ENGLISH ABSTRACT: Bio-fuels have been considered an ideal substitute for fossil fuels due to their availability and renewable nature. Bio-ethanol is currently of great market interest as an alternative fuel with the potential of supplementing petroleum as transportation fuel. Lignocellulosic biomass, a renewable energy source, can be "readily" converted to bio-ethanol. The main impediment in the conversion process is the recalcitrance of the main lignocellulosic components (cellulose, hemicelluloses and lignin) to enzymatic hydrolysis as well as the lack of available low-cost technology. Consolidated Bioprocessing (CBP) is a single process step which offers a cost-effective and economically feasible strategy for bio-ethanol production. The process requires micro-organisms that produce ethanol at high rates and titres.
Saccharomyces cerevisiae has potential as a CBP candidate due to its high ethanol yield, robustness in industrial processes, well-developed gene expression system and its safety status. Unfortunately S. cerevisiae does not degrade polysaccharides and therefore requires heterologous expression of cellulases. Genetic engineering of S. cerevisiae for cellulose hydrolysis serves as an important step in yeast strain development for CBP, and serves as a stepping stone for the commercialisation of lignocellulosic bio-ethanol. Although cellulose- utilising S. cerevisiae strains have been constructed, the cellobiose conversion is slow, hampering optimal ethanol production. β-glucosidases have been shown to be the major rate-limiting factors in cellulose saccharification as their activity determines the extent of cellulose hydrolysis, by removing excess cellobiose which causes feed-back inhibition on endoglucanase and cellobiohydrolase activities (Du Plessis et al. 2009;Lynd et al. 2002). Therefore, insufficient supply of β-glucosidase activity is detrimental to CBP and can be addressed by increasing the enzyme supply or using highly active β-glucosidases to enhance cellobiose hydrolysis.
In this study, several cellobiose fermenting S. cerevisiae strains were constructed. Extracellular fungal β-glucosidase-encoding genes were successfully expressed in S. cerevisiae under the transcriptional control of the ENO1 (enolase) promoter and terminator sequences. The recombinant enzymes produced were characterised based on pH and temperature optima as well as kinetic parameters. Bio-fuels have been considered an ideal substitute for fossil fuels due to their availability and renewable nature. Bio-ethanol is currently of great market interest as an alternative fuel with the potential of supplementing petroleum as transportation fuel. Lignocellulosic biomass, a renewable energy source, can be „readily‟ converted to bio-ethanol. The main impediment in the conversion process is the recalcitrance of the main lignocellulosic components (cellulose, hemicelluloses and lignin) to enzymatic hydrolysis as well as the lack of available low-cost technology. Consolidated Bioprocessing (CBP) is a single process step which offers a cost-effective and economically feasible strategy for bio-ethanol production. The process requires micro-organisms that produce ethanol at high rates and titres.
Saccharomyces cerevisiae has potential as a CBP candidate due to its high ethanol yield, robustness in industrial processes, well-developed gene expression system and its safety status. Unfortunately S. cerevisiae does not degrade polysaccharides and therefore requires heterologous expression of cellulases. Genetic engineering of S. cerevisiae for cellulose hydrolysis serves as an important step in yeast strain development for CBP, and serves as a stepping stone for the commercialisation of lignocellulosic bio-ethanol. Although cellulose- utilising S. cerevisiae strains have been constructed, the cellobiose conversion is slow, hampering optimal ethanol production. β-glucosidases have been shown to be the major rate-limiting factors in cellulose saccharification as their activity determines the extent of cellulose hydrolysis, by removing excess cellobiose which causes feed-back inhibition on endoglucanase and cellobiohydrolase activities (Du Plessis et al. 2009;Lynd et al. 2002). Therefore, insufficient supply of β-glucosidase activity is detrimental to CBP and can be addressed by increasing the enzyme supply or using highly active β-glucosidases to enhance cellobiose hydrolysis.
In this study, several cellobiose fermenting S. cerevisiae strains were constructed. Extracellular fungal β-glucosidase-encoding genes were successfully expressed in S. cerevisiae under the transcriptional control of the ENO1 (enolase) promoter and terminator sequences. The recombinant enzymes produced were characterised based on pH and temperature optima as well as kinetic parameters.
