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Structural, physiological and regulatory analysis of maltose transporter genes in Saccharomyces eubayanus CBS12357T.

ABSTRACT: Saccharomyces pastorianus lager brewing yeasts are domesticated hybrids of Saccharomyces cerevisiae and cold-tolerant Saccharomyces eubayanus. To better understand the contribution of both parental genomes to maltose metabolism in brewing wort, this study focuses on maltose transport in the S. eubayanus type strain CBS12357T/FM1318T. To obtain complete sequences of the MAL loci of this strain, a near-complete genome assembly was generated using the Oxford Nanopore Technology MinION sequencing platform. Except for CHRXII, all sixteen chromosomes were assembled as single contigs. Four loci harboring putative maltose transporter genes (SeMALT1-4), located in subtelomeric regions of CHRII, CHRV, CHRXIII and CHRXVI, were completely resolved. The near-identical loci on CHRV and CHRXVI strongly resembled canonical S. cerevisiae MAL loci, while those on CHRII and CHRXIII showed different structures suggestive of gene loss. Functionality of the SeMALT1-4-encoded transporters was confirmed by their ability to restore growth on maltose, but not on maltotriose, of a maltose-transport-deficient S. cerevisiae strain. Simultaneous CRISPR-Cas9-assisted deletion of SeMALT2 and SeMALT4, which shared 99.7 % sequence identity, eliminated growth of S. eubayanus CBS12357T on maltose. Transcriptome analysis of S. eubayanus CBS12357T established that, in maltose-grown cultures, SeMALT2 and SeMALT4 were expressed at much higher levels than SeMALT1 and SeMALT3, thus resolving the apparent discrepancy between heterologous expression and deletion studies. These results represent a first genomic and physiological characterization of maltose transport in S. eubayanus CBS12357T and provides a valuable resource for further industrial exploitation of this yeast.

ORGANISM(S): Saccharomyces eubayanus

PROVIDER: GSE117246 | GEO | 2018/07/18

REPOSITORIES: GEO

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Project description:Prolonged cultivation (>25 generations) of Saccharomyces cerevisiae in aerobic, maltose-limited chemostat cultures led to profound physiological changes. Maltose hypersensitivity was observed when cells from prolonged cultivations were suddenly exposed to excess maltose. This substrate hypersensitivity was evident from massive cell lysis and loss of viability. During prolonged cultivation at a fixed specific growth rate, the affinity for the growth-limiting nutrient (i.e., maltose) increased, as evident from a decreasing residual maltose concentration. Furthermore, the capacity of maltose-dependent proton uptake increased up to 2.5-fold during prolonged cultivation. Genome-wide transcriptome analysis showed that the increased maltose transport capacity was not primarily due to increased transcript levels of maltose-permease genes upon prolonged cultivation. We propose that selection for improved substrate affinity (ratio of maximum substrate consumption rate and substrate saturation constant) in maltose-limited cultures leads to selection for cells with an increased capacity for maltose uptake. At the same time, the accumulative nature of maltose-proton symport in S. cerevisiae leads to unrestricted uptake when maltose-adapted cells are exposed to a substrate excess. These changes were retained after isolation of individual cell lines from the chemostat cultures and nonselective cultivation, indicating that mutations were involved. The observed trade-off between substrate affinity and substrate tolerance may be relevant for metabolic engineering and strain selection for utilization of substrates that are taken up by proton symport. Experiment Overall Design: In a recent study (25) we analyzed glucose efflux upon exposure of S. cerevisiae to excess maltose, with yeast cells originating from âyoungâ chemostat cultures (<20 generations). In these experiments no cell lysis was observed upon exposure to excess maltose. However, in further work on this subject, we observed an apparent effect of chemostat culture age on transport capacity. The aim of the present study was to further investigate the effect of prolonged maltose-limited chemostat cultivation on the physiology of S. cerevisiae. To this end we monitored the affinity for maltose, genome-wide transcript levels, activities of key enzymes, and physiological responses to maltose excess during long-term cultivation in maltose-limited chemostat cultures. Experiment Overall Design: Jansen, M. L. A., J. H. de Winde, and J. T. Pronk. 2002. Hxt-carrier-mediated glucose efflux upon exposure of Saccharomyces cerevisiaeSaccharomyces cerevisiae to excess maltose. Appl. Environ. Microbiol. 68:4259-4265.

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Structural, physiological and regulatory analysis of maltose transporter genes in Saccharomyces eubayanus CBS12357T.

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