Application of n-dodecane as an oxygen vector to enhance the activity of fumarase in recombinant Escherichia coli: role of intracellular microenvironment

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

Fungal

and

Bacterial

Physiology

Application

of

n-dodecane

as

an

oxygen

vector

to

enhance

the

activity

of

fumarase

in

recombinant

Escherichia

coli:

role

of

intracellular

microenvironment

Sen

Zhang

a,b,∗

_,

_Ping

_Song

c

_,

_Shuang

_Li

c,∗

a_Nanjing_University_of_Chinese_Medicine,_College_of_Pharmacy,_Jiangsu_{Collaboration}_Innovation_Center_of_Chinese_Medical_Resources

Industrialization,Nanjing,People’sRepublicofChina

b_National_and_Local_{Collaborative}_Engineering_Center_of_Chinese_Medicinal_Resources_{Industrialization}_and_Formulae_Innovative

Medicine,Nanjing,People’sRepublicofChina

c_Nanjing_University_of_Technology,_College_of_{Biotechnology}_and_{Pharmaceutical}_Engineering,_State_Key_Laboratory_of_{Materials-Oriented}

ChemicalEngineering,Nanjing,People’sRepublicofChina

a

r

t

i

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Articlehistory:

Received2June2016 Accepted28September2017 Availableonline2February2018 AssociateEditor:GiseleMonteirode Souza Keywords: Oxygenvector Energy Fusionprotein Expression Redoxmetabolism

a

b

s

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t

Theeffectoftheintracellularmicroenvironmentinthepresenceofanoxygenvector dur-ingexpressionofafusionproteininEscherichiacoliwasstudied.Threeorganicsolutions atdifferentconcentrationwerechosenasoxygenvectorsforfumaraseexpression.The additionofn-dodecanedidnotinduceasignificantchangeintheexpressionoffumarase, whiletheactivityoffumaraseincreasedsignificantlyto124%at2.5%n-dodecaneadded after9hinduction.TheconcentrationofATPincreasedsharplyduringthefirst6hof induc-tion,toavalue7600%higherthanthatintheabsenceofanoxygen-vector.NAD/NADH andNADP/NADPHratioswerepositivelycorrelatedwithfumaraseactivity.n-Dodecanecan beusedtoincreasetheconcentrationofATPandchangetheenergymetabolicpathway, providingsufficientenergyforfumarasefolding.

licenses/by-nc-nd/4.0/).

Introduction

Molecular oxygen is an environmental and developmental signalthatregulatescellularbioenergetics.1,2_During_aerobic

fermentation,oxygenplaysanimportantrole asthe termi-nal electron acceptor in the respiratory chain that drives

∗ _{Corresponding}_authors.

E-mails:[email protected],[email protected](S.Zhang),[email protected](S.Li).

the energy metabolism.3 _However,_oxygen _requirements _in

aqueousgrowthmediumsystemshavebeenestimatedtobe 14% higherthan forconventional aerobicprocesses.In the case ofsubmerged fermentations,the supply ofoxygen to the aqueousphase is oneof the limiting factors. Oxygen-vectorsarehydrophobicliquidsinwhichoxygenhasahigher

https://doi.org/10.1016/j.bjm.2017.09.005

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solubilitythaninwater.4_Wang5_reported_the_oxygen

solubil-ityinn-dodecanewas54.9mg/Lat35◦Cunderatmospheric conditions,comparedwith34.9mg/Linwater.

