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RESEARCHOpenAccessAminimalgrowthmediumforthebasidiomycetePleurotussapidusformetabolicfluxanalysisMarcoAFraatz1,StefanieNaeve2,VanessaHausherr3,HolgerZorn1*andLarsMBlank4AbstractBackground:Pleurotussapidussecretesahugeenzymaticrepertoireincludinghydrolyticandoxidativeenzymesandisanexampleforhigherbasidiomycetesbeinginterestingforbiotechnology.
Thecomplexgrowthmediausedforsubmergedcultivationlimitbasicphysiologicalanalysesofthisgroupoforganisms.
Usingundefinedgrowthmedia,onlylittleinsightsintotheoperationofcentralcarbonmetabolismandbiomassformation,i.
e.
,theinterplayofcatabolicandanabolicpathways,canbegained.
Results:ThedevelopmentofachemicallydefinedgrowthmediumallowedrapidgrowthofP.
sapidusinsubmergedcultures.
AsP.
sapidusgrewextremelyslowinsaltmedium,theco-utilizationofaminoacidsusing13C-labelledglucosewasinvestigatedbygaschromatography–massspectrometry(GC-MS)analysis.
Whilesomeaminoacidsweresynthesizedupto90%invivofromglucose(e.
g.
,alanine),asparagineand/oraspartatewerepredominantlytakenupfromthemedium.
Withthisinformationinhand,adefinedyeastfreesaltmediumcontainingaspartateandammoniumnitrateasanitrogensourcewasdeveloped.
TheobservedgrowthratesofP.
sapiduswerewellcomparablewiththosepreviouslypublishedforcomplexmedia.
Importantly,fastgrowthcouldbeobservedfor4daysatleast,uptocellwetweights(CWW)of400gL-1.
Thechemicallydefinedmediumwasusedtocarryouta13C-basedmetabolicfluxanalysis,andtheinvivoreactionsratesinthecentralcarbonmetabolismofP.
sapiduswereinvestigated.
TheresultsrevealedahighlyrespiratorymetabolismwithhighfluxesthroughthepentosephosphatepathwayandTCAcycle.
Conclusions:ThepresentedchemicallydefinedgrowthmediumenablesresearcherstostudythemetabolismofP.
sapidus,significantlyenlargingtheanalyticalcapabilities.
DetailedstudiesontheproductionofextracellularenzymesandofsecondarymetabolitesofP.
sapidusmaybedesignedbasedonthereporteddata.
Keywords:Basidiomycete,Metabolicfluxanalysis,Minimalgrowthmedium,Centralcarbonmetabolism,Submergedculture,13C-fluxanalysisBackgroundHigherbasidiomycetescontributetothehumandietinmanysocietiesandareincreasinglyinvestigatedfortheirpotentialinbiotechnology.
Thelatterismainlymotivatedbythehugehydrolyticpotentialofthislargegroupoforganisms,ofwhichmanyaresaprophytic.
Applicationexamplesoffungalenzymesincludethedegradationofbiomass[1],theproductionoffinechemi-calsincludinge.
g.
norisoprenoids[2],monoterpenes[3,4],andcyathanetypediterpenoids[5].
Whiletheenzymaticrepertoireofsomehigherbasidiomyceteshasbeenin-vestigatedindetail[6],thenutritionalrequirementsofhigherbasidiomycetesareoftennot.
Themyceliumoffilamentousfungi,likePleurotussapidus,canbegrowninsubmergedculturesutilizingshakeflasksorbioreactors.
Ingeneral,glucoseactsasthemajorcarbonsourceinthegrowthmediaofhigherbasidiomycetes,whichusuallycontainadditionalcomplexingredients,likeyeastextract,maltextract,orsoyapeptone.
By-productsofthefoodin-dustrycanbeaddedtoliquidculturesofbasidiomycetesastheonlycarbonsourceandtopromotethebiotechno-logicalproductionofcomplexflavormixtures[7].
Inor-ganicsalts,aminoacids,vitamins,andtraceelementsolutionsareoftenaddedtothemedia.
Thesecomplexmediacansupportbiomassformation,withspecificgrowthratesof0.
02h1andhigher[8].
In-deed,thegrowthrateisofmajorimportanceforexperi-mentersanditisthusoptimizedtoallowrapidand*Correspondence:holger.
zorn@uni-giessen.
de1InstituteofFoodChemistryandFoodBiotechnology,JustusLiebigUniversityGiessen,Heinrich-Buff-Ring58,35392Giessen,GermanyFulllistofauthorinformationisavailableattheendofthearticle2014Fraatzetal.
