Litthauer, D.Van Heerden, E.Piater, L. A.Abbai, Nathlee Samantha2016-09-052016-09-052009-05http://hdl.handle.net/11660/4054English: The construction and screening of gene libraries prepared from DNA directly isolated from environmental samples is a recent and powerful tool for the discovery of new enzymes of biotechnological interest (Gabor et al., 2004). Standard methods based on the screening of isolated microorganisms are inherently limited to the tiny fraction of cultivable microbial species (<1%); environmental gene banks in principle provide access to the entire sequence space present in nature (Handelsman et al., 1998). Environmental libraries allow the screening of functional classes of genes from thousands of organisms and research in this area will provide an essential backdrop for understanding evolution and biochemical pathways (Rajendran et al., 2008). The metagenomics approach has been shown to be an efficient method for obtaining novel biocatalysts and useful genes from uncultured microorganisms from diverse environments. Before proceeding to the metagenome analysis, we constructed genomic libraries from a South African deep mine isolate Geobacillus thermoleovorans GE-7. The library was screened for lipolytic activity on LB tributyrin (TLB). Active clones were sequenced using 454 technology, and the sequencing results revealed the presence of the lipA and GDSL lipases, of which the latter has not yet been characterized in this organism. This family displays the characteristic G-D-S-L motif instead of the conventional G-X-S-X-G (Xdenotes any amino acid) motif. GDSL lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity and regiospecificity. They have potential for use in the hydrolysis and synthesis of ester compounds that are of interest in pharmaceutical, food, biochemical and the biological sector (Akoh et al., 2004; Lämmle et al., 2007). In addition, genes associated with fatty acid degradation, different glycolytic activities, lipolytic activity, spore germination, proper protein folding, antibiotic resistance and the cell wall were also identified in the active clones. Some of the genes identified may also aid in understanding how this organism had adapted to the environment from which it was isolated from.Biofilm collected from the Beatrix gold mine was selected for the metagenomic studies. We performed a diversity assessment of the biofilm by cloning and sequencing of the 16S (bacterial and archaeal) and 18S eukaryotic ribosomal DNA. Phylogenetic assessment of the bacterial clones indicated that clonal sequences were affiliated with at least 5 phyla of the domain bacteria.Sequences allocated to the phyla that are present in the bacterial library, have been reported to be isolated from various environments including marine environments e.g. the Sargasso Sea and sea floor basalts, sub-surface ground water, limestone caves, tar pits, alkaliphilic hot springs, mine drainage sites and biofilms (Cho and Giovanni, 2003; Kim and Crowley, 2007; Ikner et al., 2007; and Stepanauskas and Sieracki, 2007). According to the rarefaction analysis, of the 37 clones analyzed 29 different OTUs were observed. However, the rarefaction results indicate that only a portion of the richness in the bacterial community (at the ³97% sequence identity level) was surveyed with the 16S clones sequenced as the curve did not reach an asymptote. Sequence data obtained from the archaeal clonal library showed sequence identities with that of uncultured archaea present in the database, in particular a single uncultured archaeal library from a marine sample. This could be attributed to the fact that there is limited sequence data on archaea present in the databases because only 4 taxonomic groups of the domain are known thus far (Baker et al., 2003).The metagenome was screened by the sequenced-based approach for cytochrome P450 monooxygenases, in particular the CYP153 family (terminal hydroxylases and long chain alkane degraders). Cloning and sequencing of the CYP153 PCR products, revealed the presence of this family of enzymes in the metagenome P450’s are involved in a plethora of metabolic processes, both anabolic and catabolic, and collectively interact with an enormous variety of substrates (De Mot and Parret, 2002). CYP’s in bacteria are involved in