Corda Y, Lee SE, Guillot S, Walther A, Sollier J, Arbel-Eden A, Haber JE, Géli V
Mol Cell Biol. 2005 Dec;25(23):10652–64
RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and DNA damage checkpoint responses. Theirlethality can be suppressed by increasing the intracellular pool of deoxynucleotide triphosphates. We report that deletion of YKU70 or YKU80suppresses mec1Delta, but not rad53Delta, lethality. We show that suppression of mec1Delta lethality is not due to Ku--associated telomeric defects but rather results from the inability of Ku- cells to efficiently repair DNA double strand breaks by nonhomologous end joining. Consistent with these results, mec1Delta lethality is also suppressed by lif1Delta, which like yku70Delta and yku80Delta, prevents nonhomologous end joining. The viability of yku70Delta mec1Delta and yku80Delta mec1Delta cells depends on the ATM-related Tel1 kinase, the Mre11-Rad50-Xrs2 complex, and the DNA damage checkpoint protein Rad9. We further report that this Mec1-independent pathway converges with the Rad53/Dun1-regulated checkpoint kinase cascade and leads to the degradation of the ribonucleotide reductase inhibitor Sml1.
Chu Z, Li J, Eshaghi M, Karuturi RKM, Lin K, Liu J
BMC Genomics. 2007;8:323
BACKGROUND:
DNA polymerase gamma(Pol-gamma) has been shown to be essential for maintenance of the mitochondrial genome (mtDNA) in the petite-positive budding yeast Saccharomyces cerevisiae. Budding yeast cells lacking mitochondria exhibit a slow-growing or petite-colony phenotype. Petite strains fail to grow on non-fermentable carbon sources. However, it is not clear whether the Pol-gamma is required for mtDNA maintenance in the petite-negative fission yeast Schizosaccharomyces pombe.
RESULTS:
We show that disruption of the nuclear gene pog1+ that encodes Pol-gamma is sufficient to deplete mtDNA in S. pombe. Cells bearing pog1Delta allele require substantial growth periods to form petite colonies. Mitotracker assays indicate that pog1Delta cells are defective in mitochondrial function and EM analyses suggest that pog1Delta cells lack normal mitochondrial structures. Depletion of mtDNA in pog1Delta cells is evident from quantitative real-time PCR assays. Genome-wide expression profiles of pog1Delta and other mtDNA-less cells reveal that many genes involved in response to stimulus, energy derivation by oxidation of organic compounds, cellular carbohydrate metabolism, and energy reserve metabolism are induced. Conversely, many genes encoding proteins involved in amino acid metabolism and oxidative phosphorylation are repressed.
CONCLUSION:
By showing that Pol-gamma is essential for mtDNA maintenance and disruption of pog1+ alters the genome-wide expression profiles, we demonstrated that cells lacking mtDNA exhibit adaptive nuclear gene expression responses in the petite-negative S. pombe.
Xue C, Liu T, Chen L, Li W, Liu I, Kronstad JW, Seyfang A, Heitman J
MBio. 2010 May 18;1(1)
Cryptococcus neoformans and Cryptococcus gattii are globally distributed human fungal pathogens and the leading causes of fungal meningitis. Recent studies reveal that myo-inositol is an important factor for fungal sexual reproduction. That C. neoformans can utilize myo-inositol as a sole carbon source and the existence of abundant inositol in the human central nervous system suggest that inositol is important for Cryptococcus development and virulence. In accord with this central importance of inositol, an expanded myo-inositol transporter (ITR) gene family has been identified in Cryptococcus. This gene family contains two phylogenetically distinct groups, with a total of 10 or more members in C. neoformans and at least six members in the sibling species C. gattii. These inositol transporter genes are differentially expressed under inositol-inducing conditions based on quantitative real-time PCR analyses. Expression of ITR genes in a Saccharomyces cerevisiae itr1 itr2 mutant lacking inositol transport can complement the slow-growth phenotype of this strain, confirming that ITR genes are bona fide inositol transporters. Gene mutagenesis studies reveal that the Itr1 and Itr1A transporters are important for myo-inositol stimulation of mating and that functional redundancies among the myo-inositol transporters likely exist. Deletion of the inositol 1-phosphate synthase gene INO1 in an itr1 or itr1a mutant background compromised virulence in a murine inhalation model, indicating the importance of inositol sensing and acquisition for fungal infectivity. Our study provides a platform for further understanding the roles of inositol in fungal physiology and virulence.
Jaeger PA, McElfresh C, Wong LR, Ideker T.
Appl Environ Microbiol. 2015 Aug 15;81(16):5639-49. doi: 10.1128/AEM.01327-15. Epub 2015 Jun 12.
