Homologous Recombination: A GRAS Yeast Genome Editing Tool

The fermentation industry is known to be very conservative, relying on traditional yeast management. Yet, in the modern fast-paced world, change comes about in facets such as climate change altering the quality and quantity of harvests, changes due to government regulations e.g., the use of pesticides or SO2, the need to become more sustainable, and of course by changes in consumer preferences. As a silent companion of the fermentation industry, the wine yeast Saccharomyces cerevisiae has followed mankind through millennia, changing from a Kulturfolger, into a domesticated species for the production of bread, beer, and wine and further on into a platform strain for the production of biofuels, enzymes, flavors, or pharmaceuticals. This success story is based on the ‘awesome power of yeast genetics’. Central to this is the very efficient homologous recombination (HR) machinery of S. cerevisiae that allows highly-specific genome edits. This microsurgery tool is so reliable that yeast has put a generally recognized as safe (GRAS) label onto itself and entrusted to itself the life-changing decision of mating type-switching. Later, yeast became its own genome editor, interpreted as domestication events, to adapt to harsh fermentation conditions. In biotechnology, yeast HR has been used with tremendous success over the last 40 years. Here we discuss several types of yeast genome edits then focus on HR and its inherent potential for evolving novel wine yeast strains and styles relevant for changing markets.

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Synapsis-mediated fusion of free DNA ends forms inverted dimer plasmids in yeast.

Genetics ◽

10.1093/genetics/124.1.67 ◽

1990 ◽

Vol 124 (1) ◽

pp. 67-80 ◽

Cited By ~ 2

Author(s):

S Kunes ◽

D Botstein ◽

M S Fox

Keyword(s):

Homologous Recombination ◽

Plasmid Dna ◽

Genomic Dna ◽

Chromosomal Rearrangement ◽

Yeast Genome ◽

Homologous Pairing ◽

Yeast Saccharomyces Cerevisiae ◽

Free Dna ◽

Sister Chromatids ◽

Dna Ends

Abstract When yeast (Saccharomyces cerevisiae) is transformed with linearized plasmid DNA and the ends of the plasmid do not share homology with the yeast genome, circular inverted (head-to-head) dimer plasmids are the principal product of repair. By measurements of the DNA concentration dependence of transformation with a linearized plasmid, and by transformation with mixtures of genetically marked plasmids, we show that two plasmid molecules are required to form an inverted dimer plasmid. Several observations suggest that homologous pairing accounts for the head-to-head joining of the two plasmid molecules. First, an enhanced frequency of homologous recombination is detected when genetically marked plasmids undergo end-to-end fusion. Second, when a plasmid is linearized within an inverted repeat, such that its ends could undergo head-to-tail homologous pairing, it is repaired by intramolecular head-to-tail joining. Last, in the joining of homologous linearized plasmids of different length, a shorter molecule can acquire a longer plasmid end by homologous recombination. The formation of inverted dimer plasmids may be related to some forms of chromosomal rearrangement. These might include the fusion of broken sister chromatids in the bridge-breakage-fusion cycle and the head-to-head duplication of genomic DNA at the sites of gene amplifications.

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Rearrangements occurring adjacent to a single Ty1 yeast retrotransposon in the presence and absence of full-length Ty1 transcription.

Genetics ◽

10.1093/genetics/131.4.833 ◽

1992 ◽

Vol 131 (4) ◽

pp. 833-850

Author(s):

P R Sutton ◽

S W Liebman

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Transposable Element ◽

Gene Conversion ◽

Genetic Background ◽

Repetitive Sequences ◽

Full Length ◽

Yeast Genome ◽

Yeast Saccharomyces Cerevisiae ◽

Ty1 Retrotransposon

Abstract The structures of two unusual deletions from the yeast Saccharomyces cerevisiae are described. These deletions extend from a single Ty1 retrotransposon to an endpoint near a repetitive tRNA(Gly) gene. The deletions suggest that unique sequences flanked by two nonidentical repetitive sequences, or bordered on only one side by a transposable element, have the potential to be mobilized in the yeast genome. Models for the formation of these two unusual deletions were tested by isolating and analyzing 32 additional unusual deletions of the CYC1 region that extend from a single Ty1 retrotransposon. Unlike the most common class of deletions recovered in this region, these deletions are not attributable solely to homologous recombination among repetitive Ty1 or delta elements. They arose by two distinct mechanisms. In an SPT8 genetic background, most unusual deletions arose by transposition of a Ty1 element to a position adjacent to a tRNA(Gly) gene followed by Ty1-Ty1 recombination. In an spt8 strain, where full-length Ty1 transcription and, therefore, transposition are reduced, most deletions were due to gene conversion of a 7-kb chromosomal interval flanked by a Ty1 element and a tRNA(Gly) gene.

