Natural Yeast Isolates Research Articles

Discovery of a rapidly evolving yeast defense factor, KTD1, against the secreted killer toxin K28

Secreted protein toxins are widely used weapons in conflicts between organisms. Elucidating how organisms genetically adapt to defend themselves against these toxins is fundamental to understanding the coevolutionary dynamics of competing organisms. Within yeast communities, "killer" toxins are secreted to kill nearby sensitive yeast, providing a fitness advantage in competitive growth environments. Natural yeast isolates vary in their sensitivity to these toxins, but to date, no polymorphic genetic factors contributing to defense have been identified. We investigated the variation in resistance to the killer toxin K28 across diverse natural isolates of the Saccharomyces cerevisiae population. Using large-scale linkage mapping, we discovered a novel defense factor, which we named KTD1. We identified many KTD1 alleles, which provided different levels of K28 resistance. KTD1 is a member of the DUP240 gene family of unknown function, which is rapidly evolving in a region spanning its two encoded transmembrane helices. We found that this domain is critical to KTD1's protective ability. Our findings implicate KTD1 as a key polymorphic factor in the defense against K28 toxin.

Screening of xylose utilizing and high lipid producing yeast strains as a potential candidate for industrial application

BackgroundSustainable production of oil for food, feed, fuels and other lipid-based chemicals is essential to meet the demand of the increasing human population. Consequently, novel and sustainable resources such as lignocellulosic hydrolysates and processes involving these must be explored. In this paper we screened for naturally-occurring xylose utilizing oleaginous yeasts as cell factories for lipid production, since pentose sugar catabolism plays a major role in efficient utilization of lignocellulosic feedstocks. Glycerol utilization, which is also beneficial in yeast-based oil production as glycerol is a common by-product of biodiesel production, was investigated as well. Natural yeast isolates were studied for lipid accumulation on a variety of substrates, and the highest lipid accumulating strains were further investigated in shake flask cultivations and fermenter studies on xylose and hydrolysate.ResultsBy collecting leaves from exotic plants in greenhouses and selective cultivation on xylose, a high frequency of oleaginous yeasts was obtained (> 40%). Different cultivation conditions lead to differences in fatty acid contents and compositions, resulting in a set of strains that can be used to select candidate production strains for different purposes. In this study, the most prominent strains were identified as Pseudozyma hubeiensis BOT-O and Rhodosporidium toruloides BOT-A2. The fatty acid levels per cell dry weight after cultivation in a nitrogen limited medium with either glucose, xylose or glycerol as carbon source, respectively, were 46.8, 43.2 and 38.9% for P. hubeiensis BOT-O, and 40.4, 27.3 and 42.1% for BOT-A2. Furthermore, BOT-A2 accumulated 45.1% fatty acids per cell dry weight in a natural plant hydrolysate, and P. hubeiensis BOT-O showed simultaneous glucose and xylose consumption with similar growth rates on both carbon sources. The fatty acid analysis demonstrated both long chain and poly-unsaturated fatty acids, depending on strain and medium.ConclusionsWe found various natural yeast isolates with high lipid production capabilities and the ability to grow not only on glucose, but also xylose, glycerol and natural plant hydrolysate. R. toruloides BOT-A2 and P. hubeiensis BOT-O specifically showed great potential as production strains with high levels of storage lipids and comparable growth to that on glucose on various other substrates, especially compared to currently used lipid production strains. In BOT-O, glucose repression was not detected, making it particularly desirable for utilization of plant waste hydrolysates. Furthermore, the isolated strains were shown to produce oils with fatty acid profiles similar to that of various plant oils, making them interesting for future applications in fuel, food or feed production.

Gene loss and compensatory evolution promotes the emergence of morphological novelties in budding yeast.

Deleterious mutations are generally considered to be irrelevant for morphological evolution. However, they could be compensated by conditionally beneficial mutations, thereby providing access to new adaptive paths. Here we use high-dimensional phenotyping of laboratory-evolved budding yeast lineages to demonstrate that new cellular morphologies emerge exceptionally rapidly as a by-product of gene loss and subsequent compensatory evolution. Unexpectedly, the capacities for invasive growth, multicellular aggregation and biofilm formation also spontaneously evolve in response to gene loss. These multicellular phenotypes can be achieved by diverse mutational routes and without reactivating the canonical regulatory pathways. These ecologically and clinically relevant traits originate as pleiotropic side effects of compensatory evolution and have no obvious utility in the laboratory environment. The extent of morphological diversity in the evolved lineages is comparable to that of natural yeast isolates with diverse genetic backgrounds and lifestyles. Finally, we show that both the initial gene loss and subsequent compensatory mutations contribute to new morphologies, with their synergistic effects underlying specific morphological changes. We conclude that compensatory evolution is a previously unrecognized source of morphological diversity and phenotypic novelties.

