Files_used_for_figures
This folder includes all the relevant files used to generate the main Figures in the paper.
DNA repair↗
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Stable hypermutators revealed by the genomic landscape of genes involved in genome stability among yeast species
Mutator phenotypes are short-lived due to the rapid accumulation of deleterious mutations. Yet, recent observations reveal that certain fungi can undergo prolonged accelerated evolution after losing genes involved in DNA repair. Here, we surveyed 1,154 yeast genomes representing nearly all known yeast species of the subphylum Saccharomycotina (phylum Ascomycota) to examine the relationship between reduced gene repertoires broadly associated with genome stability functions (e.g., DNA repair, cell cycle) and elevated evolutionary rates. We identified three distantly related lineages—encompassing 12% of species—that had both the most streamlined sets of genes involved in genome stability (specifically DNA repair) and the highest evolutionary rates in the entire subphylum. Two of these “faster-evolving lineages” (FELs)—a subclade within the order Pichiales and the Wickerhamiella/Starmerella (W/S) clade (order Dipodascales)—are described here for the first time, while the third corresponds to a previously documented Hanseniaspora FEL. Examination of genome stability gene repertoires revealed a set of genes predominantly absent in these three FELs, suggesting a potential role in the observed acceleration of evolutionary rates. In the W/S clade, genomic signatures are consistent with a substantial mutational burden, including pronounced A|T bias and endogenous DNA damage. Interestingly, we found that the W/S clade also contains DNA repair genes possibly acquired through horizontal gene transfer, including a photolyase of bacterial origin. These findings highlight how hypermutators can persist across macroevolutionary timescales, potentially linked to the loss of genes related with genome stability, with horizontal gene transfer as a possible avenue for partial functional compensation.
DNA repair↗
Unique trajectory of gene family evolution from genomic analysis of nearly all known species in an ancient yeast lineage
Gene gains and losses are a major driver of genome evolution; their precise characterization can provide insights into the origin and diversification of major lineages. Here, we examined gene family evolution of 1154 genomes from nearly all known species in the medically and technologically important yeast subphylum Saccharomycotina. We found that yeast gene family evolution differs from that of plants, animals, and filamentous ascomycetes, and is characterized by smaller overall gene numbers yet larger gene family sizes for a given gene number. Faster-evolving lineages (FELs) in yeasts experienced significantly higher rates of gene losses—commensurate with a narrowing of metabolic niche breadth—but higher speciation rates than their slower-evolving sister lineages (SELs). Gene families most often lost are those involved in mRNA splicing, carbohydrate metabolism, and cell division and are likely associated with intron loss, metabolic breadth, and non-canonical cell cycle processes. Our results highlight the significant role of gene family contractions in the evolution of yeast metabolism, genome function, and speciation, and suggest that gene family evolutionary trajectories have differed markedly across major eukaryotic lineages.
Comparative Genomics↗
Specialization Restricts the Evolutionary Paths Available to Yeast Sugar Transporters
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 sugar 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.
59 BASIC BIOLOGICAL SCIENCES↗
Discovery of additional ancient genome duplications in yeasts
Whole-genome duplication (WGD) has had profound macroevolutionary impacts on diverse lineages, preceding adaptive radiations in vertebrates, teleost fish, and angiosperms. In contrast to the many known ancient WGDs in animals, and especially plants, we are aware of evidence for only four WGDs in fungi. The oldest of these occurred ∼100 million years ago (mya) and is shared by ∼60 extant Saccharomycetales species, including the baker’s yeast Saccharomyces cerevisiae. Notably, this is the only known ancient WGD event in the yeast subphylum Saccharomycotina. The dearth of ancient WGD events in fungi remains a mystery. Some studies have suggested that fungal lineages that experience chromosome and genome duplication quickly go extinct, leaving no trace in the genomic record, while others contend that the lack of known WGDs is due to an absence of data. Under the second hypothesis, additional sampling and deeper sequencing of fungal genomes should lead to the discovery of more WGD events. Coupling hundreds of recently published genomes from nearly every described Saccharomycotina species, with three additional long-read assemblies, we discovered three novel WGD events. Although the functions of retained duplicate genes originating from these events are broad, they bear similarities to the well-known WGD that occurred in the Saccharomycetales. In conclusion, our results suggest that WGD may be a more common evolutionary force in fungi than previously believed.
convergent evolution↗
Convergent evolution of aerobic fermentation through divergent mechanisms acting on key shared glycolytic genes
As the tree of life becomes increasingly accessible to molecular investigations, describing mechanisms underlying evolutionary convergence and constraint will be crucial to understanding diversification. The lineage including the model yeast Saccharomyces cerevisiae evolved aerobic fermentation in part through an ancient whole genome duplication and retention of glycolytic genes. To evaluate glycolytic rates across diverse yeasts, we developed and deployed an extracellular acidification rates (ECAR) assay on 299 species that span more than 400 million years of evolution and identified a clade in the genus Saturnispora that convergently evolved aerobic fermentation. Through comparative genomics and transcriptomics, we found that several glycolytic genes had higher expression and novel cis-regulatory elements in aerobically fermenting Saturnispora species. When the transcription factor required for their activation was deleted in Saturnispora dispora, the mutants had reduced glycolytic rates and increased respiration. Intriguingly, many of the upregulated genes are orthologous to duplicated glycolytic genes in S. cerevisiae. These divergent genetic mechanisms affecting the same set of genes suggest that there are strong evolutionary constraints on how aerobic fermentation can arise.
