Cytochrome P450 monooxygenases from extremophiles
| dc.contributor.advisor | Smit, M. S. | |
| dc.contributor.advisor | Van Heerden, E. | |
| dc.contributor.advisor | Albertyn, J. | |
| dc.contributor.advisor | Piater, L. A. | |
| dc.contributor.author | Muller, Walter Joseph | |
| dc.date.accessioned | 2018-05-08T08:55:14Z | |
| dc.date.available | 2018-05-08T08:55:14Z | |
| dc.date.issued | 2012-01 | |
| dc.description.abstract | English: Only five CYP450s from extremophiles have been studied, while genome sequence information indicate that CYP450s are prevalent in members of the bacterial phylum Deinococcus- Thermus as well as the archaeal family Halobacteriaceae that belong to the phyulm Euryarchaeota. A property shared by these phylogenetically distant extremophiles is the production of carotenoid pigments. It became the purpose of this study to use genome sequence information to clone and study new CYP450s from the genera Thermus and Halobacterium and to explore the role of these CYP450s in pigment production. The non-pigmented thermophilic bacterium Thermus scotoductus SA-01 was screened by PCR for the presence of a cytochrome P450 monooxygenase (CYP450). No CYP450 could be found and subsequent genome sequencing confirmed this finding. However, a CYP450 gene (CYP175A) was isolated from the closely related yellow pigmented strain Thermus sp. NMX2.A 1 using oligonucleotides based on the DNA sequence of the β-carotene gene cluster from three Thermus strains. The genome sequence of T. scotoductus SA-01, revealed a ferredoxin (Fdx) and ferredoxin reductase (FNR) that were almost identical to those of Thermus thermophilus HB27. In T. thermophilus HB27 the Fdx and FNR are the native redox partners for CYP175A 1, a β- carotene hydroxylase. After heterologous expression in Escherichia coli, we attempted to hydroxylate β-carotene with the CYP450 from Thermus sp. NMX2.A 1 and the redox partners of T. scotoductus SA-01 using cell free extracts, but no products were detected. Thirty two CYP450s have been identified in the sequenced genomes of thirteen extremely halophilic archaea. Initial attempts to clone and heterologously express a CYP174A2- homologue from a HaloarcuIa LK-1 strain in E. coli and Pseudomonas fluorescens were unsuccessful. In order to study the physiological role of CYP450s in halophilic archaea and to create a strain that can be used for heterologous expression of CYP450s from halophiles CYP174A 1 was deleted from H. salinarum R1. CYP174A 1 is the only CYP450 in H. salinarum R1 and H. salinarum R1 is a genetically tractable strain. Upon culturing the wildtype and deletion strains, a difference in red pigmentation of stationary phase cultures was observed; implying that CYP174A1 might play a role in carotenoid synthesis. Microarray analyses revealed that the bop gene, which codes for bacterioopsin (BO) was severely repressed in stationary phase cultures of the deletion strain and sucrose gradient experiments showed a consequent loss of purple membrane (PM) in the deletion strain. The classical causes of bop repression e.g. insertion elements in the bop open reading frame as well as in the brz gene was ruled out by PCR screening. In addition to bop repression, the neighboring vng1459 and vng1468 genes (both part of the bopregulon) were also down regulated, but the genes normally involved in regulation of the bop gene were not affected. Currently the functions of vng1459 and vng1468 are unknown. Retinal, together with BO, is a key component of bacteriorhodopsin (BR) and essential for PM synthesis. Retinal is formed by the central cleavage of β-carotene which can be achieved by monooxygenases or dioxygenases.The Blh and Brp proteins in H. bacterium salinarum are very closely related to a confirmed bacterial 15,15'-β-carotene dioxygenase and studies have shown that deletion of both brp and blh results in complete abolishment of retinal and BR. It is therefore unlikely that CYP174A 1 plays a role in retinal biosynthesis. Another possible function for CYP174A 1 might be the hydroxylation of β-carotene, since it is known that H. salinarum strains produce hydroxylated carotenoids such as transastaxanthin, but no genes encoding typical β-carotene hydroxylases or ketolases have been identified in the genomes of H. salinarum strains. This will imply that hydroxylated carotenoids play a role in the regulation of bop. | en_ZA |