Agar, a seaweed extract, has been the standard support matrix for microbial experiments for over a century. Recent developments in high-throughput genetic screens have created a need to reevaluate the suitability of agar for use as colony support, as modern robotic printing systems now routinely spot thousands of colonies within the area of a single microtiter plate. Identifying optimal biophysical, biochemical, and biological properties of the gel support matrix in these extreme experimental conditions is instrumental to achieving the best possible reproducibility and sensitivity. Here we systematically evaluate a range of gelling agents by using the yeast Saccharomyces cerevisiae as a model microbe. We find that carrageenan and Phytagel have superior optical clarity and reduced autofluorescence, crucial for high-resolution imaging and fluorescent reporter screens. Nutrient choice and use of refined Noble agar or pure agarose reduce the effective dose of numerous selective drugs by >50%, potentially enabling large cost savings in genetic screens. Using thousands of mutant yeast strains to compare colony growth between substrates, we found no evidence of significant growth or nutrient biases between gel substrates, indicating that researchers could freely pick and choose the optimal gel for their respective application and experimental condition.
Pozo MI, Herrera CM, Lachance MA, Verstrepen K, Lievens B, Jacquemyn H.
Environ Microbiol. 2015 Sep 3. doi: 10.1111/1462-2920.13037. [Epub ahead of print]
Identifying the ecological processes that underlie the distribution and abundance of species in microbial communities is a central issue in microbial ecology and evolution. Classical trade-off based niche theories of resource competition predict that co-occurrence in microbial communities is more likely when the residing species show trait divergence and complementary resource use. We tested the prediction that niche differentiation explained the co-occurrence of two yeast species (Metschnikowia reukaufii and M. gruessii) in floral nectar. Assessment of the phenotypic landscape showed that both species displayed a significantly different physiological profile. Comparison of utilization profiles in single versus mixed cultures indicated that these two species did not compete for most carbon and nitrogen sources. In mixed cultures, M. reukaufii grew better in sucrose solutions and in the presence of the antimicrobial compound digitonin than when grown as pure culture. M. gruessii, on the other hand, grew better in mixed cultures in glucose and fructose solutions. Overall, these results provide clear evidence that M. reukaufii and M. gruessii frequently co-occur in nectar and that they differ in their phenotypic response to variation in environmental conditions, suggesting that niche differentiation and resource partitioning are important mechanisms contributing to species co-occurrence in nectar yeast communities.
Cairns TC1, Sidhu YS1, Chaudhari YK1, Talbot NJ1, Studholme DJ1, Haynes K2.
Fungal Genet Biol. 2015 Jun;79:110-7. doi: 10.1016/j.fgb.2015.04.013.
Targeted gene deletion has been instrumental in elucidating many aspects of Zymoseptoria tritici pathogenicity. Gene over-expression is a complementary approach that is amenable to rapid strain construction and high-throughput screening, which has not been exploited to analyze Z. tritici, largely due to a lack of available techniques. Here we exploit the Gateway® cloning technology for rapid construction of over-expression vectors and improved homologous integration efficiency of a Z. tritici Δku70 strain to build a pilot over-expression library encompassing 32 genes encoding putative DNA binding proteins, GTPases or kinases. We developed a protocol using a Rotor-HDA robot for rapid and reproducible cell pinning for high-throughput in vitro screening. This screen identified an over-expression strain that demonstrated a marked reduction in hyphal production relative to the isogenic progenitor. This study provides a protocol for rapid generation of Z. tritici over-expression libraries and a technique for functional genomic screening in this important pathogen.
Mertens S, Steensels J, Saels V, De Rouck G, Aerts G, Verstrepen KJ.
Appl Environ Microbiol. 2015 Dec;81(23):8202-14. doi: 10.1128/AEM.02464-15. Epub 2015 Sep 25.
Lager beer is the most consumed alcoholic beverage in the world. Its production process is marked by a fermentation conducted at low (8 to 15°C) temperatures and by the use of Saccharomyces pastorianus, an interspecific hybrid between Saccharomyces cerevisiae and the cold-tolerant Saccharomyces eubayanus. Recent whole-genome-sequencing efforts revealed that the currently available lager yeasts belong to one of only two archetypes, "Saaz" and "Frohberg." This limited genetic variation likely reflects that all lager yeasts descend from only two separate interspecific hybridization events, which may also explain the relatively limited aromatic diversity between the available lager beer yeasts compared to, for example, wine and ale beer yeasts. In this study, 31 novel interspecific yeast hybrids were developed, resulting from large-scale robot-assisted selection and breeding between carefully selected strains of S. cerevisiae (six strains) and S. eubayanus (two strains). Interestingly, many of the resulting hybrids showed a broader temperature tolerance than their parental strains and reference S. pastorianus yeasts. Moreover, they combined a high fermentation capacity with a desirable aroma profile in laboratory-scale lager beer fermentations, thereby successfully enriching the currently available lager yeast biodiversity. Pilot-scale trials further confirmed the industrial potential of these hybrids and identified one strain, hybrid H29, which combines a fast fermentation, high attenuation, and the production of a complex, desirable fruity aroma.
