Design of Artificial Alcohol Oxidases: Alcohol Dehydrogenase–NADPH

University of Groningen

Design of artificial alcohol oxidases Aalbers, Friso; Fraaije, Marco

Published in: ChemBioChem

DOI: 10.1002/cbic.201800421

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Citation for published version (APA): Aalbers, F., & Fraaije, M. (2019). Design of artificial alcohol oxidases: alcohol dehydrogenase - NADPH oxidase fusions for continuous oxidations. ChemBioChem, 20(1), 51-56. [cbic.201800421]. https://doi.org/10.1002/cbic.201800421

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Very Important Paper Design of Artificial Alcohol Oxidases:Alcohol Dehydrogenase–NADPH Oxidase Fusions for Continuous Oxidations Friso S. Aalbers and MarcoW.Fraaije*[a]

To expandthe arsenal of industrially applicable oxidative en- the enzymes can also catalyzeketone reductionsbyusing a zymes, fusions of alcohol dehydrogenases with an NADPH-ox- reduced nicotinamide cofactor. idase were designed. Three different alcohol dehydrogenases Amajor challenge in applying ADHsfor alcohol oxidations is (LbADH, TbADH, ADHA) were expressed with athermostable their dependenceonthe nicotinamide cofactor.Because the NADPH-oxidase fusion partner (PAMO C65D) and purified. The cofactoristoo expensive to be applied in stoichiometric resultingbifunctional biocatalysts retained the catalytic proper- amounts, arecycling system is necessary to enable alcohol ties of the individual enzymes,and acted essentially like alco- oxidations in an economically feasible manner.[6,7] In addition, hol oxidases:transforming alcohols to ketones by using dioxy- alcohol oxidation with NAD(P) + is thermodynamically less gen as mild oxidant, while merely requiring acatalytic amount favorable than the reverse reaction, thus efficient recycling is of NADP+.Insmall-scale reactions, the purified fusion enzymes neededtopush against the equilibrium. One typicalNAD(P)+ show good performances, with 69–99 %conversion, 99% ee -recycling approach is the addition of an excess of asacrificial with aracemic substrate, and high cofactor and enzyme total ketonesubstrate, like acetone,that is readily reduced by the turnover numbers. As the fusion enzymes essentially act as same ADH. This approach keeps the system simple, thoughit oxidases, we found that commonly used high-throughput has somedrawbacks, such as difference in pH optimum for the oxidase-activity screening methods can be used. Therefore, if two reactions, occupation of the active sites by different sub- needed, the fusion enzymes could be easily engineered to strates, which leads to inhibition, and poor atom efficiency due tune their properties. to the excessofsacrificial substrate. Another downside is the inhibition causedbythe product from the sacrificial ketone. Al- Alcohol oxidations are vital for the synthesis of various carbon- ternatively,anNAD(P)H oxidase (NOX, EC 1.6.3) can be used to yl compounds.[1,2] In particular,acatalystthat features strict regenerate NAD(P) +.[6,7] NOXs typicallycontain atightly bound enantioselectivity can be used for the kinetic resolution of flavin cofactor andefficiently oxidize NAD(P)H by using molec- alcohols.[3] Enzymes can catalyze highly selective alcohol oxida- ular oxygen, thereby forming hydrogen peroxide (type 1NOX), tions, and such biocatalytic oxidations have arelatively low en- or water (type 2NOX). vironmental impact compared to chemically catalyzed oxida- Another type of flavin-containing enzyme is the Baeyer–Vil- tions.[3–6] liger monooxygenase (BVMO, EC 1.14.13), which can also bind The two main classes of enzymes that catalyzealcohol oxi- NAD(P)H and oxygen, but which is used to catalyze Baeyer–Vil- dationsare alcoholoxidases (EC 1.1.3) and alcohol dehydro- liger oxidations or otheroxygenations.[8] One property of this genases (ADHs,EC1.1.1.1). Although oxidases are attractive class of enzymesisthat, after binding of NAD(P)H and oxygen biocatalysts, because they depend on molecular oxygen as and formation of the peroxyflavin intermediate, anuncoupling electron acceptor,there are not many alcohol oxidases avail- reactioncan occur.During this reaction, the reactive peroxy- able. On the other hand, there is alarge array of characterized flavin shunts back to the oxidized state, H2O2 is formed, and ADHs with various substrate specificities. The dehydrogenation NADP+ is released. Amutant of aBVMO, phenylacetone mon- reactionthat the ADHs catalyzetypicallyinvolves oxidized nic- ooxygenase (PAMO), was found to have agreatlyenhanced otinamide adenine dinucleotide(phosphate)(NAD(P) +)aselec- uncoupling rate, therebyacting as an NADPH oxidase.[9] De- tron acceptorplus an alcohol substrate, andtransforms these spite aprofoundchange in activity,the C65D mutant PAMO into NAD(P)H and an aldehyde or ketoneproduct.Inversely, was found to be as stable as the wild-type PAMO, which is one

