Comparative proteomic analyses of Hyphozyma roseonigra ATCC 20624 in response to sclareol

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Biotechnology

and

Industrial

Microbiology

Comparative

proteomic

analyses

of

Hyphozyma

roseonigra

ATCC

20624

in

response

to

sclareol

Xiuwen

Wang

a

_,

_Xiaohua

_Zhang

a

_,

_Qingshou

_Yao

a

_,

_Dongliang

_Hua

b

_,

_Jiayang

_Qin

a,∗

a_{BinzhouMedicalUniversity,Yantai,China}

b_{ShandongAcademyofSciences,EnergyResearchInstitute,KeyLaboratoryforBiomassGasificationTechnologyofShandongProvince,}

Jinan,China

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Articlehistory:

Received29July2017 Accepted3April2018 Availableonline24April2018 AssociateEditor:SolangeI. Mussatto Keywords: Comparativeproteomic Hyphozymaroseonigra Sclareol Sclareolglycol Ambroxide

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b

s

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t

Sclareolisanimportantintermediateforambroxidesynthesisindustries.Hyphozyma roseon-igraATCC20624wastheonlyreportedstraincapableofdegradingsclareoltothemain productofsclareolglycol,whichistheprecursorofambroxide.Todate,knowledgeis lack-ingabouttheeffectsofsclareoloncellsandtheproteinsinvolvedinsclareolmetabolism. ComparativeproteomicanalyseswereconductedonthestrainH.roseonigraATCC20624by usingsclareolorglucoseasthesolecarbonsource.Atotalof79up-regulatedproteinspots witha>2.0-folddifferenceinabundanceon2-Dgelsundersclareolstressconditionswere collectedforfurtheridentification.Seventyspotsweresuccessfullyidentifiedandfinally integratedinto30proteins.Theup-regulatedproteinsundersclareolstressareinvolvedin carbonmetabolism;andnitrogenmetabolism;andreplication,transcription,and transla-tionprocesses.Eighteenup-regulatedspotswereidentifiedasaldehydedehydrogenases, whichindicatingthataldehydedehydrogenasesmightplayanimportantroleinsclareol metabolism.Overall,thisstudymaylaythefundamentalsforfurthercellengineeringto improvesclareolglycolproduction.

©2018PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileirade Microbiologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Sclareol,aditerpenealcoholisolatedfromSalviasclarea(Clary sage),canbeappliedtomedicine,cosmetics,healthproducts, flavorsandfragrances,andpesticides.1–3 _Themain

applica-tion of sclareol is forsynthesizing high-end substitutes of ambergrisintheperfumeindustry.4_{Giventhesupplyshortage}

andpriceinflationofambergris,anumberofsubstituteshave

∗ _{Correspondingauthorat:CollegeofPharmacy,BinzhouMedicalUniversity,Yantai264003,China.}

E-mail:qinjy@bzmc.edu.cn(J.Qin).

been developed.Ambroxideisthemostappreciated substi-tuteofambergrisandisobtainedfromthesemi-synthesisof sclareol.5–7_{Sclareolcanbetransformedtoambroxidethrough}

redoxandcyclization,duringwhichsclareolglycoland sclare-olideareimportantintermediates(Fig.1).Thus,thebiological productionofsclareol glycolorsclareolide tocomplete the biosynthesispathwayhasgained muchinterestdue tothe increasingdemandofnaturalfragrances.

Numerousstrainsreportedlytransformsclareol,whereas most strains merely introduce hydroxyl at the main ring

https://doi.org/10.1016/j.bjm.2018.04.001

1517-8382/©2018PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

sclaroel

sclaroelide

ambroxide

CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 OH OH OH A TCC 20918 chem ical con version chemical con v ersion Cr yptococcus albidus OH O O O Hyphozyma roseonigra ATCC 20624 H3C _H 3C H3C H3C

sclaroel glycol

Fig.1–Diagramofconversionfromsclareoltoambroxide.

ratherthanmodifythebranchedchain.Onlyfewstrainscan transformsclareoltosclareolglycolorsclareolide.

