Alternative sulphur metabolism in the fungal pathogen Candida parapsilosis.

Candida parapsilosis is an opportunistic fungal pathogen commonly isolated from the environment and associated with nosocomial infection outbreaks worldwide. We describe here the construction of a large collection of gene disruptions, greatly increasing the molecular tools available for probing gene function in C. parapsilosis. We use these to identify transcription factors associated with multiple metabolic pathways, and in particular to dissect the network regulating the assimilation of sulphur. We find that, unlike in other yeasts and filamentous fungi, the transcription factor Met4 is not the main regulator of methionine synthesis. In C. parapsilosis, assimilation of inorganic sulphur (sulphate) and synthesis of cysteine and methionine is regulated by Met28, a paralog of Met4, whereas Met4 regulates expression of a wide array of transporters and enzymes involved in the assimilation of organosulfur compounds. Analysis of transcription factor binding sites suggests that Met4 is recruited by the DNA-binding protein Met32, and Met28 is recruited by Cbf1. Despite having different target genes, Met4 and Met28 have partial functional overlap, possibly because Met4 can contribute to assimilation of inorganic sulphur in the absence of Met28.

Draft genome sequence of the yeast Schwanniomyces capriottii strain UCD805, isolated from soil in Ireland.

Schwanniomyces capriottii is a member of the Debaryomycetaceae family in the order Saccharomycetales. Here, we present the genome sequence of S. capriottii UCD805, which was isolated from soil in Dublin, Ireland. This genome is 12.2 Mb and was assembled into 14 scaffolds plus a mitochondrial genome scaffold.

Genome sequences of two isolates of the yeast Candida zeylanoides: UCD849 from soil in Ireland, and AWD from an African wild dog.

We report genome sequences of two new isolates of the budding yeast Candida zeylanoides. Strain UCD849 from soil in Ireland was assembled into eight complete chromosomes. Strain AWD from an African Wild Dog in a US zoo was sequenced to draft level. The assemblies are 10.6 Mb and 99.57% identical.

Identification of genetic variants of the industrial yeast Komagataella phaffii (Pichia pastoris) that contribute to increased yields of secreted heterologous proteins.

The yeast Komagataella phaffii (formerly called Pichia pastoris) is used widely as a host for secretion of heterologous proteins, but only a few isolates of this species exist and all the commonly used expression systems are derived from a single genetic background, CBS7435 (NRRL Y-11430). We hypothesized that other genetic backgrounds could harbor variants that affect yields of secreted proteins. We crossed CBS7435 with 2 other K. phaffii isolates and mapped quantitative trait loci (QTLs) for secretion of a heterologous protein, ß-glucosidase, by sequencing individual segregant genomes. A major QTL mapped to a frameshift mutation in the mannosyltransferase gene HOC1, which gives CBS7435 a weaker cell wall and higher protein secretion than the other isolates. Inactivation of HOC1 in the other isolates doubled ß-glucosidase secretion. A second QTL mapped to an amino acid substitution in IRA1 that tripled ß-glucosidase secretion in 1-week batch cultures but reduced cell viability, and its effects are specific to this heterologous protein. Our results demonstrate that QTL analysis is a powerful method for dissecting the basis of biotechnological traits in nonconventional yeasts, and a route to improving their industrial performance.

Identification of European isolates of the lager yeast parent Saccharomyces eubayanus.

Lager brewing first occurred in Bavaria in the 15th century, associated with restrictions of brewing to colder months. The lager yeast, Saccharomyces pastorianus, is cold tolerant. It is a hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus, and has been found only in industrial settings. Natural isolates of S. eubayanus were first discovered in Patagonia 11 years ago. They have since been isolated from China, Tibet, New Zealand, and North America, but not from Europe. Here, we describe the first European strains UCD646 and UCD650, isolated from a wooded area on a university campus in Dublin, Ireland. We generated complete chromosome level assemblies of both genomes using long- and short-read sequencing. The UCD isolates belong to the Holarctic clade. Genome analysis shows that isolates similar to the Irish strains contributed to the S. eubayanus component of S. pastorianus, but isolates from Tibet made a larger contribution.

A widespread inversion polymorphism conserved among Saccharomyces species is caused by recurrent homogenization of a sporulation gene family.

