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Environmental
Microbiology
Heterologous
expression
of
a
rice
metallothionein
isoform
(OsMTI-1b)
in
Saccharomyces
cerevisiae
enhances
cadmium,
hydrogen
peroxide
and
ethanol
tolerance
Zahra
Ansarypour,
Azar
Shahpiri
∗IsfahanUniversityofTechnology,CollegeofAgriculture,DepartmentofBiotechnology,Isfahan,Iran
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c
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o
Articlehistory:
Received1May2016 Accepted25October2016 Availableonline4February2017 AssociateEditor:GiseleMonteirode Souza Keywords: Cadmium Ethanol Hydrogenperoxide Ricemetallothionein S.cerevisiae
a
b
s
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c
t
Metallothioneinsareasuperfamilyoflow-molecular-weight,cysteine(Cys)-richproteins thatarebelievedtoplayimportantrolesinprotectionagainstmetaltoxicityandoxidative stress.Themainpurposeofthisstudywastoinvestigatetheeffectofheterologous expres-sionofaricemetallothioneinisoform(OsMTI-1b)onthetoleranceofSaccharomycescerevisiae
toCd2+,H
2O2andethanolstress.ThegeneencodingOsMTI-1bwasclonedintop426GPDas
ayeastexpressionvector.ThenewconstructwastransformedtocompetentcellsofS. cere-visiae.AfterverificationofheterologousexpressionofOsMTI-1b,thenewstrainandcontrol weregrownunderstressconditions.Incomparisontocontrolstrain,thetransformedS. cerevisiaecellsexpressingOsMTI-1bshowedmoretolerancetoCd2+andaccumulatedmore
Cd2+ionswhentheyweregrowninthemediumcontainingCdCl
2.Inaddition,the
heterolo-gousexpressionofGST-OsMTI-1bconferredH2O2andethanoltolerancetoS.cerevisiaecells.
TheresultsindicatethatheterologousexpressionofplantMTisoformscanenhancethe toleranceofS.cerevisiaetomultiplestresses.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
The yeast, Saccharomyces cerevisiae, has several properties which have established it as an important tool in the
Abbreviations: MT,metallothionein;Os,Oryzasativa;GST,glutathione-S-transferase;ROS,reactiveoxygenspecies.
∗ Correspondingauthorat:DepartmentofBiotechnology,CollegeofAgriculture,IsfahanUniversityofTechnology,Isfahan84156-83111,
Iran.
E-mail:a.shahpiri@cc.iut.ac.ir(A.Shahpiri).
expression of foreign proteins for research, industrial or medical use.1,2 As afood organism, it ishighly acceptable forthe productionof pharmaceuticalproteins. Incontrast,
Escherichia colihastoxiccellwallpyrogens andmammalian cells maycontainoncogenicorviralDNA,sothatproducts
http://dx.doi.org/10.1016/j.bjm.2016.10.024
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
eukaryote,andcanbemanipulatedalmostasreadilyasE.coli.
