Expression and characterization of exoglucanases in Saccharomyces cerevisiae

Van Wyk, Niel (2010-03)

Thesis (PhD (Microbiology))--University of Stellenbosch, 2010.

Thesis

ENGLISH ABSTRACT: Currently a world-wide tendency exists to shift away from relying on fossil fuels as a primary energy source and to focus on sustainable, environmentally-friendly alternatives. Ethanol is one such alternative and shows potential to replace petroleum as a transport fuel. Plant biomass, deemed a renewable energy source, can be converted to ethanol. The process of conversion via biologicallymediated events is problematic mainly due to the recalcitrance of the chief components of plant biomass namely cellulose, hemicellulose and lignin towards enzymatic degradation. A concept of consolidated bioprocessing (CBP) aims to make the process of bioconversion of plant biomass to ethanol cost-effective. For such a bioconversion, a biocatalyst is needed which can depolymerize the complex carbohydrates i.e. the cellulose and hemicellulose to their respective monomers for concurrent fermentation to ethanol. Saccharomyces cerevisiae shows potential as a candidate CBPbiocatalyst due to its high ethanol productivity, general robustness and amenability to genetic manipulation. However, S. cerevisiae does not possess the ability to break down the abovementioned carbohydrates. This study attempted to address certain aspects of yeast strain development for CBP. Genes encoding cellulases responsible for major crystalline cellulose hydrolysis i.e. exoglucanases were expressed in S. cerevisiae and the recombinant proteins were characterized. Further work involved exploring different ways of increasing the cellulolytic capability of recombinant S. cerevisiae. Both the cel9A of Thermobifida fusca and Npcel6A of Neocallimastix patriciarum were functionally expressed in S. cerevisiae. Expression of cel9A enabled S. cerevisiae to grow on phosphoric acid swollen cellulose reaching an aerobic growth rate (mMAX) of 0.088 h-1. This is the first report of S. cerevisiae growing on such a substrate while producing only one heterologous protein. An increase in the cellulolytic capability of recombinant S. cerevisiae was observed when cel9A was coexpressed with Trcel6A, cel7A and cel7B of Trichoderma reesei. These results proved that the synergy between cellulases can contribute towards increasing the cellulolytic capability of recombinant S. cerevisiae. NpCel6A has the highest reported individual activity on a crystalline cellulose substrate. However, expression of Npcel6A by S. cerevisiae resulted in lower levels of exoglucanase activity on Avicel of 0.540±0.062 mU/gDCW compared to the recombinant S. cerevisiae strains that produces Cel6A of T. reesei (4.101±0.243 mU/gDCW). This observation could be ascribed to glycosylation of the catalytic domain of NpCel6A. The replacement of the carbohydrate-binding module (CBM) and asparagine-rich linker of NpCel6A with the CBM and serine/threonine-rich linker of TrCel6A resulted in a decrease in recombinant cellulolytic activity produced by S. cerevisiae. In contrast, when the CBM and linker of NpCel6A were appended to the N-terminus of the catalytic domain of TrCel6A, significantly higher levels of cellulase activity were observed when produced by S. cerevisiae. This observation was largely attributed to the difference in glycosylation of the linkers. These results showed the value of domain swapping for obtaining increased cellulase secretion by S. cerevisiae. The native S. cerevisiae genes PSE1 and SOD1, were individually overexpressed in the S. cerevisiae strain producing NpCel6A, Cel3A of Saccharomycopsis fibuligera and Cel7B of Trichoderma reesei. The DDI1 gene of S. cerevisiae was also disrupted in the strain producing NpCel6A. In all cases, transformants were identified which displayed higher levels of cellulase activity compared to the original strain. This demonstrated the potential of S. cerevisiae to be considered as a “chassis”- strain that can, with the help of metabolic engineering, produce more recombinant cellulases. The swelling factor protein called swollenin, a contributor in the disruption of the crystallinity of cellulose, was co-expressed with cel9A and Npcel6A individually in S. cerevisiae. Even though functionality of swollenin was confirmed, no noteworthy increase in the levels of cellulase activity was observed for recombinant strains. The recombinant yeast strains generated during this study represent significant progress towards developing S. cerevisiae as a CBP organism.