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Molecular Basis of Fructose Utilization by the Wine Yeast Saccharomyces cerevisiae: a Mutated HXT3 Allele Enhances Fructose Fermentation

Applied and Environmental Microbiology ◽

10.1128/aem.02269-06 ◽

2007 ◽

Vol 73 (8) ◽

pp. 2432-2439 ◽

Cited By ~ 67

Author(s):

Carole Guillaume ◽

Pierre Delobel ◽

Jean-Marie Sablayrolles ◽

Bruno Blondin

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Basis ◽

Alcoholic Fermentation ◽

Wine Yeast ◽

Glycolytic Flux ◽

Hexose Transporter ◽

Wine Yeasts ◽

Yeast Saccharomyces Cerevisiae ◽

High Fructose ◽

Fructose Fermentation

ABSTRACT Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Δ strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.

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The Fitness Advantage of Commercial Wine Yeasts in Relation to the Nitrogen Concentration, Temperature, and Ethanol Content under Microvinification Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.03405-13 ◽

2013 ◽

Vol 80 (2) ◽

pp. 704-713 ◽

Cited By ~ 19

Author(s):

Estéfani García-Ríos ◽

Alicia Gutiérrez ◽

Zoel Salvadó ◽

Francisco Noé Arroyo-López ◽

José Manuel Guillamon

Keyword(s):

Abiotic Factors ◽

Nitrogen Concentration ◽

Wine Yeast ◽

Wine Industry ◽

Wine Yeasts ◽

Simple Method ◽

Fitness Advantage ◽

Yeast Strains ◽

Industrial Strains ◽

Nitrogen Concentrations

ABSTRACTThe effect of the main environmental factors governing wine fermentation on the fitness of industrial yeast strains has barely received attention. In this study, we used the concept of fitness advantage to measure how increasing nitrogen concentrations (0 to 200 mg N/liter), ethanol (0 to 20%), and temperature (4 to 45°C) affects competition among four commercial wine yeast strains (PDM, ARM, RVA, and TTA). We used a mathematical approach to model the hypothetical time needed for the control strain (PDM) to out-compete the other three strains in a theoretical mixed population. The theoretical values obtained were subsequently verified by competitive mixed fermentations in both synthetic and natural musts, which showed a good fit between the theoretical and experimental data. Specifically, the data show that the increase in nitrogen concentration and temperature values improved the fitness advantage of the PDM strain, whereas the presence of ethanol significantly reduced its competitiveness. However, the RVA strain proved to be the most competitive yeast for the three enological parameters assayed. The study of the fitness of these industrial strains is of paramount interest for the wine industry, which uses them as starters of their fermentations. Here, we propose a very simple method to model the fitness advantage, which allows the prediction of the competitiveness of one strain with respect to different abiotic factors.

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Some enological properties of Czechoslovak wine yeast strains.

Kvasny Prumysl ◽

10.18832/kp1977034 ◽

1977 ◽

Vol 23 (9) ◽

pp. 207-212

Author(s):

E. MINÁRIK

Keyword(s):

Wine Yeast ◽

Yeast Strains

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The genetic improvement of grapevine cultivars and wine yeast strains: Novel approaches to the ancient art of winemaking

Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie ◽

10.4102/satnt.v22i1.208 ◽

2003 ◽

Vol 22 (1) ◽

pp. 31-43

Author(s):

I. S. Pretorius

Keyword(s):

