Development of a yeast-based system to remove and recover heavy metals

Heavy metals contamination has become an environmental concern due to its toxicity and non-degradability. Looking for eco-friendly solutions, bioremediation using microorganisms has emerged as a promising alternative. Among different microorganisms, Saccharomyces cerevisiae stands out for its ease of handling, rapid growth, and low nutritional requirements, making it a cost-effective option for large biomass production. Previous studies have highlighted that the metal-binding capabilities of yeast cells can be enhanced by the Yeast Surface Display (YSD) genetic technique, that enables the expression of specific proteins on the cell surface. Over time, this strategy has been applied to express a variety of peptides and proteins on the yeast cell wall, contributing to processes such as biocatalysis and biofuel production. However, experimental investigations involving YSD have been focused only to laboratory strains of S. cerevisiae, which represent a small group of domesticated strains ideal for research purposes. In contrast, S. cerevisiae strains isolated from natural environment possess unique characteristics shaped by selective pressure, that could improve YSD efficiency. Despite the potential, there are no documented examples of YSD-engineered natural strains in the literature. The main goal of this PhD project was to develop and optimize the molecular tools required to perform the YSD protein expression in yeast strains isolated from natural environments. Specifically, S. cerevisiae natural strains have been selected from the large collection provided by the project partner company. Through a series of functional and biochemical assays, experimental evidence supported that natural strains can be genetically modified to efficiently express on their cell wall different protein-of-interest, further proven to retain their biological competence, providing the proof-of-evidence that natural strains are suitable for YSD-based applications. Noteworthy, YSD was successfully performed in S.cerevisiae cells endowed with very different features, and phylogenetically highly divergent, as the strains employed for the production of wine, bread, whisky, or animal food additives. Therefore, data collectively indicated that the YSD system developed here may be used in S. cerevisiae strains isolated from natural environments, independently of the genetic background. Additional experiments have been then performed to improve YSD efficiency by introducing site-specific genetic modifications, as the inactivation of several genes implicated in cell wall biosynthesis, but no substantial differences were revealed between mutant and control yeast cells. Interestingly, data demonstrated that YSD-engineered strains expressing metal-binding peptide/proteins on surface were significantly enhanced in metal adsorption properties, pointing to the possible use in bioremediation. Notably, yeast cells displaying either exa-histidine peptide, metallothionein, or calmodulin, were able to specifically target nickel, copper, and calcium ions, respectively. However, when applied to real matrices, as grape must or contaminated river water, metal-binding ability of the yeast cells was markedly reduced, likely due to matrix composition, where competing molecules could interfere with protein-ion binding. Nevertheless, to increase the yeast biomass, large-scale propagation and industrial production were carried out for the YSD-engineered natural IB1 strain expressing the yCup1 metallothionein. However, comparison of the ability to remove and/or recover copper ions between wild-type and YSD-yCup1 yeast strains, failed to reveal relevant differences, indicating that the large-scale production process irreversibly damaged or denatured the surface protein. Future investigations are required to optimize yeast large-scale production preserving surface-exposed proteins functionality, improving the overall performance of yeast-based systems in environmental applications.