Low impact strategies to improve ligninolytic enzyme production in filamentous fungi: the case of laccase in Pleurotus ostreatus

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Review/revue

Low

impact

strategies

to

improve

ligninolytic

enzyme

production

in

ﬁlamentous

fungi:

The

case

of

laccase

in

Pleurotus

ostreatus

Vincenzo

Lettera

*

,

Claudia

Del

Vecchio,

Alessandra

Piscitelli,

Giovanni

Sannia

DepartmentofOrganicChemistryandBiochemistry,UniversityofNaplesFedericoII,ComplessoUniversitarioMonteS.Angelo-ViaCinthia4,80126Napoli,Italy

1. Introduction

Thewhite-rotbasidiomycetefungusPleurotusostreatus isoneofthemostactivemicroorganismsdegradinglignin,a complexaromaticbiopolymerthatisextremelyrecalcitrant todegradation[1].Thisfungusproducesdifferentoxidative enzymes,withbroadsubstratespeciﬁcity,whichcanalsobe usedtodegradeavastrangeoftoxicaromaticpollutants

[2,3]. Among these enzymes, the production of several laccase (E.C. 1.10.3.2) isoenzymes is prominent [4]. The varietyoflaccaseisoenzymesisrelatedtothediversityof their roles: lignin synthesis/degradation [1,5], fruiting bodies development [6], pigment production [7], cell detoxiﬁcation [8], etc. [9]. Moreover, laccases result in biotechnologicallyrelevantproductsbecauseoftheirability tooxidizebothphenolicandnon-phenolic ligninrelated compounds as well ashighlyrecalcitrant environmental pollutants.Thesefeaturesaresuitableforseveraldifferent applicationsinindustrialefﬂuentsdisposal,medical

diag-nostics,bioremediationtodegradingpesticidesand explo-sivesinsoils,delignificationprocessesinpaperindustries andincosmeticsformulationasadditive[10].Owingtothe successfuluseoflaccasesintheabove-mentioned biotech-nologicalapplications,theever-increasingdemandrequires theproductionoflargequantitiesofenzymeatlowcost.Asa fact,severalproductionstrategieshavebeenadoptedalong with process optimization to achieve better process economics. Concurrently, studies on laccase producing organismshavebeen intensifiedintherecentyears.The overexpressioninsuitablehostsandoptimizationoflaccase productionfromdifferentmicroorganismswouldprovide meanstoachievehightiters.Onthe otherhand, several methods have been used for strain improvement in Pleurotusspp. includingselection,hybridizationandgene transformation [11–14]. Based on current legislation (European Directive 2001/18/CE), genetic transformation andmutagenictreatmentsproducestrainsnotsuitablefor ‘‘naturalorsafeprocesses’’.Therefore,theconstructionof genetically modified organisms cannot be chosen to improvetheaddressedqualityofthefungus,andbreeding shouldbebasedonclassicalgeneticapproaches.Thislast

ARTICLE INFO Keywords: White-rotfungi Laccase Autoinduction Spentmedium Classicalbreeding ABSTRACT

Theever-increasingdemandoflaccasesforbiodeligniﬁcation,industrialoxidativeprocesses andenvironmentalbioremediationrequirestheproductionoflargequantitiesofenzymesat lowcost.Thepresentworkwascarriedouttoreducelaccaseproductioncostsinliquid fermentationsofthewhite-rotfungusPleurotusostreatusthroughtwodifferentapproaches. Intheﬁrst,screeningoffungalspentmediaasnaturallaccaseinducerwasperformed, eliminatingthepresenceofpotentiallytoxic/recalcitrantandexpensiveexogenousinducers intheculturebroth.Inthelatter,breedingofdifferentstrainsofP.ostreatus,screenedfor their laccase productivity, was performed by cross-hybridisation, avoiding genetic transformationandmutagenictreatmentsthatcouldproduceorganismsnotsuitablefor ‘‘naturalorsafeprocesses’’.Alaccaseproductionlevelcloseto80,000U/Lbycombiningthe twoapproacheswasachieved.Autoinductionandclassicalbreedingrepresentpromising toolsfortheimprovementoffungalfermentationwithoutaffectingthedisposablecoststhat alsodependontheeco-compatibilityofthewholeprocess.

