Andreea Perpelea

• Utilizing agro-industrial waste as a raw material for the production of chemicals, fuels and materials could support a circular bioeconomy, helping to minimize carbon and energy loss. Pectin-rich biomass is a generally under-utilized feedstock, and includes residues such as citrus peel, apple pomace and sugar beet pulp. It offers itself as a feedstock for biotechnological production processes using microorganisms as a cell factory. Unlike other biomass residues, this waste is advantageous due to its high sugar and low lignin content. One key component of pectin is D-galacturonic acid (D-GalUA), an oxidized substrate that S. cerevisiae cannot metabolize naturally. To enable its consumption, S. cerevisiae was equipped with the catabolic pathway naturally present in filamentous fungi. However, this did not allow D-GalUA to serve as sole carbon and energy source. The inability to grow on D-GalUA was thought to be due to a lack of electrons, as the pathway requires NAD(P)H. In this work, this problem was addressed by providing a co-substrate - glycerol - a by-product of the biodiesel industry. The electrons provided in this way were supposed to not only enable D-GalUA utilization but also support its fermentation to ethanol. Therefore, S. cerevisiae was equipped with both the fungal pathway for D-GalUA catabolism and the ‘DHA pathway’ for glycerol utilization – the latter channeling electrons from glycerol oxidation into cytosolic NADH. The resulting strain not only consumed D-GalUA at a high specific rate, but also co-fermented the substrates into ethanol, achieving a maximum yield of 71% of the theoretical maximum. Additionally, the native Gcy1, a non-specific aldo-keto reductase, was found to convert D-GalUA into L-galactonate, an intermediate of the D-GalUA catabolic pathway. By providing valuable insights into the co-utilization of glycerol and D-GalUA, this study lays the foundation for future endeavors towards the valorization of these two industrial by-products.