Characterization of melatonin production and physiological functions in yeast

Motlhalamme, Thato (2020-03)

Thesis (PhDAgric)--Stellenbosch University, 2020.

Thesis

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.

AFRIKAANSE OPSOMMING: Melatonien is ’n molekule wat in alle lewende organismes voorkom en wat verskeie funksies het, soos die regulering van sirkadiese ritmes in diere en groeistimulasie in plante. Studies stel voor dat melatonien as ’n antioksidant in alle lewende organismes optree. Die biosintetiese weë en biologiese funksies daarvan is in plante en diere gekarakteriseer, maar min is bekend oor hierdie prosesse in mikroörganismes. Gis is gevind om melatonien onder verskeie toestande te sintetiseer, soos tydens fermentasie, uithongering en aërobiese groei, maar produksiepatrone is gevind om inkonsekwent te wees. Die doel van hierdie studie was om die fisiologiese funksies van melatonien in gis in groter besonderhede te ondersoek deur ’n groot getal groeitoestande te evalueer om die toestande te bepaal wat konsekwente melatonienproduksie sal ontlok. Die studie het gebruik gemaak van Saccharomyces cerevisiae as ’n modelorganisme om die impak van melatonien op sellulêre fisiologie te bestudeer en om biosintese- en melatonien-responsiewe gene te identifiseer. Geen van die toestande wat in hierdie studie ondersoek is, het gelei tot konsekwente melatonienproduksie nie. Wanneer dit bespeur is, was die konsentrasies melatonien baie laag (ng/107 selle) en het hulle grootliks tussen biologiese herhalings verskil. In plante en diere ossilleer melatonienproduksie in reaksie op dagsiklusse; hierdie ossillerende patroon is egter nie bespeur nie. ’n Analise van die tussenloperweë stel voor dat verskeie ensiemreaksies moontlik in die sintese van melatonien in gis betrokke is en dat die gis nie ’n toegewyde sinteseweg blyk te hê nie. Die afwesigheid van ortoloë van die ensieme wat in die biosintetiese weg in gis betrokke is, ondersteun hierdie gevolgtrekking. Die chaotiese produksiepatrone in gis stel voor dat melatonien moontlik ’n produk is van nie-spesifieke ensimatiese reaksies of oorvloei-metabolisme. Hierdie studie het ’n verskillende benadering gevolg tot die evaluering van die reaksie van kulture op oksidatiewe stres deur eksperimente in deurlopende kultuurtoestande te onderneem in plaas daarvan om van lotkultuur gebruik te maak. Mettertyd het die aantal differensieel uitgedrukte gene vinniger verminder, tesame met gisherstel van die stres, in melatonienbehandelde kulture as in melatonien-onbehandelde kulture. Die transkriptomiese analise van S. cerevisiae wat vóór en ná H2O2-geïnduseerde oksidatiewe stres met melatonien behandel is, dui daarop dat dit nie deur enige spesifieke stresresponsiewe weg funksioneer nie en dat die aktiwiteit daarvan nie aan enige spesifieke genetiese interaksies of regulering gekoppel kan word nie. In die afwesigheid van stres, verhoog eksogene melatonien die uitdrukking van die gene van die sulfaat-assimilasieweg. Hierdie weg lei tot die vorming van metionien en sisteïen, wat betrokke is in die produksie van glutatioon, en die respons berei dus moontlik selle vir die gevolglike stres voor. Toe S. cerevisiae met verskillende reaktiewe suurstofspesies wat stressors genereer, gestres is, het melatonienaanvulling die gevolglike oorlewing van die kulture verbeter op ’n soortgelyke manier as ander antioksidante deur die uitdrukking van verskeie gene wat die algemene antioksidantrespons ondersteun, te verhoog.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/107867
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