AFRIKAANSE OPSOMMING: Tans heers daar wêreldwyd ‘n tendens om weg te beweeg vanaf fossielbrandstowwe as ‘n primêre energiebron en om te fokus op volhoubare, omgewingsvriendelike alternatiewe. Etanol is een só ‘n alternatief en toon potensiaal om petroleum as ‘n vervoerbandstof te vervang. Plantbiomassa, wat as ‘n hernubare energiebron beskou word, kan na etanol omgeskakel word. Die proses van omskakeling via biologies-gefasiliteerde gebeurtenisse is problematies hoofsaaklik aangesien die hoofkomponente van plantbiomassa naamlik sellulose, hemisellulose en lignien weerstandig is teen ensimatiese afbraak. ‘n Konsep genaamd gekonsolideerde bioprosessering (CBP) poog om die proses van bio-omskakeling van plantbiomassa na etanol koste-effektief te maak. Vir só ‘n bioomskakeling, word ‘n biokatalis benodig om die komplekse koolhidrate i.e. die sellulose en hemisellulose te kan depolimeriseer tot hul onderskeie monomere en tegelykertyd te fermenteer na etanol. Saccharomyces cerevisiae toon potensiaal as ‘n kandidaat CBP-biokatalis vanweë sy hoë etanol-produktiwiteit, algehele robuustheid en geskiktheid vir genetiese manipulering. S. cerevisiae besit egter nie die vermoë om bogenoemde koolhidraatpolimere af te breek nie. Hierdie studie het gepoog om sekere aspekte van gisrasontwikkeling vir CBP te adresseer. Sellulasekoderende gene wat in staat is om kristallyne sellulose te hidroliseer naamlik eksoglukanases, is in S. cerevisiae uitgedruk en die rekombinante proteïene is gekarakteriseer. Verdere werk het behels die verkenning van verskillende maniere om die sellulolitiese vermoëns van rekombinante S. cerevisiae te verbeter. Beide die cel9A van Thermobifida fusca en Npcel6A van Neocallimastix patriciarum is funksioneel uitgedruk in S. cerevisiae. Uitdrukking van cel9A het S. cerevisiae die vermoë gegee om op fosforsuur-geswelde sellulose te groei teen ’n groeitempo (mMAX) van 0.088 h-1. Dit is die eerste melding van S. cerevisiae wat kon groei op so ‘n substraat deur net een heteroloë proteïen te produseer. Daar is ook ‘n toename in die sellulolitiese vermoëns van S. cerevisiae waargeneem toe cel9A saam met Trcel6A, cel7A en cel7B van Trichoderma reesei uitgedruk is wat bewys dat die sinergie tussen sellulases kan bydra tot ‘n toename in die sellulolitiese vermoëns van S. cerevisiae. NpCel6A het die hoogste aangemelde indiwiduele aktiwiteit op ‘n kristallyne sellulose substraat. Rekombinante uitdrukking van Npcel6A in S. cerevisiae het egter laer eksoglukanase aktiwiteit op Avicel getoon (0.540±0.062 mU/gDCW) in vergelyking met die rekombinante S. cerevisiae ras wat Cel6A van T. reesei produseer (4.101±0.243 mU/gDCW). Die waarneming kan aan die glikosilering van die katalitiese domein van NpCel6A toegeskryf word. Die vervanging van die koolhidraatbindingsmodule (CBM) en asparagien-ryke koppeleenheid van NpCel6A met die CBM en serien/treonien-ryke koppeleenheid van TrCel6A het tot ‘n afname in rekombinante sellulase aktiwiteit deur S. cerevisiae gelei. In teenstelling, toe NpCel6A se CBM en koppeleenheid voor die katalitiese domein van TrCel6A geplaas is, het dit tot ‘n beduidende hoër sellulase aktiwiteit deur S. cerevisiae gelei. Dié waarneming is grootliks toegeskryf aan die verskil in glikosilering van die koppeleenhede. Hierdie resultate bewys die waarde wat domein-vervanging kan hê om ‘n toename in sellulase aktiwiteit waar te neem in rekombinante S. cerevisiae. Gene eie aan S. cerevisiae, PSE1 en SOD1, is indiwidueel ooruitgedruk in die S. cerevisiae ras wat NpCel6A, Cel3A van Saccharomycopsis fibuligera en Cel7B van T. reesei produseer. Die DDI1 geen van S. cerevisiae is ook ontwrig in die ras wat NpCel6A produseer. In alle gevalle is S. cerevisiae transformante geïdentifiseer wat hoër vlakke van sellulase aktiwiteit getoon het. Dit wys die potensiaal om S. cerevisiae as ‘n “onderstel”-organisme te beskou waarvolgens dit, met behulp van metaboliese ingenieurswese, meer heteroloë sellulases kan produseer. Die swelfaktorproteïen swollenin, wat bydra tot die ontwrigting van die kristalliniteit van sellulose, is saam met cel9A en Npcel6A onderskeidelik in S. cerevisiae uitgedruk. Die funksionaliteit van swollenin is bevestig, maar geen noemenswardige toename in sellulase aktiwiteit vir die rekombinante rasse is gevind nie. Die rekombinante gisrasse wat tydens hierdie studie gegenereer is, dui op beduidenswaardige vordering in die ontwikkeling van S. cerevisiae as ‘n CBP organisme.

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