Biology subjects

Cubillos, F. A.

Publications and source records attributed to Cubillos, F. A..

13 recordsLinked to original sources

bioRxiv2026

Host-shift adaptation shapes genome architecture in S. eubayanus

The host environment can profoundly shape the genome architecture of microbial species, and Saccharomyces eubayanus, the wild progenitor of lager yeast, provides a natural system to study this process. Most populations are associated with Nothofagus trees across Patagonia, whereas related Holarctic strains occur in the Northern Hemisphere and are associated with non-Nothofagus hosts. The evolutionary events leading to the emergence of these northern populations on a novel host remain unclear. Here, we analyzed 471 genomes from eight countries and different hosts, Nothofagus in the Southern Hemisphere and non-Nothofagus tree species in the Northern Hemisphere. Phylogenomic analysis identified eight Patagonian lineages and revealed that Holarctic strains derived from recent admixture among Patagonian ancestors, generating the genomic background of the lager-yeast mother lineage. Long-read assemblies showed that non-Nothofagus-associated strains harbor an elevated burden of structural variants (SVs), particularly in subtelomeric MAL and IMA regions, involved in sugar metabolism. Phenotypic tests confirmed that Nothofagus isolates efficiently metabolize maltose, while non-Nothofagus strains do not, a pattern linked to recurrent SVs and loss-of-function mutations in MAL33. Consistently, bark-sugar profiling revealed that maltose is abundant in Nothofagus but absent in non-Nothofagus hosts, providing an ecological context for these genomic and phenotypic differences. These results support a model in which northward dispersal of Patagonian lineages into non-Nothofagus forests enriched admixed genotypes, generating genomic mosaics that accumulated structural changes and losses in maltose utilization. This interplay between gene flow and genome flexibility enabled host switching and global expansion, illustrating how ecological transitions reorder genomes and drive microbial diversification.

evolutionary biology↗

bioRxiv2025

Contrasting evolutionary forces of specialization and admixture underlie the genomic and phenotypic diversity of Yarrowia lipolytica

Microbial diversity emerges from evolutionary processes that shape genomic and phenotypic traits in response to complex environmental pressures. Deciphering these dynamics is key to understanding microbial ecology and advancing biotechnological applications. Here, we use the yeast Yarrowia lipolytica to study genomic signatures of adaptation across a broad range of environments and to illustrate how population-level data can inform targeted bioprospecting for industrial traits. Whole-genome and phenotypic analyses of 126 isolates from natural and anthropogenic environments reveal a complex population structure in this species, shaped by both niche specialization and admixture events. Structured lineages exhibit ecological filtering, reduced genetic diversity, and distinct gene content, consistent with adaptation to substrates like dairy, hydrocarbons, or industrial substrates. In contrast, admixed populations display greater genetic diversity and broader phenotypic capacity, including enhanced stress tolerance and metabolic flexibility. Genome plasticity, reflected in pangenome and CNV variation, aligns with ecological origin, while trait assays link phenotypic divergence to underlying genetic variation. For instance, better growth performance on acetate, an ecological and industrially relevant trait, is associated with hydrocarbon-adapted strains and likely linked to variation in regulatory regions of acetate metabolism genes. Together, our results reflect how divergent evolutionary trajectories--ranging from ecological specialization to genomic plasticity through admixture-- underpin the species ecological success and provide a framework for harnessing its natural diversity in microbial bioprospecting.

evolutionary biology↗

bioRxiv2025

Transcriptional Reprogramming Drives Cold Adaptation During Long-Term Starvation in S. eubayanus

The ability of microorganisms to survive prolonged periods of nutrient scarcity is essential for their survival. Yet, the underlying adaptive mechanisms remain partly understood, especially in non-model eukaryotes. Here, we examined how the cryotolerant yeast Saccharomyces eubayanus adapts to 60 days of cold (4{degrees}C) starvation, focusing on the roles of genetic and transcriptional changes. We find that the primary engine of the long-term adaptation is a stable, reprogrammed transcriptional state, rather than the selection of point mutations. Aged isolates exhibited improved growth performance and cryotolerance, a phenotype that remained stable for [~]40 generations. This specialist adaptation involved a trade-off with tolerance to other stresses. Whole-genome sequencing revealed few fixed mutations, indicating that genetic variation did not drive the phenotype. Transcriptomic analysis revealed a significant physiological reprogramming, with cells shifting from anabolic activity to a catabolic, scavenging state driven by enhanced respiration and activation of the General Stress Response. This work highlights that a stable transcriptional state drives long-term cold adaptation, providing the foundation for the superior phenotype of aged isolates that persist through generations.