Esther Meersmana, Jan Steenselsa, Tinneke Paulusa, Nore Struyfa, Veerle Saelsa, Melissa Mathawanc, Leen Allegaertc, Gino Vranckenc and Kevin J. Verstrepen
Accepted manuscript posted online 6 July 2015, doi: 10.1128/AEM.00133-15
Cocoa pulp fermentation is a spontaneous process during which the natural microbiota present at the cocoa farms is allowed to ferment the pulp surrounding the cocoa beans. Because such spontaneous fermentations are inconsistent and contribute to product variability, there is a growing interest in a microbial starter culture that could be used to inoculate cocoa pulp fermentations. Previous studies have revealed that many different fungi are recovered from different batches of spontaneous cocoa pulp fermentations, whereas the variation in the prokaryotic microbiome is much more limited. In this study, we therefore aimed to develop a suitable yeast starter culture that is able to outcompete wild contaminants and consistently produces high-quality chocolate. Starting from specifically selected Saccharomyces cerevisiae strains, we developed robust hybrids with characteristics that allow them to efficiently ferment cocoa pulp, including improved temperature tolerance and fermentation capacity. We conducted several laboratory and field trials to show that these new hybrids often outperform their parental strains and are able to dominate spontaneous pilot-scale fermentations, which results in much more consistent microbial profiles. Moreover, analysis of the resulting chocolate shows that some of the cocoa batches that were fermented with specific starter cultures yielded superior chocolate. Taken together, these results describe the development of robust yeast starter cultures for cocoa pulp fermentations that can contribute to improving the consistency and quality of commercial chocolate production.
Gabriele Romagnoli, Theo A. Knijnenburg, Gianni Liti, Edward J. Louis, Jack T. Pronk, Jean-Marc Daran
13 May 2014, DOI: 10.1002/yea.3015
Phenylethanol has a characteristic rose-like aroma that makes it a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbour an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source, such as glucose, provides an economically attractive alternative for phenylalanine bioconversion. In this study, synthetic genetic array (SGA) screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to underlie the transcriptional upregulation of ARO10 during growth, with ammonium sulphate as the sole nitrogen source. Physiological characterization revealed that the aro8Δ mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8Δ mutation also stimulated phenylethanol production when combined with other, previously documented, mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced >3 mm phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products.
Snoek T1, Picca Nicolino M1, Van den Bremt S2, Mertens S1, Saels V1, Verplaetse A2, Steensels J1, Verstrepen KJ1.
Biotechnol Biofuels. 2015 Feb 26;8:32. doi: 10.1186/s13068-015-0216-0. eCollection 2015.
During the final phases of bioethanol fermentation, yeast cells face high ethanol concentrations. This stress results in slower or arrested fermentations and limits ethanol production. Novel Saccharomyces cerevisiae strains with superior ethanol tolerance may therefore allow increased yield and efficiency. Genome shuffling has emerged as a powerful approach to rapidly enhance complex traits including ethanol tolerance, yet previous efforts have mostly relied on a mutagenized pool of a single strain, which can potentially limit the effectiveness. Here, we explore novel robot-assisted strategies that allow to shuffle the genomes of multiple parental yeasts on an unprecedented scale.
RESULTS:
Screening of 318 different yeasts for ethanol accumulation, sporulation efficiency, and genetic relatedness yielded eight heterothallic strains that served as parents for genome shuffling. In a first approach, the parental strains were subjected to multiple consecutive rounds of random genome shuffling with different selection methods, yielding several hybrids that showed increased ethanol tolerance. Interestingly, on average, hybrids from the first generation (F1) showed higher ethanol production than hybrids from the third generation (F3). In a second approach, we applied several successive rounds of robot-assisted targeted genome shuffling, yielding more than 3,000 targeted crosses. Hybrids selected for ethanol tolerance showed increased ethanol tolerance and production as compared to unselected hybrids, and F1 hybrids were on average superior to F3 hybrids. In total, 135 individual F1 and F3 hybrids were tested in small-scale very high gravity fermentations. Eight hybrids demonstrated superior fermentation performance over the commercial biofuel strain Ethanol Red, showing a 2 to 7% increase in maximal ethanol accumulation. In an 8-l pilot-scale test, the best-performing hybrid fermented medium containing 32% (w/v) glucose to dryness, yielding 18.7% (v/v) ethanol with a productivity of 0.90 g ethanol/l/h and a yield of 0.45 g ethanol/g glucose.
CONCLUSIONS:
We report the use of several different large-scale genome shuffling strategies to obtain novel hybrids with increased ethanol tolerance and fermentation capacity. Several of the novel hybrids show best-parent heterosis and outperform the commonly used bioethanol strain Ethanol Red, making them interesting candidate strains for industrial production.