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UCLAUCLAPreviouslyPublishedWorksTitleEngineeringmetabolicsystemsforproductionofadvancedfuelsPermalinkhttps://escholarship.
org/uc/item/8bn1w585JournalJournalofIndustrialMicrobiology&Biotechnology:OfficialJournaloftheSocietyforIndustrialMicrobiology,36(4)ISSN1476-5535AuthorsYan,YajunLiao,JamesC.
PublicationDate2009-04-01DOI10.
1007/s10295-009-0532-0PeerreviewedeScholarship.
orgPoweredbytheCaliforniaDigitalLibraryUniversityofCaliforniaJIndMicrobiolBiotechnol(2009)36:471–479DOI10.
1007/s10295-009-0532-0123REVIEWEngineeringmetabolicsystemsforproductionofadvancedfuelsYajunYan·JamesC.
LiaoReceived:7December2008/Accepted:14January2009/Publishedonline:7February2009TheAuthor(s)2009.
ThisarticleispublishedwithopenaccessatSpringerlink.
comAbstractThedepletingpetroleumstorageandincreasingenvironmentaldeteriorationarethreateningthesustainabledevelopmentofhumansocieties.
Assuch,biofuelsandchemicalfeedstocksgeneratedfromrenewablesourcesarebecomingincreasinglyimportant.
AlthoughpreviouseVortsledtogreatsuccessinbio-ethanolproduction,higheralco-hols,fattyacidderivativesincludingbiodiesels,alkanes,andalkenesoVeradditionaladvantagesbecauseoftheircompatibilitywithexistinginfrastructure.
Inaddition,someofthesecompoundsareusefulchemicalfeedstocks.
Sincenativeorganismsdonotnaturallyproducethesecom-poundsinhighquantities,metabolicengineeringbecomesessentialinconstructingproducingorganisms.
Inthisarti-cle,webrieXyreviewthefourmajormetabolicsystems,thecoenzyme-Amediatedpathways,theketoacidpathways,thefattyacidpathway,andtheisoprenoidpathways,thatallowproductionofthesefuel-gradechemicals.
IntroductionThedepletingpetroleumreserve,recurringenergycrisis,andglobalclimatechangearereignitingtheenthusiasmforseekingsustainabletechnologiesforreplacingpetroleumasasourceoffuelandchemicals.
Inthepastfewdecades,eVortsinthedevelopmentofbio-ethanolasanalternativefuelhaveledtosigniWcantsuccess[14–16,19].
In2007,6.
5billiongallonsofbio-ethanolwasproducedintheUnitedState[5].
However,bio-ethanolexhibitssomelimitations,suchaslowenergydensity,highvaporpressure,andcorrosiveness,whichpreventitswidespreadutilizationgiventheexistinginfrastructure.
Higheralcohols(withmorethantwocarbons),biodie-sels,andfattyacidderivativesarethoughttobemoresuit-ablefuels.
Theirphysicochemicalpropertiesaremorecompatiblewithgasoline-basedfuelsandallowdirectutili-zationofexistinginfrastructureforstorageanddistribution.
Furthermore,someofthesefuelmoleculesalsoserveasimportantchemicalfeedstocks.
Althoughtheindividualbiochemicalstepsforsynthesizingthesecompoundsinmicrobeshavebeendescribedpreviously,eVortsinputtingtogetherhighlyproductivemetabolicsystemshaveonlybegunrecently.
Inthisarticle,weWrstsummarizethemeta-bolicnetworksforproducingthesecompoundsandthenrevieweVortsinengineeringthenon-nativeproducingorganism,Escherichiacoli.
Themetabolicnetworksdis-cussedincludethetraditionalbutanolpathwayinClostrid-iumspecies,theketoacidpathwaysforhigheralcohols,theisoprenoidpathways,andthefattyacidbiosynthesis.
Thecoenzyme-A-dependentfermentativepathwaysAmongthehigheralcohols,n-butanolandisopropanolaretheonlytwothatareoverproducedinnaturebyClostrid-iumspecies.
n-ButanolhasbeenproducedbyClostridiuminacetone–butanol–ethanol(ABE)fermentation.
Thefer-mentativepathway(Fig.
1)inthisorganismstartsfromacetyl-CoA.
Theenzymeacetyl-CoAacetyltransferase,alsoknownasthiolase,condensestwomoleculesofacetyl-CoAtoonemoleculeofacetoacetyl-CoA.
Fromthismolecule,thepathwaybranchesintoisopropanolandn-butanol.