;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/4.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Fraatzetal.
FungalBiologyandBiotechnology2014,1:9http://www.
fungalbiolbiotech.
com/content/1/1/9reproducibleexperiments.
However,thecomplexnatureofthegrowthmediausuallyusedmakesitdifficulttode-terminesubstrateuptakeratesandhence,thetruede-mandofthemyceliumgrowninsubmergedcultureismainlyunknown.
Intheliterature,onlyveryfewreportsarefoundcoveringfluxanalysisofbasidiomycetes[9,10].
Noneofthemcoversfilamentousspecies,butthebasidio-mycetousyeastPhaffiarhodozymahasbeenexamined.
Therefore,achemicallydefinedmediumthatallowedhighgrowthratesandhencemetabolicstudieswasdevel-oped.
Withthismedium,theintracellularfluxdistribu-tioninahigherbasidiomycetebymeansof13C-tracerbasedfluxanalysiswasestimatedforthefirsttime.
Theresultsrevealedahighlyrespiratorymetabolism,withsig-nificantcontributionofglucosecatabolismviathepen-tosephosphatepathway.
Theanalyticalpossibilitiesreportedhereopennewpotentialsforhigherbasidio-mycetebio(techno)logy.
ResultsanddiscussionDevelopmentofaminimalmediumforthesubmergedcultivationofP.
sapidusBasidiomyceteslikeP.
sapidusaretypicallygrownsub-mergedincomplexculturemedia.
Toinvestigategrowthkineticsandcellularphysiologyindetail,min-imalmediaarethefirstchoiceinmanyareasofmicro-biology.
ForhigherfungilikeP.
sapidusminimalmediawerenotreadilyavailable.
Hence,adefinedminimalmediumwasdevelopedstartingfromacommonlyusedcomplexmediumcalledstandardnutritionsolution(SNL-H3-G30,cf.
Table1).
SNL-H3-G30isderivedfromSprecher'smedium[11]byadditionofyeastextract.
Likemanyotherbasidiomycetes,P.
sapidusgrowsonlypoorlyinunmodifiedSpecher'smediumbutverywellinthemodifiedone.
Tobenchmarkgrowth,P.
sapiduswasthereforecultivatedinSNL-H3-G30.
DuringthefirstfourdaysofgrowthP.
sapidusconsumedap-proximately15gL-1glucose.
Reducingthesugarconcen-trationoftheculturemediumbyafactoroftwodidnotinfluencethebiomassproductionsignificantly(Figure1).
Afterreplacingthestandardnutritionsolution's(SNL-H3-G15)nitrogensourceasparaginebyammoniumnitrate(NL-H3-G15,Table1)thegrowthratewasinitiallyhighercomparedtoSNL-H3-G15,butstalledafter48h(Figure2).
Toinvestigateiftheavailabilityofnitrogencausedreducedgrowth,differentammoniumnitrateconcentra-tions(1.
2-7.
2gL-1)wereevaluated.
TheproductionofbiomassofP.
sapiduswasnoteffected(datanotshown).
Incontrast,theconcentrationofyeastextractinthecul-turemediumcorrelateddirectlywiththebiomassproduc-tionofP.
sapidus(Figure3).
Withouttheadditionofyeastextract(SNL-H0-G15)verylimitedgrowthwasobservedinstandardnutritionsolution(Figure3),aswellasinmediumwithammoniumnitrateasthenitrogensource(NL-H0-G15)(Table2).
Inaddition,theinfluenceofthiamine,avitaminmix-ture(after[12]),aswellasofdifferenttraceelementso-lutions(after[11]and[12],respectively)onthegrowthratewasinvestigated.
Nosignificanteffectsontherateofgrowthorthefinalbiomassconcentrationswereob-served(datanotshown).
TodeterminewhichaminoacidsareusedbyP.
sapidusasco-substratesandtowhichextent,thebasidiomycetewasgrowninyeastcontainingstandardnutritionsolutionTable1CompositionofculturemediaGlc[gL-1]Asn[gL-1]Asp[gL-1]NH4NO3[gL-1]KH2PO4[gL-1]MgSO4[gL-1]yeast[gL-1]TE[mLL-1]vit[mLL-1]SNL-H3-G3030.
