Haploid Strains Research Articles

The Frequency of Survivorship in Heterozygous Diploids of Cdc13-1exo1Δ Mutants of S. <i>cerevisiae</i> Is One Survivor Cell in 72 Cells/Generation at 36°C

Telomeres cap ends of eukaryotic chromosomes prevent them from degradation and ensure genomic stability. Cdc13 is an essential telomere recruitment and maintenance protein. A temperature-sensitive point mutation in cdc13 gene leads to telomere impairment, giving rise to cdc13-1 mutants that suffer lethality at enhanced temperatures. Deleting Exo1 gene from these mutants, however, leads to the emergence of temperature-tolerant mutants called survivors. Yeasts are known to exist as either diploids or haploids. These yeast genotypes generate survivors. The frequency of survivorship in the haploid genotype is one cell in 104 cells/generation at 36°C, however, the frequency at which they emerge in their diploid counterparts at the same temperature is not known. In this study, we investigated the frequency of Survivorship in heterozygous diploids of cdc13-1exo1Δ mutants of S. cerevisiae at 36°C. Diploids were constructed by mating haploid strains of opposite mating type cdc13-1 exo1:LEU strains with strains of cdc13-1 exo1:HIS. The crosses were 1296 × 3181, 2561 × 3182, 1296 × 3182 and 2561 × 3181. Genetic markers and phenotypic appearance were considered while mating the mutant cells. Using a stick, a smear of one haploid strain was made on each YEPD plate labelled C2, C8, C9, D1, D14, and D15. A smear of another opposite mating type was made on the previous strain. They were mixed and allowed to mate overnight, before culturing on media lacking Luecine and Histidine (-L and -H). Survivors were generated by culturing these diploids at 36°C. Using SPSS 20.0 software for windows SPSS, 2011, the frequency was determined as one Survivor cell in 72 cells/generation, as their frequency of survivorship averaged 5.9 × 10-5 ± 0.04.

Isolation and characterization of Zygosaccharomyces sp. yeast strains from miso.

There is currently great interest in the salt-tolerant yeast strains used to produce miso and soy sauce. Since the isolation of Zygosaccharomyces sp. strain from Japanese miso more than 60 years, several hybrid strains have been identified in fermented foods. Studies have shown that the active mating-type locus of the original Zygosaccharomyces sp. yeast strain is located between the T-subgenome sequence and the P-subgenome sequence. In this study, 32 salt-tolerant Zygosaccharomyces sp. yeast strains were isolated from five miso factories in Hiroshima Prefecture, Japan. Analysis by flow cytometry revealed that 27 strains were diploid and five strains were haploid. PCR analysis indicated that the 27 diploid strains had the same chromosomal structure of the active mating-type (MAT) locus as the original yeast strain isolated from miso 60 years ago. In addition, the 27 diploid strains were allodiploid, namely, natural hybrids of Z. rouxii and a related species, while the five haploid strains were all Z. rouxii. We found that cells of yeast strains isolated from miso changed morphologically when co-cultured with a yeast strain of opposite mating-type under nitrogen starvation conditions. The DNA sequence of the active mating-type locus and the results of cell morphology changes by co-culture were consistent with the mating type of each strain shown in the mating experiments. These findings will be useful for the future production of miso and soy sauce.

PLA2A gene from Arabidopsis thaliana in response to infection by Ustilago maydis

In this work, U. maydis-Arabidopsis pathosystem was used to evaluate the PLP2A Arabidopsis gene roll in response to U. maydis infection. For this, both wild-type and pla2a mutant plants were inoculated with a haploid strain of U. maydis, and the progress of the infection was followed, results show that transcript plays an important role in the establishment of the infection being evident in the mutant plant compared to wild-type plants. On the other hand, the orthologous gene was found in corn, the natural host of U. maydis, with 69% identity to the AtPLA2A gene. The PLP2A gene expression was analyzed by RT-PCR assays and the results showed that this gene is induced from the first day after inoculation, the transcript increases as the infection progresses; this supports the theory that the transcript AtPLA2A is involved in the mechanisms of resistance and susceptibility to the pathogen. The use of Arabidopsis has made it possible to understand the defense responses in the plant and the pathogenic process of U. maydis, therefore, the results obtained can clarify the role of PLA2A in a comparative and integrative way in host and non-host plants of U. maydis.