of the mostthermostable BVMOs characterized(Tm =608C). [a] F. S. Aalbers, Prof. Dr.M.W.Fraaije With this favorable stability and the more alkaline pH optimum Molecular EnzymologyGroup,University of Groningen of the mutant (pH 8.0) compared to that of some other natural Nijenborgh 4, 9747AG Groningen (The Netherlands) [7] E-mail:[email protected] NOXs, PAMO C65D is an attractive biocatalyst to apply for + Supporting information and the ORCID identification numbers for the NADP recycling. authors of this article can be found under https://doi.org/10.1002/ Considering the combination of an ADH with aNOX for per- cbic.201800421. forming alcoholoxidations, we explored the approach of 2019 The Authors. Published by Wiley-VCH Verlag GmbH&Co. KGaA. fusing these two enzymes together (Scheme1). With this ap- This is an open access article under the terms of the Creative Commons At- proach,the enzymes can be produced and purified in one go, tribution-NonCommercial-NoDerivs License, whichpermitsuse and distribu- tion in any medium, provided the originalwork is properly cited, the use is and, as the enzymes are colocalized, the NOX could support non-commercial and no modifications or adaptations are made. the rapid regenerationofNADP+ .Inrecent years, the possibili-

ChemBioChem 2019, 20,51–56 51 2019 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Communications

Table 1. Alcohol dehydrogenase–NADPH-oxidase fusionsproduced.

Enzyme N-terminal LinkerC-terminal MW [kDa] NOX-APAMO C65D SGSAAG ADHA 90.3 NOX-LPAMO C65D SGSAAG LbADH 90.5 T-NOX TbADH SGSAAG PAMO C65D 101.4

Scheme1.Alcoholdehydrogenases (ADHs) can catalyze alcohol oxidations A: ADHA, L: LbADH from L. brevis;T:TbADHfrom T. brockii,NOX:PAMO and ketonereductions. By fusing an ADH with aNOX enzyme, which can ox- mutant C65D. idize the reduced nicotinamide cofactor NADPHbyusing oxygen, the equilibrium is driven toward catalyzingalcohol oxidations. In essence, the fusion of the two enzymesacts like an alcohol oxidase:analcohol substrate is converted at the costofoxygen, and hydrogen peroxide is produced. zyme per liter culture could be obtained. For wild-type PAMO, 40 mg of purified enzyme per liter culture was reported.[20] The UV/Vis absorbance spectra of the fusion enzymes were ties and advantages of enzymefusions have been explored for different from the spectrumofthe single NOX. In atypical variousenzyme types, including fusions of redox enzymes.[10–12] spectrum of oxidized FADinNOX, the two absorbance maxima For instance, to enable NADPH-dependentenzymes to be re- at 350–385nmand at 440–460 nm have roughly the same cycled, variousBVMOs and aP450 monooxygenase werefused height; forthe fusions the 350–385 nm peak was more to phosphite dehydrogenase.[13–15] Some studies showed that, pronounced. This an indication of the presence of some fully rather than using asacrificial substrate like phosphite, it is pos- reduced and/or semiquinoneflavin. It is difficulttopinpoint sible to fuse ADHs with acyclohexanone monooxygenase the cause of this change;possibly the cells experienced more (CHMO) to enable cascadereactions from alcohols to esters,[16] oxidative stress duringthe expression of these fusions, and or from cyclohexanoltocaprolactone,[17] for which the fusions this affected the oxidation state of the NOX. To fully oxidize were more efficient than the separate enzymes. Another the flavin cofactor in the purifiedfusion enzymes,they were recent example of enzymefusions is the combination of oxi- incubated overnight with 10 mm potassium ferricyanide dases with aperoxidase to produce fusions that can be used (K3[Fe(CN)6]), which was subsequently removed by gel-filtration for cascade reactions, as the hydrogen peroxide that is pro- chromatography.After this treatment, the spectra from the duced by the oxidasecan be used directly by the fused per- fusion enzymesresembled the spectrum of the individual oxidase.[1] NOX, thus indicating that the FADisinoxidizedstate (Fig- The aim of this study is to investigate whether fusing an ure S3). ADH with aNOX produces abifunctional enzyme that can be As mentioned before,the fusion of aprotein to the Nor appliedfor dioxygen-driven alcohol oxidations by facilitating Cterminus of an enzyme can greatlyinfluence the activity of the regenerationofNADP + (Scheme 1). Fusionswere made by that enzyme. Therefore, we determined the kinetic parameters pairing the PAMO C65D mutant (NOX) with three NADP+ - of the ADH–NOX fusions and the single enzymes (Table 2). Cy- dependentalcohol dehydrogenases: LbADH (R-selective) from clohexanol was chosen as model substrate for the alcohol oxi- Lactobacillus brevis, TbADH from Thermoanaerobacter brockii, dation activity,asitisaknown substrate for each of the three and acommercial ADH (ADHA). ADHs.The alcohol oxidationresults show minor differencesin