04.
50.
00.
01.
51.
03.
01.
00.
0SNL-H5-G1515.
04.
50.
00.
01.
51.
05.
01.
00.
0SNL-H4-G1515.
04.
50.
00.
01.
51.
04.
01.
00.
0SNL-H3-G1515.
04.
50.
00.
01.
51.
03.
01.
00.
0SNL-H2-G1515.
04.
50.
00.
01.
51.
02.
01.
00.
0SNL-H1-G1515.
04.
50.
00.
01.
51.
01.
01.
00.
0SNL-H0-G1515.
04.
50.
00.
01.
51.
00.
01.
00.
0NL-H3-G1515.
00.
00.
02.
41.
51.
03.
01.
00.
0NL-H2-G1515.
00.
00.
02.
41.
51.
02.
01.
00.
0NL-H1-G1515.
00.
00.
02.
41.
51.
01.
01.
00.
0NL-H0-G1515.
00.
00.
02.
41.
51.
00.
01.
00.
0NL-D5-G3015.
00.
04.
82.
41.
51.
00.
01.
010.
0BMENL-D5-G1515.
00.
04.
82.
41.
51.
00.
01.
010.
0BMENL-D0.
4-G1515.
00.
00.
42.
41.
51.
00.
01.
01.
0VERThepHwasadjustedto6.
0with1MNaOHpriortosterilization;Glc:D-glucosemonohydrate,Asn:L-asparagine,Asp:L-asparticacid,MgSO4:magnesiumsulfateheptahydrate,yeast:yeastextract,TE:traceelementssolution(5mgL-1CuSO4·5H2O,80mgL-1FeCl3·6H2O,90mgL-1ZnSO4·7H2O,30mgL-1MnSO4·H2O,and0.
4gL-1EDTA),vit:BMEvitaminssolutionorafter[12](VER),mediumNL-D5-G15(highlightedinbold)wasusedforfluxanalysis(cf.
Figure6).
Fraatzetal.
FungalBiologyandBiotechnology2014,1:9Page2of8http://www.
fungalbiolbiotech.
com/content/1/1/9(SNL-H3-G15)withamixtureof[U-13C]-glucoseandun-labeledglucose(50:50,w/w)asitscarbonsource.
Afterharvestingthefungus,hydrolysis,andderivatization,thefractionallabelingoftheaminoacids(Ala,Asx,Glx,Gly,His,Ile,Leu,Lys,Met,Phe,Pro,Ser,Thr,Tyr,andVal)wasdeterminedbyGC-MS.
Thedenovosynthesisofaminoacidsfromglucosewasbetween22%(Asx)and92%(Ala)(Figure4).
Thus,allaminoacidsweremetabo-lizedbyP.
sapidus,howevertoverydifferentproportions.
Tofurthersimplifythemedium,singleaminoacidsaswellasselectedcombinationsofaminoacidsweretestedfortheirgrowthratepromotioninyeastfreemediacon-tainingammoniumnitrateasanadditionalnitrogensource.
Allcombinationswithoutaspartateresultedinpoorgrowthratesandbiomassconcentrations(datanotshown).
Therefore,mediumNL-D5-G15containingaspartate(4.
8gL-1),salts(NH4NO3,KH2PO4,andMgSO4),vitamins,traceelements,and15gL-1glucosewasselectedasthesimplestchemicallydefinedminimalmediumforfurtherinvestigations.
Underallconditionstested,P.
sapidusgrewfilamentous.
Themyceliumrapidlyformedpellets,whichincreasedovertimeinsize(cf.
Figure3).
Strategiestoavoidpelletformationarediscussedintheliterature[13,14]andmightbeappliedtothenewlydevelopedgrowthmediuminfu-ture.
Theresultingsaltmediumwiththesingleaminoacidaspartateallowedforaconsistentandrapidgrowthfor6to8days(Figure5),andcouldthereforebeusedforquan-titativephysiologicalexperiments.
UseoftheminimalmediumforquantitativephysiologyofP.
sapidusForthesoleadditionofaspartate,thedenovosynthesisofaminoacidswasquantifiedby13C-labelingofglucoseFigure1GrowthkineticsofP.
sapidusincomplexstandardmedium.
Initialglucoseconcentrationofstandardmedium30gL-1(SNL-H3-G30)and15gL-1(SNL-H3-G15),respectively,BM:biomass,Glc:glucose,cf.