ß2011Acade´miedessciences.PublishedbyElsevierMassonSAS.Allrightsreserved.

* Correspondingauthor.

E-mailaddress:vincenzo.lettera@unina.it(V.Lettera).

ContentslistsavailableatScienceDirect

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Rendus

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1631-0691/$–seefrontmatterß2011Acade´miedessciences.PublishedbyElsevierMassonSAS.Allrightsreserved.

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technique is based on the mating of two monokaryotic compatiblestrainsofinterest,whosehyphaeareabletofuse andgiverisetoadikaryotic myceliuminwhichthetwo parentalnucleiremainindependent[13,14].Productionof the monokaryotic strain, germinating from uninucleate basidiospores,isachievedwhenthe fungusentersintoa reproductivephasetriggeringbasidiocarpformation:during basidia formation, karyogamy takes place immediately beforetheonsetofthemeiosisgivingrisetofouruninucleate basidiospores. Chaudhary et al. [15] developed single sporeisolates from the white-rotfungiPleurotus djamor, P. ostreatus var. ﬂorida, Pleurotus citrino pileatus and Hypsizygusulmarius.Thehybridsshowedimprovedmycelial growthratecomparedtoparentalstrains.Inanotherwork, SawasheandSawant[16]developedhybridcultureswhich required a signiﬁcantly shorterperiod for spawn run as comparedtotheparentspecies.

Selecting newhybrid strains for enzyme production couldbeviewedasasolutiontomaketheentireprocess cost effective, and further enhancement using inducers maybeaddedtothebeneﬁt.

Useofinducerstoenhancelaccaseproductionhasbeen widelypracticed infungiespeciallyinwhite-rots, where inductionoflaccaseproductionbyaromaticcompoundsis wellestablished[17].Increasinginlaccaseproductionhas beenalsoachieved inpresence ofother compoundslike aminoacids[18],plantextracts[19]andcopper[20,21]in the growth medium. However, useof above-mentioned inducersenhance productioncost,because oftheirprice andtheirlikelytoxicitywhichcouldnegativelyaffectcost ofwastewaterdisposable.Inordertoavoidtheseproblems, studies on fungal autoinduction mechanisms [22] are beingcarriedoutbyseveralgroups.Autoregulations,and the signal molecules involved in, have been clearly elucidated in dimorphic fungi like Candida albicans and Saccharomycescerevisiae[23].However,severalcompounds havebeencorrelatedtotheregulationofdifferentaspectsof fungalphysiologyindiverseclassesofthekingdommycota

[22].Schimmeletal.[24]reportedaspeciﬁcautoinduction effectonlovastatinsynthesisinAspergillusterreus,whena spent medium solution extracted from the fungal sub-mergedcultureisusedtoconditionanewfreshgrowthof thesamestrain.Conversely,intheliterature,noinformation aboutlaccaseautoinducersisavailable.

Theaimofthepresentworkhasbeentoexploitboth approaches: conditioning P. ostreatus growth by spent medium solution extracted from liquid culture and breedingthefungalstrainsbyclassicalcrossing.Finally, thepotentialexploitableeffectofspentmediumsolutions on laccase production was combined with improved capabilitiesofnewhybridsderivedfromthebreedingof twoP.ostreatusvarieties.

2. Materialsandmethods

2.1. Organisms

All P. ostreatus monokaryotic and dikaryotic strains weremaintainedthroughperiodicserialtransfersandkept at 48C on agar plates in the presence of 2.4% potato dextroseand0.5%yeastextract(PDY)(Difco).

Monokaryoticprogenywereidentiﬁedbyaprogressive numberfollowedbythe(lowercase)letteroftheparent strains (strains no.‘‘f’’ and no.‘‘o’’, respectively). New dikaryoticvarieties wereclassiﬁedbyprogressive num-bersfollowedbythetwolowercaselettersandseparated bythe‘‘X’’(no.fXno.o).

