Actinomycetes From Unexplored Environmental Niches in Ethiopia and Their Biotechnological Potentials for Antimicrobial Compound Production

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Addis Ababa University


Actinomycetes have a widely recognized potential for the production of significant bioactive compounds. The major aim of this study was to isolate, screen and evaluate the biotechnological potential of selected actinomycete isolates particularily for antimicrobial compound production using standard bioassays methods, LC-MS, high resolution mass spectrometry (HR-MS) and NMR techniques. It consists of six distinct chapters. In the first chapter, general introduction, statement of the problem and major objectives are presented. The second chapter deals with the review of related literatures. The rest four chapters (3-6) are the experimental sections of the work. Hence, the third chapter concentrated on the isolation, screening, bioactivity detection and phylogenetic analysis of promising actinomycetes capable of producing bioactive secondary metabolites from various unexplored niche habitats in Ethiopia. Among the 416 isolates screened for bioactivities, 101 (24%) isolates were inhibiting the growth of C. albicans, and 88 (21%) isolates were inhibiting both C. albicans (ATCC 62376) and C. neoformans (clinical isolate). Ten isolates having considerable activities were chosen for further investigation and taxonomic identification studies. The polyphasic identification results of these isolates found to be in consistent with the genus Streptomyces described in Bregay’s manual of systematic bacteriology. Identification of the isolates have been verified by the analysis of the 16s rRNA gene sequence. The phylogenetic relationships of the isolates to type strains and best matches based on BLAST search were inferred using the Maximum Likelihood algorithm in MEGA 7 software and confirmed that all the isolates belong to genus Streptomyces. The fourth chapter deals with the cultivation of five promising isolates namely Ac-029, Ac-125, Rv-355, Ac-464 and Go-475 for bioactive secondary metabolite production and subsequently evaluation of SSF process parameters on metabolite yield. Depending on the types of the isolates, variations were observed ii in optimal fermentation process parameters on bioactive secondary metabolite production. It was demonstrated that wheat bran in the presence of supplementary nutrients, an initial moisture content of 65%, a pH value of 7.5, incubation temperature of 30 oC, an inoculum size of 3x107 CFU/mL and incubation period of 12 days were the optimal SSF conditions for most of the isolates studied. The fifth chapter focused on antimicrobial potential of Streptomyces sp. Rv-355 cultivated in submerged culture. In its bioactivity profile, Streptomyces sp. Rv-355 produced antimicrobial compounds with wider spectrum of activities against yeasts, Gram positive and Gram negative bacterial pathogens. It was found that biomass production and bioactivity profiles of Streptomyces sp. Rv- 355 are positively correlated. Bioactivity guided analysis of the crude extract from Streptomyces sp. Rv-355 using TLC, column chromatography, HPLC, LC-MS showed the presence of potential compounds. The partially purified extract showed MIC values of 50μg/mL against Candida albicans and 100μg/mL against Bacillus subtilis. The result is found to be a prelude for further analysis of the crude extract from Rv-355 using HR-MS, and NMR methods. The sixth chapter was targeted on the bioactivity guided identification and structural elucidation of members of benz[a]anthraquinone antibiotics, 8-O-methyltetrangomycin and 8-O-methyltetrangulol from Streptomyces sp. Go-475 extracts using LC-MS, HR-MS/MS and 1H NMR 13C NMR methods. Streptomyces sp. Go-475 displayed potent activity against both yeasts and Gram-positive bacteria with MIC values of the crude extracts 100μg/mL and 50μg/mL against Candida albicans ATCC62376 and Bacillus subtilis ATCC6633 respectively. The analysis revealed that Streptomyces sp. Go-475 is able to produce at least three known secondary metabolites (4-Methoxy-1(3H)-isobenzofuranone, 3-Phenylpropionic acid or 1, 2- Benzenediol and Dehydrocineromycin B) that were not detected in the SmF extract. However, betaine was detected in both SSF and SmF extracts of this isolate. Two important anti-bacterial iii compounds were purified from methanol extract of Streptomyces sp. Go-475 and their structures were elucidated by NMR and HR-MS/MS as 8-O-methyltetrangomycin and 8-Omethyltetrangulol. Besides, many potentially novel metabolites were detected, the majority of which were produced in SSF method. The findings enable us to conclude that Streptomyces sp. Go-475 and other isolates from Ethiopian soil have the capacity to produce potentially new antifungal secondary metabolites and warrant further investigations. The results also proved that SSF as promising economical and best option to produce potential bioactive secondary metabolites from Streptomyces spp. The genome sequence of Streptomyces sp. Go-475 was obtained using a hybrid assembly approach of high quality Illumina short read and low quality Oxford Nanopore long read data. The complete linear chromosome of 8,570,609 bp, featuring a G+C content of 71.96%, contains 7,571 predicted coding sequences, 83 t(m)RNA genes, and six rrn operons. Analysis of the genome for secondary metabolite biosynthesis gene clusters allowed us to connect certain clusters with experimentally confirmed molecules. The findings also verified great potential of Streptomyces sp. Go-475 for the production of chemically diverse secondary metabolites.



Actinomycetes, Antimicrobial Bioassay, Biosynthetic Genes, Ethiopian Soils, Genome Analyses, High Resolution Mass Spectrometry, Nmr-Assisted Structure Elucidation, Solid State Fermentation, Streptomyces