the biosynthesis of secondary metabolites such as antibiotics and in the utilization of hydrophobic low molecular weight compounds such as alkanes and aromatics (Kubota et al., 2005). The biocatalytic production of the anticancer drug perillyl alcohol from limonene has been reported to involve a CYP153 cytochrome from Mycobacterium sp. thereby indicating the potential application of this enzyme in the clinical setting (Urlacher and Eiben, 2006).For the function-based approach, both small and large-insert metagenomic libraries were constructed. The libraries were screened for lipolytic, amylase, protease as well as antibacterial and antibiotic resistant genes. Only lipolytic active clones were obtained. Sequence analysis of selected TLB active clones revealed the presence of three different lipolytic enzymes (isochorismatase, sulfatase and phosholipase, patatin family protein).Only the phospholipase, patatin protein was further characterized. Sequence analysis revealed the presence of the classical esterase motif (G-X-S-X-G) and conserved aspartatic acid and histidine residues in the patatin. According to phylogenetic analysis phospholipase, patatin proteins constitute a new family of lipolytic enzymes, since they do not form part of any of the eight classes representative of lipolytic enzymes. The patatin was heterologously expressed in E. coli. Biochemical analysis of the partially purified protein showed that the enzyme had a preference for shorter carbon chained substrates, indicating that patatin displays esterase rather than lipase activity and functioned optimally at 30°C and pH 8 which was expected considering that the enzyme was isolated from a mesophilic environment.Afrikaans: Die konstruksie en sifting (‘screening’) van genoombiblioteke wat voorberei is vanaf DNS, geïsoleer direk uit die omgewing is ‘n eietydse en kragtige tegniek vir die ontdekking van nuwe ensieme met biotegnologiese waarde (Gabor et al., 2004). Standaard metodes gebaseer op die sifting van geïsoleerde mikro-organismes is inherent beperk tot die klein breukdeel van kweekbare mikrobiese spesies (<1%). Omgewings genoombiblioteke, daarenteen, lewer meer toegang tot die totale DNS basispaar opeenvolging wat daardie omgewing bied (Handelsman et al. 1998). Omgewings genoombiblioteke bied die navorser die geleentheid om te sif vir funksionele klasse van ensieme vanaf duisende organismes en navorsing in hierdie studieveld sal bydra tot ons huidige insig en begrip van die evolusie van metaboliese we¸ (Short, 1997; Rajendran et al., 2008). Dit is bewys dat die metagenoom aanslag ‘n effektiewe tegniek is vir die isolering van nuwe biokataliste en bruikbare gene van onkweekbare mikro-organismes afkomstig vanaf diverse omgewings. Voordat daar met die metagenoomanalise begin is, het ons ‘n genoombiblioteek berei uit ‘n bakteriese isolaat afkomstig van ’n diep myn: Geobacillus thermoleovorans GE-7. Die biblioteek is gesif vir lipolitiese aktiwiteit m.b.v. LB tributirien plate (TLB). Klone wat aktiwiteit getoon het, se DNS basispaar opeenvolging is bepaal d.m.v. 454 tegnologie en die data het getoon dat die lipA en GDSL lipases teenwoordig was. Die laasgenoemde lipase is nog nie gekaraktiriseer in hierdie organisme nie. Hierdie lipase familie vertoon die kenmerkende G-D-S-L aminosuur motief in plaas van die konvensionele G-X-S-X-G motief (X = dui enige amino suur aan).GDSL lipases is hidrolitiese ensieme met multifunksionele eienskappe soos bv. ‘n wye substraatspesifisiteit en regiospesifisiteit. Hierdie ensieme het ook die potensiaal om die hidrolise en sintese van ester verbindings te bewerkstellig wat van belang is in die farmaseutiese, voedsel, biochemiese en biologiese sektore (Akoh et al., 2004; Lämmle et al., 2007). Aktiewe klone van hierdie genoom biblioteek het ook gene opgelewer wat ge-assosieer word met vetsuur afbraak, verskillende glikolitiese aktiwititeite, lipolitiese aktiwiteite, spoor ontkieming, protein vouïng, antibiotika weerstandbiedendheid en die selwand. Sommige van hierdie gene se funksies mag lei tot beter insig oor hoe hierdie organisme aangepas het in die omgewing waarvan dit geïsoleer was.Biofilm vanaf die Beatrix goud myn is gekies vir metagenomiese studies. ‘n Diversiteits studie van hierdie biofilm is gedoen m.b.v. die klonering en basispaar volgorde