scholarly journals Overexpression of RAD51 Enables PCR-Based Gene Targeting in Lager Yeast

2019 ◽  
Vol 7 (7) ◽  
pp. 192 ◽  
Author(s):  
Beatrice Bernardi ◽  
Yeseren Kayacan ◽  
Madina Akan ◽  
Jürgen Wendland
Keyword(s):  
Gene Targeting ◽  
Gene Function ◽  
Genetic Alterations ◽  
Selection Marker ◽  
Lager Yeast ◽  
Diagnostic Pcr ◽  
Local Selection ◽  
Lager Beer ◽  
Low Efficiency ◽  
Saccharomyces Eubayanus

Lager beer fermentations rely on specific polyploid hybrids between Saccharomyces cerevisiae and Saccharomyces eubayanus falling into the two groups of S. carlsbergensis/Saaz-type and S. pastorianus/Frohberg-type. These strains provide a terroir to lager beer as they have long traditional associations and local selection histories with specific breweries. Lager yeasts share, based on their common origin, several phenotypes. One of them is low transformability, hampering the gene function analyses required for proof-of-concept strain improvements. PCR-based gene targeting is a standard tool for manipulating S. cerevisiae and other ascomycetes. However, low transformability paired with the low efficiency of homologous recombination practically disable targeted gene function analyses in lager yeast strains. For genetic manipulations in lager yeasts, we employed a yeast transformation protocol based on lithium-acetate/PEG incubation combined with electroporation. We first introduced freely replicating CEN/ARS plasmids carrying ScRAD51 driven by a strong heterologous promoter into lager yeast. RAD51 overexpression in the Weihenstephan 34/70 lager yeast was necessary and sufficient in our hands for gene targeting using short-flanking homology regions of 50 bp added to a selection marker by PCR. We successfully targeted two independent loci, ScADE2/YOR128C and ScHSP104/YLL026W, and confirmed correct integration by diagnostic PCR. With these modifications, genetic alterations of lager yeasts can be achieved efficiently and the RAD51-containing episomal plasmid can be removed after successful strain construction.

scholarly journals Hybridization of Saccharomyces cerevisiae Sourdough Strains with Cryotolerant Saccharomyces bayanus NBRC1948 as a Strategy to Increase Diversity of Strains Available for Lager Beer Fermentation

2020 ◽  
Author(s):  
Martina Catallo ◽  
Fabrizio Iattici ◽  
Cinzia Randazzo ◽  
Cinzia Caggia ◽  
Kristoffer Krogerus ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Yeast Culture ◽  
Culture Collections ◽  
Lager Yeast ◽  
Ethyl Hexanoate ◽  
Brewing Yeast ◽  
Saccharomyces Bayanus ◽  
Lager Beer ◽  
Saccharomyces Eubayanus

The search for novel brewing strains from non-brewing environments represents an emerging trend to increase genetic and phenotypic diversities in brewing yeast culture collections. Another valuable tool is hybridization, where beneficial traits of individual strains are combined in a single organism. This has been used successfully to create de novo hybrids from parental brewing strains by mimicking natural Saccharomyces cerevisiae ale x Saccharomyces eubayanus lager yeast hybrids. Here, we integrated both these approaches to create synthetic hybrids for lager fermentation using parental strains from niches other than beer. Using a phenotype-centered strategy, S. cerevisiae sourdough strains and the S. eubayanus x Saccharomyces uvarum strain NBRC1948 (also referred to as Saccharomyces bayanus) were chosen for their brewing aptitudes. We demonstrated that, in contrast to S. cerevisiae x S. uvarum crosses, hybridization yield was positively affected by time of exposure to starvation, but not by staggered mating. In laboratory-scale fermentation trials at 20°C, one triple S. cerevisiae x S. eubayanus x S. uvarum hybrid showed a heterotic phenotype compared with the parents. In 2L wort fermentation trials at 12°C, this hybrid inherited the ability to consume efficiently maltotriose from NBRC1948 and, like the sourdough S. cerevisiae parent, produced appreciable levels of the positive aroma compounds 3-methylbutyl acetate (banana/pear), ethyl acetate (general fruit aroma) and ethyl hexanoate (green apple, aniseed, and cherry aroma). Based on these evidences, the phenotype-centered approach appears promising for design of de novo lager beer hybrids and may help to diversify aroma profiles in lager beers.

scholarly journals A Mutated Cytosine Deaminase Gene, codA (D314A), as an Efficient Negative Selection Marker for Gene Targeting in Rice

2014 ◽  
Vol 55 (3) ◽  
pp. 658-665 ◽  
Author(s):  
Keishi Osakabe ◽  
Ayako Nishizawa-Yokoi ◽  
Namie Ohtsuki ◽  
Yuriko Osakabe ◽  
Seiichi Toki
Keyword(s):  
Gene Targeting ◽  
Negative Selection ◽  
Cytosine Deaminase ◽  
Selection Marker ◽  
Negative Selection Marker ◽  
Cytosine Deaminase Gene

scholarly journals An efficient and adaptable workflow for editing disease-relevant single nucleotide variants using CRISPR/Cas9

