Magister Scientiae - MSc (Biotechnology)

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Browsing by Subject "Actinobacteria"

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    Actinobacterial diversity of the Ethiopian Rift Valley lakes
    (University of the Western Cape, 2011) Du Plessis, Gerda; Cowan, D.A.; Tuffin, Marla
    The class Actinobacteria consists of a heterogeneous group of filamentous, Gram-positive bacteria that colonise most terrestrial and aquatic environments. The industrial and biotechnological importance of the secondary metabolites produced by members of this class has propelled it into the forefront of metagenomics studies. The Ethiopian Rift Valley lakes are characterized by several physical extremes, making it a polyextremophilic environment and a possible untapped source of novel actinobacterial species. The aims of the current study were to identify and compare the eubacterial diversity between three geographically divided soda lakes within the ERV focusing on the actinobacterial subpopulation. This was done by means of a culture-dependent (classical culturing) and culture-independent (DGGE and ARDRA) approach. The results indicate that the eubacterial 16S rRNA gene libraries were similar in composition with a predominance of α-Proteobacteria and Firmicutes in all three lakes. Conversely, the actinobacterial 16S rRNA gene libraries were significantly different and could be used to distinguish between sites. The actinobacterial OTUs detected belonged to both the Rubrobacterales and Actinomycetales orders with members of the genus Arthrobacter being found in all three lakes. Geochemical properties were significantly different between the lakes, although more than one property attributed to the variance between community compositions. The diversity detected in the culture-based study differed significantly and all isolates belonged to the genus Streptomyces. Two novel strains were characterized by means of phylogenetic (16S rRNA gene sequence), physiological, morphological and biochemical analyses. Both novel isolates were capable of growing under "extreme" conditions- pH 12, 10% NaCl and 45°C. Partial enzyme characterization revealed that both strains produced xylanase enzymes that were active at pH 6.5 and 8.5 with an increase in activity up to 45°C. The results obtained revealed a previously undetected diversity of actinobacteria in the Ethiopian Rift Valley with a potentially novel subpopulation adapted to haloalkaline conditions. The low 16S rRNA sequence similarity of a substantial proportion of the libraries suggests that culture-based isolation may play a vital role in deciphering the community fingerprint.
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    Biological and enzymatic activity of actinobacteria associated with aloe ferox
    (University of the Western Cape, 2023) Isaacs, Nasreen; Kirby-McCullough, Bronwyn
    Antimicrobial resistance is a global crisis that has been on the rise for decades, threatening the health and safety of many nations. This highlights the need to discover novel antimicrobial compounds to combat already resistant pathogens, as well as newly emerging pathogens. Historically soil bacteria, particularly actinobacteria, have been a source of novel bioactive compound, however, the constant re-isolation of known actinobacterial strains led to the need to explore unique environments. Higher plants, especially medicinal plants, represent a unique niche for the isolation of rare, bioactive actinobacterial species.
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    Development of an actinobacteria based in vitro transcription and translation systems
    (University of the Western Cape, 2015) Maake, Takalani Whitney; Tuffin, Marla; van Zyl
    Heterologous metagenomic screening strategies have relied largely on the construction of DNA libraries and screening in Escherichia coli to access novel enzymes. There is an increased demand for the identification of novel lignocellulose degrading enzymes with enhanced biochemical properties which are suitable for applications in industrial processes; biofuels being one of them. The use of heterologous gene expression in function based metagenomic studies has resulted in the discovery of enormous novel bioactive compounds. However, there are limitations associated with using E. coli as a heterologous host which does not allow transcription and translation of all genes in the metagenome. E. coli can only express 40% of the environmental DNA because of promoter recognition, codon usage, and host toxicity of gene products. Therefore alternative strategies for expressing or producing novel enzymes are needed, which can also be employed in metagenomic gene discovery. In vitro protein synthesis is an important tool in molecular biology and used to obtain proteins from genes for functional and expression studies. These systems may hold the key to unlock more of the potential in metagenomic DNA. The broader aim of the study is to develop non- E. coli based cell-free protein synthesis systems to further the metagenomics screening. In this study, Rhodococcus erythropolis H8 was evaluated for its suitability in cell-free expression. Crude extracts containing the macromolecular components (70S or 80S ribosomes, tRNAs, initiation, elongation and termination factors) fromR. erythropolis were prepared using existing crude extract based cell-free protein synthesis (CFPS) protocols. Three genes were selected and used as templates for synthesis: cell11, xp12 and acetyl xylan esterase (axe10), all previously isolated from metagenomic libraries screened inE. coli. As judged by zymograms and enzyme assays, all enzymes were successfully expressedfrom their native promoters and in recombinants clones using the PtipA promoter, and wereactive. Furthermore, the amounts of XP12 protein produced using pFos-XP_12 was 1.2mg/mlfrom E. coli and 1.67mg/ml from R. erythropolis CFPS, showing that the R. erythropolismachinery was more efficient in the expression of XP12 than the E. coli machinery. To the best of our knowledge this is the first demonstration of a cell-free expression using an actinomycete.