microbiology↗

bioRxiv2025

Assessing the molecular and phenotypic contribution of a horizontally acquired region to yeast adaptation

Horizontal Gene Transfer (HGT) is the movement of genetic material across species. In Saccharomyces cerevisiae, a DNA segment known as Region B was acquired horizontally from a distant yeast species. This region ([~]17 Kb) encodes 5 genes and is present in the genomes of yeast strains from different phylogenetic clades, with its contribution to yeast niche-specific adaptation remaining unclear. In this work, the genomic structure of Region B was analyzed in yeast strains from the ScRAP (Saccharomyces cerevisiae Reference Assembly Panel) collection, identifying 10 variants that maintain a circular continuity. To assess the role of Region B in yeast adaptation, we performed a high-throughput phenotyping of the ScRAP collection under different growth conditions, identifying that Region B is potentially associated with higher tolerance to oxidative stress. Then, we selected a single yeast strain from the ScRAP collection for characterization of the transcriptional activity of each gene within Region B using a fluorescent reporter. The results revealed that gene expression depends on the hosts genetic background and transcription factors encoded within Region B. To identify the genetic determinants involved in Region B expression within different genetic backgrounds, three expression Quantitative Trait Loci (eQTLs) were mapped and validated. Finally, by performing Region B deletion, we determine the contribution of this region to different fermentative phenotypes, including fermentation rate and amino acid consumption. Altogether, our results suggest a complex regulatory interaction between the horizontally acquired genes and the host genome that contributes to yeast adaptation under fermentation conditions.

molecular biology↗

bioRxiv2025

Allelic variation in MAL33 drives ecological adaptation of maltose metabolism in Saccharomyces eubayanus

Maltose is one of the most abundant sugars in brewers 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), and 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 MAL33ChrV 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 QC18s own MAL33ChrV 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 lead to differences in maltose consumption between strains. 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 evolutionary plasticity, representing a key evolutionary mechanism that influences maltose adaptation among natural Saccharomyces isolates.

evolutionary biology↗

bioRxiv2025

Oxidative stress and vacuolar variants influence paraquat response in Saccharomyces cerevisiae of different genetic variants

The biological effects of paraquat (PQ), an herbicide linked to Parkinsons disease (PD) risk in humans can be studied in Saccharomyces cerevisiae due to its evolutionary conservation with mammals. To understand how genetic background influences PQ toxicity, we treated four yeast strains (NA, SA, WA, and WE) with PQ and assessed physiological (growth curves), molecular (superoxide and peroxide levels) and cellular consequences (vacuolar disaggregation) on each genetic background. PQ significantly reduced specific growth rates (Max) in WE and WA, while SA and NA remained unaffected. Furthermore, PQ increased superoxide and peroxide levels across the strains, but to different extents, being the SA and WE the most affected. PQ also influenced vacuolar morphologies strain-dependently, shifting from one large organelle to small disaggregate vacuoles, with WE being the most susceptible. Interestingly, we found an inverse association between superoxide levels and Max. Given the known involvement of lysosomal dysfunction in pesticide-induced PD, we investigated correlations between predicted missense variants in vacuolar genes and PQ responses across the yeast strains. We identified associations between fen2 variants, the human orthologue of SLC17A5, and vacuolar disaggregation. To validate this, we exposed a fen2-deleted strain to PQ, which exhibited increased vacuolar disaggregation. In conclusion, our findings demonstrate that the strain-specific susceptibility to PQ is associated with superoxide levels and that fen2 likely plays a role in the vacuolar adaptive response to PQ exposure. We speculate that the most resistant strains may facilitate the development of novel therapeutics for humans exposed to PQ.

