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Preprint:Pleasenotethatthisarticlehasnotcompletedpeerreview.

IsolatingpromotersfromCorynebacteriumammoniagenesATCC6871andapplicationinCoAsynthesisCURRENTSTATUS:ACCEPTED

YingshuoHouInstituteofMicrobiologyChineseAcademyofSciences

SiyuChenInstituteofMicrobiologyChineseAcademyofSciences

JianjunWangInstituteofMicrobiologyChineseAcademyofSciences

GuizhenLiuKaipingGenuineBiochemicalPharmaceuticalCo.Ltd

ShengWuInstituteofMicrobiologyChineseAcademyofSciences

shengwu@im.ac.cnCorrespondingAuthorORCiD:https://orcid.org/0000-0001-8205-7753

YongTaoInstituteofMicrobiologyChineseAcademyofSciences

DOI:10.21203/rs.2.10395/v3

SUBJECTAREASBiotechnologyandBioengineering

KEYWORDSCorynebacteriumammoniagenes·transcriptome·promoter·CoenzymeA.

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AbstractBackground�Corynebacteriumammoniagenesisanimportantindustrialorganismthatiswidelyused

toproducenucleotidesandthepotentialforindustrialproductionofcoenzymeAbyC.ammoniagenes

ATCC6871hasbeenshown.However,theyieldofcoenzymeAneedstobeimproved,andthe

availableconstitutivepromotersareratherlimitedinthisstrain.Results�Inthisstudy,20putative

DNApromotersderivedfromgeneswithhightranscriptionlevelsand6promotersfrommolecular

chaperonegeneswereidentified.Toevaluatetheactivityofeachpromoter,redfluorescenceprotein

(RFP)wasusedasareporter.Wesuccessfullyisolatedarangeofpromoterswithdifferentactivity

levels,andamongtheseafragmentderivedfromtheupstreamsequenceofthe50Sribosomal

proteinL21(Prpl21)exhibitedthestrongestactivityamongthe26identifiedpromoters.

Furthermore,typeIIIpantothenatekinasefromPseudomonasputida(PpcoaA)wasoverexpressedin

C.ammoniagenesunderthecontrolofPrpl21,CoAyieldincreasedapproximately4.4times.

Conclusions�Thisstudyprovidesaparadigmforrationalisolationofpromoterswithdifferent

activitiesandtheirapplicationinmetabolicengineering.Thesepromoterswillenrichtheavailable

promotertoolkitforC.ammoniagenesandshouldbevaluableincurrentplatformsformetabolic

engineeringandsyntheticbiologyfortheoptimizationofpathwaystoextendtheproductspectrumor

improvetheproductivityinC.ammoniagenesATCC6871forindustrialapplications.

BackgroundCorynebacteriumammoniagenes(formerlyknownasBrevibacteriumammoniagenes)isaGram-

positive,non-pathogenicsoilbacteriumwithhighguanine–cytosine(GC)DNAcontent.Duetothe

divergentevolutionofC.ammoniagenes,differentstrainsareusedtoproducedifferentmetabolites.

Forexample,C.ammoniagenesATCC6872isusedintheproductionofnucleotidesandnucleosides

[1],whileC.ammoniagenesATCC6871showsarelativelyhighsynthesiscapacityofcoenzymeA

(CoA),whichisaubiquitousandessentialcofactorfoundinallthreedomainsoflifeandisinvolvedin

numerousmetabolicpathways[2-4].

SequencingoftheC.ammoniagenesgenomehashelpedtoidentifykeyenzymesfordivertingcarbon

flowfrommetabolicpathwaystootherproducts[5,6].However,amajorbreakthroughingenetic

3

manipulationtopreciselyregulatetheexpressionofindividualbiosyntheticgenesisstillinprocess.

ProgressinthemanipulationofC.ammoniagenesgeneshasbeenachievedthroughthedevelopment

ofeffectivetransformationprotocolsandcloningvectors[7].MostvectorsarebasedontherelatedC.

glutamicumendogenouscrypticplasmidsthathaveincorporatedE.colielementsfortransfer

betweenthespecies[8-10].AlmostallthesevectorshaveadoptedE.colipromotersorC.

ammoniagenesnativepromoterstoexpressforeignproteinsinC.ammoniagenes[11-13].Although

someofthesepromotersareactiveinC.ammoniagenes,theactivitiesarequitelow[14].

