CRISPR-PCDup: a novel approach for simultaneous segmental chromosomal duplication in Saccharomyces cerevisiae

Bioethanol production from lignocellulosic hydrolysates requires a producer strain that tolerates both the presence of growth and fermentation inhibitors and high ethanol concentrations. Therefore, we constructed heterozygous intraspecies hybrid diploids of Saccharomyces cerevisiae by crossing two natural S. cerevisiae isolates, YIIc17_E5 and UWOPS87-2421, a good ethanol producer found in wine and a strain from the flower of the cactus Opuntia megacantha resistant to inhibitors found in lignocellulosic hydrolysates, respectively. Hybrids grew faster than parental strains in the absence and in the presence of acetic and levulinic acids and 2-furaldehyde, inhibitors frequently found in lignocellulosic hydrolysates, and the overexpression of YAP1 gene increased their survival. Furthermore, although originating from the same parental strains, hybrids displayed different fermentative potential in a CO2 production test, suggesting genetic variability that could be used for further selection of desirable traits. Therefore, our results suggest that the construction of intraspecies hybrids coupled with the use of genetic engineering techniques is a promising approach for improvement or development of new biotechnologically relevant strains of S. cerevisiae. Moreover, it was found that the success of gene targeting (gene targeting fidelity) in natural S. cerevisiae isolates (YIIc17_E5α and UWOPS87-2421α) was strikingly lower than in laboratory strains and the most frequent off-targeting event was targeted chromosome duplication.

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Population Analysis and Evolution of Saccharomyces cerevisiae Mitogenomes

Microorganisms ◽

10.3390/microorganisms8071001 ◽

2020 ◽

Vol 8 (7) ◽

pp. 1001

Author(s):

Daniel Vieira ◽

Soraia Esteves ◽

Carolina Santiago ◽

Eduardo Conde-Sousa ◽

Ticiana Fernandes ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Yeast Species ◽

Phenotypic Diversity ◽

Population Analysis ◽

Nuclear Genome ◽

Reference Sequence ◽

Evolutionary Analysis ◽

Novel Approach ◽

History Of ◽

First Time

The study of mitogenomes allows the unraveling of some paths of yeast evolution that are often not exposed when analyzing the nuclear genome. Although both nuclear and mitochondrial genomes are known to determine phenotypic diversity and fitness, no concordance has yet established between the two, mainly regarding strains’ technological uses and/or geographical distribution. In the current work, we proposed a new method to align and analyze yeast mitogenomes, overcoming current difficulties that make it impossible to obtain comparable mitogenomes for a large number of isolates. To this end, 12,016 mitogenomes were considered, and we developed a novel approach consisting of the design of a reference sequence intended to be comparable between all mitogenomes. Subsequently, the population structure of 6646 Saccharomyces cerevisiae mitogenomes was assessed. Results revealed the existence of particular clusters associated with the technological use of the strains, in particular regarding clinical isolates, laboratory strains, and yeasts used for wine-associated activities. As far as we know, this is the first time that a positive concordance between nuclear and mitogenomes has been reported for S. cerevisiae, in terms of strains’ technological applications. The results obtained highlighted the importance of including the mtDNA genome in evolutionary analysis, in order to clarify the origin and history of yeast species.

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Enforced Mutualism Leads to Improved Cooperative Behavior between Saccharomyces cerevisiae and Lactobacillus plantarum

Microorganisms ◽

10.3390/microorganisms8081109 ◽

2020 ◽

Vol 8 (8) ◽

pp. 1109

Author(s):

