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Heterologous expression of fungal LPMOs and CDHs in Saccharomyces cerevisiae
Thesis (MSc)--Stellenbosch University, 2022.
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Stellenbosch : Stellenbosch University
2022
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| _version_ | 1867613763503390720 |
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| access_status_str | Open Access |
| author | Smuts, Ivy Elizabeth |
| author2 | Van Zyl, Willem Heber |
| author_browse | Smuts, Ivy Elizabeth Van Zyl, Willem Heber |
| author_facet | Van Zyl, Willem Heber Smuts, Ivy Elizabeth |
| author_sort | Smuts, Ivy Elizabeth |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/125072 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:41:19.170Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/125072 Heterologous expression of fungal LPMOs and CDHs in Saccharomyces cerevisiae Smuts, Ivy Elizabeth Van Zyl, Willem Heber Viljoen-Bloom, Marinda Stellenbosch University. Faculty of Science. Dept. of Microbiology. Saccharomyces cerevisiae -- Biotechnology Yeast Saccharomyces cerevisiae -- Effect of stress on Cellulosic ethanol Lignocellulose -- Biotechnology Ethanol as fuel Biomass energy Enzyme inhibitors UCTD Thesis (MSc)--Stellenbosch University, 2022. ENGLISH ABSTRACT: Lignocellulosic biomass is one of the most abundant resources on earth and can be used in the production of bioethanol and other value-added products. Consolidated bioprocessing (CBP) has the potential to combine and simplify the processing steps involved in the production of cellulosic ethanol and other industrially important products. It is also more cost-effective and less energy-intensive than other bioprocessing strategies such as simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF). The yeast Saccharomyces cerevisiae is well-suited for consolidated bioprocessing as it can be used for ethanol production at an industrial scale. However, ethanol production from cellulosic biomass by a CBP organism requires exogenous cellulases, which S. cerevisiae strains do not produce naturally. Strains of S. cerevisiae have been used successfully as a recombinant host to produce cellulases such as cellobiohydrolases, endoglucanases and β-glucosidases. Unfortunately, lignocellulose is recalcitrant to microbial hydrolysis, and factors such as the crystallinity, available surface area, pore size, and degree of polymerization limit enzymatic hydrolysis and confine larger cellulase enzymes to the surface of the substrate. Lytic polysaccharide monooxygenases (LPMOs) are a class of auxiliary active enzymes that can increase the number of binding sites in cellulose by oxidizing the glycosidic bonds in the crystalline region of the substrate. This boosts the activity of other cellulases by increasing the accessibility of the substrate and thus the overall rate of hydrolysis. The LPMOs are metalloenzymes with a copper ion as their active centre and regioselectively cleave the glycosidic bond at either (or both) the C₁ or C₄ atoms of the cellulose crystal. LPMOs act through either an oxygenase or peroxygenase mechanism to cleave the glycosidic bonds in the substrate but recent findings suggest that the peroxygenase mechanism dominates in LPMOs. At the start of the reaction, the LPMOs are found in their resting state and need to be reduced to become active. Cellobiose dehydrogenase (CDH) is commonly used as a multifunctional redox partner for LPMOs. CDHs have two domains, namely a cytochrome domain (CYT) and a flavodehydrogenase domain (DH). Electrons are donated from the DH domain via the CYT domain to the LPMO. In this study, the aim was to produce recombinant fungal LPMOs in the S. cerevisiae Y294 laboratory strain to assist with the degradation of recalcitrant lignocellulose whilst simultaneously producing two CDHs as a redox partner for the LPMOs. Plasmids containing either the Neurospora crassa cdh1 or Myceliophthora thermophila CDH2, or one of four LPMOs, namely Podospora anserina gh61A, Podospora anserina gh61B, Myceliophthora thermophila gh61B and Chaetomium thermophilum gh61 genes were expressed in the S. cerevisiae Y294 laboratory strain. The extracellular production of the CDH and LPMO enzymes was confirmed by SDS-PAGE analysis. Enzyme assays confirmed CDH activity, producing 16.08 U/L for CDH2 and 7.36 U/L for CDH3, but the LPMO assay were unsuccessful. HPLC analysis was used to indirectly check for LPMO activity and in vitro cooperation between a combination of LPMOs, CDHs, two cellobiohydrolases, endoglucanase and a β-glucosidase, as indicated by glucose production through hydrolysis of Whatman filter paper. AFRIKAANSE OPSOMMING: Lignosellulolitiese biomassa is een van die mees volop hulpbronne op aarde en kan vir die produksie van bioethanol en ander