Actinomycetes From Unexplored Environmental Niches in Ethiopia and Their Biotechnological Potentials for Antimicrobial Compound Production
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Date
2018-06-04
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Addis Ababa University
Abstract
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
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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
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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.
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Keywords
Actinomycetes, Antimicrobial Bioassay, Biosynthetic Genes, Ethiopian Soils, Genome Analyses, High Resolution Mass Spectrometry, Nmr-Assisted Structure Elucidation, Solid State Fermentation, Streptomyces