Smit, M. S.Albertyn, J.Pohl-Albertyn, C. H.Opperman, D. J.Kuloyo, Oluwasegun Olalekan2015-10-282015-10-282014-06http://hdl.handle.net/11660/1475English: Cytochrome P450 monooxygenases (CYP450) are heme-thiolate proteins which are present in all biological kingdoms. They catalyse the addition of a single oxygen atom to a non-activated carbon atom. The metabolic system of filamentous fungi is maintained by numerous enzymes catalysing the production of bioactive compounds. Genome sequencing projects have revealed the presence of a large collection of CYP450 families within the fungal kingdom. Although some fungal CYP450s are involved in essential reactions such as xenobiotic degradation and membrane ergosterol synthesis, there is still a large collection of uncharacterized fungal CYP450s. The aim of this study was to accomplish and optimize expression and then investigate the substrate specificity of the self-sufficient Aspergillus terreus CYP505E3 as well as five CYP450s from Cryptococcus neoformans. In the case of the Cryptococcus CYP450s the ultimate aim was to establish their possible role in Cryptococcal prostaglandin production. Self-sufficient A. terreus CYP450s were reported by Vatsyayan and co-workers (2008) to show activity towards alkanes, alkane derivatives, alcohols, aromatic compounds, organic solvents and steroids. The genome of A. terreus comprises 125 CYP450s with two probable self-sufficient CYP450s namely CYP505A19 and CYP505E3. Omissions within the CYP505A19 sequence makes it unlikely to be active leaving the CYP505E3 as probably the only functional self-sufficient CYP450 in A. terreus. The nucleotides on the N-terminus of A. terreus CYP505E3 were modified to enhance the expression level in Escherichia coli. The recovery of CYP505E3 in the soluble fraction of cell free extract was significantly improved by using expression conditions established through a number of Plackett-Burman experiments. A low temperature, low FeCl3, high culture volume and prolonged incubation time contributed to the improved recovery. The CYP505E3 within the whole cells catalysed sub-terminal hydroxylation of hexylbenzoic acid (HBA) to produce ω-1 OH-HBA, ω-2 OH-HBA and ω-4 OH-HBA. However, no hydroxylated products were produced from the other substrates tested. The activity of self-sufficient A. terreus CYP505E3, towards HBA was similar to that CYP505E3, towards HBA was similar to that of Bacillus megaterium CYP102A1 and Fusarium oxysporum CYP505A1 indicating that CYP505E3 is probably also a fatty acid hydroxylase. The opportunistic yeast pathogen Cryptococcus neoformans is known to produce authentic immunomodulatory prostaglandin E2 from arachidonic acid (AA). However, because of the absence of cyclooxygenase (COX) and lipoxygenase (LOX) homologues in its genome, the mechanism used by the organism to produce prostaglandins remains unclear. Five CYP450s and a CYP450 reductase (CPR) are present within the genome of the organism. The probable roles of these CYP450s in Cryptococcal prostaglandin production were investigated. The C. neoformans CYP450 genes were cloned into pET28 and into a pETDuet plasmid into which the CPR had already been cloned. These plasmids were used to investigate the expression of the CYP450s as well as the CPR using E. coli BL21 (DE3) as well as strains carrying plasmids for the co-expression of tRNAs that are rare in E. coli or for the molecular chaperones groES and groEL. The CO difference spectra of all the cloned CYP450s did not produce the expected absorbance peak at 450 nm. However, SDS-PAGE analysis indicated possible expression with protein bands which corresponded with the expected sizes. Three of the CYP450s gave acceptable expression when they were expressed using just pET28 without co-expression of the additional tRNA or molecular chaperones. The co-expression of C. neoformans CYP450 with groES-groEL molecular chaperones eliminated low molecular weights protein bands produced in the case of the CYP450s and the CPR. The C. neoformans CYP450s co-expressed with the CPR and groES-groEL molecular chaperones had barely detectable or no activity when tested with arachidonic acid and hexylbenzoic acid. The challenges encountered with heterologous expressions of C. neoformans CYP450s in an E. coli host, could be resolved using an alternative host such as Saccharomyces cerevisiae.Afrikaans: Sitochroom P450 monoöksigenases (CYP450’s) is heem-tiolaat proteïene wat voorkom in alle biologiese koninkryke. Hulle kataliseer die toevoeging van ʼn enkele