Project description:The Crabtree phenotype defines whether a yeast can perform simultaneous respiration and fermentation under aerobic conditions at high growth rates, a phenomenon that resembles the Warburg effect in cancer cells. Whole genome duplication, global promoter rewiring and loss of respiratory complex I are the main molecular events that contributed to the evolution of Crabtree effect. Here we show that overexpression of a single Gal4-like transcription factor is sufficient to convert Crabtree-negative Komagataella phaffii (Pichia pastoris) into a Crabtree positive yeast. We report the transcriptome profile (RNASeq) of the Δgal4-like and Gal4-like overexpression K. phaffii strains. Upregulation of the glycolytic genes and a significant increase in glucose uptake rate due to the overexpression of the Gal4-like transcription factor caused an overflow metabolism, triggering both short-term and long-term Crabtree phenotypes. This indicates that a single mutation leading to overexpression of one gene may have been sufficient as a first molecular event towards respiro-fermentative metabolism in the course of yeast evolution.
Project description:Adaptation to alkalinization of the medium in fungi involves an extensive remodeling of gene expression. Komagataella phaffii is an ascomycetous yeast that has become an organism widely used for heterologous protein expression. We explore here the transcriptional impact of moderate alkalinization in this yeast. In spite of a minor effect on growth, shifting the cultures from pH 5.5 to 8.0 or 8.2 provokes significant changes in the mRNA levels of over 700 genes. Functional categories such as arginine and methionine biosynthesis, non-reductive iron uptake and phosphate metabolism are enriched in induced genes, whereas many genes encoding iron-sulfur proteins or members of the respirasome were repressed. We also show that alkalinization is accompanied by oxidative stress and we propose this circumstance as a common trigger of a subset of the observed changes. PHO89, encoding a Na+/Pi cotransporter, appears among the most potently induced genes by high pH. We demonstrate that this response is mainly based on two calcineurin-dependent response elements located in its promoter, thus indicating that alkalinization triggers a calcium-mediated signal in K. phaffii. Further characterization of alkaline pH responsive promoters may lead to developing novel pH-controlled systems for heterologous protein expression in this fungus.