Fortheisopropanolbiosynthesis,anacetoace-tyl-CoAtransferase(ACoAT)transferstheCoAgroupY.
Yan·J.
C.
Liao(&)DepartmentofChemicalandBiomolecularEngineering,UniversityofCaliforniaatLosAngeles,5531BoelterHall,420WestwoodPlaza,LosAngeles,CA90095,USAe-mail:Liaoj@ucla.
edu472JIndMicrobiolBiotechnol(2009)36:471–479123awayfromacetoacetyl-CoAtoacetateorbutyrate,form-ingacetoacetate.
Theacetyl-CoAisrecycledbacktoace-tatebythecombinedphosphotransacetylaseandacetatekinasereaction.
Further,acetoacetateisdecarboxylatedtoacetonebyanacetoacetatedecarboxylase(ADC).
ThenacetoneisreducedtoisopropanolbyaNADPH-depen-dentsecondaryalcoholdehydrogenase(SADH)[12].
Forn-butanolbiosynthesis,acetoacetatehastogothroughfourstepsofNADH-dependentreductionandonestepofdehydration.
AcetoacetateisWrstreducedto3-hydroxybutyryl-CoAby3-hydroxybutyryl-CoAdehydro-genase(HBD).
Then,3-hydroxybutyryl-CoAisdehydratedtocrotonyl-CoAbyacrotonase(CRT).
Third,abutyryl-CoAdehydrogenase(BCD)catalyzesthereductionofcrotonyl-CoAtobutyryl-CoA.
Finally,analdehyde/alcoholdehydrogenase(AADH)convertsbutyryl-CoAton-butanolthroughtwoconsecutivereductionreactions.
IsopropanolproductioninEscherichiacoliThesecondaryalcohol,isopropanol,isbothadesirablefuelandanimportantchemicalfeedstockinthepetrochemicalindustry.
Itsdehydratedproduct,propylene,servesasthemonomerformakingpolypropylene.
Inaddition,itcanbeusedasanadditivetopetroleum-basedfuels.
Replacingmeth-anolwithisopropanolintheesteriWcationprocessoffatandoilcouldgeneratecrystallization-resistantbiodiesels[12].
Asdescribedabove,isopropanolisproducedbyClos-tridiumspeciesinnature.
However,asanativemetabolite,itcanonlybeproducedinalimitedamountforthehosts'ownbeneWtsasadetoxiWcationresponsetolowpHcondi-tions.
Themaximumtiterreportedinitsnativeproducer,Clostridium,was1.
8g/l[9].
Toimprovetheproductionofisopropanol,thefullycharacterizedisopropanolbiosyn-theticpathway(Fig.
1)wasreconstructedinthegenetictractablehostE.
coli[12].
Escherichiacolihasbeenreportedtoproduceacetone[6],theimmediateprecursorofisopropanol,byexpressingtheintactpathwayfromClostridiumacetobutylicumATCC824consistingoftheacetyl-CoAacyltransferase,ACoAT,ADCencodedbythethl,ctfAB,andadcgenes,respectively.
Thereportedtiterwasaround5.
4g/l,similartotheyieldofnativehostforacetone.
Furthermore,withaSADHco-expressedwiththeacetonepathwayinE.
coli,theisopropanolproductionwasachieved[12].
ThepathwayeYciencywastunedbyusinggenesfromdiVerentorgan-isms,abio-prospectingapproach.
SincethegenesfromClostridiumusuallyhavealowGCcontent,whichmayleadtopoorexpression,theE.
colinativegenesatoBandatoAD,encodingacetyl-CoAacyltransferaseandACoAT,werealsotestedaspathwaycomponents.
Additionally,twogenesfromC.
beijerinckiiNRRLB593andThermoanae-robacterbrockiiHTD4,encodingSADHs,weretotallysynthesizedwithcodonoptimizationandinstalledintothepathwaytotestforproduction.
WiththeseeVorts,thestrainwithacombinationofC.
acetobutylicumthl,E.
coliatoAD,C.
acetobutylicumadc,andC.
beijerinckiiadhachievedthehighesttiter(5.
0g/l).
Theresultispromising,sinceitdemonstrates43.
5%(mol/mol)conversionratio.
Thetheo-reticalyieldis1molisopropanolpermoleglucose.
Theproductionofisopropanolfromglucoseisnotredox-balanced.
FourmolesofNADHisproduced,whileFig.
1Metabolicpathwaysforisopropanoland1-butanolproductioninengineeredE.
coli.
