Meiosis-Based Laboratory Evolution of the Thermal Tolerance in Kluyveromyces marxianus

Kluyveromyces marxianus is the fastest-growing eukaryote and a promising host for producing bioethanol and heterologous proteins. To perform a laboratory evolution of thermal tolerance in K. marxianus, diploid, triploid and tetraploid strains were constructed, respectively. Considering the genetic diversity caused by genetic recombination in meiosis, we established an iterative cycle of “diploid/polyploid - meiosis - selection of spores at high temperature” to screen thermotolerant strains. Results showed that the evolution of thermal tolerance in diploid strain was more efficient than that in triploid and tetraploid strains. The thermal tolerance of the progenies of diploid and triploid strains after a two-round screen was significantly improved than that after a one-round screen, while the thermal tolerance of the progenies after the one-round screen was better than that of the initial strain. After a two-round screen, the maximum tolerable temperature of Dip2-8, a progeny of diploid strain, was 3°C higher than that of the original strain. Whole-genome sequencing revealed nonsense mutations of PSR1 and PDE2 in the thermotolerant progenies. Deletion of either PSR1 or PDE2 in the original strain improved thermotolerance and two deletions displayed additive effects, suggesting PSR1 and PDE2 negatively regulated the thermotolerance of K. marxianus in parallel pathways. Therefore, the iterative cycle of “meiosis - spore screening” developed in this study provides an efficient way to perform the laboratory evolution of heat resistance in yeast.

Metabolic and evolutionary engineering of diploid yeast for the production of first-and second-generation ethanol

Background: Saccharomyces cerevisiae has been widely used in the fermentation of plant-derived sugars to produce ethanol, called first-generation (1G) bioethanol, but made an impact on global food markets. Significant efforts have been therefore to employ non-food lignocellulosic feedstocks for bioethanol production, known as second-generation (2G) bioethanol. However, S. cerevisiae cannot naturally utilize xylose, a major component in lignocellulosic hydrolysates, and it has low tolerance to common carboxylic acid inhibitors present in lignocellulosic hydrolyzates. Metabolic engineering and evolutionary engineering have shown great power in strain improvement, which were also adopted here to solve these limiting factors in developing 2G bioethanol.Results: An efficient expression of a six-gene cluster, including XYL1/XYL2/XKS1/TAL1/PYK1/MGT05196, was achieved in the evolved S. cerevisiae diploid strain A21Z, showing the ability to use mixed glucose and xylose. The engineered strain A21Z expressing the six-gene cluster displayed a high xylose consumption after 96 h, reaching 90.7% of the theoretical yield in ethanol production. To investigate its industrial characteristics, A31Z was obtained by direct evolution of A21Z under the treatment of industrial hydrolysate from wheat straw. Under different fermentation conditions with 1G and 2G feedstock candidates, A31Z showed a markedly improved xylose fermentation performance. A31Z could produce more ethanol and less glycerol compared to the control Angel from corn starch during 120 h, with a final ethanol production at 122.32 g/L. The ability to produce higher ethanol production was also found under the fermentation using carbon source from hydrolysis of Dried Distillers Grains with Solubles (DDGS) or whole corn.Conclusions: Here, we report an effective strategy to improve xylose fermentation with an evolutionary engineering in the industrial S. cerevisiae diploid strain A31Z. This study demonstrated that a constructed A31Z has the higher xylose consumption and efficient ethanol production in mixed glucose and xylose with acetate. A31Z also gave a good ethanol production in 1G and 2G industrial feedstocks, indicating its significant contribution in the transition stage from the 1st generation to the 2nd generation bioethanol.