Saccharomyces genomes are highly collinear and show relatively little structural variation, both within and between species of this yeast genus. We investigated the only common inversion polymorphism known in S. cerevisiae, which affects a 24-kb 'flip/flop' region containing 15 genes near the centromere of chromosome XIV. The region exists in two orientations, called reference (REF) and inverted (INV). Meiotic recombination in this region is suppressed in crosses between REF and INV orientation strains such as the BY x RM cross. We find that the inversion polymorphism is at least 17 million years old because it is conserved across the genus Saccharomyces. However, the REF and INV isomers are not ancient alleles but are continually being re-created by re-inversion of the region within each species. Inversion occurs due to continual homogenization of two almost identical 4-kb sequences that form an inverted repeat (IR) at the ends of the flip/flop region. The IR consists of two pairs of genes that are specifically and strongly expressed during the late stages of sporulation. We show that one of these gene pairs, YNL018C/YNL034W, codes for a protein that is essential for spore formation. YNL018C and YNL034W are the founder members of a gene family, Centroid, whose members in other Saccharomycetaceae species evolve fast, duplicate frequently, and are preferentially located close to centromeres. We tested the hypothesis that Centroid genes are a meiotic drive system, but found no support for this idea.

Draft Genome Sequence of the Yeast Torulaspora quercuum Strain UCD657, Isolated from Soil in Ireland.

Torulaspora quercuum is an ascomycete yeast first isolated in 2009. Here, we present the genome sequence of T. quercuum isolate UCD657, which was isolated from soil in Ireland. This genome is 10.4 Mb and was assembled into 8 chromosome-sized scaffolds of >1 Mb in size, plus a mitochondrial genome scaffold.

Draft Genome Sequence of the Yeast Blastobotrys aristata Strain UCD613, Isolated from Soil in Ireland.

Blastobotrys aristata is a member of the Trichomonascaceae family in the order Saccharomycetales. Here, we present the genome sequence of B. aristata UCD613, which was isolated from soil in Dublin, Ireland. This genome is 13.3 Mb and was assembled into 4 chromosome-size scaffolds of >2.2 Mb in size plus a mitochondrial genome scaffold.

Draft Genome Sequence of a Diploid and Hybrid Candida Strain, Candida sanyaensis UCD423, Isolated from Compost in Ireland.

Candida sanyaensis is a CUG-Ser1 clade yeast that is associated with soil. Assembly of short-read and long-read data shows that C. sanyaensis has a diploid and hybrid genome, with approximately 97% identity between the haplotypes. The haploid genome size is approximately 15.4 Mb.

Draft Genome Sequence of the Yeast Ogataea degrootiae Strain UCD465, Isolated from Soil in Ireland.

Ogataea degrootiae is an ascomycete yeast that was first isolated in the Netherlands in 2017. It is a member of the Pichiaceae clade. Here, we present the genome sequence of O. degrootiae UCD465, which was isolated from soil in Ireland. This genome is 14.6 Mb and haploid.

The yeast mating-type switching endonuclease HO is a domesticated member of an unorthodox homing genetic element family.

The mating-type switching endonuclease HO plays a central role in the natural life cycle of Saccharomyces cerevisiae, but its evolutionary origin is unknown. HO is a recent addition to yeast genomes, present in only a few genera close to Saccharomyces. Here we show that HO is structurally and phylogenetically related to a family of unorthodox homing genetic elements found in Torulaspora and Lachancea yeasts. These WHO elements home into the aldolase gene FBA1, replacing its 3' end each time they integrate. They resemble inteins but they operate by a different mechanism that does not require protein splicing. We show that a WHO protein cleaves Torulaspora delbrueckii FBA1 efficiently and in an allele-specific manner, leading to DNA repair by gene conversion or NHEJ. The DNA rearrangement steps during WHO element homing are very similar to those during mating-type switching, and indicate that HO is a domesticated WHO-like element.