ToefficientlyengineerC.ammoniagenes,itisnecessarytoscreenendogenouspromoters.Paikand

coworkersisolated22representativepromotersfromthegenomeofC.ammoniagenesATCC6872

andtheactivityofoverexpressedchloramphenicoltransacetylasefromthestrongestpromoterIJ73

was2.85U[7].SimilarstudieshavebeenperformedbyParkandcoworkers,wherethey

overexpressedtheattenuatorbindingproteininC.ammoniagenesATCC6872usingtheendogenous

promoterCJ1[15].However,theabovepromotershavenotbeenuniversallyappliedinC.

ammoniagenesATCC6871(seetext),probablyduetothedivergentevolutionofC.ammoniagenes

genomes.Forinstance,thesequenceidentitiesofpromotersIJ59andIJ73fromC.ammoniagenes

ATCC6872withthehomologouspromotersfromC.ammoniagenesATCC6871are93%and89%,

respectively.Therefore,duetotheimportanceofCoA,itisnecessarytofindpromotersthatare

effectiveinC.ammoniagenesATCC6871.

Endogenouspromoterscanbeobtainedbygenomicdissectionorpromotertrappinget.al[16].

Nonetheless,withtheadventofgenomicsandtranscriptomics,wecanisolatepromotersmore

rationally.Studieshaveshownthatstrongpromotersareusuallyobtainedfromthepromotersof

essentialgeneswhosetranscriptionlevelsarepresumedtobehighandconstant[17].Inaddition,we

foundthatsomecommonly-usedstrongpromoterssuchasPdnakinGluconobacteroxydansandPgro

inCorynebacteriumglutamicumarepromotersofmolecularchaperones[18,19].Inthisstudy,the

promotersfor20geneswithhightranscriptionlevelsand6molecularchaperone-encodinggenesare

identifiedbybioinformaticmethodsandtheiractivitieswerefurtherexaminedexperimentally.Among

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them,Prpl21wasfoundtobethestrongestpromoter,whichwasusedinthebiosynthesisofCoA,

increasingtheyieldby4.4times.

ResultsScreeningpromotersandconstructionofprobe-vector

C.ammoniagenesATCC6871cellsgrowninLBmediumandfermentationmediumtotheexponential

phase(OD600nm≈2.0)werecollectedandsenttoMegagenomics(Beijing,China)forRNA-seq.Atotal

of2411geneswereidentifiedfromthetranscriptomicdata.Consideringthattherearemanycasesin

whichgenesareinthesameoperon,geneticlocianalysiswasperformedandtheresultsshowedthat

1508genesin547operonswereexpressed.Ingeneral,genesinthesameoperonsshareacommon

promoter,sothefirstgeneineachoperonwasselected.Thus,thetotalnumberofremaininggenes

was1450(2411-1508+547).Transcriptionabundanceanalysiswasusedtoselect71geneswith

transcriptionlevelsinthetop100inbothRNA-seqprofiles.Sequencesupstreamofthetop20genes

werepredictedandscoredusingtheonlinepromoterpredictiontoolasdescribedbelow.The

annotationsandRPKMvaluesforthesegenesareshowninTable1.Atranscriptomicdataanalysis

flowchartandthetranscriptionlevelsofgenesareshowninFigure1.

Previousstudieshaveshownthatmostpromotersofmolecularchaperonesexhibithightranscription

levelsinprokaryotes,soall6annotatedmolecularchaperonesintheC.ammoniagenesATCC6871

genome(AccessionNumber:NZ_CP009244)(twoGroEL,twoDnaJ,oneDnaK,andoneCo-chaperone

GroES)wereidentifiedandtheirpromoterswerepredictedasdescribedinmaterialsandmethods.

TheinformationofthesegenesandtranscriptomicRPKMvaluesarealsolistedinTable1.By

predictingthepromoterregions,26promoterswereconstructedaccordingtotherulesdescribed

belowandtheircorrespondingRFPexpressionplasmidswerenamedaspXMJ190-Pn.Thesequences

ofthe26promotersarelistedinTableS3.

Foracomprehensivecomparison,twoconservedhomologouspromotersCJ1andIJ59thatareactive

inC.ammoniagenesATCC6872werealignedagainstthegenomeofC.ammoniagenesATCC6871by

BLASTandwereclonedintopXMJ190togivepXMJ190-CJ1andpXMJ190-IJ59[7,15],respectively.

