ACKNOWLEDGMENTS

I wish to express my gratitude to my supervisor Professor Giovanni Sannia, for his constant support and inspiration to do research.

I would like to thank Dr. Alessandra Piscitelli and Dr. Cinzia Pezzella for their precious help.

Finally, I like to convey my heartfelt thanks to Dr. Dietmar Schlosser for his valuable encouragement and support during the period spent in Leipzig.

A special thank goes to Lucia Guarino, Paola Cicatiello, Pamela di Pasquale and Francesca de Maria for their friendship.

My sincere thanks go to all the people I have met in the lab during these years. A very big thank goes to them, for having provided a fun and nice laboratory environment to work in.

I would like to thank my parents, my sister Angela, my roommates Margherita, Maria Teresa and Fulvia, and my boyfriend Giuliano for the love, the support and the encouragement that I always received from them.

Review/Revue

Fungal

laccases:

Versatile

tools

for

lignocellulose

transformation

AlessandraPiscitelli, ClaudiaDel Vecchio, VincenzaFaraco, PaolaGiardina, Gemma Macellaro,Annalisa Miele, CinziaPezzella,Giovanni Sannia*

DepartmentofOrganicChemistryandBiochemistry,UniversityofNaplesFedericoII,viaCinthia4,80126Napoli,Italy

1. Introduction

Lignocelluloseisacomplexofcarbohydratepolymers (celluloseandhemicellolose)tightlyboundtolignin,andis amajorconstituentofawidevarietyofmaterialsincluding waste materials from agriculture, forestry, wood-based industries,andmunicipalsolidwaste[1].Thesematerials areproducedinabundance,andrepresentagoodoption forconversiontouseful,highvalueproducts.Lignocellu- loseconversionrequiresapre-treatmentsteptodegrade or loosen the recalcitrant and heterogeneous lignin fraction. Thismulti-facetedchallengeisbeingaddressed by an ever-increasing suite of ligninolytic enzymes isolated from various sources. Among these, fungal laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2)are known toplay an importantrole in lignin degradation/modification processes. These enzymes can besuccessfullyappliedtopapermanufacturing,enhance-

mentoffibreproperties,productionofimprovedforages and pre-treatment of lignocellulosic biomasses for fuel production.

Biotechnologycan contribute to thedevelopment of ‘‘green tools’’ for the transformation of lignocellulosic feedstocksbyprovidingtailor-madebiocatalystsbasedon the oxidative enzymes responsible for lignin attack in nature[1].Withthispurpose,laccasesarecurrentlybeing improved using (rational and random-based) protein engineering[2].

Laccaseisoneoftheoldestenzymesreportedanditis arousing great interest in the scientific community becauseofitsverybasic requirements(itjust needsair toworkand its onlyreleasedby-product is water)and hugecatalyticcapabilities,makingitoneofthe‘‘greenest’’ enzymesofthe21stcentury[3].Thisenzymeisproduced byvariousfungi,plants,andcertainbacteriaorinsects[4]. Laccaseisabletocatalyzedirectoxidationofortho-and para-diphenols,aminophenols, polyphenols, polyamines, andaryldiaminesaswellassomeinorganicions.Itcouples the four single-electron oxidations of the reducing substrate tothefour electronreductive cleavageof the

C.R.Biologies334(2011)789–794

ARTICLE INFO Keywords:

Highredoxpotentiallaccases Lignin

Directedevolution Industrialapplication

ABSTRACT

Conversionoflignocellulosicmaterialstouseful,highvalueproductsnormallyrequiresa

pre-treatmentsteptotransformordeconstructtherecalcitrantandheterogeneouslignin

fraction.The development of ‘‘green tools’’ forthe transformation of lignocellulosic

feedstocksisinhighdemandforasustainableexploitationofsuchresources.Thismulti-

facetedchallengeisbeingaddressedbyanever-increasingsuiteofligninolyticenzymes

isolatedfrom various sources. Among these, fungal laccases are known to play an

important role in lignin degradation/modification processes. The white-rot fungus

Pleurotusostreatusexpressesmultiplelaccasegenesencodingisoenzymeswithdifferent

properties.TheavailabilityofestablishedrecombinantexpressionsystemsforP.ostreatus

laccaseisoenzymeshasallowedtofurtherenrichthepanelofP.ostreatuslaccasesbythe

construction of mutated, ‘‘better performing’’ enzymes through molecular evolution

techniques.Newoxidativecatalystswithimprovedactivityandstabilityeitherathigh

temperatureandatacidicandalkalinepHhavebeenisolatedandcharacterized.