Farnesol secretion as a possible driving force for maintaining Candida albicans as a diploid

Candida albicans is a pathogenic dimorphic fungus which is invariably found as a diploid in patients. C. albicans secretes the sesquiterpene farnesol both as a quorum sensing molecule which blocks the yeast to hypha conversion and as a virulence factor for pathogenicity. 20-25 μM farnesol kills other competing yeasts and fungi, often by triggering apoptosis, and yet wild type diploid C. albicans tolerates 300-500 μM farnesol. The recent availability of 10 haploid strains of C. albicans (5 mating type aand 5 mating type α) allowed us to compare their production of and sensitivity to farnesol. On average, the heterozygous diploid strains of C. albicans were 2.4 times more resistant to 20-40 μM farnesol than MTLa haploid cells and 4.6 times more resistant than MTLα haploid cells. Furthermore, the MTLa haploids produce approximately 10 times more farnesol than do the MTLα haploids. Prior work concluded that haploid strains exhibited such low fitness that C. albicans was thought to be an obligate diploid. We now suggest that increased farnesol secretion by the MTLa haploids and increased farnesol sensitivity of the MTLα haploids is a mechanism for maintaining the dominant heterozygous diploid status of C. albicans. This idea is based on the observation that the a-factor peptide pheromone is farnesylated but the α-factor pheromone is not farnesylated. Our working hypothesis is that farnesol is secreted in part via Ste6 and imported in part via Ste3, the proteins which export and import the farnesylated a-pheromone. We also examined whether farnesol was excreted in extracellular vesicles.

Fur4-mediated uracil-scavenging to screen for surface protein regulators.

Cell surface membrane proteins perform diverse and critical functions and are spatially and temporally regulated by membrane trafficking pathways. Although perturbations in these pathways underlie many pathologies, our understanding of these pathways at a mechanistic level remains incomplete. Using yeast as a model, we have developed an assay that reports on the surface activity of the uracil permease Fur4 in uracil auxotroph strains grown in the presence of limited uracil. This assay was used to screen a library of haploid deletion strains and identified mutants with both diminished and enhanced comparative growth in restricted uracil media. Factors identified, including various multisubunit complexes, were enriched for membrane trafficking and transcriptional functions, in addition to various uncharacterized genes. Bioinformatic analysis of expression profiles from many strains lacking transcription factors required for efficient uracil‐scavenging validated particular hits from the screen, in addition to implicating essential genes not tested in the screen. Finally, we performed a secondary mating factor secretion screen to functionally categorize factors implicated in uracil‐scavenging.

Recombinant Yarrowia lipolytica strains for the heterologous expression of multi-component enzyme systems: Expression of mammalian steroidogenic proteins

New Yarrowia lipolytica strains for the co-expression of steroidogenic mammalian proteins were obtained in this study. For this purpose, a two-step approach for constructing recombinant strains that permits the simple introduction of several expression cassettes encoding heterologous proteins into the yeast genome was successfully applied. This study tested two series of integrative multi-copy expression vectors containing cDNAs for the mature forms of P450scc system components (cytochrome P450scc (CYP11A1), adrenodoxin reductase, adrenodoxin, or fused adrenodoxin-P450scc) or for P45017α (CYP17A1) under the control of the isocitrate lyase promoter pICL1, which were constructed using the basic plasmids p64PT or p67PT (rDNA or the long terminal repeat (LTR) zeta of Ylt1 as integration targeting sequences and ura3d4 as a multi-copy selection marker). This study demonstrated the integration of up to three expression vectors containing different heterologous cDNA via their simultaneous transformation into haploid recipient strains. Additionally, further combinations of the different expression cassettes in one strain were obtained by subsequent diploidisation using selected haploid multi-copy transformants. Thus, recombinant strains containing three to five different expression cassettes were obtained, as demonstrated by Southern blotting. Expression of P450scc system proteins was identified by western blotting. The presented method for recombinant strain construction is a useful tool for the heterologous expression of multi-component enzyme systems in Y. lipolytica.

