Bioethanol a Microbial Biofuel Metabolite

27 Pages·2017·2.4 MB·English

fermentation
Review
Bioethanol a Microbial Biofuel Metabolite;
New Insights of Yeasts Metabolic Engineering
KhaledA.Selim1,*,DinaE.El-Ghwas1,2,SaadiaM.Easa3and
MohamedI.AbdelwahabHassan4,5,6
1 PharmaceuticalandDrugIndustriesResearchDivision,NationalResearchCentre,33-El-BohouthSt.(former
ElTahrirSt.),Dokki,P.O.Box12622Giza,Egypt;[email protected]
2 BiologyDepartment,FacultyofScience,UniversityofJeddah,Jeddah21577,SaudiArabia
3 MicrobiologyDepartment,FacultyofScience,AinShamsUniversity,11566Cairo,Egypt;
[email protected]
4 JenaMicrobialResourceCollection,LeibnizInstituteforNaturalProductResearchandInfection
Biology—HansKnöllInstitute,07745Jena,Germany;[email protected]
5 DepartmentMicrobiologyandMolecularBiology,InstituteofMicrobiology,FacultyofBiologicalScience,
Friedrich,SchillerUniversityJena,07743Jena,Germany
6 NationalResearchCentre,33-El-BohouthSt.(formerElTahrirSt.),Dokki,P.O.Box12622Giza,Egypt
* Correspondence:[email protected]
Received:7January2018;Accepted:23February2018;Published:8March2018
Abstract: Scarcityofthenon-renewableenergysources,globalwarming,environmentalpollution,
and raising the cost of petroleum are the motive for the development of renewable, eco-friendly
fuelsproductionwithlowcosts. Bioethanolproductionisoneofthepromisingmaterialsthatcan
subrogatethepetroleumoil,anditisconsideredrecentlyasacleanliquidfueloraneutralcarbon.
Diversemicroorganismssuchasyeastsandbacteriaareabletoproducebioethanolonalargescale,
whichcansatisfyourdailyneedswithcheapandapplicablemethods. Saccharomycescerevisiaeand
Pichiastipitisaretwoofthepioneeryeastsinethanolproductionduetotheirabilitiestoproducea
highamountofethanol. Therecentfocusisdirectedtowardslignocellulosicbiomassthatcontains
30–50%celluloseand20–40%hemicellulose,andcanbetransformedintoglucoseandfundamentally
xylose after enzymatic hydrolysis. For this purpose, a number of various approaches have been
usedtoengineerdifferentpathwaysforimprovingthebioethanolproductionwithsimultaneous
fermentationofpentoseandhexosessugarsintheyeasts. Theseapproachesincludemetabolicand
fluxanalysis,modelingandexpressionanalysis,followedbytargeteddeletionsortheoverexpression
ofkeygenes. Inthisreview,wehighlightanddiscussthecurrentstatusofyeastsgeneticengineering
forenhancingbioethanolproduction,andtheconditionsthatinfluencebioethanolproduction.
Keywords: bioethanol; fermentation; metabolic engineering; biofuel; Saccharomyces cerevisiae;
Pichiastipitis
1. Introduction
Theexcessiveusageoffossilfuelstosatisfytherapidincreaseofenergydemandhascreated
severeenvironmentalproblems, suchasairpollution, acidrain, andglobalwarming[1]. Allover
the world, Humans are constantly based on biomass-derived energy and carbon for survival and
nutrition. Also, in late history for the commodity fuels and chemicals we became dependent on
petroleum-derivedenergyandcarbon. Inthenon-renewablepetroleum-basedcarbon, thecarbon
issittinginbiomass,wherethebiomassisatemporarystoreunitforthesunlightandatmospheric
carbon derived energy. In the last century, there are rising requests to implement and to improve
strategiesforproductionofcommodityfuelsandchemicalsfrombiomasscomparedtopetroleum.
Fermentation 2018,4,16;doi:10.3390/fermentation4010016 www.mdpi.com/journal/fermentation
Fermentation 2018,4,16 2of27
Tocompetewiththeexistingprocessofpetroleumproduction,thefermentationprocessshouldbemore
efficient,orcomparableformaximumproductionofthetargetedchemical. Therefore,theproduction
of petrochemical and biofuels by means of microbial fermentation of agriculture wastes could be
ouralternativeway. Yeastshaveadvantagesoverbacteriaforcommercialfermentationduetothe
thicknessoftheircellwalls,lessstringentnutritionalrequirements,largesizes,utmostresistanceto
contamination,andbettergrowthatacidicpH.SaccharomycescerevisiaeandPichiastipitisareoneofthe
mostpromisingmodelorganismsinmicrobiologicalfermentationduetotheirabilitiestoproducea
highamountofethanol.Theyareabletoutilizediversesubstratesthatarefullofcarbohydratesthrough
fermentationprocesses. Besides,theyareresistanttohighethanolconcentrations. Therecentfocusis
directedtowardslignocellulosicbiomass,whichcontains30–50%celluloseand20–40%hemicellulose,
andcanbetransformedintoglucoseandfundamentallyxyloseafterenzymatichydrolysis. Innature,
thesecondmostabundantcarbohydrateisxylose,anditcouldsupplyanalternativefuelsourcefor
itsabilitytobecommerciallyfermentedintoethanol. S.cerevisiaecountsasthemainandthemost
importantsourceofbioethanolproductionattheindustriallevel. However,severalattemptswere
implementedtoengineerS.cerevisiaetotransportandfermentxylosebecausenaturallyitdoesnot
use xylose. Moreover, genetic engineering can upgrade the fermentative activities of some native
xylose-metabolizingyeastlikeP.stipitis. Inthisreview,wepointoutthecurrentstatusofyeastsgenetic
engineeringapproachesforenhancingbioethanolproduction,anddiscussingthefactorsthataffect
positively/negativelyonthefermentationprocess.
Theprocessesofbioethanolproductionfromtheutilizationofstarch,sugarcane,andmicrobial
fermentationofsugarcompoundsconsidersasthemainsourcetodispensewithclassicalfuelwhich
causesnoticeablepollutionforourplanet. Researcherswhoworkinthefieldoffermentationgive
moreattentionforlignocellulose,asitisanalternativeandpromisingwayforbioethanolproduction
in our future research [2]. It is known as feedstock that is more plentiful and substantial issue in
ourenvironmentanddoesnotcompetewiththesourcesoffoodsupplies[3]. Although,thecentral
predicamentforbioethanolproductionfromlignocelluloseconversioniseconomicstatuswherethe
productioncostshouldbereasonable. Thestatusthatreducestheperformanceefficiencyofyeastis
thatithasmanydrawbacksasincreasingtheconcentrationofharmfulprocessconditionsandtoxic
chemicalswithlowtolerancecapacity,whichispreventedenergeticallyandeconomically[4].