Cryptococ-cusalbidusATCC 20918converts sclareoltosclareolide ata

highyieldofmorethan 100g/Landhasbeen exploitedfor industrialization.8_{Todate,HyphozymaroseonigraATCC20624,}

which can exist inboth yeast-like and filamentous forms, isthe onlyreported straincapableofdegrading sclareol to the mainproduct ofsclareol glycol.9 _{However, the explicit}

conversionmechanism from sclareolto sclareolglycol and otherproductshasnotbeenreported.Knowledgeisalso lack-ingabouttheeffectsofsclareoloncellgrowth,considering thatsclareolisalabdanediterpenewithahighantimicrobial activity.10_{Theseproblemshinderthefurtherimprovementof}

conversionrateandtheexpandedapplicationofthe biotech-nology.H.roseonigraATCC20624cansurviveinbasicinorganic saltmediumwithsclareolasthesole carbonsourceand is thereforeagood candidateforexploringthe mechanismto someextent.

Inthepresentstudy,comparativeproteomicanalyseswere conductedonthestrainH.roseonigraATCC20624,withsclareol orglucoseasthesolecarbonsource.Thedifferenceinprotein expressionbetweenthetwoconditionsisexpectedtoenrich ourknowledgeonsclareolstressinmicroorganisms.

Material

and

methods

Microorganismandcultivationconditions

H.roseonigraATCC20624waspurchasedfromATCC(MD,USA)

andgrownonyeast-maltmedium(perliter:3gyeastextract, 3gmaltextract,10gglucose,and5gtryptoneatpH6.2)at24◦C and180rpm.Theseedculturewasincubatedintwokindsof basicinorganicsaltmedium(BSM)containingsclareol(BSMS) orglucose(BSMG)tomonitorthecellgrowthrates.

The modified BSM medium contained (per liter) 2.44g KH2PO4,14.04gNa2HPO4·12H2O,2gNH4Cl,0.2gMgCl2·6H2O,

1mgCaCl2·2H2O, 0.01g yeastextract, 5mL metal ion

mix-ture,and0.2mLvitaminmixture.Meanwhile,themetalion mixtureincluded(perliter)0.5gFeCl2·4H2O,0.5gMnCl2,0.1g

NaMoO4·2H2O, 0.05g CuCl2, and 120mM HCl.The vitamin

mixturecontained(perliter)2gcalciumpantothenate,1g cre-atine,2gnicotinicacid,2gpyridoxamine,2.5mgcobalamin,

and1gpara-aminobenzoicacid.

Tween 80 (0.8%,v/v)and sclareol (2mM)were added to BSMmediumtopreparetheBSMSmedium,whereasglucose (1.33mM)wasaddedtoBSMmediumtoproducethe BSMG medium.CellsgrownintheBSMGmediumwereruninparallel withthesamplesintheBSMSmedium.

Preparationofproteinextractsandcomparativeproteomic analysis

Cellsintheexponentialgrowthphasewere harvestedfrom 50mLculturesbycentrifugationat4000×gfor10minat4◦C. Thecellpelletswerefrozeninliquidnitrogenandstoredat −80◦Cuntilproteomeanalysis.

Comparative proteomic analysis by 2-Dgel was carried outasdescribedinliterature.11_{Cellsweresuspendedinlysis}

bufferof2DE(SangonCo.,Shanghai,China)andultrasonically disruptedat80–100Wfor3min.Thedebriswasremovedby centrifugationandthesupernatantwasincubatedovernight withice-coldacetone.Aftercentrifugationat12,000×g and 4◦Cfor30min,theprecipitatedproteinsweresolubilizedin samplelysisbuffer.Totalproteinconcentrationswere deter-minedusingtheNon-interferenceProteinAssayKit(Sangon Co.,Shanghai,China).Proteinspotsonthegelswere visual-izedbysilverstaininginaccordancewiththeprotocol.12_Each

setofsampleswasindependentlyanalyzedintriplicate. Analysesof2-Dgels

2-Dgelswerescannedataresolutionof300dotsperinch(dpi) byanImageScannerIII(GEhealthcare).Statistically signifi-cantdifferenceswerediscernedbyperformingStudent’sttest. Protein spots showingsignificant changes in up-regulation and down-regulation were extracted, digested, rehydrated, and identified in a 5800 Plus MALDI TOF/TOFTM analyzer (AppliedBiosystems).Datawereacquiredinapositivemass spectrometry(MS)reflectorbyusingaCalMix5standardto calibratetheinstrument(ABI5800CalibrationMixture).