In the same way as a sperm from a male and an egg from a female join together to form an embryo in most animals, yeast cells have two sexes that coordinate how they reproduce. These are called "mating types" and, rather than male or female, an individual yeast cell can either be mating type "a" or "alpha". Every yeast cell contains the genes for both mating types, and each cell's mating type is determined by which of those genes it has active. Only one mating type gene can be 'on' at a time, but some yeast species can swap mating type on demand by switching the corresponding genes 'on' or 'off'. This switch is unusual. Rather than simply activate one of the genes it already has, the yeast cell keeps an inactive version of each mating type gene tucked away, makes a copy of the gene it wants to be active and pastes that copy into a different location in its genome. To do all of this yeast need another gene called HO. This gene codes for an enzyme that cuts the DNA at the location of the active mating type gene. This makes an opening that allows the cell to replace the 'a' gene with the 'alpha' gene, or vice versa. This system allows yeast cells to continue mating even if all the cells in a colony start off as the same mating type. But, cutting into the DNA is risky, and can damage the health of the cell. So, why did yeast cells evolve a system that could cause them harm? To find out where the HO gene came from, Coughlan et al. searched through all the available genomes from yeast species for other genes with similar sequences and identified a cluster which they nicknamed "weird HO" genes, or WHO genes for short. Testing these genes revealed that they also code for enzymes that make cuts in the yeast genome, but the way the cell repairs the cuts is different. The WHO genes are jumping genes. When the enzyme encoded by a WHO gene makes a cut in the genome, the yeast cell copies the gene into the gap, allowing the gene to 'jump' from one part of the genome to another. It is possible that this was the starting point for the evolution of the HO gene. Changes to a WHO gene could have allowed it to cut into the mating type region of the yeast genome, giving the yeast an opportunity to 'domesticate' it. Over time, the yeast cell stopped the WHO gene from jumping into the gap and started using the cut to change its mating type. Understanding how cells adapt genes for different purposes is a key question in evolutionary biology. There are many other examples of domesticated jumping genes in other organisms, including in the human immune system. Understanding the evolution of HO not only sheds light on how yeast mating type switching evolved, but on how other species might harness and adapt their genes.

Genomic diversity and meiotic recombination among isolates of the biotech yeast Komagataella phaffii (Pichia pastoris).

BACKGROUND: Komagataella phaffii is a yeast widely used in the pharmaceutical and biotechnology industries, and is one of the two species that were previously called Pichia pastoris. However, almost all laboratory work on K. phaffii has utilized strains derived from a single natural isolate, CBS7435. There is little information about the sequence diversity of K. phaffii or the genetic properties of this species. RESULTS: We sequenced the genomes of all the known isolates of K. phaffii. We made a genetic cross between derivatives of two isolates that differ at 44,000 single nucleotide polymorphism sites, and used this cross to analyze the rate and landscape of meiotic recombination. We conducted tetrad analysis by making use of the property that K. phaffii haploids do not mate in rich media, which enabled us to isolate and sequence the four types of haploid cell that are present in the colony that forms when a tetra-type ascus germinates. CONCLUSIONS: We found that only four distinct natural isolates of K. phaffii exist in public yeast culture collections. The meiotic recombination rate in K. phaffii is approximately 3.5 times lower than in Saccharomyces cerevisiae, with an average of 25 crossovers per meiosis. Recombination is suppressed, and genetic diversity among natural isolates is low, in a region around centromeres that is much larger than the centromeres themselves. Our work lays a foundation for future quantitative trait locus analysis in K. phaffii.

Draft Genome Sequence of a Red Basidiomycete Yeast, Symmetrospora coprosmae Strain UCD350, Isolated from Soil in Ireland.

Symmetrospora coprosmae is a red yeast from the subphylum Pucciniomycotina in the phylum Basidiomycota. Here, we present the first genome sequence of S. coprosmae strain UCD350, from an isolate collected from soil in Ireland. The genome size is 20.2 Mb.

Draft Genome Sequences of Two Isolates of the Yeast Kazachstania servazzii Recovered from Soil in Ireland.

We sequenced two isolates of Kazachstania servazzii, UCD13 and UCD335, from soil in Ireland. Heterozygosity in these diploid genomes differs 19-fold between the two strains. Most currently available K. servazzii genome sequences come from Korean kimchi isolates, so our data will facilitate analysis of diversity in this species.

Draft Genome Sequence of the Birch Tree Fungal Pathogen Taphrina betulina UCD315.

Taphrina betulina is the ascomycete yeast that causes the formation of witches' brooms in birch trees. Here, we report the first draft genome sequence of T. betulina, from strain UCD315, isolated from soil in Ireland. The genome is haploid and 12.5 Mb long.

The Methylotroph Gene Order Browser (MGOB) reveals conserved synteny and ancestral centromere locations in the yeast family Pichiaceae.

The yeast family Pichiaceae, also known as the 'methylotrophs clade', is a relatively little studied group of yeasts despite its economic and clinical relevance. To explore the genome evolution and synteny relationships within this family, we developed the Methylotroph Gene Order Browser (MGOB, http://mgob.ucd.ie) similar to our previous gene order browsers for other yeast families. The dataset contains genome sequences from nine Pichiaceae species, including our recent reference sequence of Pichia kudriavzevii. As an example, we demonstrate the conservation of synteny around the MOX1 locus among species both containing and lacking the MOX1 gene for methanol assimilation. We found ancient clusters of genes that are conserved as adjacent between Pichiaceae and Saccharomycetaceae. Surprisingly, we found evidence that the locations of some centromeres have been conserved among Pichiaceae species, and between Pichiaceae and Saccharomycetaceae, even though the centromeres fall into different structural categories-point centromeres, inverted repeats and retrotransposon cluster centromeres.