The organizationofthe fusions was inspired by our previous kcat and KM values betweenthe fused and nonfused ADH en- study on ADH/CHMO fusions,inwhich we found acleardiffer- zymes;atmost 1.5- to 2-fold differencesinkinetic parameters. ence in ADH activity depending on the orientation:ADH– This indicates that the activity of the ADH enzymes was unaf- CHMO or CHMO–ADH.[17] The findings from that studyindicate that short-chain dehydrogenases/reductases (SDRs) lose activity as N-terminal fusions (ADH–BVMO), possibly through pertur- Table 2. Alcohol and NADPHoxidation kineticsofthe fusion enzymes.[a] bation of the dimer/tetramer formation of the SDRs. Although LbADH was not investigated in that study,other studies found EnzymeCyclohexanol oxidation NADPHoxidation [19] kcat KM kcat/KM kcat KM kcat/KM that aC-terminal His tag was detrimental to the activity. For 1 1 1 1 1 1 [sÀ ][mm][sÀ mÀ ][sÀ ][mm][sÀ mmÀ ] ADHA,wehave similar evidence(unpublished results). Consid- NOX –––5.0[b] 3.5[b] 1400 ering their classification as SDRs, we presumed that they ADHA 0.26 19 14 ––– would be activeasC-terminal fusions (BVMO–ADH). Therefore, NOX-A 0.56 10 56 5.1 27 190 we designed the fusion enzymes NOX-A, NOX-L, and T-NOX LbADH 2.2 31 71 ––– (Table 1), each with an N-terminal His tag. NOX-L 2.0 29 69 4.4 5.8 760 TbADH8.3 3.7 2200 ––– The three fusion constructs were first cloned and then trans- T-NOX 5.7 5.8 980 2.8 5.7 490 formed into Escherichia coli for recombinantexpression. The expression levels were found to be similar to those of the indi- [a] The kineticsweredetermined by measuring the change in absorbance at 340 nm at various concentrationsofsubstrate (5–10 different concen- vidual ADH and NOX enzymes, based on SDS-PAGE with sam- trations, in duplicateortriplicate, FiguresS4and S5).The alcohol oxida- ples from the cell-free extracts (Figures S1 and S2 in the Sup- tion rateswere measuredin20mm KPO4 (pH 7.5), and NADPHoxidation porting Information) and the amountofpurified enzyme. After was measured in 50 mm Tris·HCl (pH 8.0), both at 258C. [b] Data taken affinity chromatography purification,40–150 mg of fusion en- from ref. [9] (in 50 mm Tris·HCl (pH 7.5)).

