Transcriptional response to lactic acid stress in the hybrid yeast Zygosaccharomyces parabailii

Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH, and a fermentative metabolism with a fast growth rate. Here we used RNA-seq to analyze Z. parabailii's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than in control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/2, previously described as lactic acid-responsive in Saccharomyces cerevisiae. Furthermore, formate dehydrogenase (FDH) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii as compared to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production.Importance Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression in conditions of interest, and relating gene expression patterns to protein functions. However, hybrid organisms present a challenge to the standard use of mRNA sequencing (RNA-seq) to study transcriptional responses to stress, because their genomes contain two similar copies of almost every gene. Here we used stringent mapping methods and a high-quality genome sequence to study the transcriptional response to lactic acid stress in Zygosaccharomyces parabailii ATCC60483, a natural interspecies hybrid yeast that contains two complete subgenomes that are approximately 7% divergent in sequence. Beyond the insights we gained into lactic acid tolerance in this study, the methods we developed will be broadly applicable to other yeast hybrid strains

Ploidy variation in Kluyveromyces marxianus separates dairy and non-dairy isolates

Kluyveromyces marxianus is traditionally associated with fermented dairy products, but can also be isolated from diverse non-dairy environments. Because of thermotolerance, rapid growth and other traits, many different strains are being developed for food and industrial applications but there is, as yet, little understanding of the genetic diversity or population genetics of this species. K. marxianus shows a high level of phenotypic variation but the only phenotype that has been clearly linked to a genetic polymorphism is lactose utilisation, which is controlled by variation in the LAC12 gene. The genomes of several strains have been sequenced in recent years and, in this study, we sequenced a further nine strains fromdifferent origins. Analysis of the Single Nucleotide Polymorphisms (SNPs) in 14 strains was carried out to examine genome structure and genetic diversity. SNP diversity in K. marxianus is relatively high, with up to 3% DNA sequence divergence between alleles. It was found that the isolates include haploid, diploid, and triploid strains, as shown by both SNP analysis and flow cytometry. Diploids and triploids contain long genomic tracts showing loss of heterozygosity (LOH). All six isolates from dairy environments were diploid or triploid, whereas 6 out 7 isolates from non-dairy environment were haploid. This also correlated with the presence of functional LAC12 alleles only in dairy haplotypes. The diploids were hybrids between a non-dairy and a dairy haplotype, whereas triploids included three copies of a dairy haplotype

EDUCACIÓN AMBIENTAL Y SOCIEDAD. SABERES LOCALES PARA EL DESARROLLO Y LA SUSTENTABILIDAD

Este texto contribuye al análisis científico de varias áreas del conocimiento como la filosofía social, la patología, la educación para el cuidado del medio ambiente y la sustentabilidad que inciden en diversas unidades de aprendizaje de la Licenciatura en Educación para la Salud y de la Maestría en Sociología de la SaludLas comunidades indígenas de la sierra norte de Oaxaca México, habitan un territorio extenso de biodiversidad. Sin que sea una área protegida y sustentable, la propia naturaleza de la región ofrece a sus visitantes la riqueza de la vegetación caracterizada por sus especies endémicas que componen un paisaje de suma belleza