Asaeukaryote,S.cerevisiaeisasuitablehostorganismforthe high-levelproductionofsecretedaswellassolublecytosolic proteins.5
Industrialyeaststrainsduringfermentationareexposed tovarious stressessuchas osmoticshock, oxidativestress andtoxicityofsecondarymetaboliteswhichleadtotheloss ofbiologicalproducts.6,7Molecularoxygenisrelatively unre-active and harmless in its ground state, but can undergo partialreductiontoformanumberofreactiveoxygenspecies (ROS),includingthesuperoxideanionandhydrogenperoxide (H2O2),which canfurtherreacttoproducethehighly reac-tivehydroxylradical.8ROSaretoxicagentsthatcandamage awidevarietyofcellularcomponentsresultinginlipid per-oxidation,proteinoxidation,andgeneticdamagethroughthe DNAmodification.8,9YeastcellsmaybeexposedtoROS gener-atedbyneutrophilsandmacrophagesduringimmunological defense mechanisms and followingexposure tonumerous exogenousagentsincludingxenobiotics,carcinogens,UVand ionizingradiation.7
S.cerevisiae,likeallorganisms,contains effective antiox-idant defense mechanisms, which detoxify ROS as they are generated and maintain the intracellular redox envi-ronment in a reduced state.9 In response to an oxidant challenge, yeast cells increase the synthesis of a num-ber of antioxidant enzymes involved in the detoxification ofROSincluding glutathionereductase, superoxide dismu-tase, catalase, glutathione peroxidase and thioredoxins.9,10 Inadditiontoantioxidantenzymes,growingamountof evi-dences show aclose relationship between oxidativestress andmetallothioneins(MTs);the lowmolecularweight Cys-richproteinswhich are ubiquitouslypresent ineukaryotes and prokaryotes. In fact a variety of physical and chemi-cal stresses associated with oxidative injury increase MTs synthesis.11–13
MTs belong to a superfamily of intracellular metal-binding proteins which is composed of 15 families.14,15 MTs bind metals through the thiol groups of their Cys residues.14,16 Induction of MTs synthesis by heavy met-als is a very well-known phenomenon and accordingly MTs are regarded as biomarker to reflect heavy metal exposures in ecotoxicological assessment.15,17 MTs have a strong effect in scavenging free radicals, which are pro-duced under various stress conditions.13 In Arabidopsis, it has been demonstrated that different types of MTs exhibitdistinctandoverlappingfunctionsinmaintainingthe homoeostasisofessentialtransitionmetals,detoxificationof toxicmetals, and protectionagainst intracellular oxidative stresses.18–21 Transgenictobaccoandyeastthatoverexpress MTfromcottondisplayedincreasedtoleranceto environmen-talstresses,indicatingtheroleofMTinresponsetoabiotic stresses.22
Previously we heterologously expressed the isoform OsMTI-1b,a riceMT type 1,in E. colias carboxyl-terminal extensionsofglutathione-S-transferase(GST).15,23 Thecells expressing OsMTI-1b showed increased tolerance to Ni2+,
cells.
Inthisstudy,thegeneencodingOsMTI-1bwas heterolo-gouslyexpressedinS.cerevisiae.ThetoleranceoftransgenicS. cerevisiaetocadmium,hydrogenperoxideaswellasethanol wasexaminedandtheaccumulationofcadmiuminthecells wasdetermined.
Materials
and
methods
CloningofgenesencodingGST-OsMTI-1b
In order to clone the gene encoding GST-OsMTI-1b, the plasmid pET41a-OsMTI-1b was used as template.15 The DNA fragment containing GST-OsMTI-1b was ampli-fied using Pfu DNA polymerase (Thermo Scientific) in a reaction mixture containing template plasmid, deoxy nucleotides,reactionbufferandtheprimers5-ATATAAGCTTG CGGATAACAATTCCCCTCT-3,whichcarriesanHindIII restric-tion site atthe 5 end (underlined)of forwardprimer, and 5-ATTTCTCGAGTTAGCAGTTGCAAGGGTT-3 with a restric-tion siteXhoI (underlined) atthe 5 end ofreverse primer. Anadditionoffourbaseswasincludedatthe5 endineach oligonucleotideprimer.Thethermalprofilewasasfollows:1 cycleat92◦Cfor5min;35cyclesat92◦Cfor1min;63◦Cfor 1.5min;72◦Cfor2minand1cycleat72◦Cfor10min.ThePCR productwasthendigestedwithenzymesHindIIIandXhoIand ligated usingT4ligaseinto p426GPD asayeastexpression vector,24afterlinearizationwithHindIIIandXhoI.Theligation mix was used to transform competent E. coli cells (DH5␣). Transformantswere selected onLBmediumsupplemented withampicillin(50mgL−1).Theresultingplasmidwastermed GPD-GST-OsMTI-1b.Theresultingplasmidswerethenverified bysequencing.