pharmacology and toxicology↗

bioRxiv2024

Understanding brewing trait inheritance in de novo lager yeast hybrids

Hybridization between Saccharomyces cerevisiae and Saccharomyces eubayanus resulted in the emergence of S. pastorianus, a crucial yeast for lager fermentation. However, our understanding of hybridization success and hybrid vigour between these two species remains limited due to the scarcity of S. eubayanus parental strains. Here, we explore hybridization success and the impact of hybridization on fermentation performance and volatile compound profiles in newly formed lager hybrids. By selecting parental candidates spanning a diverse array of lineages from both species, we reveal that the Beer and PB-2 lineages exhibit high rates of hybridization success in S. cerevisiae and S. eubayanus, respectively. Polyploid hybrids were generated through rare mating techniques, revealing a prevalence of triploids and diploids over tetraploids. Despite the absence of heterosis in fermentative capacity, hybrids displayed phenotypic variability, notably influenced by maltotriose consumption. Interestingly, ploidy levels did not significantly correlate with fermentative capacity, although triploids exhibited greater phenotypic variability. The S. cerevisiae parental lineages primarily influenced volatile compound profiles, with significant differences in aroma production. Interestingly, hybrids emerging from the Beer S. cerevisiae parental lineages exhibited a volatile compound profile resembling the corresponding S. eubayanus parent. This pattern may result from the dominant inheritance of the S. eubayanus aroma profile, as suggested by the over-expression of genes related to alcohol metabolism and acetate synthesis in hybrids including the Beer S. cerevisiae lineage. Our findings suggest complex interactions between parental lineages and hybridization outcomes, highlighting the potential for creating yeasts with distinct brewing traits through hybridization strategies.

microbiology↗

bioRxiv2024

Wild Patagonian yeast improve the evolutionary potential of novel interspecific hybrid strains for Lager brewing

ABSTRACTLager yeasts are limited to a few strains worldwide, imposing restrictions on flavour and aroma diversity and hindering our understanding of the complex evolutionary mechanisms during yeast domestication. The recent finding of diverse S. eubayanus lineages from Patagonia offers potential for generating new Lager yeasts and obtaining insights into the domestication process. Here, we leverage the natural genetic diversity of S. eubayanus and expand the Lager yeast repertoire by including three distinct Patagonian S. eubayanus lineages. We used experimental evolution and selection on desirable traits to enhance the fermentation profiles of novel S. cerevisiae x S. eubayanus hybrids. Our analyses reveal an intricate interplay of pre-existing diversity, selection on species-specific mitochondria, de-novo mutations, and gene copy variations in sugar metabolism genes, resulting in high ethanol production and unique aroma profiles. Hybrids with S. eubayanus mitochondria exhibited greater evolutionary potential and superior fitness post-evolution, analogous to commercial Lager hybrids. Using genome-wide screens of the parental subgenomes, we identified genetic changes in IRA2, SNF3, IMA1 and MALX genes that influence maltose metabolism, and increase glycolytic flux and sugar consumption in the evolved hybrids. Functional validation and transcriptome analyses confirmed increased maltose-related gene expression, influencing grater maltotriose consumption in evolved hybrids. This study demonstrates the potential for generating industrially viable Lager yeast hybrids from wild Patagonian strains. Our hybridization, evolution, and mitochondrial selection approach produced hybrids with high fermentation capacity, expands Lager beer brewing options, and deepens our knowledge of Lager yeast domestication.

microbiology↗

bioRxiv2023

Domestication signatures in the non-conventional yeast Lachancea cidri

Evaluating domestication signatures beyond model organisms is essential for thoroughly understanding the genotype-phenotype relationship in wild and human-related environments. Structural variations (SVs) can significantly impact phenotypes playing an important role in the physiological adaptation of species to different niches, including during domestication. A detailed characterization of the fitness consequences of these genomic rearrangements, however, is still limited in non-model systems, largely due to the paucity of direct comparisons between domesticated and wild isolates. Here, we used a combination of sequencing strategies to explore major genomic rearrangements in a Lachancea cidri yeast strain isolated from cider (CBS2950) and compared them to those in eight wild isolates from primary forests. Genomic analysis revealed dozens of SVs, including a large reciprocal translocation ([~]16 kb and 500 kb) present in the cider strain, but absent from all wild strains. Interestingly, the number of SVs was higher relative to single-nucleotide polymorphisms in the cider strain, suggesting a significant role on the strains phenotypic variation. The set of SVs identified directly impacts dozens of genes, and likely underpins the greater fermentation performance in the L. cidri CBS2950. Additionally, the large reciprocal translocation affects a proline permease (PUT4) regulatory region, resulting in higher PUT4 transcript levels, which agrees with higher ethanol tolerance, improved cell growth when using proline, and higher amino acid consumption during fermentation. These results suggest that SVs are responsible for the rapid physiological adaptation of yeast to an anthropogenic habitat and demonstrate the key contribution of SVs in adaptive fermentative traits in non-model species. ImportanceThe exploration of domestication signatures associated with anthropogenic niches has predominantly focused on studies conducted on model organisms, such as Saccharomyces cerevisiae, overlooking the potential for comparisons across other non-Saccharomyces species. In our research, employing a combination of long- and short-read data, we found domestication signatures in L. cidri, a non-model species recently isolated from fermentative environments in cider in France. The significance of our study lies in the identification of large array of major genomic rearrangements in a cider strain compared to wild isolates, which underly several fermentative traits. These domestication hallmarks result from structural variants, which are likely responsible for the phenotypic differences between strains, providing a rapid path of adaptation to human-related environments.