2.2. Fructiﬁcationandbasidiosporesisolation

Mushrooms of two commercial dikaryotic strain of P. ostreatus, P. ostreatus variant Florida (strain F) and P.ostreatusvariantostreatus(strainO),werecultivatedin 500mLjarscontaining400gofwheat-straw(65%water content),whichweresterilizedbysteamheated(1218C)in autoclave for 1h at 1218C. Sterilizationprocedure was repeatedasecondtimeafteranincubationtimeof24hat roomtemperature.Eachjarwasinoculatedwithfouragar plug (13mmdiameter), andlefttogrowat288Cfor 30 daysinthedark.Fructiﬁcationwaspromotedbyopening the jars,and placing them in presence of daylight in a chamberat1558Cand90%relativehumidity.Primordia appearedafterafurther15daysofgrowth,andbasidiocarps wereharvested7dayslaterandweighed[25].

P.ostreatusbasidiosporeswerecollectedbysporalprint onaglassPetridish,previouslysterilisedinautoclave(1h at1218C).

A spore suspension was prepared in 1ml sterile physiologicalsaltsolution(0.9%NaCl).Sporal concentra-tionwasestimatedbycountingin aThomachamber on opticalmicroscopy.

2.3. Matingtest

ThebasidiosporessuspensionwasplatedonPDYagar mediumafterappropriatedilution.Vegetativemycelium colonieswereexaminedbyphase-contrastmicroscopyfor clamp connections, the appearance of colony characte-risticsspeciﬁcfordikaryon.Colonieslackingclampswere subculturedinPDYagarslantsat288Candinoculatedin pairson2%maltextractagarplates,sothattheirmycelia wouldfuse.Compatiblemonokaryonswereidentiﬁedby theproductionofclampconnections.

2.4. Cultureconditionsinliquidculture

2.4.1. Condition1(C1),forspentmediapreparation MyceliumofvarietyP.ﬂoridawasgrownin1lshaken ﬂasks(125rpm)containing300mlofGYM(Glucose,Yeast extract, Mineral solution) formulated as follow: 10g/l glucose;3,8g/lyeastextract(Difco)2g/lH2KPO4;0.5g/l

MgSO47H2O;0,1g/lCaCl22H2O;biotin10mg/l;thiamine

10mg/land10mlofmineralstocksolution(0.5g/lMnSO4

5H2O;1g/lNaCl;0,1g/lFeSO47H2O;0.1g/lCoCl26H2O;

0,1g/l ZnSO4 7 H2O; 0,01g/l CuSO4 5 H2O; 0,01g/l

AlK(SO4)2;0.01g/lH3BO3;0.01g/lNaMoO42H2O);ﬁnal

pH5.Exceptwhereindicated,allchemicalswereobtained fromSigmaChemicalCo.5-day-oldculturewere homoge-nized by Ultra-Turrax1

T25 Basic interconnected with S18N-19G dispersing tool (3 ﬂashes of 30seconds at 24,000rpmseparatedby30secondsofstand-by)and1mlof homogenatewastransferredin1-lﬂaskscontaining300ml

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ofGYMbroth.Theculturesweregrowninshakenﬂasksat 125rpmandincubatedat288Cinthedarkfor17days. 2.4.2. Condition2(C2),forhybridstraingrowth

Submerged cultivation was carried out in 100ml Erlenmeyer ﬂasks containing 30ml of PDY with copper sulphate (ﬁnal concentration 150

m

M) on rotary shaker (125rpm).Theﬂaskswereinoculatedwithfouragarplugs (8mmdiameter)cutfromtheactivelygrowingpartofthe colonyonaPetridishandincubatedforatleast17daysat 288Cinthedark.

2.5. Liquid–liquidextractionofspentmedia

Extractions were performed on P.ﬂorida samplesby adding ultra pure chloroform (Carlo Erba reagents) to 300mlofP.ostreatusharvestedgrowthmedium(condition 1) using a 1:1v/v ratio. The mixture wassubjected to horizontalandrotaryshakingfor2min.Theprocedurewas repeatedtwiceforeachsample.After10mindecantation, organicphasewasremovedandconcentratedupto1000 timesusingaHeidolphLaborota4000rotaryevaporator. The liquid-liquidextraction wasused toprepare condi-tioning solution SM7, SM10, SM13 and SM16 (spent medium7,10,13and16daysold,respectively). 2.6. Conditioningbyspentmediumsolutions

The liquid-liquid extraction of 300ml spent GYM medium were concentrated upto 1000 times,sterilized byﬁltermembrane(cut-off0,22

m

m,Millipore1

)andused tocondition300mlofbasalmediumofafreshgrowth(C1). ConditioningofGYMbasalmediumwasperformedusing extracted spent medium solutions supplemented at the timeofinoculation.

