Improving the protein secretion capacity of Saccharomyces cerevisiae with strain engineering

Kroukamp, Heinrich (2015-03)

Thesis (PhD)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae is frequently chosen for the production of industrial and pharmaceutical proteins, due to its rapid growth, microbial safety, eukaryotic post-translational processing and high-density fermentation capability. With the development of recombinant DNA technologies and efficient expression systems, this yeast also gained a prominent new role as a protein production host, due to its ease of genetic manipulation. Improving the production and secretion of recombinant proteins, whether for pharmaceutical, agricultural or industrial application, has the benefit of reducing the production costs and promoting accessibility to these technologies. This also holds true for the second generation biofuel production, where high levels of hydrolytic enzymes are required to break down the complex carbohydrates in lignocellulose. While high copy number expression vector systems, strong promoters and efficient secretion signals resulted in significant enhancement of protein production yields, these strategies are often limited by bottlenecks in the yeast secretion pathway. Although protein characteristics and host restrictions are likely to contribute to these bottlenecks, these factors are still poorly understood. Nonetheless, strain engineering approaches have shown great potential as a means to relieve these protein secretion bottlenecks and advancing recombinant protein production to new levels. In this study, the potential of strain engineering, with regards to enhancing cellulase secretion for second generation bioethanol production, was evaluated. Further work involved the identification of novel genetic elements enhancing protein secretion and the elucidation of possible mechanisms involved in high cellulase secretion by S. cerevisiae. When the native PSEI gene was expressed under the transcriptional control of the PGK1 promoter in S. cerevisiae, the secreted yields of recombinantly produced Neocallimastix patriciarum Cel6A, Trichoderma reesei Cel7B and Saccharomycopsis fibuligera Cel3A were increased 1.15, 1.25 and 3.70 fold, respectively. The overexpression of SOD1 did not increase any of the above mentioned cellulases. When SOD1 was overexpressed in combination with PSE1, a synergistic enhancement in secreted Cel3A was obtained. To our knowledge, this is the first reported case where SOD1 overexpression in S. cerevisiae resulted in higher heterologous protein secretion. The effect of disrupting protein N-glycosylation elongation at various steps, on the secretion of heterologously produced Neosartorya fischeri Cel12A (lacking N-glycosylation sites) and the S. fibuligera Cel3A, was investigated. The deletion of the MNN2 gene was shown to increase the extracellular Cel12A by 1.30 fold, while the deletion of MNN11 resulted in a 1.26 fold increase in extracellular Cel3A. These results implicate the cell wall as a possible barrier to protein secretion. It was also found that Cel3A with shorter N-glycosylation chains had reduced cell wall retention, compared to enzymes resembling the native glycosylation pattern. The removal of the PMR1 gene product (predicted to result in a general decrease in Golgi mannosyltranferase activities) was the only modification which enhanced the cell specific activities of both reporter cellulases, although this mutant's poor growth makes it an unlikely candidate for industrial application. The S. cerevisiae M0341 strain that secretes high levels of recombinant Talaromyces emersonii Cel7A, was mated to the Y294 control strain to produce the H3 hybrid strain. Several high secreting progeny were selected after sporulating the H3 strain. Through genome shuffling and single nucleotide polymorphism (SNP) analysis of pooled segregants, five genomic regions of the M0341 strain were identified that contain putative alleles that are beneficial to Cel7A secretion. Identifying these alleles proved problematic due to strain instability. When the T. emersonii CEL7A, N. fischeri CEL12A and S. fibuligera CEL3A were expressed in selected H3 progeny on episomal plasmids, these strains had up to ~3.5 fold increased Cel7A secretion compared to the M0341 parental strain, but no increase were observed for the other cellulase. It was also shown that cell flocculation could improve secretion. The strains constructed in this study represent a step toward efficient cellulase secreting yeasts for second generation biofuel production and presents a novel strategy to identify secretion enhancing elements for the protein production industry.