ß2011Acade´miedessciences.PublishedbyElsevierMassonSAS.Allrightsreserved.

* Correspondingauthor.

E-mailaddress:[email protected](G.Sannia).

ContentslistsavailableatScienceDirect

Comptes

Rendus

Biologies

w ww . sc i e nce d i re ct . co m

1631-0691/$–seefrontmatterß2011Acade´miedessciences.PublishedbyElsevierMassonSAS.Allrightsreserved. doi:10.1016/j.crvi.2011.06.007

dioxygenbond,usingfour Cuatoms distributedagainst three sites, defined according to their spectroscopic properties.Typicalmetalcontentoflaccaseincludesone type-1(T1) copper,andonetype-2(T2)and twotype-3 (T3)copperions,withT2andT3arrangedinatrinuclear cluster(TNC)[4].

Fungallaccasesexhibitasimilarmoleculararchitecture organizedinthreesequentiallyarrangedcupredoxin-like domains.EachofthemhasaGreekkeyb-barreltopology

[4].T1is locatedindomain3,whilsttheTNCclusteris embeddedbetweendomains1and3withbothdomains providingresiduesforcoppercoordination.Thestructure isstabilizedbytwodisulfidebridgesbetweendomains1 and3andbetweendomains1and2.

Laccasesarecommonlyclassifiedaslow-mediumand highredoxpotentiallaccases(HRPLs)accordingtotheir redoxpotential at theT1 site rangingfrom+430mV in bacterial and plant laccases to+790mV in somefungal laccases.Thelatterarebyfarthemostimportantfroma biotechnological point of view [5]. HRPLs are typically secreted by ligninolytic basidiomycetes, the so-called white-rotfungi[3].

The white-rot fungus Pleurotus ostreatus expresses multiplelaccasegenesencodingisoenzymeswithdifferent properties, being the physiological significance of this multiplicitystillunknown[6].Amongthese,POXA1b,in additiontoitshighredoxpotential(+650mV)[7],ishighly stableathightemperatureandinthepHintervalof7to10

[8].Thus, thisenzyme isa suitablescaffold fordirected evolutionexperiments,since thelikelihoodof achieving requiredimprovementswithout affectingits stabilityis high.

Thisarticlereportstheoptimizationofthefunctional properties of POXA1b laccase expressed in the yeast Saccharomycescerevisiae [9]. We integrate these results with a structural analysis of some of the generated mutantsthatallowedustosuggestsomeofthereasons, atamolecularlevel,fortheirenhancedactivity.

2. Experimental

2.1. Strains,media,andplasmids

TheS.cerevisiaestrainusedforheterologousexpression wasW303-1A(MATade2-1,his3-11,15,leu2-3,112,trp1- 1, ura3-1, can1-100). The plasmidused for S. cerevisiae expressionwaspSAL4(copper-inducibleCUP1promoter). S. cerevisiae was grown in selective medium (6.7gL1

yeast nitrogen base w/o amino acids and ammonium sulfate; 5gL1 _{casaminoacids; 30mgL}1 _adenine;

40mgL1 _{tryptophane;50mM succinatebufferpH 5.3;}

20gL1_glucose).

2.2. Randommutagenesis

Randommutationswereintroducedwithlow,medium, andhighfrequencyofmutation,intothePOXA1bencoding cDNAs using GeneMorphTM PCR Mutagenesis Kit (Stratagene, La Jolla, CA). EP-PCR was performed with primers POXA1bfw (ATAAAAGCTTGAATTCATGGCGGTTG-

CATTCG)andPOXA1brev(TAAGGATCCAAGCTTTTATAAT- CATGCTTC).