2021 ◽  
Author(s):  
Inga Usher ◽  
Lorena Ligammari ◽  
Sara Ahrabi ◽  
Emily Hepburn ◽  
Calum Connolly ◽  
...  
Keyword(s):  
Genetic Research ◽  
Bone Cancer ◽  
Illumina Miseq ◽  
Genetic Alterations ◽  
Single Nucleotide Variants ◽  
Experimental Conditions ◽  
Single Nucleotide ◽  
Droplet Pcr ◽  
Low Efficiency

Single nucleotide variants are the commonest genetic alterations in the human genome. At least 60,000 have been reported to be associated with disease. The CRISPR/Cas9 system has transformed genetic research, making it possible to edit single nucleotides and study the function of genetic variants in vitro. While significant advances have improved the efficiency of CRISPR/Cas9, the editing of single nucleotides remains challenging. There are two major obstacles: low efficiency of accurate editing and the isolation of these cells from a pool of cells with other editing outcomes. We present data from 85 transfections of induced pluripotent stem cells and an immortalised cell line, comparing the effects of altering CRISPR/Cas9 design and experimental conditions on rates of single nucleotide substitution. We targeted variants in TP53, which predispose to several cancers, and in TBXT which is implicated in the pathogenesis of the bone cancer, chordoma. We describe a scalable and adaptable workflow for single nucleotide editing that incorporates contemporary techniques including Illumina MiSeq sequencing, TaqMan qPCR and digital droplet PCR for screening transfected cells as well as quality control steps to mitigate against common pitfalls. This workflow can be applied to CRISPR/Cas9 and other genome editing systems to maximise experimental efficiency.

scholarly journals Deletion of the DNA Ligase IV Gene in Candida glabrata Significantly Increases Gene-Targeting Efficiency

2015 ◽  
Vol 14 (8) ◽  
pp. 783-791 ◽  
Author(s):  
Yuke Cen ◽  
Alessandro Fiori ◽  
Patrick Van Dijck
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Candida Glabrata ◽  
The United States ◽  
Genetic Alterations ◽  
Nonhomologous End Joining ◽  
Specific Gene ◽  
Phylogenetic Distance ◽  
End Joining ◽  
Content Type

ABSTRACTCandida glabratais reported as the second most prevalent human opportunistic fungal pathogen in the United States. Over the last decades, its incidence increased, whereas that ofCandida albicansdecreased slightly. One of the main reasons for this shift is attributed to the inherent tolerance ofC. glabratatoward the commonly used azole antifungal drugs. Despite a close phylogenetic distance toSaccharomyces cerevisiae, homologous recombination works with poor efficiency inC. glabratacompared to baker's yeast, in fact limiting targeted genetic alterations of the pathogen's genome. It has been shown that nonhomologous DNA end joining is dominant over specific gene targeting inC. glabrata. To improve the homologous recombination efficiency, we have generated a strain in which theLIG4gene has been deleted, which resulted in a significant increase in correct gene targeting. The very specific function of Lig4 in mediating nonhomologous end joining is the reason for the absence of clear side effects, some of which affect theku80mutant, another mutant with reduced nonhomologous end joining. We also generated aLIG4reintegration cassette. Our results show that thelig4mutant strain may be a valuable tool for theC. glabrataresearch community.

scholarly journals Measuring Food Anticipation in Mice

2018 ◽  
Vol 1 (1) ◽  
pp. 65-74
Author(s):  
Tomaz Martini ◽  
Jürgen Ripperger ◽  
Urs Albrecht
Keyword(s):  
Body Temperature ◽  
Gene Function ◽  
Metabolic Disorders ◽  
Genetic Alterations ◽  
Circadian System ◽  
Physiological Adaptation ◽  
Pharmacological Intervention ◽  
Feeding Time ◽  
Evolutionary Advantage ◽  
Internal Body

The interplay between the circadian system and metabolism may give animals an evolutionary advantage by allowing them to anticipate food availability at specific times of the day. Physiological adaptation to feeding time allows investigation of animal parameters and comparison of food anticipation between groups of animals with genetic alterations and/or post pharmacological intervention. Such an approach is vital for understanding gene function and mechanisms underlying the temporal patterns of both food anticipation and feeding. Exploring these mechanisms will allow better understanding of metabolic disorders and might reveal potential new targets for pharmacological intervention. Changes that can be easily monitored and that represent food anticipation on the level of the whole organism are a temporarily restricted increase of activity and internal body temperature.

scholarly journals A cucumber green mottle mosaic virus vector for virus-induced gene silencing in cucurbit plants

2019 ◽  
Author(s):  
Mei Liu ◽  
Zhiling Liang ◽  
Miguel A. Aranda ◽  
Ni Hong ◽  
Liming Liu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Gene Silencing ◽  
Mosaic Virus ◽  
Gene Function ◽  
Nicotiana Benthamiana ◽  
Phytoene Desaturase ◽  
Virus Induced Gene Silencing ◽  
Economic Significance ◽  
Potential Alternative ◽  
Low Efficiency

