This study focuses on understanding the mechanisms of expression control of a phospholipid biosynthetic gene, INO1. This study also includes investigation into transcriptional regulation of SNA3, a gene in tandem upstream of INO1. INO1 expression is a prevailing model for transcription studies. INO1 is repressed under growth conditions with inositol and derepressed by two transcription activators, Ino2 and Ino4, when inositol is absent. Coordination of the centromeric binding factor, Cbf1, with Ino2 and Ino4 is required for efficient derepression of INO1. Transcription of the INO1 adjacent SNA3 gene is also influenced by inositol. INO1 and SNA3 are co-regulated by Cbf1, Ino2 and Ino4. However, the mechanism of this co-regulation is not fully understood. A separate aspect of INO1 expression is its growth phase regulation. Under inositol depleted conditions, the expression of INO1 increases during log phase and decreases during stationary phase. Most genes in yeast are believed to be expressed at a constant level through all growth phases. It is unclear how INO1 growth phase regulation takes place. The first part of my work focused on exploring the mechanism through which Cbf1, Ino2 and Ino4 control the inositol-mediated regulation of INO1 and SNA3. This included determining the necessity of the Cbf1 binding site for Ino2 and Ino4 binding, as well as for the inositol mediated regulation of INO1 and SNA3. The second part of my work focused on understanding the growth phase regulation of INO1. This includes examining the expression of INO1 in individual cells in a growing population.

Yeasts in their natural environment form structured colonies. This allows them to better adapt to environmental conditions, but also to more easily resist various types of yeast infection inhibitors. The metabolism of phospholipids is closely related to the morphology of colonies. An important gene involved in phospholipid metabolism is INO1, which encodes inositol-3- phosphate synthase. Expression of the INO1 gene is regulated by the Opi1p negative transcription factor, which also affects a number of other genes for phospholipid metabolism enzymes, is also necessary for the expression of the FLO11 gene, encoding Flo11p, which is essential to the formation of a structured colony. The main aim of my work was to investigate the correlation between colony morphology of a natural strain of Saccharomyces cerevisiae and phospholipid metabolism. I have found that changes in INO1 gene expression and colony morphology are influenced by carbon source, selenate activity and the inhibitor of β-oxidation, 2-bromooctanoic acid. Although the INO1 gene is not essential for cell viability, its deletion or overexpression causes changes in phospholipid metabolism and colony morphology. Selenate and 2-bromooctanoic acid also alter expression of the FLO11 gene, which is reflected in colony structure. Thus, 2-...

Myo-inositol is ubiquitous in nature and is found at the structural core of a diverse range of biologically important derivatives, including phosphatidylinositols, inositol phosphates and mycothiol. The synthesis of myo-inositol derivatives is notoriously difficult due to the need to control both regio- and enantioselectivity. As a result, synthetic routes to derivatives of this type are often lengthy and low yielding. The first biosynthetic step in the production of all myo-inositol metabolites is the isomerisation of D-glucose 6- phosphate to L-myo-inositol 1-phosphate as mediated by L-myo-inositol 1-phosphate synthase (INO1). For the protozoan parasite Trypanosoma brucei, INO1 is essential for survival and its version of the enzyme (TbINO1) has a high turnover. This makes TbINO1 an attractive candidate for the biocatalytic production of L-myo-inositol 1- phosphate, and a potential starting point for drastically shortened syntheses of important myo-inositol derivatives. The production of L-myo-inositol 1-phosphate by TbINO1 has been optimised to achieve complete conversion in reaction conditions that facilitate product isolation. Due to problems with an in-batch process, the TbINO1 enzyme was immobilised and the process was transferred to a flow system. This has allowed for production of significant quantities of L-myo-inositol 1-phosphate with a high level of purity. L-myo-inositol 1- phosphate obtained from the flow system has been used to prepare mycothiol glycosylation acceptor, 1,2,4,5,6-penta-O-acetyl-D-myo-inositol, in a concise synthesis with a greatly improved yield over the literature.