2.3. Constructionofmutantlibrary

ThecDNAresultingfromEP-PCRonpoxa1bcDNAwere clonedinpSAL4expressionvector,digestedwithSmaIand BglIIrestrictionenzymes,byusinghomologousrecombina- tionexpressionsystemofS.cerevisiae.Yeasttransformation andselectionwasperformedasalreadyreported[10]. 2.4. Libraryscreening

Singleclonesgrownonplatewerepickedandtransferred into96-wellplatescontaining30mLofselectivemedium perwell.Plateswereincubatedat288C,250rpmfor24h. After24h,130mLofselectivemediumwasaddedtoeach wellandthe plateswereincubatedat288C,250rpmfor 24h.Thirtymicrolitersofeachculturewastransferredtoa new96-wellplatetomeasuretheOD600value.Theplates

were then centrifugedfor 10minat 1500g, 48C, and a suitablevolumeofsupernatantwastransferredtoanew96- wellplatetoperformlaccaseassay.Phenoloxidaseactivity was assayed at 258C using 2mM 2,20_{-azino-bis (3-}

ethylbenzothiazoline-6-sulfonicacid)(ABTS)in0.1Msodi- umcitratebuffer,pH3.0.OxidationofABTSwasfollowedby absorbanceincreaseat420nm(e36,000M1_cm1_),using

Benchmark Plus microplate spectrophotometer (BioRad, Hercules,CA).Enzymeactivitywasexpressedininterna- tional units (U). Cultures in shaken flasks were also performed.Pre-cultures(10mL)were grownonselective mediumat288Conarotaryshaker(150rpm).Avolumeof suspensionsufficienttoreachafinalOD600valueof0.5was

thenusedtoinoculate250mLErlenmeyerflaskscontaining 50mLofselectivemediumandcellswerethengrownona rotaryshaker.Opticaldensityandlaccaseactivitydetermi- nationweredailyassayed.

2.5. Screeningoflibraryforstability

The collectionof 3300 mutantsobtained by random mutagenesis of POXA1b laccase was analysed in three different screenings. First and second screening were effectuatedin96-wellplate,whilethethirdscreeningwas effectuatedinshakenflask.Inthefirstscreeningthelibrary wasanalysedafterone-daygrowthin96-wellplate.The supernatant was incubated for 48hours at pH 3 in Robinson and Britton buffer, and then activity towards ABTSassayed.Thepositivecloneswerefurtheranalysed during a three days growth in 96-well plate. Cellular densityandlaccaseactivityproductionwerefollowedfor threedays.Everydaythesupernatantwasincubatedfor 96hoursat378CatpH3inRobinsonandBrittonbuffer. Laccaseactivitywasanalysedevery24hours,inthisphase onlyclonesthatshowedgreaterstabilityifcomparedto thewild-typeenzymeatpH3wereselected.

2.6. Molecularmodeling

The structureof POXA1bwasobtained by homology modelingfromthecrystalstructureofTrametesversicolor

A.Piscitellietal./C.R.Biologies334(2011)789–794 790

(1GYC pdb entry), with which it shares 60% sequence identity. Thelast 16 residuesof POXA1bweremodeled usingthecoordinatesofthecorrespondingresiduesatthe C-terminus ofthe crystal structure of theMelanocarpus albomyces laccase (1GWOpdb entry). 3D model and in silico mutants were generated using the SWISS-MODEL webserverbymeansoftheprojectmodeoptionthatallow toselectthetemplateandcontrolthegapplacementinthe alignment.Refinementofthemodelshasbeenperformed by molecular dynamics simulations. Simulationson the wild-typePOXA1bandontheinsilicogeneratedmutants 1M9B and 3M7C were performed with the GROMACS packageasalreadydescribed[10].

2.7. Stabilityassays

Stability at pH values was measured using citrate- phosphatebufferadjustedatpH3,5and7.0,andTris–HCl bufferadjustedatpH10.

2.8. r4cDNAconstruction

Toobtainthelaccaser4cDNAthe30_{terminalportion}

(fromthenucleotide 453)ofthecDNAcodingfor3M7C was ligated to pSAL4 vector containing the 50 _terminal

portion (thefirst50 _{terminal452nucleotides)of1M10B}

encoding cDNA, after KpnI digestion of pSAL4 vectors containingthetwocDNAs.

2.9. DNAsequencing

Sequencingbydideoxychain-terminationmethodwas performedbythePrimmSequencingService(Naples,Italy) usingspecificoligonucleotideprimers.