S. Christine du Toit ◽

Debra Rossouw ◽

Maret du Toit ◽

Florian F. Bauer

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Lactobacillus Plantarum ◽

Species Interactions ◽

Cooperative Behavior ◽

Synthetic Medium ◽

Grape Juice ◽

Growth Patterns ◽

Microbial Interactions ◽

Novel Approach

Saccharomyces cerevisiae and Lactobacillus plantarum are responsible for alcoholic and malolactic fermentation, respectively. Successful completion of both fermentations is essential for many styles of wine, and an understanding of how these species interact with each other, as well as the development of compatible pairings of these species, will help to manage the process. However, targeted improvements of species interactions are difficult to perform, in part because of the chemical and biological complexity of natural grape juice. Synthetic ecological systems reduce this complexity and can overcome these difficulties. In such synthetic systems, mutualistic growth of different species can be enforced through the reciprocal exchange of essential nutrients. Here, we implemented a novel approach to evolve mutualistic traits by establishing a co-dependent relationship between S. cerevisiae BY4742Δthi4 and Lb. plantarum IWBT B038 by omitting different combinations of amino acids from a chemically defined synthetic medium simulating standard grape juice. After optimization, the two species were able to support the growth of each other when grown in the absence of appropriate combinations of amino acids. In these obligatory mutualistic conditions, BY4742Δthi4 and IWBT B038 were co-evolved for approximately 100 generations. The selected evolved isolates showed improved mutualistic growth and the growth patterns under non-selective conditions indicate the emergence of mutually beneficial adaptations independent of the synthetic selection pressure. The combined use of synthetic ecology and co-evolution is a promising strategy to better understand and biotechnologically improve microbial interactions.

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Novel approach in developing Saccharomyces cerevisiae hybrid bioethanol producers by mating of natural isolates having desirable traits

Journal of Biotechnology ◽

10.1016/j.jbiotec.2018.06.294 ◽

2018 ◽

Vol 280 ◽

pp. S89

Author(s):

Bojan Zunar ◽

Anamarija Stafa ◽

Antonio Zandona ◽

Marina Svetec Miklenic ◽

Bozidar Santek ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Novel Approach ◽

Natural Isolates

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The stress-inducible peroxidase TSA2 enables Chromosome IV duplication to be conditionally beneficial in Saccharomyces cerevisiae

10.1101/101139 ◽

2017 ◽

Author(s):

Robert A. Linder ◽

John P. Greco ◽

Fabian Seidl ◽

Takeshi Matsui ◽

Ian M. Ehrenreich

Keyword(s):

Oxidative Stress ◽

Saccharomyces Cerevisiae ◽

Copy Number ◽

Molecular Mechanisms ◽

Protective Effects ◽

Beneficial Effects ◽

Mapping Strategy ◽

Single Stress ◽

Chromosomal Duplication ◽

Inducible Gene

AbstractAlthough chromosomal duplications are often deleterious, in some cases they enhance cells’ abilities to tolerate specific genetic or environmental challenges. Identifying the genes that cause particular chromosomal duplications to confer these conditionally beneficial effects can improve our understanding of the genetic and molecular mechanisms that enable certain aneuploidies to persist in cell populations and contribute to disease and evolution. Here, we perform a screen for spontaneous mutations that improve the tolerance of haploid Saccharomyces cerevisiae to hydrogen peroxide. Chromosome IV duplication is the most frequent mutation, as well as the only change in chromosomal copy number, seen in the screen. Using a genetic mapping strategy that involves systematically deleting segments of a duplicated chromosome, we show that the Chromosome IV duplication’s effect is largely due to the generation of a second copy of the stress-inducible cytoplasmic thioredoxin peroxidase TSA2. This finding is consistent with a growing literature indicating that the conditionally beneficial effects of chromosomal duplications tend to reflect the contributions of small numbers of genes that enhance tolerance to specific stresses when their copy number is increased.Article summaryChanges in karyotype play an important role in evolution and health. Although these aneuploidization events are usually deleterious, in some instances they show conditionally beneficial effects by enabling cells to tolerate specific mutations or environmental stresses. The mechanisms underlying these protective effects of aneuploidization are not fully understood. To provide insights into this problem, we identify and characterize a conditionally beneficial chromosomal duplication that makes haploid yeast more tolerant to oxidative stress. We determine that the effect of the chromosomal duplication on oxidative stress tolerance is largely explained by duplication of a single stress-inducible gene.