Results

GrowthcharacteristicsofH.roseonigraATCC20624

ThegrowthratesofH.roseonigraATCC20624onmedia con-tainingdifferentsclareolconcentrations(2,6,and10mM)were investigated.ThegrowthcurvesofH.roseonigraATCC20624 onmediacontaining2and6mMsclareolweresimilar,and theopticaldensity(OD600)of2.23±0.11for2mMsclareoland

2.41±0.05for6mMsclareolwereobtainedat120h.Duringthe first50hincubation,H.roseonigraATCC20624grewwellonthe mediumcontaining10mMsclareol,andprolongedcultivation timesresultedinretardedgrowthrates(Fig.2A).Glucoseis acarbon sourcepreferred bymostmicroorganisms. Hence,

B

A

2.5 1.5 2.0 2 mM BSMS 1.33 mM BSMG 10 mM 6 mM 2 mM 1.0 OD 600 OD 600 0.6 0.4 0.2 0.0 10 20 30 5 15 25 0 0.0 100 140 Time (h) Time (h) 120 80 60 40 20 0 0.5 0.8

Fig.2–Effectsofsclareolstressonthecellgrowthof

HyphozymaroseonigraATCC20624.(A)Effectsofsclareolon

cellgrowth.(B)GrowthcurvesofH.roseonigraATCC20624 inBSMSandBSMGmedia.Experimentswereperformedin triplicate.Errorbarsrepresentthestandarddeviationsof themeansofthreeindependentexperiments.

thecellgrowthonthemediumcontaining1.33mMglucose wasalsoinvestigated;thisconcentrationwassettoprovide the same number of moles of carbon asthat lost if 2mM sclareolwascompletelytransformedtosclareolglycol.The cellmassofsclareolculturesreachedthesamevalueobserved asthoseintheglucoseculturesduringthefirst12h cultiva-tion.H.roseonigraATCC20624alsogrewmuchfasteronthe mediumwithglucosethaninthatwithsclareolfrom12hto 15h(Fig.2B).However,furtherincubationinglucosedecreased cellgrowth,andtheOD600valueofH.roseonigraATCC20624

ontheglucose-containingmediumreached0.53±0.04at24h. Interestingly,thegrowthofH.roseonigraATCC20624 contin-uedtoincreaseafter15hofcultivationinBSMSmedium.This resultindicatesthepredominantsclareol-tolerantcapacityof

H.roseonigraATCC20624.CellsculturedinBSMGmediumand

BSMSmediumwerecollectedat12handusedforsubsequent proteomicanalyses.Atthistimepoint,sclareolhadnotbeen completelyconvertedtosclareolglycol(Supplementarydata, Fig.I).

Proteinexpressionalterationsinresponsetosclareolstress TheglobalproteomicresponseofH.roseonigraATCC20624to sclareolwasprofiledby2-Dgelelectrophoresis.Glucose cul-tureswerethenusedasthecontrolgroup,whereassclareol cultureswere adoptedas the experimentalgroup.In three independentexperiments,proteinabundanceswereanalyzed usingthePDQuestsoftware.Proteinabundanceswere signif-icantlyaltered(p<0.05)in117spots(TableS1andFig.3)from theBSMSandBSMGmedia.Thischangesuggeststhatsclareol affectedthefungus’scellularphysiology.Amongthesespots, 79up-regulatedspotswitha>2.0-folddifferenceinabundance on 2-D gels were collected for furthermass spectrometric

identification. Seventy spots were successfully recognized, whereasseveralspotswereidentifiedasthesameproteinafter OmicsBeananalysis.Finally,70proteinspotswereintegrated into30proteins(Table1).