Moreover,previousstudieshaveshownpromotersthatworkwellinC.glutamicummayalsobeactive

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inC.ammoniagenes[20].Thus,astrongendogenousC.glutamicumpromoternamedPgro(the

promoterofgroESgene)wasclonedaspXMJ190-Pgro[19].Inaddition,themostwidelyusedtac

promoterandconservedSDsequencewasalsoselectedandclonedintopXMJ190aspXMJ190-Ptac

[21].ThepXMJ190wasusedasnegativecontrol.Aflowchartforconstructionoftheprobevectorsto

identifypromotersisshowninFigure2.Alloftheaboveconstructedplasmidswerevalidatedby

sequencingandthentransformedintoC.ammoniagenesATCC6871.

AnalysisandcomparisonofpromotersinC.ammoniagenesATCC6871

Toanalyzetheactivitiesofourclonedpromoters,wefirstmadeavisualassessmentofallC.

ammoniagenesATCC6871strainsharboringpXMJ190,pXMJ190-CJ1,pXMJ190-IJ59,pXMJ190-Pgro,

pXMJ19-PtacandpXMJ190-PnseriesplasmidsonLBplatescontaining20μg/mlchloramphenicol.Red

fluorescencewasobservedbymicroscopy,withdifferentintensitiesindifferentcolonies,indicating

thattherewasvariableexpressionofRFPfromthe26putativepromoters(Figure3a).Amongthese

promoters,Prpl21,Prpl10,PgroELB,andPdnaKexhibitedstrongactivityinC.ammoniagenesATCC6871,

withPrpl21beingthestrongestpromoter.Theremainingpromotershadverylowornon-existentred

fluorescence.

Toquantifytheactivitiesoftheclonedpromoters,growth-normalizedfluorescenceintensitieswere

measuredusingamicroplateassayandthedataareshowninFigure3bandTableS2,theresults

showedthatwesuccessfullyisolated20endogenouspromoterswithdifferentactivities.Amongthe

20promoters(PrrlA-Psbp)fromgeneswithhightranscriptionlevels,Ptmr,Pnat,PatpG,PgpdⅠ,Pacnare

essentiallyinactive,whilePgroELAfromthe6molecularchaperonepromoterswasalsoinactive.

Meanwhile,fourstrongpromoters,includingthePrpl21with43433RFU/ODintensityandfollowedby

PdnaK(19125RFU/OD),PgroELB(8166RFU/OD)andPrpl10(6490RFU/OD)wereidentifiedwhose

fluorescencelevelswereconsistentwiththoseobservedonplates.Thus,arangeofpromoterswith

differenttranscriptionalactivities,includingfourstrongpromoters,wereidentifiedandcanbeused

directlyinfurtherapplications.Accordingtotheobtaineddata,onlytwopromoters(Prpl21andPrpl10)

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fromthe20geneswithhighlevelsoftranscriptionhadstrongactivities,whiletwopromoters(PgroELB

andPdnaK)sourcedfromthesixmolecularchaperonegenesexhibitedrelativelyhighactivities,which

mayindicatethatisolatingpromotersfrommolecularchaperonesmaybeamoreefficientstrategy.

AlthoughmostofthepromotersidentifiedfromtheRNA-seqprofilesexhibitedlowlevelexpressionor

weresilent,theredfluorescenceintensityofPrpl21wasalmost2.3timesthatofthehighestpromoter

(PdnaK)derivedfromupstreamregionsofthemolecularchaperones.Thissuggestsisolatingpromoters

fromgeneswithhightranscriptionlevelsmayyieldstrongpromoters.

Whenitcomestotheknownpromoters,thepromoterCJ1hadsomecertainactivity(4171RFU/OD),

theIJ59(2866RFU/OD)promoterhadalmostnonactivity,andtheactivityofPgro(5872RFU/OD)was

slightlyhigherthanthePtac(5683RFU/OD)promoterbutstilllowerthanpromoterPrpl10(6490

RFU/OD).Thisdemonstratesthepromotersthatworkwellinonespeciesorhomologousstrainsmay

notpossessthesamecharacteristicsinanother.