Mutagenicity in haploid yeast meiosis resulting from repair of DSBs by the sister chromatid.

Mutations in diploid budding yeast occur in meiosis at higher frequencies than in cells grown vegetatively. Such meiotic mutations are thought to result from the repair of double-strand breaks (DSBs) in meiosis, during the process of recombination. Here, we report studies of mutagenicity in haploid strains that may undergo meiosis due to the expression of both mating-type alleles, MATa and MATα. We measure the rate of mutagenicity in the reporter gene CAN1, and find it to be fivefold higher than in mitotic cells, as determined by fluctuation analysis. This enhanced meiotic mutagenicity is shown to depend on the presence of SPO11, the gene responsible for meiotic DSBs. Mutations in haploid meiosis must result from repair of the DSBs through interaction with the sister chromatid, rather than with non-sister chromatids as in diploids. Thus, mutations in diploid meiosis that are not ostensibly associated with recombination events can be explained by sister-chromatid repair. The spectrum of meiotic mutations revealed by Sanger sequencing is similar in haploid and in diploid meiosis. Compared to mitotic mutations in CAN1, long Indels are more frequent among meiotic mutations. Both, meiotic and mitotic mutations are more common at G/C sites than at A/T, in spite of an opposite bias in the target reporter gene. We conclude that sister-chromatid repair of DSBs is a major source of mutagenicity in meiosis.

Role for nitrate assimilatory genes in virulence of Ustilago maydis

Ustilago maydis can utilize nitrate as a sole source of nitrogen. This process is initiated by transporting nitrate from the extracellular environment into the cell by a nitrate transporter and followed by a two-step reduction of nitrate to ammonium via nitrate reductase and nitrite reductase enzymes, respectively. Here, we characterize the genes encoding nitrate transporter, um03849 and nitrite reductase, um03848 in U. maydis based on their roles in mating and virulence. The deletion mutants for um03848, um03849 or both genes were constructed in mating compatible haploid strains 1/2 and 2/9. In addition, CRISPR-Cas9 gene editing technique was used for um03849 gene to create INDEL mutations in U. maydis mating strains. For all the mutants, phenotypes such as growth ability, mating efficiency and pathogenesis were examined. The growth of all the mutants was diminished when grown in a medium with nitrate as the source of nitrogen. Although no clear effects on haploid filamentation or mating were observed for either single mutant, double Δum03848 Δum03849 mutants showed reduction in mating, but increased filamentation on low ammonium, particularly in the 1/2 background. With respect to pathogenesis on the host, all the mutants showed reduced degrees of disease symptoms. Further, when the deletion mutants were paired with wild type of opposite mating-type, reduced virulence was observed, in a manner specific to the genetic background of the mutant's progenitor. This background specific reduction of plant pathogenicity was correlated with differential expression of genes for the mating program in U. maydis.

A 20-kb lineage-specific genomic region tames virulence in pathogenic amphidiploid Verticillium longisporum.

Amphidiploid fungal Verticillium longisporum strains Vl43 and Vl32 colonize the plant host Brassica napus but differ in their ability to cause disease symptoms. These strains represent two V. longisporum lineages derived from different hybridization events of haploid parental Verticillium strains. Vl32 and Vl43 carry same‐sex mating‐type genes derived from both parental lineages. Vl32 and Vl43 similarly colonize and penetrate plant roots, but asymptomatic Vl32 proliferation in planta is lower than virulent Vl43. The highly conserved Vl43 and Vl32 genomes include less than 1% unique genes, and the karyotypes of 15 or 16 chromosomes display changed genetic synteny due to substantial genomic reshuffling. A 20 kb Vl43 lineage‐specific (LS) region apparently originating from the Verticillium dahliae‐related ancestor is specific for symptomatic Vl43 and encodes seven genes, including two putative transcription factors. Either partial or complete deletion of this LS region in Vl43 did not reduce virulence but led to induction of even more severe disease symptoms in rapeseed. This suggests that the LS insertion in the genome of symptomatic V. longisporum Vl43 mediates virulence‐reducing functions, limits damage on the host plant, and therefore tames Vl43 from being even more virulent.

