Biotechnological applications of yeasts: stress responses of Spathaspora passalidarum and mode of action of the biocontrol agent Pseudozyma flocculosa
Authors:   , Albuini Fernanda Matias 
Published: November 2023

Yeasts are biodiverse microorganisms that present several biotechnological applications, such as in bioethanol production and biological control of plants against fungal pathogens. The first two chapters of this thesis focused on studying the mechanisms underlying the stress responses of Spathaspora passalidarum, a yeast capable of fermenting xylose into ethanol, and the third chapter aimed to investigate the mode of action of Pseudozyma flocculosa, a natural antagonistic yeast of powdery mildew fungi. In the first study, an RNA-seq experiment was performed with S. passalidarum cells exposed to 2 h of 4% (v/v) ethanol stress. The bioinformatics analyses supported that S. passalidarum activated responses regarding protein folding and antioxidant stress. However, the osmotic stress response of this yeast appeared impaired, and genes encoding proteins from lipid metabolism, transporters, and enzymes from glycolysis and fermentation pathways showed downregulation. Additionally, ethanol-treated cells presented a pseudo-hyphal morphology. Changes in fatty acid profile were only observed after 12 h of ethanol exposure, coinciding with the yeast recovery of its xylose consumption ability. Thus, the halt in nutrient acquisition and fermentation, the lack of fast membrane adjustments, and the limited osmotic stress response were the main aspects identified underlying the low ethanol tolerance of S. passalidarum. In the second study, comparative genomic analyses were performed with Spathaspora passalidarum and Saccharomyces cerevisiae searching for clues regarding their distinct robustness to stresses. The results showed major differences in transcription factor sequences while signaling pathways and responsive proteins appeared conserved between the yeasts. The evolutionary differences in transcription factors might contribute to distinct stress responses and, ultimately, different physiological profiles. Indeed, our results showed that S. passalidarum and S. cerevisiae present distinct tolerance profiles to ethanol, oxidative, and osmotic stresses. The overexpression of the MSN-like transcription factor of S. passalidarum in an S. cerevisiae msn2 msn4 double-deleted strain indicated that the MSN-like only partially complemented the function of their orthologs in S. cerevisiae under stress, corroborating our hypothesis that transcription factors have diverged in S. passalidarum and it might have impacted the yeasts stress responses. In the third study, the role of the Pf2826 effector of Pseudozyma flocculosa was investigated in the yeast biocontrol activity against barley powdery mildew. This gene is highly expressed only during the P. flocculosa- barley-powdery mildew tripartite interaction. The knockout of this gene using the CRISPR-Cas9 mediated genome edition tool showed that the mutant line developed less aggressively and did not overtake the powdery mildew colonies, indicating that this gene is essential for the full biocontrol activity of P. flocculosa. The yeast-two- hybrid methodology was used to validate the interaction of the Pf2826 effector with seven putative interactors previously identified by a pull-down assay. The results confirmed the interaction of the Pf2826 effector with a barley pathogenesis-related protein that has putative roles in plant defense against biotic stress, and with a powdery mildew effector, which might be involved in the pathogen’s aggressiveness. These interactions probably destabilize the effector-induced host immunity suppression and boost the plant defenses, resulting in the collapse of the powdery mildew colonies. Keywords: Yeast biotechnology. Bioethanol production. Biological control.