Interplay of Chimeric Mating-Type Loci Impairs Fertility Rescue and Accounts for Intra-Strain Variability in Zygosaccharomyces rouxii Interspecies Hybrid ATCC42981

The pre-whole genome duplication (WGD) Zygosaccharomyces clade comprises several allodiploid strain/species with industrially interesting traits. The salt-tolerant yeast ATCC42981 is a sterile and allodiploid strain which contains two subgenomes, one of them resembling the haploid parental species Z. rouxii. Recently, different mating-type-like (MTL) loci repertoires were reported for ATCC42981 and the Japanese strain JCM22060, which are considered two stocks of the same strain. MTL reconstruction by direct sequencing approach is challenging due to gene redundancy, structure complexities, and allodiploid nature of ATCC42981. Here, DBG2OLC and MaSuRCA hybrid de novo assemblies of ONT and Illumina reads were combined with in vitro long PCR to definitively solve these incongruences. ATCC42981 exhibits several chimeric MTL loci resulting from reciprocal translocation between parental haplotypes and retains two MATa/MAT\u3b1 expression loci, in contrast to MAT\u3b1 in JCM22060. Consistently to these reconstructions, JCM22060, but not ATCC42981, undergoes mating and meiosis. To ascertain whether the damage of one allele at the MAT locus regains the complete sexual cycle in ATCC42981, we removed the MAT\u3b1 expressed locus by gene deletion. The resulting MATa/- hemizygous mutants did not show any evidence of sporulation, as well as of self- and out-crossing fertility, probably because incomplete silencing at the chimeric HML\u3b1 cassette masks the loss of heterozygosity at the MAT locus. We also found that MAT\u3b1 deletion switched off a2 transcription, an activator of a-specific genes in pre-WGD species. These findings suggest that regulatory scheme of cell identity needs to be further investigated in Z. rouxii protoploid yeast

Draft Genome Sequences of the Highly Halotolerant Strain Zygosaccharomyces rouxii ATCC 42981 and the Novel Allodiploid Strain Zygosaccharomyces sapae ATB301T Obtained Using the MinION Platform

Here, we report draft genome sequences of the halotolerant and allodiploid strains Zygosaccharomyces rouxii ATCC 42981 and Zygosaccharomyces sapae ABT301T. Illumina and Oxford Nanopore MinION sequencing revealed genome sizes of 20.9 and 24.7\u2009Mb, respectively. This information will be useful for deciphering the genetics of hybrid adaptation to high salt and sugar concentrations in nonconventional yeasts

Recent advances in understanding yeast genetics of sex determination.

Sex determination is among the most fascinating areas of study in modern genetics and encompasses many topics, such as developmental mechanisms, behaviour, sex chromosome biology, population evolution and diversity. Yeasts from the subphylum Saccharomycotina are of great importance to humans, not only as pathogens but for numerous essential ecosystem services and serve as model to study evolution in action. The recent advent of inexpensive sequence information and other new tools has led to notable advances in our understanding of reproductive mechanisms over a broad range of species, revealing that genetic sex determination occurs in different ways with a myriad of outcomes in yeast

The sex determination system and the role of chimeric a1/alpha2 heterodimer in the sterility of an allodiploid Zygosaccharomyces yeast