Substrate, temperature, and geographical patterns among nearly 2000 natural yeast isolates.

Yeasts have broad importance as industrially and clinically relevant microbes and as powerful models for fundamental research, but we are only beginning to understand the roles yeasts play in natural ecosystems. Yeast ecology is often more difficult to study compared to other, more abundant microbes, but growing collections of natural yeast isolates are beginning to shed light on fundamental ecological questions. Here, we used environmental sampling and isolation to assemble a dataset of 1962 isolates collected from throughout the contiguous United States of America (USA) and Alaska, which were then used to uncover geographic patterns, along with substrate and temperature associations among yeast taxa. We found some taxa, including the common yeasts Torulaspora delbrueckii and Saccharomyces paradoxus, to be repeatedly isolated from multiple sampled regions of the USA, and we classify these as broadly distributed cosmopolitan yeasts. A number of yeast taxon-substrate associations were identified, some of which were novel and some of which support previously reported associations. Further, we found a strong effect of isolation temperature on the phyla of yeasts recovered, as well as for many species. We speculate that substrate and isolation temperature associations reflect the ecological diversity of and niche partitioning by yeast taxa.

Computational analysis of GAL pathway pinpoints mechanisms underlying natural variation.

Quantitative traits are measurable phenotypes that show continuous variation over a wide phenotypic range. Enormous effort has recently been put into determining the genetic influences on a variety of quantitative traits with mixed success. We identified a quantitative trait in a tractable model system, the GAL pathway in yeast, which controls the uptake and metabolism of the sugar galactose. GAL pathway activation depends both on galactose concentration and on the concentrations of competing, preferred sugars such as glucose. Natural yeast isolates show substantial variation in the behavior of the pathway. All studied yeast strains exhibit bimodal responses relative to external galactose concentration, i.e. a set of galactose concentrations existed at which both GAL-induced and GAL-repressed subpopulations were observed. However, these concentrations differed in different strains. We built a mechanistic model of the GAL pathway and identified parameters that are plausible candidates for capturing the phenotypic features of a set of strains including standard lab strains, natural variants, and mutants. In silico perturbation of these parameters identified variation in the intracellular galactose sensor, Gal3p, the negative feedback node within the GAL regulatory network, Gal80p, and the hexose transporters, HXT, as the main sources of the bimodal range variation. We were able to switch the phenotype of individual yeast strains in silico by tuning parameters related to these three elements. Determining the basis for these behavioral differences may give insight into how the GAL pathway processes information, and into the evolution of nutrient metabolism preferences in different strains. More generally, our method of identifying the key parameters that explain phenotypic variation in this system should be generally applicable to other quantitative traits.

Selection of Potential Yeast Probiotics and a Cell Factory for Xylitol or Acid Production from Honeybee Samples.

Excessive use of antibiotics has detrimental consequences, including antibiotic resistance and gut microbiome destruction. Probiotic-rich diets help to restore good microbes, keeping the body healthy and preventing the onset of chronic diseases. Honey contains not only prebiotic oligosaccharides but, like yogurt and fermented foods, is an innovative natural source for probiotic discovery. Here, a collection of three honeybee samples was screened for yeast strains, aiming to characterize their potential in vitro probiotic properties and the ability to produce valuable metabolites. Ninety-four isolates out of one-hundred and four were able to grow at temperatures of 30 °C and 37 °C, while twelve isolates could grow at 42 °C. Fifty-eight and four isolates displayed the ability to grow under stimulated gastrointestinal condition, at pH 2.0–2.5, 0.3% (w/v) bile salt, and 37 °C. Twenty-four isolates showed high autoaggregation of 80–100% and could utilize various sugars, including galactose and xylose. The cell count of these isolates (7–9 log cfu/mL) was recorded and stable during 6 months of storage. Genomic characterization based on the internal transcribed spacer region (ITS) also identified four isolates of Saccharomyces cerevisiae displayed good ability to produce antimicrobial acids. These results provided the basis for selecting four natural yeast isolates as starter cultures for potential probiotic application in functional foods and animal feed. Additionally, these S. cerevisiae isolates also produced high levels of acids from fermented sugarcane molasses, an abundant agricultural waste product from the sugar industry. Furthermore, one of ten identified isolates of Meyerozyma guilliermondiii displayed an excellent ability to produce a pentose sugar xylitol at a yield of 0.490 g/g of consumed xylose. Potentially, yeast isolates of honeybee samples may offer various biotechnological advantages as probiotics or metabolite producers of multiproduct-based lignocellulosic biorefinery.