Effects of pyruvate decarboxylase (pdc1, pdc5) gene knockout on the production of metabolites in two haploid Saccharomyces cerevisiae strains

Saccharomyces cerevisiae has good reproductive ability in both haploid and diploid forms, a pyruvate decarboxylase plays an important role in S. cerevisiae cell metabolism. In this study, pdc1 and pdc5 double knockout strains of S. cerevisiae H14-02 (MATa type) and S. cerevisiae H5-02 (MATα type) were obtained by the Cre/loxP technique. The effects of the deletion of pdc1 and pdc5 on the metabolites of the two haploid S. cerevisiae strains were consistent. In S. cerevisiae H14-02, the ethanol conversion decreased by 30.19%, the conversion of glycerol increased by 40.005%, the concentration of acetic acid decreased by 43.54%, the concentration of acetoin increased by 12.79 times, and the activity of pyruvate decarboxylase decreased by 40.91% compared to those in the original H14 strain. The original S. cerevisiae haploid strain H14 produced a small amount of acetoin but produced very little 2,3-butanediol. However, S. cerevisiae H14-02 produced 1.420 ± 0.063 g/L 2,3-BD. This study not only provides strain selection for obtaining haploid strains with a high yield of 2,3-BD but also lays a foundation for haploid S. cerevisiae to be used as a new tool for genetic research and breeding programs.

Engineered Polyploid Yeast Strains Enable Efficient Xylose Utilization and Ethanol Production in Corn Hydrolysates.

The reported haploid Saccharomyces cerevisiae strain F106 can utilize xylose for ethanol production. After a series of XR and/or XDH mutations were introduced into F106, the XR-K270R mutant was found to outperform others. The corresponding haploid, diploid, and triploid strains were then constructed and their fermentation performance was compared. Strains F106-KR and the diploid produced an ethanol yield of 0.45 and 0.48 g/g total sugars, respectively, in simulated corn hydrolysates within 36 h. Using non-detoxicated corncob hydrolysate as the substrate, the ethanol yield with the triploid was approximately sevenfold than that of the diploid at 40°C. After a comprehensive evaluation of growth on corn stover hydrolysates pretreated with diluted acid or alkali and different substrate concentrations, ethanol yields of the triploid strain were consistently higher than those of the diploid using acid-pretreatment. These results demonstrate that the yeast chromosomal copy number is positively correlated with increased ethanol production under our experimental conditions.

Postzygotic reproductive isolation among three Saccharomyces yeast species.

We have previously isolated heterothallic haploid strains from original homothallic diploids of two yeast species in the family Saccharomycetaceae. In this study, heterothallic haploid strains were isolated from an original homothallic diploid of Saccharomyces kudriavzevii type strain, followed by investigation of sexual interactions among these yeast strains, in addition to S. cerevisiae laboratory strains. It has been shown that prezygotic reproductive isolation was observed between Kazachstania naganishii and S. cerevisiae with α-factor mating pheromones representing crossaction with each other beyond the genus boundary. Using heterothallic strains, postzygotic reproductive isolation system was shown to reside in the genus Saccharomyces by mass mating and cell-cell contact experiments. In mass mating experiments, crossaction of α-factor and a-factor mating pheromones and sexual agglutination effectively occurred beyond species boundaries among S. kudriavzevii, S. paradoxus, and S. cerevisiae. When the fates of cell-cell pairs from these Saccharomyces yeast species were systematically chased one by one, interspecific F1 hybrids were effectively produced, while sporulations were partially prohibited, with spore germination perfectly blocked in the hybrids. These results indicated that postzygotic reproductive isolation definitively resides among these Saccharomyces yeast species and that disorder of chromosome organization had to some extent occurred in interspecific F1 hybrids.

