Genetics and biochemistry of coenzyme A biosynthesis in the yeast Saccharomyces cerevisiae

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Since coenzyme A (CoA) and its acylation products (mainly acetyl-CoA) are of central importance for metabolism in all organisms, biosynthesis of CoA is an essential cellular function. By identification of structural genes CAB1-CAB5 (coenzyme A biosynthesis) and construction of a hyperactive variant of key enzyme pantothenate kinase (CAB1 gene product) we obtained yeast cells with a concentration of acetyl-CoA which was about 6-fold increased, compared to a wild-type. Since acetyl-CoA has a fundamental role in metabolism and functions as a signalling molecule for initiation of a new cell division cycle we wish to investigate the implications of the increased amount of acetyl-CoA for the cellular proteome and part of its metabolome (focusing on fatty acids and ergosterol). By varying gene expression of genes ACS2 (acetyl-CoA synthetase) and LEU5 (mitochondrial CoA importer) we plan to modify the ratio of CoA and acetyl-CoA as well as the intracellular distribution of CoA. Results of these studies should allow us to define favourable conditions for CoA-dependent processes in biotechnology. We were able to demonstrate that gene products Cab2-Cab5 interact with each other and that they form a CoA-synthesizing protein complex (CoA-SPC) with Cab3 as a key subunit for mediating interactions. In the future we plan to investigate CoA-SPC more precisely, possibly defining subunits not yet identified and its stoichiometry. To do this, Cab3 will be epitope-tagged and used for affinity-purification of the complex which will be subsequently analyzed by mass spectrometry. Results obtained for CoA-SPC of yeast will be finally compared with the situation in higher eukaryotes.