Horianopoulos, Linda C. [Great Lakes Bioenergy Res↗
Convergent evolution of aerobic fermentation through divergent mechanisms acting on the same key glycolytic genes
Expression profiling by high throughput sequencing The model yeast Saccharomyces cerevisiae has evolved aerobic fermentation in part through a whole genome duplication and subsequent retention of glycolytic genes. To identify independent yeast lineages with a high glycolytic rate phenotype we developed an assay measuring extracellular acidification rates (ECAR) to evaluate the glycolytic rates across diverse yeast species. This assay identified a novel group of yeasts in the genus Saturnispora with rapid glycolytic rates. Through comparative approaches, we found that several glycolytic genes encoding hexokinases and enzymes in lower glycolysis had higher expression and modifications in promoter sequences in rapid ECAR species. Intriguingly, many of the upregulated genes are the same genes that are duplicated in S. cerevisiae. When the transcription factor required for their activation was deleted, the mutants had a slow glycolytic rate and more respiratory phenotype. This work shows how the aerobic fermentation phenotype has convergently evolved in Saturnispora through transcriptional rewiring. This divergent mechanism nevertheless impacts the same glycolytic genes, which suggests that there are evolutionary constraints on how aerobic fermentation can arise.
Horianopoulos, Linda C. [Department of Food Scienc↗
Machine learning identifies novel signatures of antifungal drug resistance in Saccharomycotina yeasts
Antifungal drug resistance is a major challenge in fungal infection management. Numerous genomic changes are known to contribute to acquired drug resistance in clinical isolates of specific pathogens, but whether they broadly explain natural resistance across entire lineages is unknown. We leveraged genomic, ecological, and phenotypic trait data from naturally sampled strains from nearly all known species in subphylum Saccharomycotina to examine the evolution of resistance to eight antifungal drugs. The phylogenetic distribution of drug resistance varied by drug; fluconazole resistance was widespread, while 5-fluorocytosine resistance was rare, except in Lipomycetales. A random forest algorithm trained on genomic data predicted drug-resistant yeasts with 54–75% accuracy. Fluconazole resistance was consistently predicted with the highest accuracy (75.2%). Furthermore, fluconazole resistance prediction accuracy was similar between models trained on genome-wide variation in the presence and number of InterPro protein annotations across Saccharomycotina (75.2%) and those trained on amino acid sequence alignment data of Erg11, a protein known to be involved in fluconazole resistance (74.3-74.9%). Interestingly, the top Erg11 residues for predicting fluconazole resistance across Saccharomycotina do not overlap with, are not spatially close to, and are less conserved than those previously linked to resistance in clinical isolates of Candida albicans. In silico deep mutational scanning of the C. albicans Erg11 protein reveals that amino acid variants implicated in clinical cases of resistance are almost universally destabilizing while variants in our most informative residues are energetically more neutral, explaining why the latter are much more common than the former in natural populations. Importantly, previous experimental analyses of C. albicans Erg11 have shown that amino acid variation in our most informative residues, despite having never been directly implicated in clinical cases, can directly contribute to resistance. Our results suggest that studies of natural resistance in yeast species never encountered in the clinic will yield a fuller understanding of antifungal drug resistance.
Harrison, Marie-Claire [Vanderbilt Univ., Nashvill↗
Orthogonal replication with optogenetic selection evolves yeast JEN1 into a mevalonate transporter
Abstract The in vivo continuous evolution system OrthoRep (orthogonal replication) is a powerful strategy for rapid enzyme evolution inSaccharomyces cerevisiaethat diversifies genes at a rate exceeding the endogenous genome mutagenesis rate by several orders of magnitude. However, it is difficult to neofunctionalize genes using OrthoRep partly because of the way selection pressures are applied. Here we combine OrthoRep with optogenetics in a selection strategy we call OptoRep, which allows fine-tuning of selection pressure with light. With this capability, we evolved a truncated form of the endogenous monocarboxylate transporterJEN1 (JEN1t)into a de novo mevalonate importer. We demonstrate the functionality of the evolvedJEN1t(JEN1t Y180C/G ) in the production of farnesene, a renewable aviation biofuel, from mevalonate fed to fermentation media or produced by microbial consortia. This study shows that the light-induced complementation of OptoRep may improve the ability to evolve functions not currently accessible for selection, while its fine tunability of selection pressure may allow the continuous evolution of genes whose desired function has a restrictive range between providing effective selection and cellular viability.
