The effects of native Saccharomyces cerevisiae snare gene over expression on Heterologous cellulase secretion

Van Zyl, J. H. D. (2015-12)

Thesis (PhD)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: The budding yeast Saccharomyces cerevisiae has been successfully utilized in several industrial sectors and has over the last decade emerged as a promising host for the production of valuable heterologous proteins. As with the development of most biologicallybased production systems, there are invariably hurdles to overcome, the most pressing being the sub-optimal production yields for many heterologous proteins. The low protein secretion capacity of S. cerevisiae has been attributed to a great number of factors including various unknown secretory bottlenecks within the secretion pathway that collectively result in secretory titers that are often lower than 1% of the theoretical estimates. Increased secretory titers for the industrially significant fungal cellulases in the S. cerevisiae protein production host would greatly contribute to the economic feasibility of second generation bioethanol production. Improved titers will also benefit the production of commercially important biopharmaceutical proteins. SNAREs (Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment REceptor proteins) represent a class of membrane proteins that are required for the majority of membrane fusion events in the cell, including fusion of the protein secretory vesicles with the cis-Golgi and the plasma membrane. In this study, we attempted to elucidate whether the overproduction of some of these SNARE components at the cis-Golgi interface (BOS1, BET1, SEC22 and SED5) and at the plasma membrane (SNC1, SNC2, SSO1, SSO2 and SEC9) could increase the efficiency of the protein secretion process in S. cerevisiae for two industrially significant fungal cellulases – the Saccharomycopsis fibuligera Cel3A (β-glucosidase) and the Talaromyces emersonii Cel7A (cellobiohydrolase I). Our investigation further attempted to elucidate other physiological effects that these genetic modifications could bring about, both in terms of growth vigor and response to secretory stress. The exocytic t-SNARE Sso1p yielded the most improved secretory phenotype for Sf-Cel3A, with an improvement of approximately 43%, whilst the Snc1p v-SNARE component yielded the largest improvement in Te-Cel7A secretion of 71% (relative to the parental strain). The improvements for this reporter protein could be semi-quantitatively illustrated using SDSPAGE and densitometry analysis. Simultaneous overexpression of exocytic SNARE genes led to a moderate improvement of 52% and 48% for the secretion of Te-Cel7A and Sf-Cel3A, respectively, whilst simultaneous SNARE-overexpression in the strains producing the Sf-Cel3A led to measurable decreases in ethanol and osmotolerance, as well as a decreased growth vigor. For the Endoplasmic Reticulum (ER)-to-Golgi SNAREs, it was the t-SNARE Sed5p that yielded the biggest improvements in the secretion of Sf-Cel3A (22%) and Te-Cel7A (68%). However, overexpression of Sed5p did lead to decreases in ethanol and osmotolerance for strains harboring either of the heterologous cellulases expressed on episomal plasmids, in addition to slight decreases in growth vigor. Simultaneous ER-to-Golgi SNARE overexpression led to less significant secretory improvements for Te-Cel7A and decreased secretory titers for Sf-Cel3A, whilst the yeast could not maintain cell viability upon simultaneous overexpression of the ER-to-Golgi SNAREs in the presence of the beforementioned reporter protein. Co-overexpression of the most promising ER-to-Golgi and exocytic SNARE components identified for the improvement of Sf-Cel3A secretion (SED5 and SSO1, respectively) led to a significant improvement in extracellular activity of 130%. The production of Sf-Cel3A led to a measurably increased unfolded protein response (UPR), a mechanism proportionately induced by the buildup of folded and misfolded proteins in the ER. When Sed5p, which led to an improved secretion phenotype for Sf-Cel3A, was overexpressed in conjunction with the aforementioned reporter protein, the UPR activation was notably diminished. This suggests that a higher dosage of Sed5p may improve ER-to- Golgi protein transport to such an extent that the UPR response diminished. Overexpression of the exocytic SNAREs proved more effective for the improvement of native invertase secretion, with Sso1p and Snc1p leading to improvements of 53% and 32%, respectively. However, Sed5p only yielded a 15% improvement. This study suggests that SNAREs fulfill a prominent role within a larger cascade of secretory pathway components that hold potential as secretory-enhancing factors for the S. cerevisiae heterologous protein production host. The positive effects that overexpression of SNAREs introduced for the secretion of heterologous and native proteins (such as invertase) indicate that these components may be implicated in secretory bottlenecks at the cis-Golgi and/or plasma membrane interface.

