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Maltose is one of the most abundant sugars in brewer’s wort, and its efficient utilization is critical for successful fermentation. However, maltose consumption varies naturally among Saccharomyces eubayanus strains isolated from different host trees, such as Quercus and Nothofagus. To identify the genetic determinants underlying these phenotypic differences, we performed bulk segregant analysis (BSA) and quantitative trait loci (QTL) mapping using an F₂ offspring derived from QC18 (Quercus-associated) and CL467.1 (Nothofagus-associated) strains. QTL mapping identified two significant genomic regions on subtelomeric loci of chromosomes V-R and XVI-L, each containing complete MAL loci composed of MAL32 (encoding maltase), MAL31 (transporter), andmore » MAL33 (transcriptional activator) genes. Comparative polymorphism analyses identified mutations in MAL32 and MAL33 of QC18, including frameshift mutations resulting in premature stop codons. Functional validation demonstrated that the heterologous expression of MAL33_ChrV from CL467.1 fully restored maltose utilization in QC18, indicating the functional presence of MAL33 cis-regulatory sequences and MAL32 and MAL31 genes in QC18. While structural protein predictions identified truncation and impaired functionality in the maltose-responsive activation domain of Mal33p from QC18, overexpression of QC18’s own MAL33_ChrV allele also improved maltose metabolism, suggesting dosage-dependent transcriptional limitations rather than complete functional loss. These results indicate that allelic variations in the maltose-responsive activation domain of Mal33p result in differences in maltose consumption between strains. Here, we hypothesized that reduced maltose metabolism in QC18 is an adaptive response to the distinct sugar composition in Quercus robur bark, contrasting with the starch-rich environment of Nothofagus pumilio. These findings highlight subtelomeric MAL gene diversity as a reservoir of genetic variation, representing a key evolutionary mechanism that influences maltose adaptation among natural Saccharomyces isolates.« less

Many remarkable phenotypes have repeatedly occurred across vast evolutionary distances. When convergent traits emerge on the tree of life, they are sometimes driven by the same underlying gene families, while other times, many different gene families are involved. Conversely, a gene family may be repeatedly recruited for a single trait or many different traits. To understand the general rules governing convergence at both genomic and phenotypic levels, we systematically tested associations between 56 binary metabolic traits and gene count in 14,785 gene families from 993 Saccharomycotina yeasts. Using a recently developed phylogenetic approach that reduces spurious correlations, we found thatmore » gene family expansion and contraction were significantly linked to trait gain and loss in 45/56 (80%) traits. While 595/739 (81%) significant gene families were associated with only one trait, we also identified several “keystone” gene families that were significantly associated with up to 13/56 (23%) of all traits. Strikingly, most of these families are known to encode metabolic enzymes and transporters, including all members of the industrially relevant MAL tose fermentation loci in the baker’s yeast Saccharomyces cerevisiae. These results indicate that convergent evolution on the gene family level may be more widespread across deeper timescales than previously believed.« less

Biofuels derived from renewable and sustainable lignocellulosic biomass, such as switchgrass, offer a promising means to limit greenhouse gas emissions. However, switchgrass grown under drought conditions contains high levels of chemical compounds that inhibit microbial conversion to biofuels. Fermentation of drought switchgrass hydrolysates by engineered Saccharomyces cerevisiae and Zymomonas mobilis results in lower ethanol production than does fermentation of hydrolyzed switchgrass from a typical rainfall year. Here, it is demonstrated that this inhibitory effect can be alleviated by altering the pH of drought switchgrass hydrolysates produced by two different pretreatment methods: Ammonia Fiber Expansion (AFEX) and Soaking in Aqueous Ammoniamore » (SAA). Fermentation rates and biofuel production by Saccharomyces cerevisiae and Zymomonas mobilis were higher at pH 5.8 than at pH 5.0 from all feedstock years and following both pretreatment methods. SAA pretreatment of drought switchgrass furthermore enabled increased fermentation rates and biofuel titers compared to AFEX pretreatment. A synthetic mimic of switchgrass hydrolysate was developed and identified relief from pH-dependent inhibition by lignocellulose-derived inhibitors as the cause of increased biofuel production above a pH of 5.0. These results demonstrate that SAA pretreatment and pH adjustment can significantly improve fermentation and biofuel production from inhibitory feedstocks by industrial microorganisms.« less

Functional innovation at the protein level is a key source of evolutionary novelties. The constraints on functional innovations are likely to be highly specific in different proteins, which are shaped by their unique histories and the extent of global epistasis that arises from their structures and biochemistries. These contextual nuances in the sequence–function relationship have implications both for a basic understanding of the evolutionary process and for engineering proteins with desirable properties. Here, we have investigated the molecular basis of novel function in a model member of an ancient, conserved, and biotechnologically relevant protein family. These Major Facilitator Superfamily sugarmore » porters are a functionally diverse group of proteins that are thought to be highly plastic and evolvable. By dissecting a recent evolutionary innovation in an α-glucoside transporter from the yeast Saccharomyces eubayanus, we show that the ability to transport a novel substrate requires high-order interactions between many protein regions and numerous specific residues proximal to the transport channel. To reconcile the functional diversity of this family with the constrained evolution of this model protein, we generated new, state-of-the-art genome annotations for 332 Saccharomycotina yeast species spanning ~400 My of evolution. By integrating phylogenetic and phenotypic analyses across these species, we show that the model yeast α-glucoside transporters likely evolved from a multifunctional ancestor and became subfunctionalized. The accumulation of additive and epistatic substitutions likely entrenched this subfunction, which made the simultaneous acquisition of multiple interacting substitutions the only reasonably accessible path to novelty.« less