on Luria-Bertani agar plate with ampicillin (100 µg/mL). The single colony was grown in 5 mL of Luria-Bertani medium supplemented with ampicillin (100 µg/mL) with shaking at 170 rpm overnight while maintaining 37 ◦C. The aliquots of cell culture (1% v/v) were inoculated in 50 mL of Terrific Broth medium supplemented with ampicillin (100 µg/mL). The cells were grown at 37 ◦C with shaking at 170 rpm to an OD600 of approximately 0.6–0.8. Then, isopropyl-β-d-thiogalactopyranoside (0.5 mM) and δ-aminolevulinic acid (1.0 mM) were added for enzyme expression. After the cultures were allowed to grow at 20 or 25 ◦C with 150 rpm, OD600 and CO spectra were measured at culture times of 12, 16, 20, 24, and 28 h. CYP102A1 (P450) concentrations of whole cells were determined from the CO-difference spectra using an extinction molecular coefficient, ε = 91 mM/cm [26].

3.3. CYP102A1 Mutants Used to Screen Highly Active Naringin DC Hydroxylases An extensive set of CYP102A1 mutants was generated in previous work [12,14,19,22–25], and the WT BM3 and 60 mutants were used for screening highly active mutants towards naringin DC. To make more active mutants having naringin DC hydroxylase activity, a random mutagenesis was performed to make a DNA library of the M16V2 heme domain. The chimeric protein M16V2 was originally made by exchanging the reductase domain of M16 with that of CYP102A1 natural variant V2, as described [21,24]. The error-prone PCR was performed on the CYP102A1 heme domain (1st–430th amino acid residues) of the M16V2 to make a DNA library. Oligonucleotide primers were used to introduce the BamHI/SacI restriction sites: BamHI forward, 50-ataGGATCCatgacaattaaagaaatg cctc-30 and SacI reverse, 50-ataGAGCTCgtagtttgtatgatcttcaaagtcaaag tg-30. DNA libraries of random mutants were constructed using a reaction mixture (50 µL) of 10 pmol of each primer, 0.2 mM dNTP (0.05 mM each of dATP, dGTP, dCTP, and dTTP), Taq DNA polymerase (5 units/µL), MgCl2 (2.5 or 5 mM), and MnCl2 (0.1 or 0.15 mM) in 10 mM Tris-HCl containing 50 mM KCl (pH 8.4, 25 ◦C). The PCR reaction was started at 95 ◦C for 5 min and run through 26 thermocycles of 95 ◦C for 60 s, 58 ◦C for 60 s, and 72 ◦C for 90 s. After completing the reaction, the reaction medium was held at 72 ◦C for 5 min and subsequently soaked at 4 ◦C. The amplified PCR library fragments were purified and cloned into the pCWBM3M16V2/BamHI/SacI vector using the restriction sites of BamHI and SacI. The mutation rate (2.9 mutations per 1290 bp) was validated by sequencing 12 randomly selected clones before screening of expression level and activity. The size of the screened mutant library was approximately 1.0 106. × Seven mutants selected based on a blue colony-based colorimetric method and hydroxylation activity toward naringin DC were expressed in the E. coli strain DH5αF’-IQ. The CYP102A1 is expressed in cytosol and partially purified as supernatant lysate after removal of cell debris and membrane fractions [24]. The lysate was used to determine the CYP102A1 (P450) concentrations from the CO-difference spectra [26]. For the M16V2 and its mutants, a typical culture yielded 300 to 700 nM of P450. Catalysts 2020, 10, 823 10 of 14

3.4. Hydroxylation of Naringin Dihydrochalcone by CYP102A1 Mutants The reaction mixtures contained 200 µM naringin DC substrate in 100 mM potassium phosphate buffer (pH 7.4) and 0.20 µM CYP102A1. An aliquot of a NADPH-generating system (10 mM glucose-6-phosphate, 0.5 mM NADP+, and 1.0 UI yeast glucose-6-dehydrogenase/mL) was added to the initial reaction. The reaction mixture was incubated for 30 min at 37 ◦C and stopped by 600 mL ice-cold ethyl acetate. The naringin DC and its products were analyzed by HPLC using a Gemini C18 column (4.6 150 mm, 5 µm, 110 Å; Phenomenex, Torrance, CA, USA) with the mobile phase A × (water containing 0.5% methanol and 0.1% formic acid) and the mobile phase B (acetonitrile) [52]. The flow rate of the elution column was 1.0 mL/min by a gradient pump (LC-20AD; Shimadzu, Kyoto, Japan) with the following gradient: 0–3 min controlled at 9% mobile phase B, 3–20 min gradually increased reaching to 30% mobile phase B, 20–21 min decreased to 9% mobile phase B, and 21–30 min controlled at 9% mobile phase B and detected by UV at 285 nm. The kinetic parameters of CYP102A1 mutants were determined by reaction, including 10–500 µM of naringin DC in 100 mM potassium phosphate buffer (pH 7.4) and 0.20 µM enzymes. The NADPH-generating systems were added to the initial reaction and the reaction mixtures were incubated for 30 min at 37 ◦C. A stock of substrate solution was prepared in methanol and diluted in the enzymatic reactions to the final organic solvent concentration of <1% (v/v). The kinetic parameter results were analyzed using GraphPad Prism software (Graph, San Diego, CA, USA). The TTNs of CYP102A1 mutants were determined by reaction contained in 500 µM naringin DC in 100 mM potassium phosphate buffer (pH 7.4) and 0.40 µM enzymes. The NADPH-generating systems were added to the initial reaction and the reaction mixtures were incubated for 20 min, 30 min, 1 h, 2 h, and 4 h at 37 ◦C.