ApplicationofPrpl21inC.ammoniagenesforimprovingtheproductionofCoA

IthasbeenreportedthatCoA,aubiquitousandessentialcofactorinbiochemicalreactions,canbe

producedbyC.ammoniagenesATCC6871[25].IntheCoAbiosyntheticpathway,thecommittedstep

catalyzedbypantothenatekinase(coaA)issubjecttofeedbackinhibitionbyCoAandacyl-CoAs[26].

BacterialcoaAproteinsarecategorizedbasedontheiraminoacidsequencesintothreetypes,namely

typeI,II,andIII.C.ammoniagenescarriesatypeIcoaAwhichishighlyregulatedbyCoAandits

derivatives.Incontrast,typeIIandIIIenzymesareinsensitivetoCoAanditsthioesters[27,28],

therefore,inordertoreducefeedbackinhibitionandincreasetheproductionofCoA,thetypeIII

pantothenatekinasefromPseudomonasputida(PpcoaA)wasselectedandoverexpressedbythe

controlofthestrongestpromoterPrpl21inC.ammoniagenesATCC6871[29,30].

ConsideringtherearenodirectenzymeassaysfortypeIIIpantothenatekinase,RFPwasco-expressed

withPpcoaA.AsshowninFigure4a,brightredfluorescencewasobservedinthetubewithcells

containingpXMJ190-Prpl21-PpcoaA-RFP,suggestingthatRFPandPpcoaAweresuccessfullyco-

expressed.Bymeasuringtheredfluorescenceintensity,thestrainharboringpXMJ190-Prpl21-PpcoaA-

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RFPhadupto7560RFU/OD.ThisvaluewaslowerthanthatobservedwhenRFPexpressedalone

underthecontrolofPrpl21,possiblyduetothelongdistancefromthetranscriptioninitiationsiteand

theincreasedcellularburdencausedbyco-expressionoftwoproteins.TheexpressionofRFPand

PpcoaAwasalsobeobservedbySDS-PAGE.AsshowninFigure4b,significantbandsofapproximately

27kDaand32kDawereobservedindicatingthatRFPandPpcoaAwereoverexpressedandthatthe

Prpl21promoterfunctionedwellinC.ammoniagenes.Furthermore,threecrucialsubstrates

(pantothenicacid(2mM),L-cysteine(2mM),ATP(6mM))wereaddedtothereactionmixture

containingacertainnumberofbacterialcells(OD600nm≈40)at39℃.CoenzymeAproduction

increasedtoasatisfactoryyieldofapproximately315U/mLin6h(Figure4c),whichwasalmost4.1

timeshigherthanthesameconditionswithoutpXMJ190-Prpl21-PpcoaA-RFPinthecells(76U/mL).The

resultsindicatedthatPpcoaAwassuccessfullyoverexpressedunderthecontrolofpromoterPrpl21and

increasedtheanabolicflowofCoA.TofurtherimprovetheCoAproduction,theRFPgenewas

removedfromtheplasmidpXMJ190-Prpl21-PpcoaA-RFPforeliminatingtheburdenofRFPexpressionin

cells.WiththePpcoaAoverexpressedaloneinC.ammoniagenes,theCoAproductionwasreached

332±13U/mL,whichwasslightlyincreasedthanthePpcoaAandRFPco-expressionalsystem.

DiscussionC.ammoniagenesATCC6871isaproducerofCoA,however,itsgeneticoperationhasbeenrarely

reported,anditsindustrialapplicationpotentialstillneedstobeimproved.Inordertofurther

increasetheproductionofCoA,endogenouspromotersfromC.ammoniagenesATCC6871were

screenedinthisstudy.Firstly,20putativepromotersbasedongeneswithhightranscriptionlevels

and6promotersupstreamofmolecularchaperoneswereidentifiedandcharacterizedfor

transcriptionalactivitybyusingRFPasthereportergene.Amongthe26putativepromoters,20with

differenttranscriptionalactivitieswereisolatedincluding15fromthehightranscriptionlevelgenes

and5frommolecularchaperonegenes.Theoutputefficiencydemonstratesthatisolatingpromoters

frommolecularchaperones(5/6)mightbeamoreefficientstrategythanusingRNA-seq(15/20).

