Evolution of a novel chimeric maltotriose transporter in Saccharomyces eubayanus from parent proteins unable to perform this function

Abstract

At the molecular level, the evolution of new traits can be broadly divided between changes in gene expression and changes in protein structure. For proteins, the evolution of novel functions is generally thought to proceed through sequential point mutations or recombination of whole functional units. In Saccharomyces, the uptake of the sugar maltotriose into the cell is the primary limiting factor in its utilization, but maltotriose transporters are relatively rare, except in brewing strains. No known wild strains of Saccharomyces eubayanus, the cold-tolerant parent of hybrid lager-brewing yeasts (Saccharomyces cerevisiae x S. eubayanus), are able to consume maltotriose, which limits their ability to fully ferment malt extract. In one strain of S. eubayanus, we found a gene closely related to a known maltotriose transporter and were able to confer maltotriose consumption by overexpressing this gene or by passaging the strain on maltose. Even so, most wild strains of S. eubayanus lack native maltotriose transporters. To determine how this rare trait could evolve in naive genetic backgrounds, we performed an adaptive evolution experiment for maltotriose consumption, which yielded a single strain of S. eubayanus able to grow on maltotriose. We mapped the causative locus to a gene encoding a novel chimeric transporter that was formed by an ectopic recombination event between two genes encoding transporters that are unable to import maltotriose. In contrast to classic models of the evolution of novel protein functions, the recombination breakpoints occurred within functional domains. Thus, the ability of the new protein to carry maltotriose was likely acquired through epistatic interactions between independently evolved substitutions. By acquiring multiple mutations at once, the transporter rapidly gained a novel function, while bypassing potentially deleterious intermediate steps. This study provides an illuminating example of how recombination between paralogs can establish novel interactions among substitutions to create adaptive functions.\n\nAuthor summaryHybrids of the yeasts Saccharomyces cerevisiae and Saccharomyces eubayanus (lager-brewing yeasts) dominate the modern brewing industry. S. cerevisiae, also known as bakers yeast, is well-known for its role in industry and scientific research. Less well recognized is S. eubayanus, which was only discovered as a pure species in 2011. While most lager-brewing yeasts rapidly and completely utilize the important brewing sugar maltotriose, no strain of S. eubayanus isolated to date is known to do so. Despite being unable to consume maltotriose, we identified one strain of S. eubayanus carrying a gene for a functional maltotriose transporter, although most strains lack this gene. During an adaptive evolution experiment, a strain of S. eubayanus without native maltotriose transporters evolved the ability to grow on maltotriose. Maltotriose consumption in the evolved strain resulted from a chimeric transporter that arose through recombination between genes encoding parent proteins that were unable to transport maltotriose. Traditionally, functional chimeric proteins are thought to evolve by recombining discrete functional domains or modules, but the breakpoints in the chimera studied here occurred within modular units of the protein. These results support the less well-recognized role of recombination between paralogous sequences in generating novel proteins with adaptive functions.