HeterologousexpressionofOsMTI-1binS.cerevisae
The competent cells from S. cerevisiae strain CEN.PK 113-5D (5D)were providedbyusing lithiumacetatemethodas previouslydescribed.25Thenewconstructs GPD-GST-OsMTI-1b was transformed to competent cells of5D to givenew strain5D-2527.Acontrolstrainwasprovidedbytransforming emptyGPDtocompetentcells.Transformantswereselected onplatescontainingSC-URAmediumafterincubationat30◦C for2–3days.
Thenewstrain5D-2527andcontrolweregrowninshake flasks containing10mLappropriateminimal medium[10% (NH4)2SO4,12%KH2PO4,5%MgSO4,tracemetals1mLL−1,2% glucose andvitamins]at30◦Cundervigorous agitationfor overnight.Then4mLoftheovernightculturesofcellswere inoculatedin80mLofminimalmediumsupplementedwith 2%glucose.WhentheO.D.600ofcultureswereabout2.5and 3.5,1mLsamplesofculturemediumwereharvestedby cen-trifugationat12,000×gfor15min,andfrozenat−20◦Cuntil use.
A
B
GPD promoter
XhoI
HindIII
ATG GST.Tag His.Tag S.Tag ATG OsMTI-1b TAA
Fig.1–Aminoacidsequenceandexpressionvectormaps.(A)TheaminoacidsequenceofOsMTI-1b.TheCysresiduesare shownasbold.(B)ThemapofGPD-GST-OsMTI-1b.ThepositionofHis.tag,S.tagandGST.tagisshowningraybox.
Extractionandidentificationofrecombinantproteinswith westernblotanalyses
Theextractionoftotalproteinwasperformedasexplained previously.26Forextractionoftotalproteins,thefrozenpellets werefirstpre-treatedwith2.0MLiAcfor10minatroom tem-perature.Aftercentrifugation(12,000×gfor10min,4◦C),the remainingpelletswereresuspendedin0.4MNaOHfor5min onice,followedbycentrifuging(12,000×gfor10min,4◦C), theremainingpelletswereresuspendedin100LSDS sam-plebuffer(0.06MTris–HC1pH6.8,10%(v/v)glycerol,2%(w/v) sodium dodecyl sulfate (SDS), 5% (v/v) 2-mercaptoethanol, 0.0025%(w/v)bromophenolblue)andboiledfor5min.Then, thecelllysatewascentrifugedtoremovethecelldebris.The totalproteinsrecoveredinsupernatantphasewereanalyzed by12%SDS-PAGEandstainedbyCoomasieBrilliantBlue R-250.27
Forwesternblotting,proteinsweretransferredtoaPVDF membrane (Roche AppliedScience) according tothe man-ufacturer’s instructions. The blots were blocked overnight in blocking buffer containing 5% (w/v) skimmed milk in TBST(10mmolL−1NaCl,25mmolL−1 Tris–HCl,pH7.5,0.1% (v/v)Tween20).TherabbitHis-Tagantibody(GenScript),was diluted1:1000inTBST.Theantigen–antibodyinteractionwas carried out at room temperature for 1h. The blots were washed(3× 10min)withTBSTfollowedby(1×10min) TBS (10mmolL−1NaCl,25mmolL−1Tris–HCl,pH7.5).Blotswere thenprobedwithgoat-anti-rabbitIgGconjugatedwithhorse radishperoxidase(GenScript)diluted1:2000inTBSTas sec-ondary antibody. The membranes were washed again, as explainedbefore.Afterwashing,theimmuneblotswere devel-oped using 0.5mgmL−1 diaminobenzidine in 50mmolL−1 Tris–HCl,pH7and0.22%hydrogenperoxide.
