Key amino acid residues of the AGT1 permease required for maltotriose consumption and fermentation by Saccharomyces cerevisiae.

AIMS: The AGT1 gene encodes for a general α-glucoside-H+ symporter required for efficient maltotriose fermentation by Saccharomyces cerevisiae. In the present study, we analysed the involvement of four charged amino acid residues present in this transporter that are required for maltotriose consumption and fermentation by yeast cells. METHODS AND RESULTS: By using a knowledge-driven approach based on charge, conservation, location, three-dimensional (3D) structural modelling and molecular docking analysis, we identified four amino acid residues (Glu-120, Asp-123, Glu-167 and Arg-504) in the AGT1 permease that could mediate substrate binding and translocation. Mutant permeases were generated by site-directed mutagenesis of these charged residues, and expressed in a yeast strain lacking this permease (agt1∆). While mutating the Arg-504 or Glu-120 residues into alanine totally abolished (R504A mutant) or greatly reduced (E120A mutant) maltotriose consumption by yeast cells, as well as impaired the active transport of several other α-glucosides, in the case of the Asp-123 and Glu-167 amino acids, it was necessary to mutate both residues (D123G/E167A mutant) in order to impair maltotriose consumption and fermentation. CONCLUSIONS: Based on the results obtained with mutant proteins, molecular docking and the localization of amino acid residues, we propose a transport mechanism for the AGT1 permease. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results present new insights into the structural basis for active α-glucoside-H+ symport activity by yeast transporters, providing the molecular bases for improving the catalytic properties of this type of sugar transporters.

Characterization of maltotriose transporters from the Saccharomyces eubayanus subgenome of the hybrid Saccharomyces pastorianus lager brewing yeast strain Weihenstephan 34/70.

The genome from the Saccharomyces pastorianus industrial lager brewing strain Weihenstephan 34/70, a natural Saccharomyces cerevisiae/Saccharomyces eubayanus hybrid, indicated the presence of two different maltotriose transporter genes: a new gene in the S. eubayanus subgenome with 81% of homology to the AGT1 permease from S. cerevisiae, and an amplification of the S. eubayanus MTY1 maltotriose permease previously identified in S. pastorianus yeasts. To characterize these S. eubayanus transporter genes, we used a S. cerevisiae strain deleted in the AGT1 permease and introduced the desired permease gene(s) into this locus through homologous recombination. Our results indicate that both the MTY1 and AGT1 genes from the S. eubayanus subgenome encode functional maltotriose transporters that allow fermentation of this sugar by yeast cells, despite their apparent differences in the kinetics of maltotriose-H(+) symport activity. The presence of two maltotriose transporters in the S. eubayanus subgenome not only highlights the importance of sugar transport for efficient maltotriose utilization by industrial yeasts, but these new genes can be used in breeding and/or selection programs aimed at increasing yeast fitness for the efficient fermentation of brewer's wort.