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Overexpression of transcriptional activators in Saccharomyces cerevisiae for improved recombinant protein production
Thesis (MSc)--Stellenbosch University, 2019.
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Stellenbosch : Stellenbosch University
2019
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| _version_ | 1867614050345549824 |
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| access_status_str | Open Access |
| author | Martin, Jade Kyle |
| author2 | Viljoen-Bloom, Marinda |
| author_browse | Martin, Jade Kyle Viljoen-Bloom, Marinda |
| author_facet | Viljoen-Bloom, Marinda Martin, Jade Kyle |
| author_sort | Martin, Jade Kyle |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2019. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/105698 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:45:52.267Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/105698 Overexpression of transcriptional activators in Saccharomyces cerevisiae for improved recombinant protein production Martin, Jade Kyle Viljoen-Bloom, Marinda Rose, Shuanita Stellenbosch University. Faculty of Science. Dept. of Microbiology. Saccharomyces cerevisiae Promoters Transcriptional activators Recombinant protein production Gene expression -- Regulation Proteins UCTD Thesis (MSc)--Stellenbosch University, 2019. ENGLISH ABSTRACT: The yeast, Saccharomyces cerevisiae, possesses multiple characteristics that classify it as an excellent host organism for the recombinant production of industrial and medical proteins. This includes its ability to process and secrete heterologous eukaryotic proteins at far higher levels than native proteins, which significantly simplifies downstream protein purification. However, it has had limited success in industry due to limitations in the yeast’s recombinant protein production capability. Gene expression in S. cerevisiae is tightly regulated and metabolic engineering is therefore required to direct cellular resources and metabolic energy towards heterologous gene expression. Promoters are genetic elements that control gene expression and specific DNA sequences within promoters act as binding sites for transcriptional activators. Transcriptional activators upregulate gene expression, but they are also subject to regulation that could limit the level of heterologous gene expression. Increasing the cellular concentration of such activators could further upregulate gene expression and consequently, increase recombinant protein production. The aim of this study was to develop a tool for the overproduction of recombinant proteins in S. cerevisiae and increase its competitiveness as a microbial cell factory in industry. Therefore, the effect of overexpressing two promoter-specific transcriptional activators (RAP1 and GCR1) on the strength of three strong constitutive promoters (ENO1P, ATEF1P and STEF1P) in the S. cerevisiae Y294 laboratory strain, was evaluated. Reporter genes coding for raw starch-degrading enzymes were used as they have a large industrial significance in the food, fermentation, textile, paper, detergent and pharmaceutical industries. The abovementioned transcriptional manipulations were evaluated in terms of the extracellular enzyme activity and protein production under aerobic and fermentative conditions. The best candidates were further tested in raw corn starch fermentations in terms of ethanol production and carbon conversion. While GCR1 overexpression had no effect, RAP1 overexpression resulted in a three-fold increase in the extracellular protein concentration. Under aerobic conditions, the volumetric activity of the recombinant AteA α-amylase was increased by 81%, 28% and 46%, when the gene was expressed under the transcriptional control of the ENO1, ATEF1 and STEF1 promoters, respectively. In addition, improvements of 99%, 29% and 69% were observed under fermentative conditions for the three respective promoters. RAP1-overexpressing strains were subsequently used for the construction of an amylolytic co-culture (S. cerevisiae Y294[ENO1_TemA_RAP1] + Y294[ENO1_TemG_RAP1]) that exhibited an improved fermentation profile during the bioconversion of 200 g/l raw corn starch. The ethanol yield produced after 48 hours was comparable to that obtained from a commercial enzyme preparation (STARGENTM 002) and 54% higher than the benchmark co-culture (S. cerevisiae Y294[ENO1_TemA] + Y294[ENO1_TemG]). More encouraging, was that the ethanol yield from the S. cerevisiae Y294[TemA_TemG_RAP1] strain (co-expressing the TemA and TemG genes) was 111% higher with RAP1 overexpression and 201% higher than that of the S. cerevisiae Y294[AmyA_GlaA] reference strain after 48 hours. Furthermore, RAP1 overexpression resulted in a fermentation profile in the S. cerevisiae Y294[TemA_TemG_RAP1] laboratory strain that was comparable to that of a recombinant S. cerevisiae industrial strain (S. cerevisiae Ethanol Red[TemA_TemG]). This study has confirmed the potential of RAP1 overexpression as a tool to increase recombinant protein production in S. cerevisiae, thus increasing the economic viability of S. cerevisiae as a microbial cell factory. AFRIKAANSE OPSOMMING: Die gis, Saccharomyces cerevisiae, bevat vele eienskappe wat dit 'n uitstekende mikrobiese gasheer vir die produksie van rekombinante