Complex modifier landscape underlying genetic background effects

The phenotypic consequence of a given mutation can be influenced by the genetic background. For example, conditional gene essentiality occurs when the loss of function of a gene causes lethality in one genetic background but not another. Between two individual Saccharomyces cerevisiae strains, S288c and Σ1278b, ∼1% of yeast genes were previously identified as "conditional essential." Here, in addition to confirming that some conditional essential genes are modified by a nonchromosomal element, we show that most cases involve a complex set of genomic modifiers. From tetrad analysis of S288C/Σ1278b hybrid strains and whole-genome sequencing of viable hybrid spore progeny, we identified complex sets of multiple genomic regions underlying conditional essentiality. For a smaller subset of genes, including CYS3 and CYS4, each of which encodes components of the cysteine biosynthesis pathway, we observed a segregation pattern consistent with a single modifier associated with conditional essentiality. In natural yeast isolates, we found that the CYS3/CYS4 conditional essentiality can be caused by variation in two independent modifiers, MET1 and OPT1, each with roles associated with cellular cysteine physiology. Interestingly, the OPT1 allelic variation appears to have arisen independently from separate lineages, with rare allele frequencies below 0.5%. Thus, while conditional gene essentiality is usually driven by genetic interactions associated with complex modifier architectures, our analysis also highlights the role of functionally related, genetically independent, and rare variants.

The Genetic Basis of Mutation Rate Variation in Yeast.

Mutations are the root source of genetic variation and underlie the process of evolution. Although the rates at which mutations occur vary considerably between species, little is known about differences within species, or the genetic and molecular basis of these differences. Here, we leveraged the power of the yeast Saccharomyces cerevisiae as a model system to uncover natural genetic variants that underlie variation in mutation rate. We developed a high-throughput fluctuation assay and used it to quantify mutation rates in seven natural yeast isolates and in 1040 segregant progeny from a cross between BY, a laboratory strain, and RM, a wine strain. We observed that mutation rate varies among yeast strains and is heritable (H2 = 0.49). We performed linkage mapping in the segregants and identified four quantitative trait loci underlying mutation rate variation in the cross. We fine-mapped two quantitative trait loci to the underlying causal genes, RAD5 and MKT1, that contribute to mutation rate variation. These genes also underlie sensitivity to the DNA-damaging agents 4NQO and MMS, suggesting a connection between spontaneous mutation rate and mutagen sensitivity.

Genomic and transcriptomic analysis of Saccharomyces cerevisiae isolates with focus in succinic acid production.

Succinic acid is a platform chemical that plays an important role as precursor for the synthesis of many valuable bio-based chemicals. Its microbial production from renewable resources has seen great developments, specially exploring the use of yeasts to overcome the limitations of using bacteria. The objective of the present work was to screen for succinate-producing isolates, using a yeast collection with different origins and characteristics. Four strains were chosen, two as promising succinic acid producers, in comparison with two low producers. Genome of these isolates was analysed, and differences were found mainly in genes SDH1, SDH3, MDH1 and the transcription factor HAP4, regarding the number of single nucleotide polymorphisms and the gene copy-number profile. Real-time PCR was used to study gene expression of 10selected genes involved in the metabolic pathway of succinic acid production. Results show that for the non-producing strain, higher expression of genes SDH1, SDH2, ADH1, ADH3, IDH1 and HAP4 was detected, together with lower expression of ADR1 transcription factor, in comparison with the best producer strain. This is the first study showing the capacity of natural yeast isolates to produce high amounts of succinic acid, together with the understanding of the key factors associated, giving clues for strain improvement.

De novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer.