Therefore,isolatingpromotersfrommolecularchaperonegenesismoreeffortlessandcost-effective

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thanRNA-seq.TheseresultsmayascribetothattheRPKMvaluesofthegenesonlyrepresentthe

abundanceofRNAanddonotequivalenttoitspromoteractivities,whichmightbecausedbymultiple

factors,suchasthecodonusageofgenes,copynumbersofgenes,thehalf-livesofmRNAand

limitationsinherentintranscriptomics[22-24].Itthus,isolatingpromotersbasedonRNA-Seqrequires

furtherverificationbyexperiments.

Nevertheless,weshouldnotethattheredfluorescenceintensityofthehighestpromoter(Prpl21)

identifiedfromRNA-seqwasalmost2.3timesthanthehighestpromoter(PdnaK)derivedfrom

molecularchaperonegenes,soisolatingpromotersbasedonthegenestranscriptionaldatamaybe

morelikelytoyieldpromoterswithhighestactivity.Toourbestknowledge,Prpl21fromtheupstream

sequenceofthe50SribosomalproteinL21isthestrongestC.ammoniagenesATCC6871promoter

reportedsofar.ThepromotersobtainedinthisworkwillenrichtheavailablepromotertoolkitforC.

ammoniagenesandshouldbevaluableincurrentplatformsformetabolicengineeringandsynthetic

biologyfortheoptimizationofpathwaystoextendtheproductspectrumorimprovetheproductivity

inC.ammoniagenesATCC6871.

TofurtherverifythecapacityofPrpl21,thekeygenePpcoaAinthebiosyntheticpathwayofCoAwas

overexpressedinC.ammoniagenesATCC6871withtheaimofreducingthefeedbackinhibitionand

increasingtheproductionofCoA.TheCoAproductionofmanipulatedC.ammoniageneswas

approximate4.4timestothecontrol,whichconfirmedthatPpcoaAoverexpressedandfunctionalized

successfullyinC.ammoniagene.Theseresultsprovethattheselectedpromoterforthe

overexpressionofforeigngenesinC.ammoniagenescouldbeusedasanefficienttoolforimproving

theyieldofmajorproductsinC.ammoniagenes.However,eliminatingtheburdenofco-expressing

RFPonlyslightlyincreasetheCoAproduction,possiblyduetotheundiscoveredlimitationsexistinthe

CoAsyntheticpathway.Thus,moreeffortsshouldbefocusontheresolvingofrate-limitingstepsof

CoAsynthesisinC.ammoniagenes.

ConclusionsInsummary,thisstudyprovidesarationalstrategytoisolateendogenouspromotersfromC.

ammoniagenesATCC6871,whichmaybehelpfulinothersimilarscientificresearch.Throughthis

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strategy,wesuccessfullyisolatedarangeofpromoterswithdifferenttranscriptionalactivitiesandthe

strongestonewasappliedtoimprovetheCoAproductioninC.ammoniagenesATCC6871,raising

hopeforfurtherimprovingtheindustrialproductionlevelofCoA.

MethodsBacterialstrains,media,andgrowthconditions

TheC.ammoniagenesATCC6871waspurchasedfromChinaGeneralMicrobiologicalCulture

CollectionCenter(CGMCC,Beijing,China).E.coliTop10(Tsingke,Beijing,China)wascultivatedin

Luria-Bertani(LB)brothoronLBplateswith2%(w/v)agarat37℃asahostfortransformation.C.

ammoniagenesATCC6871growninNCMmediumat30℃wasusedasthetransformationhostforE.

coli/Corynebacteriavectorsusedinthisstudy.Aftertransformationbyelectroporation(1.8kV),

transformantswereplatedonBHISplateswith20μg/mLchloramphenicolat30℃.Fermentation

medium(glucose100g/L,peptone12g/L,yeastpowder8g/L,MgSO4·7H2O10g/L,KH2PO410g/L,

K2HPO410g/L,urea7g/L,pH7.5-7.8)wasusedtocultivatestrainsproducingCoA.Cultivationfor

expressionanalysiswasperformedinatleastbiologicalduplicates.

RecombinantDNAtechniques

C.ammoniagenesgenomicDNAwasisolatedusingagenomicDNAisolationkit(Tiangen,Beijing,

China).Kitsforplasmidisolation,extractionofDNAfromagarosegelsandPCRproductpurification

werealsopurchasedfromTiangen(Beijing,China).I-52×High-FidelityMasterMixandTrelief™

SoSooCloningKitwerepurchasedfromTsingke(Beijing,China)andusedforroutinemolecular

biologyapplications.

