Smit, M. S.Nthangeni, M. B.Ramagoma, Faranani2017-03-152017-03-152011-03http://hdl.handle.net/11660/5819English: Yarrowia lipolytica is a non-conventional yeast which is considered to be suitable for production of industrially important proteins at commercial scale. The yeast is non pathogenic and is generally regarded as safe and as such can be used to produce biotechnological products for human consumption and applications. The genetic tools for the manipulation of the yeast have been developed over the years. This includes chemical and genetic based mutagenesis techniques to develop and isolate Y. lipolytica strains with enhanced properties in the production of endogenous and heterologous proteins. Protein expression systems comprised of a variety of inducible and constitutive promoters are available; secretion signals for extracellular production of proteins and a choice of replicative or integrative expression systems have also been developed. Auxotrophic and antibiotic based selectable markers are available some of which result in single or multiple copies of the integrative expression cassettes containing the target gene. The Y. lipolytica yeast can utilise a wide variety of carbon sources and its fermentation processes in bioreactors are well established. The property of Y. lipolytica as a prolific producer of the endogenous extracellular Lip2p was exploited for the development of an expression system for production of therapeutic peptides. To this end, the 38 amino acid long RANTES I peptide with therapeutic applications in the treatment of HIV/AIDS was selected as a model peptide for co-expression with Lip2p. The sequence encoding the peptide was cloned downstream of the complete LIP2 gene. The therapeutic peptide was separated from the LIP2 gene by the sequences encoding for 6X His and the DDDDK sequence recognized by the enterokinase proteolytic enzyme. The expression cassette was under the hp4d quasi constitutive promoter with the rDNA as a target for multi copy integration using the defective Ura3d4 integration cassette. The amount of RANTES I produced by the expression system was estimated to be 0.274 μg.L-1 as determined by the CCL5/RANTES immunoassay method. The RANTES I peptide produced in this study was reactive to polyclonal antibodies raised against human RANTES and functionality assays showed that the peptide inhibited the binding of the pseudoviral particles to the TZM-bl cells through the CD4/CCR5 receptors. Another section of the study sought to identify genetic elements responsible for enhanced secretion of the extracellular Lip2p in Y. lipoyltica. Lipase hyperproducing mutants were generated using the zeta based mutagenesis system which integrates randomly within the Y. lipolytica genome. One mutant denoted Y. lipolytica Y12 which showed lipase hyperproducing phenotype was found to be disrupted in the function of the GPI7 gene, a protein encoding a polypeptide of 830 amino acids. The GPI7 protein is 33% identical to the S. cerevisiae GPI7 known to play a role in the maintenance of the yeast cell wall integrity. Consistent with the disruption of cell wall integrity through deletion of GPI7, the Y. lipolytica strain was more sensitive to cell wall hydrolysing enzymes, had defects in cell separation and a daughter cell-specific growth defect at the non-permissive temperature and exhibited hypersensitivity to Calcofluor white and Congo red which are known to interfere with yeast cell wall composition. While the Y. lipolytica Yl12 and the ”wild-type” Po1d strains grew similarly in shake flask cultures, the Y. lipolytica Yl12 strain produced 7 times more extracellular lipase activity in liquid shake flask cultures than the control Y. lipolytica Po1d strain. To investigate the mode of GPI7 action in the