Background: Oxford Nanopore Technologies Ltd (Oxford, UK) have recently commercialized MinION, a small single-molecule nanopore sequencer, that offers the possibility of sequencing long DNA fragments from small genomes in a matter of seconds. The Oxford Nanopore technology is truly disruptive; it has the potential to revolutionize genomic applications due to its portability, low cost, and ease of use compared with existing long reads sequencing technologies. The MinION sequencer enables the rapid sequencing of small eukaryotic genomes, such as the yeast genome. Combined with existing assembler algorithms, near complete genome assemblies can be generated and comprehensive population genomic analyses can be performed. Results: Here, we resequenced the genome of the Saccharomyces cerevisiae S288C strain to evaluate the performance of nanopore-only assemblers. Then we de novo sequenced and assembled the genomes of 21 isolates representative of the S. cerevisiae genetic diversity using the MinION platform. The contiguity of our assemblies was 14 times higher than the Illumina-only assemblies and we obtained one or two long contigs for 65 % of the chromosomes. This high contiguity allowed us to accurately detect large structural variations across the 21 studied genomes. Conclusion: Because of the high completeness of the nanopore assemblies, we were able to produce a complete cartography of transposable elements insertions and inspect structural variants that are generally missed using a short-read sequencing strategy. Our analyses show that the Oxford Nanopore technology is already usable for de novo sequencing and assembly; however, non-random errors in homopolymers require polishing the consensus using an alternate sequencing technology.

Rapid evolutionary adaptation to growth on an 'unfamiliar' carbon source.

BackgroundCells constantly adapt to changes in their environment. When environment shifts between conditions that were previously encountered during the course of evolution, evolutionary-programmed responses are possible. Cells, however, may also encounter a new environment to which a novel response is required. To characterize the first steps in adaptation to a novel condition, we studied budding yeast growth on xylulose, a sugar that is very rarely found in the wild.ResultsWe previously reported that growth on xylulose induces the expression of amino acid biosynthesis genes in multiple natural yeast isolates. This induction occurs despite the presence of amino acids in the growth medium and is a unique response to xylulose, not triggered by naturally available carbon sources. Propagating these strains for ~300 generations on xylulose significantly improved their growth rate. Notably, the most significant change in gene expression was the loss of amino acid biosynthesis gene induction. Furthermore, the reduction in amino-acid biosynthesis gene expression on xylulose was tightly correlated with the improvement in growth rate, suggesting that internal depletion of amino-acids presented a major bottleneck limiting growth in xylulose.ConclusionsWe discuss the possible implications of our results for explaining how cells maintain the balance between supply and demand of amino acids during growth in evolutionary ‘familiar’ vs. ‘novel’ conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3010-x) contains supplementary material, which is available to authorized users.

Table wine from tropical fruits utilizing natural yeast isolates.

An attempt was made to utilize few widely available tropical fruits to develop wine with the objective of comparing the fermentation efficiency (along with progress in fermentation) of two efficient yeast isolates with commercially available strain. Fruit wine from juices of fully ripe mango, jackfruit and pineapple alone and in blended combinations of all three fruit juice (2: 1: 2) was prepared using two different yeasts (Y4 and Y7) isolated from natural plain date palm juice and one standard Saccharomyces cerevisiae (MTCC-170) collected from IMTECH, Chandigar. Juices were extracted by using pectinase enzyme at 0.15-0.20% of pulp. Changes in °Brix, titratable acid content, pH, total viable yeast count were recorded and rate of fermentation, sugar use efficiency were determined at every 24-hour interval up to the completion (6days after inoculation) of fermentation. Considering all the quality parameter as well as fermentation efficiency, yeast isolate Y7 was found superior followed by Y4 as fermenting agent and pineapple juice as sole substrate found to be the most suitable medium for production of wine followed by fruit juice blending. In interpreting the efficacy of fruit and yeast in combination, pineapple juice inoculated with Y7 found to be the best in reducing the degree Brix to its lowest from initial 24 degree.

Screening of natural yeast isolates under the effects of stresses associated with second-generation biofuel production

Robust microorganisms are required for sustainable second-generation biofuel production. We evaluated the growth and fermentation performance of six natural isolates that were derived from grape wine and medicinal herbs using a wide range of carbon sources, rice and wheat straw hydrolysates as well as stress conditions associated with second-generation ethanol production. Sequence analysis of the 5.8S internal transcribed spacer (ITS) and species-specific PCR amplification of the HO gene region assigned the natural isolates to Saccharomyces cerevisiae. Restriction fragment length polymorphism (RFLP) analysis of the mitochondrial DNA revealed that natural yeast isolates are genetically closer to the laboratory strain BY4741 than to the CEN.PK strains. Dextrose fermentation by a natural isolate, MTCC4780, under semi-anaerobic conditions produced maximum ethanol yields of 0.44 g/g and 0.39 g/g, respectively, with and without the stresses encountered during lignocellulosic ethanol fermentation. However, MTCC4780 produced ethanol yields of 0.48 g/g, 0.42 g/g and 0.45 g/g, respectively, with glucose, rice and wheat straw enzymatic hydrolysate fermentation in a bioreactor. The isolates MTCC4781 and MTCC4796 showed higher growth and fermentation performance than did MTCC4780 in the presence of elevated temperature and pre-treatment inhibitors. Taken together, the MTCC4780, MTCC4781 and MTCC4796 strains have the potential to serve as a platform for lignocellulosic ethanol production under stresses associated with second-generation biofuel production.