Table1fordetailedmediumcomposition.
Figure2GrowthkineticsofP.
sapidusindependenceonAsnsupplementation.
BM:biomass,Glc:glucose,SNL-H3-G15:standardmedium,NL-H3-G15:withoutAsn,cf.
Table1fordetailedmediumcomposition.
Fraatzetal.
FungalBiologyandBiotechnology2014,1:9Page3of8http://www.
fungalbiolbiotech.
com/content/1/1/9(Figure4)attwodifferentaspartateconcentrations(NL-D0.
4-G15,NL-D5-G15,Table1).
Theadditionofonly0.
4gL-1aspartateresultedinminoruseofthisaminoacidasadditionalcarbonsource.
OnlyAsx,Ile,Pro,andThrwerepartially(about20%)synthesizedfromaspar-tate,whileglucosewasthemainsourceoftherespectivecarbonskeleton.
Indeed,aspartateistheprecursorforthreonineandisoleucinesynthesis.
Theabsenceofun-labeledcarboninglycinesuggeststhatathreoninealdol-ase,catalyzingthesynthesisofglycinefromthreoninewhileproducingacetaldehydeisnotpresentornotac-tiveinP.
sapidusundertheinvestigatedconditions.
Theaminoacidsderivedfromketoglutarate(Glx,Pro)werepartiallysynthesizedfromaspartate.
AspartateisreadilydeaminatedtooxaloacetateexplainingthecontributiontoTCAcycleintermediates.
Nounlabeledcarbonwasobservedinpyruvatederivedaminoacids(e.
g.
,Ala,Val),indicatingthatgluconeogenicreactionsareabsentdur-inggrowthonglucose.
Thecontributionof10%ofas-partatetothemainlypyruvatederivedaminoacidleucineisnotreadilyexplained,aspyruvateisfullyla-beled(e.
g.
,Ala).
Inaddition,twocarbonatomsofleucineoriginatefromacetyl-CoA.
Acetyl-CoAcaneitherorigin-atefromcytosolicormitochondrialpyruvate.
Thelatterissynthesizedviathepyruvatedehydrogenasecomplex,al-thoughcontributionsfromthemalicenzyme(malatetopyruvate)werereportedforascomycetous[10,15,16],butnotbasidiomycetousyeasts.
Indeed,whenperforminga13C-basedmetabolicfluxanalysis,amalicenzymeactivitywasobservedinP.
sapidus(Figure6).
Ingeneral,within-creasedaspartateconcentrations(4.
8gL-1),thecontribu-tiontoaminoaciddenovosynthesisincreasedslightly.
TheexceptionwasthesynthesisofAsx,whichoriginatedtomorethan80%fromaspartatetakenupfromthemedium,andlessdistinctthesynthesisofisoleucineandthreonine(about40%).
Performinga13C-fluxanalysisexperimentwiththede-finedmediumNL-D5-G15(Table1)allowedforthequan-tificationoftheglucoseuptakerateandspecificgrowthratewith0.
24mmolg-1h-1and0.
048h-1,respectively.
Thesevaluesarelowwhencomparedtopreviousreportsonascomycetes.
Glucosewascatabolizedviaglycolysisandupto35%viathepentosephosphatepathway.
Intheba-sidiomycetousyeastPhaffiarhodozyma,glucosewascatab-olizedviathepentosephosphatepathwayupto65%[10].
Noby-productslikeethanol,acetateorglycerolwereob-servedinP.
sapiduscultures(datanotshown).
Thesere-sultincombinationwithahighlyactiveTCAcycle(morethan80%oftheoxaloacetateoriginatedfromtheTCAcycle,whilelessthan20%originatedfromtheanapleroticreactioncatalyzedbythepyruvatecarboxylase)stronglyin-dicatedthatthemetabolismofP.
sapidusisfullyrespira-toryunderthegrowthconditionstestedhere.
Theabsolutefluxesindicatedaconsiderablefluxtobiomass.
ThisisalsoinagreementwiththefluxthroughthepentosephosphatepathwayasnotonlybiomassFigure3GrowthofP.
sapidusindependenceonyeastextractsupplementation.
Left:GrowthofP.
sapidusindependenceonyeastextractsupplementation;H0-H5equatesto0–5gL-1yeastextract,cf.
Table1fordetailedmediumcomposition.