Bioethanolisthemostcommonsourceofthebiofuel, andabout90%oftotalbiofuelusageis
basedonbioethanolproduction. Theprocessofconventionalbioethanolproductioniswellknownto
bedependentontheenzymatictransformationofstarchybiomassintosugarsand/orfermentation
of 6-carbon sugars with final distillation of ethanol to fuel grade. Ethanol can be produced from
variousfeedstockssuchascorn(maize),sugarbeets,othercerealcrops,sugarcane,cassava,sorghum,
and potatoes. Animal feed (co-products) helps in decreasing the production cost as well. Also,
bagasse (crushed stalk) is used to supply power and heat for energy applications. Additionally,
the biggest bulk of agriculture biomass is composed of lignin, cellulose, and hemicelluloses,
whichcouldextendoursustainableandrenewableresourcesforusageinethanolproduction[5,6].
According to the two largest ethanol producers USA and Brazil, the major sources of ethanol
productionaremaizestarchandsucrosefromsugarcane[5,7]. Thefermentablesugarslikemannose,
glucose, hexoses, arabinose and xylose can result from hemicelluloses and cellulose fractions of
agro-industrialresidueseitherbychemicalhydrolysesorenzymaticones[8]. Currently,researchers
in the field of fermentation exert more effort and focus on enhancing the production of ethanol
through the bioconversion of lignocellulose for reducing the dependence on the non-renewable
energy. Lignocelluloseisoneofthepromisingandamplesources,whichconsistsoflignin,cellulose,
and hemicellulose [9]. Conversion, the hemicellulose and cellulose to ethanol, occurred through
severalprocessesthatinitiatewithdifferentpre-treatment(mechanical,chemical,orboth).
An effective transformation of all sugars sitting in lignocellulosic hydrolysates to ethanol
is necessary for maximizing the profitability of the industrial process and for reducing the
competitivenesscostofbioethanolproduction[10]. Bio-productionofethanolfromlignocellulosic
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materialhastobeatanacceptablerate, inhighyield, andatconcentrationsthatareeconomically
retrievable. The major impediment of cellulose fermentation is the enzymatic saccharification.
For hemicellulosic, the utilization of multiple sugars of arabinose, rhamnose, xylose, galactose,
mannose,andglucoseinthepresenceofferulicandaceticacids,alongwithdiversedisintegration
productsfromthermalandchemicalpretreatment,isthelimitingfactorofthefermentationprocess.
Phosphorylationofhexosescompoundisoccursoncegettinginthecell;diversebiochemicalstages
shouldbeappliedforhemicellulosiccomponentsbeforephosphorylation[11].
Thepentosesmicrobialbiotransformationhasbeenrecognizedasthemainresearchchallenge
forthesecond-generationethanoltechnologybecauseitrepresentsabout25–40%ofthetotalsugars
transformation[6]. ThemajorlignocellulosicbiomasscomponentisD-Xylose. Forefficienteconomical
lignocellulosesbioconversion,thealcoholicfermentationofthisrenewablecarbonsources(D-Xylose)is
required[12]. ThepentosesugarxylosefromplanthydrolysatescannotbefermentedbySaccharomyces
cerevisiae (wild types strains), which is the communally used for ethanol production. Therefore,
tosolvethisproblem,manyattemptstogenerategeneticallyrecombinantstrainsofS.cerevisiaeand
P.stipitis,toimprovethecapabilityofxylosefermentationintoethanol,havebeenreported. However,
thelarge-scaleindustrialprocessofbioethanolproductionislimitedduetopoorlyunderstanding
of yeasts metabolic regulations [13]. Additionally, our knowledge for the efficient utilization of
lignocelluloseshydrolysateswithsuchyeaststrainsundertheindustrialconditionsisstilllimiting[14].
Theproductionofbiofuelsfromlignocellulosicbiomassremainsanunsolvedchallengeinthe
industrialbiotechnology. Effortstouseyeastforbiofuelsproductionfaceseveralquestions: ofwhich
organismtobeuse,counterbalancingtheeaseofgeneticmanipulationwiththepromiseofrobust
industrialphenotypes. Saccharomycescerevisiaeremainsthepremierhostformetabolicengineeringof
biofuelpathways,duetotheavailabilityofmanysystemsandsyntheticbiologytoolstoforgenetic
manipulation[15]. Whencomparedtoothertypesofmicrooganisms,Saccharomycescerevisiaeisthe
commonmicrobesemployedinethanolproductionduetoitshighethanolproductivity,highethanol
toleranceandabilityoffermentingwiderangeofsugars. However,therearestillsomechallengesin
yeastfermentationthatinhibitethanolproduction,suchasthehightemperature,thehighethanol
concentration,andtheabilitytofermentpentosesugars[16].
Yeasts such as Pachysolen tannophilus, P. stipitis, Candida tropicalis, and Candida shehatae have
beendetectedtofermentoneofthemostabundantpentosessugars(xylose)toethanol. However,
thewild-typestrainsoftheseyeastsareunabletofermentsimultaneouslythehexosesandpentoses
sugars. Therefore,thecreationofgeneticallymodifiedstrainshasbeennotifiedasspecialfermentation
strategiestointroducenewpathwaystostimulatetheabilityforsimulationstransformationofpentoses
and hexose sugars [17]. Co-culture of Zymomonas mobilis bacterium and yeasts was also studied
to prove the potential synergistic utilization of different microorganism metabolic pathway [18].
Forexamples,co-cultureofP.stipitisandS.cerevisiaeimmobilizedwithZ.mobilishasbeenstudied.
Also,co-cultivationofP.tannophilusandZ.mobilishasbeeninvestigatedusingsyntheticmediawitha
differentcombinationofxyloseandglucose[10,19,20]. Thewild-typestrainofZ.mobiliswasisolated
originallyfromalcoholicliquidsinnaturalenvironmentscontainingfermentablesugars,andcanonly
utilizealimitedcarbonsource,includingglucose,fructose,andsucrose[21].