bepaling van 16S (baketerie¸ en archaea) en 18S eukariotiese ribosomale DNS-gene. Filogenetiese analise van die bakteriële klone het getoon dat hulle DNS basispaar opeenvolging gegroepeer het met ten minste 5 fila vanaf die bakeri¸le koninkryk. Die 5 fila is afkomstig vanaf baie diverse omgewings soos bv. die Sargasso see en see basaltvloere, ondergrondse water, kalksteen grotte, teerputte, ‘n alkaliese warmwater bron, dreinerings areas van myne en biofilms (Cho en Giovanni, 2003; Kim en Crowley, 2007; Ikner et al., 2007 en Stepanauskas en Sieracki, 2007). Analise (‘Rarefaction analysis’) van 37 klone het getoon dat daar 29 verskillende OTE (‘operasionele taksonomiese eenhede’) was. Dit moet gemeld word dat die analise wel aangetoon het dat slegs ‘n klein gedeelte van die bakteri¸le gemeenskap se diversiteit (gebaseer op ‘n 97 % DNS basispaar opeenvolgingidentiteit) bestudeer is, m.b.v. die 16S klone, aangesien die grafiek nooit die asimptoot bereik het nie. DNS basispaar opeenvolgingdata afkomstig vanaf die archaea genoombiblioteek, het identiteite getoon met dié van ‘n onkweekbare archaea in die databasis en in besonder die van ‘n enkele onkweekbare archaea genoombiblioteek vanaf ‘n seewater monster. Hierdie kan toegeskryf word aan die feit dat daar tot op hede slegs beperkte archaea DNS basispaar opeenvolgingdata beskikbaar is en dat daar nog net 4 taksonomiese groepe uit hierdie koninkryk aan ons bekend is (Baker et al., 2003).Die metagenoom was gesif m.b.v. ‘n DNS basispaar opeenvolging-gebaseerde benadering vir sitokroom P450 mono-oksigenases (CYP450s) met spesiale klem op die CYP153 familie, wat verantwoordelik is vir die terminale hidroksilering van lang-ketting alkane. Klonering en opeenvolgingbepaling van hierdie CYP153 PKR (‘PCR’) produk het die teenwoordigheid van hierdie ensiem familie in die metagenoom getoon en dat hulle betrokke is by ‘n wye verskeidenheid metaboliese prosesse, diverse substraatvoorkeure het en beide kataboliese en anaboliese reaksies kataliseer (De Mot en Parret, 2002). CYP450s in bakterie¸ is verantwoordelik vir die biosintese van sekondêre metaboliete soos bv. antibiotika en die verbruik van lae molekulere massa, hidrofobiese verbindings soos alkane en aromatiese verbindings (Kubota et al., 2005). Daar is bewyse dat die CYP153 vanaf Mycobacterium sp. betrokke is by die biokatalise van die teenkanker middel: periliel alkohol vanaf limonien wat dui op die potensi¸le toepassing van hierdie ensiem in ‘n kliniese milieu (Urlacher en Eiben, 2006).Funksie-gebaseerde sifting was m.b.v. genoombiblioteke wat klein en groot invoegings bevat het, uitgevoer. Die biblioteke was gesif vir lipolitiese, amilase, protease en antibakteriese gene. Slegs lipolities-aktiewe klone kon geïdentifiseer word. Klone wat aktiwiteit op TLB-plate getoon het se basispaar opeenvolging was bepaal. Die basispaar opeenvolgingdata het getoon dat daar 3 verskillende lipolitiese ensieme teenwoordig was (isokorismatase, sulfatase en fosfolipase patatien familie proteïne). Slegs die fosfolipase patatein proteïen is verder bestudeer. Getransleerde DNS basispaar opeenvolging data het getoon dat hierdie ensiem die klassieke esterase motief (G-X-S-X-G) sowel as die gekonserveerde histidien en aspartiensuur aminosure het. Filogenetiese analises het getoon dat fosfolipase patatien proteïene ‘n nuwe familie lipolitiese ensieme is, aangesien hulle nie deel uitmaak van die huidige 8 klasse lipoliliese ensieme nie. Die patatiengeen is heteroloog uitgedruk in Escherichia coli en biochemiese analise van die semi-suiwer ensiem het getoon dat die ensiem ‘n voorkeur het vir kort koolstof-ketting subtrate. Hierdie was ‘n sterk aanduiding dat patatien esterase aktiwiteit toon en nie lipase aktiwiteit nie. Die ensiem het ook optimaal gefunksioneer by 30°C en by ‘n pH van 8. Hierdie was te wagte gewees aangesien die ensiem afkomstig was vanaf ‘n mesofiliese omgewing.enMetagenomeDiversity assessmentSequenced-based screeningCytochrome P450 alkane hydroxylasesLibrary constructionFunctional-based screeningLipolytic activityPhospholipase patatinHeterologous expressionBiochemical characterizationCatalysisGene librariesMicrobial ecologyEnzymesThesis (Ph.D. (Microbial, Biochemical and Food Biotechnology)--University of the Free State, 2009ThesisUniversity of the Free State