Saccharomyces cerevisiae quiescent cells: cadmium resistance and adaptive response

The budding yeast Saccharomyces cerevisiae is a widely used model organism to investigate the changes occurring in the eukaryotic cell and to predict its possible ‘reaction’ to different environmental factors. Recently it was also shown that these microorganisms possess another advantageous ability: to ‘enter’ into quiescent state (G0). Yeast G0 cells have similar physiological characteristics to those of higher eukaryotes making them a better model for toxicology studies. As cadmium could affect severely human health, the main aim of the present study was to use Saccharomyces cerevisiae quiescent cells to investigate the resistance potential and corresponding adaptive response of eukaryotic cells to elevated cadmium concentrations. Both diploid and haploid yeast strains in logarithmic, quiescent and non-quiescent state were exposed to different concentrations of Cd(NO3)2. The half-maximal inhibitory concentration (IC50) for all tested cell types was 100 µmol/L Cd(NO3)2. The deleterious effects of cadmium on intracellular macromolecule structures were analyzed through evaluating the levels of accumulated reactive oxygen species (ROS) and carbonylated proteins. The highest ROS concentration was measured in logarithmic cells: up to 50% versus about 8% in G0 cells. Significant damage in protein molecules was observed in haploid cells where the protein carbonylation reached levels of 25 µmol/mg. Studying the adaptive response to elevated Cd2+ concentrations revealed that quiescent and non-quiescent cells respond with increased expression of key elements from the antioxidant defense system: reduced glutathione, superoxide dismutase (SOD) and catalase. Furthermore, an additional SOD izoenzyme was detected when diploid and haploid cell populations were exposed to Cd2+.

Provocation: all yeast cells are born equal, but some grow to be more equal than others

Yeast is central to the brewing process and as such deserves great care and attention to ensure it is fit-for-purpose. It is widely accepted that assessment based on simple viability testing is useful but far too blunt an instrument to give much useful information regarding the physiological state of the live yeast cells within the population. The need to address this issue has spawned a plethora of procedures collectively termed ‘vitality tests’. The concept of yeast vitality is nebulous, and this goes some way to explain why no single test has been universally adopted. There is, however, another major flaw in the underlying assumptions of the majority of vitality tests. This is, most are based on bulk samples which ignore heterogeneities within the population of cells being examined. The asymmetric mode of proliferation of Saccharomyces yeasts means that mother and daughter cells are always unequal. Individual cells have finite lifespans, old cells die and must be replaced with new fitter progeny. We are fortunate in that budding yeasts are widely used as a model cell for studies into ageing in higher eukaryotes and as a result there is a huge and ever-expanding body of literature devoted to this subject. This has shown that yeast populations comprise several distinct sub-populations which respond differently to external messengers. These messengers may be generated by one sub-set of the population and received and acted upon by another indicating a degree of cooperation designed to ensure survival of the whole. Death of individuals within populations can be as a result of the application of lethal external stresses but more usually is a programmed process in which targeted cells commit suicide in an expression of altruism for the greater good of the whole. On reflection, perhaps this is unexpected but perfectly logical behaviour for single celled organisms to adopt. Most yeast population studies have been performed using haploid laboratory strains cultivated under relatively defined conditions. The industrial scale model is, of course, far removed from this. Brewing yeast strains are very different, and the complexities of serial fermentations and associated yeast handling processes introduce added layers of complexity. Nevertheless, there is every reason to believe that similar population heterogeneities and cooperative behaviour exist within the context of brewing. The thrust of the arguments presented in this article is that we should embrace this and consider individual cells within yeast populations within a fermenter, for example, as being individual free-living components of a multicellular organism. Further, intercellular cooperation is managed by a network of messenger molecules many of which are likely to trigger the global shifts associated in metabolic activities which accompany the reversible passage from growth to quiescence and which coincidentally are likely to be of importance in beer flavour. The purpose of this article is to argue that the majority of current yeast vitality tests are not fit for their intended purpose. They do not provide outputs that are predictive of fermentation performance or an indication of the metabolic status of the population as a whole. Simple viability tests based on differential responses to biological stains, including methylene blue, are of equal value in commercial brewing. This situation can be improved by the introduction of tests which give information of how sub-populations develop and interact with each other. Probably dual staining techniques which probe membrane competence and detect cells in the early stages of programmed cell death, as discussed in this article, are promising candidates. © 2021 The Institute of Brewing & Distilling

Anther-Based Regeneration of African Violet (Saintpaulia ionantha).

