Albertyn, J.Smit, M. S.Müller, Walter Joseph2015-11-112015-11-112006-012006-012006-01http://hdl.handle.net/11660/1600English: The dimorphic fungus Yarrowia lipolytica is an n-alkane assimilating yeast. During nalkane oxidation toxic fatty aldehydes are formed that are further oxidized by fatty aldehyde dehydrogenases (FALDH) to carboxylic acids that then enter the β-oxidation pathway. Very little research emphasis has been placed on FALDHs in yeasts and their precise role in n-alkane metabolism. This study aimed at contributing to the limited knowledge of yeast FALDHs and in particular the four putative Y. lipolytica FALDHs (YlFALDH1 - 4) that were recently identified in the fully sequenced genome of Y. lipolytica E150. The contribution made from this study to YlFALDHs was with reference to their promoter expression and subcellular localization. The promoter and terminator of each YlFALDH was initially used to construct reporter cassettes in conjunction with β-galactosidase (lacZ) by utilizing the Sticky-end PCR (SEP) and Enzyme-free cloning methods. These two methods proved to be unsuitable for the expression study. The promoter region of each YlFALDH was then cloned into the pINA781 expression vector containing lacZ to study the expression further. With the aid the pINA781 integrative vector and qualitative plate assays, with 5-bromo-4-chloro-3- indolyl-β-D-galactosidase (X-gal), it was observed that the promoters of YlFALDH1 and 2 were inducible by dodecane and hexadecane but not by oleic acid, glucose or glycerol. The promoter of YlFALDH2 also seemed to display the same level of transcriptional strength as the inducible POX2 promoter. Induction of the YlFALDH3 and 4 promoters was not observed. Localization of the YlFALDH proteins was studied with the aid of green fluorescent protein (GFP) from Aequorea victoria and putative localization sequences (LS) from each YlFALDH isozyme. The putative Y. lipolytica LSs comprised of the last 150 bp of the COOH-terminal of the YlFALDH proteins. These LSs were modeled from Rattus norvegicus FALDH that possesses a 35 amino acid hydrophobic protein anchor at its COOH-terminal. For localization studies, chimerical JMP5 molecules were created with an inducible ICL1 promoter, GFP and putative Y. lipolytica LS from each isozyme. Chimerical molecules were also constructed with a pKOV136 vector that contained a constitutive TEF promoter, a GFP-LS fragment (from a JMP5-chimera) and LIP2 terminator. No fluorescence was observed with epifluorescence or confocal laser microscopy when either of the JMP5- or pKOV136-chimeras were transformed into Y. lipolytica E150 and Po1g respectively. Consequently the subcellular localization could not be identified.Afrikaans: Die dimorfiese fungus Yarrowia lipolytica is ‘n n-alkaan assimilerende gis. Tydens oksidasie van n-alkane word toksiese vet aldehiede gevorm wat verder ge-oksideer word deur vet aldehied dehidrogenase (VALDH) om karboksielsure te vorm wat dan die β- oksidasie weg binne gaan. Betreklik min klem is in die verlede geplaas op VALDHs in giste en hulle presiese rol in die metabolisme van n-alkane. Hierdie studie het gepoog om ’n bydrae te lewer tot die beperkte kennis van gis VADLH deur die vier putatiewe Y. lipolytica VADLHs (YlVADLH1-4) te bestudeer wat onlangs in die volkome bepaalde nukleotied volgorde van Y. lipolyitca E150 geïdentifiseer is. Die bydrae tot hierdie beperkte kennis van gis VADLH is gemaak m.b.t. uitdrukkings studies van die YlVALDH gene en die sellulêre lokalisering van die YlFALDH proteïene in Y. lipolytica. Die promoter en termineerder van elke YlFALDH was aanvanklik gebruik om rapporterings kassette te konstrueer m.b.v. die β-galaktosidase geen (lacZ) deur die ”Sticky-end PCR” (SEP) en die ”Enzyme-free cloning” metodes te gebruik. Die laasgenoemde twee metodes het nie herhaalbare en bevredigende resultate gelewer nie. Die promoter en termineerder van elke YlVALDH was toe gekloneer in die pINA781 uitdrukkings vektor (wat ’n lacZ geen bevat) om die uitdrukking van hierdie YlVALDHs verder te bestudeer. Met behulp van die pINA781 uitdrukkings vektor en kwalitatiewe plaat proewe, met 5-brom-4-chloro-3-indoliel-β-D-galaktosidase (X-gal), het ons vasgestel dat die promoters van YlVALDH1 en 2 geïnduseer word deur dodekaan en heksadekaan, maar nie deur oleïensuur, glukose of gliserol nie. Die promoter van YlVALDH2 blyk dieselfde vlak van uitdrukkings sterkte te vertoon as die van die sterk induseerbare POX2 promoter. Induksie van die YlVALDH3 en YlVALDH4 promoters kon nie waargeneem word nie. Lokalisering van die YlVALDH proteiene was bestudeer m.b.v. ”Green Fluorescent Protein” (GFP) van die jellievis Aequorea victoria en putatiewe lokaliserings volgordes (LV) van elke YlVALDH wat gekloneer was aan die 3′-end van die GFP. Die putatiewe Y. lipolytica LVs het bestaan uit die laaste 150 basis pare van die COOH-terminus van die YlVALDH gene. Hierdie LVs van die YlVALDHs was gemodelleer op grond van die laaste 35 amino sure (aan die COOH-terminus) van Rattus norvegicus se VALDH wat dien as ’n hidrofobiese anker vir die proteïen in die membraan van die ER. Die lokalerings studies was uitgevoer met chimere van JMP5 en pKOV136. Die JMP5- chimere het bestaan uit die induseerbare ICL1 promoter, die ”GFP” en ’n putatiewe YlVALDH LV. Die pKOV136 –chimere het bestaan uit ’n konstitiewe TEF promoter, ’n ”GFP”-LV fragment (van ’n JMP5-chimeer) en ’n LIP2 termineerder. Geen fluoresensie van die ”GFP” kon waargeneem word toe beide die JMP5- en pKOV136-chimere onderskeidelik in Y. lipolytica E150 en Po1g getransformeer is nie. Gevolglik kon lokalisering ook nie geïdentifiseer word nie.enDissertation (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2006Yeast fungiAldehyde dehydrogenaseFALDH promotersN-alkanesExpression studiesFatty aldehyde dehydrogenase (FALDH)Yarrowia lipolyticaSubcellular localizationLocalization sequences (LS)DissertationUniversity of the Free State