Biochemistry & Molecular Biology↗
Data for Engineering and Evolution of Yarrowia lipolytica for Producing Lipids from Lignocellulosic Hydrolysates
Yarrowia lipolytica , an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.
Hydrolysate↗
The macroevolution of filamentation morphology across the Saccharomycotina yeast subphylum
Saccharomycotina is a subphylum of ascomycete fungi with diverse asexual growth morphologies. Filamentous growth can comprise linear and branched budding cells that do not undergo cell separation, termed pseudohyphae, or tubular filaments with septa that perforate allowing movement of organelles, termed true hyphae. We integrated phenotypic, genomic, metabolic, and environmental data on isolation sources from 1051 species to examine the variation and evolutionary history of filamentation across Saccharomycotina and determine whether these data could predict filamentation types. We found that 63.37% of strains can form filaments; 6.56% true hyphae, 42.40% pseudohyphae, and 14.39% both true hyphae and pseudohyphae. The distributions of species that can produce true hyphae or filament were more strongly correlated with the yeast phylogeny than the distribution of species with pseudohyphae. Ancestral state reconstruction suggested that true hyphal and pseudohyphal morphologies evolved several times, that most yeast ancestors likely produced pseudohyphae or lacked filaments, and that the Saccharomycotina last common ancestor likely produced pseudohyphae but not true hyphae. Machine learning models trained on genomic and metabolic features predicted filament morphologies with ∼70% accuracy. Connecting the evolution of morphologies to their genomic, physiological, and ecological characteristics will enrich our understanding of how the diversity of lifestyles evolved in Saccharomycotina.
Saccharomycotina↗
Ancient co-option of LTR retrotransposons as yeast centromeres
Here we map and characterize centromeres in the sister order Saccharomycodales and identify evolutionarily related “proto-point” centromeres that bridge repeat-rich and point centromeres.
centromeres↗
Engineering and evolution of Yarrowia lipolytica for producing lipids from lignocellulosic hydrolysates
Yarrowia lipolytica, an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.
60 APPLIED LIFE SCIENCES↗
raogroupuiuc/yl_variantcalling
NGS analysis for mutation analysis in Yarrowia lipolytica evolved strains. Yarrowia lipolytica, an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.
Deewan, Anshu↗
Bulk segregant and QTL analysis of maltose metabolism in Saccharomyces eubayanus
This study investigates natural variation in maltose utilization among Saccharomyces eubayanus strains isolated from different tree hosts. An F2 population was generated from a cross between strain QC18 (Quercus-associated) and strain CL467.1 (Nothofagus-associated). Bulk segregant analysis (BSA) was performed on the F2 offspring, and QTL mapping was conducted by whole-genome sequencing of two pools of 30 segregants each, representing the lowest and highest growth in high maltose concentrations. For QTL analysis, the parental strains QC18 and CL467.1 were also sequenced.
evolutionary plasticity↗
A trait syndrome ties cell morphology to glycolysis across the yeast subphylum
Co-variation of traits provides fundamental insights into constraints governing their evolution. An inverse correlation between glucose uptake rates (GURs) and cell surface area-to-volume (SA:V) ratios across 11 yeast species was recently reported. Here, we expand the analysis to 282 species to assess the generalizability of this correlation across Saccharomycotina yeasts and the contribution of shared evolutionary history to the co-variation of these traits. Using phylogenetic regression models, we found extracellular acidification rates (ECARs, used as a proxy for GURs) were weakly, but significantly, correlated with SA:V across Saccharomycotina. ECARs were also correlated with genome sizes and growth rates. Our findings support the reported correlation between GURs and SA:V ratios, but suggest other associated traits, including genome sizes. Specifically, yeasts that consume glucose faster tend to have lower SA:V, faster growth rates, and larger genomes, suggesting a trait syndrome governing several metabolic, genomic, and morphological traits across the yeast subphylum.
biological sciences↗
Convergent expansions of keystone gene families drive metabolic innovation in Saccharomycotina yeasts
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 that 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.
59 BASIC BIOLOGICAL SCIENCES↗
YeastWGD2025
Supplementary data for Discovery of additional ancient genome duplications in yeasts wgd_syn / - directory containing wgd syn output for all contiguous genomes [dataset] Tree - phylogeny [dataset]Duplications - duplication table from OrthoFinder output KOannotations - KEGG annotations used for enrichment analysis IPRannotations - InterPro annotations used for enrichment analysis DipodascalesOrthogroups - formatted orthogroup assignments for Dipodascales genes.fa and .gff3 files for each new genome assembly are also provided, those these are not required to replicate the analysis
Genomics↗