Right:VisualcomparisonofP.
sapidusgrownfor4daysinstandardnutritionmediumwith3gL-1(SNL-H3-G15,top)and0gL-1(SNL-H0-G15,bottom)yeastextract.
Table2Biomass(cellwetweight)ofP.
sapidusafter4dayswhengrowninculturemediawithammoniumnitrateasnitrogensourceanddifferentyeastextractconcentrationsMediumnameYeastextract[gL-1]Cellwetweight[gL-1]NL-H0-G150.
031NL-H1-G151.
083NL-H2-G152.
0157NL-H3-G153.
0192Cf.
Table1fordetailedmediumcomposition.
Fraatzetal.
FungalBiologyandBiotechnology2014,1:9Page4of8http://www.
fungalbiolbiotech.
com/content/1/1/9precursorslikeriboseanderythrose-4Pfornucleicandaminoacidssynthesis,respectively,butalsotheanabolicdemandforNADPHcanbemetviatheoxidativebranchofthispathway.
Indeed,thefluxthroughthepentosephosphatepathwaywaspreviouslylinkedtothebiomassyieldinascomycetes[16].
ConclusionsThepresentedresultsallowforexperimentswithP.
sapidusgrowingsubmergedinachemicallydefinedmedium.
ThisenablesresearcherstostudythebiologyofP.
sapidus(andpossiblyothermushrooms)inthecontextofmetabolism,significantlyenlargingtheana-lyticalcapabilities.
Whiletheinformationoftherespiratorycapabilitiesishighlyinteresting,thelowoverallmetabolicactivitymostlikelyrequiresmodifica-tionsofP.
sapidusasaproductionhostinindustrialbiotechnology.
Withthisinformationinhand,e.
g.
,de-tailedinductionstudiesofhydrolyticenzymesofP.
sapiduscanbedesigned.
MethodsChemicalsCopper(II)sulfatepentahydrate,iron(III)chloridehexa-hydrate,andzincsulfateheptahydratewerepurchasedfromAppliChem(Darmstadt,Germany),D-glucose[U-13C]fromEURISO-TOP(Gif-sur-Yvette,France);D-glucosemonohydrateandL-asparticacidwereobtainedFigure4DenovosynthesisofaminoacidsinP.
sapidus.
Thebarsrepresenttherelativeamountofdenovosynthesizedaminoacidsduringgrowthinstandardnutritionsolution(SNL-H3-G15)andtwochemicaldefinedmediawithdifferentaspartateconcentrations(0.
4gL-1:NL-D0.
4-G15;4.
8gL-1:NL-D5-G15).
Thecontributionofdenovosynthesiswasestimatedfromtheamountoflabelmeasuredintheaminoacids,whichoriginatedfrom50%[U-13C]-labeledglucoseasmaincarbonsource.
Errorbarsrepresenttherangeofduplicates.
Figure5GrowthkineticsofP.
sapidusindevelopedminimalmedium(NL-D5-G15)incomparisontostandardnutritionsolution(SNL-H3-G30).
BM:biomass,Glc:glucose,cf.
Table1fordetailedmediumcomposition.
Fraatzetal.
FungalBiologyandBiotechnology2014,1:9Page5of8http://www.
fungalbiolbiotech.
com/content/1/1/9fromCarlRothGmbH(Karlsruhe,Germany),EDTAfromFluka(Buchs,Germany);agar,L-asparagine,magnesiumsulfateheptahydrate,manganese(II)sulfatemonohydrate,andyeastextractwerepurchasedfromServa(Heidelberg,Germany);BasalMediumEagle(BME)vitaminssolutionwaspurchasedfromSigma(Steinheim,Germany).
Figure6AbsolutemetabolicfluxesofP.
sapidus.
P.
sapiduswasgrowninmediumNL-D5-G15,cf.
Table1fordetailedmediumcomposition.
Fraatzetal.
FungalBiologyandBiotechnology2014,1:9Page6of8http://www.
fungalbiolbiotech.
com/content/1/1/9MicroorganismThefilamentousfungusPleurotussapiduswasobtainedfromtheGermanCollectionofMicroorganismsandCellCultures(DSMZ8266),Brunswick,Germany.
CultivationofP.
sapidusStockculturesweremaintainedonagarplatescontain-ingstandardnutritionsolution(SNL-H3-G30,Table1)and15gL-1agar.
Thestockcultureswerestoredat4°Cuntilusage.