Indeed anumber ofresearchers haveused oxygen vec-tors to enhance oxygen solubility successfully,6 _and _as _a

consequence,oxygenvectorshavebeenappliedinseveral dif-ferentculturesystems.5–7_Examples_of_common_hydrocarbon

oxygen vectorsincluden-dodecane, n-hexadecane, perﬂuo-rocarbons and numerous vegetable oils.8–10 _However, _it _is,

notuncommonthatoverexpressedrecombinantproteinsin

Escherichiacoli(E.coli)showadiscrepancybetweenhigh spe-ciﬁcactivityandincreasedexpressionlevel.11_The_expression

of proteinis a complicated process which is regulated by multiple-signalingpathways,includingonesaffectedbythe availabilityofnutrients,growthfactors,andtheintracellular microenvironment(ATP,NADHandNADPH).12,13

Moststudieshaveconcernedtheapplicationofoxygento enhancethe yieldoftargetproduct; howeverthe effectsof theintracellularmicroenvironment(ATP,NADHandNADPH) inthepresenceofoxygenvectorduringexpressionofafusion proteininE.colihavebeenless extensivelystudied. There-fore,inthepresentstudy,theintracellularmicroenvironment responseton-dodecaneinE.coliwasinvestigated,especially theeffectsontheexpressionoffumarase.

Materials

and

methods

Microorganisms,plasmidsandgrowthconditions

GrowthconditionsandtherecombinantE.coli BL21-pET22b-fumR,whichproducesRhizopusoryzae fumarase,havebeen previously described.14 _The _expression _of _fumarase _was

inducedbyadding IPTG(0h,ﬁnal concentration1mmol/L). Cellsweregrownforadditional6hat30◦Candwereharvested bycentrifugation.

Oxygenvector

WhenfumaraseexpressionwasinducedbyaddingIPTG, oxy-gen vector (n-hexane, n-dodecane and n-hexadecane) was immediatelyaddedtothesterilefermentationbrothat dif-ferentvolumetricfractionsasappropriate.

Preparationofcell-freeextractsandenzymeassays

Fumarasewaspuriﬁedaccordingtoourpreviouslyresearch,14

andassayedaccordingtothemethoddescribedbyKanarek andHill.15 _Protein_{concentration}_was_determined_using_the

Bradfordassay.16_One_unit_of_fumarase_activity_was_deﬁned_as

theproductionof1␮moloffumarateorl-malateperminute understandardreactionconditions.

Gelelectrophoresis

To determine the amount of soluble fumarase, the super-natant and the pellet fraction after cell disruption were analyzedbySDS-PAGEona12.5% separationgel.17 _A

Mini-PROTEANTearCell(Bio-RAD,Hercules,CA,USA)wasusedfor electrophoresis.

Table1–Effectsofdifferentoxygenvectorsonfumarase activity. Oxygen-vectors Concentrationof oxygen-vectors (%,v/v) OD600 Fumarase activity(U/mg) Control 0 2.61±0.11 6.44±0.22 n-Hexane 0.6 1.23±0.06 9.32±0.36 n-Dodecane 1.5 2.76±0.12 12.47±0.59 n-Hexadecane 1.5 2.90±0.13 10.01±0.49

Molecular weightmarkerswere purchasedfrom TaKaRa BioGroup.Afterelectrophoresis,theproteinbandswere visu-alizedwithCoomassieBrilliantbluestaining.14

IntracellularATP

The intracellular ATP concentration was analyzed by an HPLCsystem(DionexP680pump,Chromeleoncontroller,and DionexUVD170UDetector;DionexCorporation,CA,USA).18

Onemillilitercellsuspensionwasharvestedbycentrifugation at15,000×gfor1minat−8◦_C_and_the_harvested_cells_were

mixedwith1mLmethanolat−40◦_C._A_reagent _containing

50%perchloricacidwasusedtoextractATP.Thesolutionwas ﬁlteredthrougha0.22␮mmembrane,ifnecessary,and20␮L was injectedintothe HPLCsystem.Themobile phase com-prisedphosphatebuffersolution/acetonitrile/water(86:4:10) ataﬂowrateof1.0mL/min.Thephosphate buffersolution was composedofNaH2PO4 10.93g/L,Na2HPO4 3.04g/L, and

tetrabutylammoniumbromide3.22g/L(pH6.5).ASepax HP-C18column(250mm×4.6mm)wasusedat35◦C.Thecolumn efﬂuentwasmonitoredat254nm.