3.5. LC-MS Analysis To identify the minor and major products of naringin DC produced by CYP102A1 mutants, a liquid chromatography–mass spectrometry (LC–MS) analysis was performed, and the LC profile and fragmentation patterns of the authentic compounds (naringin DC and neoeriocitrin DC) were compared on a Thermo Scientific AccelaTM and TSQ QuantumTM Access MAX system with the heated electrospray ionization interface with HESI II probe (Thermo Fisher Scientific, Waltham, MA, USA) (Figure5). The oxidation reaction of naringin DC by CYP102A1 was performed as described above. The separation was performed on a ZorBax SB-C18 (4.6 250 mm, 5 µm, 80 Å; Agilent Technologies, × Santa Clara, CA, USA); the gradient mobile phase was 0.5% (v/v) methanol and 0.1% formic acid (v/v) in water (A) in acetonitrile (B), delivered at a flow rate of 1.0 mL/min. The initial composition of mobile phase B was 9%; after 3 min the mobile phase B composition increased to 30% over 17 min, decreased to 9% over 2 min, and finally re-equilibrated to the initial conditions over 13 min. Thus, the total run time was 35 min. The temperatures of the column and autosampler were kept at 40 and 4 ◦C, respectively, and the injection volumes were 5 µL for all samples tested here. The electrospray ionization procedure was performed in the negative ion mode. The spray voltage was 3500 V and vaporizer temperature was 300 ◦C. Capillary temperature was 200 ◦C. Nitrogen sheath gas and auxiliary gas pressures were 40 psi and 12 psi, respectively. All data were acquired with full scan mass spectrometry (full scan) or single ion monitoring (SIM) in the negative ion detection mode using XcaliburTM 3.0 software. To confirm the purity of the naringin DC product produced by the CYP102A1 mutants and purified by preparative HPLC, an LC–MS analysis of products was performed to compare LC profiles and fragmentation patterns with those of the authentic compounds (naringin DC and neoeriocitrin DC) (Figure6). The mutant M221 was included with 200 µM naringin DC for 50 min at 37 ◦C with the NADPH-generating system and performed using Applied Biosystems’ QTRAP-3200 mass spectrometer (Waltham, MA, USA) having LC-MS solution software. Catalysts 2020, 10, 823 11 of 14

3.6. NMR Spectroscopy After the major product of naringin DC was produced by the M221, separated by preparative HPLC (C18 column, 10 150 mm, gradient from 10% to 100% methanol, 3 mL/min), and collected in × an ice bucket, the solvent was then removed by freezer-dryer. NMR investigations were performed at ambient temperature on a JNM-ECA600 600MHz FT-NMR spectrometer (JEOL Ltd., Tokyo, Japan). CD3OD was used as solvent, and chemical shifts for proton NMR spectra were measured in parts per million (ppm) relative to tetramethylsilane.

3.7. Spectral Binding Titration

Spectral determinations of Kd values for the binding of substrates to the P450s were performed as described [53]. The binding affinity of naringin DC to four CYP102A1 mutants was determined (at 23 ◦C) by titrating 1.0 µM enzyme in 100 mM potassium phosphate buffer (pH 7.4). The absorption difference between 350 and 500 nm was plotted against the substrate concentration (0–20 µM). The Kd values were estimated using GraphPad Prism software (GraphPad Software, San Diego, CA, USA).

4. Conclusions An enzymatic strategy for the eﬃcient synthesis of a potentially valuable product from naringin DC, a glycoside of phloretin, was developed using Bacillus megaterium CYP102A1 monooxygenase. At present, no enzymatic or chemical methods to make products of naringin DC by hydroxylation reactions have not been reported. In this study, a set of CYP102A1 mutants was used to catalyze the hydroxylation of naringin DC. We found that the major product is neoeriocitrin DC by NMR and LC-MS analyses. Sixty seven mutants of CYP102A1 were tested for hydroxylation of naringin DC to produce neoeriocitrin DC. Six mutants with high activity were selected to determine the kinetic 1 parameters and total turnover numbers (TTNs). The kcat value of the most active mutant was 11 min− 1 and its TTN was 315. The productivity of neoeriocitrin DC production increased up to 1.1 mM h− , 1 1 which corresponds to 0.65 g L− h− . We achieved an eﬃcient regioselective hydroxylation of naringin DC to produce neoeriocitrin DC, a catechol product.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/10/8/823/s1, Figure S1. 1H NMR spectra of the major product, neoeriocitrin DC; Figure S2. 2D HMBC NMR spectra of the major product, neoeriocitrin DC; Figure S3. 2D COSY NMR spectra of the major product, neoeriocitrin DC; Table S1: The amino acid sequence of M16V2 and CYP102A1 mutants. Author Contributions: Conceptualization, S.-J.Y., H.-S.K., and C.-H.Y.; investigation, T.H.H.N., S.-M.W., N.A.N., and G.-S.C.; writing—original draft preparation, T.H.H.N. and C.-H.Y.; supervision, S.-J.Y., H.-S.K., and C.-H.Y.; funding acquisition, H.-S.K. and C.-H.Y. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Next-Generation BioGreen 21 program (SSAC, grant no.: PJ01333101); Rural Development Administration and the Basic Research Lab Program (NRF-2018R1A4A1023882); National Research Foundation of Korea, Republic of Korea. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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