Recent developments of Metabolic Control Analysis include the derivation of a general method (the ‘universal method’) which aims to define the conditions under which a pre-determined change in the flux to a specific end-point metabolite may be achieved without changing other fluxes or metabolic concentrations. This thesis describes the application of this method in the yeast, <i>Saccharomyces cerevisiae</i>, by over-expressing genes of the shikimate and tryptophan pathways and, by analysing the outcomes, attempts an evaluation of the method’s validity. A single-copy plasmid, pPH28, bearing the 4 genes encoding enzymes of the shikimate pathway was constructed and introduced into a strain along with a multi-copy plasmid, pME554, bearing the 5 genes of the tryptophan pathway. In this way the activity of the shikimate enzymes and tryptophan enzymes were over-expressed by factors of about 2-5 and 20-60, respectively. The stability of the plasmids, both separately and together, under selective and non-selective conditions in different media was studied, along with enzyme activities. Results showed higher losses of plasmids when grown without selection, greater loss of the 2-micron plasmid than for the centromeric plasmid, and, in general, corresponding rates of loss of enzyme activities. The effects of the plasmids upon the production of tryptophan, phenylalanine and tyrosine were studied. pME554 and, subsequently, pPH28 had no observable, effect upon the total flux to tryptophan, phenylalanine and tyrosine. However, fluxes to tryptophan, phenylalanine and tyrosine pools were altered. pME554 increases intracellular concentrations of tryptophan and decreases those of phenylalanine and tyrosine, all by small amounts. When pPH28 is also included, intracellular tryptophan, phenylalanine and tyrosine concentrations increase by factors of 2-3. There are also corresponding changes in the amounts excreted into the medium.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:652721 |
| Date | January 2002 |
| Creators | Hunt, Paul |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/15059 |
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