Pantothenate Auxotrophy in a Naturally Occurring Biocontrol Yeast

The genus Hanseniaspora is characterized by some of the smallest genomes among budding yeasts. These fungi are primarily found on plant surfaces and in fermented products and represent promising biocontrol agents against notorious fungal plant pathogens. In this work, we identify pantothenate auxotrophy of a Hanseniaspora meyeri isolate that shows strong antagonism against the plant pathogen Fusarium oxysporum. Furthermore, strong biocontrol activity in vitro required both pantothenate and biotin in the growth medium. We show that the H. meyeri isolate APC 12.1 can obtain the vitamin from plants and other fungi. The underlying reason for the auxotrophy is the lack of two key pantothenate biosynthesis genes, but six genes encoding putative pantothenate transporters are present in the genome. By constructing and using a Saccharomyces cerevisiae reporter strain, we identified one Hanseniaspora transporter that conferred pantothenate uptake activity to S. cerevisiae. Pantothenate auxotrophy is rare and has been described in only a few bacteria and in S. cerevisiae strains that were isolated from sake. Such auxotrophic strains may seem an unexpected and unlikely choice as potential biocontrol agents, but they may be particularly competitive in their ecological niche and their specific growth requirements are an inherent biocontainment strategy preventing uncontrolled growth in the environment. Auxotrophic strains, such as the H. meyeri isolate APC 12.1, may thus represent a promising strategy for developing biocontrol agents that will be easier to register than prototrophic strains, which are normally used for such applications. IMPORTANCE As a precursor of the essential coenzyme A (CoA), pantothenate is present in all organisms. Plants, bacteria, and fungi are known to synthesize this vitamin, while animals must obtain it through their diet. Pantothenate auxotrophy has not been described in naturally occurring, environmental fungi and is an unexpected property for an antagonistic yeast. Here, we report that yeasts from the genus Hanseniaspora lack key enzymes for pantothenate biosynthesis and identify a transporter responsible for the acquisition of pantothenate from the environment. Hanseniaspora isolates are strong antagonists of fungal plant pathogens. Their pantothenate auxotrophy is a natural biocontainment feature that could make such isolates interesting candidates for new biocontrol approaches and allow easier registration as plant protection agents than prototrophic strains.ISSN:0099-2240ISSN:1098-533

Coverage-Versus-Length Plots, a Simple Quality Control Step for de Novo Yeast Genome Sequence Assemblies

Illumina sequencing has revolutionized yeast genomics, with prices for commercial draft genome sequencing now below $200. The popular SPAdes assembler makes it simple to generate a de novo genome assembly for any yeast species. However, whereas making genome assemblies has become routine, understanding what they contain is still challenging. Here, we show how graphing the information that SPAdes provides about the length and coverage of each scaffold can be used to investigate the nature of an assembly, and to diagnose possible problems. Scaffolds derived from mitochondrial DNA, ribosomal DNA, and yeast plasmids can be identified by their high coverage. Contaminating data, such as cross-contamination from other samples in a multiplex sequencing run, can be identified by its low coverage. Scaffolds derived from the bacteriophage PhiX174 and Lambda DNAs that are frequently used as molecular standards in Illumina protocols can also be detected. Assemblies of yeast genomes with high heterozygosity, such as interspecies hybrids, often contain two types of scaffold: regions of the genome where the two alleles assembled into two separate scaffolds and each has a coverage level C, and regions where the two alleles co-assembled (collapsed) into a single scaffold that has a coverage level 2C. Visualizing the data with Coverage-vs.-Length (CVL) plots, which can be done using Microsoft Excel or Google Sheets, provides a simple method to understand the structure of a genome assembly and detect aberrant scaffolds or contigs. We provide a Python script that allows assemblies to be filtered to remove contaminants identified in CVL plots

Evolutionary restoration of fertility in an interspecies hybrid yeast, by whole-genome duplication after a failed mating-type switch - Fig 5

