Browsing by Author "Motlhalamme, Thato"

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    Characterization of melatonin production and physiological functions in yeast
    (Stellenbosch : Stellenbosch University, 2020-03) Motlhalamme, Thato; Bauer, Florian; Prior, Bernard; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.
    ENGLISH ABSTRACT: Melatonin is a molecule that is found in all living organisms with numerous functions such as the regulation of circadian rhythms in animals and growth stimulation in plants. Studies suggest that melatonin acts as an antioxidant in all living organisms. Its biosynthetic pathways and biological functions are characterised in plants and animals but very little is known about these processes in microorganisms. Yeasts have been found to synthesise melatonin under various conditions such as fermentation, starvation and aerobic growth, but production patterns were found to be inconsistent. The purpose of this study was to investigate the physiological functions of melatonin in yeast in more detail by evaluating a large number of growth conditions to determine conditions that would elicit consistent melatonin production. The study used Saccharomyces cerevisiae as a model organism to study the impact of melatonin on cellular physiology and identify biosynthesis- and melatonin-responsive genes. None of the conditions investigated in this study resulted in consistent melatonin production. When detected, concentrations of melatonin were very low (ng/107 cells) and varied greatly between biological repeats. In plants and animals, melatonin production oscillates in response to diurnal cycles; however, this oscillatory pattern was not observed in the current study. An analysis of the pathway intermediates suggest that multiple enzyme reactions may be involved in the synthesis of melatonin in yeast and that yeast appears not to possess a dedicated synthesis pathway. The absence of orthologs of the enzymes involved in the biosynthetic pathway in yeast supports this conclusion. The chaotic production pattern in yeast suggests that melatonin may be a product of non-specific enzymatic reactions or overflow metabolism. This study took a different approach to evaluating the response of cultures to oxidative stress by conducting experiments in continuous culture conditions instead of batch culture. Over time, the number of differentially expressed genes decreased more rapidly together with yeast recovery from stress in melatonin treated cultures compared to melatonin untreated cultures. Transcriptomic analysis of S. cerevisiae treated with melatonin pre- and post-H2O2 induced oxidative stress suggests that it does not act through any specific stress-responsive pathway and its activity could not be linked to any specific genetic interaction or regulation. However, in the absence of stress, exogenous melatonin enhanced the expression of sulphate assimilation pathway genes. This pathway leads to the formation of methionine and cysteine which are involved in the production of glutathione, and the response therefore may prime cells for subsequent stress. When S. cerevisiae was stressed with various reactive oxygen species generating stressors, melatonin supplementation improved the survival of the cultures in a similar manner to other antioxidants, by increasing the expression of several genes that support the general antioxidant response.