Thedashedlineindicatesomittedsteps.
Isopropanolpathwayconsistsoffourenzymaticstepsfromacetyl-CoA.
1-Butanolpathwayconsistsofsixenzymaticsteps.
aceEFandlpdencodepyruvatedehydrogenase;atoB/thlencodesacetyl-CoAacetyltransferase;ctfAB/atoADencodesacetoacetyl-CoAtransferase;adc,acetoacetatedecarboxylase;sadhencodessecondaryalcoholdehydrogenase;hbdencodes3-hydroxybutyryl-CoAdehydrogenase;crtencodescrotonase;bcdencodesbutyryl-CoAdehydrogenase;etfencodeselectrontransferXavoprotein;adhE2encodesaldehyde/alcoholdehydrogenaseGlucose2Acetyl-CoAAcetoacetyl-CoAAcetoacetateAcetoneIsopropanol3-Hydroxybutyryl-CoACrotonyl-CoAButyryl-CoAButyraldehyden-Butanol2NAD+2NADH2Pyruvate2NAD+2NADH2CO2CoAAcetateAcetyl-CoACO2NADPHNADP+aceEFlpdatoB/thlctfAB/atoADadcsadhNADHNAD+hbdcrtH2ObcdetfNADHNADHNADHNAD+NAD+NAD+adhE2adhE2JIndMicrobiolBiotechnol(2009)36:471–4794731231molofNADPHisconsumedpermoleofisopropanol.
Therefore,anexternalelectronacceptorisrequiredorabyproductisservedasanelectronacceptor.
n-ButanolproductioninE.
colin-Butanolwasproposedtobeoneofthebettersubstitutesforgasoline-basedtransportationfuel,becauseofitshighenergydensityandhydrophobicity.
Itsenergycontent(27MJ/l)issimilartothatofgasoline(32MJ/l).
ThehighhydrophobicityenablesitstransportationandstorageusingexistingpetrochemicalinfrastructurewithminimalmodiW-cation.
Inaddition,n-butanolhasalowvaporpressureof4mmHgat20°C,whichallowsitsmixingwithgasolineatanyratiowithoutexceedingairqualityspeciWcations.
Themicrobialproductionofn-butanolhasahistoryofover100years.
Traditionally,n-butanolisproducedbyClostridiumspeciesthroughtheABEfermentation.
How-ever,n-butanolproductionviathisprocedureisdiYculttocontrolandoptimize,particularlybecauseClostridiumexhibitscomplexphysiologicalfeatures,suchasoxygensensitivity,slowgrowthrate,andspore-forminglifecycles.
Thus,itisdesirabletocreatenewn-butanolproducingorganismsusingmetabolicengineeringtechniques.
Recently,n-butanolproductioninaheterologoushost,E.
coli,usingthetraditionalCoA-dependentpathwayorigi-natedfromC.
acetobutylicum(Fig.
1)wasreportedfortheWrsttime[2].
Atsumietal.
createdtwosyntheticoperonscarryingalltheessentialgenes(thl,hbd,crt,bcd,etfAB,andadhE2)involvedinthepathway.
Co-expressionofthetwooperonsinE.
coliledtotheinitialproductionofn-butanolat14mg/lanaerobicallyusingglucoseassolecar-bonsource.
Tooptimizethepathway,alternativeenzymesofdiVerentoriginswereevaluated.
MorespeciWcally,withE.
coliatoBgeneinplaceofC.
acetobutylicumthl,amorethanthreefoldincreaseofn-butanolproductionwasobserved.
However,replacingtheoriginalenzymesforconversionfromcrotonyl-CoAtobutyryl-CoAwithhomologuesandisoenzymefromMegasphaeraelsdeniiorStreptomycescoelicolorresultedinamuchloweryieldofn-butanolinE.
coli.
Nevertheless,thisresultdoesnotexcludethepossibilityoftheexistenceofothergenesthatmightimproven-butanolproductioninE.
coli.
Furthermore,n-butanolproductiondoesnotsimplyrelyontheenzymeactivities.
TheproductformationalsoneedssuYcientcarbonprecursor,acetyl-CoA,andreducingpower,NADH.
Tofurtherimprovedn-butanolproduction,thehostE.
colistrainwasengineeredbydeletingthenativepathwaycompetingforbothcarbonXuxandreducingpower.
Thebeststraincandidate,namedJCL88,withthedeletionofldhA,adhE,frdBC,pta,andfnr,allowedamorethantwofoldincreaseinn-butanolproduction,accompa-niedbythedramaticdropintheformationoflactate,acetate,ethanol,andsuccinate.