<p>(A) Organization of <i>MAT</i>, <i>HML</i>, and <i>HMR</i> loci in <i>Z</i>. <i>parabailii</i> ATCC60483. The genome contains 6 <i>MAT</i>-related regions, with 1 <i>MAT</i>, 1 <i>HML</i>, and 1 <i>HMR</i> locus derived from each of the A and B parents. Pink and green backgrounds indicate sequences from the A- and B-subgenomes, respectively. The <i>MAT</i> locus in the A-subgenome (position 294 kb on chromosome 7) is intact and expressed. The <i>MAT</i> locus of the B-subgenome has been broken into 2 parts by cleavage by HO endonuclease. All 6 copies of the X repeat region (654 bp) are identical in sequence, as are all 6 copies of the Z repeat region (266 bp). Gray triangles indicate the disruption of the splicing of intron 2 in <i>MAT</i>α2 and <i>HML</i>α2 of the B-subgenome. The binding sites for primers A–F used for PCR amplification are indicated by gray arrows. (B) Sequences at the <i>MAT</i> locus breakpoint. Red, <i>MAT</i>α1-derived sequences. The HO cleavage site (CGCAGCA, giving a 4-nucleotide 3′ overhang) is highlighted in gray. Blue, the <i>GDA1-YEF1</i> intergenic region from the equivalent region of <i>Z</i>. <i>bailii</i> CLIB213^T and homologous sequences from the A-subgenome on <i>Z</i>. <i>parabailii</i> chromosomes (chrs.) 2 and 16. A 5-bp sequence (ACAAC) that became duplicated during the rearrangement is underlined. (C) Sequences of <i>MAT</i>α2 intron 2 (lowercase) from the A- and B-subgenomes. An AG-to-AC mutation (red) at the 3′ end of the intron moved the splice site by 2 bp in the B-subgenome, causing a frameshift and premature translation termination. The splice sites in both genes were identified from RNA sequencing (RNA-Seq) data.</p

Saccharomyces cerevisiae CEN.PK113-7D: Anaerobically evolved fungi that harbor Class II dihydroorotate dehydrogenase enzymes that function independently of the respiratory chain

The goal of this study was elucidate the respiratory chain dependency of the abovementioned Class II DHODs. The URA9 genes of Anaeromyces robustus, Sch. japonicus, D. bruxellensis, O. parapolymorpha and K. marxianus were expressed in a ura1 null mutant of S. cerevisiae and growth was studied under anaerobic conditions. The subcellular localization of the enzymes was determined by fluorescence microscopy of GFP-fused Ura9 proteins. Finally, alternative electron acceptors were determined for these proteins by enzyme assays using cell free extracts and mitochondrial extracts of S. cerevisiae ura1 null mutants expressing the URA9 genes from a multicopy plasmid

Saccharomyces cerevisiae CEN.PK113-7D: Anaerobically evolved fungi that harbor Class II dihydroorotate dehydrogenase enzymes that function independently of the respiratory chain

The goal of this study was elucidate the respiratory chain dependency of the abovementioned Class II DHODs. The URA9 genes of Anaeromyces robustus, Sch. japonicus, D. bruxellensis, O. parapolymorpha and K. marxianus were expressed in a ura1 null mutant of S. cerevisiae and growth was studied under anaerobic conditions. The subcellular localization of the enzymes was determined by fluorescence microscopy of GFP-fused Ura9 proteins. Finally, alternative electron acceptors were determined for these proteins by enzyme assays using cell free extracts and mitochondrial extracts of S. cerevisiae ura1 null mutants expressing the URA9 genes from a multicopy plasmid

Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis

Abstract Background In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete Anaeromyces robustus and the yeasts Schizosaccharomyces japonicus and Dekkera bruxellensis) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD. Results Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (Arura9, SjURA9, DbURA9) in an S. cerevisiae ura1Δ strain supported aerobic as well as anaerobic pyrimidine prototrophy. A strain expressing DbURA9 showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. GFP-tagged SjUra9 and DbUra9 were localized to S. cerevisiae mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. Expression of SjURA9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9. Conclusions Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in S. cerevisiae, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure–function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in S. cerevisiae strains expressing an anaerobically active DHOD from Sch. japonicus

Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis

Abstract Background In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete Anaeromyces robustus and the yeasts Schizosaccharomyces japonicus and Dekkera bruxellensis) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD. Results Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (Arura9, SjURA9, DbURA9) in an S. cerevisiae ura1Δ strain supported aerobic as well as anaerobic pyrimidine prototrophy. A strain expressing DbURA9 showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. GFP-tagged SjUra9 and DbUra9 were localized to S. cerevisiae mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. Expression of SjURA9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9. Conclusions Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in S. cerevisiae, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure–function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in S. cerevisiae strains expressing an anaerobically active DHOD from Sch. japonicus