2.7. Protein,biomassandglucoseconcentration determinations.

ProteinconcentrationwasdeterminedusingtheBioRad proteinassaykit(BioRad,Hercules,California),following themanufacturer’sinstructions,withbovineserum albu-minasstandard.Biomasswasdriedbydryingovenat658C overnightandestimatedgravimetrically.Glucose concen-trationsweredeterminedbytheglucoseoxidizemethod

[26].Eachassaywasperformedintriplicate.

2.8. Enzymeassays

Phenol-oxidaseactivitywasassayedat258Cusing2,20_–

azinobis-(3-ethylbenzothiazoline-6-sulfonicacid)(ABTS)as substrate[27].Theassaymixturecontained2mMABTSand 0.1Msodium-citratebuffer,pH3,0.OxidationofABTSwas followedbyabsorbanceincreaseat420nm(

e

=36,000M1

cm1₎_for₁_minute._Enzyme_activity_was_expressed_in_UI._All

measurementswererepeatedatleastintriplicate.

2.9. Nativepolyacrylamidegelelectrophoresis

Polyacrylamidegelelectrophoresis(PAGE)wascarried outatalkalinepHundernon-denaturingconditions.The

resolvingandstackinggelscontained9%and4%acrylamide, respectively.Thebuffersolutionusedfortheresolvinggel contained50mMTris-Cl(pH9.5),andthebuffersolution usedforthestackinggelcontained18mMTris-Cl(pH7.5). Theelectrodereservoirsolutioncontained25mMTris-Cl and190mMglycine(pH8.4).Gelswerestainedtovisualize laccaseactivitybyusingABTSassubstrate,insodiumcitrate buffer0.1MpH3.Samplescontaining0.015laccaseunities wereloadedoneachlane.

3. Resultsanddiscussion

3.1. AnalysisofPleurotusostreatusgrowthmodelinliquid culture

Avoidingfalsepositiveinductionderivedfrom biotrans-formation of unknowncompounds (generallypresentin complex media), P.ostreatus var.ﬂorida wasgrown ina semisyntheticmediumcontainingglucoseasmaincarbon source,andyeastextract,asnitrogensource.Asshownin

Fig.1,threephasesofthebasalgrowth,formingthetypical behaviourofﬁlamentousfungiinliquidcultures,havebeen detected.Startingfromtheinoculationtime(t=0day),the lagphasewasdisplayedfor2days.Duringthisphase,no relevantglucoseconsumptioninthemediumwasdetected. Increasing of glucose consumption rate occurred in the trophophase up tothe complete depletion of the main carbonsource(7thday).Duringthesametime,thefungal culturereachedhighestcelldensity,measuredasmycelial dryweight(7.20.5g/L).However,betweenthe5th_and_the

7th_fermentation_day,_the_rapid_increase_of_glucose_uptake_and

thedecrease of growthrateindicatethe beginning ofthe idiophase,whentheanabolicpathwaysoftheculturedfungus are altered to produce different biomolecular compounds (secondarymetabolites).Infact,thebeginningofexponential growthisrelatedtothestart-pointofglucoseconsumption, whilecellularlyses(Fig.2)inthelastphaseofgrowthshouldbe aconsequenceofcarbonstarvation.

Timecourse analysisof extracellular laccase activity productionwascarriedoutandtheproductionproﬁlewas evaluatedinparallelwiththefungalgrowth.Aspreviously reported [28], laccase synthesis does not appear to be relatedonly tothehyphal growth, becausetheenzyme activity does not parallel biomass production (Fig. 1). Laccase activity peaked on the 5th _and _the ₁₃th _day,

reaching 4000and 3000U/Lrespectively,anddecreased dramatically thereafter. The ﬁrst laccase production increment,rising upto the5th _day, _is _probably_related

tothedevelopment offungal biomassin liquid culture, whereasthelatterisprobablyconnectedtotheidiophase phenomena, where activation of secondary metabolism andcellularautolysisoccurs(Fig.2).