NAD(P)-NAD(P)Hpoolisolationandcyclingassay

Sampleswereremovedfromaerobiccultures,andthe dinu-cleotideswereextractedbythemethoddescribedbyVemuri.19

Recycling assays of the extracts containing speciﬁc dinu-cleotidespecieswereperformedintriplicate.20

Results

and

discussion

Effectsofoxygenvectorsonfumaraseactivity

Three oxygen vectors (n-hexane, n-dodecane and

n-hexadecane) at a ﬁnal concentration of 1.5% (v/v) were immediately added tothe fermentation broth.The oxygen vectorsaddedinthisstudywereallorganicsolvents,which havetoxicityeffectsoncells athighconcentrations.21–23 _In

this study,1.5% n-hexanewas foundto be toxic, and was observed to inhibit growth of the cell and cause cell lysis after3h.Asa consequence,theconcentrationofn-hexane

wasdecreasedto0.6%.Theeffectsofoxygenvectorsoncell growth andfumarase activitywereinvestigatedafter6hof induction(Table1).Thebiomassincreasedafteradding1.5%

n-dodecaneandn-hexadecane,whileitwasstillinhibitedby the0.6%n-hexane.

Enhancementoffumaraseactivitywasdetected.With1.5%

n-dodecane, fumaraseactivityreachedthemaximumvalue of12.47U/mg,whichincreasedby94% comparedwith

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con-Table2–Effectsofconcentrationsofn-dodecaneon fumaraseactivity. Concentrationof n-dodecane(%, v/v) OD600 Fumarase activity(U/mg) 0 2.92±0.15 6.33±0.32 0.5 3.02±0.15 7.09±0.24 1 3.12±0.16 8.96±0.54 1.5 3.21±0.11 10.32±0.52 2 3.07±0.14 12.68±0.59 2.5 3.03±0.14 14.21±0.61 3 2.86±0.13 11.33±0.56 3.5 2.54±0.14 1.32±0.07

Fig.1–Effectof2.5%(v/v)n-dodecaneonthetime-course proﬁlesoffumaraseactivity.,Fumaraseactivitywithout IPTGorn-dodecane;䊉,fumaraseactivitywithIPTGbut withoutn-dodecane;,fumaraseactivitywithIPTGand

n-dodecane;,OD600withoutIPTGorn-dodecane;,

OD600withIPTGbutwithoutn-dodecane;,OD600with

IPTGandn-dodecane.

trolexperiments.Therefore,thisvectorwasselectedforthe subsequentinvestigations.

Theconcentrations(0–3.5%,v/v)ofthen-dodecanewere added to investigate their inﬂuences on cell growth and fumarase activity. Table 2 shows that the addition of

n-dodecaneasanoxygenvectoratlowconcentrationsincreased biomassproductionandfumaraseactivity.Atconcentrationof 2.5%n-dodecane,theactivitywashigherthanthatobserved intheconcentrationsof1,1.5and2,despitelowerbiomass, whichmayindicateabetterfoldingoftheenzyme.However, atvolumetricfractionsofn-dodecanehigherthan%,celllysis anddecreasedfumaraseactivitywereobserved,whichmight havebeenbecausehigherconcentrationsofn-dodecaneare toxictocells.

Effectsofconcentrationsof2.5%(v/v)n-dodecaneonthe time-courseproﬁlesoffumaraseactivity

Theeffectofthepresenceof2.5%n-dodecaneisshown in

Fig.1illustratesthatexpressionofthefumRgenewasinduced by adding IPTG; meanwhile the biomass was inhibited by IPTG.TheOD600andfumaraseactivityduringtheinduction

Fig.2–Effectofn-dodecaneontheexpressionoffumR gene.SDSPAGEanalysisoftheexpressionlevelsofthe fumRat37.Theexpressionofthefumarasewasinduced byaddingIPTG(ﬁnalconcentration1mM).Cellswere grownforadditional6hat180rpmand37andwere harvestedbycentrifugation.Thecellswerenormalizedto theirendOD600.M:M:proteinmarker;1:

pET-22b-fumR,-IPTG,–n-dodecane;2:pET-22b-fumR,+IPTG, –n-dodecane:3:pET-22b-fumR,+–IPTG,+n-dodecane.

process were depicted that then-dodecane keepthe activ-ity andbiomassathigherlevel comparedwithn-dodecane

absence.After9hofinduction,thebiomassandthefumarase activity both reached the maximum value. The 2.5%

n-dodecaneadditionincreasedfumaraseactivityto14.21U/mg, whichis124%enhancementcomparedwiththeabsenceof

n-dodecane.