ChemBioChem 2019, 20,51–56 www.chembiochem.org 52 2019 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Communications fected by the fusion. For the NOX activity,the differences were in asimilarly small range, thoughthere was aremarkable increaseinKM for NADPH of the NOX-A fusion. Still, the KM,NADPH for NOX-A is in the micromolar range. Overall,wefound that the fused enzymes largely retainedtheir catalytic properties, and proceeded to investigate their utility as biocatalysts. To assess the applicabilityofthe fusion enzymes for alcohol oxidations, the conversion of cyclohexanol by NOX-Awas Scheme2.Kinetic resolution of rac-1-phenylethanol with the NOX-ADH fu- tested. The reactionmixtures consisted of fusion enzyme, sions. The reaction would ideally yield 50 %acetophenone, and 50%of99% ee (R)- or (S)-1-phenylethanol. buffer,substrate, and NADP+.Initially,only moderate conversions could be achieved, ranging from 32 to 76%(data not shown). Possibly,the hydrogen peroxide that is formed during the reaction was inactivating one or both enzymes. Another Table 4. Conversions of cyclohexanol and racemic 1-phenylethanol by complication could be the loss of FADfrom the NOX fusion. To ADH/NOX fusion enzymes. evaluatewhether the addition of catalase or FADcould im- Substrate EnzymeConversion ee[a] TTN TTN prove the level of conversion,each additive was added sepa- [%] [%] (enzyme)(cofactor) rately (Table 3). Both additives significantly improved the level cyclohexanolNOX-A 95 n.a.31666 475 NOX-L 99 n.a.33000 495 T-NOX69n.a. 23000 345 Table 3. Effect of additives on conversion by NOX-A. rac-1-phenylethanol NOX-A 94 99 (R)31333 470 NOX-A 56[b] 99 (R)28000 140 Enzyme AdditiveConversion [%] TTN[a] (enzyme) TTN[a] (cofactor) NOX-L 50 99 (S)16666 250 NOX-A –75 39500 395 Reaction conditions: 50 mm substrate with 1.5 mm of fusion enzymein + NOX-A FAD93 46500 465 50 mm Tris·HCl(pH 8.5), 100 mm NADP ,64hat 248C, 500 rpm (Thermo- NOX-A catalase 89 44500 445 Mixer Eppendorf);10mm FAD, 1000 Ucatalase. Reactions with rac-1-phe- NOX-A FAD +catalase 95 47500 475 nylethanolincluded 2% DMSO.[a] Enantiomeric excess of the remaining alcohol substrate (Figure S6), [b] 0.5 mm NOX-A, 25 mm substrate, 100 mm [a] TTN:total turnover number (amountofsubstrate converted per fusion NADP+,50mm N-cyclohexyl-2-aminoethanesulfonic acid (CHES;pH9.0) enzyme). Reactionconditions: 50 mm cyclohexanol with 1 mm of NOX-A for 24 hat248C. in 50 mm Tris·HCl (pH 8.5), 100 mm NADP +,64hat 248C, 500 rpm (Ther- moMixer Eppendorf);10mm FAD, 1000 Ucatalase. Experiments were performed in duplicate. oxidation (Table 2), it showedthe worst conversion (69%) of the three fusions enzymes,with only 23000 turnovers of conversion. For the catalase, the reasonsfor the improve- (Table 4). Still, the conversion was significantly better than the ment are fairly straightforward: removing the hydrogen per- conversion without FADand catalase (only 51%conversion), oxide prevents damage to the enzymes while it regenerates thus suggesting that this fusion in particularsuffered from the oxygen that can be used by the NOX. The beneficial effect of formation of hydrogen peroxide. Despite the addition of asub- additional FADmight lie in the stabilizing effect of forcingthe stantialamount of catalase, some hydrogen peroxide can still enzyme to remain in its holo (FAD-bound) state. Arecent accumulate because the affinity of catalase towards hydrogen study found that the addition of FADindeed improves the peroxide is rather poor (KM = >10 mm). TbADH is amedium- stabilityofFAD-dependent monooxygenases.[22] In that study, chain dehydrogenase that features acysteine-coordinated zinc superoxide dismutase (SOD) was also found to improve bio- that is involved in catalysis, which could make it more sensitive catalystperformance. Yet, when we added 40 UofSOD, no im- to peroxide-induced inactivation. provement was found. For the conversions of 1-phenylethanol, it was gratifying to Based on the results, FADand catalase were included in all note that NOX-L retained the strict enantioselectivity of the subsequentconversionsofcyclohexanol and 1-phenylethanol. native LbADH, with 50 %conversion to acetophenone and We also included 1-phenylethanol as an additional test sub- yielding 99 % ee of the (S)-1-phenylethanol. On the other hand, strate to explorekinetic resolutions of this chiral alcohol NOX-A showed astrong preference for the (S)-substrate. De- (Scheme 2). As TbADH is not active towards 1-phenylethanol, pending on the duration of the reaction, one could achieve the Tb-NOX fusion was not tested for this substrate. (44%) (R)-1-phenylethanol of 99% ee,orprimarily acetophe- The results clearly show that the fusions can be used for none (94 %). effective alcohol oxidations, with high total turnover numbers With the developed fusion approach, dioxygen-driven self- (TTN) for both the enzyme and the cofactor (Table 4). Both sufficient alcohol dehydrogenases weregeneratedthat could metrics are of interestfrom an industrial perspective, as the be appliedfor selective alcohol oxidations. Essentially,asthe biocatalyst and the cofactor have high cost contributions.[7] overall reaction only consumes molecular oxygen and produ- However,the performance of T-NOX was considerably poorer ces hydrogen peroxide, these fusion enzymes can be regarded than those of the other two fusion enzymes. Even thoughT- as artificialalcohol oxidases.With that in mind, we were inter- NOX displayed the best kinetic parameters for cyclohexanol ested to see whether such “alcoholoxidases” are also suitable