ToleranceofS.cerevisiaecellstoCd2+,H
2O2andethanol Tolerance of new strain and control to metals, H2O2 and ethanol inthe growth mediumwas examined indifferent concentrationsofCdCl2·H2O (0.3 and0.9mM), H2O2 (1 and 3mM)andethanol(7(v/v)%and10(v/v)%).Forthese analy-ses,5mLsamplesoftheovernightcultureswereinoculated in 80mL of minimal medium supplemented with 2% glu-cose.Thecultureswereincubatedat30◦Cwithshaking.The
culturemediacontaining5D-2527orcontrolstrainwere sup-plementedwithcadmiumatconcentrationsof0.3and0.9mM whenO.D.600reached3.3.Thegrowthwasmonitoredupto14h byO.D.600measurements.Eachdatarepresentsthemean±SD obtainedfromthreeindependentexperimentswithtwo repli-cates. For the analysis ofcadmium in medium,cells from 10mLofcultureat0(T0)and5haftercadmiumaddition(T1) wereprecipitatedbycentrifugationat6000×gfor20min.The supernatantwasanalyzedforCd2+usinginductivelycoupled plasmaatomicabsorptionspectroscopy(PerkinElmer AAna-lyst700).Thecadmiumconcentrationchangesinthemedium of controland 5D-2527 between T1and T0(CT0–CT1)were calculated.Eachdataisthe mean±SDobtainedfromthree independentexperimentswithtworeplicates.Differencesat the5%levelwereconsideredsignificantasanalyzedbythe pairedstudent’st-test(p<0.05).
For analysis of H2O2 and ethanol tolerance, H2O2 and ethanol were added to the culture media after 7h when O.D.600 reached 3.6. The growth was monitored up to 14h by O.D.600 measurements. Each data represents the mean±SD obtained from three independent experiments with two replicates. Differences atthe 5%level were con-sideredsignificantasanalyzedbythepairedstudent’st-test
(p<0.05).
Results
HeterologousexpressionofGST-OsMTI-1binS.cerevisiae
Thefull-lengthcodingsequenceofOsMTI-1bconsistsof219bp encoding a protein with 72 amino acids and theoretical molecular weight (kDa)/pI of 7.13/5.08. The predicted pro-tein consistsof sixC–X–C motifsequally distributed in N-andC-terminals,asotherplanttype1MTproteins(Fig.1A). Theproteinwasexpressedascarboxyl-terminalextensions ofGST.tag,6His.tagandS.tagwhichweentirelynamedGST inthisstudy(Fig.1B).TheuseofGSThastheadvantagesof highsolubilityandstabilityagainstproteolyticdegradation.28 Therefore,theproteinGST-OsMTI-1bispredictedtohave the-oreticalmolecularweight(kDa)/pIof39.94/5.93.Westernblot analysisshowedasharpproteinbandofexpectedmolecular mass(Fig.2,lanes3and4).
35
25
45
35
25
SDS-Page
Western
blot
Fig.2–SDS-PAGEandwesternblotanalysisoftotal proteinsextractedfromcontrolstrain(lanes1and2)and 5D-2527(lanes3and4)whentheO.D.600ofcellcultures
were2.5(lanes1and3)and3.5(lanes2and4).Thepurified GST-OsMTI-1bobtainedfromheterologousexpressionin
E.coli15wasruninlaneP.
TolerancetoH2O2andethanol
Thecomparisonofgrowthcurvesofstrain5D-2527andcontrol revealsthatintheH2O2/ethanolfreemedium,themaximum O.D.600forcontrolstrain(O.D.max6006.6±0.2)isalmosthigher than that forthe strain 5D-2527 (O.D.max600 6±0.2). How-ever,inthepresenceof1mMH2O2themaximumO.D.600of strain5D-2527(O.D.max 6.2±0.24)washigherthanthatfor thecontrolstrain(O.D.max6004.6±0.2,Fig.3A).Inthe pres-enceof3mMH2O2themaximumO.D.600ofstrains5D-2527 andcontrolstrainwere4.8±0.2and 3.8±0.17,respectively. Inaddition,inthepresenceof7and10%ethanolthe maxi-mumO.D.600valuesforthecontrolstrainwere4.6±0.2and 4.2±0.2, respectively. These valueswere reached5.6±0.18 and 5.1±0.25for 5D-2527inthe same conditions (Fig.3B). Theseresultssuggestthattheheterologousexpressionof GST-OsMTI-1bconfersH2O2andethanoltoleranceinS.cerevisiae cells.