Preculturesweregrownaerobicallyinstandardnutri-tionsolution(SNL-H3-G30,Table1)aftertransferring1cm2agarplugsfromtheleadingmycelialedgeofthestockculturesfollowedbyhomogenizationusinganUltraTurraxhomogenizer(IKA,Staufen,Germany).
Thesubmergedcultures(200mLmedium)werekeptonarotaryshaker(25mmshakingdiameter;Multitron,Infors,Einsbach,Germany)at150rpmand24°CinErlenmeyerflasks(500mL)for4daysindarkness.
Theprecultureswerecentrifugedfor10min(3375*g,4°C),andthesupernatantwasdecanted.
Theremainingpelletswereresuspendedinthesamevolumeofdistilledwaterandcentrifugedagainfor10min(3375*g,4°C).
Thisprocedurewasrepeatedtwice.
Subsequentlytothelastcentrifugationstepthepelletsweredispersedinthemainculturemedium(Table1)andhomogenizedusinganUltraTurraxhomogenizer.
Forthemaincultures40mLmediumwasinoculatedwith4mLhomogenizedpre-culturebrothinErlenmeyerflasks(100mL)andincu-batedonarotaryshaker(25mmshakingdiameter,150rpm,24°C)for4to10days.
13C-basedcarbonfluxanalysisTheGC-MSdatarepresentsetsofionclusters,eachshowingthedistributionofmassisotopomersofagivenaminoacidfragment.
Foreachfragmentα,onemassiso-topomerdistributionvector(MDV)wasassigned,MDVam0m1m2…mn266664377775withXmi11withm0beingthefractionalabundanceofthelowestmassandmi>0theabundancesofmoleculeswithhighermasses.
Toobtaintheexclusivemassisotopedis-tributionofthecarbonskeleton,correctionsfornatur-allyoccurringisotopesinthederivatizationreagentandtheaminoacidswereperformedasdescribedpreviously[17,18],followedbythecalculationsofthemassdistri-butionvectorsoftheaminoacids(MDVAA)andtheme-tabolites(MDVM).
MetabolicfluxratioswerecalculatedfromtheMDVMasdescribedindetailbyNanchenetal.
[19]usingFiatFlux[20].
AbsolutevaluesofintracellularfluxeswerecalculatedwithafluxmodelfromtheyeastSaccharomycescerevisiaethatcomprisedallmajorpath-waysofcentralcarbonmetabolism[21].
TheerrorminimizationwascarriedoutasdescribedbyFischeretal.
[18].
AnalyticalmethodsDeterminationofcellwetweightTheculturebrothwascentrifugedfor10min(3375*g,4°C),andthesupernatantwasreplacedbythesamevol-umeofdistilledwater.
Themyceliumwasresuspendedandcentrifuged.
Thiswashingstepwasrepeatedtwice.
Afterwards,thesupernatantwasdiscarded,andtheweightoftheremainingmyceliumwasdetermined.
DeterminationofglucoseTheD-glucoseconcentrationintheculturesupernatantwasdeterminedenzymaticallybymeansofanenzymaticD-glucoseassay(R-BiopharmAG,Darmstadt,Germany)accordingtomanufacturer'sinstruction.
Determinationofthe13C-labelingpatternsoftheproteinogenicaminoacidsTheglucoseusedinshakeflasksexperimentswasamix-tureof50%(n/n)uniformlylabeled[U-13C]-glucoseand50%(n/n)naturallylabeledglucose.
Thebiomasswaswashedtwicewith0.
9%NaClandhydrolyzedwith150μLof6MHClfor15–24hat105°C.
Thehydroly-zatewasdriedbyheatingthevialto85°Cunderacon-stantflowofair.
Thehydrolyzatewasdissolvedin50μLdimethylformamideandtransferredtoanewvial.
Theaminoacidsweresilylatedbyadditionof50μLN-methyl-N(tert-butyldimethylsilyl)-trifluoroacetamideandsubse-quentlyincubatedat85°Cfor60min.
OneμLofthismixturewasinjectedintoaVarianGC3800gaschro-matograph,equippedwithaVarianMS/MS1200triplequadrupolemassspectrometer(VarianDeutschland,Darmstadt,Germany).
ThederivatizedaminoacidswereseparatedonaFactorFourVF-5mscolumn(30m*0.
25mmID,0.
25μmfilmthickness;VarianDeutschland)ataconstantflowrateof1mLhelium(5.
0)min-1.