The CYPome of the model xenobiotic-biotransforming fungus Cunninghamella elegans.

The fungus Cunninghamella elegans is recognised as a microbial model of mammalian drug metabolism owing to its ability to catabolise xenobiotic compounds in an analogous fashion to animals. Its ability to produce phase I (oxidative) metabolites of drugs is associated with cytochrome P450 (CYP) activity; however, almost nothing is known about these enzymes in the fungus. In this paper we report the in silico analysis of the genome sequence of C. elegans B9769, which contains 32 genes putatively coding for CYPs. Based on their predicted amino acid sequences these were classified as belonging to CYP509, 5203, 5208, 5313, 5210, 61 and 51 families. Reverse transcription-quantitative PCR revealed that the gene coding for CYP5313D1 was significantly upregulated when C. elegans DSM1908 was cultivated in sabouraud dextrose in contrast to its expression in cells grown in Roswell Park Memorial Institute medium. This corresponded to the fungus' xenobiotic biotransformation ability when grown in the two media. Heterologous expression of cyp5313D1 in Pichia pastoris resulted in a recombinant strain that biotransformed flurbiprofen to 4'-hydroxyflurbiprofen, the same metabolite generated by C. elegans cultures. This is the first report of a xenobiotic-biotransforming CYP from this biotechnologically important fungus.

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.

Comparative Analysis of Oomycete Genome Evolution Using the Oomycete Gene Order Browser (OGOB).

The oomycetes are a class of microscopic, filamentous eukaryotes within the stramenopiles-alveolates-rhizaria eukaryotic supergroup. They include some of the most destructive pathogens of animals and plants, such as Phytophthora infestans, the causative agent of late potato blight. Despite the threat they pose to worldwide food security and natural ecosystems, there is a lack of tools and databases available to study oomycete genetics and evolution. To this end, we have developed the Oomycete Gene Order Browser (OGOB), a curated database that facilitates comparative genomic and syntenic analyses of oomycete species. OGOB incorporates genomic data for 20 oomycete species including functional annotations and a number of bioinformatics tools. OGOB hosts a robust set of orthologous oomycete genes for evolutionary analyses. Here, we present the structure and function of OGOB as well as a number of comparative genomic analyses we have performed to better understand oomycete genome evolution. We analyze the extent of oomycete gene duplication and identify tandem gene duplication as a driving force of the expansion of secreted oomycete genes. We identify core genes that are present and microsyntenically conserved (termed syntenologs) in oomycete lineages and identify the degree of microsynteny between each pair of the 20 species housed in OGOB. Consistent with previous comparative synteny analyses between a small number of oomycete species, our results reveal an extensive degree of microsyntenic conservation amongst genes with housekeeping functions within the oomycetes. OGOB is available at https://ogob.ie.

Population genomics shows no distinction between pathogenic Candida krusei and environmental Pichia kudriavzevii: One species, four names.

We investigated genomic diversity of a yeast species that is both an opportunistic pathogen and an important industrial yeast. Under the name Candida krusei, it is responsible for about 2% of yeast infections caused by Candida species in humans. Bloodstream infections with C. krusei are problematic because most isolates are fluconazole-resistant. Under the names Pichia kudriavzevii, Issatchenkia orientalis and Candida glycerinogenes, the same yeast, including genetically modified strains, is used for industrial-scale production of glycerol and succinate. It is also used to make some fermented foods. Here, we sequenced the type strains of C. krusei (CBS573T) and P. kudriavzevii (CBS5147T), as well as 30 other clinical and environmental isolates. Our results show conclusively that they are the same species, with collinear genomes 99.6% identical in DNA sequence. Phylogenetic analysis of SNPs does not segregate clinical and environmental isolates into separate clades, suggesting that C. krusei infections are frequently acquired from the environment. Reduced resistance of strains to fluconazole correlates with the presence of one gene instead of two at the ABC11-ABC1 tandem locus. Most isolates are diploid, but one-quarter are triploid. Loss of heterozygosity is common, including at the mating-type locus. Our PacBio/Illumina assembly of the 10.8 Mb CBS573T genome is resolved into 5 complete chromosomes, and was annotated using RNAseq support. Each of the 5 centromeres is a 35 kb gene desert containing a large inverted repeat. This species is a member of the genus Pichia and family Pichiaceae (the methylotrophic yeasts clade), and so is only distantly related to other pathogenic Candida species.