ThecellsexpressingOsMTI-1bhavemoretoleranceto cadmium
Inthepresenceof0.3and0.9mMCd2+themaximumO.D. 600 forcontrol strainwere 3.2±0.25and 3.4±0.2,respectively. Thesevaluesreached4±0.2and4.7±0.15forthestrain 5D-2527whenitwasgrowninthesameconditions.Theseresults suggest that the heterologous expressionofGST-OsMTI-1b conferscadmiumtoleranceinS.cerevisiaecells.
Todeterminewhetherthe enhancedoftoleranceof 5D-2527tocadmiumwasduetoincreasedaccumulationofthis metalioninthe cells,theconcentrationsofcadmiumions intheculturemediainoculatedwiththe5D-2527andcontrol strainsweredeterminedbyatomicabsorptionat0h(T0)and 5h (T1)aftertheadditionofcadmiumtothe cultures.The concentrationofcadmiumionsatT1wassimilartothatatT0
Discussion
S. cerevisiaeisthe mostimportantindustrial yeastand the main microorganismemployedin bioethanolproduction.29 Industrial yeaststrainsduringfermentationare exposedto variousstressessuchasosmoticshock,oxidativestressand toxicity of secondary metabolites which lead to the loss ofbiological products.6,29 During oxidativestress inS.
cere-visiaetheproteinYap1pasatranscriptionfactorplaysakey role in activationoftranscription ofmany genes including
TRX2,30encodingthioredoxin,TRR1–thioredoxinreductase,31
GSH2 – glutathione synthase, GPX2 – glutathione peroxi-dase232andTSA1–thioredoxinperoxidase133,34viabinding withspecificDNAsequenceslocalizedintheirpromoters.35 Correspondingly the over expression of genes TRX2 and
TRR1 enhanced the resistance of S. cerevisiae to oxidative stress.30,31
EthanolproductionbyS.cerevisiaeisoneofthefirst biotech-nologicalcommoditiesformanyyears.Howevertheinhibitory effect ofethanol accumulationonyeastgrowth during fer-mentation is still an important challenge for bio-ethanol production.36 Depending on yeast strain and condition, ethanolinhibitiononyeastgrowthbeginsatconcentrations of less than 5%.37 The mechanisms that yeasts cope with ethanolstressareverycomplicatedandnotfullyunderstood. Thequantificationofthegrowthbehaviorofallavailable sin-glegenedeletionstrainsofS.cerevisiaeunderethanolstress revealed thatthegrowth of446deletionstrains was defec-tiveindicatingthatmanygenesareinvolvedintheethanol resistanceinyeast.Thedeletionofsomeofthesegeneslike
PEXgeneswhoseproductsareinvolvedinperoxisome trans-portexhibitedtheseverestsensitivitytoethanol.38Recently thegenerationofnewtransgenicS.cerevisiaestrainshasbeen successfullyimplementedtoimproveethanoltolerance.39–42 Forinstance,theoverexpressionofgenesINO1,DOG1,HAL1
and truncated form of MSN2 in S. cerevisiae resulted in remarkably increased tolerance to high concentrations of ethanol.41
In the present work the gene encoding OsMTI-1b was transferredtoS.cerevisiae.MTsareCys-richandlow molec-ularweightproteinscharacterizedasimportantintracellular factorsindetoxificationofheavymetalsinthecellsof prokary-otes and eukaryotes.14,43 However the role of MTs in the protectionofplantandanimalcellsagainstoxidativestress hasbeenrecentlydemonstrated.22,44,45Theisoform OsMTI-1bisknownasariceMTtype1duetothepresenceofsix CysresiduesinN-and C-terminals.Previouslythis isoform was heterologously expressed in E. coli. Transformed cells showedincreasedtolerancetoheavymetalscadmium,zinc andnickelandaccumulatedmoremetalsascomparedtothe controlstrain.15,23 Furthermore,theheterologousexpression ofOsMTI-1bconferredhydrogenperoxidetolerancetoE.coli