Laccaseswere analyzed by native PAGE and stained withABTS.Analysisofsampleswithdrawnfromthemedia at different growth times indicates that the activity is associatedmainlytotheproductionofthreeisoenzymes POXA3,POXA1BandPOXC[4],asreported inFig.3.The same isoenzymatic pattern wasobserved in correspon-denceofthetwomaximumproductionlevels,althoughthe bandintensitieschangedduringthetimecourse: detect-ablelevelsofPOXA1BandPOXA3activityproductionwere

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onlyfoundincorrespondencewiththeﬁrstmaximumof laccase production (4–5th day), while no signiﬁcant difference in the relative amount of POXC isoenzyme wasdetectedatdifferenttimesofgrowth(4th_,₅th_and₁₃th

day).

Takingintoaccountthewholedataacquiredduringthe timecourseanalysis,secondarymetabolismactivatedin theidiophasecouldbeassociatedtothesecondmaximum oflaccaseproduction,althoughacause-effectcorrelation cannotbeformulatedatthisstage.Ithasbeenpreviously reported that P. ostreatus produceseveral natural com-poundsduringfermentationinsubmergedcultureduring the idiophase [29,30]. The reasons why fungi produce secondarymetabolitesarestillunknownandmostofthese moleculeshavenot beencreditedwitha biologicalrole

[30]. Although no comparative analysesabout dynamic variation of exo-metabolites during fermentation in submergedculturewasperformed,itisevidentthatunder conditions of nutrient limitation, morphological altera-tions and mycelium changes variations in secondary metabolismdynamicallyoccur.Moreover,manyevidences

strongly indicate that Pleurotus spp. displaysthe ability to synthesize lignin-related compounds [31,32]. These extracellular metabolitescould regulate laccase expres-sionsimilarlytootherchemical-relatedinducers[32],as ferulicacid[33].

3.2. IncreasingoflaccaseexpressioninPleurotusostreatusby spentmediumsolution

Inordertoinvestigatetheabove-mentioned hypothe-sis, metabolitesexcreted by P. ostreatus wereextracted from the fungal fermented broth, starting from the beginningoftheidiophase(7th_day)._Spent_media_solution

derivedfromtheculturebrothat7,10,13,and 16days (SM7,SM10,SM13andSM16,respectively)wereusedto conditionliquidfermentationofP.ostreatusgrowninbasal condition(C1).

Analyseswereperformedmonitoringbiomassgrowth, totalsecretedproteinandlaccaseproductionproﬁlesafter adding spentmedium solutions at theinoculation time (t=0 days).Conditioninggrowthmediaby SM10,SM13

days 0 1 2 3 4 5 6 7 8 9 10 11 12 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 5,000 4,000 3,500 4,500 3,000 2,000 1,500 2,500 1,000 0 500 glucose g/L ; biom ass g/L laccase acitivity U /L; total secret ed prot ein µg/L

Fig. 1.Pleurotusostreatus 17daysfermentation proﬁlein basalcondition:extracellular laccase activity,secretedprotein concentration, glucose consumptionandbiomassincreasingarereportedasU/ml( ),10*mg/l( );g/l( ),g/l( )respectively.

Fig.2.Lightmicroscopyobservationoffungalpelletatthe5th

(A)andthe12th

(B)dayofgrowthinliquidbasalmedium.InFig2B,itispossibletoappreciate vacuolizationandcelllyses.Bar,12mm.

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and SM16causedanincreaseoflaccaseactivityupto5 foldsincorrespondenceoftheﬁrstpeakatthe4th_and_the

5th_day₍_Fig.₄_),_whereas_no_induction_of_laccase_expression

wasinducedatthe13th_day._No_signiﬁcant_variation_of_the

otherparameterswasobserved(p<0.005).

No relevant difference in the enzymatic pattern of samplescollectedat4th_,₅th_and₁₃th_day,_from_basal_and

conditionedgrowthswasobserved.Suchdataindicatethat thepresenceofspentmediumsolutionsinliquidculture affectsgeneralmechanismsoflaccaseexpressionand/or secretionandthattheincreaseoflaccaseactivityatthe4th

andthe5th_day_does_not_depend_on_the_{over-expression}_of

aspeciﬁcisoenzyme.Thisbehaviourdoesnotcorrespond to the laccase expression trend observed in the basal growthduring the secondphase of enzyme production (latepartoftheidiophase).Asafact,activitydramatically decreasesafterthe13th_day._After_the₁₃th_day,_when_the

lastphaseofcarbonstarvationstresstakesplace,different mechanismscoulddeactivate/repressanybiological syn-thesis,includinglaccaseexpression.Asafact,quiescence orlysesofhyphaecouldcauseaninsensitivityofthefungal cellstothepresenceofanyinducer.

