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Strategies to enhance production of a recombinant lignin peroxidase in Pichia pastoris
Thesis (PhD)--Stellenbosch University, 2023.
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Stellenbosch ; Stellenbosch University
2023
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| _version_ | 1867613982124146688 |
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| access_status_str | Open Access |
| author | Biko, Odwa Dinilesizwe Vuyani |
| author2 | Van Zyl, Willem Heber |
| author_browse | Biko, Odwa Dinilesizwe Vuyani Van Zyl, Willem Heber |
| author_facet | Van Zyl, Willem Heber Biko, Odwa Dinilesizwe Vuyani |
| author_sort | Biko, Odwa Dinilesizwe Vuyani |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/128401 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:44:46.833Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Stellenbosch ; Stellenbosch University |
| publisherStr | Stellenbosch ; Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/128401 Strategies to enhance production of a recombinant lignin peroxidase in Pichia pastoris Biko, Odwa Dinilesizwe Vuyani Van Zyl, Willem Heber Viljoen-Bloom, Marinda Stellenbosch University. Faculty of Science. Dept. of Microbiology. Recombinant proteins Lignin -- Peroxidation Peroxidase Enzymes -- Biotechnology Pichia pastoris -- Biotechnology UCTD Thesis (PhD)--Stellenbosch University, 2023. ENGLISH ABSTRACT: The biorefinery concept utilizes lignocellulosic biomass as raw material to produce bio-based fuels, materials and value-added chemicals, similar to petroleum refineries that use finite fossil fuel-based resources. The technologies currently used to deconstruct lignocellulosic substrates require expensive pretreatment processes. Lignin peroxidase (LiP) enzyme technology has emerged as an alternative strategy to degrade lignin or convert inhibitory lignin-derived compounds to valuable chemicals. Insufficient production titers from native and heterologous expression hosts have limited the potential industrial and biotechnological applications of LiP. The methylotrophic yeast Komagataella phaffii or K. pastoris (Pichia pastoris) has been successfully used to produce many heterologous proteins for application in numerous industrial sectors, such as biopharmaceuticals, biofuels, and food and animal feed. However, to obtain sufficient titers of recombinant LiP (rLiP) in P. pastoris, several challenges need to be addressed. In this study, medium optimization and strain engineering was investigated to improve recombinant LiP production of the Phanerochaete chrysosporium lipH8 gene in P. pastoris. Different cultivation conditions and parameters were evaluated to improve the production and secretion of rLiP in P. pastoris. The adjustment of the pH to 4 increased extracellular rLiP activity by 5.5-fold, whereas co-feeding with sorbitol and methanol increased rLiP activity by 5.9-fold compared to the control conditions. Adding 1 mM FeSO4 increased LiP activity a further 6.0-fold during the induction phase. Combining several optimal growth parameters increased the extracellular LiP activity more than 10-fold higher relative to the standard growth conditions. The rLiP-producing P. pastoris strain was further engineered to overexpress two native genes involved in the heme biosynthesis pathway (HEM1 and HEM2) and two molecular chaperones involved in protein folding and protein translocation (PDI1 and KAR2). Overexpression of HEM2 and PDI1 decreased extracellular rLiP activity. The KAR2 gene increased rLiP activity by 3.33- fold, whilst HEM1 increased rLiP activity by 1.25-fold relative to the parental strain. The impact of transcription factors (TF) was also evaluated, namely overexpression of HAC1, involved in the regulation of the unfolded protein response (UPR) and MXR1, MIT1 and TRM1, positive regulators of the alcohol oxidase I promoter (PAOX1). The overexpression of HAC1, MIT1 and TRM1 showed the best results, with enzyme activities that were 4.02-, 4.36- and 4.68-fold higher than for the parental strain, respectively. These findings clearly illustrate that overexpression of transcription factors significantly benefits rLiP production in P. pastoris. The in vitro enzymatic activity of rLiP on ‘real’ substrates was demonstrated by its ability to decolorize several synthetic dyes and convert inhibitory by-products such as 5- hydroxymethylfurfural (HMF) into high-value chemicals. The rLiP converted up to 95% HMF to oxidized products. The work presented here illustrates that medium composition is pivotal for rLiP production levels in P. pastoris. In addition, strain engineering by overexpressing targeting genes involved in different cellular functions, such as heme biosynthesis, protein translocation, UPR and PAOX1 - regulation, improved rLiP production in P. pastoris. These strategies will assist in developing large- scale production strategies for rLiP for industrial and biotechnological applications. AFRIKAANSE OPSOMMING: Die bioraffinadery-konsep gebruik lignosellulose biomassa as grondstof om bio-gebaseerde brandstof, materiale en waardetoegevoegde chemikalieë te