ChemBioChem 2019, 20,51–56 www.chembiochem.org 53 2019 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Communications for established oxidase-based, activity-screening methods. One nies that expressed the NOX-A fusion quickly turned an intense commonly used method for screening activity with oxidases is blue, butnot the colonies that express only NOX(Figure 2) or the horseradish peroxidase (HRP)-coupled assay.[21] When the the ADH (not shown). The blue colonies could be picked and oxidases transform alcohols and concomitantly produce hydro- used for growth and plasmidisolation, relyingonthe cells that gen peroxide, the HRP can use the peroxide to oxidize fluoro- or chromogenic substrates, by which an easily detectable product is formed. First, it wastested whether oxidaseactivity could be measured by meansofacommonly used HRP-based method. The assay included 4-aminoantipyrine(AAP) and 3,5-dichloro-2- hydroxybenzenesulfonic acid (DCHBS) which,upon peroxidase- catalyzed oxidation, form astable pink product. When using cell-free extract (after growth andexpression of NOX-A)and 30 mm cyclohexanol, the reactionmixture turnedpink after a few minutes, whereas the control reactions, which excluded one of the components, remained colorless (Figure 1). It Figure 2. The fusion of an ADH with aNOX enablesthe detection of alcohol oxidation activity in colonies. The plates containcolonies that expressed A) NOX-A or B) NOX. Only the colonies that produce the fusion enzyme turn dark blue after addition of the assay mix.

survived the freeze–thawing step. Thisapproach could be val- uable when engineering an ADH/NOX towards anew substrate,ascolonies would only turn blue when it has activity toward the substrate that is in the assay mix. In previous studies that used this method for screening of oxidase mutants, it was shown to be extremelyuseful for identifying improved variants,for example, in the case of engineering amine oxidase [24] Figure 1. With an HRP-coupled assay,the alcohol oxidation activity of the variants. NOX-A fusion can be detected without any additionofNADP + (3). The re- The designed NOX–ADH fusions presented in this study are action mixture included buffer (50 mm Tris·HCl pH 7.5), HRP (0.8 U), AAP not only suitable for performing alcohol oxidations with high (0.1 mm)and DCHBS (1 mm), cell-freeextract containingNOX-A (10 % v/v) and 30 mm cyclohexanol.Controls:1)nosubstrate and 2) no cell-free ex- total turnover numbers, the fusion of the two enzymes also tract. enabled oxidase-based activity screening.All three ADH en- zymeswere active in fusion with aNOX, and each enzymere- tained its catalytic properties and level of expression. Although should be made clear that no NADP +/NADPH wasadded to the hydrogen peroxide-forming NOX might not be suitable for the assay mixture, this shows that the amount of nicotinamide some ADHs,very good conversionscould be attainedwith ad- cofactor in the extract wasenough to support catalysis by dition of catalase. In this regard, ADH fusions with awater- NOX-A. E. coli cells contain roughly100 mm of NADP +/ forming NOX could be more appealing from an industrialper- NADPH.[23] Even thoughADH activity can be monitored by de- spective.[7] An alternative approachcould be atriple fusion: tecting NAD(P)H formation,this peroxidase-basedassayoffers addingacatalase as afusion partner.However,sofar catalase acheap (no cofactor needed), facile and rapid method to mea- fusion proteins have been found to be problematic,asthey sure or detectADH/NOX oxidase activity. could not be expressed (unpublished results). Considering that Aside from screening the activityofcell-free extracts, anoth- current screening of ADHmutantsisquite labor intensive and er peroxidase-based method has been developed that allows costly,itis worth notingthat the NOX fusion partner opens the screening of oxidaseactivity in colonies.[24] Such ahigh- doors to arapid and cheap qualitative activity screening, and a throughput approach is extremely well suited to enzyme convenient qualitative colony-based screening. The NOX–ADH engineering strategies. This approach is highlyappealing for fusion approachcould be avaluable tool for the development making large mutantlibraries, as positive mutants can be of useful androbust biocatalysts. selected based on the color of the colonies. We explored this option with the NOX-A fusion. After the cells had been trans- formed with aconstruct for expression of the NOX-Afusion, Experimental Section they were plated onto apermeable membrane that wason Chemicals, reagents, enzymes and strains: Chemicals, medium top of alayer of LB agar containing arabinose for expression. components, and reagents were obtained from Sigma–Aldrich, After growing for 24 hat308C, the membrane with the colo- Merck, Acros Organics, Alfa Aesar,Thermo Fisher,and Fisher Scien- nies was transferred to an empty plate, frozen at 208C, tific. Oligonucleotides, superoxide dismutase (bovine SOD, re- À thawed for partial lysis, and submerged in an assay mix. Colo- combinant), catalase (Micrococcus lysodeikticus), and HRP were ob-