Theformationofcellinhibitorysubstanceslikephenoliccompounds,furanderivativesandweak
acids is another important factor implicated in the production of bioethanol (second-generation),
theformationofthosesubstancesalwaysoccursduringthechemicalhydrolysesoflignocellulosic
materials [22,23]. High level of the toxic byproduct compounds that form during the production
oflignocellulosichydrolysate, ischallengingaswell. Thereleasedaceticacidandfurfuralduring
sugarcanehydrolysiscanreachupto5g/L[24], whileaftertheprocessingofchippedpinewood
hydrolysate,hydroxymethylfurfural(HMF)canbereachedup6g/L[25]. Theoverallcellphysiology
isaffectedbysuchinhibitorsandleadstodecreaseinthestrainsproductivity,ethanolyieldsandcell
viability[26]. Forexample,furanathighconcentrationscausesaninhibitoryeffectonS.cerevisiae,
Fermentation 2018,4,16 4of27
interfering with macromolecules syntheses and glycolytic enzymes. Whereas, in the process of
lignocellulosichydrolysis,theaceticacidreleased,hascytotoxiceffectsatelevatedconcentrations[27].
Theinterestinstudyingcatabolicpathwayofxyloseutilizationhasbeengrown[28–30],withthe
discovery of pentose-fermenting yeast strains [31,32]. The bio-renewable chemicals and fuels
fermentativeproductionneedthebiocatalystsengineeringthatcanquicklyandefficientlytransform
sugars to the target products with lower cost than the existing petrochemical-based processes.
Forinstance,foreightknownreactionsrequiredforconversionofxylosetoethanolbySaccharomyces
cerevisiae,LatimerandDueberin(2017)screenedseveralkeyenzymesforfunctionalexpressionin
S.cerevisiae,followedbyacombinatorialexpressionanalysistoachieveapathwayfluxbalancingand
toidentifytheratelimitingenzymaticsteps[33].
Scientistshavechangedthebiocatalystsprocessesslowlybychoosingdesirabletraitswithout
understanding the biological mechanisms. However, after the evaluation of recombinant DNA
technology and the elucidation of the biological code, we became able to do more than choosing
the naturally or evolved strains under the selective evolutionary pressures. Nowadays, scientists
coulddesignandevaluateproteins,metabolicpathways,andevenengineerwholedesirablestrains.
Therefore,themodernthinkingisdirectedintheformofmetabolicengineering. Bailey(1991)and
StephanopoulosandVallino(1991and1998)definedthemetabolicengineeringasthedirectadvance
technologyforformationandproductionofdesirablecellularpropertiesthroughthealterationof
biochemicalpathwaysorthroughusingtherecombinantDNAtechnologyfortheintroductionofnew
traits[34–36].
Fortheproductionofatargetcompound,thesyntheticbiologycanbeexpressedasasequence
of the following events: (1) designing the phenotypic properties and the metabolic pathways of
the wanted system. What are the required products and substrates? What are the predictable
environmentalstressors? (2)Selectingthesuitablehostorganismsdependingonthefollowingcriteria.
Which organisms display some of the wanted properties? The characters of these organisms and
the availability of molecular biology tools for the modulation of this chassis. (3) Formulating an
implementationapproach. Whatmodulationsareneededtoobtainthepropertiesandpathwayinthe
firststep? Doesthetargetedmetabolicpathwayrequiretoberemoved,addedortuned? Doesthe
requiredphenotypeorpathwayexistinnatureordoesitneedtobeprepareddenovo? (4)Optimizing
oftheredesignedsystemandassessingthepropertiesofthesystemrelativetotheidealprocesses.
Also,asimplebiocatalystlikeEscherichiacoli,whichisthelaboratoryworkhorse,isacomplex
system of an estimated 2077 reactions, 4603 genes, and 1039 unique metabolites [37,38]. However,
thestepsoutlinedabovearerelativelysimple,itishardtorapidlyengineerabiocatalysttoperform
thedesirablebehaviours[39]. Themetabolicevolution,thestandardizationofbiologicalsystemsand
thesystemsbiologyareallimportanttocompensateforthedisconnectingbetweenactualbiocatalyst
behavioursandtheexpectedone. Throughthecombinationofallofthesepowerfultechniques,the
biocatalystsredesignedfortheproductionofchemicalsandcommodityfuelscanbeachieved(Figure1).
Forheterologousgeneexpression,yeastsbecomeimportanthosts[40]. Itisnecessarytocontrol
geneexpressionquantitativelyforoptimalmetabolicengineering. WilliamsandBowles(2004)found
theco-expressionofneighboringgenesinArabidopsis[41]. Thistypeofexpressionhasbeenobserved
inmanyeukaryoticgenomes[42–44]. Thesameorientationofthegeneshasbeenproposedasoneof
themechanisticexplanationforco-expression[41,45]. Forinstance,theeffectsoftandemorientationor
divergentgeneorientationcanexistinco-expressedgenesinplants[46,47]. PrescottandProudfoot
(2002)recognizedthetranscriptionalrepressionofconvergentlyarrangedgenesinyeast,whichwas
explainedbythetranscriptionalcollision[48]. Thesefindingssuggestedthatintergenicregionsamong
adjacentgenes,whichhavesignificantrolesintranscription. Scratchingofregionsbetweenthetwo
ORFscausedreductionofyeasttranscript[49,50]. TheintergenicregioninS.cerevisiaewasfoundto
havearegulatoryfunction[51,52],andaccordingtotheorientationofneighboringORFs,theyhave
differentbasecompositions[53].
Fermentation 2018,4,16 5of27
Fermentation 2017, 3, x 5 of 26

Figure 1. Overview of tools for the metabolic redesign.
Figure1.Overviewoftoolsforthemetabolicredesign.