Wine Yeast ◽

Wine Industry ◽

Market Demand ◽

Wine Production ◽

Yeast Strains ◽

Quantum Leap ◽

Grape Quality ◽

The Cost ◽

Grape Varieties ◽

Grape Cultivars

The widening gap between wine production and wine consumption, the shift of consumer preferences away from basic commodity wine to top quality wine, and the gruelling competition brought about by economic globalisation call for a total revolution in the magical world of wine. In the process of transforming the wine industry from a production-driven industry to a market-orientated enterprise, there is an increasing dependence on, amongst others, biotechnological innovation to launch the wine industry with a quantum leap across the formidable market challenges of the 21st century. Market-orientated designer grape cultivars and wine yeast strains are currently being genetically programmed with surgical precision for the cost-competitive production of high quality grapes and wine with relatively minimal resource inputs and a low environmental impact. With regard to Grapevine Biotechnology, this entails the establishment of stress tolerant and disease resistant varieties of Vitis vinifera with increased productivity, efficiency, sustainability and environmental friendliness, especially regarding improved pest and disease control, water use efficiency and grape quality. With regard to Wine Yeast Biotechnology, the emphasis is on the development of Saccharomyces cerevisiae strains with improved fermentation, processing and biopreservation abilities, and capacities for an increase in the wholesomeness and sensory quality of wine. The successful commercialisation of transgenic grape cultivars and wine yeasts depends on a number of scientific, technical, safety, ethical, legal, economic and marketing factors, and it therefore will be unwise to entertain high expectations in the short term. However, in the light of the phenomenal potential advantages of tailor-made grape varieties and yeast strains, it would be equally self-destructive in the long term if this strategically important “life insurance policy” is not taken out by the wine industry. This overview highlights the most important examples of the way in which V. vinifera grape varieties and S. cerevisiae wine yeast strains are currently being designed with surgical precision on the basis of market demand for the cost-effective, sustainable and environmentally friendly production of healthy, top quality grapes and wine.

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Genomics and biochemistry of Saccharomyces cerevisiae wine yeast strains

Biochemistry (Moscow) ◽

10.1134/s0006297916130046 ◽

2016 ◽

Vol 81 (13) ◽

pp. 1650-1668 ◽

Cited By ~ 10

Author(s):

M. A. Eldarov ◽

S. A. Kishkovskaia ◽

T. N. Tanaschuk ◽

A. V. Mardanov

Keyword(s):

Saccharomyces Cerevisiae ◽

Wine Yeast ◽

Yeast Strains

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DYNAMICS OF SACHAROMYCES CEREVISIAE YEAST CYTOMORPHOMETRIC CHARACTERISTIC CHANGES DURING FRUIT AND BERRY WORT FERMENTATION

BIOTECHNOLOGY: STATE OF THE ART AND PERSPECTIVES ◽

10.37747/2312-640x-2020-18-401-403 ◽

2020 ◽

pp. 401-403

Author(s):

A. Shitova ◽

I. Anisimova

Keyword(s):

Saccharomyces Cerevisiae ◽

Wine Yeast ◽

Cell Area ◽

Black Currant ◽

Yeast Saccharomyces Cerevisiae ◽

Sacharomyces Cerevisiae

The activity of wine yeast Saccharomyces cerevisiae used for black currant, cherry and cranberry fermentation was studied using cell area dynamics calculated by computer cytomorphometry method.

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Use of Pseudocereals Preferment Made with Aromatic Yeast Strains for Enhancing Wheat Bread Quality

Foods ◽

10.3390/foods8100443 ◽

2019 ◽

Vol 8 (10) ◽

pp. 443 ◽

Cited By ~ 9

Author(s):

Păucean ◽

Man ◽

Chiş ◽

Mureşan ◽

Pop ◽

...

Keyword(s):

Yeast Strain ◽

Wine Yeast ◽

Wheat Bread ◽

Bakery Products ◽

Total Polyphenols ◽

White Wheat ◽

Yeast Strains ◽

Wine Yeast Strain ◽

Nutritional Qualities ◽

Beer Yeast

Usually, aromatic yeasts are designed to ferment wheat substrates for baking purposes but identification of new substrates for these strains and consequently new formulations for dough could lead to diversified bakery products with improved nutritional qualities and specific sensorial properties. The purpose of our study was to optimize the fermentation of quinoa and amaranth flours with non-conventional yeast strains in order to obtain a preferment with high potential in enhancing nutritional, textural and sensorial features of white wheat bread. Two biotypes of Saccharomyces cerevisiae yeast—a wine yeast strain and a beer yeast strain—commercialized for their aromatic properties were used. Both aromatic yeast strains revealed good performance on fermenting pseudocereal substrates. Utilization of the obtained preferment in white wheat breadmaking led to bread with higher protein, fibres, mineral, total polyphenols content, with specific texture and aroma profile and high consumers’ acceptability.

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