Toni Rendulić

• Succinic acid (SA) is one of the most promising bio-based platform chemicals, as it can serve as a precursor in the synthesis of many industrially relevant chemicals and polymers. Yeasts are particularly desirable microbial hosts for SA production because they tolerate low environmental pH, allowing the direct production of the undissociated form of the acid, which results in a significantly simpler and more cost-effective overall process. The fermentation performance of yeast cell factories heavily depends on the activity of membrane transporters responsible for substrate import, metabolite exchange between intracellular compartments, and SA export from the cell. As such, membrane transporters represent key targets in metabolic engineering efforts and are the primary focus of this work. The first part of this thesis provides new insights into plasma membrane transporters from the acetate uptake transporter (AceTr) family, namely Ato1 and SatP, by identifying amino acid residues critical for their specificity and activity. This includes the engineering and characterisation of Ato1 and SatP variants capable of transporting SA in S. cerevisiae. The impact of these transporter variants on extracellular SA accumulation is assessed and compared with that of Dct-02, a known fungal SA exporter, revealing that AceTr homologues and Dct-02 have opposing effects on SA production in S. cerevisiae under industrially relevant conditions. The second part of this thesis optimizes SA production from glycerol via the CO₂-fixing reductive TCA pathway in S. cerevisiae. To increase carbon flux through this cytosolic pathway, mitochondrial membrane transporters are identified as particularly attractive engineering targets. Overall, this work expands the understanding of membrane transporters relevant for SA production and improves the fermentation performance of existing SA-producing yeast cell factories.