waardetoegevoegde produkte gebruik word. Gekonsolideerde bioprosesseering (“CBP”) het die potensiaal om die prosessering-stappe in die produksie van sellulose-etanol en ander industrieël-belangrike produkte te kombineer en te vereenvoudig. Dit is ook meer koste-effektief en minder energie-intensief as ander bioprosseseerings strategieë soos gelyktydige versuikering en fermentasie (“SSF”) en afsonderlike hidrolise en fermentasie (“SHF”). Die gis Saccharomyces cerevisiae is geskik vir gekonsolideerde bioprosseseering omdat dit vir etanolproduksie op ‘n industriële vlak gebruik kan word. Etanolproduksie vanaf sellulose-biomassa deur ‘n CBP-organisme benodig egter eksogeniese sellulases, wat S. cerevisiae rasse nie natuurlik kan produseer nie. Rasse van S. cerevisiae is reeds suksesvol as ʼn rekombinante gasheer gebruik vir die produksie van sellulases soos sellobiohidrolases, endoglukanase en β-glukosidases. Ongelukkig is lignosellulose weerstandbiedend teen mikrobiese hidrolise en faktore soos kristalliniteit, beskikbare oppervlak-area, porie grootte en die graad van polimerisasie beperk ensiematiese hidroliese, en die groter sellulase ensieme tot die oppervlak van die substraat. Litiese polisakkaried mono-oksigenases (“LPMOs”) is ʼn klas ensieme wat die aantal bindings in die sellulose kan vermeerder deur glukosidiese bindings in die kristallyne area van die substraat te oksideer. Dit verhoog die aktiwiteit van ander sellulases deur toeganklikheid tot die substraat te verhoog en dus algemene hidroliese te versnel. LPMOs is metallo- ensieme met ʼn koper ioon as hul aktiewe kern en kloof die glukosidiese binding by óf (of albei) die C1 of C₄ atome van die sellulose kristal. LPMOs het ʼn oksigenase en ʼn peroksigenase meganisme wat die glukosidiese bindings in die substraat breek, maar nuwe resultate suggesteer dat die peroksigenase meganisme in LPMOs domineer. Aan die begin van die reaksie is LPMOs gewoonlik in ʼn rustende toestand en benodig reduksie vir aktivering. Sellobiose-dehidrogenases (“CDHs”) word algemeen gebruik as ʼn multifunksionele redoks-vennoot vir LPMOs. CDHs het twee domeine, naamlik ʼn sitochroom domein (CYT) en ʼn flavodehidrogenase domein (DH) wat elektrone vanaf die DH domein deur die CYT domein na die LPMO oordra. Die doel van hierdie studie was om rekombinante swam LPMOs in ʼn S. cerevisiae Y294 laboratorium-ras te produseer om met die afbraak van weerstandige lignosellulose te help, met die gelyktydige uitdrukking van twee CDHs wat as ʼn redoksvennoot vir die LPMOs optree. Plasmiede wat óf die Neurospora crassa cdh1 óf Myceliophthora thermophila CDH2 uitdruk, óf een van vier LPMO's, naamlik Podospora anserina gh61A, Podospora anserina gh61B, Myceliophthora thermophila gh61B en Chaetomium thermophilum gh61 gene bevat, is uitgedruk in die S. cerevisiae Y294 laboratorium-ras. Die ekstrasellulêre produksie van die CDH en LPMO ensieme is deur SDS-PAGE analise bevestig. Ensiemtoetse het suksesvolle CDH aktiwiteit bevestig, met 'n volumetriese aktiwiteit van 16,08 U/L vir CDH2 en 7,36 U/L vir CDH3, maar die ensiemtoets vir die LPMOs was onsuksesvol. HPLC-analise is gedoen om indirek vir LPMO-aktiwiteit en in vitro-samewerking tussen 'n kombinasie van LPMO's, CDH's, twee sellobiohidrolases, 'n endoglukanase en 'n β-glukosidase na te gaan, soos verklap deur die vrystelling van glukose weens hidroliese van Whatman-filtreerpapier. Masters 2022-03-09T18:24:00Z 2022-04-29T12:52:45Z 2022-03-09T18:24:00Z 2022-03 Thesis http://hdl.handle.net/10019.1/125072 en_ZA Stellenbosch University application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Saccharomyces cerevisiae -- Biotechnology Yeast Saccharomyces cerevisiae -- Effect of stress on Cellulosic ethanol Lignocellulose -- Biotechnology Ethanol as fuel Biomass energy Enzyme inhibitors UCTD Smuts, Ivy Elizabeth Heterologous expression of fungal LPMOs and CDHs in Saccharomyces cerevisiae |
| title | Heterologous expression of fungal LPMOs and CDHs in Saccharomyces cerevisiae |
| title_full | Heterologous expression of fungal LPMOs and CDHs in Saccharomyces cerevisiae |
| title_fullStr | Heterologous expression of fungal LPMOs and CDHs in Saccharomyces cerevisiae |
| title_full_unstemmed | Heterologous expression of fungal LPMOs and CDHs in Saccharomyces cerevisiae |
| title_short | Heterologous expression of fungal LPMOs and CDHs in Saccharomyces cerevisiae |
| title_sort | heterologous expression of fungal lpmos and cdhs in saccharomyces cerevisiae |
| topic | Saccharomyces cerevisiae -- Biotechnology Yeast Saccharomyces cerevisiae -- Effect of stress on Cellulosic ethanol Lignocellulose -- Biotechnology Ethanol as fuel Biomass energy Enzyme inhibitors UCTD |
| url | http://hdl.handle.net/10019.1/125072 |
| work_keys_str_mv | AT smutsivyelizabeth heterologousexpressionoffungallpmosandcdhsinsaccharomycescerevisiae |