genetics↗

bioRxiv2022

Natural Variation in Diauxic Shift between Patagonian Saccharomyces eubayanus Strains

The study of natural variation can untap novel alleles with immense value for biotechnological applications. Saccharomyces eubayanus Patagonian isolates exhibit differences in the diauxic shift between glucose and maltose, representing a suitable model to study their natural genetic variation for novel strains for brewing. However, little is known about the genetic variants and chromatin regulators responsible for these differences. Here, we show how genome-wide chromatin accessibility and gene expression differences underlie distinct diauxic shift profiles in S. eubayanus. We identified two strains with a rapid diauxic shift between glucose and maltose (CL467.1 and CBS12357) and one strain with a remarkably low fermentation efficiency and longer lag phase during diauxic shift (QC18). This is associated in the QC18 strain to lower transcriptional activity and chromatin accessibility of specific genes of maltose metabolism, and higher expression levels of glucose transporters. These differences are governed by the HAP complex, which differentially regulates gene expression depending on the genetic background. We found in the QC18 strain a contrasting phenotype to those described in S. cerevisiae, where hap411, hap511 and cin511 knockouts significantly improved the QC18 growth rate in the glucose-maltose shift. The most profound effects were found between CIN5 allelic variants, suggesting that Cin5p could strongly activate a repressor of the diauxic shift in the QC18 strain, but not necessarily in the other strains. The differences between strains could originate from the tree host from which the strains were obtained, which might determine the sugar source preference and the brewing potential of the strain. ImportanceThe diauxic shift has been studied in the budding yeast under laboratory conditions, however, few studies have addressed the diauxic shift between carbon sources under fermentative conditions. Here, we study the transcriptional and chromatin structure differences that explain the natural variation in fermentative capacity and efficiency during diauxic shift of natural isolates of S. eubayanus. Our results show how natural genetic variants in transcription factors impact sugar consumption preferences between strains. These variants have different effects depending on the genetic background, with a contrasting phenotype to those previously described in S. cerevisiae. Our study shows how relatively simple genetic/molecular modifications/editing in the lab can facilitate the study of natural variation of microorganisms for the brewing industry.

microbiology↗

bioRxiv2021

Uncovering divergence in gene expression regulation in the adaptation of yeast to nitrogen scarcity

Saccharomyces cerevisiae rewires its transcriptional output to survive stressful environments, such as nitrogen scarcity under fermentative conditions. Although divergence in nitrogen metabolism has been described among natural yeast populations, the impact of regulatory genetic variants modulating gene expression and nitrogen consumption remains to be investigated. Here, we employed an F1 hybrid from two contrasting S. cerevisiae strains, providing a controlled genetic environment to map cis factors involved in the divergence of gene expression regulation in response to nitrogen scarcity. We used a dual approach to obtain genome-wide allele-specific profiles of chromatin accessibility, transcription factor binding, and gene expression through ATAC-seq and RNA-seq. We observed large variability in allele-specific expression and accessibility between the two genetic backgrounds, with a third of these differences specific to a deficient nitrogen environment. Furthermore, we discovered events of allelic bias in gene expression correlating with allelic bias in transcription factor binding solely under nitrogen scarcity, where the majority of these transcription factors orchestrates the Nitrogen Catabolite Repression regulatory pathway and demonstrates a cis x environment-specific response. Our approach allowed us to find cis variants modulating gene expression, chromatin accessibility and allelic differences in transcription factor binding in response to low nitrogen culture conditions. ImportanceHistorically, coding variants were prioritized when searching for causal mechanisms driving adaptation of natural populations to stressful environments. However, the recent focus on non-coding variants demonstrated their ubiquitous role in adaptation. Here we performed genome-wide regulatory variation profiles between two divergent yeast strains when facing nitrogen nutritional stress. The open chromatin availability of several regulatory regions changes in response to nitrogen scarcity. Importantly we describe regulatory events that deviate between strains. Our results demonstrate a widespread variation in gene expression regulation between naturally occurring populations in response to stressful environments.