Moreover, conditioned fungal cultures result not responsive totheinduction duringtheidiophase, while a laccase production increase during the trophophase occurs. Probably, chemicals contained in the spent mediumsolutions, that weresupplemented atthetime ofinoculation,weremetabolizedordegradedbythefungal enzymaticactivities.

3.3. IncreasingoflaccaseexpressioninPleurotusostreatusby classicalbreeding

Thebreedingstrategiesofnewvarietiesofindustrially usefulfungi likeP.ostreatus aredeﬁned bythe breeding objectivesandthelegalconstraintsimposedtothebreeding technology used. This last aspect is of the greatest importanceinthecaseofwild-typemicroorganismswhich areconsiderededible, GRASandeco-compatibles. Inthe frameworkoflocalandinternationallegislations,infact,the use of genetically modiﬁed microorganisms (GMMs) in industrialbioprocessescouldincreasethewastedisposable costandprecludethepotentialconversionofbiomassin bioproducts as animal fodder (European Communities

days 0 5000 10000 15000 20000 2500 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 A B C D E laccase acitivity U /L

Fig.4.LaccaseactivityperlitreofcultureinPleurotusostreatusgrowthmedium(C1)inabsence(lineA)andinthepresenceofSM7(lineB),SM10(lineC), SM13(lineD)andSM16(lineE).

Fig.3.Zymogramsoflaccaseisoenzymesinthebasalcondition(C1). Samplescontaining0.015Uoflaccaseactivitycollectedatdifferenttimes (4th_,₅th_,_and₁₃th_day)_were_used.

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Guidancenotesforriskassessmentoutlinedinannex3of council directive 90/219/EEC on the contained use of genetically modiﬁed microorganisms). This prevents the useofgenetic-engineeringbasedtechnologiesforbreeding. Consequently,ourworkwasfocusedonclassicbreeding,in ordertoevaluatethecapabilityofthistechniquetoimprove safestrainsforlaccaseproduction.Asreportedinliterature

[34], heterozygosity in genes responsible for laccase expressiontriggershighvariabilityofenzymeproduction andincreasesaverageproductioninbasidiosporesderived monokaryonsobtainedfromasingledikaryoticstrain.In fact,certain monokaryoticisolatesproducemuch higher titresofenzymesthantheparentalstrain.

P.ostreatus var.ﬂorida (strainF)andP.ostreatusvar. ostreatus(strainO) arethedikaryoticfungi usedin the presentwork.Thesevarietiesdifferinseveral morphologi-calandphysiologicalfeatureslikesize,colour,temperature tolerance,etc.Bothstrainshavebeenextensively charac-terized in previous studies for their ability to produce oxidative enzymes[4,35,36], hydrophobins[37,38], and natural compounds [29]. In order to produce new dikaryotichybridswithincreasedproductioncapabilities, basidiospores-derivedmonokaryons,obtainedfromboth strains,wereisolatedandanalysed.Collectedsporesfrom

the two differentbasidiocarpsweresuccessfully germi-nated in solid medium. Microcolonies progeny was microscopically analyzed and monokaryotic state was conﬁrmedbytheabsenceofmycelialclampconnections. Monokaryotic strains ‘‘f’’ and ‘‘o’’, isolated among twenty-eightrandomlychosengerminatingspores,were inoculatedinthePDYproductionmedium(C2)inorderto enhance laccaseproduction duringfungal fermentation. Time courses analysis of extracellular laccase activities showedhighvariabilityamongthestrainsandamaximum ofproductionbetweenthe9th_and_the₁₀th_day_of_growth

(Fig.5).ThreemonokaryonsfromtheparentalstrainF(5f, 6f,18f)andthreemonokaryonsfromtheparentalstrainO (3o, 9o, 11o) exhibit higher or comparable production levelsthanthetwoparentalstrains.Asalsoreportedby Eichlerov´a and Homolka [34], the isolates differ also in morphologyand growthratewhen subculturedonagar mediainPetridishes(datanotshown).However,colony appearance,whosemorphologicalpatternsaretransient, couldnotbeconnectedwithlaccaseproductionlevels.

