Characterisation of the malate transporter and malic enzyme from Candida utilis

Saayman, Maryna (2011-10)

Dissertation (PhD)--University of Stellenbosch, 2005.

Thesis

ENGLISH ABSTRACT: Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. The fission yeast Schizosaccharomyces pombe effectively degrades L-malate, but only in the presence of an assimilable carbon source. In contrast, the yeast Saccharomyces cerevisiae is unable to effectively degrade L-malate, which is ascribed to the slow uptake of L-malate by diffusion. In contrast, the yeast Candida utilis can utilise L-malate as the only source of carbon and energy, but this is subject to substrate induction and catabolite repression. Very little research has been done on a molecular level in C. utilis and only a few of its genes have been studied. In this study, we have shown that the yeast C. utilis effectively degraded extracellular L-malate and fumarate, but in the presence of glucose or other assimilable carbon sources, the transport and degradation of these dicarboxylic acids was repressed. The transport of both dicarboxylic acids was shown to be strongly inducible by either L-malate or fumarate and kinetic studies suggest that the same transporter protein transports the two dicarboxylic acids. In contrast, S. pombe effectively degraded extracellular L-malate, but not fumarate, only in the presence of glucose or other assimilable carbon sources. The S. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of L-malate. In order to clone the C. utilis dicarboxylic acid transporter, a cDNA library from C. utilis was constructed using a number of strategies to ensure representativeness and high transformation frequencies. The cDNA library was transformed in a S. cerevisiae strain carrying a plasmid containing the S. pombe malic enzyme gene (mae2) to allow screening for a malate-degrading S. cerevisiae clone. However, no positive clones that would indicate the successful cloning of the C. utilis malate transporter were obtained. The C. utilis malic enzyme gene, CuME, was subsequently isolated from the cDNA library based on conserved sequence homologies with the genes of S. cerevisiae and S. pombe, and characterised on a molecular and biochemical level. Sequence analysis revealed an open reading frame of 1926 bp, encoding a 641 amino acid polypeptide with a predicted molecular weight of 70.2 kDa. The optimum temperature for the C. utilis malic enzyme was 52°C and the enzyme was stable at 50°C for 2 hours. The inferred amino acid sequence showed significant homology with the malic enzymes of S. pombe and S. cerevisiae. Expression of the CuME gene is subject to glucose repression and substrate induction, as was observed for the dicarboxylic acid transporter from C. utilis. The CuME gene was successfully coexpressed with the S. pombe malate permease gene (mae1), resulting in a recombinant strain of S. cerevisiae able to effectively degrade L-malate.

AFRIKAANSE OPSOMMING: Daar is ’n merkwaardige verskil in die vermoë van verskillende gisspesies om ektrasellulêre dikarboksielsure af te breek en dit as enigste bron van koolstof te benut. Die splitsingsgis Schizosaccharomyces pombe kan L-malaat effektief afbreek, maar slegs in die teenwoordigheid van ’n ander benutbare koolstofbron. In teenstelling hiermee is dit vir die gis Saccharomyces cerevisiae onmoontlik om L-malaat effektief af te breek en te benut, wat hoofsaaklik toegeskryf kan word aan die stadige opname van L-malaat deur middel van diffusie. Die gis Candida utilis kan egter L-malaat as die enigste bron van koolstof en energie benut, maar dit is onderhewig aan substraat-induksie en kataboliet onderdrukking. Baie min navorsing op molekulêre vlak is tot hede in C. utilis uitgevoer en slegs ’n paar gene in hierdie gis is al bestudeer. In hierdie studie het ons aangetoon dat die gis C. utilis L-malaat en fumaraat effektief afbreek, maar dat glukose of ander benutbare koolstofbronne die opname en afbraak van hierdie dikarboksielsure onderdruk. Die opname van beide dikarboksielsure is sterk induseerbaar deur L-malaat óf fumaraat, terwyl kinetiese studies toon dat beide dikarboksielsure deur dieselfde transporter-proteïen vervoer word. In teenstelling hiermee kan S. pombe ekstrasellulêre L-malaat, maar nie fumaraat nie, in die teenwoordigheid van glukose of ’n ander benutbare koolstofbron effektief afbreek. Die S. pombe L-malaat transporter was nie in staat om fumaraat te vervoer nie, alhoewel fumaraat die opname van L-malaat onderdruk het. Ten einde die dikarboksielsuur transporter van C. utilis te kloneer, is verskeie strategieë gevolg ten einde ’n cDNA-biblioteek van C. utilis te konstrueer wat verteenwoordiging en hoë transformasie-frekwensies kan verseker. Die cDNA-biblioteek is getransformeer in ’n S. cerevisiae ras wat die S. pombe malaatensiem geen (mae2) bevat om die sifting van ’n S. cerevisiae kloon wat malaat effektief kan afbreek, moontlik te maak. Geen positiewe klone wat dui op die klonering van die C. utilis malaat transporter kon egter gevind word nie. Die C. utilis malaatensiem geen, CuME, is vervolgens van uit die cDNA biblioteek geïsoleer deur van gekonserveerde DNA-homologie met S. cerevisiae en S. pombe gebruik te maak, en op molekulêre en biochemiese vlak gekarakteriseer. DNA-volgordebepaling het ’n oopleesraam van 1926 bp onthul, wat kodeer vir ’n 641 aminosuur polipeptied met ’n verwagte molekulêre gewig van 70.2 kDa. Die optimale temperatuur van die C. utilis malaatensiem was 52°C en die ensiem was vir 2 ure stabiel by 50°C. Die afgeleide aminosuurvolgorde het beduidende homologie met die malaatensieme van S. pombe en S. cerevisiae getoon. Die CuME geen is suksesvol saam met die S. pombe malaat permease geen (mae1) uitgedruk om ’n rekombinante S. cerevisiae ras te genereer wat in staat is om L-malaat effektief af te breek.

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