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-coding sequence. 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 a single functional domain. 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.

Fig 1. Alignment of AGT1-like genes.

A) Tables highlighting the nucleotide (nuc) and amino acid (aa) percent identities between members of the AGT1 family. Darker colors indicate greater sequence similarity. B) Multiple sequence alignment between nucleotide sequences of tbAGT1, lgAGT1, and ncAGT1. Black lines indicate nucleotide differences. C) Multiple sequence alignments between protein sequences of tbAGT1, lgAGT1, and ncAGT1. White gaps indicate amino acid differences.

Fig 2. Phylogeny of Saccharomyces MALT genes.

ML phylogenetic tree of MALT genes described in S. cerevisiae, S. eubayanus, and lager-brewing hybrids. The scale bar equals the number of nucleotide substitutions per site. Black “*” indicate genes characterized as encoding proteins capable of transporting maltotriose. Gray “*” indicates genes encoding transporters whose ability to transport maltotriose is ambiguous.

Fig 3. Evolution and validation of the chimeric maltotriose transporter Malt434.

A) After continuous culturing on maltotriose with a small amount of added glucose, WI-Seub (yHKS210), which was originally unable to use maltotriose (MalTri-), evolved the ability to consume maltotriose (MalTri+). B) Strain yHEB1593, which is a backcross between yHKS210 and yHEB1505, was also MalTri+. C) To test the inheritance of maltotriose utilization, yHEB1593 was sporulated. The panel shows a subset of tetrads screened growing on SC + 2% maltotriose. Examples of MalTri- spores in Tetrad 1 are circled in red, and MalTri+ examples are circled in green. Whole genome sequencing of MalTri+ and MalTri- pools showed that maltotriose utilization perfectly correlated with the presence/absence of MALT434. D) Reciprocal hemizygosity test [81] of the MALT4/MALT434 locus in the backcross strain yHEB1593. E) Table of initial and day-three OD₆₀₀ (OD) readings of yHKS210, yHEB1505, yHEB1593, yHEB1853, and yHEB1854 on SC + 2% maltotriose as the sole carbon source. N = 3, standard deviation in parentheses. * Control grown in SC + 0.04% glucose to reflect the approximate amount of growth expected from contamination with other carbon sources when using 98% pure maltotriose.

Fig 4. Sequence architecture of MALT434.

A) Schematic of the origin of MALT434. B) Line graphs representing the identity between nucleotide sequences of MALT3 and MALT4 from WI-Seub (yHKS210) to MALT434 over 10-bp sliding windows. C-D) Segment of the alignment of the chimeric region between Malt3, Malt4, Malt434, scAgt1, and lgAgt1. The region highlighted in yellow in the Malt434 sequence indicates the chimeric region. The regions underlined with a red dashed line are predicted transmembrane domains. The amino acids highlighted in red are predicted maltose-binding residues. The residues highlighted in blue were experimentally found to be important for maltotriose transport by Smit et al. 2008.

Fig 5. Heterologous expression of MALT434.

A) Evolution of non-maltotriose utilizing strain (MalTri-), WI-Seub (yHKS210), to maltotriose utilizing (MalTri+) strain, yHEB1505, by serial passing on maltotriose containing media (same as Fig 3A). B) Insertion of MALT434 into vector pBM5155 for doxycycline-inducible heterologous expression in MalTri- strains. C) Transformation of MALT434 expression plasmid in MalTri- S. eubayanus strains yHKS210 and NC-Seub (yHRVM108). D) Table of initial and day-six OD₆₀₀ (OD) measurements of parent strains and strains carrying the MALT434 expression plasmid grown in SC media with maltotriose as the sole carbon and doxycycline to induce plasmid expression. N = 3, standard deviation in parentheses. * Control grown in SC + 0.04% glucose + doxycycline to reflect the approximate amount of growth expected from contamination with other carbon sources when using 98% pure maltotriose.