AFRIKAANSE OPSOMMING: Die gis Saccharomyces cerevisiae is oor die afgelope dekade suksesvol vir die produksie van ‘n groot verskeidenheid heteroloë proteïene in verskeie industriële sektore benut. Soos vir die ontwikkeling van ander biologiese produksiesisteme, is daar altyd struikelblokke om te oorkom en een van die mees prominente uitdagings t.o.v. hierdie gis is sy suboptimale heteroloë sekresiekapasiteit. Die lae heteroloë sekresiekapasiteit in S. cerevisiae is al aan ‘n reeks veranderlikes toegeskryf, insluitende ‘n groot hoeveelheid onbekende sekresiebottelnekke wat gesamentlik tot sekresieopbrengste lei wat dikwels laer as 1% van die teoretiese skatting is. Verbeterde opbrengste vir waardevolle heteroloë sellulases in S. cerevisiae sal ‘n merkwaardige bydrae tot die realisering van ‘n bekostigbare tweedegenerasie bio-etanol alternatief lewer, terwyl ‘n verbetering in dié produksieorganisme ‘n bydrae tot die produksie van kommersiële biofarmaseutiese proteïene kan lewer. SNAREs (“Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment REceptor proteins”) verteenwoordig ‘n klas membraanproteïene wat essensieël vir die meerderheid van membraan samesmeltingsreaksies in die sekresiepadweg van die eukariotiese sel is. Hierdie reaksies sluit die samesmeltings van die proteïensekresievesikels met die cis-Golgi en die plasmamembraan, onderskeidelik, in. In hierdie studie het ons probeer bepaal of die oorproduksie van sommige van hierdie SNARE-komponente by die cis-Golgi intervlak (BOS1, BET1, SEC22 en SED5) en plasmamembraan (SNC1, SNC2, SSO1, SSO2 en SEC9) die effektiwiteit van die sekresieproses in S. cerevisiae vir twee heteroloë sellulases, naamlik die Saccharomycopsis fibuligera Cel3A (β-glukosidase) en Talaromyces emersonii Cel7A (sellobiohydrolase I), verbeter. Verder het ons ook probeer bepaal watter ander fisiologiese effekte dié genetiese modifikasies kon meebring, beide in terme van groei en sekresiestres. Die eksositiese t-SNARE-komponent Sso1p het die grootste impak op die sekresie van Sf- Cel3A gelewer, met ‘n verbetering van 43%, terwyl die Snc1p v-SNARE-komponent die grootste verbetering vir Te-Cel7A (71%) (relatief tot die ouerras) gelewer het. Die verbeteringe vir die Te-Cel7A kon semi-kwantitatief m.b.v. SDS-PAGE- en densitometrieanaliese geïllustreer word. Gesamentlike ooruitdrukking van eksositiese SNARE gene het matige verbeteringe van 52% en 48% vir onderskeidelik die Te-Cel7A and Sf-Cel3A opgelewer, hoewel dié strategie tot vertraagde groei en verlaagde etanol- en osmotoleransie gelei het. Die ER-to-Golgi t-SNARE Sed5p- komponent het tot die grootste verbetering in die sekresie van Sf-Cel3A (22%) en Te-Cel7A (68%) gelei. Die ooruitdrukking van Sed5p het ook tot ‘n verswakking in groeikapasiteit gelei, asook verlaagde etanol- en osmotoleransie in rasse wat een van die die heteroloë sellulases vanaf episomale plasmiede uitgedruk het. Gesamentlike ooruitdrukking van die ER-tot-Golgi-SNAREs het tot slegs matige verbeteringe in die sekresie van Te-Cel7A gelei, maar het die sekresie van Sf-Cel3A negatief beïnvloed. Die gis kon ook nie die gelyktydige ooruitdrukking van al vier komponente onderhou in kombinasie met die laasgenoemde heteroloë proteïen. Die gelyktydige ooruitdrukking van die mees effektiewe ER-tot-Gogli en eksositiese SNARE-komponente vir die verbetering van Sf-Cel3A produksie (SED5 en SSO1) het tot ‘n merkwaardige verbetering van ongeveer 130% gelei. Die heteroloë produksie van Sf-Cel3A het tot ‘n beduidende induksie van die ontvouproteïenreaksie (UPR) gelei, ‘n spesifieke reaksiemeganisme wat die gis onder ERstres- geïnduseerde omstandighede aktiveer wanneer die opeenhoping van gevoude en misgevoude proteïene in die ER toeneem. Wanneer Sed5p, wat Sf-Cel3A sekresie verbeter, tesame met Sf-Cel3A ooruitgedruk word, is ‘n verlaging in hierdie UPR waargeneem, wat aandui dat verbeterde proteïenverkeer tussen die ER en die Golgi apparaat deur ‘n verhoogde dosis van Sed5p bewerkstellig is, met ‘n gevolglike verlaging in die UPR-reaksie. Ooruitdrukking van die eksositiese SNAREs (Snc1p en Sso1p) was meer effektief vir die verbetering van die gis se eie invertasesekresie in vergelyking met die mees prominente ERto- Golgi alternatief, (Sed5p) met verbeteringe van 32%, 53% en 15%, onderskeidelik. Hierdie studie stel voor dat SNAREs ‘n prominente rol binne ‘n breë kaskade van sekresiewegkomponente uitmaak wat potensiële voordele vir die heteroloë sekresiekapasiteit van S. cerevisiae kan inhou. Die positiewe effekte van SNAREooruitdrukking op die sekresie van heteroloë sellulases en eie proteïene soos invertase, impliseer dat SNAREs ‘n rol in die sekresiebottelnekke by die cis-Golgi en plasmamembraan speel.

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