industriële en medies-toepaslike proteïene maak. Dit sluit in die vermoë om heteroloë eukariotiese proteïene te prosesseer en teen baie hoër vlakke as eie proteïene uit te skei, wat stroom-af suiwering van proteïene beduidend vereenvoudig. Dit het egter tot dusver beperkte sukses in die industrie gehad weens beperkinge in die gis se rekombinante proteïenproduksievermoë. Geenuitdrukking in S. cerevisiae word streng gereguleer en metaboliese ingenieurswese word dus vereis om sellulêre hulpbronne en metaboliese energie na heteroloë geenuitdrukking te herlei. Promotors is genetiese elemente wat geenuitdrukking beheer en spesifieke DNA- volgordes in promoters funksioneer as bindingsetels vir transkripsionele aktiveerders. Transkripsionele aktiveerders verhoog geenuitdrukking, maar hulle is self ook aan regulering onderworpe wat die vlak van heteroloë geenuitdrukking kan beperk. 'n Verhoging in die sellulêre konsentrasie van sulke aktiveerders kan moontlik geenuitdrukking bevorder en dus die vlakke van rekombinante proteïenproduksie verhoog. Die doel van hierdie studie was om gereedskap vir die oorproduksie van rekombinante proteïene in S. cerevisiae te ontwikkel en sodoende hierdie gis se mededingendheid as 'n mikrobiese selfabriek in die bedryf te verhoog. Die effek van die ooruitdrukking van twee promotor-spesifieke transkripsionele aktiveerders (RAP1 en GCR1) op die sterkte van drie sterk konstitutiewe promotors (ENO1P, ATEF1P en STEF1P) is in die S. cerevisiae Y294 laboratoriumras geëvalueer. Verklikkergene wat vir rou stysel-hidroliserende ensieme kodeer is hiervoor gebruik, aangesien hulle groot industriële waarde in die voedsel-, fermentasie-, tekstiel-, papier-, skoonmaakmiddel- en farmaseutiese bedryf het. Bogenoemde transkripsionele manipulasies is in terme van die ekstrasellulêre ensiemaktiwiteit en proteïenproduksie onder aërobiese en fermentatiewe toestande geëvalueer. Die beste kandidate is verder in rou mieliestysel fermentasies in terme van etanolproduksie en koolstof- omskakeling getoets. Terwyl GCR1-ooruitdrukking geen effek gehad het nie, het RAP1-ooruitdrukking die rekombinante proteïenkonsentrasie tot drievoudig verhoog. Onder aërobiese toestande is die volumetriese aktiwiteit van die rekombinante AteA α-amilase met 81%, 28% en 46% verhoog toe die geen onderskeidelik onder die transkripsionele beheer van die ENO1, ATEF1 en STEF1 promotors uitgedruk is. Verder is verbeterings van 99%, 29% en 69% vir die onderskeie promotors onder fermentatiewe toestande waargeneem. Ooruitdrukking van RAP1 is gevolglik vir die konstruksie van 'n amilolitiese kombinasiekultuur (S. cerevisiae Y294 [ENO1_TemA_RAP1] + Y294[ENO1_TemG_RAP1]) gebruik wat gedurende die bio- omskakeling van 200 g/l rou mieliestysel 'n verbeterde fermentasieprofiel getoon het. Die etanolopbrengs was vergelykbaar met 'n kommersiële ensiempreparaat (STARGENTM 002) en 54% hoër as die maatstaf kombinasiekultuur (S. cerevisiae Y294[ENO1_TemA] + Y294[ENO1_TemG]). Meer bemoedigend, was die feit dat die etanolopbrengs van die S. cerevisiae Y294[TemA-TemG_RAP1] ras (wat die TemA and TemG gene gesamentlik uitdruk) na 48 ure 111% hoër met RAP1 ooruitdrukking en 201% hoër as die S. cerevisiae Y294[AmyA-GlaA] verwysingsras was. Verder het die ooruitdrukking van RAP1 ’n fermentasieprofiel in die S. cerevisiae Y294[TemA_TemG_RAP1] laboratoriumras geskep wat vergelykbaar met 'n industriële ras (S. cerevisiae Ethanol Red[TemA_TemG]) was. Hierdie studie het die potensiaal van RAP1-ooruitdrukking as 'n hulpmiddel vir verhoogde rekombinante proteïenproduksie in S. cerevisiae bevestig, wat sodoende die ekonomiese lewensvatbaarheid van S. cerevisiae as 'n mikrobiese selfabriek kan verhoog. Masters 2019-02-27T07:15:31Z 2019-04-17T08:08:57Z 2022-03-03T03:00:12Z 2019-04 Thesis http://hdl.handle.net/10019.1/105698 en_ZA Stellenbosch University xi, 118 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Saccharomyces cerevisiae Promoters Transcriptional activators Recombinant protein production Gene expression -- Regulation Proteins UCTD Martin, Jade Kyle Overexpression of transcriptional activators in Saccharomyces cerevisiae for improved recombinant protein production |
| title | Overexpression of transcriptional activators in Saccharomyces cerevisiae for improved recombinant protein production |
| title_full | Overexpression of transcriptional activators in Saccharomyces cerevisiae for improved recombinant protein production |
| title_fullStr | Overexpression of transcriptional activators in Saccharomyces cerevisiae for improved recombinant protein production |
| title_full_unstemmed | Overexpression of transcriptional activators in Saccharomyces cerevisiae for improved recombinant protein production |
| title_short | Overexpression of transcriptional activators in Saccharomyces cerevisiae for improved recombinant protein production |
| title_sort | overexpression of transcriptional activators in saccharomyces cerevisiae for improved recombinant protein production |
| topic | Saccharomyces cerevisiae Promoters Transcriptional activators Recombinant protein production Gene expression -- Regulation Proteins UCTD |
| url | http://hdl.handle.net/10019.1/105698 |
| work_keys_str_mv | AT martinjadekyle overexpressionoftranscriptionalactivatorsinsaccharomycescerevisiaeforimprovedrecombinantproteinproduction |