AbstractCucurbits produce fruits or vegetables that have great dietary importance and economic significance worldwide. The published genomes of at least 11 cucurbit species are boosting gene mining and novel breeding strategies, however genetic transformation in cucurbits is impractical as a tool for gene function validation due to low transformation efficiencies. Virus-induced gene silencing (VIGS) is a potential alternative tool. So far, very few ideal VIGS vectors are available for cucurbits. Here, we describe a new VIGS vector derived from cucumber green mottle mosaic virus (CGMMV), a monopartite virus that infects cucurbits naturally. We show that the CGMMV vector is competent to induce efficient silencing of the phytoene desaturase (PDS) gene in the model plant Nicotiana benthamiana and in cucurbits, including watermelon, melon, cucumber and bottle gourd. Infection with the CGMMV vector harboring PDS sequences of 69-300 bp in length in the form of sense-oriented or hairpin cDNAs resulted in photobleaching phenotypes in N. benthamiana and cucurbits by PDS silencing. Additional results reflect that silencing of the PDS gene could persist for over two months and the silencing effect of CGMMV-based vectors could be passaged. These results demonstrate that CGMMV vector could serve as a powerful and easy-to-use tool for characterizing gene function in cucurbits.One sentence summaryA CGMMV-based vector enables gene function studies in cucurbits, an extremely low efficiency species for genetic transformation.

scholarly journals Creation and Characterization of Industrially Applicable de Novo Lager Yeast Hybrids with a Unique Genomic Architecture

2020 ◽  
Author(s):  
Zachari Turgeon ◽  
Thomas Sierocinski ◽  
Cedric A. Brimacombe ◽  
Yiqiong Jin ◽  
Brittany Goldhawke ◽  
...  
Keyword(s):  
De Novo ◽  
Phenotypic Diversity ◽  
Breeding Strategy ◽  
Lager Yeast ◽  
Genomic Architecture ◽  
Group I ◽  
Saccharomyces Pastorianus ◽  
Lager Beer ◽  
Group Ii ◽  
Beer Production

Lager beer is produced by Saccharomyces pastorianus, which is a natural allopolyploid hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus. Lager strains are classified into two major groups based largely on genomic composition: Group I and Group II. Group I strains are allotriploid, whereas Group II are allotetraploid. A lack of phenotypic diversity in commercial lager strains has led to substantial interest in the reconstitution of de novo allotetraploid lager strains by hybridization of S. cerevisiae and S. eubayanus strains. Such strategies rely on the hybridization of wild S. eubayanus isolates, which carry unacceptable traits for commercial lager beer such as phenolic off-flavours and incomplete utilization of carbohydrates. Using an alternative breeding strategy, we have created de novo lager hybrids containing the domesticated S. eubayanus subgenome from an industrial S. pastorianus strain by hybridizing diploid meiotic segregants of this strain to a variety of S. cerevisiae ale strains. Five de novo hybrids were isolated which had fermentation characteristics similar to those of prototypical commercial lager strains but with unique phenotypic variation due to the contributions of the S. cerevisiae parents. Genomic analysis of these de novo lager hybrids identified novel allotetraploid genomes carrying three copies of the S. cerevisiae genome and one copy of the S. eubayanus genome. Most importantly, these hybrids do not possess the negative traits which result from breeding wild S. eubayanus. The de novo lager strains produced using industrial S. pastorianus in this study are immediately suitable for industrial lager beer production. IMPORTANCE All lager beer is produced using two related lager yeast types: Group I and Group II, which are highly similar resulting in a lack of strain diversity for lager beer production. To date, approaches for generating new lager yeasts have generated strains possessing undesirable brewing characteristics which render them commercially inviable. We have used an alternative approach that circumvents this issue and created new lager strains that are directly suitable for lager beer production. These novel lager strains also possess a unique genomic architecture, which may lead to a better understanding of industrial yeast hybrids. We propose that strains created using our approach be classified as a third group of lager strains (Group III). We anticipate that these novel lager strains will be of great industrial relevance, and that this technique will be applicable to the creation of additional novel lager strains that will help broaden the diversity in commercial lager beer strains.

scholarly journals A Large Set of Newly Created Interspecific Saccharomyces Hybrids Increases Aromatic Diversity in Lager Beers

2015 ◽  
Vol 81 (23) ◽  
pp. 8202-8214 ◽  
Author(s):  
Stijn Mertens ◽  
Jan Steensels ◽  
Veerle Saels ◽  
Gert De Rouck ◽  
Guido Aerts ◽  
...  
Keyword(s):  
Large Scale ◽  
Alcoholic Beverage ◽  
Pilot Scale ◽  
Temperature Tolerance ◽  
Large Set ◽  
Lager Yeast ◽  
Content Type ◽  
High Attenuation ◽  
Saccharomyces Pastorianus ◽  
Lager Beer

ABSTRACTLager 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 ofSaccharomyces pastorianus, an interspecific hybrid betweenSaccharomyces cerevisiaeand the cold-tolerantSaccharomyces 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 ofS. cerevisiae(six strains) andS. eubayanus(two strains). Interestingly, many of the resulting hybrids showed a broader temperature tolerance than their parental strains and referenceS. pastorianusyeasts. 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.

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