Technical, economic, and political considerations are the driving force for improving the
Tfeercmhennitcaatlio,ne coof nxoymloisce, taon edthapnoolli.t iFcaolr cinosntasnidcee,r athtieo nresneawreabtlhe efuderl ivprinodgucfotirocne ufsoinrgi mbipolroogviicnalg the
fermebniotamtiaosns aosf xay fleoesdesttoocekt hcaann olle.aFdo troi nas dtaencrceea,steh einr etnhee wacacbulmefuulaetliopnro odfu gcrteioennhuosuisneg gbaisosleosg [i5c4a–l5b7i]o. mass
asafTeheedrsetfoorcek, cthaen Eleuardopteoana dUenciorena sreecionmtmheenadcecdu mthuatl abtiioofnueolsf gshreoeunldh oauppseeagr a2s%se sby[5 420–0557 ]o.fT thoetarle fore,
transportation fuel consumption and 5.75% by 2010 [58]. Moreover, the agricultural policies of the
theEuropeanUnionrecommendedthatbiofuelsshouldappear2%by2005oftotaltransportation
European Union, Brazil, and United States are directed towards the ethanol industries for the
fuelconsumptionand5.75%by2010[58]. Moreover,theagriculturalpoliciesoftheEuropeanUnion,
commercial production of ethanol from sugar cane, grains, and other feedstock. The chance for the
Brazil,andUnitedStatesaredirectedtowardstheethanolindustriesforthecommercialproductionof
processing residues and the bioconversion of harvested agriculture materials are increasing along
ethanolfromsugarcane,grains,andotherfeedstock. Thechancefortheprocessingresiduesandthe
with these markets. For instance, the usage of sugarcane bagasse, corn cobs, grain hull, and corn
biococnovbesr sbiyopnroodfuhcatsr vteos tiendcraeagsreic tuhletu prreomduactteiroina losfa reethianncorle afrsoinmg tahleo nwgawsteit hsttrheaemses mbeacrokmetess. Ffeoarsiibnlset ance,
the u[s5a9g,6e0]o. fWshuiglea rthcae nmeabjoarg caosmsep,ocnoernnt ocof bthse, grernaeinwahbulell ,baionmdascso rfenedcsotbosckb iysp trhoed xuycltosset,o thinucs,r etahsee the
produteccthionnicaolf beatrhraienro flofrr coommmtheercwiaal sbtieosctornevaemrssiobne coof mfeeedssfteoacski bisl eth[e5 9a,c6ti0v]e. Wutihliizlaetitohne omf axjyolrosceo [m61p,6o2n]e. ntof
thereHnaelwf oafb lteheb iwoomrdas asgfreiecudlsttuorcek pihsytthoemxayslso csoen,ttahiunisn,gth leigtneocchenlliuclaolsbica rcrroiepr rfeosridcoumes m[6e3r]c, iaanldb iwocitohnovuet rsion
offeecdosmtopcektiinsgt hweiathct iovtehuert illiizgantoicoenlloufloxsyicl-obsaes[e6d1 ,i6n2d]u.sHtraielfs,o fcothnesiwdeorradblaeg rfricauctlitounrse pcahny tboem raescsocvoenretdai ning
[56,64]. In woody angiosperms, xylose constitutes is about 17% of the total dry weight, and in
lignocellulosiccropresidues[63],andwithoutcompetingwithotherlignocellulosic-basedindustries,
herbaceous angiosperms, it can be up to 31% [65,66]. The sulfite-pulping of hardwood is the source
considerablefractionscanberecovered[56,64]. Inwoodyangiosperms,xyloseconstitutesisabout17%
of xylose [67]. The pretreatment of lignocellulosic residues can recover 80–95% of the xylose,
ofthetotaldryweight,andinherbaceousangiosperms,itcanbeupto31%[65,66]. Thesulfite-pulping
depending on the reaction conditions and the substrate [24,68,69].
of hardwood is the source of xylose [67]. The pretreatment of lignocellulosic residues can recover
Native S. cerevisiae strains do not use xylose as carbon sources. Also, the yeast Candida utilis is
80–95s%tricotflyt haeerxoybilco saen,dd ceapne gnrdoiwn gono nxytlhoeser, ebauctt iito ins cnoont adnit eiothnasnaonl dprtohdeusciunbgs sttrraatien.[ 2E4a,r6ly8 ,i6n9 1].980, after
NidaentitvifeicaSt.iocner tehvei saiabeilisttyr aoifn Sschdizoosnacocthaursoemyxcyelso psoemabes, cSa. rcebroevnissiaoeu arncde so.thAerl syoe,atsht estryaeinass ttoC faenrmdiednat utilis
isstrDic-tXlyylaoesreo tbo iecthaanndolc [a7n0,7g1r]o, wa lootn ofx syclroeseen,inbgu etffiotrits rnapoitdalyn deetthecatnedo lthparto sdoumcei nstgrasitnrsa cinou.lEd acrolnyvienrt 1980,
afterxiydleonset ifitoc aettihoannothl eimambielditiyateolfy Sucnhdizeors aocxcyhgaerno-mliymcietsinpgo morb ea,erSo.bcice recvoinsdiaiteioannsd [3o2t,h72e]r. yNeoawstadsatryasi,n s to
fermePnicthDia- Xstyipliotisse, Cto. eshthehaantaoel, [a7n0d,7 P1]a,chaylsootleonf tsacnrneoepnhiilnugs eaftftroarctt raa pliodt loyf daettteencttieodn thasa tthseo mbeests tkranionwsnc ould
natural xylose-fermenting yeasts [73–75]. Although the technical and economical biotransformation
convertxylosetoethanolimmediatelyunderoxygen-limitingoraerobicconditions[32,72]. Nowadays,
of pentoses sugars to ethanol are still challenging [12,76–79], many achievements in genetic
Pichia stipitis, C. shehatae, and Pachysolen tannophilus attract a lot of attention as the best known
engineering have been done to ferment arabinose and xylose to lactic acid and ethanol.
naturalxylose-fermentingyeasts[73–75]. Althoughthetechnicalandeconomicalbiotransformationof
pento sessugarstoethanolarestillchallenging[12,76–79],manyachievementsingeneticengineering
havebeendonetofermentarabinoseandxylosetolacticacidandethanol.
Fermentation 2018,4,16 6of27
The development of yeast strain mainly focuses on the genetic engineering of S. cerevisiae.
HoweFveremre,nftaotironx 2y0l1o7s, 3e, xfe rmentation,P.stipitishasalsobeenmodified. Metabolicengineeringca6n ofc 2h6 ange
the pentose phosphate pathway, sugar transport, assimilation, the terminal steps of fermentation,
The development of yeast strain mainly focuses on the genetic engineering of S. cerevisiae.
glycolysisandtheinterlayer betweenfermentationandrespirationthat influencetheintracellular
However, for xylose fermentation, P. stipitis has also been modified. Metabolic engineering can
redoxbalance. Whereas,fewalternationscouldmakexyloseutilizationinS.cerevisiaepossible,where;
change the pentose phosphate pathway, sugar transport, assimilation, the terminal steps of
inS.cerevisiae,thegenesforxyloseassimilationexist,buttheyarenotexpressedataconsiderable
fermentation, glycolysis and the interlayer between fermentation and respiration that influence the
levelthatcanbesufficientforxylosefermentation. EngineeringofP.stipitishasbeenmorerestricted;
intracellular redox balance. Whereas, few alternations could make xylose utilization in S. cerevisiae
however,someprincipleshavebeenidentifiedinthisyeast.