Constructionofprobe-vectorpXMJ190

TheshuttlevectorpXMJ19(akindgiftfromProfessorDong,Figure2a)wasusedasthebackbonefor

vectorsconstructedinthisstudy.Toeliminateinterferencefromthepromoteralreadypresentin

pXMJ19,thetacpromoterandlacoperatorwereremoved.Ageneencodingredfluorescentprotein

(RFP)wasinsertedintotheMCStoformaprobe-vectornamedpXMJ190(Figure2b).Gibsonassembly

wasusedtoassembleDNAfragmentsupstreamofthereportergenerfp,resultinginseamless

ligationbetweenfragmentsandtheprobe-vector[31,32].

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GeneticmanipulationofC.ammoniagenes

Toincreasetransformationefficiency,recipientsweregrownin100mLofNCMmediumat30°Cuntil

anOD600nmofapproximately1.0.Cellswereincubatedonicefor20minandharvestedby

centrifugationinapolypropylenetubeat4000rpmfor10minat4°C.Afterwashingtwiceincold

distilledwaterandtwowashesinice-cold10%glycerol,cellswereresuspendedin1mLof10%

glycerol.Forelectroporation,100μLofthefreshlypreparedelectro-competentcellsweremixedwith

3μLplasmid(50ng/μLinddH2O)inacoldsterileelectroporationcuvette(1mmelectrodegap)and

pulsedimmediatelywithaMicroPulserelectroporator(Bio-RadLaboratories,Inc.,Hercules,CA).The

electroporatorwasusuallysetatavoltageof1.8kV.Cellsweresubsequentlyresuspendedin0.9mL

ofBHIS,heatedat46°Cfor6minutesandwithdrawnimmediatelyforrecoverybyincubatingfor3h

at30°Candthenplatedforselectionoftransformants.

Constructionandanalysisofselectedpromoters

ThetranscriptionallevelsofgenescanbeestimatedbyReadsPerKilobaseperMillionmappedreads

(RPKM),sothegeneswererankedbyRPKMvaluebasedontranscriptionaldatafromC.

ammoniagenesandthetop20geneswereselected.Allofthegeneswereidentifiedfromthegenome

andtheirpromoterswerepredictedasbelow.Sixannotatedmolecularchaperonesinthegenome

weresortedandtheirpromoterswerepredictedasoutlinedbelow.Accordingtopreviousstudies,the

promoter-5’-UTRjunctioninfluencesmRNAandproteinlevels[23,33].Therefore,weintegratedthe

corresponding5’-UTRintoeachpromoter.

PromotersequenceswerepredictedusingtheNeuralNetworkPromoterPredictiononlinetool

(http://www.fruitfly.org/seq_tools/promoter.html)[34].Thepromoterpredictionscorethresholdwas

setto0.7.Moreover,duetothepossibilityoftandempromoters,alleligiblepromotersequences

within300bpupstreamofthestartcodonwereconsidered[35].Forcorrecttranscriptioninitiation,

thecomplete“promoter”consistedof5’-UTR,predictedpromoterregionandShine-Dalgarno

sequence,whichtypicallycorrespondtotheregion60bpupstreamofapredictedpromoterand1bp

upstreamofthestartcodon.The60bpextensionupstreamofthepromoteraccountsforpotential

11

UP-elements[36,37].

PromoterswereamplifiedfromC.ammoniagenesATCC6871andC.glutamicumATCC13032

genomicDNAwiththecorrespondingprimers.Oligonucleotideprimersusedinthisworkarelistedin

TableS1.PCRproductswereligatedintothevectorpXMJ190usingGibsonassemblyasshownin

Figure2c.AllplasmidswereconstructedinE.coliTOP10andthentransformedintoC.ammoniagenes

ATCC6871forsubsequentanalysis.

Fluorescenceintensityassay

ToevaluateRFPexpressionunderthecontrolofthevariouspromoters,C.ammoniagenesstrains

harboringvariousvectorsweregrownovernightonLBplatescontaining20μg/mlchloramphenicol.

FluorescencewasobservedusingaLUYOR-3430stereomicroscopewithafluorescenceexcitation

source(LUYOR,USA)setat501nmandmatchinglensestodetectRFP.Pictureswerecapturedwitha

camera.

