Ascomycete yeasts are metabolically diverse, with great potential for biotechnology. Here, we report the comparative genome analysis of 29 taxonomically and biotechnologically important yeasts, including 16 newly sequenced. We identify a genetic code change, CUG-Ala, in Pachysolen tannophilus in the clade sister to the known CUG-Ser clade. Our well-resolved yeast phylogeny shows that some traits, such as methylotrophy, are restricted to single clades, whereas others, such as L-rhamnose utilization, have patchy phylogenetic distributions. Gene clusters, with variable organization and distribution, encode many pathways of interest. Genomics can predict some biochemical traits precisely, but the genomic basis of others, such as xylose utilization, remains unresolved. Our data also provide insight into early evolution of ascomycetes. We document the loss of H3K9me2/3 heterochromatin, the origin of ascomycete mating-type switching, and panascomycete synteny at the MAT locus. These data and analyses will facilitate the engineering of efficient biosynthetic and degradative pathways and gateways for genomic manipulation.genomics | bioenergy | biotechnological yeasts | genetic code | microbiology Y easts are fungi that reproduce asexually by budding or fission and sexually without multicellular fruiting bodies (1, 2). Their unicellular, largely free-living lifestyle has evolved several times (3). Despite morphological similarities, yeasts constitute over 1,500 known species that inhabit many specialized environmental niches and associations, including virtually all varieties of fruits and flowers, plant surfaces and exudates, insects and other invertebrates, birds, mammals, and highly diverse soils (4). Biochemical and genomic studies of the model yeast Saccharomyces cerevisiaeessential for making bread, beer, and wine-have established much of our understanding of eukaryotic biology. However, in many ways, S. cerevisiae is an oddity among the yeasts, and many important biotechnological applications and highly divergent physiological capabilities of lesser-known yeast species have not been fully exploited (5). Various species can grow on methanol or n-alkanes as sole carbon and energy sources, overproduce vitamins and lipids, thrive under acidic conditions, and ferment unconventional carbon sources. Many features of yeasts make them ideal platforms for biotechnological processes. Their thick cell walls help them survive osmotic shock, and in contrast to bacteria, they are resistant to viruses. Their unicellular form is easy to cultivate, scale up, and harvest. The objective of this study was, therefore, to put yeasts with diverse biotechnological applications in a phylogenomic context and relate their physiologies to genomic SignificanceThe highly diverse Ascomycete yeasts have enormous biotechnological potential. Collectively, these yeasts convert a broad range of substrates into useful compounds, such as ethanol, lipids, and vitamins, and can grow in extremes of temperature, salinity, and pH. We compared 29 yeast genome...

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.

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 from different 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.

The genetic code used in nuclear genes is almost universal, but here we report that it changed three times in parallel during the evolution of budding yeasts. All three changes were reassignments of the codon CUG, which is translated as serine (in 2 yeast clades), alanine (1 clade), or the ‘universal’ leucine (2 clades). The newly discovered Ser2 clade is in the final stages of a genetic code transition. Most species in this clade have genes for both a novel tRNASer(CAG) and an ancestral tRNALeu(CAG) to read CUG, but only tRNASer(CAG) is used in standard growth conditions. The coexistence of these alloacceptor tRNA genes indicates that the genetic code transition occurred via an ambiguous translation phase. We propose that the three parallel reassignments of CUG were not driven by natural selection in favor of their effects on the proteome, but by selection to eliminate the ancestral tRNALeu(CAG).

Centromere organization has evolved dramatically in one clade of fungi, the Saccharomycotina. These yeasts have lost the ability to make normal eukaryotic heterochromatin with histone H3K9 methylation, which is a major component of pericentromeric regions in other eukaryotes. Following this loss, several different types of centromere emerged, including two types of sequence-defined (“point”) centromeres, and the epigenetically defined “small regional” centromeres of Candida albicans. Here we report that centromeres of the methylotrophic yeast Komagataella phaffii (formerly called Pichia pastoris) are structurally defined. Each of its four centromeres consists of a 2-kb inverted repeat (IR) flanking a 1-kb central core (mid) region. The four centromeres are unrelated in sequence. CenH3 (Cse4) binds strongly to the cores, with a decreasing gradient along the IRs. This mode of organization resembles Schizosaccharomyces pombe centromeres but is much more compact and lacks the extensive flanking heterochromatic otr repeats. Different isolates of K. phaffii show polymorphism for the orientation of the mid regions, due to recombination in the IRs. CEN4 is located within a 138-kb region that changes orientation during mating-type switching, but switching does not induce recombination of centromeric IRs. Our results demonstrate that evolutionary transitions in centromere organization have occurred in multiple yeast clades.

BackgroundKomagataella 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.ResultsWe 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.ConclusionsWe 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.

Centromere organization has evolved dramatically in one clade of fungi, the Saccharomycotina. These yeasts have lost the ability to make normal eukaryotic heterochromatin with histone H3K9 methylation, which is a major component of pericentromeric regions in other eukaryotes. Following this loss, several different types of centromere emerged, including two types of sequence-defined ("point") centromeres, and the epigenetically defined "small regional" centromeres of Candida albicans. Here we report that centromeres of the methylotrophic yeast Komagataella phaffii (formerly called Pichia pastoris) are structurally defined. Each of its four centromeres consists of a 2-kb inverted repeat (IR) flanking a 1-kb central core (mid) region. The four centromeres are unrelated in sequence. CenH3 (Cse4) binds strongly to the cores, with a decreasing gradient along the IRs. This mode of organization resembles Schizosaccharomyces pombe centromeres but is much more compact and lacks the extensive flanking heterochromatic otr repeats. Different isolates of K. phaffii show polymorphism for the orientation of the mid regions, due to recombination in the IRs. CEN4 is located within a 138-kb region that changes orientation during mating-type switching, but switching does not induce recombination of centromeric IRs. Our results demonstrate that evolutionary transitions in centromere organization have occurred in multiple yeast clades.

Riboswitches are non-coding RNA molecules that regulate gene expression by binding to specific ligands. They are primarily found in bacteria. However, one riboswitch type, the thiamin pyrophosphate (TPP) riboswitch, has also been described in some plants, marine protists and fungi. We find that riboswitches are widespread in the budding yeasts (Saccharomycotina), and they are most common in homologs of DUR31, originally described as a spermidine transporter. We show that DUR31 (an ortholog of N. crassa gene NCU01977) encodes a thiamin transporter in Candida species. Using an RFP/riboswitch expression system, we show that the functional elements of the riboswitch are contained within the native intron of DUR31 from Candida parapsilosis, and that the riboswitch regulates splicing in a thiamin-dependent manner when RFP is constitutively expressed. The DUR31 gene has been lost from Saccharomyces, and may have been displaced by an alternative thiamin transporter. TPP riboswitches are also present in other putative transporters in yeasts and filamentous fungi. However, they are rare in thiamin biosynthesis genes THI4 and THI5 in the Saccharomycotina, and have been lost from all genes in the sequenced species in the family Saccharomycetaceae, including S. cerevisiae.