Anastomosisinduction,followedbyformationofclamp connections, indicated compatibility among two strains and the formation of the corresponding dikaryon. All compatiblecrossings,amongthesixmonokaryoticstrains

Fig.5.Timecourseoflaccaseactivitysecretedbymonokaryoticstrains‘‘f’’and‘‘o’’inPDYmedium(C2).EnzymaticactivityofparentalvarietiesFandOare alsoreported.Standarddeviation<20%.

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wereperformed.Crossingselectedstrains,newdikaryotic hybrids were obtained, excepting for thepairs coming fromthesameparent(fXfandoXo)andthecrossing5fX 9o,thatresultedincompatible(Table1).Thesedikaryons were studied for their laccase production yields when growninproductionmedium(C2).Fourhybridsreached laccaseexpressionlevelshigherthanthoseofcomparedto therelatedmonokaryons(Fig.6)upto70,000U/L. 3.4. Laccaseinductionbynewdikaryotichybridsthrough spentmediumsolution

As mentioned above, because laccase production is regulated by multifactorial and mutiallelic expression systemswhicharedependentonextra-andintracellular regulations [4,39], the effect of SM16 on the partially characterized dikaryotic hybrids was tested. Results indicate that hybrids grown submerged cultures could differentiallyrespondtotheautoinductionmechanisms, increasing enzymaticproduction.Asa fact,addition of theinducersolutiontotheculturebroth(C2)atthetime

oftheinoculationincreaseslaccaseproductionlevels,for allbutoneoftheselectedhybrids,upto3times(Fig.7). Surprisingly, the best laccase producer among the selected hybrids results insensitive to the presence of theinducer.Theinsensitivestrainseemsthereforetobe deregulated.

This study has allowed us to get new insights in improvingfungallaccasesproduction:classicalbreeding andautoinductionmechanisms.Theserepresent promis-ing tools for the improvement of fungal fermentation withoutaffectingwastedisposablecostthatalsodepend onthesafeandecocompatibilityofthewholeprocess.

Disclosureofinterest

The authors declare that they have no conﬂicts of interestconcerningthisarticle.

0 10000 20000 30000 40000 50000 60000 70000 80000 12 11 10 9 8 7 6 5 4 3 2 1 0

5f X 3o 5f X 9o 5f X 11o 6f X 3o 6f X 9o 6f x 11o 18f x 3o 18f X 11o PARENT F PARENT O Fig.6.LaccaseactivityofPleurotusostreatusselectedhybridsinPDYculture(C2)after12daysofgrowth.

Table1

Matingtastebetweenselectedmonokaryonsderivedfromtheparent strainF(5f,6fand18f)andO(3o,9oand11o).

5f 6f 18f 3o 9o 11o 5f – – – + + + 6f – – – + – + 18f – – – + + + 3o + + + – – – 9o + – + – – – 11o + + + – – –

+: indicates a compatible interaction, clamp connection formed; –: indicatesanincompatibleinteraction,noclampconnectionformed.

Fig.7.MaximumlaccaseactivitysecretedbyPleurotusostreatusselected hybridsinPDYmedium(C2)inabsence(light)andinpresence(dark)of SM16laccaseinducer.Standarddeviation<20%.

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Acknowledgements

ThisworkwassupportedbytheEuropeanCommission, SixthFrameworkProgram(QUORUM,032811),bygrants fromtheMinistero dell’Universita` edellaRicerca Scien-tifica (Progetti di Rilevante Interesse Nazionale, PRIN), from Compagnia di San Paolo, Turin, Italy, project ‘‘Sviluppo di procedure di biorisanamento di reflui industriali (BIOFORM)’’,fromCOST ActionFP0602 ‘‘Bio-technologyforlignocellulosebiorefineries’’(BIOBIO),and fromtheMinisteroDegliAffaridegliEsteridiIntesaconil Ministerodell’Universita` edellaRicerca(Progettidiricerca dibaseetecnologicaapprovatineiprotocollidi coopera-zionescientificaetecnologicabilateralecomeprevistodal protocollobilateraletraItaliaeTurchia).

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