Joeline Xiberras

• Glycerol is an attractive carbon source in industrial biotechnology because its conversion into the glycolytic intermediate pyruvate via the glycolytic pathway produces twice the amount of reducing equivalents (per carbon equivalent) compared to glucose. This enables higher maximum theoretical yields of metabolic products whose production pathways require more reducing equivalents in the form of NADH such as succinic acid (SA). Since Saccharomyces cerevisiae does not naturally produce SA at high levels, extensive metabolic engineering is necessary to increase SA production. Therefore, the first part of the thesis consists of a review of the state-of-the-art regarding the S. cerevisiae growing on glycerol, which highlights the importance of gaining more knowledge about the carbon catabolism during growth of S. cerevisiae on this carbon source. This thesis also presents novel findings regarding the anaplerotic reactions active during growth of S. cerevisiae on glycerol. In comparison to glucose or ethanol as carbon sources, S. cerevisiae shows a higher metabolic flexibility with regard to the active anaplerotic reactions during growth on glycerol. The knowledge gained from investigating the anaplerotic reactions proved to be essential for engineering S. cerevisiae for SA production through the redox-neutral pathway, i.e. glycerol catabolism via the NAD+-dependent pathway and SA production through the cytosolic reductive branch of the TCA cycle (referred to as ‘SA module´). The data presented in this thesis shows for the first time SA production from glycerol in S. cerevisiae. Although the SA was not produced via the initially envisaged pathway, i.e. ‘SA module’, the experimental data provides a solid basis for future work focusing on further metabolic engineering to channel the carbon flux through the desired pathway for achieving redox-neutral production of SA.