Formoreaccuratecomparisons,thefluorescenceintensitiesofbacteriaharboringdifferentplasmids

weremeasuredusingaSynergyH4microplatereader(BioTek,USA).Inordertoexcludeother

interferingfactors,harvestedcellswerewashedoncewithPBS(pH7.4)andthenresuspendedinPBS

(pH7.4)atanOD600nmofapproximately1.0.TheexcitationwavelengthforRFPwassetat554nm

andemissionwassetat586nm.FluorescenceintensitieswerenormalizedbyOD600nmandwere

usedtoindicatetheactivitiesofthepromoters.BacteriaharboringpXMJ19-Ptacwereinducedwith1

mMIPTG.

ConstructionoftherecombinantplasmidpXMJ190-Prpl21-PpcoaA-RFP

Tofurtherexaminethefunctionoftheisolatedpromoters,atypeIIIpantothenatekinasefromP.

putidaKT2440(PpcoA)andRFPwereco-expressedunderthecontrolofthestrongestpromoterPrpl21.

ThePpcoaAgenewasamplifiedbyPCRfromthegenomicDNAofP.putidaKT2440,andtheShine-

DalgarnosequencefortranslationofRFPwascalculatedusingtheRBScalculatoronlinetool

(https://www.denovodna.com/software/).AllprimersusedinthissectionarelistedinTableS1.The

recombinantplasmidpXMJ190-Prpl21-PpcoaA-RFPwasconstructedwithGibsonassemblyandpositive

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colonieswereconfirmedbyDNAsequencing(Tsingke,China).Recombinantplasmidswere

transformedintoC.ammoniagenesATCC6871forfurtherexperiments

SDS-PAGEanalysis

C.ammoniagenesstrainswerepreculturedin10mLLBmediumat30°Candshakenat220rpmfor

24h.Tenpercentoftheculturewasinoculatedina250mLshakeflaskcontaining100mL

fermentationmedium.After24hcultivation,cellsampleswereharvestedbycentrifugationat12,000

rpmfor10min.Fortymicrogramsofcelllysatewereloadedperlane.ThePpcoaAandRFPexpression

wasanalyzedby15%(v/w)polyacrylamidegelelectrophoresis(PAGE)withcell-freeextractunder

denaturingconditions.Mini-ProteanIIIElectrophoresisSystem(Bio-Rad,USA)wasutilizedtoperform

theoperation.CoomassieBrilliantBlueR-250(0.2%,w/v)wasutilizedtostainproteinonthegel.

AnalysisofCoAproduction

CoenzymeAcontentwasdeterminedaccordingtothemodifiedphosphotransacetylasemethod[38,

39].AllreagentswerepurchasedfromNationalInstitutesforFoodandDrugControl.Briefly,3.0mLof

Trisbuffer(pH7.6),0.1mLofacetylphosphatedilithiumsalt(15.2g/L)and0.1mLofthetest

solutionwereaddedintoa1cmcuvetteandmixed.Absorbanceat233nmwasrecordedasE0;and

then0.01mLofthephosphotransacetylase(30-40U/mL)solutionwasadded,mixedwellandthe

highestabsorbancewithin3to5minuteswastakenasE1.Finally,another0.01mLof

phosphotransacetylasesolutionwasadded,mixedwellandtheabsorbancewasreadasE2.The

numberofCoAunitspermilliliterwascalculatedasU=(2E1-E0-E2)×5.55×413.

AbbreviationsCoA:coenzymeA;IPTG:Isopropylβ-D-1-thiogalactopyranoside

DeclarationsEthicsapprovalandconsenttoparticipate

Notapplicable.

Consenttopublication

Notapplicable.

Availabilityofdataandmaterials

13

AlldatageneratedoranalyzedduringthisstudyareincludedinthismanuscriptandtheAdditional

filesassociatedwithit.

Competinginterests

Theauthorsdeclarethattheyhavenocompetinginterests.

Funding

ThisstudywasfundedbyNationalNaturalScienceFoundationofChina(31570077).Thefundingbody

didnotplayanyroleinthedesign,execution,analysis,andinterpretationsofdataorinwritingthe

manuscript.

Authors’contributions

YH,SW,YTdesignedalltheexperiments;YHandSCcarriedouttheexperiments,YHorganizedand

interpretedthedata,anddraftedthemanuscript.JW,GL,SWandYTcontributedtodiscussion,

revisedandcorrectedthemanuscript.Allauthorsreadandapprovedthefinalmanuscript.