Defining Molecular Basis for Longevity Traits in Natural Yeast Isolates.

Background:The budding yeast has served as a useful model organism in aging studies, leading to the identification of genetic determinants of longevity, many of which are conserved in higher eukaryotes. However, factors that promote longevity in a laboratory setting often have severe fitness disadvantages in the wild.Aims and Methods:To obtain an unbiased view on longevity regulation, we analyzed how a replicative lifespan is shaped by transcriptional, translational, metabolic, and morphological factors across 22 wild-type Saccharomyces cerevisiae isolates.Results:We observed significant differences in lifespan across these strains and found that their longevity is strongly associated with up-regulation of oxidative phosphorylation and respiration and down-regulation of amino- acid and nitrogen compound biosynthesis.Conclusions:As calorie restriction and TOR signaling also extend the lifespan by adjusting many of the identified pathways, the data suggest that the natural plasticity of yeast lifespan is shaped by the processes that not only do not impose cost on fitness, but also are amenable to dietary intervention.

Hsp12p and PAU genes are involved in ecological interactions between natural yeast strains.

The coexistence of different yeasts in a single vineyard raises the question on how they communicate and why slow growers are not competed out. Genetically modified laboratory strains of Saccharomyces cerevisiae are extensively used to investigate ecological interactions, but little is known about the genes regulating cooperation and competition in ecologically relevant settings. Here, we present evidences of Hsp12p-dependent altruistic and contact-dependent competitive interactions between two natural yeast isolates. Hsp12p is released during cell death for public benefit by a fast-growing strain that also produces a killer toxin to inhibit growth of a slow grower that can enjoy the benefits of released Hsp12p. We also show that the protein Pau5p is essential in the defense against the killer effect. Our results demonstrate that the combined action of Hsp12p, Pau5p and a killer toxin is sufficient to steer a yeast community.

Genetic variation in the cysteine biosynthesis pathway causes sensitivity to pharmacological compounds.

Complex traits are the product of multiple genes with effects that depend on both the genetic and environmental background. Although this complexity makes a comprehensive genetic analysis difficult, identification of even a single gene provides insight into the biochemical and/or signaling pathway underlying a trait. However, it is unknown whether multiple pathways, and consequently multiple genes, must be identified to adequately understand a trait's molecular basis. Using crosses between three natural isolates of Saccharomyces cerevisiae, we mapped sensitivity to a number of pharmacologically active compounds to a single nonsynonymous polymorphism in cystathione-beta-synthase (CYS4), which is required for the first committed step in the cysteine biosynthesis pathway. Drug sensitivity is mediated by a deficiency in cysteine and consequently glutathione production, because drug sensitivity is abrogated by cysteine or glutathione supplementation. Within a diverse panel of 60 natural yeast isolates, the drug-sensitive CYS4 allele is rare, and glutathione supplementation failed to alleviate drug-dependent growth defects in two other drug-sensitive strains. These results implicate the cysteine/glutathione biosynthesis pathway as a significant, but not the sole contributor to pharmacological variation in yeast.

System of Centromeric, Episomal, and Integrative Vectors Based on Drug Resistance Markers for SacCharomyces Cerevisiae

Integrative, centromeric, and episomal plasmids are essential for easy, fast, and reliable genetic manipulation of yeast. We constructed a system of shuttle vectors based on the widely used plasmids of the pRS series. We used genes conferring resistance to Geneticin (kanMX4), nourseothricin (natNT2), and hygromycin B (hphNT1) as markers. The centromeric and episomal plasmids that we constructed can be used the same way as the traditional auxotrophic marker-based shuttle vectors (pRS41x and pRS42x series). Additionally, we created a set of nine yeast integrative vectors with the three dominant markers. These plasmids allow for direct integration in the LEU2, URA3, and HIS3 locus of any yeast strain and the concomitant partial deletion of the gene. This prevents multiple integrations and allows for the rapid identification of correct integrants. The set of new vectors considerably enhances the flexibility of genetic manipulations and gene expression in yeast. Most notably, the new vectors allow one to work with natural yeast isolates, which do not contain auxotrophic markers.