ChemBioChem 2019, 20,51–56 www.chembiochem.org 54 2019 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Communications

tained from Sigma. T4 ligase and the restriction enzyme BsaI were yellow elute was then incubated with KFe(CN)6 (100 mm)for 16 hat ordered from New England Biolabs. The PfuUltra Hotstart PCR 48Conarocking table to oxidize any reduced FAD. The yellow so- master mix was purchased from Agilent Technologies. E. coli NEB lution was applied to aPG-10 desalting column that was pre-equi- 10-beta (New England Biolabs) chemically competent cells were librated with Tris·HCl (50 mm,pH8.0), and the fusion enzyme was used as host for cloning the recombinant plasmids, and for protein eluted with the same buffer.Purified protein was analyzed by spec- expression. Precultures were grown in glass tubes with lysogeny trophotometry (200–700 nm) and SDS-PAGE. broth (LB);for the subsequent main culture terrific broth (TB) was SDS-PAGE and UV/Vis spectra: During the purification, small sam- used in baffled flasks. ples were taken before each step. SDS loading dye was added to Golden Gate cloning: All fusion constructs were cloned with the these samples, and the mixture was incubated at 958Cfor 5min, Golden Gate cloning approach. Before commencing the amplifica- then centrifuged at 13 000g for 1min. The samples were loaded tion and assembly of the genes into the vector,the pamo gene onto aprecast SDS-PAGE gel (GenScript, USA), and the gels were (TFU_RS07375) was mutated through QuikChange to remove a run in aMini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad) BsaI site, and to introduce the C65D mutation. Henceforth, the mu- with the current being applied by using aPowerPac HC High-Cur- tated pamo gene will be referred to as nox. Primers were designed rent Power Supply (Bio-Rad) set at 120 V. When the blue front of that contained aflanking region coding for the BsaI restriction site. the loading dye reached the bottom of the gel, after about 70 min, These primers were used for PCR to amplify the nox and three the gel was removed from the chamber,and stained with Coomas- alcohol dehydrogenase genes: Tbadh (X64841.1), Lbadh sie InstantBlue (Expedeon, US). An UV/Vis absorption spectrum (AJ544275.1), and adhA. In addition to the BsaI sites, linker regions from 200 to 700 nm (V-330 Spectrophotometer,JASCO) was taken were added to reverse primers of the first gene and the forward of each purified fusion protein diluted 1:10 in buffer (50 mm primer of the second gene of aconstruct. These introduced linker Tris·HCl, pH 8) in aquartz cuvette (Hellma Analytics, Germany). The 1 1 [20] regions together code for ashort peptide linker (SGSAAG) after li- protein concentration (e441 =12.4 mmÀ cmÀ ) was calculated by gation of the PCR products. The primers were designed such that using the values at 441 nm. the PCR products from one adh gene and the nox gene could be Activity measurements and determination of kinetic parame- inserted into apBAD vector.The pBAD vector contains two BsaI ters: Kinetic measurements were made by following the formation restriction sites, with an upstream region coding for an N-terminal or depletion of NADPH at 340 nm. After the enzyme ( 0.1 mm) His6 tag, an AraC promoter,and an ampicillin resistance gene. The had been mixed with substrate in buffer (50 mm Tris·HCl pH 8.0), fusion constructs were produced by incubating together two PCR NADP or NADPH (100 mm)was added, briefly mixed in acuvette, products (an adh gene and nox),Golden Gate pBAD vector,BsaI re- and then the reaction was followed (V-330 Spectrophotometer, striction enzyme, T4 ligase, ligation buffer,and sterile Milli-Q water. JASCO). For ADH activity,cyclohexanol was used as substrate;for The incubation temperature alternated between 168C(for 5min) the NOX activity,only NADPH was used, although in different con- and 37 8C(for 10 min) for 30 