Thesplitratiowas1:25andtheinlettemperaturewassetto250°C.
ThetemperatureoftheGCovenwaskeptconstantfor2minat150°Candafterwardsincreasedto250°Cwithagradientof3°Cmin-1.
Thetemperaturesofthetransferlineandthesourcewere280°Cand250°C,respectively.
Ionizationwasperformedbyelectronimpactionizationat-70eV.
Forenhanceddetection,aselectedionmoni-toringtimesegmentwasdefinedforeveryaminoacid[22].
GC-MSrawdatawereanalyzedusingtheWork-stationMSDataReview(VarianDeutschland).
Fraatzetal.
FungalBiologyandBiotechnology2014,1:9Page7of8http://www.
fungalbiolbiotech.
com/content/1/1/9DeterminationofdenovoaminoacidsynthesisIntracellulardenovoaminoacidsynthesiswasdeter-minedfortheaminoacidsalanine,aspartate,glutamate,glycine,histidine,isoleucine,leucine,lysine,methionine,phenylalanine,proline,serine,threonine,tyrosine,andvalineaspreviouslyreportedin[23].
Thepercentagesofdenovosynthesizedaminoacidscorrespondtothe13C-labelingintheaminoacidsderivedfrom13Clabeledglu-cose.
Theunlabeledfractioncorrespondstotheamountofunlabeledaminoacid,whichwastakenupfromthemedium.
GC-MSanalysisbasedonproteinogenicaminoacidsisabletodetect15ofthe20proteinogenicaminoacids.
Argininewasomittedbecauserearrangementsdur-ingelectronimpactionizationobscureitsfragmentationpattern.
Cysteineandtryptophanareoxidativelydestroyedduringacidhydrolysis,andasparagineandglutaminearedeamidatedtoaspartateandglutamate,respectively[24].
Themixturesofasparagine/aspartateandglutamine/glutamateweresubsequentlyreferredtoasASXandGLX,respectively.
LabelingpatternswereanalyzedusingthesoftwareFiatFlux[20].
AbbreviationsAla:Alanine;Asx:Asparagine/aspartate;Asn:Asparagine;Asp:Asparticacid;BME:Basalmediumeagle;CWW:Cellwetweight;EDTA:Ethylenediaminetetraaceticacid;GC:Gaschromatography;GC-MS:Gaschromatography–massspectrometry;Glc:Glucose;Glx:Glutamine/glutamate;Gly:Glycine;His:Histidine;ID:Innerdiameter;Ile:Isoleucine;Leu:Leucine;Lys:Lysine;MDV:Massisotopomerdistributionvector;Met:Methionine;Phe:Phenylalanine;Pro:Proline;Ser:Serine;SNL:Standardnutritionsolution;TCA:Tricarboxylicacidcycle;Thr:Threonine;TE:Traceelementssolution;Tyr:Tyrosin;Val:Valine;vit:Vitaminssolution.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsMAFcoordinatedtheexperimentsandwrotethemanuscript,SNandVHperformedandanalyzedtheexperiments,HZcoordinatedthestudyandcriticallyreviewedthemanuscript,LMBcoordinatedandanalyzedthemetabolicfluxexperimentsandwrotethemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsTheauthorswouldliketothankAndreasSchmidforlaboratoryaccessaswellasJanHeylandandRahulDeshpandeforassistancewithGC-MSmeasurements.
HZisgratefultotheHessianMinistryofScienceandArtforagenerousgrantfortheLOEWEresearchfocus'IntegrativeFungalResearch'.
Authordetails1InstituteofFoodChemistryandFoodBiotechnology,JustusLiebigUniversityGiessen,Heinrich-Buff-Ring58,35392Giessen,Germany.
2LaboratoryofTechnicalBiochemistry,TUDortmund,44221Dortmund,Germany.
3IfADo-LeibnizResearchCenterforWorkingEnvironmentandHumanFactors,Ardeystr.
67,44139Dortmund,Germany.
4iAMB-InstituteofAppliedMicrobiology,ABBt-AachenBiologyandBiotechnology,RWTHAachenUniversity,WorringerWeg1,52074Aachen,Germany.
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doi:10.
1186/s40694-014-0009-4Citethisarticleas:Fraatzetal.
:AminimalgrowthmediumforthebasidiomycetePleurotussapidusformetabolicfluxanalysis.
FungalBiologyandBiotechnology20141:9.
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