A simple, cheap, and robust protocol for the identification of mating type in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an exceptional genetic system, with genetic crosses facilitated by its ability to be maintained in haploid and diploid forms. Such crosses are straightforward as long as the mating type and ploidy of the strains are known. Haploid S. cerevisiae cells are either MATa or MATα mating type. Several techniques can be used to determine mating type (or ploidy), but are typically time-consuming, require specialized components, and/or the results are inconsistent and transient. Here we validated a simple, cheap and robust method to enable rapid identification of S. cerevisiae mating types. When cells of opposite mating type are mixed in liquid media, they creep up culture vessel sides, a phenotype that can easily be detected visually. In contrast, mixtures of cells of the same mating type or with a diploid strain(s) simply settle out. The method does not require specialized equipment, and is robust to different media, cell densities, temperatures and strains. It can be performed in 96-well plates, and the phenotype is observable for several days. The simplicity and robustness of this method makes it ideal for routine verification of S. cerevisiae mating type, and it could be used to screen for genes underlying the creeping phenotype.

Whole-Genome Sequence of an Isogenic Haploid Strain, Saccharomyces cerevisiae IR-2idA30(MATa), Established from the Industrial Diploid Strain IR-2

We present the draft genome sequence of an isogenic haploid strain, IR-2idA30(MAT a), established from Saccharomyces cerevisiae IR-2. Assembly of long reads and previously obtained contigs from the genome of diploid IR-2 resulted in 50 contigs, and the variations and sequencing errors were corrected by short reads.

Draft Genome Sequence of a Highly Heterozygous Yeast Strain from the Metschnikowia pulcherrima Subclade, UCD127

ABSTRACT Metschnikowia strain UCD127 was isolated from soil in Ireland and sequenced. It is a highly heterozygous diploid strain with 385,000 single nucleotide polymorphisms (SNPs). Its ribosomal DNA has the highest similarity to that of M. chrysoperlae, but its ACT1 and TEF1 loci and mitochondrial genome show affinity to those of M. fructicola, whose genome is significantly larger.

Redesigning chromosomes for optimized Hi-C assay provides insights on loop formation and homologs pairing during meiosis

In all chromosome conformation capture based experiments the accuracy with which contacts are detected varies considerably because of the uneven distribution of restriction sites along genomes. In addition, repeated sequences as well as homologous, large identical regions remain invisible to the assay because of the ambiguities they introduce during the alignment of the sequencing reads along the genome. As a result, the investigation of homologs during meiosis prophase through 3C studies has been limited. Here, we redesigned and reassembled in yeast a 145kb region with regularly spaced restriction sites for various enzymes. Thanks to this Syn-3C design, we enhanced the signal to noise ratio and improved the visibility of the entire region. We also improved our understanding of Hi-C data and definition of resolution. The redesigned sequence is now distinguishable from its native homologous counterpart in an isogenic diploid strain. As a proof of principle, we track the establishment of homolog pairing during meiotic prophase in a synchronized population. This provides new insights on the individualization and pairing of homologs, as well as on their internal restructuration into arrays of loops during meiosis prophase. Overall, we show the interest of redesigned genomic regions to explore complex biological questions otherwise difficult to address.

Essential Gene Discovery in the Basidiomycete Cryptococcus neoformans for Antifungal Drug Target Prioritization