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A novel approach for the improvement of ethanol fermentation by Saccharomyces cerevisiae

Canadian Journal of Microbiology ◽

10.1139/w10-032 ◽

2010 ◽

Vol 56 (6) ◽

pp. 495-500 ◽

Cited By ~ 5

Author(s):

Lihua Hou ◽

Xiaohong Cao ◽

Chunling Wang

Keyword(s):

Saccharomyces Cerevisiae ◽

Ethanol Production ◽

Genome Engineering ◽

Ethanol Conversion ◽

Mitotic Chromosomes ◽

Gene Encoding ◽

Very High Gravity ◽

Novel Approach ◽

Very High Gravity Fermentation ◽

Tetraploid Strain

Fermentation properties under the control of multiple genes are difficult to alter with traditional methods in Saccharomyces cerevisiae . Here, a novel genome engineering approach is developed to improve ethanol production in very high gravity fermentation with 300 g/L glucose as the carbon source. This strategy involved constructing aneuploid strains on the base of tetraploid cells. The tetraploid strain was constructed by using the plasmid YCplac33-GHK, which harbored the HO gene encoding the site-specific Ho endonucleases. The aneuploid strain, WT4-M, was selected and screened after the tetraploid cells were treated with methyl benzimidazole-2-yl-carbamate to induce loss of mitotic chromosomes. It was found that aneuploid strain WT4-M not only exhibited an increase in ethanol production and osmotic and thermal tolerance, but also an improvement in the sugar–ethanol conversion rate. Notably, WT4-M provided up to 9.8% improvement in ethanol production compared with the control strain. The results demonstrated that the strategy of aneuploidy was valuable for creating yeast strains with better fermentation characteristics.

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PRECISE MAPPING OF THE HOMOTHALLISM GENES HML AND HMR IN SACCHAROMYCES CEREVISIAE

Genetics ◽

10.1093/genetics/96.2.315 ◽

1980 ◽

Vol 96 (2) ◽

pp. 315-320

Author(s):

Amar J S Klar ◽

Jean McIndoo ◽

James B Hicks ◽

Jeffrey N Strathern

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Control Mechanism ◽

Negative Control ◽

Switching Function ◽

Novel Approach ◽

Precise Mapping ◽

Chromosome Iii ◽

The Right ◽

Saccharomyces Yeasts

ABSTRACT The HML and HMR loci carry unexpressed copies of MAT a and MATα information, and a replica of that information is transposed to MAT during mating-type interchange in Saccharomyces yeasts. A negative control mechanism keeps silent the information located at the HML and HMR loci. We mapped these loci by constructing strains in which these loci are expressed. In these strains, the mating type of the segregants is dependent upon the allele at HML and HMR. This novel approach is independent of their switching function. HML is located on the left arm of chromosome III distal to his4 by about 26.8 centimorgans (cM). HMR maps on the right arm of the same chromosome distal to thr4 by about 39.8 cM and proximal to MAL2 by about 1.0 cM. The results allow the exact placement of these loci and are in accord with the observations made by Harashima and Oshima (1976).

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Engineering of Yeast Old Yellow Enzyme OYE3 Enables Its Capability Discriminating of (E)-Citral and (Z)-Citral

Molecules ◽

10.3390/molecules26165040 ◽

2021 ◽

Vol 26 (16) ◽

pp. 5040

Author(s):

Tairan Wang ◽

Ran Wei ◽

Yingting Feng ◽

Lijun Jin ◽

Yunpeng Jia ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Rational Design ◽