cycles, was then set to 558Cfor centrations. The slopes of the initial 20 swere used to calculate 10 min, and finally to 808Cfor 20 min to inactivate the enzymes. the activity rates. The obtained slope value is expressed in absorp- To transform host cells with the fusion constructs, the Golden Gate 1 tion change per minute [AbsminÀ ]. This value was then divided reaction mixture (3 mL) was added to chemically competent E. coli 1 1 by the extinction coefficient of NADPH (e =6.22 mmÀ cmÀ )in cells, and aheat shock (428C) was applied for 30 s. After overnight 340 accordance with the Beer–Lambert law to give avalue in mm per growth on an LB agar plate with ampicillin, colonies were picked minute. With that value and the enzyme concentration, the activity and grown in liquid LB, then the plasmids were isolated and sent rate was calculated. All measurements were made in duplicate or for sequencing (GATC, Germany) to confirm correct ligation of the triplicate. Activity data were fitted with GraphPad Prism 6.0 (Graph- genes. Pad;Figures S4 and S5). Culture growth and protein purification: Fusion enzymes were Conversions: Small-scale biotransformations were performed in produced and purified as described previously.[17] The E. coli cells 2mLEppendorf tubes, with 0.5 mL of reaction mixture. The mix- harboring the plasmids were grown in LB (5 mL) with ampicillin + 1 ture consisted of fusion enzyme (1–5 mm), cofactor (200 mm NADP (50 mgmLÀ ;378C, 135 rpm, 16–24 h). From this preculture, an ali- ), substrate, and buffer (50 mm Tris·HCl, pH 8.5, unless stated other- quot (2 mL, 4%, v/v)was used to inoculate TB (50 mL), which con- 1 wise). Acontrol without fusion enzyme was run in parallel. The tained ampicillin (50 mgmLÀ )and 0.02%filtered l-arabinose. The tubes with the reaction mixtures were incubated (248C, 600 rpm, cells were grown in a250-mL baffled flask (Sigma) at 248Cand ThermoMixer C, Eppendorf), the the samples were extracted three 135 rpm for 40 h. After harvesting by centrifugation (3000g,48C, times with an equivalent volume of ethyl acetate. The pooled 20 min), the cells were stored at 208Cfor several days. To purify À extract (1.5 mL) was dried over magnesium sulfate, and then ana- the enzymes, the cells were first resuspended in buffer (10 mL; lyzed by using GC-MS (HP-5 column, injection temperature:2508C, 50 mm Tris·HCl pH 8, 5mm imidazole) supplemented with FAD 1 oven temperature gradient:40–308C, 58CminÀ )orbyusing chiral (10 mm)and PMSF (phenylmethylsulfonyl fluoride, 100 mm). Then GC (Hydrodex b-TBDAc column (Aurora Borealis, The Netherlands), the cell suspension was cooled in ice water and subjected to soni- injection temperature:2508C, oven temperature gradient:60– cation (5 son/off, 10 min);subsequently the lysate was centrifuged 1 908C, 58CminÀ ). for 45 min at 18514g and 48C(Eppendorf F-34-6-38 rotor in 5810 Rcentrifuge). The supernatant was filtered (pore size 0.45 mm) into Oxidase activity assay: After expression of the NOX-A in E. coli, 2+ agravity flow column containing Ni Sepharose resin (1 mL;GE the cells were harvested, dissolved in buffer,and lysed by sonica- Healthcare), which was then closed and incubated for 60 min at tion. Only asmall volume (e.g.,500 mL) of solubilized cells is 48Conarocking table. The flow-through was collected, and the needed for this assay.The insoluble fraction was separated by cen- resin was washed with five column volumes of two solutions con- trifugation (18514g,Eppendorf F-34-6-38 rotor in 5810 Rcentri- sisting of buffer (Tris·HCl;50mm,pH8.0) and imidazole 10 and fuge, 30 min, 48C). In awell of a96-well plate, the soluble fraction 20 mm.Subsequently,the bound proteins were eluted by applying after lysis (20 mL), AAP (1.0 mm,20mL), DCHBS (10 mm,20mL), HRP imidazole (500 mm)inTris·HCl (50 mm,pH8.0). The obtained (4 U, 4 mL), substrate, and buffer to atotal volume of 200 mLwere

