Positive Selection Evidence in Xylose-Related Genes Suggests Methylglyoxal Reductase as a Target for the Improvement of Yeasts' Fermentation in Industry

Abstract

Xylose assimilation and fermentation are important traits for second generation ethanol production. However, some genomic features associated with this pentose sugar's metabolism remain unknown in yeasts. Comparative genomics studies have led to important insights in this field, but we are still far from completely understanding endogenous yeasts' xylose metabolism. In this work, we carried out a deep evolutionary analysis suited for comparative genomics of xylose-consuming yeasts, searching for of positive selection on genes associated with glucose and xylose metabolism in the xylose-fermenters' clade. Our investigation detected positive selection fingerprints at this clade not only among sequences of important genes for xylose metabolism, such as xylose reductase and xylitol dehydrogenase, but also in genes expected to undergo neutral evolution, such as the glycolytic gene phosphoglycerate mutase. In addition, we present expansion, positive selection marks, and convergence as evidence supporting the hypothesis that natural selection is shaping the evolution of the little studied methylglyoxal reductases. We propose a metabolic model suggesting that selected codons among these proteins caused a putative change in cofactor preference from NADPH to NADH that alleviates cellular redox imbalance. These findings provide a wider look into pentose metabolism of yeasts and add this previously overlooked piece into the intricate puzzle of oxidative imbalance. Although being extensively discussed in evolutionary works the awareness of selection patterns is recent in biotechnology researches, rendering insights to surpass the reached status quo in many of its subareas.

Keywords: Saccharomycotina; comparative genomics; natural selection; phylogenetics; xylitol dehydrogenase; xylose reductase.

Fig. 1.

—Maximum likelihood species phylogenetic inference for 18 yeasts. S. pombe was used as outgroup of the 17 species from Saccharomycotina subphylum. Tree was reconstructed in RAxML using 1,255 single-copy ortholog groups and branch supports were obtained using 5,000 bootstrap randomizations. The group referred to as fermenters’ clade is highlighted. Phylogeny is scaled in substitution per positions.

Fig. 2.

—Rate of gene gains and losses along species of Saccharomycotina. Inferences were made by maximum likelihood estimates based on gene birth and death models of evolution. Branch rates were calculated as the difference ratio between gene gains and losses to its inferred ancestor gene number. The tree branches’ width and colors indicate the percentage of gene gain/loss as shown in caption. Two families were significantly expanded in the fermenters’ ancestral branch, shown by the dark gray box. The actual number of genes is indicated in the side table. Light gray box highlights xylose-fermenters. Phylogeny is scaled in substitutions per site.

Fig. 3.

—Methylglyoxal Bayesian gene tree inference (fam7 and fam167). Columns show amino acid alignment for sites which have evidence for positive selection or parallel evolution. Highlighted boxes represent clades with positive selection evidence on fermenters’ group. Red gene names indicate gene copies from fam167, and red amino acids indicate that for this gene the respective amino acid is distinct from others at the alignment site. Amino acids with convergence between families on the alignment are shown in orange. Branches are colored relative to their posterior probabilities. Tree is scaled by substitutions per site.

Fig. 4.

—Phylogenetic inference for 179 genes (not shown) from 3 gene families of putative xylose reductase (black dots) genes and 4 families of xylitol dehydrogenase genes (gray dots), carried out with maximum likelihood inference on RAxML with branch support obtained with 1,000 bootstraps. Tree is scaled in substitutions per site.

Fig. 5.

—Xylose reductase Bayesian gene tree inference of fam634. Posterior probabilities are shown above the branches. The three columns show amino acid alignment for sites which have positive selection evidence. Red amino acids are distinct when compared with other species. Highlighted boxes represent clades with positive selection evidence on fermenter groups. Tree is scaled by substitutions per site.

Fig. 6.

—Inferred probabilities of evolutionary models along protein sequences. Plots of posterior probabilities (in y axis) for each tested evolutionary model (negative selection, neutral evolution, positive selection) in codeml model 2, for each protein site (x axis). Sites with higher probability than 0.95 for positive selection are indicated with the model code above graphs. Cofactor binding sites and proton donor sites known for S. cerevisiae are also shown.

Fig. 7.

—Saccharomyces cerevisiae metabolic pathways related to xylose and glucose consumption. Enzymes are colored according to omega (dN/dS, CODEML model 2, NsSites 1–2) values (light red—low values, dark red—high values). (A) and (B) refer to reactions where the cofactor preference is modified in S. passalidarum or S. stipitis. (C) refers to methylglyoxal reductase hypothetical cofactor preference change in paralog proteins with evidence for positive selection in our analysis.