Molecular and physiological aspects of alcohol dehydrogenases in the ethanol metabolism Saccharomyces cerevisiae

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Date
2007-05
Authors
De Smidt, Olga
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University of the Free State
Abstract
English: When Saccharomyces cerevisiae is grown on a fermentable carbon source such as glucose, the fermentative alcohol dehydrogenase, ADH I , catalyses the regeneration of NAD+ from NADH and produces ethanol from acetaldehyde. When the fermentable carbon source is depleted, a variety of other enzymes are derepressed in order to utilise the previously excreted ethanol via oxidative respiration and gluconeogenesis . To provide both the carbon source and energy for this system, the yeast cell requires an efficient method for oxidising this previously excreted ethanol. ADH II is a catabolite repressible isoenzyme which primarily functions in the cell to oxidise ethanol to acetaldehyde, which can be metabolised via the tricarboxylic acid cycle or act as intermediate product in gluconeogenesis. ADH III is a mitochondrial isoenzyme participating in the respiratory metabolism by forming part of the ethanol-acetaldehyde shuttle that is important for shuttling mitochondrial reducing equivalents to the cytosol under anaerobic conditions. The physiological roles and regulation of ADH1, ADH2, ADH3, ADH4 and ADH5 were investigated by monitoring transcription levels in chemostat and batch cultivations with Northern blotting and real-time RT-PCR. ADH I was shown to be the key enzyme in the reduction of acetaldehyde to ethanol and also demonstrated ample ability to oxidise ethanol. ADH2 transcription was inhibited by glucose and ethanol in chemostat cultures pulsed with both these carbon sources, but only glucose repression was evident in batch cultures. Northern blot analysis showed that the ADH3 gene was induced during the ethanol phase of the pulses suggested that the mitochondrial ADH III enzyme could also be involved in the first step in ethanol utilisation. The growth kinetics of a strain expressing only ADH III demonstrated that the ADH3 gene product could fulfil the same function as ADH II. ADH4 transcription was detected for the first time in batch cultures and was shown not to be involved in the production or assimilation of ethanol. ADH5 transcription was also demonstrated for the first time and data suggest that ADH V is not involved in ethanol production in a adh1-adh4 deletion mutant.
Afrikaans: Wanneer Saccharomyces cerevisiae op ‘n fermenteerbare koolstofbron soos glukose geweek word, kataliseer die fermentatiewe alkohol dehidrogenase, ADH I, die regenerering van NAD+ vanaf NADH en produseer etanol vanaf asetaldehied. Sodra die fermenteerbare koolstofbron uitgeput is, word ’n aantal ander ensieme se onderdrukking opgehef om die voorheen-uitgeskeide etanol via oksidatiewe respirasie en glukoneogenese te verbruik. Om beide die koolstofbron en energie vir hierdie sisteem te verskaf, het die gissel ’n effektiewe metode nodig om die voorheen-uitgeskeide etanol te oksideer. ADH II is ’n kataboliet-onderdukbare iso-ensiem wat in die sel primêr funksioneer om etanol na asetaldehied te oksideer, wat dan weer via die trikarboksielsuur-siklus gemetaboliseer kan word, of ook kan optree as ’n intermediêre produk in glukoneogenese. ADH III is ’n mitochondriale isoensiem wat deel is van respiratoriese metabolisme deur deelname aan die etanol-asetaldehied pendelmeganisme wat belangrik is om mitochondriale reduserende ekwivalente na die sitosol te vervoer onder anaërobe toestande. Die fisiologiese rolle en regulering van ADH1, ADH2, ADH3, ADH4 en ADH5 is ondersoek deur transkripsievlakke in chemostaat en lotkulture te monitor met die Northern kladtegniek en reële-tyd RT-PCR. Dit is aangetoon dat ADH I die sleutelensiem in die reduksie van asetaldehied na etanol is, asook dat dit etanol kan oksideer. ADH2 transkripsievlakke is onderdruk deur glukose en etanol in chemostaatkulture wat met beide hierdie koolstofbronne gepuls is, maar slegs glukose-onderdrukking was duidelik in lotkulture. Northern klad-analise het gewys dat die ADH3 geen gedurende die etanol-fase van die puls geïnduseer word, wat ’n aanduiding kan wees dat die mitochondriale ADH III ensiem ook betrokke kan wees in die eerste stap van etanolverbruik. Die groeikinetika van ‘n stam waar slegs ADH III uitgedruk word, het aangedui dat die ADH3 geenproduk dieselfde funksie as ADH II kan vervul. ADH4 transkripsie is waargeneem vir die eerste keer in lotkulture en dit is bewys dat dit nie betrokke was by die produksie of opname van etanol nie. ADH5 transkripsie is ook vir die eerste keer aangetoon en data dui aan dat ADH V nie by etanol produksie in ‘n adh1-adh4 delesiemutant betrokke is nie.
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Keywords
Thesis (Ph.D. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2007, Saccharomyces cerevisiae, Alcohol dehydrogenase, Enzymes
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