possible, where; in S. cerevisiae, the genes for xylose assimilation exist, but they are not expressed at a
Pc.onstsiipdietriasbhlea slevaels ethtaot fcapnh byes isoulfofigciiecnatl fcohr axryalcotseer fsertmhaenttmatiaokne. Eintgvinereyerihnegl poff uPl. sftoipritliisg hnaosc beelleunl oses
biocomnvoerer srieosntr.icIttedh;a hsoawhevigerh, scoampea cpirtiyncfioprlexsy hlaovsee bfeeernm iednentatitfiioend .inA tlhsios ,yietaisst. a bletofermentcellobiose,
glucose,mPa. nstnipoisties, hanasd ag aselat cotof spehaylsoionlgogwiciatlh cxhyalraanctoerlsig tohmate mrsaakned itm vaenryn ahne.lpTfhule rfeofro lrieg,niotciesllcuolnosseids ered
as a bpiootceonntveorsrigoann. iIst mhasf oar hsigimh ucalptaancietoy ufosr oxrylohsyed freormlyesnattaetiofenr. mAelsnot, aitt iiosn abalne dto sfaercmcheanrt icfiecllaotbioionse(, SSF).
glucose, mannose, and galactose along with xylan oligomers and mannan. Therefore, it is considered
Afterglucose,thesecondmostabundanthemicellulosiccomponentisxyloseinfast-growinghardwood
as a potent organism for simultaneous or hydrolysate fermentation and saccharification (SSF). After
speciesandagricultureresidues.Ontheotherhand,theprimarysugarformedinenzymatichydrolysis
glucose, the second most abundant hemicellulosic component is xylose in fast-growing hardwood
iscellobiose[78].
species and agriculture residues. On the other hand, the primary sugar formed in enzymatic
Hemicellulosesugarsareeasilyrecoveredafterthetreatmentofagriculturewasteswithdilute
hydrolysis is cellobiose [78].
acid,alkaliorautohydrolysis,whichiscrucialfortheeconomicalbioconversion.TheabilityofP.stipitis
Hemicellulose sugars are easily recovered after the treatment of agriculture wastes with dilute
to useaccide,l laolbkiaolis eori saustioghnyifidrcoalnytsiisn, wShSiFchb eisc acruusceiatlh feorc othme meceorncoimalicparle bpiaorcaotnivoenrssioonf. cTehlleu laabsileitay roef aPlw. ays
incomstpipleitties tion uβs-eg lcueclloosbiidosaes eist rseigantmifiecanntst iann SdStFh ebeacgagusree gtahtei ocnomomfceerlclioabl iporseepainrahtiiobnitss ocfe lcleulllualsaesea catriev ities.
Theaabliwliatyyso ifnPc.osmtipplietties tion uβ-sgeluocliogsoidmaseer itcresautmgaernstsi sanndec tehses aargygrbeegcaatiuosne otfh ceemlloibldioasec iidnhpirbeittsr ecaetlmluleansets can
bypasascttihveitifeosr. mTahtei oanbioliftysu ogfa rP.d setgipriatdisa ttoio nuspe rooldiguocmtse,rbicu tscuagnarasl siso rneescoelsusatiroyn b1e5c–a5u0se% tohfe tmheilsdo laucbidil ized
pretreatments can bypass the formation of sugar degradation products, but can also resolution
hemicellulosesinanoligomericform[78].
15–50% of the solubilized hemicelluloses in an oligomeric form [78].
Hemicellulosicsugarsareunderutilized,however;theycanbereadilyrecoveredatalowercost
Hemicellulosic sugars are underutilized, however; they can be readily recovered at a lower cost
thanglucosefromcellulosewithhigheryields. Moreover, theycanbetransformedintoanumber
than glucose from cellulose with higher yields. Moreover, they can be transformed into a number of
ofusefulproductslikelacticacid, xylitolandotherchemicals. Ethanolistheproductthathasthe
useful products like lactic acid, xylitol and other chemicals. Ethanol is the product that has the
largest potential market. Ethanol production from lignocellulosic sources will keep increasing as
largest potential market. Ethanol production from lignocellulosic sources will keep increasing as a
a concsoenqsueqeunecneceo forfe rceecnetnta gagrircicuullttuurree aanndd eenneerrggyy ppoolilciiceise stot orerdeudcue ccearcbaornb odniodxiidoex iedmeiessmioinsss ioannds and
alternaaltteivrnearteivnee rwenaebwleabfulee fluse[l7s8 []7.8].
ReseaRrecsheearrcshienrsm ina nmyanlayb loabraotroartoiersiesh ahvavees sccrroouunnggeedd ggeenneess frformom P.P s.tisptiitpisi taisnda nodtheort hfeerrmfeenrmtateivneta tive
micromorigcraonoirsgmasnitsomasl tteor Sal.tceerr eSv. icseiareevfiosirace eflolur lcoeslelu,lxoysleo, sxey,loorsex,y olarn xmylaenta bmoeltiasmbo.liHsmow. Hevoewr,evtoers,o tmo esoemxtee ntof
extent of success, efficient xylose utilization has been achieved by engineered S. cerevisiae strains
success,efficientxyloseutilizationhasbeenachievedbyengineeredS.cerevisiaestrainsmostlyatalowrate
mostly at a low rate of xylose assimilation. The most of biochemical steps required for xylose
ofxyloseassimilation.Themostofbiochemicalstepsrequiredforxyloseutilizationareunderstandable;
utilization are understandable; however, a more thorough knowledge of how local
however,amorethoroughknowledgeofhowlocalxylose-fermentingyeasts,likeP.stipitis,adjusttheir
xylose-fermenting yeasts, like P. stipitis, adjust their metabolism would improve the field on many
metabolism would improve the field on many fronts [78]. In general, the assimilation pathways for
fronts [78]. In general, the assimilation pathways for hexoses and pentoses, mannose, glucose and
hexosesandpentoses,mannose,glucoseandgalactosearesummarizedinFigure2.
galactose are summarized in Figure 2.

FigurFeig2u.rAe s2s.i Amsisliamtiiolantipona tphawthawyasyfso frohr ehxeoxosesessa anndd ppeennttoosseess, ,mmaannnnosoes, eg,lgucluosceo saenda ngdalagcatolascet, ossheo,wsihnogw ing
the interconversion of pentoses and pentitols by NAD (PH)-mediated oxidoreductases [11,80].
theinterconversionofpentosesandpentitolsbyNAD(PH)-mediatedoxidoreductases[11,80].