In Saccharomyces cerevisiae diploids, the a1/alpha2 protein heterodimer acts as repressor of haploid-specific genes, such as MATalpha1 and HO genes, and allows a/alpha diploid cells to undergo sporulation. The regulatory routes governing the cell type and fate have been not yet studied in the yeasts belonging to the Zygosaccharomyces rouxii complex. These yeasts are relevant in foodstuff elaboration and spoilage due to a wide repertoire of tolerances to osmotic stress. Their genetic variability favors phenotypic diversity and adaptation to hostile environments by ectopic recombination of mating-type (MTL) loci through an error-prone switching mechanism. Among them, the allodiploid strain ATCC42981 exhibits hybrid vigor and multi-stress tolerance, but it is unable to undergo sexual reproduction. Aim of this work was to characterize allodiploid strain ATCC42981 for the genetic organization and the transcriptional expression of mating type-like (MTL) loci and HO genes. Genetic dissection of ATCC42981 sex determinants revealed a MATa/MATalpha genotype with a redundant number of partial divergent HMR cassettes. MATa expression locus contained Z. rouxii-related MATa1 and MATa2 genes, whereas MATalpha expression locus contained Z. sapae-related MATalpha1 and MATalpha2 genes (termed MATalpha1 and MATalpha2 copy 2, respectively). Both MAT expression loci are linked to phylogenetically congruent HML silent cassettes on non-homologous chromosomes. Other than a \u201chybrid\u201d and redundant three-cassette system, ATCC42981 possesses two divergent HO genes. Consistently with this gene organization, ATCC42981 expressed MATa1 and MATalpha2 copy 2 genes under standard conditions, whereas aand \u3b1-idiomorph genes from HMR and HML cassettes were silent. Differently from S. cerevisiae diploids, ATCC42981 did not repress either MATalpha1 or HO gene transcription. Under hypersaline stress (which should be induce meiosis in Zygosaccharomyces cells by turning on the haploid gene-specific program), ATCC42981 was not able to undergo meiosis and over-expressed HO copy 2, MATa1 and MATalpha1 copy 2. We hypothesized that the partial incompatibility between ATCC42981 Z. rouxii-like a1 and Z. sapae-like alpha2 subunits in MATa1/alpha2 heterodimer causes a defective silencing of haploid-specific genes, including meiosis inhibiting factors, leading to clonality as the only possible reproduction strategy for this allodiploid yeas

Molecular two-step strategy to select inter-species Saccharomyces hybrids

Hybridization is a common tool to improve yeast for wine industry. Using non-GE hybridization techniques a lot of attempts lead to failed mating which is mainly caused by low spore viability and haplo-selfing. This work proposed a two-step molecular strategy to validate inter-species Saccharomyces hybrids rapidly

Chimeric Sex-Determining Chromosomal Regions and Dysregulation of Cell-Type Identity in a Sterile Zygosaccharomyces Allodiploid Yeast.

Allodiploidization is a fundamental yet evolutionarily poorly characterized event, which impacts genome evolution and heredity, controlling organismal development and polyploid cell-types. In this study, we investigated the sex determination system in the allodiploid and sterile ATCC 42981 yeast, a member of the Zygosaccharomyces rouxii species complex, and used it to study how a chimeric mating-type gene repertoire contributes to hybrid reproductive isolation. We found that ATCC 42981 has 7 MAT-like (MTL) loci, 3 of which encode α-idiomorph and 4 encode a-idiomorph. Two phylogenetically divergent MAT expression loci were identified on different chromosomes, accounting for a hybrid a/α genotype. Furthermore, extra a-idimorph-encoding loci (termed MTLa copies 1 to 3) were recognized, which shared the same MATa1 ORFs but diverged for MATa2 genes. Each MAT expression locus was linked to a HML silent cassette, while the corresponding HMR loci were located on another chromosome. Two putative parental sex chromosome pairs contributed to this unusual genomic architecture: one came from an as-yet-undescribed taxon, which has the NCYC 3042 strain as a unique representative, while the other did not match any MAT-HML and HMR organizations previously described in Z. rouxii species. This chimeric rearrangement produces two copies of the HO gene, which encode for putatively functional endonucleases essential for mating-type switching. Although both a and α coding sequences, which are required to obtain a functional cell-type a1-α2 regulator, were present in the allodiploid ATCC 42981 genome, the transcriptional circuit, which regulates entry into meiosis in response to meiosis-inducing salt stress, appeared to be turned off. Furthermore, haploid and α-specific genes, such as MATα1 and HO, were observed to be actively transcribed and up-regulated under hypersaline stress. Overall, these evidences demonstrate that ATCC 42981 is unable to repress haploid α-specific genes and to activate meiosis in response to stress. We argue that sequence divergence within the chimeric a1-α2 heterodimer could be involved in the generation of negative epistasis, contributing to the allodiploid sterility and the dysregulation of cell identity

A set of plasmids carrying antibiotic resistance markers and Cre recombinase for genetic engineering of nonconventional yeast Zygosaccharomyces rouxii

The so‐called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732T. By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMXR and ClonNATR as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase‐mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX–loxP module in Z. rouxii ATCC 42981 G418‐resistant mutants previously constructed by replacing the MATαP expression locus with the loxP–kanMX–loxP cassette

Ectopic recombination in sex-determination system as a source of genetic variation in the diploid yeast Zygosaccharomyces sapae.