2.10. Decolourizationexperiments

Batch decolourization experiments have been per- formedincubating(1mLfinalvolumeofreaction) crude preparation of laccase containing different enzyme amounts(0.1U,1Uand3U)inthreewastewatermodels: Acid (0.1gL1 _{Acid blue 62, 0.1gL}1 _{Acid Yellow 49,}

0.1gL1_{AcidRed266,2gL}1_Na

2SO4,pH5),Direct(1gL1

Directblue71,1gL1_{DirectYellow106,1gL}1_DirectRed

80, 5gL1_{NaCl,pH9)and Reactive(1.25gL}1_Reactive

blue 222, 1.25gL1 _{Reactive Yellow 145, 1.25gL}1

Reactive Red 195, 1–25gL1 _{Reactive Black 5, 70gL}1

Na2SO4,pH10)[11].

Performancesofselectedlaccasesinmodelwastewater decolourizationwereevaluatedbyrecordinglightabsorp- tionspectra between280and 800nmatdifferenttimes (10min,20min,1h,2h,3hand24h),andcomparingthem with the corresponding spectra of controls (the waste incubated with the supernatant of yeastcultures trans- formedwiththeemptyexpressionvector).Decolourization wascalculatedastheextentofdecreaseofspectrumarea recordedbetween380and740nmwithrespecttoacontrol sample.Allspectrawererecordedafter1:100dilutionofthe sampleinmilliQwater.Allexperimentswerecarriedoutin duplicates,andthemeanvaluesweretaken.Thestandard deviationfortheexperimentswaslessthan5%.

3. Resultsanddiscussion

The white-rot fungus P. ostreatus expresses multiple laccasegenesencodingisoenzymeswithdifferentproper- ties,beingthephysiologicalsignificanceofthismultiplicity still unknown [6]. Investigation of the recently released P. ostreatus genome (http://www.jgi.doe.gov/sequencing/ why/50009.html) has disclosed a complex multicopper oxidasefamilyofuptotwelvemembers.Theavailabilityof establishedrecombinantexpressionsystemsforP.ostreatus POXA1b [9] has allowed the improvement of enzymes features through a combination of rational and random mutagenesis [10,12,13]. Ourstarting pointwas the high redox potential laccase POXA1b [14], which exhibits remarkablestability at alkaline pH[8]. The ideabehind theevolutionstrategyistocreateanidealbiocatalyst,ableto oxidise a wideassortmentof substrates,andstable ina broadrangeofpH.Thus,differentscreeningcriteriawere appliedtosearchforsuchcatalysts.

3.1. Firstgeneration

Alibraryofalmost1100mutantswithlow,medium and high range of mutations was generated by error- pronePCR(EP-PCR)usingpoxa1bcDNAastemplate[10]. Screeningthislibraryforvariantswithimprovedactivity at pH 3 using the non-phenolic substrate ABTS has allowed the selection of one mutant, named 1M9B. It showsasinglemutation(L112F)leadingtoanimprove- mentofactivitybutadecreaseofstabilitywithrespectto the wild-type enzyme (POXA1b) in all the analyzed conditions.Inposition112,thereisagenerallyconserved leucinein alllaccases frombasidiomycetes,althougha phenylalanine seems to be conserved in laccase sequencesfromascomycetes.POXA1b3Dmodelshows thattheresidue 112is locatedin thechannel through whichthesolventhasaccesstotheoxygen-reducingT2/ T3 site. To elucidatethe roleplayed by this mutation, MolecularDynamic(MD)simulationswereperformedon the modelof the mutant and compared with thoseof POXA1b. The analyses show a movement of the sub- domain around position 112 as a consequence of a conformationalrearrangementduetothepresenceofthe bulkier residue of phenylalanine. A significant effect generatedbythemutationisobservedinthepermeabili- tytowateroftheT2/T3channel.ResidueF112islocated atthe entranceof thechannelanditssteric hindrance affectsthepassageofwatermoleculestowardtheTNC.As afact,alargernumberofwatermoleculesintheT2/T3 channelhasbeenobservedfor1M9B. Thesedatacould suggest an increased affinity of this mutant toward oxygenmolecules, thusjustifyingitsimproved specific activity.