Acknowledgements

WearegratefultoProfessorZhiyangDong(InstituteofMicrobiology,ChineseAcademyofSciences,

China)forgenerouslyprovidingtheplasmidpXMJ19.ThisworkwassupportedbyGrantfromNational

NaturalScienceFoundationofChina(31570077toS.W.).

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TablesTable1.IdentifiedmolecularchaperonegenesorthosewithhighlevelexpressionfromRNA-seqdata.

Name DownstreamProduct AverageRPKM Score

PrrlA 23SribosomalRNA 988873 0.99

18

PrrlB 23SribosomalRNA 913696 0.97

Ptmr Transfer-messengerRNA 853640 0.96

Pfer Ferritin 46832 0.92

PraiA Ribosomalsubunitinterfaceprotein 44446 0.91

Pusp Universalstressprotein 28915 0.83

Prpl29 50SribosomalproteinL29 24596 0.89

Pnat N-acetyltransferase 18830 0.96

PatpG F0F1ATPsynthasesubunitgamma 16820 0.80

Ptuf ElongationfactorTu 14228 0.98

Prpl21 50SribosomalproteinL21 13153 0.81

Pfnt NarK/NasAfamilynitratetransporter 12650 0.94

PgpdⅠ TypeⅠglyceraldehyde-3-phosphatedehydrogenase 12223 0.83

Phfp Hemoglobinflavoprotein 12078 0.86

Pacn Aconitatehydratase 10439 0.84

Prpl10 50SribosomalproteinL10 10398 1.00

Pabm Antibioticbiosynthesismonooxygenase 9935 0.73

19

PfbaⅡ ClassⅡfructose-bisphosphatealdolase 9740 0.77

Prpl11 50SribosomalproteinL11 9473 0.82

Psbp ABCtranspoter,substrate-bindingprotein 9120 0.99

PgroELA MolecularchaperoneGroEL 6594 0.89

PgroELB MolecularchaperoneGroEL 6261 0.87

PdnaJA MolecularchaperoneDnaJ 239 0.85

PdnaJB MolecularchaperoneDnaJ 703 0.87

PgroES Co-chaperoneGroES 2215 1.00

PdnaK MolecularchaperoneDnaK 4523 0.98

Figures

20

Figure1

RationalselectionofpromotersformRNA-seqprofile.a:Theflowchartforselectionof

promotersfromthetranscriptomicdata.b:Thetranscriptionlevelsofselected20genesin

twodifferentsamples

21

Figure2

Flowchartfortheconstructionofprobe-vectorthatusedforscreeningpromoters.a:The

shuttlevectorpXMJ19servesasthebackbone.b:Theprobe-vectorpXMJ190withreporterof

RFPanddeletionofPtacandOlac.c:pXMJ190-Pnrepresentvectorswiththe26putative

promotersinsertedintheupstreamofRFP.

22

Figure3

Differentactivitiesofselectedpromoters.a:Observedredfluorescenceofbacteriausinga

LUYOR-3430stereomicroscopewithafluorescenceexcitationsource(LUYOR,USA)setat

501nm.b:MeasuredfluorescenceintensitiesofbacteriausingaSynergyH4microplate

reader.CellswerewashedoncewithPBS(pH7.4)andthenresuspendedinPBS(pH7.4)at

anOD600nmofapproximately1.0.TheexcitationwavelengthforRFPwassetat554nm

andemissionwassetat586nm.Errorbarsshowthestandarddeviationofthree

measurements.

23

Figure4

ApplicationofPrpl21inC.ammoniagenesforimprovingtheproductionofCoA.a:The

comparisonoffluorescenceintensitiesbetweencellsharboringpXMJ190-Prpl21-PpcoaA-RFP

andwildtypecells.b:SDS–PAGEanalysisofRFPandPpcoaAco-expressioninC.

ammoniagenes.Sampleswerepreparedwithanequalconcentrationofcells,and40μgof

celllysatewereloadedperlane.LaneM:proteinmarker.c:TheCoAproductionofcells

harboringpXMJ190-Prpl21-PpcoaA-RFPandcontrol.Errorbarsshowthestandarddeviation

ofthreemeasurements.

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