Sexual reproduction increases genetic variation, which is strongly advantageous under harsh environmental conditions, as it allows natural selection to proceed more effectively. The yeasts of the Zygosaccharomyces rouxii complex are relevant in food elaboration and spoilage due to their ability to cope with low water activity environments and are characterized by gene copy number variation, genome instability, and aneuploidy/allodiploidy (Solieri et al. 2013). The mating-type locus (MAT) is a hotspot for chromosome rearrangement in yeasts. Here, we investigated the genetic architecture of sex determinants, including MAT loci and HO endonuclease in Zygosaccharomyces sapae diploid strain ABT301T, belonging to the Z. rouxii complex. We cloned these genes through a DNA walking strategy and a characterization of the flanking regions, while the chromosome assignment was performed combining Southern blotting and PFGE-karyotyping. We identified three divergent mating type-like (MTL) α-idiomorph sequences, designated as ZsMTLα copies 1, 2, and 3, which encoded homologues of Z. rouxii CBS 732T MATα2 (aa sequence identity from 67.0 to 99.5%) and MATα1 (identity 81.5-99.5%). Cloning of MATa-idiomorph yielded one ZsMTLa locus encoding two Z. rouxii-like proteins, MATa1 and MATa2. ABT301T possesses two divergent HO genes encoding distinct endonucleases. Based on the cloned ZsMTLα and ZsMTLa idiomorphs flanking regions we discovered that Z. sapae ABT301T displays an aααα genotype lacking the HMR silent cassette. Additionally, four putative HML cassettes were identified, two harbouring the ZsMTLα copy 1 and the remaining containing ZsMTLα copies 2 and 3. In conclusion, our results show that the mating-type switching is responsible for hyper-mutation in Z. rouxii complex. The ectopic recombination underlying this process is an error-prone mechanism, which represents a possible source of genetic variation providing yeast progeny with phenotypic variability and adaptation to hostile environments

Exploitation of an evolution strategy to select yeast strains improved in glutathione production.

Yeasts have been largely explored as cell factories to produce substances for food and industrial biotechnological applications. Among these chemicals, glutathione (GSH) is an important antioxidant molecule involved in several processes, including the control of redox potential, protection against oxidative stress, detoxification and transport of organic sulfur. Due to its functional roles, GSH is widely used in the pharmaceutical, food and cosmetic industries. Recently, GSH has received growing attention also in the winemaking field, to control oxidative spoilage damage; to limit the amount of browning pigments; to avoid the formation atypical aging characters; and to exert a protective effect on various aromatic compounds. At present GSH is successfully produced on an industrial scale through fermentation by high GSH-producing Saccharomyces cerevisiae strains, and several methodological tools have been reported for increasing efficiency and yield of the bioprocess. In this study, we have applied an evolution-based strategy that combines the sexual recombination of spores with the application of molybdate Mo(VI), a sulfate analogue toxic for the cells at high concentration, as specific selective pressure, to generate evolved S. cerevisiae strains with enhanced GSH production. To achieve this aim we used the 21T2 wine strain from the Unimore Microbial Culture Collection (UMCC) and we exploited its resistance to Mo(VI) as a rapid and high-throughput screening method for the selection of the evolved strains improved in GSH production. By this strategy, we obtained two evolved strains, Mo21T2-5 and Mo21T2-12, both able to enhance GSH content in wine with an increase of 100% and 36%, respectively, compared with the parental strain 21T2, and 120% and 50% compared with initial GSH content in the must. Our strategy, unlike the standard evolutionary approaches, has the advantage of not requiring multiple rounds of screening and extensive cultivation periods because the evolved strains are recognized through a selectable phenotype. The Mo(VI) resistance has proved to be effective for the selection of the desired evolved strains, probably by activating the yeast common metal response that involves sulfur assimilation and GSH biosynthesis