Discussions

Many ofthedifferentiallyexpressedproteinsinTable1are commontoseveralcategories.Approximately27%ofthe dif-ferentiallyexpressedproteinsbelongedtocarbonmetabolism, followedby10%belongingtothemetabolicpathwayand10% belonging toproteinprocessingin theendoplasmic reticu-lum. Meanwhile, 3%of the proteinseach belonged to the cellcycle,phagosomeformation,DNAreplication,glutathione metabolism, peroxisome formation, lysine degradation, ␤-alanine metabolism, antibioticbiosynthesis, and glyoxylate metabolism.

Theproteinsinvolvedincarbonmetabolism(including gly-colysisandthecitricacidcycle),suchasfructosebisphosphate aldolase (FBA),enolase(ENO),citratesynthase(CIT),malate dehydrogenase (MDH), succinate dehydrogenase (SDH) and phosphoenolpyruvatecarboxykinase,wereallup-regulatedin responsetosclareolstress.Notably,thefoldchangeofMDH, whichcatalyzestheconversionofmalateandoxaloacetate,13

wasthelargest,atapproximately111folds.Theobserved up-regulationoftheseproteinsindicatedthattheH. roseonigra

ATCC 20624 cells expressedadditionalenzymes forcarbon metabolismtometabolizesclareolmoreefficientlythanusual. Additionally, sclareol stress induced significant changes in theexpressionofaldehydedehydrogenases(ALDHs).ALDHs, generally considered as a superfamily of NADP-dependent enzymesandparticipatinginaldehydemetabolism,catalyze the oxidation of exogenous aldehydes into corresponding carboxylic acids.14 _{Theup-regulated ALDHsmay protectH.}

roseonigraATCC20624fromdamageinducedbyactive

alde-hydes.Eighteenup-regulatedspotswereidentifiedasALDHs, whichindicatedthat ALDHsmay playanimportantrolein sclareolmetabolism.

Besidestheabove-mentionedproteinnarrative,twoother proteins, that is, heatshock proteins SSB2 and SSA2, were found up-regulated (2.58 and 7.96 fold increases, respec-tively). SSB2and SSA2aremolecularchaperonesbelonging tothe70kDaheat-shockproteins(Hsp70s).Thetwoenzymes exhibited low intrinsic ATPase activities inthe native pro-tein,whereastheisolatedATPasedomainsmanifestedmuch greater ATPase activities.15 _{Both proteinsparticipateinthe}

transportofpolypeptidesbutfunctiondistinctly.Furthermore, proteinsactingoncellularprocesses,suchasthenicotinicacid transporter(TNA),meioticrecombinationprotein(REC),DNA replicationlicensingfactor(MCM),andeukaryotictranslation initiation factor(HYP) wereup-regulated.Thisfinding indi-catesthatsclareolstressalsoforcedH.roseonigraATCC20624 tostrengthenitslifeactivities.

H.roseonigraATCC20624alsoexpressedadditional

struc-tural proteins, such as actins, structural maintenance of chromosomes protein4,and flocculation proteinFLO11,to surviveundersclareolstress.Threeproteins,thatis,78kDa glucose-regulated protein homolog, GTPase, and ATP syn-thasesubunit␣,werealsoup-regulated.Thisproteinhomolog

Fig.3–ComparativeproteomicprofilingofH.roseonigraATCC20624exposedto(A)1.33mMglucoseand(B)2mMsclareol. Thedifferentiallyexpressedproteinswerelabeledwithspotnumbers.Experimentswereperformedintriplicate.

Table1–Proteinsidentifiedbymassspectrometry.