ABSTRACTFungal diseases represent a major burden to health care globally. As with other pathogenic microbes, there is a limited number of agents suitable for use in treating fungal diseases, and resistance to these agents can develop rapidly.Cryptococcus neoformansis a basidiomycete fungus that causes cryptococcosis worldwide in both immunocompromised and healthy individuals. As a basidiomycete, it diverged from other common pathogenic or model ascomycete fungi more than 500 million years ago. Here, we reportC. neoformansgenes that are essential for viability as identified through forward and reverse genetic approaches, using an engineered diploid strain and genetic segregation after meiosis. The forward genetic approach generated random insertional mutants in the diploid strain, the induction of meiosis and sporulation, and selection for haploid cells with counterselection of the insertion event. More than 2,500 mutants were analyzed, and transfer DNA (T-DNA) insertions in several genes required for viability were identified. The genes include those encoding the thioredoxin reductase (Trr1), a ribosome assembly factor (Rsa4), an mRNA-capping component (Cet1), and others. For targeted gene replacement, theC. neoformanshomologs of 35 genes required for viability in ascomycete fungi were disrupted, meiosis and sporulation were induced, and haploid progeny were evaluated for their ability to grow on selective media. Twenty-one (60%) were found to be required for viability inC. neoformans. These genes are involved in mitochondrial translation, ergosterol biosynthesis, and RNA-related functions. The heterozygous diploid mutants were evaluated for haploinsufficiency on a number of perturbing agents and drugs, revealing phenotypes due to the loss of one copy of an essential gene inC. neoformans. This study expands the knowledge of the essential genes in fungi using a basidiomycete as a model organism. Genes that have no mammalian homologs and are essential in bothCryptococcusand ascomycete human pathogens would be ideal for the development of antifungal drugs with broad-spectrum activity.IMPORTANCEFungal infections are very common in humans but may be neglected due to misdiagnosis and inattention.Cryptococcus neoformansis a yeast that infects mainly immunocompromised people, causing high mortality rates in developing countries. The fungus infects the lungs, crosses the blood-brain barrier, and invades the cerebrospinal fluid, causing fatal meningitis.C. neoformansinfections are treated with amphotericin B, flucytosine, and azoles, all developed decades ago. However, problems with antifungal agents highlight the urgent need for more-effective drugs to treatC. neoformansand other invasive fungal infections. These issues include the negative side effects of amphotericin B, the spontaneous resistance ofC. neoformansto azoles, and the inefficacy of the echinocandin antifungals. In this study, we report the identification ofC. neoformansessential genes as targets for the development of novel antifungals. Because of the level of evolutionary divergence betweenC. neoformansand the ascomycetes, a subset of these genes is likely essential in all fungi. Genes identified in this study represent an excellent starting point for the future development of new antifungals by pharmaceutical companies.

Cohesin Irr1/Scc3 is likely to influence transcription in Saccharomyces cerevisiae via interaction with Mediator complex.

The evolutionarily conserved proteins forming sister chromatid cohesion complex are also involved in the regulation of gene transcription. The participation of SA2p (mammalian ortholog of yeast Irr1p, associated with the core of the complex) in the regulation of transcription is already described. Here we analyzed microarray profiles of gene expression of a Saccharomyces cerevisiae irr1-1/IRR1 heterozygous diploid strain. We report that expression of 33 genes is affected by the presence of the mutated Irr1-1p and identify those genes. This supports the suggested role of Irr1p in the regulation of transcription. We also indicate that Irr1p may interact with elements of transcriptional coactivator Mediator.

Vegetative Compatibility Groups and Parasexual Segregation in Colletotrichum acutatum Isolates Infecting Different Hosts

Heterokaryosis is an important mechanism which provides genetic variability increase in filamentous fungi. In order to assess the diversity of vegetative compatibility reactions existing among Colletotrichum acutatum isolates derived from different hosts, complementary nit mutants of each isolate were obtained and paired in all possible combinations. Vegetative compatibility groups (VCG) were identified among the isolates according to their ability to form viable heterokaryons. Seven VCGs were identified among the isolates, one of which contained isolates from different hosts. VCGs 2 and 6 contained two and three members, respectively; VCG-3 contained four members, and four VCGs (1, 4, 5, and 7) contained a single one. This study shows, for the first time, the isolation and the parasexual segregation of a heterozygous diploid sector derived from the heterokaryon formed with nit mutants from VCG-6. Diploid, named DE-3, showed nit+ phenotype and growth rate similar to the parental wild isolate. When inoculated in the presence of the haploidizing agent benomyl, the diploid strain produced parasexual haploid segregants exhibiting the nit phenotypes of the crossed mutants. Since viable heterokaryons and diploid may be formed among vegetative compatible isolates of C. acutatum, this study suggests that the parasexual cycle may be an alternative source of genetic variability in C. acutatum isolates.