Theaddition ofn-dodecanedidnotinducea signiﬁcant change in the expression of fumR gene (Fig. 2), while the fumarase activity wasincreased signiﬁcantly.It ispossible thattheaccuratefoldingcausedthehigheractivity.Protein foldingandexpressionarecomplicatedprocessesthatare reg-ulatedbymultiple-signalingpathwaysincludingoneaffected bytheavailability ofnutrients,growthfactors,intracellular ATPlevels,internalredoxstate(NADH/NAD),24_and

environ-mental stress.12 _The_addition_of _an_{oxygen-vector}_possibly

inﬂuenced oxygen transportthrough therespiratory chain, subsequentlychangingtheintracellularmicroenvironment.

ChangesofintracellularATP

TheuniversalmolecularcarrierforbiologicalenergyisATP, andbothproteinbiosynthesisandfoldingrequireATP hydrol-ysis.Theeffectofn-dodecaneandIPTGonATPconcentration are shown in Fig. 3. With the absence of IPTG, the con-centrationofATPwas continuouslyrising.While IPTG and

n-dodecanewerebothused,theconcentrationofATPreached themaximumvalueat6h.Andduringtheﬁrst6hof induc-tion,the concentrationofATPincreasedsharplytoavalue 7700%higherthanthatobtainedwithoutanoxygen-vector, andthenitdroppedsigniﬁcantly.However,fumaraseactivity

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Fig.3–TheeffectofdifferentoxygenvectorsonATP concentration.,-IPTG-oxygenvector;,-IPTG-oxygen

vector;,+n-dodecane;,+n-dodecane+IPTG.

(Fig.1)wasincreasedcontinuouslywhiletheexpressionlevel almostkeptthesameafter6hinduction(Fig.2).Ithasbeen suggestedthatmolecularchaperonesactivelyfacilitate pro-teinfoldingbyusingtheenergyofATPhydrolysistoalterthe accessibleconformationsforanon-nativeprotein.25,26

Chaperonesareessentialformaintainingprotein-folding homeostasisandplayimportantrolesinthecellularstress response.By bindingto aggregation-sensitivefolding inter-mediates,chaperonesinhibitaberrantinteractions between proteins.The mostintensively studied foldingchaperones, suchastheGroEL–ESandDnaKsystems,facilitatesubstrate proteinfoldingthroughATP-andcofactor-driven conforma-tionalchanges.25–27_Although_a_number_of_{ATP-independent}

chaperoneshavebeenreported,mostchaperonesusecyclesof ATPbindingandhydrolysistoassistproteinfoldingwerewell studied.27,28 _The_mechanism_of_nucleotide_sensing_in_group

IIchaperoninsineukaryoteshasbeenrevealed. Nucleotide-sensing loop (NSL) was studied as an ATP binding site. Functionalanalysis usingNSL mutantsshowsa signiﬁcant decreaseinATPaseactivity,suggestingthattheNSLisinvolved in timing ofthe protein folding cycle. Thebinding ofATP andsubsequenthydrolysispromotetheclosureofthe multi-subunitringswhereproteinfoldingoccurs.29

Changesofintracellularredoxstate

NAD, NADH, NADP and NADPH are important cofactors involvedinbiological redoxreactions. Thesecofactorsplay animportantroleinpreservingandregulating the intercel-lularredoxstate.Inthisstudy,theconcentrationchangesof those cofactorswere investigatedafteradding IPTGand

n-dodecane.AfteraddingIPTG,theconcentrationofNADand NADHdeclined(Fig.4A).Duringthe induction,the concen-tration of NAD and NAD/NADH ratio gradually increased, reaching the maximum values at 9h, and subsequently decreased,whiletheconcentrationofNADHincreasedslowly. The addition of oxygen vectors enable the intracellular NAD/NADHratioincreasemoresigniﬁcantly.Inaddition,the sametrendoccurredinthechangesinconcentrationofNADP, NADPH, and NADP/NADPH ratio (Fig. 4B). NAD/NADH and NADP/NADPHratioswerepositivelycorrelatedwithfumarase activity.