enhancement of protein production, a Y. lipolytica YlHmA25 strain expressing intracellular epoxide hydrolase under control of the hp4d promoter was deleted for the GPI7 gene to create a Y. lipolytica YlHmA25ΔGPI7 strain. The YlHmA25ΔGPI7 strain contained epoxide hydrolase activity in the extracellular in contrast to the Y. lipolytica YlHmA25. In addition, the Y. lipolytica YlHmA25ΔGPI7 accumulated more total extracellular protein than Y. lipolytica YlHmA25 an indication that the strain accumulates more proteins extracellulary in comparison with the Y. lipolytica YlHmA25 strain. Taken together, the results suggested that the disruption of GPI7 affects the integrity of the yeast cell wall which in turn results in leakage or “enhanced” secretion of proteins to the extracellular. The challenge in developing a host system with broad applications in heterologous production of proteins including therapeutics lies in the elimination of the endogenous hyper-mannosylated yeast glycans. The OCH1 gene encoding a protein with α-1,6- mannosyltransferase activity was deleted from the GPI7 deleted mutant of Y. lipolytica. The OCH1 and GPI7 double deletion mutant of Y. lipolytica showed similar growth patterns to the GPI7 null mutant. Glycan analyses revealed that the most abundant glycans are the Man8GlcNAc2 and Man9GlcNAc2 which are acceptable substrates of the Trichoderma reesei α-1,2-mannosidase I the enzyme widely used in the engineering of yeast glycosylation pathway to produce Man5GlcNAc2, a prerequisite intermediate for the synthesis of complex glycans in human cells. The OCH1 and GPI7 double deletion mutant of Y. lipolytica is a good candidate for further glycoengineering to enable production of extracellular therapeutic proteins with human-like glycans.Afrikaans: Yarrowia lipolytica is ‘n onkonvensionele gis wat beskou word as geskik vir die produksie van proteïene van industriële belang op kommersiële skaal. Die gis is niepatogenies en word algeneem beskou as veilig (generally regarded as safe) en kan gevolglik gebruik word vir die produksie van biotegnologiese produkte vir menslike verbruik. Die genetiese gereedskap vir die manupilasie van gis is oor die jare ontwikkel. Dit sluit in chemies- en geneties-gebasseerde mutagenese tegnieke om Y. lipolytica rasse te ontwikkel en isoleer met verbeterde eienskappe vir produksie van endogene en heteroloë proteïene. Proteïenuitdrukkingsisteme, bestaande uit ‘n verskeidenheid induseerbare en konstitutiewe promotors, is beskikbaar; sekresieseine vir ekstrasellulêre produksie van proteïene asook ‘n keuse van replikatiewe of integrerende uitdrukkingsisteme is ook al ontwikkel. Ouksotrofiese en antibiotika gebasseerde selekteerbare merkers is beskikbaar, waarvan sommige lei tot integrasie van enkel of veelvuldige kopieë van die integrerende uitdrukkingskasette wat die teikengeen bevat. Die Y. lipolytica gis kan ‘n wye verskeidenheid koolstofbronne benut en het ‘n goed vasgestelde fermentasieproses in bioreaktors. Die eienskap van Y. lipolytica as ‘n effektiewe produseerder van endogeniese ekstrasellulêre Lip2p is gebruik vir die ontwikkeling van ‘n uitdrukkingsisteem vir die produksie van terapeutiese proteïene. Vir hierdie doel is die 38 aminosuur lange RANTES I peptied, met terapeutiese toepassings in die behandeling van HIV/VIGS, gekies as ‘n model peptied vir ko-uitdrukking met die Lip2p. Die volgorde wat kodeer vir die peptied is stroom-af van die volledige LIP2 geen gekloneer. Die terapeutiese peptied is geskei van die LIP2 geen deur die volgordes wat kodeer vir 6X His en die DDDK volgorde wat deur enterokinase ensiem herken word. Die uitdrukkingskaset is onder beheer van die hp4d quasi-konstitutiewe promotor met die rDNA as ‘n teiken vir veelvuldige integrasie deur gebruik te maak van