Spotnumbera _{Proteinidentification Foldchange}b

2007,5722,6117,7208,7209,7210,7315, 4720,5122,7113,2017,2019,7712,5719, 7608,3111,5817,6109

Aldehydedehydrogenase(ALD5) 11.62,3.37,33.63,2.40,2.91,2.97,5.00, 2.85,3.23,3.67,12.66,9.35,4.30,8.81,2.70, 6.52,4.82,3.86

5806,6119,7108 Nicotinicacidtransporter(TNA1) 2.91,8.25,3.50

8114 Meioticrecombinationprotein(REC114) 4.43

3109 Polyamineoxidase(FMS1) 2.33

6014 Histone-lysineN-methyltransferase

(SET1)

3.76

6212 Peroxisomal2,4-dienoyl-CoAreductase

(SPS19)

4.89

6213 Enolase1(ENO1) 3.54

6215,6306 Transcriptionalregulator(URE2) 4.10,4.16

6313 Citratesynthase,mitochondrial(CIT1) 3.35

6319,7114,7118,6320 DNAreplicationlicensingfactor(MCM3) 11.15,4.81,8.72,10.35

7014 MitochondrialperoxiredoxinPrx1(PRX1) 6.12

119,6118 Actin(ACT1) 2.89,2.89

5115 Heatshockprotein(SSB2) 2.58

3003,122,313,121 Fructose-bisphosphatealdolase(FBA1) 3.59,3.74,3.16,3.91

134 Saccharopepsin(PEP4) 3.16

1215 78kDaGlucose-regulatedprotein

homolog(KAR2)

6.97

2218 Structuralmaintenanceofchromosomes

protein(SMC4)

2.60

3816 GTPase(MTG2) 6.52

4005 Heatshockprotein(SSA2) 7.96

1312,4409,4818,5010,7110,6312 Malatedehydrogenase(MDH1) 3.10,4.58,6.37,110.69,7.80,6.54

5117 Flocculationprotein(FLO11) 3.40

7812 PeroxisomalcatalaseA(CTA1) 3.89

8112 Acetyl-coenzymeAsynthetase2(ACS2) 5.39

2006,6118,8314,1223 NADP-dependent3-hydroxyacid

dehydrogenase(YMR226C)

2.40,2.40,16.42,4.60

1312 Succinatedehydrogenase(SDH1) 6.19

2117 Eukaryotictranslationinitiationfactor

(HYP2) 3.48 5716 Glutaminesynthetase(GLN1) 6.13 6009 Cerevisin(PRB1) 17.27 28,2712,6012,7012,7013 Phosphoenolpyruvatecarboxykinase (PEPCK1) 13.54,5.45,15.45,13.45,9.74

7108,7112 ATPsynthasesubunit(ATP1) 5.59,16.42

a _{ConsistentwiththeproteinnumberasshowninFig.3.}

b _{FoldchangesinproteinexpressionwerecalculatedusingthecellsfromtheBSMGmediumasreference.}

is active as a homodimer and exhibits ATPase activity. Meanwhile, GTPase is required for mitochondrial protein synthesis.16Theup-regulationoftheseproteinsimpliesthat

H.roseonigraATCC20624cangenerateadditionalATPorGTPby

expressingadditionalenzymesrelatedtoenergymetabolism whenexposedtosclareolstress.

Conclusions

In summary, the results indicated that proteins involved in carbon metabolism; nitrogen metabolism; and replica-tion,transcription,andtranslationprocessesareup-regulated whensclareolisthesolecarbonsourcerelativetothatwhen glucose is the sole carbon source. Some of the identified proteins are known to serve heat-inductive functions and play general roles in cross-protecting cells against diverse stressconditions.These cross-protectingproteinsmay play

importantrolesinthesclareoltoleranceofH.roseonigraATCC 20624.Overall,thepresentstudymaylaythefundamentalsfor furthercellengineeringtoimprovesclareolglycolproduction.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

This work was supported bythe National Natural Science Foundation of China (31200078), the Shandong Province Science and Technology Project (No. ZR2017MC023 and 2015GSF117034),andtheJinanYouthScienceandTechnology StarProject(No.201406021).

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2018.04.001.

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