Fermentation 2018,4,16 7of27
2. NewYeastforLignocellulosesBioconversion
Intellectualmetabolicengineeringrequiresadetailedunderstandingofthegenetic,physiology
and biochemistry of the desired strain to be engineered. It is potentially possible only when the
main mechanisms and pathways are known. Genetic and biochemical characterization of xylose
fermentation by P. stipitis has been underway for at least 22 years since the evaluation of mating
systems[79]anddevelopinggenetictransformationtools[81–83]. Moreover,theresearchersarestill
aimingtoimproveandenhancethexyloseutilizationbyP.stipitis,despitethefactthatthecapability
ofthenativestrainstoproducehighamountsofethanolthananyotherstudiedyeast,includingthe
geneticallyengineeredS.cerevisiae[84,85].
The native P. stipitis strains were originally isolated from insect larvae. Phylogenetically, it is
relatedtotheendosymbiontsgutmicrobiota,whichinhabitsthepassalidbeetlesgut[86]thatdegrades
whiterottedhardwood[87]. Geneticallyandsystematically,P.stipitisispredominantlyhomothallic,
haploidandhemiascomycetousyeast[79,88–90]thatformsbudsalongwithpseudomyceliaduring
vegetativegrowth,andtwohat-shapedascosporesfromeachascus. Fed-batchculturesofP.stipitis
produceupto47g/Lofethanolfromxyloseat30◦C[85]withethanolyieldsof0.35–0.44g/Lxylose[91].
Moreover,theyareabletofermentsugarsfromhemicellulosicacidhydrolysateswithayieldequalto
about80%ofthemaximumtheoreticalconversionefficiency[92,93].
The genomes of P. stipitis strains are encoding for xylanase, mannases, cellulases, and other
degradative enzymes that enable them to grow and survive in the insect-gut environment of
wood-inhabiting[86]. P.stipitishastheabilitytofermentxylan, xylose[94,95], andcellobioseand
touseallthemainsugarsthatfoundinwoodcontainingrhamnoseandarabinose[96]. Therefore,
P.stipitisgenomewasconsideredtobethepopulargenessourceforxylosemetabolismtoengineer
S.cerevisiae[80].
Additionally, P. stipitis has many other bioconversion related traits: forming various esters
and aroma components [97], reducing acyclic enones to the corresponding alcohols [98], modifying
low-molecular-weight lignin moieties [99], and can be engineered to produce xylitol [100] or lactic
acid[101]inhighyields.Also,P.stipitisstrainsareresistanttohydroxyl-methylfurfuralandfurfural[102].
Adaptive evolution and metabolic engineering of S. cerevisiae for fermentation of xylose were
successful to certain degrees [103–105]. Engineering S. cerevisiae with the basic xylose assimilatory
machinerywithexpressingxylosereductases,xyloseisomerase[13,106]andxylitoldehydrogenases[107]
cantheoreticallyenhancetheethanolyield. However,itisunclearuntilnowwhichapproachwould
demonstratemoresuccesstoimprovethefermentationcapabilityofS.cerevisiae[108].
Likewise,theoverexpressionofP.stipitisandotherfungalsugartransporterscanenhancethe
performanceofengineeredS.cerevisiaetogrowanduptakexylose[109–112]. However,additional
regulatory steps are required to engineer an efficient ethanol producer S. cerevisiae strain, because
themechanismforproductionofethanolinresponsetoxyloseinS.cerevisiaeismissing[113]. Thus,
nowadaysthebiochemical,physiological,andgeneticregulationsofP.stipitisandotheryeaststhatuse
theunusualsubstratesnatively,haveattractedalotofattention,forengineeringS.cerevisiaestrains
whichhavecapabilitytofermentrhamnose,cellulose,xylose,arabinose,xylan[96],andothersugars
residue. Conversely,theS.cerevisiaemechanismsforxylosefermentationcanbeusedfordeveloping
theperformanceofP.stipitis.
P.stipitismaneuversmostofitsmetabolicfluxintoethanolproductionandverysmallamount
of xylitol can be formed as well. However, its fermentation rate on xylose is low when compared
tothatofS.cerevisiaeonglucose. Xyloseandglucosearenotequalfermentationsformanyreasons,
butincreasingthecapacityofP.stipitisforrapidxylosefermentationcangreatlydevelopitsusefulness
incommercialapplications. OneoffermentativeregulatorymachineryisthatS.cerevisiaeregulates
fermentationprocessbysensingtheexistenceofglucose,butP.stipitispromotesfermentativeactivity
inresponsetooxygenlimitation[114–117]. Universalexpressionarrayanalysishasshownspecial
responsepatternsforrahamnose,cellobiose,arabinose,xyloseandotherlignocellulosicsubstrates. But,
itisnotfullyunderstooduntilnowwhetherthesepatternswererelatedtospecificinductionorcarbon
Fermentation 2018,4,16 8of27
catabolitedepression. Additionally,termitesareknownasxylophages,becausetheyareabletofeed
onthFeervmaenrtiaotiuons 2s0o17u, r3c, xe s oflignocellulosicbiomassaswood,whicharecharacterizedbymassiv8e ofa 2m6 ount
oflignin. Thiscapabilitycomesfromthesymbioticrelationshipsbetweenthetermitesandtheirgut
induction or carbon catabolite depression. Additionally, termites are known as xylophages, because
microbiomes. Termite-gutmicrobiomescanbeutilizedforenhancingbiomassconversionprocesses
they are able to feed on the various sources of lignocellulosic biomass as wood, which are
invitro [118]. Interestingly, Physisporinus vitreus depends on unprecedented versatile peroxidase
characterized by massive amount of lignin. This capability comes from the symbiotic relationships
(VP)strategythathelpedtocharacterizenoveldelignificationdelineation. Thisstrategyplayedvital
between the termites and their gut microbiomes. Termite-gut microbiomes can be utilized for
roleiennheannhcainngc ibniogmoaxsisd cizoantvieornsiobno tphropchesesneos liinc vaintrdo n[1o1n8]p. hInetneorelisctinugnliyt,s Pohfysliisgpnorininuasn vditbreruesa dkeinpgenvdas rious
linkaogne siunnlpirgenciend.eTntheids revceernsattdileis copvereorxyidsahseed t(hVePl)i ghsttraotnegtyh etvhaaltu ehoeflpVedP intoe ncohramraocutesriazpep lnicoavteiol nsof
biotedchelnigonloifgiciactaiolnp rdoecliensesaetsio[n1.1 T9]h.is strategy played vital role in enhancing oxidization both phenolic
and nonphenolic units of lignin and breaking various linkages in lignin. This recent discovery shed
