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Exploration of the genetic makeup and expression of the glycolytic and fermentative pathways within the Saccharomyces genus

ABSTRACT: The ability of the yeast Saccharomyces cerevisiae to convert glucose, even in the presence of oxygen, via glycolysis and the fermentative pathway to ethanol has played an important role in its domestication. Despite the extensive knowledge on these pathways in S. cerevisiae, relatively little is known about these pathways in other industrially-relevant Saccharomyces yeast species. In this study we explore the diversity of the glycolytic and fermentative pathways within the Saccharomyces genus using S. cerevisiae, S. kudriavzevii and S. eubayanus as paradigms. Sequencing data revealed a highly conserved genetic makeup of the glycolytic and fermentative pathways in the three species in terms of number of paralogous genes. Although promoter regions were less conserved between the three species as compared to coding sequences, binding sites for Rap1, Gcr1 and Abf1, main transcriptional regulators of glycolytic and fermentative genes, were highly conserved. Transcriptome profiling of these three strains grown in aerobic batch cultivation in chemically defined medium with glucose as carbon source, revealed a remarkably similar expression of the glycolytic and fermentative genes across species, and the conserved classification of genes into major and minor paralogs. Furthermore, transplantation of the promoters of major paralogs of S. kudriavzevii and S. eubayanus into S. cerevisiae demonstrated not only the transferability of these promoters, but also the similarity of their strength and response to various environmental stimuli. The relatively low homology of S. kudriavzevii and S. eubayanus promoters to their S. cerevisiae relatives makes them very attractive alternatives for strain construction in S. cerevisiae, thereby expanding S. cerevisiae molecular toolbox.

ORGANISM(S): Saccharomyces kudriavzevii Saccharomyces eubayanus Saccharomyces cerevisiae

PROVIDER: GSE117404 | GEO | 2018/07/21

REPOSITORIES: GEO

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Project description:As a result of ancestral whole genome and small-scale duplication events, the genome of Saccharomyces cerevisiaeM-bM-^@M-^Ys, and of many eukaryotes, still contain a substantial fraction of duplicated genes. In all investigated organisms, metabolic pathways, and more particularly glycolysis, are specifically enriched for functionally redundant paralogs. In ancestors of the Saccharomyces lineage, the duplication of glycolytic genes is purported to have played an important role leading to S. cerevisiae current lifestyle favoring fermentative metabolism even in the presence of oxygen and characterized by a high glycolytic capacity. In modern S. cerevisiae, the 12 glycolytic reactions leading to the biochemical conversion from glucose to ethanol are encoded by 27 paralogs. In order to experimentally explore the physiological role of this genetic redundancy, a yeast strain with a minimal set of 14 paralogs was constructed (MG strain). Remarkably, a combination of quantitative, systems approach and of semi-quantitative analysis in a wide array of growth environments revealed the absence of phenotypic response to the cumulative deletion of 13 glycolytic paralogs. This observation indicates that duplication of glycolytic genes is not a prerequisite for achieving the high glycolytic fluxes and fermentative capacities that are characteristic for S. cerevisiae and essential for many of its industrial applications and argues against gene dosage effects as a means for fixing minor glycolytic paralogs in the yeast genome. MG was carefully designed and constructed to provide a robust, prototrophic platform for quantitative studies, and is made available to the scientific community. The goals of the present study are to experimentally explore genetic redundancy in yeast glycolysis by cumulative deletion of minor paralogs and to provide a new experimental platform for fundamental yeast research by constructing a yeast strain with a functional M-bM-^@M-^Xminimal glycolysisM-bM-^@M-^Y. To this end, we deleted 13 minor paralogs, leaving only the 14 major paralogs for the S. cerevisiae glycolytic pathway. The cumulative impact of deleting all minor paralogs was investigated by two complementary approaches. A first, quantitative analysis focused on the impact on glycolytic flux under a number of controlled cultivation conditions that, in wild-type strains, result in different glycolytic fluxes. These quantitative growth studies were combined with transcriptome, enzyme-activity and intracellular metabolite assays to capture potential small phenotypic effects. A second, semi-quantitative characterization explored the phenotype of the M-bM-^@M-^Xminimal glycolysisM-bM-^@M-^Y strain under a wide array of experimental conditions to identify potential context-dependent phenotypes

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Exploration of the genetic makeup and expression of the glycolytic and fermentative pathways within the Saccharomyces genus

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