| dc.description.abstract | Afrikaans: Tot op hede is nog net vyf CYP450s vanuit ekstremofiele bestudeer ondanks die feit dat genoomvolgordebepaling data aandui dat CYP450s volop teenwoordig is in lede van die bakteriese filum Deinococcus- Thermus sowel as die archaea familie Halobacteriaceae wat behoort aan die filum Euryarchaeota. Hierdie twee ver vewante filogenetiese ekstremofiele het wel een fenotipe in gemeen naamlik: die sintese van karoteen pigmente. Die doelwit van hierdie studie was om nuwe CYP450s van die Thermus en Halobacterium genera te kloneer en bestudeer en sodoende hulle rol in pigment metabolism te ondersoek. Die pigmentlose, termofiliese bakterium Thermus scotoductus SA-01 was ge-evalueer vir die teenwoordigheid van 'n sitokroom P450 mono-oksigenase (CYP450) deur middel van PKR analise. Geen CYP450 kon gevind word nie en die daaropvolgende genoomvolgordebepaling het hierdie bevinding bevestig. Daar was egter 'n CYP450 geen (CYP175A) geïsoleer vanuit 'n geneties verwante geel gepigmenteerde Thermus sp. NMX2.A 1 stam m.b.v. priem stukke wat se ONS-volgorde gebaseer was op dié van 'n β- karoteen geen groepering uit drie verskillende Thermus stamme. Vanaf die genoomvolgorde van T. scotoductus SA-01, was daar 'n ferredoksien (Fdx) en ferredoksien reduktase (FNR) geïdentifiseer wat byna identies was aan die Fdx en FNR van Thermus thermophilus HB27. In T. thermophilus HB27 is Fdx en FNR die wilde tipe redeks-paar vir CYP175A 1 ('n β-karoteen hydroksilase). Die CYP450 van Thermus sp. NMX2.A 1 tesame met die redeks-paar van T. scotoductus SA-01 was heteroloog uitgedruk in Escherichia coli en met behulp van hierdie drie komponente was daar gepoog om β-karoteen te hidroksileer m.b.v. sel-vrye ekstrakte. Hierdie eksperimente was egter onsuksesvol. Twee-en-dertig CYP450s van dertien ekstreme halofiliese archaea is al geïdentifiseer danksy inligting verkry vanuit heel genoom DNS-volgordebepaling projekte. Tot op hede is nog geen van hierdie CYP450s bestudeer nie. Aanvanklike pogings om 'n CYP174A2 geen, vanuit 'n HaloarcuIa LK-1 stam in E. coli en Pseudomonas fluorescens heteroloog uit te druk, was onsuksesvol aangesien die uitgedrukte proteïen verkeerdelik gevou en in die onoplosbare fraksie gelokaliseer het. Pogings om die onoplosbare proteïen te denatureer en hervou, was ook onsuksesvol. Daar was besluit om eerder 'n CYP450 (CYP174A1) delesie in H. salinarum R1 te maak en dan die delesie stam se transkriptoom te bestudeer m.b.v. 'microarray' analise om sodoende die fisiologiese rol van CYP174A 1 in halofiliese archaea te bepaal. CYP174A 1 is die enigste CYP450-geen in H. salinarum en is geneties maklik manipuleerbaar. Tydens groei eksperimente van die wilde tipe en delesie stamme was daar on noemenswaardige verskil in rooi pigmentasie waargeneem tydens die stasionêre fase van groei. Hierdie fenotipe was 'n aanduiding dat die CYP174A 1-delesie dalk karoteen metabolimse in Halobacterium beïnvloed het. "Microarray' ontleding het getoon dat die bop geen, wat kodeer vir bakterio-opsien (BO), erg onderdruk was in die delesie stam en daaropvolgende sukrose-gradiënt eksperimente het hierdie resultaat bevestig aangesien daar 'n verlies van pers membraan (PM) was in die delesie stam, maar nie in die wilde tipe stam nie. Die algemene oorsake van bop onderdrukking, bv. insersie-elemente in die bop oop lees raam sowel as in die brz geen was uitgeskakel d.m.v. PKR analise. Tesame met bop, was twee na-burige gene naamlik vng1459 en vng1468 ook af-waarts gereguleer. Die funksie van vng1459 en vng1468 is onbekend. Gene wat deel vorm van die "bop-regulon' wat gewoontlik gereguleer word tydens die stasionêre fase van groei was ongeaffekteerd in die delesie stam. Retinal tesame met BO, is 'n belangrike komponent van bakteriorodopsien (BR) en gevolglik, PM sintese. Retinal word gevorm deur die sentrale splyting van β-karoteen en die hierdie tipe splyting kan bewerkstellig word deur on mono-oksigenase sowel as 'n dioksigenase ensiem. In H. salinarum R1 is daar onBlh proteïen wat baie nou verwant is aan 'n bakteriële 15, 15°-β-karoteen di-oksigenase sowel as on Brp proteïen met on soortgelyke funksie. Studies het getoon dat die delesie van blh geen invloed het op instra-sellulêre retinal vlakke nie. Delesie van brp veroorsaak on afname in retinal vlakke sowel as BR vlakke, máár die delesie van beide brp sowel as blh veroorsaak 'n algehele verlies van retinal en BR en gevolglik PM. Dit is dus onwaarskynlik dat CYP174A1 In rol speel in retinal biosintese. 'n Ander moontlike funksie van CYP174A 1 is die hidroksilering van β-karoteen aangesien dit bekend is dat H. salinarum stamme gehidrosileerde pigmente sintetiseer soos bv. trans-astaxantin. Tot op hede is die gene wat kodeer vir tipiese β-karoteen hidroksilases of ketolases nog nie in die genome van H. salinarum stamme gevind nie. Hierdie dui op 'n moontlikheid dat hierdie gehidroksileerde karotene 'n rol kan speel in die regulering van bop. | en_ZA |
| dc.description.sponsorship | National Research Foundation (South Africa) | en_ZA |
| dc.description.sponsorship | TIA/BIOPAD | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11660/8252 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | CYP175A 1 | en_ZA |
| dc.subject | Ferredoxin | en_ZA |
| dc.subject | Ferredoxin reductase | en_ZA |
| dc.subject | β-carotene | en_ZA |
| dc.subject | CYP174A 1 | en_ZA |
| dc.subject | Deletion | en_ZA |
| dc.subject | Microarray | en_ZA |
| dc.subject | Purple membrane | en_ZA |
| dc.subject | Extreme environments -- Microbiology | en_ZA |
| dc.subject | Cytochrome P-450 | en_ZA |
| dc.subject | Monooxygenases | en_ZA |
| dc.subject | Thesis (Ph.D. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2012 | en_ZA |
| dc.title | Cytochrome P450 monooxygenases from extremophiles | en_ZA |
| dc.type | Thesis | en_ZA |