Thehighesttiterof552mg/lwasreportedwithoptimizedpathwayandimprovedstrain.
Althoughtheyieldwasstilllow,thisworkdemonstratedthefeasibilityofheterologousn-butanolproductionandproposedtheprinciplesforfurtheroptimization.
TheketoacidpathwaysImportinganon-nativepathwayinaheterologoushostsuchasE.
coliunavoidablyintroducesnon-nativemetabolitesandpotentialtoxicity,inadditiontodiYcultiesinexpress-ingheterologousenzymes.
Theresultingmetabolicimbal-anceandcytotoxicityposeabarrierforlargequantityproduction.
Inthiscontext,itisdesirabletoseekforthepathwayscompatibletothehost.
Aminoacidbiosynthesisgeneratesmanyketoacidintermediates.
Theseketoacidscanbeconvertedtoalcoholsbyintroducingsequentialdecarboxylationandreductioncatalyzedbybroad-sub-strate-rangeketoaciddecarboxylase(KDC)andalcoholdehydrogenase(ADH)(Fig.
2).
Forexample,theisoleucinebiosynthesispathwaygenerates2-ketobutyrateand2-keto-3-methyl-valerate(KMV),whichcanbeconvertedton-propanoland2-methyl-1-butanol(2MB),respectively.
Thevalinebiosynthesispathwayproduces2-ketoisovalerate(KIV),whichistheprecursorforisobutanol.
Theleucinebiosynthesispathwaygenerates2-keto-4-methyl-pentano-ate,whichisthesubstratefor3-methyl-1-butanol(3MB).
Thephenylalaninebiosynthesispathwayproducesphenyl-pyruvate,whichcanleadto2-phenylethanol.
Thenorvalinebiosynthesispathway,whichisnormallyatoxicside-reactionoftheleucinebiosynthesis,producesasubstrateforn-butanol,2-ketovalerate(KV)[3].
Thesepathwaysrecentlyhavebeenexploredforproductionofthecorre-spondingalcoholsinE.
coliwithencouragingresults.
IsobutanolproductioninE.
coliIsobutanolisanisomerofbutanol.
Ithassimilarphysico-chemicalpropertieston-butanol,whilehavingahigheroctanenumberthann-butanol.
Isobutanolhasbeenidenti-Wedasaminorfermentationproduct,butitshighlevelpro-ductionhasnotbeenreporteduntilrecently[3].
Toachieveisobutanolproductioninalargequantity,thenativeilvIHCDoperonfromE.
coliwasWrstoverexpressedtodivertthecarbonXuxfrompyruvatetoKIV(Fig.
3),whichledtoisobutanolproductionat1.
7g/l,aboutaWvefoldincreaseoverthestrainwithoutilvIHCDoverexpression.
Topreventcarbonleakageandreducepowerwaste,thepreviouslygeneratedknockoutstrainJCL88(adhE,ldhA,frdAB,fnr,pta)wasusedashost;aslightincreaseinisobutanolproduction(2.
2g/l)wasobserved.
Further,alsSfromBacillussubtiliswasusedtoreplace474JIndMicrobiolBiotechnol(2009)36:471–479123E.
coliilvIHforitshighaYnitytowardspyruvate,whichledtotheisobutanolproductionat3.
7g/l.
Inaddition,pXBwasdeletedinstrainJCL88toconservethepyruvateavail-abilityforKIVformation.
WiththecombinationoftheseoverexpressionsandgenomicmodiWcations,theengineeredstrainwasabletoproduceisobutanolatatiterof20g/land86%oftheoreticalyield(Fig.
4)[3].
NotethatisobutanolistoxictoE.
coliataconcentration>10g/l.
Howevertheproductionofisobutanoloccursmainlyinthenon-growingphase(Fig.
4)[3].
Thisresultindicatesthateventhoughthecellscannotgrowatthehigherconcentration,theynonethelesscontinuetoproduceandexcreteisobutanol.
Thus,eventhoughisobutanoltoxic-ityposesachallenge,theproductionlevelcanexceedthetoxicitylevelsigniWcantly.
Mutantswithhigherisobutanoltolerancehavebeenisolated[3],whichalsoimprovestheproductivity.
Suchahigh-yieldproductiondemonstratestheversatilityinexploringtheketoacidpathwaysforbio-fuelproduction.
Theproductionofisobutanol(3.
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