3. Petnhteo lsigehPt hoons tphhe avtaeluPea otfh VwPa iyn enormous applications of biotechnological processes [119].
Thebiochemicalrouteforxylosemetabolismisthepentosephosphatepathway(PPP),whichsets
3. Pentose Phosphate Pathway
inallcellularorganismswhereitsuppliesD-erythrose4-phosphateforaromaticaminoacidssynthesis,
The biochemical route for xylose metabolism is the pentose phosphate pathway (PPP), which
D-riboseforbiosynthesisofnucleicacid,andNADPHfortheanabolicreactions. ThePPPiscomposed
sets in all cellular organisms where it supplies D-erythrose 4-phosphate for aromatic amino acids
oftwofunctionallyinterrelatedbranches: theoxidativeandthenon-oxidative[120]. Theoxidative
synthesis, D-ribose for biosynthesis of nucleic acid, and NADPH for the anabolic reactions. The PPP
phasies tchoamtptorasends foofr mtwso tfhuencDti-ognlualclyo sien,tehrerexloatseed inbrtaonDch-ersib: uthleo soexi5d-apthivoes apnhda tteh,e NnoAnD-oPxHid,atpivlue s[1C20O].2 and
pentoTshee. oTxhideatnivoen -pohxaisdea tthivate tprahnassfoertmhsa tthter aDn-sgflourcmosse, Dh-erxibosuel oinsteo 5D-p-rhibouslposhea t5e-pihnotsopDha-tgel,y NceArDalP-dHe, hyde
3-phopslupsh aCteO,2D a-xnydl uploenseto5s-ep. hTohsep hnaotne-,oDx-idriabtoivsee 5p-hpahsoe spthhaat tet,raDn-sffrourcmtoss eD-6r-ipbuhloosspe h5a-tpeh,oDs-psehdatoeh einpt-ot ulose
7-phoDs-gplhycaetrea,lD-d-eehryydthe ro3s-ep4h-opsphhoastpeh, atDe-,xLy-lualroasbei no5-speh,oasnpdhaDte-X, yDlo-rsiebocsoem e5-ipnhtohsephPaPteP, ovDe-frruDc-txoyselu lose
(Figu6r-eph3o).spThhaetet,r aDn-ssefodromheapt-itounloosef D7--pxhyolospsehatote,D D-x-eyrlyutlhorsoesei n4-bpahcotesprihaatgeo, eLs-abryabwinaoyseo, faxnydl oDs-eXiysloomse erase
(xylAc)o.mIne yinea tshte, sPoPmP eoveeurk Da-rxyyolutelossaen (dFigfiularme 3e)n. Ttohues trfaunnsgfoi,rmthaistiopnro ocfe Ds-sxhyalossed oton eD-bxyylaultowseo i-nst beapctoexriida ation
goes by way of xylose isomerase (xylA). In yeast, some eukaryotes and filamentous fungi, this
andreductionthatismediatedbyxylitoldehydrogenases(Xyl2p,XYL2)andxylosereductase(Xyl1p,
process has done by a two-step oxidation and reduction that is mediated by xylitol dehydrogenases
XYL1),respectively. Therequirementofcofactorofthesetworeactionsisinfluencedbythecellular
(Xyl2p, XYL2) and xylose reductase (Xyl1p, XYL1), respectively. The requirement of cofactor of these
demandsforoxygen[121].
two reactions is influenced by the cellular demands for oxygen [121].

Figure 3. The pentose phosphate pathway (PPP) in yeasts.
Figure3.Thepentosephosphatepathway(PPP)inyeasts.
Fermentation 2018,4,16 9of27
KötterandCiracy(1993)proposedthat,theexcessivexylitolproductionbygeneticallyengineered
S. cerevisiae with xylose reductases (XYL2 and XYL1) was restricted by the dual cofactor ability of
the P. stipitis xylose reductases, the excessive activity of the oxidative PPP in S. cerevisiae, and by
theincompleteabilityofthenon-oxidativePPP[122]. Overexpressingoftransketolase(TKL1)from
P.stipitisinanengineeredS.cerevisiaecausesareductioninthegrowthrateofthenewtransformant
mutantondefinedxyloseminimalmedium[123]. TheoverexpressingstainsofP.stipitistransaldolase
(TAL1) gene, along with XYL2 and XYL1 implant faster growth than strains expressing XYL2 and
XYL1alone[124]. However,theplasmidburdenbecauseoftheoverexpressionofXYL1,XYL2and
TAL1decreasedtherateofgrowthofthetransformantstrainrelativetotheparentalstrain[125,126].
Sinorhizobium meliloti (gram-negative bacteria) defined as a model organism for investigating the
plant-microbeinteractions,becauseitparticipatesinprocessofnitrogenfixationasithassymbiotic
relationshipwithMedicagosativaandM.truncatula. Mutationinthegenetkt2,aputativetransketolase
andtal,aputativetransaldolasehadremarkableinfluenceonthenon-oxidativebranchofthepentose
phosphatepathway,acquisitionofironandseveralphysiologicalprocessessuchasthebiosynthesisof
exopolysaccharide[127].
4. XyloseIsomeraseMechanism
TheengineeringofSaccharomycescerevisiaeforxyloseutilizationisanessentialsteptowardsthe
productionofthisbiofuel. Thoughxyloseisomerase(XI)isthekeyenzymeforxyloseconversion,
almost half of the XI genes are not functional when it is expressed in S. cerevisiae. However,
XI from Piromyces sp. E2 (PirXI) can be used to equip Saccharomyces cerevisiae with the capacity
to ferment xylose to ethanol [128,129]. For xylose assimilation, the initial metabolic engineering
endeavor was achieved by the heterologous expression of xylose isomerase (XI) in S. cerevisiae.