African violet (Saintpaulia ionantha) is an herbaceous perennial of the Gesneriaceae family. Because almost all the cultivars are heterozygous, pure lines are useful for both classical and new breeding approaches. A shortcut to obtain purebred lines involves the production of doubled haploid strains produced from anther-derived haploids. In this chapter, a protocol for culturing African violet anthers is described in detail.

Polyploid engineering by increasing mutant gene dosage in yeasts.

The yeast Saccharomyces cerevisiae, widely used for ethanol production, is one of the best-understood biological systems. Diploid strains of S.cerevisiae are preferred for industrial use due to the better fermentation efficiency, in terms of vitality and endurance as compared to those of haploid strains. Whole-genome duplications is known to promote adaptive mutations in microorganisms, and allelic variations considerably contribute to the product composition in ethanol fermentation. Although fermentation can be regulated using various strains of yeast, it is quite difficult to make fine adjustment of each component in final products. In this study, we demonstrate the use of polyploids with varying gene dosage (the number of copies of a particular gene present in a genome) in the regulation of ethanol fermentation. Ethyl caproate is one of the major flavouring agents in a Japanese alcoholic beverage called sake. A point mutation in FAS2 encoding the α subunit of fatty acid synthetase induces an increase in the amount of caproic acid, a precursor of ethyl caproate. Using the FAS2 as a model, we generated and evaluated yeast strains with varying mutant gene dosage. We demonstrated the possibility to increase mutant gene dosage via loss of heterozygosity in diploid and tetraploid strains. Productivity of ethyl caproate gradually increased with mutant gene dosage among tetraploid strains. This approach can potentially be applied to a variety of yeast strain development via growth-based screening.

Different transcriptional responses of haploid and diploid S. cerevisiae strains to changes in cofactor preference of XR

BackgroundXylitol accumulation is a major barrier for efficient ethanol production through heterologous xylose reductase-xylitol dehydrogenase (XR-XDH) pathway in recombinant Saccharomyces cerevisiae. Mutated NADH-preferring XR is usually employed to alleviate xylitol accumulation. However, it remains unclear how mutated XR affects the metabolic network for xylose metabolism. In this study, haploid and diploid strains were employed to investigate the transcriptional responses to changes in cofactor preference of XR through RNA-seq analysis during xylose fermentation.ResultsFor the haploid strains, genes involved in xylose-assimilation (XYL1, XYL2, XKS1), glycolysis, and alcohol fermentation had higher transcript levels in response to mutated XR, which was consistent with the improved xylose consumption rate and ethanol yield. For the diploid strains, genes related to protein biosynthesis were upregulated while genes involved in glyoxylate shunt were downregulated in response to mutated XR, which might contribute to the improved yields of biomass and ethanol. When comparing the diploids with the haploids, genes involved in glycolysis and MAPK signaling pathway were significantly downregulated, while oxidative stress related transcription factors (TFs) were significantly upregulated, irrespective of the cofactor preference of XR.ConclusionsOur results not only revealed the differences in transcriptional responses of the diploid and haploid strains to mutated XR, but also provided underlying basis for better understanding the differences in xylose metabolism between the diploid and haploid strains.

Impact of stress-response related transcription factor overexpression on lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae environmental isolates.