produseer, soortgelyk aan petroleumraffinaderye wat beperkte fossielbrandstof-gebaseerde hulpbronne gebruik. Die tegnologieë wat tans gebruik word om lignosellulose substrate af te breek vereis duur vooraf-behandelingsprosesse. Ensiemtegnologie gebaseer op lignienperoksidase (LiP) is potensieël 'n alternatiewe strategie om lignien af te breek of inhiberende lignien-gebaseerde verbindings na waardevolle chemikalieë om te skakel. Onvoldoende produksietiters vir beide natuurlike en heteroloë uitdrukking-gashere het tot dusver die potensiële industriële en biotegnologiese toepassings van LiP beperk. Die metielotrofiese gis Komagataella phaffii of K. pastoris (Pichia pastoris) is reeds suksesvol gebruik vir die produksie van verskeie heteroloë proteïene vir toepassing in talle industriële sektore, soos biofarmaseutika, biobrandstof, en voedsel en dierevoer. Om egter voldoende titers van rekombinante LiP (rLiP) in P. pastoris te verkry, moet verskeie uitdagings aangespreek word. In hierdie studie is medium-optimering en stam-ingenieurswese ondersoek om recombinant LiP-produksie vanaf die Phanerochaete chrysosporium lipH8-geen in P. pastoris te verbeter. Verskillende groeitoestande en parameters is geëvalueer om die produksie en uitskeiding van rLiP in P. pastoris te verbeter. Die aanpassing van die pH na 4 het ekstrasellulêre rLiP-aktiwiteit 5.5- voudig verhoog, terwyl gelyktydige voer met sorbitol en metanol rLiP-aktiwiteit 5.9-voudig relatief tot die kontrole toestande verhoog het. Die byvoeging van 1 mM FeSO₄ het LiP-aktiwiteit verder 6.0-voudig tydens die induksiefase verhoog. Die kombinasie van verskeie optimale groeiparameters het die ekstrasellulêre LiP aktiwiteit meer as 10-voudig hoër verhoog relatief tot die standaard groeitoestande. Die rLiP-produserende P. pastoris stam is verder gemanipuleer om twee inheemse gene betrokke by die heem-biosintese-weg (HEM1 en HEM2) en twee molekulêre chaperones betrokke by proteïenvouïng en proteïentranslokasie (PDI1 en KAR2) teen verhoogde vlakke uit te druk. Ooruitdrukking van HEM2 en PDI1 het ekstrasellulêre rLiP-aktiwiteit verminder. Die KAR2-geen het rLiP-aktiwiteit 3.33-voudig verhoog, terwyl HEM1 rLiP-aktiwiteit 1.25-voudig relatief tot die ouerstam verhoog het. Die impak van transkripsiefaktore (TF) is ook geëvalueer, naamlik die ooruitdrukking van HAC1, betrokke by die regulering van die ontvoude proteïenrespons (UPR) en MXR1, MIT1 en TRM1, positiewe reguleerders van die alkoholoksidase I-promotor (PAOX1). Die ooruitdrukking van HAC1, MIT1 en TRM1 het die beset resultate getoon, met 4.02, 4.36 en 4.68-voudig hoër ensiemaktiwiteit as in die ouerstam, onderskeidelik. Hierdie bevindinge illustreer duidelik dat die ooruitdrukking van transkripsiefaktore rLiP-produksie in P. pastoris beduidend bevoordeel. Die in vitro ensiematiese aktiwiteit van rLiP op 'werklike' substrate is bewys deur sy vermoë om verskeie sintetiese kleurstowwe te ontkleur en inhiberende neweprodukte soos 5-hidroksimetielfurfural (HMF) na hoëwaarde-chemikalieë om te skakel. Die rLiP het tot 95% HMF verwyder, wat hoofsaaklik na furfurielalkohol omgeskakel is. Die werk wat hier aangebied word, illustreer dat mediumsamestelling 'n deurslaggewende rol in rLiP-produksievlakke in P. pastoris speel. Verder het stam-ingenieurswese deur die ooruitdrukking van teikengene betrokke by verskillende sellulêre funksies, soos heembiosintese, proteïentranslokasie, UPR en PAOX₁ regulering, rLiP-produksie in P. pastoris verbeter. Hierdie strategieë kan help met die ontwikkeling van grootskaalse produksiestrategieë vir rLiP vir industriële en biotegnologiese toepassings. Doctoral 2023-02-14T12:50:55Z 2023-08-30T13:07:05Z 2023-03 2023-02-14T12:50:55Z 2023-08-31T09:18:36Z 2023-02-14T12:50:55Z 2023-08-31T09:18:36Z 2023-03 Thesis https://scholar.sun.ac.za/handle/10019.1/128401 en Stellenbosch University application/pdf xi, 106 pages : illustrations application/pdf Stellenbosch ; Stellenbosch University |
| spellingShingle | Recombinant proteins Lignin -- Peroxidation Peroxidase Enzymes -- Biotechnology Pichia pastoris -- Biotechnology UCTD Biko, Odwa Dinilesizwe Vuyani Strategies to enhance production of a recombinant lignin peroxidase in Pichia pastoris |
| title | Strategies to enhance production of a recombinant lignin peroxidase in Pichia pastoris |
| title_full | Strategies to enhance production of a recombinant lignin peroxidase in Pichia pastoris |
| title_fullStr | Strategies to enhance production of a recombinant lignin peroxidase in Pichia pastoris |
| title_full_unstemmed | Strategies to enhance production of a recombinant lignin peroxidase in Pichia pastoris |
| title_short | Strategies to enhance production of a recombinant lignin peroxidase in Pichia pastoris |
| title_sort | strategies to enhance production of a recombinant lignin peroxidase in pichia pastoris |
| topic | Recombinant proteins Lignin -- Peroxidation Peroxidase Enzymes -- Biotechnology Pichia pastoris -- Biotechnology UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/128401 |
| work_keys_str_mv | AT bikoodwadinilesizwevuyani strategiestoenhanceproductionofarecombinantligninperoxidaseinpichiapastoris |