Thisprocesswasfeasible,giventhatS.cerevisiaewasabletogrowandfermentxylulose. However,
theconsiderableactivityofxyloseisomerasewasnotfulfilledintheoverexpressingstrain,probably
duetoproteinmisfolding[76,77]. Moreover,ifalittleamountoftheXIproteinwasproperlyfolded
and was active, the competitive inhibitor xylitol would be formed [130]. Later, the heterologous
overexpressionofXIfromthermophilicbacteriumThermusthermophilesinthebackgroundofgenetically
modifiedS.cerevisiae,attainedsuccessfullytheproductionofanactiveXIenzymetodecreasexylitol
production[131,132]. However,theoptimumtemperatureforthebestactivityofT.thermophilusXI
ishigherthantherequiredtemperaturefortheoptimumgrowthofSaccharomyces. Recentlyusing
directevolutionapproach,theactivityofXIcouldbeenhancedmorenine-foldthanpreviousstudies
at a higher temperature of 60 ◦C and for xylitol much higher inhibition constants [133]. Recently,
eukaryoticXIhasbeenidentified,followedbytheintroductionofXIfromanaerobicfungus(AraA)into
S.cerevisiae[103]. ThetransformationofS.cerevisiaewithAraAenhancedslowlytheutilizationrateof
xyloseassimilation[134]. Additionally,thefruitfuluseofXIdependsonotherfactors. Atequilibrium,
the energetic rate of the isomerization between xylulose and xylose support xylose formation by
17:83[135],therefore,anotherdrivingforceisnecessarytopromotethereaction. Additionally,arecent
studypointedouttheimportanceandrelatednessofengineeringofmetalhomeostasistometabolic
pathways for balancing metal-dependent enzymes. It focused on the role of metal interactions of
heterologousXIsinengineeringxylosemetabolisminS.cerevisiae[136].
5. PathwayofXyloseReductaseandXylitolDehydrogenase
Theexistenceofanaldose(xylose)reductase(XR)isconsideredasthemainfactorforassimilation
of xylose by native anaerobic yeasts like Pachystolon tannophilus that might utilize and use either
NADPH or NADH as a cofactor [80]. These results were confirmed by an earlier study that used
Candidautilisfortheutilizationandassimilationofxylose. Theutilizationandassimilationofglucose
bythisyeastwaseasyduringthefermentationprocess;however,thefermentationactivityrefrains
directly once the use of xylose as a substrate. Solely, the C. utilis XR utilizes NADH as a cofactor.
While,theXRofPichiastipites,PachysolentannophilusandCandidashehataecanutilizeeitherNADPHor
Fermentation 2018,4,16 10of27
NADHascofactorsthroughthefermentationprocess[80]. Theyassimilateandhavethecapability
to utilize xylose anaerobically, although they do not grow natively under such conditions [137].
Two oxidoreductase steps are essential for completing the assimilation processes of xylose, and
they are the main core for balancing the oxidoreductase reactions, so the regeneration of NAD+
and NADPH in the absence of transhydrogenase ceased the xylose assimilation under anaerobic
conditions. ThisevidencewasconfirmedbycloningandheterologousoverexpressingtheP.stipitesXR
inS.cerevisiae[80],wheretheoverexpressingmutantstrainproducedxylitolwhendifferentialsources
ofcarbonwererepresentedtoprovidereductant[137]. Theassimilation,utilizationofxylose,andthe
productionofxylitolwereinhibited,whenglucosewasusedinthemediaasco-substrate,probably
duetocompetitionfortransport[138,139]. However,P.stipitisXYL1doesnothavesuchanimpacton
xyloseassimilation[140]. Formaximumethanolproduction,itrequiresasystemwherethereduction
ofacetaldehydeunderlimitedoxygenconditionsispossible, andadditionallyoxidationofxylitol
toxylulose[80]. ThecapabilityofS.cerevisiaetoassimilateandutilizexylosewasclearlyobserved
withtheisolationandtransformationoftwoXRgenes(XYL1andXYL2)fromP.stipites,whichwere
responsiblefortheassimilationofxylose[141,142]. ThegeneticallyengineeredS.cerevisiaewiththose
twogeneshadtheabilitytoutilizexylosebyoxidationprocessesandgeneratexylitolintheabsenceof
anyadditionalco-metabolizablecarbonsourcesforxyloseassimilationprocesses. Furthermore,ina
combinationofXRwithxylitoldehydrogenase(XDH),theethanolproductionwasenhancedwhile
the xylitol production was decreased because augmentation the activity of xylitol dehydrogenase
(XDH)isproportionaltoXRactivity(XDH:XR=1.66)ascomparedtoXR,whichwasexpressedin
abundance[143]. Whereas,theoverexpressionofXYL2causesthesecretionxylulose,reflectingthat
theactivityofxylulokinase(XK)inhibitstheassimilationandutilizationofxyloseonsuchcells[144].
Candidaglycerinogenes,expressingthexylitoldehydrogenase(XYL2)gene,andcanproduceglycerol
fromxylosethathasobservableroleinpentosephosphatepathway. Minimalexpressionlevelsof
theXYL2geneoriginatedfromScheffersomycesstipitisinC.glycerinogeneshasadistinguishedrolein
enhancingtheefficiencyofxylosefermentation[145]. AnengineeredstrainthathasbothwildXRand
mutantXRshowedlowerxylitolaccumulationandfasterxyloseconsumptionthanengineeredstrains
overexpressingonlyonetypeofXRs. ThisresultemphasizedtheroleofXRgenesinenhancingthe
quantityofproducedethanol[146].
6. XyloseTransport
S.cerevisiaedidnotproperlyutilizethexyloseduetothelowaffinityofitsnaturalnonspecific
hexose-transportsystemforxylose. Theheterologousoverexpressionofaspecificxylosetransporter
hasanapparentinfluenceonbiotechnologicalethanolproduction,soalotofresearchersfocusedon
thatissue. Candidaintermediahaveseveralindicationsthatstrengthentheprobabilitythepresenceof
xylosetransporters,suchasconstitutivefacilitateddiffusionofxylose,glucose-repressibleandhigh
xylose affinity. Two transporters from this yeast already are described. They are known as GXF1
(glucose/xylosefacilitator)andGXS1(glucose/xylosesymporter)transporters. Facilitateddiffusion
does not require high energy for sugar uptake as compared to proton symport, so the facilitator
proteinconsidersaspromisingmethodespeciallyinthescarcityofATPproductionandabsenceof
oxygensources[147]. Furthermore,ageneencodingxylose/glucoseprotontransporterisolatedfrom
CandidaintermediawasintroducedtoS.cerevisiae,whichinturn,improvedthexyloseutilizationwith
K of200µMforxylose,buttheK was10foldslowerforglucose[110,147]. GXF1andGXS1are
m m
transportersthatplayanimportantroleinenhancingtheutilizationofglucose10-foldhigherthan
xylose. TheexpressionofGxs1andGxf1isdirectlyproportionaltotheconcentrationofglucose[112].
Atthehighconcentrationsofxylose,Gxf1hadnodistinguishableinfluenceonaxyloseengineered
S.cerevisiaestrain,butitboostedgrowthremarkablyatlowconcentrations[148]. Trxlt1transporter,
whichwasidentifiedinTrichodermareesei, increasedthegrowthopportunityonxylose, butnoton
glucose,whichrevealsthatitmaybeparticularlyaxylosetransporter[110].