Numerous transcription factor genes associated with stress response are upregulated in Saccharomyces cerevisiae grown in the presence of inhibitors that result from pretreatment processes to unlock simple sugars from biomass. To determine if overexpression of transcription factors could improve inhibitor tolerance in robust S. cerevisiae environmental isolates as has been demonstrated in S. cerevisiae haploid laboratory strains, transcription factors were overexpressed at three different expression levels in three S. cerevisiae environmental isolates. Overexpression of the YAP1 transcription factor in these isolates did not lead to increased growth rate or reduced lag in growth, and in some cases was detrimental, when grown in the presence of either lignocellulosic hydrolysates or furfural and 5-hydroxymethyl furfural individually. The expressed Yap1p localized correctly and the expression construct improved inhibitor tolerance of a laboratory strain as previously reported, indicating that lack of improvement in the environmental isolates was due to factors other than nonfunctional expression constructs or mis-folded protein. Additional stress-related transcription factors, MSN2, MSN4, HSF1, PDR1, and RPN4, were also overexpressed at three different expression levels and all failed to improve inhibitor tolerance. Transcription factor overexpression alone is unlikely to be a viable route toward increased inhibitor tolerance of robust environmental S. cerevisiae strains.

Genome editing in the yeast Nakaseomyces delphensis and description of its complete sexual cycle.

The environmental yeast Nakaseomyces delphensis is, phylogenetically, the closest known species to Candida glabrata, a major fungal pathogen of humans. C. glabrata is haploid and described as asexual, while N. delphensis is also haploid, but has been described as competent for mating and meiosis. Both genomes contain homologues of all the genes necessary for sexual reproduction and also the genes for Ho-dependent mating-type switching, like Saccharomyces cerevisiae. We first report the construction of genetically engineered strains of N. delphensis, including by CRISPR-Cas 9 gene editing. We also report the description of the sexual cycle of N. delphensis. We show that it undergoes Ho-dependent mating-type switching in culture and that deletion of the HO gene prevents such switching and allows maintenance of stable, separate, MATa and MATalpha haploid strains. Rare, genetically selected diploids can be obtained through mating of haploid strains, mutated or not for the HO gene. In contrast to HO/HO diploids, which behave as expected, Δho/Δho diploids exhibit unusual profiles in flow cytometry. Both types of diploids can produce recombined haploid cells, which grow like the original haploid-type strain. Our experiments thus allow the genetic manipulation of N. delphensis and the reconstruction, in the laboratory, of its entire life cycle.

Repetitive δ-integration of a cellulase-encoding gene into the chromosome of an industrial Angel Yeast-derived strain by URA3 recycling.

Genetic modification of industrial yeast strains often faces more difficulties than that of laboratory strains. Thus, new approaches are still required. In this research, the Angel Yeast-derived haploid strain Kα was genetically modified by multiple rounds of δ-integration, which was achieved via URA3 recycling. Three δ-integrative plasmids, pGδRU, pGδRU-BGL, and pGδRU-EG, were first constructed with two 167bp δ sequences and a repeat-URA3-repeat fragment. Then, the δ-integrative strains containing the bgl1 or egl2 gene were successfully obtained by one-time transformation of the linearized pGδRU-BGL or pGδRU-EG fragment, respectively. Their counterparts in which the URA3 gene was looped out were also easily isolated by selection for growth on 5´-fluoroorotic acid plates, although the ratio of colonies lacking URA3 to the total number of colonies decreased with increasing copy number of the corresponding integrated cellulase-encoding gene. Similar results were observed during the second round of δ-integration, in which the δ-integration strain Kα(δ::bgl1-repeat) obtained from the first round was transformed with a linearized pGδRU-EG fragment. After 10 rounds of cell growth and transfer to fresh medium, the doubling times and enzyme activities of Kα(δ::bgl1-repeat), Kα(δ::egl2-repeat), and Kα(δ::bgl1-repeat)(δ::egl2-repeat) showed no significant change and were stable. Further, their maximum ethanol concentrations during simultaneous saccharification and fermentation of pretreated corncob over a 7-day period were 46.35, 33.13, and 51.77g/L, respectively, which were all substantially higher than the parent Kα strain. Thus, repetitive δ-integration with URA3 recycling can be a feasible and valuable method for genetic engineering of Angel Yeast. These results also provide clues about some important issues related to δ-integration, such as the structural stability of δ-integrated genes and the effects of individual integration-site locations on gene expression. Further be elucidation of these issues should help to fully realize the potential of δ-integration-based methods in industrial yeast breeding.