Bervanakis, George, 2009 Detection and Expression of Biosynthetic Genes in Actinobacteria, Flinders University, School of Medicine
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Most microbial organic molecules are secondary metabolites which consist of diverse chemical structures and a range of biological activities. Actinobacteria form a large group of Eubacteria that are prolific producers of these metabolites. The recurrence of pathogens resistant to antibiotics and a wider use of these metabolites apart from their use as anti-infectives, has been the impetus for pharmaceutical companies to search for compounds produced by rare and existing actinobacterial cultures. Accessing microbial biosynthetic pathway diversity has been possible through the use of sensitive and innovative molecular detection methodologies. The present study evaluated the use of molecular based screening as a rational approach to detect secondary metabolite biosynthetic genes (SMBG) in uncharacterised natural Actinobacterial populations. A polymerase chain reaction (PCR) approach was selected for ease of application and high sample processivity. Rational designed screening approaches using PCR in the discovery of SMBG, involved identifying common functions in secondary metabolite biosynthetic pathways, such as condensation reactions in polyketide synthesis, genes encoding these functions, and using conserved regions of these genes as templates for the design of primers to detect similar sequences in uncharacterised actinobacteria. Design of primers involved rigorous in silico analysis followed by experimentation and validation. PCR screening was applied to 22 uncharacterised environmental isolates, eight of these displayed the presence of the ketosynthase (KS) gene belonging to the type I polyketide synthases and eight contained the ketosynthase (KS) gene belonging to the type II polyketide synthases, six of the isolates contained the presence of a presumptive dTDP-glucose synthase (strD) gene which is involved in the formation of deoxysugar components of aminoglycoside antibiotics and one isolate contained the presence of a presumptive isopenicillin N synthase (pcbC) gene involved in beta-lactam synthesis. Alignments of partially sequenced PCR products from isolates A1488 and A3023 obtained using type II PKS primers showed close similarities with KS genes from antibiotic producing actinobacteria. Similarly, alignments of sequences from isolates A1113 and A0350 showed regions of similarities to KS genes from antibiotic producing actinobacteria. Fermentation techniques were used for inducing expression of secondary metabolites from the uncharacterised actinobacteria isolates. By using antimicrobial guided screening it was determined that most of the isolates possessed the capacity to produce antimicrobial metabolites. Dominant antagonistic activity was detected against Gram positive bacteria and to a minor extent against fungi. Optimal fermentation liquid media were identified for certain isolates for the production of antimicrobial metabolites. Two alternative fermentation methods; solid-state and liquid-oil fermentations were evaluated to improve secondary metabolite production in the uncharacterised isolates. Solid-substrate fermentation showed that it could induce a complex metabolite pattern by TLC analysis, however this pattern varied according to the substrate being used. Liquid media supplemented with refined oils, showed a positive response indicated by higher antibacterial activities detected. Evaluation of semi-purified organic extracts identified two isolates A1113 and A0350 producing similar antimicrobial metabolites as detected by HPLC/UV/MS, a literature database search of similar compounds containing the same molecular weight identified the compound as belonging to the actinomycin group of compounds. A complex metabolic pattern was identified for isolate A2381, database searching identified some of the compounds as having similar molecular weights to actinopyrones, trichostatins, antibiotics PI 220, WP 3688-5 and YL 01869P. Drug discovery screening can serve to benefit from PCR detection of biochemical genotypes in initial screens, providing a rapid approach in identifying secondary metabolite producing capabilities of microorganisms prior to the commencement of costly and time consuming fermentation studies. Additionally the identification of biochemical genotypes allows a directed approach in using fermentation media designed to induce biosynthetic pathways of specific classes of compounds.
Keywords: actinobacteria, PCR screening, secondary metabolites
Subject: Medical Biotechnology thesis
Thesis type: Masters
School: School of Medicine