Structural, Physiological and Regulatory Analysis of Maltose Transporter Genes in Saccharomyces eubayanus CBS 12357T

Abstract

<i>Saccharomyces pastorianus</i> lager brewing yeasts are domesticated hybrids of <i>Saccharomyces cerevisiae</i> and cold-tolerant <i>Saccharomyces eubayanus</i>. To understand the contribution of both parental genomes to maltose metabolism in brewing wort, this study focuses on maltose transport in the <i>S. eubayanus</i> type strain CBS 12357^T/FM1318. To obtain complete sequences of the <i>MAL</i> 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 (<i>SeMALT1-4</i>), located in subtelomeric regions of CHRII, CHRV, CHRXIII, and CHRXVI, were completely resolved. The near-identical loci on CHRV and CHRXVI strongly resembled canonical <i>S. cerevisiae MAL</i> loci, while those on CHRII and CHRXIII showed different structures suggestive of gene loss. Overexpression of <i>SeMALT1-4</i> in a maltose-transport-deficient <i>S. cerevisiae</i> 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 <i>SeMALT2</i> and <i>SeMALT4</i>, which shared 99.7% sequence identity, eliminated growth of <i>S. eubayanus</i> CBS 12357^T on maltose. Transcriptome analysis of <i>S. eubayanus</i> CBS 12357^T established that <i>SeMALT1</i> and <i>SeMALT3</i>, are poorly expressed in maltose-grown cultures, while <i>SeMALT2</i> and <i>SeMALT4</i> were expressed at much higher levels than <i>SeMALT1</i> and <i>SeMALT3</i>, indicating that only <i>SeMALT2/4</i> are responsible for maltose consumption in CBS 12357^T. These results represent a first genomic and physiological characterization of maltose transport in <i>S. eubayanus</i> CBS 12357^T and provides a valuable resource for further industrial exploitation of this yeast.