AFRIKAANSE OPSOMMING: Die gis, Saccharomyces cerevisiae, word dikwels vir die produksie van industriële en farmaseutiese proteïene gekies, te danke aan sy vinnige groei tempo, veiligheid van gebruik, eukariotiese na-translasie modifisering van proteïene en hoë-digtheid fermentasie vermoë. Met die ontwikkeling van rekombinante DNS-tegnologieë en effektiewe geenuitdrukking sisteme, het die gis 'n prominente nuwe rol verwerf as 'n proteïen produksie gasheer vanweë die eenvoud waarmee dit geneties gemanulipeer kan word. Die verbetering in produksie en sekresie van rekombinante proteïene, hetsy vir farmaseutiese, landbou of industriële gebruik, bevoordeel laer produksie kostes en meer algemene toegang tot hierdie produkte. Hierdie voordele is ook belangrik vir die “tweede generasie” bio-brandstof bedryf, waar hoë vlakke hidrolitiese ensieme benodig word om die komplekse koolhidrate in lignosellulose af te breek. Hoë kopie getal uitdrukkings vektore, sterk promoters en effektiewe sekresie seine het aansienlike verbeteringe in proteïen produksie vlakke te weeg gebring, maar hierdie strategieë kan soms verstoppings in die sekresie weg veroorsaak. Alhoewel dit bekend is dat proteïen eienskappe en beperkinge van die uitdrukkings gasheer moontlik tot hierdie sekresie weg verstoppings kan bydrae, word hierdie faktore nog sleg verstaan. Nietemin, genetiese ingenieurswese van uitdrukkings rasse het die poteinsiaal om die verstoppings in die sekresie weg te verlig en sodoende die sekresie van rekombinante proteïene te verbeter. In hierdie studie was die potensiaal van genetiese ingenieurswese, met betrekking tot die verbetering van sellulase sekresie vir tweede generasie bio-etanol produksie, geëvalueer. Verder was daar gepoog om nuwe genetiese elemente te identifiseer wat proteïen sekresie kan verhoog en om insig oor die meganismes wat betrokke is by hoë sellulase sekresie in S. cerevisiae te bekom. Die ooruitdrukking van die natuurlike PSE1 geen, onder die transkriptionele beheer van die PGK1 promoter in S. cerevisiae, het die sekresie vlakke van rekombinant geproduseerde Neocallimastix patriciarum Cel6A, Trichoderma reesei Cel7B en Saccharomycopsis fibuligera Cel3A respektiwelik met 1.15, 1.25 en 3.70-voud verbeter. Die ooruitdrukking van die natuurlike SOD1 geen kon nie die sekresie van die bogenoemde sellulases verhoog nie, maar die gesamentlike ooruitdruking van SOD1 en PSE1 was wel in staat om 'n sinergistiese verhoging in Cel3A sekresie vlakke teweeg te bring. Tot ons kennis, is dit die eerste geval waar verhoogde heteroloë proteïen sekresie a.g.v. die ooruitdrukking van SOD1 in die gis S. cerevisiae, wat aangemeld is. Vervolgens het ons die effek wat die ontwrigting van proteïen N-glikosilering-ketting verlenging, gedurende verskillende stappe van die proses, op die heteroloë produksie van Neosartorya fischeri Cel12A en die S. fibuligera Cel3A ondersoek. Delesie van die MNN2 geen het 'n 1.3-voudige toename in Cel12A sekresie teweeg gebring, terwyl die delesie van MNN11 ekstrasellulêre Cel3A 1.26-voudig laat toeneem het. Hierdie resultate impliseer dat die selwand as 'n moontlike hindernis optree gedurende proteïen sekresie. Dit is gevind dat rekombinante Cel3A met korter N-glikosilerings kettings 'n laer affiniteit vir selwand assosiasie het, teenoor die ensiem met die natuurlike glikosilerings patroon. Die delesie van die PMR1 geen (wat 'n algeneme verlaging in Golgi mannosieltransferase aktiwiteit veroorsaak) was die enigste verandering wat die sel-spesifieke aktiviteite van beide Cel3A en Cel12A kon verhoog. Ten spyte van dié mutante ras se verbeterde sekresie, is dit ongeskik vir industriële toepassing weens vertraagde groei. Die S. cerevisiae M0341-ras wat hoë vlakke van die rekombinante Talaromyces emersonii Cel7A produseer, is met die Y294 kontrole ras gepaar om die H3 hibried ras te genereer. Nadat die H3-ras gesporuleer is, is menigte afstammelinge met „n hoë sekresie fenotipe geselekteer. Deur genoom skommeling en die analiese van enkel nukleotied-polimorfismes (ENPs) van die geselekteede afstammelinge, kon vyf genoom areas van die M0341 ras geïdentifiseer word wat moontlike allele bevat wat voordelig is vir die sekresie van Cel7A. Die identifikasie van die spesifieke allele was egter nie moontlik nie a.g.v. die onstabiliteit van die afstammeling genome. Dit kon wel gedemonstreer word dat sel flokkulasie die sekresie van Cel7A bevoordeel. Die uitdrukking van die T. emersonii CEL7A geen met hoë kopie getal episomale plasmiede in geseleteerde H3 afstammelinge, het 'n verhoging van tot ~3.5-voudig in vergelyking met die M0341 ras, tot gevolg gehad. Soortgelyke uitdrukking van die S. fibuligera CEL3A en N. fischeri CEL12A in hierdie afstammelinge het geen sekresie verbetering tot gevolg gehad nie. Die rasse wat tydens hierdie studie gegenereer is, verteenwoordig beduidende vordering tot effektiewe sellulase sekresie in gis vir tweede generasie bio-brandstof produksie en demonstreer 'n nuwe strategie om genetiese elemente op te spoor wat die sekresie van verskeie industriële en farmaseutiese proteïen produkte kan verhoog.

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