AFRIKAANSE OPSOMMING: Biobrandstof word beskou as die ideale plaasvervanger vir fossielbrandstof weens die beskikbaarheid en herwinbare aard daarvan. Bio-etanol wek tans groot mark-verwante belangstelling as alternatiewe brandstof weens die potensiaal om petroleum as vervoerbrandstof aan te vul. Lignosellulose biomassa, 'n hernubare energiebron, kan "maklik" tot bio-etanol omgeskakel word. Die groot struikelblok in die omskakelingsproses is die weerstandbiedendheid van die lignosellulose komponente (sellulose, hemisellulose en lignien) teen ensiematiese hidroliese asook die gebrek aan beskikbaarheid van lae koste tegnologie. Gekonsolideerde Bioprosessering (KBP) is 'n enkel stap proses wat 'n koste-effektiewe en ekonomiesvatbare strategie voorstel vir bio-etanolproduksie. Die proses benodig 'n mikroorganisme wat daartoe instaat is om etanol teen hoë vlakke en tempo te kan produseer.
Saccharomyces cerevisiae het potensiaal as 'n KBP kandidaat weens sy hoë vlakke van etanolproduksie, gehardheid in industriële prosesse, goed-ontwikkelde geenuitdrukking sisteme en veiligheidstatus. Ongelukkig kan S. cerevisiae nie polisakkariede afbreek nie en benodig derhalwe heteroloë uitdrukking van sellulases. Die genetiese manipulering van S. cerevisiae vir sellulose hidroliese dien as 'n belangrike stap in gisrasontwikkeling vir KBP en dien as 'n “stepping stone” vir die kommersialisasie van lignosellulose bio-etanol. Alhoewel sellulose-benuttende S. cerevisiae rasse reeds gekonstrueer is, is sellulose omskakeling stadig en belemmer dit optimale etanolproduksie. 'n Hoogs aktiewe glukosidase word derhalwe benodig om die hidroliese van sellobiose te versnel.
Die studie behels die konstruksie van verskeie sellobiose-fermenterende S. cerevisiae rasse. Ektrasellulêre, fungiese -glukosidase-koderende gene was suksesvol in S. cerevisiae uitgedruk onderhewig aan die transkripsionele beheer van die ENO1 (enulase) promoter en termineerder DNS-volgordes. Die geproduseerde, rekombinante ensieme is gekarakteriseer op grond van optimale pH en temperatuur, asook kinetiese eienskappe. Die intrasellulêre benutting van sellobiose is 'n ideale benadering tot sellobiose hidroliese siende dat dit die risiko van kontaminasie verminder wat veroorsaak word deur die glukose wat vrygestel word in die ekstrasellulêre omgewing. Tog beskik S. cerevisiae nie oor 'n vervoer meganisme om sellobiose in die sel in te bring nie. Derhalwe is die intrasellulêre Phanaerochaete chrysosporium -glukosidase-koderende geen suksesvol saam met die Kluyveromyces lactis laktose permease uitgedruk. Alle rekombinante rasse is vir groei op sellobiose geevalueer. Die mees belowendste esktrasellulêre -glukosidase-produserende S. cerevisiae Y294[Pccbgl1] ras toon 'n aktiwiteit van 3.85 nkat.g-1, 1.85 keer meer die aktiwiteit van die S. cerevisiae Y294[SFB] ras (2.07 nkat.g-1). S. cerevisiae Y294[Pccbgl1] het ook 'n maksimum groei tempo van 0.25 h-1 onder anearobiese kondisies in vergelyking met die 0.064 h-1 van S. cerevisiae Y294[iPcbglB+lac12] toon. Onder anaërobe kondisies het S. cerevisiae Y294[Pccbgl1] 7.95 g.l-1 sellobiose verbruik en 4.05 g. l-1 etanol geproduseer oor 'n tydperk van 116 uur, terwyl S. cerevisiae Y294[iPcbglB+lac12] 0.41 g.l-1 sellobiose verbruik het en 0.21 g.l-1 etanol oor dieselfde tydperk geproduseer het. Die rekombinante rasse wat in die studie gekonstrueer is, is 'n belangrike stap in die ontwikkeling van S. cerevisiae as KBP sellulolitiese gis.
