Abstract
Saccharomyces pastorianus lager brewing yeasts are domesticated hybrids of Saccharomyces cerevisiae and cold-tolerant Saccharomyces eubayanus. To 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 CBS 12357T/FM1318. 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. Overexpression of SeMALT1-4 in a maltose-transport-deficient S. cerevisiae strain restored growth on maltose, but not on maltotriose, indicating maltose-specific transport functionality of all four transporters. Simultaneous CRISPR-Cas9-assisted deletion of only SeMALT2 and SeMALT4, which shared 99.7% sequence identity, eliminated growth of S. eubayanus CBS 12357T on maltose. Transcriptome analysis of S. eubayanus CBS 12357T established that SeMALT1 and SeMALT3, are poorly expressed in maltose-grown cultures, while SeMALT2 and SeMALT4 were expressed at much higher levels than SeMALT1 and SeMALT3, indicating that only SeMALT2/4 are responsible for maltose consumption in CBS 12357T. These results represent a first genomic and physiological characterization of maltose transport in S. eubayanus CBS 12357T and provides a valuable resource for further industrial exploitation of this yeast.
Highlights
Saccharomyces eubayanus was first isolated from Nothofagus trees and stromata of Cyttaria harioti in North-Western Patagonia (Libkind et al, 2011)
Access to S. eubayanus strains stimulated vigorous research into de novo generation of hybrids between S. cerevisiae and S. eubayanus in the laboratory (Steensels et al, 2014; Hebly et al, 2015; Krogerus et al, 2015, 2017a; Magalhães et al, 2017) This approach has the potential to increase our understanding of the domestication process of lager brewing strains and, to strongly increase the genetic and phenotypic variety of lager yeast strains available to the brewing industry
Owing to advances in genome sequencing technology, the quality of genome sequence data of S. eubayanus CBS 12357T/FM1318 has gradually improved (Libkind et al, 2011; Baker et al, 2015; Hebly et al, 2015; Okuno et al, 2016) The currently available reference sequence is based on second generation sequencing technology (Illumina generated data), obtained from libraries with different insert sizes that were co-assembled into a 11.66 Mb genome, comprising 144 contigs forming 22 scaffolds
Summary
Saccharomyces eubayanus was first isolated from Nothofagus trees and stromata of Cyttaria harioti in North-Western Patagonia (Libkind et al, 2011). Isolation and characterization of S. eubayanus provided a strong impetus for research on S. pastorianus lager brewing yeasts. De novo constructed S. cerevisiae × S. eubayanus hybrids have been demonstrated to combine advantageous brewing-related properties of both parents (cryotolerance, maltotriose utilization, and strong flocculation) and even exhibited best parent heterosis referred to as hybrid vigor (Steensels et al, 2014; Hebly et al, 2015; Krogerus et al, 2017a,b; Peris et al, 2017). Generation of new hybrids is, by itself, not sufficient to understand the genetic basis for the exceptional performance of S. pastorianus under brewing conditions
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