Project description:Aims: We performed an analysis of maltotriose utilization by 52 Saccharomyces yeast strains able to ferment maltose efficiently and correlated the observed phenotypes with differences in the copy number of genes possibly involved in maltotriose utilization by yeast cells. Methods and Results: The analysis of maltose and maltotriose utilization by laboratory and industrial strains of the species Saccharomyces cerevisiae and Saccharomyces pastorianus (a natural S. cerevisiae/Saccharomyces bayanus hybrid) was carried out using microscale liquid cultivation, as well as in aerobic batch cultures. All strains utilize maltose efficiently as a carbon source, but three different phenotypes were observed for maltotriose utilization: efficient growth, slow/delayed growth and no growth. Through microarray karyotyping and pulsed-field gel electrophoresis blots, we analysed the copy number and localization of several maltose-related genes in selected S. cerevisiae strains. While most strains lacked the MPH2 and MPH3 transporter genes, almost all strains analysed had the AGT1 gene and increased copy number of MALx1 permeases. Conclusions: Our results showed that S. pastorianus yeast strains utilized maltotriose more efficiently than S. cerevisiae strains and highlighted the importance of the AGT1 gene for efficient maltotriose utilization by S. cerevisiae yeasts. Significance and Impact of the Study: Our results revealed new maltotriose utilization phenotypes, contributing to a better understanding of the metabolism of this carbon source for improved fermentation by Saccharomyces yeasts.
Project description:Aims: We performed an analysis of maltotriose utilization by 52 Saccharomyces yeast strains able to ferment maltose efficiently and correlated the observed phenotypes with differences in the copy number of genes possibly involved in maltotriose utilization by yeast cells. Methods and Results: The analysis of maltose and maltotriose utilization by laboratory and industrial strains of the species Saccharomyces cerevisiae and Saccharomyces pastorianus (a natural S. cerevisiae/Saccharomyces bayanus hybrid) was carried out using microscale liquid cultivation, as well as in aerobic batch cultures. All strains utilize maltose efficiently as a carbon source, but three different phenotypes were observed for maltotriose utilization: efficient growth, slow/delayed growth and no growth. Through microarray karyotyping and pulsed-field gel electrophoresis blots, we analysed the copy number and localization of several maltose-related genes in selected S. cerevisiae strains. While most strains lacked the MPH2 and MPH3 transporter genes, almost all strains analysed had the AGT1 gene and increased copy number of MALx1 permeases. Conclusions: Our results showed that S. pastorianus yeast strains utilized maltotriose more efficiently than S. cerevisiae strains and highlighted the importance of the AGT1 gene for efficient maltotriose utilization by S. cerevisiae yeasts. Significance and Impact of the Study: Our results revealed new maltotriose utilization phenotypes, contributing to a better understanding of the metabolism of this carbon source for improved fermentation by Saccharomyces yeasts. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.
Project description: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.
Project description:Investigation of whole genome gene expression level changes in three S. cerevisiae Y55 mutants, compared to the wild-type strain. The UV-induced mutations enable the mutant strains to ferment high-gravity maltose faster than the WT. The mutants analyzed in this study are further described in Baerends, R.J.S., J.L. Qiu, L. Gautier, and A. Brandt. A high-throughput system for screening of fast-fermenting Saccharomyces cerevisiae strains. Manuscript in preparation. A single-dye 12-plex array chip study using double-stranded DNA prepared from messenger RNA purified from total RNA recovered from three separate Saccharomyces cerevisiae Y55 wild-type cultures and 3x three separate cultures each corresponding to a fast-fermenting UV-induced mutant (mutant 1, 2 and 3), during fermentation of high-gravity maltose at day 2. Each array on the 12-plex chip measures the expression level of 5,777 genes from Saccharomyces cerevisiae S288C with eight 60-mer probes per gene, with three-fold technical redundancy.
Project description:Investigation of whole genome gene expression level changes in three S. cerevisiae Y55 mutants, compared to the wild-type strain. The UV-induced mutations enable the mutant strains to ferment high-gravity maltose faster than the WT. The mutants analyzed in this study are further described in Baerends, R.J.S., J.L. Qiu, L. Gautier, and A. Brandt. A high-throughput system for screening of fast-fermenting Saccharomyces cerevisiae strains. Manuscript in preparation.
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. Keywords: Evolution
Project description:Beer brewing is a well-known process that still faces great challenges, such as the total consumption of sugars present in the fermentation media. Lager-style beer, major worldwide beer type, is elaborated by Saccharomyces pastorianus (Sp) yeast which must ferment high maltotriose content worts, but its consumption represents a notable problem, especially among Sp strains belonging to group I. Factors like fermentation conditions, presence of maltotriose transporters, transporter copy number variation, and genetic regulation variations contribute to this issue. We assess the factors affecting fermentation in two Sp yeast strains: SpIB1, with limited maltotriose uptake, and SpIB2, known for efficient maltotriose transport. Here, SpIB2 transported significantly more maltose (28%) and maltotriose (32%) compared to SpIB1. Furthermore, SpIB2 expressed all MAL transporters (ScMALx1, SeMALx1, ScAGT1, SeAGT1, MTT1, and MPHx) on the first day of fermentation, while SpIB1 only exhibited ScMalx1, ScAGT1, and MPH2/3 genes. Some SpIB2 transporters had polymorphic transmembrane domains (TMD) resembling MTT1, accompanied by higher expression of these transporters and its positive regulator genes like MAL63. These findings suggest that, in addition of the factors mentioned above, positive regulators of Mal transporters contribute significatively to phenotypic diversity in maltose and maltotriose consumption among the studied lager yeast strains.
Project description:In response to limited nitrogen and abundant carbon sources, diploid Saccharomyces cerevisiae strains undergo a filamentous transition in cell growth as part of pseudohyphal differentiation. Use of the disaccharide maltose as the principal carbon source, in contrast to the preferred nutrient monosaccharide glucose, has been shown to induce a hyper-filamentous growth phenotype in a strain deficient for GPA2 which codes for a Galpha protein component that interacts with the glucose-sensing receptor Gpr1p to regulate filamentous growth. In this report, we compare the global transcript and proteomic profiles of wild-type and Gpa2p deficient diploid yeast strains grown on both rich and nitrogen starved maltose media. We find that deletion of GPA2 results in significantly different transcript and protein profiles when switching from rich to nitrogen starvation media. The results are discussed with a focus on the genes associated with carbon utilization, or regulation thereof, and a model for the contribution of carbon sensing/metabolism-based signal transduction to pseudohyphal differentiation is proposed. Keywords: Saccharomyces cerevisiae, nitrogen starvation, maltose, pseudohyphal differentiation, yeast, expression profiling
Project description:We study the genetics, including microarray karyotyping using comparative genomic hybridization to explore global changes in the genomic DNA, of four S. bayanus var uvarum strains related to traditional fermentations of very different sources comparing to the sequenced S. cerevisiae laboratory strain (S288C). Our final goal is to determine the adaptive evolution of properties of biotechnological interest in Saccharomyces yeasts. Many copy number variations (CNV) were observed, especially in genes associated to subtelomeric regions and transposon elements. Among the fermentation strains, differential CNV was observed in genes related to sugar transport and metabolism. An outstanding example of diverse CNV is the gen PUT1, involved in proline assimilation, which correlated with the adaptation of the strains to the presence of this nitrogen source in the media.