cells.15Thedatapresentedhereshowthattheheterologous expressionofOsMTI-1bwassuccessfulwhenitwasexpressed ascarboxy-terminalextensionofGST.TheS.cerevisiaecells
8 7 6 5 4 3 2 1 0 8 7 6 5 4 3 2 1 0 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Control (0 mM) Control (1 mM) 5D-2527 (0 mM) 5D-2527 (1 mM) Control (0) Control (7 %) 5D-2527 (0) 5D-2527 (7 %) Control (0) Control (10 %) 5D-2527 (0) 5D-2527 (10 %) Control (0 mM) Control (0.9 mM) 5D-2527 (0 mM) 5D-2527 (0.9 mM) Control (0 mM) Control (0.3 mM) 5D-2527 (0 mM) 5D-2527 (0.3 mM) Time (h) Time (h) 8 7 6 5 4 3 2 1 0 8 7 6 5 4 3 2 1 0 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Control (0 mM) Control (3 mM) 5D-2527 (0 mM) 5D-2527 (3 mM) OD600 OD600 OD600
A
B
C
Fig.3–EffectsofheterologousexpressionofGST-OsMTI-1bonthetoleranceofS.cerevisiaeto(A)H2O2(1and3mM),(B)
ethanol(7%and10%)and(C)Cd2+(0.3and0.9mM).Eachdataisthemean±SDobtainedfromthreeindependent
experimentswithtworeplicates.Differencesatthe5%levelwereconsideredsignificantasanalyzedbythepairedstudent’s
t-test(p<0.05).
expressingGST-OsMTI-1bwereabletoremoveefficiently cad-miumfromculturemediumand showedmoretolerancein themediumcontainingcadmium ascomparedtothe con-trol strain. In addition, the cells expressing GST-OsMTI-1b
conferredH2O2tolerancetoS.cerevisiae.Similarresultshave been previouslyreportedfortransgenicyeasts overexpress-ingcottonMTtype3(GhMT3)inresponsetoH2O2stress22or MTtype3fromTamarixhispida(ThMT3)inresponsetometal
125 120 115 110 Control (T0) 5D-2527(T0) Cd 2+ (ppm) 5D-2527 (T1) Control (T1)
Fig.4–Cadmiumconcentrationvariationinthemediumof controlstrainandstrain5D-2527betweenT1(5hafter additionCdCl2tomedium)andT0(startingpointof
additionCdCl2).Eachdataisthemean±SDobtainedfrom
threeindependentexperimentswithtworeplicates. Asterisksindicatestatisticallysignificantdifferencesfrom thecorrespondingcontrolasanalyzedbythepaired student’st-test(p<0.05).
toxicity.45 Herewe alsofound that the expression of GST-OsMTI-1benhancestheresistanceoftransgenicS.cerevisiae
againstethanol.
Taken together on the basisofour results, the heterol-ogousexpressionof isoform OsMTI-1bconferred improved toleranceagainstH2O2,metalsandethanol.Thereforeplant genesencodingMTisoformsmaybeusefulforaugmentingthe genetictoolboxforgeneratingnewstrainswithmultiplestress resistancewhichcouldbeveryefficientforbiofuelproduction.
Conflicts
of
interest
The authors declare that there is no conflict of interest regardingthepublicationofthispaper.
Acknowledgment
IranNationalScienceFoundationisthankedforfinancial sup-port(grantNo.91001342).
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