Theproperfoldingandstabilityofmanyproteinsdepend ontheformationofdisulﬁdebonds.30_The_higher_NAD/NADH

andNADP/NADPHratiosaremoresuitablefortheformation ofdisulfidebonds,resultinginproperproteinfolding.Protein DataBank(PDB)containsseveralfumarasecrystalstructures, althoughnoneofthemhasdisulfidebonds.R.oryzaefumarase presents6cysteinesresidues,beingpossibletheformationof disulfidebonds,differentlyfromtheothersfumarase.Hence, onepossibilityisthattheadditionalNADHoxidationoccurs throughelectrontransportchain(ETC)andmoreATPis pro-ducedtoprovideenoughenergyfortheformationofdisulfide

Fig.4–Effectofn-dodecaneonintracellularredoxstate.A:Effectofn-dodecaneontheNAD/NADHratio.,the

concentrationofintracellularNADHwithoutaddingn-dodecane;䊉,theconcentrationofintracellularNADwithoutadding n-dodecane;,NAD/NADHratiowithoutaddingn-dodecane;,theconcentrationofintracellularNADHaddingwith n-dodecane;,theconcentrationofintracellularNADaddingwithn-dodecane;,NAD/NADHratioaddingwith

n-dodecane.B:Effectofn-dodecaneontheNADP/NADPHratio.,theconcentrationofintracellularNADPHwithoutadding n-dodecane;䊉,theconcentrationofintracellularNADPwithoutaddingn-dodecane;,NADP/NADPHratiowithoutadding n-dodecane;,theconcentrationofintracellularNADPHaddingwithn-dodecane;,theconcentrationofintracellular NADPaddingwithn-dodecane;,NADP/NADPHratioaddingwithn-dodecane.

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bonds,andforproteinfolding.NADPHcanalsoenterthe mito-chondrialETCthroughcomplexreactions.Withthepresence ofn-dodecane,the over-expressfumarasefromR.oryzaeby recombinantE.colicausescellstoproducemoreATPforthe proteinexpressingandfolding,requiringmoreenergyfrom NADHoxidationthroughtherespiratorychain.

Conclusions

Theeffectofoxygen-vectorsdisturbanceontheintracellular microenvironmentofthesubmergefermentationoffumarase overexpressioninE.coliwasinvestigated.Inthis study,the additionofn-dodecaneincreasedfumaraseactivityandthe concentration of ATP. Redox ratios significantly increased, whileasignificantchangeintheexpressionofthefumRgene was not induced (Fig. 4). Thus, on the basis of the above phenomenon, it may be inferred that the addition of an oxygen-vectorchangedtheintracellularenergymetabolism.A moreoxidativeenvironmentcanfacilitatethecorrectfolding ofaproteinthatpresentdisulfideinthesurface.Thisfumarase possess6Cysthatcould beinvolvedinstructuraldisulfide formation.

In conclusion, application of n-dodecane as an oxygen vector can greatly enhance the expression offumarase in recombinantE.coli,andthebiologicalenergymoleculeATP may play a key role in protein expression for both high expressionlevelsandspeciﬁcactivities,whichsuggestsanew strategyintheheterologousexpressionofprotein.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

ThisworkwassupportedbytheNaturalScienceFoundation ofJiangsuProvince(BK20161048),theNationalsciencefund forcollegesanduniversitiesinJiangsuProvince(17KJB530006), andtheKeyProjectofJiangsuCollaborativeInnovationCenter ofChineseMedicinalResourcesIndustrialization.

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