3.2. Secondgeneration

1m9bcDNAwasusedastemplateforasecondroundof EP-PCRatlowandmediumfrequencyofmutation[10].A secondgenerationlibraryof1200cloneswasobtainedand screenedusingthesamecriteriumdescribedbefore.Three mutants,1L2B, 1M10B(L112F, K37Q,K51N), and 3M7C

(L112F,P494T),wereselectedshowinganactivityincrease ofuptothreefoldwithrespecttoPOXA1b.

Concerning 1M10B mutations, it finds out how directed evolution can get the same result of natural evolution, preserving theproperties of mutated amino acids. As a fact, positions 37 and 51 are generally occupiedbyamidicresidues.Themutant3M7Cdisplays ahighactivityandanuptotwofoldincreasedstability atacidicandneutralpH,aswellasathightemperature. 1M10BvariantismorestableatalkalinepH(abouttwo fold), whereasits stabilityis almostequivalentto that ofPOXA1bintheothertestedconditions.Themutation P494TislocatedintheC-terminalloopthathasalready beenascertainedtoaffectthefunctionoffungallaccases

[4]. MD simulations of this mutant and comparison withthewild-typePOXA1brevealeda lowerflexibility ofthesubdomainaroundposition112probablyrespon- sible of its higher stability. On the other hand, an increased mobility of loops forming the reducing substrate binding site, has been observed in 3M7C leadingtohigheraccessibilityofwatermoleculestothe T1 copper site, and to an increased activity of the enzyme.

Firstandsecondgenerationlibraries(2300clones)were thenscreenedforvariantswithimprovedactivityatpH5 using either the non-phenolic substrate ABTS, and the phenolicone2,6-dimethoxyphenol(DMP).

WhenDMPwasusedassubstrate,twonewvariants (2L4Aand3L7H)endowedwithhigherenzymeactivity (about three fold increase) than the wild-type laccase wereselected[12]. Bothmutantsdoubledthe stability of the wild-type enzyme at pH 5. Q272H mutation foundin2L4AmaystabilizetheproteinstructureatpH5 allowing additional interactions – electrostatic and hydrogen bonds – between the positively charged imidazolicringofHis272andthesidechainofAsp287. After screening with ABTS, one clone, 1L9A, was selected,showing an increaseof about threefold with respect to wild-type. Besides the parental mutation (L112F),1L9AalsopresentsthemutationR284H,located in the loop Gly282-Thr289. This loop may play an important role in protein stability [12]. As a fact, concerningitsproperties,themutantincreasesstability atpH5(1.5fold), whileloosing thehighcharacteristic stabilityofPOXA1bat alkalinepH.

3.3. Rationaldesign

During the engineering of POXA1b, some of the beneficial mutations discovered in the early stages of evolution were merged in the rational designed R4 mutant. Synthesis of a laccase joining mutations of 3M7C and 1M10B variants [10] was performed to combine the increased stability of 1M10B at alkaline pHandtheimprovedcatalyticefficiencyof3M7C[13]. Joining these mutationsa two-foldincrease in laccase activitywithrespecttowild-typeenzymewasobtained. Themainimprovement duetothechimerconstruction isaslightincreaseinstabilityathightemperature,and evenmore atneutral (aboutfour-fold)andalkalinepH values(abouttwo-fold).

3.4. Thirdgeneration

TheincreasedstabilityofR4makesitanappropriate scaffoldtocarryoutdirectedevolution[15].Infact,more stable enzymes should also be more susceptible to evolution since they have higher ability to tolerate functionallybeneficialbutdestabilizingmutations.There- fore, directedevolution ofR4waschosenasstrategyto improve itsperformances[13].A libraryof 1000clones withlow,mediumandhighaverageofmutationfrequency wasobtainedthroughEP-PCR.Whenthisnewcollection wasscreenedbyassayingactivitytowardsABTSatpH3, two mutants, 4M10G and 1H6C, with higher activity (aboutfour-foldincrease)thanthatofPOXA1bwild-type wereselected.Bothmutantsdisplayhigherstabilitythan POXA1batpH5(almostfour-fold).1H6CalsoretainsR4 stabilityfeaturesatpH10andatpH7.Sequenceanalyses of the selected mutantsled tothe identificationof the mutationsV126Iforthe4M10GvariantandV148Lforthe