Heterologous expression of a fungal lignin peroxidase in Pichia pastoris

Biko, Odwa Dinilesizwe (2019-03)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Considerable research has been being devoted to seek cost-effective and environmentally friendly alternatives to replace fossil fuel-based resources for energy and other essential products. Lignocellulosic biomass consisting of cellulose, hemicellulose and lignin presents the most abundant and sustainable resource for this purpose. Lignin, the non-polysaccharide constituent of lignocellulose, consists of p-coumaryl, coniferyl and sinapyl alcohol monomers that can be exploited as a source of aromatic compounds for conversion to phenol derivatives and vanillin, among others. A consortium of enzymes is required for its degradation, including lignin peroxidase, manganese peroxidase, versatile peroxidase and laccase. There is a particular interest in the development of recombinant microorganisms capable of producing these enzymes for the cost-effective conversion of technical lignins to high-value bioproducts. The poor secretion and complex protein structure of lignin peroxidase have limited the production thereof in native hosts as well as heterologous expression systems such as Escherichia coli and Saccharomyces cerevisiae. The yeast Pichia pastoris is a good alternative host for heterologous protein expression and has been used to produce many enzymes for industrial applications due to its high recombinant protein titres, inexpensive cultivation methods, Generally Regarded As Safe (GRAS) status and clear-cut downstream processes. Molecular biology and process engineering have been implemented to produce various recombinant enzymes in P. pastoris in significant amounts. In this study, three synthetic Phanerochaete chrysosporium lignin peroxidase (lipH8) genes were expressed in P. pastoris DSMZ 70382 under the control of the constitutive PGAP and inducible PAOX1 promoters. The codon sequence for the native (LiP_Nat) gene was optimised using two different indices, yielding LiP_CAI and LiP_CBI. Under the transcriptional control of the constitutive PGAP promoter, no recombinant lignin peroxidase was detected. In shake flask cultivations, the activity levels of the recombinant lignin peroxidases encoded by the LiP_Nat, LiP_CBI and LiP_CAI genes under the control of the methanol-inducible PAOX1 promoter, peaked at 597, 679 and 1169 U/L, respectively. High cell-density fermentations in a 14-L bioreactor with the best strain, P. pastoris pJ901[LiP_CAI], resulted in a 3.3-fold increase in lignin peroxidase activity (3818 U/L). The rLiPH8 constituted 68% of the total protein in the cell-free supernatant and displayed optimal activity on veratryl alcohol at 25 °C and pH 3. This study demonstrated the successful production of a recombinant fungal lignin peroxidase in P. pastoris and indicated that high cell-density fermentations can be used to increase rLiPH8 production in P. pastoris. The codon-optimised LiP_CAI and LiP_CBI genes resulted in higher extracellular LiP activity in P. pastoris than the LiP_Nat. The results provide a starting point for the optimisation of heterologous production of a fungal lignin peroxidase in P. pastoris at both the molecular and bioprocess level. The cost-effective production of enzymes such as lignin peroxidase is essential for the enzymatic upgrade of industrial lignin, as well as bioremediation applications (i.e. treatment of water). The high levels of enzyme activity coupled with the low amount of endogenous proteins obtained in P. pastoris, will minimize downstream processing (concentration and purification of rLiPH8) and may allow for direct application of the cell-free supernatant.

AFRIKAANSE OPSOMMING: Aansienlik baie navorsing word aan koste-effektiewe en omgewingsvriendelike alternatiewe vir fossielbrandstofgebaseerde hulpbronne vir energie en ander noodsaaklike produkte gewy. Lignosellulose biomassa, bestaande uit sellulose, hemisellulose en lignien, bied die mees oorvloedige en volhoubare hulpbron vir hierdie doel. Lignien, die nie-polisakkaried bestanddeel van lignosellulose, bestaan uit p-kumariel-, koniferiel- en sinapielalkohol subeenhede wat as 'n bron van aromatiese verbindings vir omskakeling na fenolderivate en vanillien gebruik word. 'n Konsortium van ensieme word vir die afbraak daarvan benodig, insluitende lignienperoksidase, mangaanperoksidase, veelsydige peroksidase en lakkase. Daar bestaan veral 'n groot aanvraag vir die ontwikkeling van 'n rekombinante mikro-organisme wat hierdie ensieme kan produseer vir die koste-effektiewe omskakeling van tegniese ligniene tot hoë-waarde bioprodukte. Die swak sekresie en komplekse proteïenstruktuur van lignienperoksidase het tot dusver die produksie daarvan in natuurlike gashere sowel as in heteroloë uitdrukkingstelsels soos Escherichia coli en Saccharomyces cerevisiae beperk. Die gis Pichia pastoris is 'n goeie alternatiewe gasheer vir heteroloë proteïenuitdrukking en is reeds gebruik om baie ensieme vir industriële toepassings te produseer danksy sy hoë rekombinante proteïentiters, goedkoop kweking, Algemeen-Veilige status en eenvoudige stroomaf prosesse. Molekulêre biologie en prosesingenieurswese is toegepas om verskeie rekombinante ensieme in P. pastoris in beduidende hoeveelhede te produseer. In hierdie studie, is drie sintetiese Phanerochaete chrysosporium lignienperoksidase (lipH8) gene in P. pastoris DSMZ 70382 onder beheer van die konstitutiewe PGAP en induseerbare PAOX1 promotors uitgedruk. Die kodonvolgorde van die natuurlike (LiP_Nat) geen is met behulp van twee verskillende indekse ge-optimiseer, wat LiP_CAI en LiP_CBI gelewer het. Onder die transkripsionele beheer van die konstitutiewe PGAP promotor is geen rekombinante lignienperoksidase waargeneem nie. In skudfles-kulture het rekombinante lignienperoksidase wat deur die LiP_Nat-, LiP_CBI- en LiP_CAI-gene onder beheer van die metanol-induseerbare PAOX1-promotor gekodeer is, ensiemaktiwiteit van onderskeidelik 597, 679 en 1169 U/L bereik. Hoë seldigtheid-fermentasies in 'n 14 L bioreaktor met die beste ras, P. pastoris pJ901[LiP_CAI], het 'n 3.3-voudige toename in uitdrukkingsvlakke (3818 U/L) gelewer. Die rLiPH8 het 68% van die totale proteïen in die selvrye bostand verteenwoordig en het optimale aktiwiteit op veratrilalkohol by 25 °C en by pH 3 getoon. Hierdie studie het die suksesvolle produksie van ʼn rekombinante swam-lignienperoksidase in P. pastoris getoon en aangedui dat hoë seldigtheid-fermentasies rLiPH8 produksie in P. pastoris kan verhoog. Die kodon-geoptimiseerde LiP_CAI en LiP_CBI gene het hoër ekstrasellulêre LiP-aktiwiteit as die LiP_Nat geen in P. pastoris gelewer. Hierdie studie bied ʼn vertrekpunt vir die optimisering van heteroloë produksie van ʼn swam-lignienperoksidase in P. pastoris op beide molekulêre en bioprosesseringsvlak. Die koste-effektiewe produksie van ensieme soos lignienperoksidase is noodsaaklik vir die ensiematiese opgradering van industriële lignien, asook bioremediëringstoepassings (bv. waterbehandeling). Die hoë vlakke van ensiemaktiwiteit tesame met die lae hoeveelheid endogene proteïene wat in P. pastoris verkry is, sal stroomaf prosessering (konsentrasie en suiwering van rLiPH8) vergemaklik en mag moontlik die direkte gebruik van die selvrye bostand toelaat.

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