Asymmetric Reduction ◽

Glucose Dehydrogenase ◽

Direct Synthesis ◽

Cascade Reaction ◽

Old Yellow Enzyme ◽

Complete Reduction ◽

Novel Approach ◽

Double Substitution

The importance of yeast old yellow enzymes is increasingly recognized for direct asymmetric reduction of (E/Z)-citral to (R)-citronellal. As one of the most performing old yellow enzymes, the enzyme OYE3 from Saccharomyces cerevisiae S288C exhibited complementary enantioselectivity for the reduction of (E)-citral and (Z)-citral, resulting in lower e.e. value of (R)-citronellal in the reduction of (E/Z)-citral. To develop a novel approach for the direct synthesis of enantio-pure (R)-citronellal from the reduction of (E/Z)-citral, the enzyme OYE3 was firstly modified by semi-rational design to improve its (R)-enantioselectivity. The OYE3 variants W116A and S296F showed strict (R)-enantioselectivity in the reduction of (E)-citral, and significantly reversed the (S)-enantioselectivity in the reduction of (Z)-citral. Next, the double substitution of OYE3 led to the unique variant S296F/W116G, which exhibited strict (R)-enantioselectivity in the reduction of (E)-citral and (E/Z)-citral, but was not active on (Z)-citral. Relying on its capability discriminating (E)-citral and (Z)-citral, a new cascade reaction catalyzed by the OYE3 variant S296F/W116G and glucose dehydrogenase was developed, providing the enantio-pure (R)-citronellal and the retained (Z)-citral after complete reduction of (E)-citral.

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Adhesion-dependent rupturing of Saccharomyces cerevisiae on biological antimicrobial nanostructured surfaces

Journal of The Royal Society Interface ◽

10.1098/rsif.2014.0999 ◽

2015 ◽

Vol 12 (102) ◽

pp. 20140999 ◽

Cited By ~ 65

Author(s):

Kyle Nowlin ◽

Adam Boseman ◽

Alan Covell ◽

Dennis LaJeunesse

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Yeast Cell ◽

Morphological Changes ◽

Wall Structure ◽

Direct Role ◽

Nanostructured Surfaces ◽

Novel Approach ◽

Nanoscale Topography ◽

Underlying Mechanisms

Recent studies have shown that some nanostructured surfaces (NSS), many of which are derived from surfaces found on insect cuticles, rupture and kill adhered prokaryotic microbes. Most important, the nanoscale topography is directly responsible for this effect. Although parameters such as cell adhesion and cell wall rigidity have been suggested to play significant roles in this process, there is little experimental evidence regarding the underlying mechanisms involving NSS-induced microbial rupture. In this work, we report the NSS-induced rupturing of a eukaryotic microorganism, Saccharomyces cerevisiae . We show that the amount of NSS-induced rupture of S. cerevisiae is dependent on both the adhesive qualities of the yeast cell and the nanostructure geometry of the NSS. Thus, we are providing the first empirical evidence that these parameters play a direct role in the rupturing of microbes on NSS. Our observations of this phenomenon with S. cerevisiae, particularly the morphological changes, are strikingly similar to that reported for bacteria despite the differences in the yeast cell wall structure. Consequently, NSS provide a novel approach for the control of microbial growth and development of broad-spectrum microbicidal surfaces.

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Targeted 2′-O Methylation at a Nucleotide within the Pseudoknot of Telomerase RNA Reduces Telomerase Activity In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.00384-10 ◽

2010 ◽

Vol 30 (18) ◽

pp. 4368-4378 ◽

Cited By ~ 9

Author(s):

Chao Huang ◽

Yi-Tao Yu

Keyword(s):

Saccharomyces Cerevisiae ◽

Triple Helix ◽

Telomerase Activity ◽

Rna Modification ◽

Telomerase Rna ◽

Telomere Shortening ◽

Pseudoknot Structure ◽

Novel Approach ◽

Telomere Lengthening

ABSTRACT Telomerase RNA is an essential component of telomerase, a ribonucleoprotein enzyme that maintains chromosome ends in most eukaryotes. Here we employ a novel approach, namely, RNA-guided RNA modification, to assess whether introducing 2′-O methylation into telomerase RNA can influence telomerase activity in vivo. We generate specific 2′-O methylation sites in and adjacent to the triple helix (within the conserved pseudoknot structure) of Saccharomyces cerevisiae telomerase RNA (TLC1). We show that 2′-O methylation at U809 reduces telomerase activity, resulting in telomere shortening, whereas 2′-O methylation at A804 or A805 leads to moderate telomere lengthening. Importantly, we also show that targeted 2′-O methylation does not affect TLC1 levels and that 2′-O-methylated TLC1 appears to be efficiently assembled into telomerase ribonucleoprotein. Our results demonstrate that RNA-guided RNA modification is a highly useful approach for modulating telomerase activity.

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