genomics↗

bioRxiv2021

A comprehensive fitness landscape model reveals the evolutionary history and future evolvability of eukaryotic cis-regulatory DNA sequences

Mutations in non-coding cis-regulatory DNA sequences can alter gene expression, organismal phenotype, and fitness. Fitness landscapes, which map DNA sequence to organismal fitness, are a long-standing goal in biology, but have remained elusive because it is challenging to generalize accurately to the vast space of possible sequences using models built on measurements from a limited number of endogenous regulatory sequences. Here, we construct a sequence-to-expression model for such a landscape and use it to decipher principles of cis-regulatory evolution. Using tens of millions of randomly sampled promoter DNA sequences and their measured expression levels in the yeast Sacccharomyces cerevisiae, we construct a deep transformer neural network model that generalizes with exceptional accuracy, and enables sequence design for gene expression engineering. Using our model, we predict and experimentally validate expression divergence under random genetic drift and strong selection weak mutation regimes, show that conflicting expression objectives in different environments constrain expression adaptation, and find that stabilizing selection on gene expression leads to the moderation of regulatory complexity. We present an approach for detecting selective constraint on gene expression using our model and natural sequence variation, and validate it using observed cis-regulatory diversity across 1,011 yeast strains, cross-species RNA-seq from three different clades, and measured expression-to-fitness curves. Finally, we develop a characterization of regulatory evolvability, use it to visualize fitness landscapes in two dimensions, discover evolvability archetypes, quantify the mutational robustness of individual sequences and highlight the mutational robustness of extant natural regulatory sequence populations. Our work provides a general framework that addresses key questions in the evolution of cis-regulatory sequences.

genomics↗

bioRxiv2019

An Out-of-Patagonia dispersal explains most of the worldwide genetic distribution in Saccharomyces eubayanus

Saccharomyces eubayanus represents missing cryotolerant ancestor of lager yeast hybrid and can be found in Patagonia in association with Nothofagus forests. The limited number of isolates and associated genomes available has prevented to resolve the S. eubayanus origin and evolution. Here, we present a sampling effort at an unprecedented scale and report the isolation of 160 strains from ten sampling sites along 2,000 km distance in South America. We sequenced the genome of 82 strains and, together with other 25 available genomes, performed comprehensive phylogenetic analysis. Our results revealed the presence of three main Patagonia-B lineages together with dozens of admixed strains distributed in three mosaic clusters. The PB-1 lineage isolated from Tierra del Fuego exhibited the highest genetic diversity, lowest LD blocks and highest Fis values compared to the other lineages, suggesting a successful adaptation to cold temperatures in extreme environments and greater inbreeding rates in Tierra del Fuego. Differences between lineages and strains were found in terms of aneuploidy and pangenome content, evidencing a lateral gene transfer event in PB-2 strains from an unknown donor species. Overall, the Patagonian lineages, particularly southern populations, showed a greater global genetic diversity compared to Holarctic and Chinese lineages, supporting the scenario of a S. eubayanus colonization from Patagonia and then spread towards northern and western regions, including the Holarctic (North America and China) and New Zealand. Interestingly, fermentative capacity and maltose consumption resulted negatively correlated with latitude, indicating a better fermentative performance in norther populations. Our genome analysis together with previous reports in the sister species S. uvarum strongly suggests that the S. eubayanus ancestor could have originated in Patagonia or the Southern Hemisphere, rather than China, yet further studies are needed to resolve this conflicting scenario. Understanding S. eubayanus evolutionary history is crucial to resolve the unknown origin of the lager yeast and might open new avenues for biotechnological applications.

genomics↗