Actinomycetes From Unexplored Environmental Niches In Ethiopia And Their Biotechnological Potentials For Antimicrobial Compound Production

Actinomycetes have a widely recognized potential for the production of significant bioactiverncompounds. The major aim of this study was to isolate, screen and evaluate the biotechnologicalrnpotential of selected actinomycete isolates particularily for antimicrobial compound productionrnusing standard bioassays methods, LC-MS, high resolution mass spectrometry (HR-MS) andrnNMR techniques. It consists of six distinct chapters. In the first chapter, general introduction,rnstatement of the problem and major objectives are presented. The second chapter deals with thernreview of related literatures. The rest four chapters (3-6) are the experimental sections of thernwork. Hence, the third chapter concentrated on the isolation, screening, bioactivity detection andrnphylogenetic analysis of promising actinomycetes capable of producing bioactive secondaryrnmetabolites from various unexplored niche habitats in Ethiopia. Among the 416 isolates screenedrnfor bioactivities, 101 (24%) isolates were inhibiting the growth of C. albicans, and 88 (21%)rnisolates were inhibiting both C. albicans (ATCC 62376) and C. neoformans (clinical isolate).rnTen isolates having considerable activities were chosen for further investigation and taxonomicrnidentification studies. The polyphasic identification results of these isolates found to be inrnconsistent with the genus Streptomyces described in Bregay’s manual of systematic bacteriology.rnIdentification of the isolates have been verified by the analysis of the 16s rRNA gene sequence.rnThe phylogenetic relationships of the isolates to type strains and best matches based on BLASTrnsearch were inferred using the Maximum Likelihood algorithm in MEGA 7 software andrnconfirmed that all the isolates belong to genus Streptomyces. The fourth chapter deals with therncultivation of five promising isolates namely Ac-029, Ac-125, Rv-355, Ac-464 and Go-475 forrnbioactive secondary metabolite production and subsequently evaluation of SSF processrnparameters on metabolite yield. Depending on the types of the isolates, variations were observedrniirnin optimal fermentation process parameters on bioactive secondary metabolite production. It wasrndemonstrated that wheat bran in the presence of supplementary nutrients, an initial moisturerncontent of 65%, a pH value of 7.5, incubation temperature of 30 oC, an inoculum size of 3x107rnCFU/mL and incubation period of 12 days were the optimal SSF conditions for most of thernisolates studied. The fifth chapter focused on antimicrobial potential of Streptomyces sp. Rv-355rncultivated in submerged culture. In its bioactivity profile, Streptomyces sp. Rv-355 producedrnantimicrobial compounds with wider spectrum of activities against yeasts, Gram positive andrnGram negative bacterial pathogens. It was found that biomass production and bioactivity profilesrnof Streptomyces sp. Rv- 355 are positively correlated. Bioactivity guided analysis of the crudernextract from Streptomyces sp. Rv-355 using TLC, column chromatography, HPLC, LC-MSrnshowed the presence of potential compounds. The partially purified extract showed MIC valuesrnof 50μg/mL against Candida albicans and 100μg/mL against Bacillus subtilis. The result isrnfound to be a prelude for further analysis of the crude extract from Rv-355 using HR-MS, andrnNMR methods. The sixth chapter was targeted on the bioactivity guided identification andrnstructural elucidation of members of benz[a]anthraquinone antibiotics, 8-O-methyltetrangomycinrnand 8-O-methyltetrangulol from Streptomyces sp. Go-475 extracts using LC-MS, HR-MS/MSrnand 1H NMR 13C NMR methods. Streptomyces sp. Go-475 displayed potent activity against bothrnyeasts and Gram-positive bacteria with MIC values of the crude extracts 100μg/mL andrn50μg/mL against Candida albicans ATCC62376 and Bacillus subtilis ATCC6633 respectively.rnThe analysis revealed that Streptomyces sp. Go-475 is able to produce at least three knownrnsecondary metabolites (4-Methoxy-1(3H)-isobenzofuranone, 3-Phenylpropionic acid or 1, 2-rnBenzenediol and Dehydrocineromycin B) that were not detected in the SmF extract. However,rnbetaine was detected in both SSF and SmF extracts of this isolate. Two important anti-bacterialrniiirncompounds were purified from methanol extract of Streptomyces sp. Go-475 and their structuresrnwere elucidated by NMR and HR-MS/MS as 8-O-methyltetrangomycin and 8-Omethyltetrangulol.rnBesides, many potentially novel metabolites were detected, the majority ofrnwhich were produced in SSF method. The findings enable us to conclude that Streptomyces sp.rnGo-475 and other isolates from Ethiopian soil have the capacity to produce potentially newrnantifungal secondary metabolites and warrant further investigations. The results also proved thatrnSSF as promising economical and best option to produce potential bioactive secondaryrnmetabolites from Streptomyces spp. The genome sequence of Streptomyces sp. Go-475 wasrnobtained using a hybrid assembly approach of high quality Illumina short read and low qualityrnOxford Nanopore long read data. The complete linear chromosome of 8,570,609 bp, featuring arnG+C content of 71.96%, contains 7,571 predicted coding sequences, 83 t(m)RNA genes, and sixrnrrn operons. Analysis of the genome for secondary metabolite biosynthesis gene clusters allowedrnus to connect certain clusters with experimentally confirmed molecules. The findings alsornverified great potential of Streptomyces sp. Go-475 for the production of chemically diversernsecondary metabolites.

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