suurstofatoom aan ʼn nie-geaktiveerde koolstofatoom. Die metaboliese sisteem van filamentagtige fungi word in stand gehou deur menigte ensieme wat die produksie van bio-aktiewe verbindings kataliseer. Genoom basispaaropeenvolgingbepalings projekte het gedui op die teenwoordigheid van ʼn groot versameling CYP450 families in die fungale koningryk. Alhoewel sekere fungale CYP450’s betrokke is in essensiële reaksies soos die afbreek van xenobiotiese produkte en membraan ergosterol sintese, is daar steeds ʼn groot hoeveelheid ongekarakteriseerde fungale CYP450s. Die doel van hierdie studie was om die self-onderhoudende Aspergillus terreus CYP505E3, asook vyf CYP450’s van Cryptococcus neoformans, uit te druk en die uitdrukking te optimiseer, sowel as om hul substraatspesifisiteit te bepaal. In die geval van die Cryptococcus CYP450’s was die uiteindelike doel om hul moontlike rol in Kriptokokkale prostaglandien produksie te bepaal. Vatsyayan en medewerkers (2008) het aangedui dat self-onderhoudende A. terreus CYP450’s aktief was teenoor alkane, alkaan-afgeleide produkte, alkohole, aromatiese verbindings, organiese oplosmiddels en steroïdes. Die A. terreus genoom bevat 125 CYP450’s met twee moontlike self-onderhoudende CYP450’s, naamlik CYP505A19 en CYP505E3. Weglatings binne die basispaar volgorde van CYP505A19 laat dit heel waarskynlik onaktief, menende CYP505E3 is heel waarskynlik die enigste funksionele, self-onderhoudende CYP450 in A. terreus. Die N-terminale nukleotides van die A. terreus CYP505E3 was verander om die uitdrukkingsvlak in Escherichia coli te verhoog. Die vlak van oplosbare CYP505E3 in die sel-vrye ekstrak was noemenswaardig verhoog deur die uitdrukking kondisies te optimiseer met behulp van Plackett-Burman eksperimente. ʼn Lae temperatuur, lae FeCl3, hoë kultuurvolume en verlengde inkubasie tyd het bygedra tot die verhoogde vlak van oplosbare CYP505E3 Heelselekstrakte wat CYP505E3 bevat het, het die subterminale hidroksilasie van heksielbensoësuur (HBA) gekataliseer om ω-1 OH-HBA, ω-2 OH-HBA en ω-4 OH-HBA te produseer. Geen gehidroksileerde produkte is geproduseer van enige ander getoetse substrate nie. Die aktiwiteit van die self-onderhoudende A. terreus CYP505E3 teenoor HBA was vergelykbaar met die van Bacillus megaterium CYP101A1 en Fusarium oxysporum CYP505A1, wat daarop dui dat CYP505E3 heel waarskynlik ook ʼn vetsuur hidroksilase is. Die opportunistiese gispatogeen Cryptococcus neoformans produseer die immuunmoduleerder prostaglandien E2 vanaf arachidoonsuur (AA). As gevolg van die afwesigheid van siklooksigenase (COX) en lipoksigenase (LOX) homoloë in die genoom, is die meganisme van prostaglandien produksie deur die organisme onbekend. Die genoom bevat vyf CP450’s en ‘n CYP450 reduktase (CPR). Die waarskynlike rolle van hierdie CYP450’s in prostaglandien produksie is ondersoek. Die C. neoformans CYP450 gene was ingekloneer in die pET28 plasmied, asook in die pETDuet plasmied waarin die CPR geen reeds teenwoordig was. Die plasmiede was gebruik om die uitdrukking van die CYP450’s, asook die CPR, in E. coli BL21(DE3) te ondersoek, asook stamme wat plasmiede bevat vir die gesamentlike uitdrukking van tRNA’s wat skaars is in E. coli, en vir die molekulêre chaperones groES en groEL. Die CO verskil spektra van die gekloneerde CYP450’s het nie die verwagte absorbansiepiek by 450nm getoon nie. Tog het SDS-PAGE analise gedui op moontlike uitdrukking, aangesien proteïen bande teenwoordig was met die verwagte groottes. Drie van die CYP450’s het aanvaarbare uitdrukkingsvlakke gehad deur slegs die pET28 vektor te gebruik, sonder gesamentlike uitdrukking met addisioneel toegevoegde tRNA molekules of molekulêre chaperones. Uitdrukking van die C. neoformans CYP450 saam met die groES-groEL molekulêre chaperones het die lae molekulêre massa proteïenbande wat geproduseer is, geëlimineer in die geval van die CYP450’s en die CPR. Die C. neoformans CYP450’s wat saam uitgedruk is met die CPR en groES-groEL molekulêre chaperone, het baie lae of geen aktiwiteit getoon teenoor arachidoonsuur en heksielbensoësuur nie. Die uitdagings wat gebied word deur die heteroloë uitdrukking van die C. neoformans CYP450s in E. coli kan moontlik oorkom word deur ʼn alternatiewe gasheer te gebruik, soos Saccharomyces cerevisiae.enAspergillus terreusCryptococcus neoformansCytochrome P450Self-sufficient CYP450Hexylbenzoic acidGroES-groELArachidonic acidCYP505E3HydroxylationMonooxygenases.Dissertation (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2014DissertationUniversity of the Free State