die defektiewe Ura3d4 integrasiekaset. Die hoeveelheid RANTES I geproduseer deur hierdie uitdrukkingsisteem is beraam as 0.274 μg.L-1 soos bepaal deur die CCL5/RANTES immuno-essai metode. Die RANTES I peptied geproduseer in hierdie studie het reageer met poliklonale teenliggame opgewek teen menslike RANTES en aktiwiteitstoetse het gewys die peptied verhoed binding van die pseudovirale partikels aan die TZM-bl selle deur die CD4/CCR5 reseptore. ‘n Ander gedeelte van die studie het beoog om genetiese elemente te identifiseer wat verantwoordelik is vir verbeterde sekresie van die ekstrasellulêre LIP2 in Y. lipolytica. Lipase hiperproduserende mutante is gegenereer deur die zeta-gebasseerde mutagenese sisteem te gebruik wat ewekansig in die Y. lipolytica genoom integreer. Een mutant, genaamd Y. lipolytica Y12, wat die lipase hiperproduserende fenotipe toon is gevind om ontwrig te wees in die funksie van die GPI7 geen wat kodeer vir ‘n polipeptied van 830 aminosure. Die ontwrigte GPI7 proteïen het ‘n 33% similaritiet met die Saccharomyces cerevisiae GPI7 wat bekend is om ‘n rol te speel in die instandhouding van die integriteit van die gis selwand. Ooreenstemmend met die ontwrigting van die selwand integriteit deur verwydering van GPI7 was die Y. lipolytica ras meer sensitief vir selwand hidroliserende ensieme, het defekte gehad in selskeiding met ‘n dogtersel-spesifieke groeidefek by die nie-permissiewe temperatuur en hipersensitiwiteit getoon teenoor Calcofluor wit en Kongo rooi, wat bekend is om in te meng met die samestelling van die gis selwand. Alhoewel Y. lipolytica Y12 en wilde tipe Po1d eenders gegroei het in skudfleskulture, het Y12 sewe keer meer extrasellulêre aktiwiteit getoon in hierdie kulture as die kontrole, Po1d. Om die werking van GPI7 in die verhoging van proteïen produksie te ondersoek is ‘n Y. lipolytica YlHmA25 ras, wat intrasellulêre epoksied hidrolase onder beheer van die hp4d promotor produseer, se GPI7 geen verwyder om ‘n Y. lipolytica YlHmA25ΔGPI7 ras te produseer. Die YlHmA25ΔGPI7 ras het ekstrasellulê epoksied hidrolase aktiwiteit getoon in teenstelling met Y. lipolytica YlHmA25. Verder het Y. lipolytica YlHmA25ΔGPI7 meer totale ekstrasellulêre proteïen opgehoop as Y. lipolytica YlHmA25, ‘n aanduiding dat hierdie ras meer proteïene ekstrasellulêr ophoop as Y. lipolytica YlHmA25. Saamgevat dui die resultate aan dat die ontwrigting van GPI7 die integriteit van die gis selwand beïnvloed wat gevolglik ‘n lekkasie of “verhoogde” sekresie van proteïene na die ekstrasellulêre omgewing veroorsaak. Die uitdaging in die ontwikkeling van ‘n gasheersisteem met wye toepassings vir heteroloë produksie van proteïene, insluitende terapeutiese proteïene, lê in die eliminering van endogene hiper-mannosileerde gis glikane. Die OCH1 geen, wat kodeer vir ‘n proteïen met α-1,6-mannosieltransferase aktiwiteit, was verwyder van die GPI7-verwyderde mutant van Y. lipolytica. Die OCH1 en GPI7 dubbel-delesie mutant het eenderse groeipatrone getoon as die GPI7 nul mutant. Glikaan analise wys Man8GlcNAc2 and Man9GlcNac2 is die volopste glikane, wat aanvaarbare substrate is vir Trichoderma reesei α-1,2-mannosidase I, die ensiem wat algeneem gebruik word in die manipulasie van die gis glikosileringsweg om Man5GlcNAc2 te produseer, ‘n vereiste tussenganger vir die sintese van komplekse glikane in menslike selle. Die OCH1 en GPI7 dubbel-delesie mutant van Y. lipolytica is ‘n goeie kandidaat vir verdere gliko-manipulasie vir die produksie van ekstrasellulêre terapeutiese proteïene met menslike glikane.enThesis (Ph.D. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2011SaccharomycetaceaeYeast fungi -- Genetic engineeringProteins -- SynthesisFungal proteins -- Therapeutic usePeptides -- SynthesisThesisUniversity of the Free State