ChemBioChem 2019, 20,51–56 www.chembiochem.org 55 2019 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim Communications combined. As the lysate will contain NADP+/NADPH, it is not nec- mann, C.E.Paul, M. Pesic, S. Schmidt, Y. Wang,S.Younes, W. Zhang, essary to add the cofactor.The formation of apink product (as a Angew.Chem. Int. Ed. 2018, 57,9238 –9261; Angew.Chem. 2018, 130, result of peroxide production) can be followed over time to obtain 9380 –9404. 1 1 [4] R. D. Schmid, V. Urlacher in Modern Biooxidation:Enzymes,Reactions areaction rate (e =26 mmÀ cmÀ ). 515 and Applications (Eds.:R.D.Schmid, V. Urlacher), Wiley-VCH, Weinheim, Colony-screening assay: The assay was performed as described 2007,pp. XIII –XIV. previously,[24] with some adjustments. Cells harboring the plasmid [5] M. Pickl, M. Fuchs, S. M. Glueck, K. Faber, Appl. Microbiol. Biotechnol. for expression of NOX-A were plated onto aporous membrane (ni- 2015, 99,6617 –6642. trocellulose, Amersham, UK) on an LB agar plate that contained [6] J. Liu, S. Wu, Z. Li, Curr. Opin.Chem.Biol. 2018, 43,77–86. 1 [7] G. Rehn, A. T. Pedersen, J. M. Woodley, J. Mol. Catal. B 2016, 134,331 – ampicillin (50 mgmLÀ )and arabinose (0.02%). After 40 hofgrowth 339. at 308C, or when the colony-size large enough to pick multiple [8] D. E. Torres PazmiÇo, H. M. Dudek, M. W. Fraaije, Curr.Opin. Chem. Biol. times, the membrane was transferred to an empty Petri dish and 2010, 14,138–144. incubated at 20 8Cfor 1h.After the membrane with the colonies [9] P. B. Brondani, H. M. Dudek, C. Martinoli, A. Mattevi, M. W. Fraaije, J. Am. À had been thawed at room temperature for 1h,the assay mix Chem. Soc. 2014, 136,16966–16969. (20 mL), which contained melted agarose (10 mL, 2% w/v), potassi- [10] R. J. Conrado, J. D. Varner,M.P.DeLisa, Curr.Opin. Biotechnol. 2008, 19, um phosphate buffer (10 mL, 100 mm,pH7.5), HRP (100 U), 4- 492–499. chloro-1-naphthol (2 mm), cyclohexanol (50 mm;substrate), NADP+ [11] S. Elleuche, Appl. Microbiol. Biotechnol. 2015, 99,1545 –1556. (100 mm), was gently poured onto the membrane. The plate was [12] H. Yang, L. Liu, F. Xu, Appl.Microbiol. Biotechnol. 2016, 100,8273–8281. [13] D. E. Torres PazmiÇo, R. Snajdrova, B.-J. Baas, M. Ghobrial, M. D. Mihovi- incubated at 30 C, and after minutes/hours, the colonies with 8 lovic, M. W. Fraaije, Angew.Chem.Int. Ed. 2008, 47,2275 –2278; Angew. active “oxidases” developed adark blue color. Chem. 2008, 120,2307–2310. [14] D. E. Torres PazmiÇo, A. Riebel, J. de Lange, F. Rudroff, M. D. Mihovilovic. M. W. Fraaije, ChemBioChem 2009, 10,2595 –2598. Acknowledgements [15] N. Beyer, J. K. Kulig, A. Bartsch, M. A. Hayes, D. B. Janssen,M.W.Fraaije, Appl.Microbiol. Biotechnol. 2017, 101,2319–2331. This researchreceived fundingfrom the European Union (EU) [16] E. Y. Jeon, A. H. Baek, U. T. Bornscheuer,J.B.Park, Appl. Microbiol. Bio- technol. 2015, 99,6267–6275. project ROBOX (grant agreement no. 635734)under the EU’sHo- [17] F. S. Aalbers, M. W. Fraaije, Appl. Microbiol.Biotechnol. 2017, 101,7557 – rizon 2020 Program Research and Innovation actions H2020-LEIT 7565. BIO-2014-1. The views and opinions expressed in this article are [18] D. I. Colpa,N.Loncˇar,M.Schmidt, M. W. Fraaije, ChemBioChem 2017, 18, only those of the authors and do not necessarily reflect those of 2226 –2230. the European Union Research Agency.The European Union is not [19] C. Peters, F. Rudroff, M. D. Mihovilovic, U. T. Bornscheuer, Biol. Chem. 2017, 398,31–37. liable for any use that may be made of the information con- [20] M. W. Fraaije, J. Wu, D. P. H. M. Heuts, E. W. van Hellemond,J.H.L.Spel- tained herein. berg, D.B.Janssen, Appl. Microbiol.Biotechnol. 2005, 66,393–400. [21] A. R. Ferrari, Y. Gaber,M.W.Fraaije, Biotechnol. Biofuels 2014, 7,37. [22] L. C. P. Goncalves, D. Kracher, S. Milker,M.J.Fink, F. Rudroff, R. Ludwig, Conflict of Interest A. S. Bommarius, M. D. Mihovilovic, Adv.Synth. Catal. 2017, 359,2121 – 2131. [23] B. D. Bennett, E. H. Kimball, M. Gao, R. Osterhout, S. J. VanDien, J. D. Ra- The authors declare no conflict of interest. binowitz, Nat. Chem. Biol. 2009, 5,593–599. [24] M. Alexeeva, A. Enright, M. J. Dawson, M. Mahmoudian,N.J.Turner, Angew.Chem. Int. Ed. 2002, 41,3177 –3180; Angew.Chem. 2002, 114, Keywords: alcoholdehydrogenases · biocatalysis · enzyme 3309 –3312. engineering · fusion enzymes · oxidases

[1] T. Mallat,A.Baiker, Chem. Rev. 2004, 104,3037–3058. Manuscript received:July 24, 2018 [2] R. A. Sheldon, Catal. Today 2015, 247,4–13. [3] a) F. Hollmann,I.W.C.E.Arends, K. Buehler,A.Schallmey,B.Bühler, Acceptedmanuscript online:September 5, 2018 Green Chem. 2011, 13,226–265;b)J.Dong, E. Fernµndez-Fueyo, F. Holl- Version of record online:October 4, 2018

ChemBioChem 2019, 20,51–56 www.chembiochem.org 56 2019 The Authors. PublishedbyWiley-VCH Verlag GmbH &Co. KGaA, Weinheim