Identification of the genes encoding enzymes involved in the synthesis of the biopolymer paramylon from Euglena gracilis

Mackay, Stephen (2010-12)

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2010.

Includes bibliography.

Title page: Dept. of Genetics, Faculty of Science

Thesis

ENGLISH ABSTRACT: Recent advances in medical pharmacology have identified the immune-potentiating effects of β-1,3-glucans on mammalian immune systems. Extensive research has identified and described the mechanisms of action and receptor binding specificity of different β-1,3-glucans as well as their structural and functional relationships. Molecular mass, solubility, structural order, degree of branching as well as chemical modification all determine the effectiveness of the β-1,3-glucan immune-modulating activities, which typically include; macrophage activation, antibody adjuvant activities, reduction of LDL-cholesterol, leukocyte mitogenic activities, cytokine and chemokine production as well as antiviral and antitumor activities. Currently β-1,3-glucans have been sold commercially under the name β-glucan, mostly in the form of Betafectin, a genetically modified yeast derived β-1,3-glucan. Recent studies of different β-1,3-glucans have identified the pharmacological activities of paramylon, a Euglena derived β-1,3-glucan. Although paramylon has relatively low immune-stimulating activities, chemical modification of the paramylon granule increased immune-potentiation with specific antimicrobial and anti-HIV activities. Due to these specific immune-potentiating activities, paramylon is novel in terms of both structure as well as functional activity. In terms of biotechnological application, paramylon is greatly favoured as it is synthesized as an insoluble membrane bound granule in the cytosol of Euglena where most plant and fungal β-1,3-glucan synthases are cell membrane bound highly regulated multifunctional complexes, synthesizing β-1,3-glucan as cell wall components. Due to the novel granular nature of paramylon, expression in other systems with genetic modification could potentially further increase immuno-potentiating activities. In this study, different approaches were attempted in order to identify the genes involved in paramylon synthesis including; constructing and screening a Euglena gracilis cDNA library, sequence analysis of the purified proteins as well as transcription analysis of the sequenced transcriptome and genome of E. gracilis. Putative candidates that encode subunits of the paramylon synthase complex have been identified.

AFRIKAANSE OPSOMMING: Onlangse vordering in mediese farmakologie het die immuun-stimulerende effek van β-1,3-glukane op die soogdier immuunsisteem geïdentifiseer. Intensiewe navorsing het die meganisme van die werking en reseptor bindingspesifisiteit van verskillende β-1,3-glukane, asook hulle struktuur en funksionele verhoudings, geïdentifiseer. Die molekulêre massa, oplosbaarheid, strukturele orientasie, mate van vertakking asook chemiese modifikasies bepaal almal die effektiwiteit van die β-1,3-glukaan immuun-modulerende aktiwiteite. Tipiese immuno-moduleringsaktiwiteite sluit makrofaag aktivering, teenliggaampie adjuvant aktiwiteite, verlaging van LDL-cholesterol, leukosiet mitogeniese aktiwiteite, sitokien en chemokien produksie asook anti-virale en antitumor aktiwiteite in. Huidiglik word β-1,3-glukane onder die naam β-glukaan verkoop meestal in die vorm van Betafectin, ‘n geneties gemodifiseerde gis wat van β-1,3-glukaan afkomstig is. Onlangse studies van verskillende β-1,3-glukane het die farmakologiese aktiwiteit van paramylon, ‘n Euglena afkomstige β-1,3-glukaan geïdentifiseer. Alhoewel paramylon relatiewe lae immuun-stimulerende aktiweite toon, verhoog chemiese modifikasies van die paramylon granules immuun-stimulering, spesifiek die anti-mikrobiese en anti-MIV aktiwiteite. Weens hierdie spesifieke immuun-stimulerende aktiweite, word paramylon as nuut beskou veral in terme van beide struktuur asook funksionele aktiwiteit. In terme van biotegnologiese toepassing, verkry paramylon voorkeur aangesien dit as ‘n onoplosbare membraangebonde granule in die sitosol van Euglena gesintetiseer word terwyl meeste plant en fungus β-1,3-glukaan sintases hoogs gereguleerde multifunksionele selmembraan gebonde komplekse is wat β-1,3-glukaan asook ander selwand komponente sintetiseer. Weens die unieke granulêre natuur van paramylon, sal uitdrukking in ander sisteme ‘n moontlike industrie skep waar deur die transgeniese uitdrukking van granulêre paramylon verdere verbetering van die immuun-stimulerings aktiwiteite kan lei. In hierdie studie is verskillende benaderings aangewend om die gene wat by paramylon sintese betrokke is te identifiseer, dit sluit in die konstruksie en sifting van ‘n E. gracilis cDNS biblioteek, aminosuur volgorde analise van gesuiwerde proteiene asook die transkripsionele analise van die volgorde van die transkriptoom en genoom van E. gracilis. Moontelike kandidate wat vir die subeenhede van die paramylon syntase kompleks kodeer is geïdentifiseer.

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