The regulatable<i>MAL32</i>promoter in<i>S. cerevisiae</i>: characteristics and tools to facilitate its use

Meurer, Matthias; Chevyreva, Veronika; Cerulus, Bram; Knop, Michael

doi:10.1101/061127

Cited by 5 publications

(7 citation statements)

References 28 publications

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“…The heterologous regulator ScMAL13 , expressed from the constitutive ScPGK1 promoter, displayed the same expression in glucose- and maltose-grown cultures. Although ScMAL13 was efficiently expressed on glucose, none of the nine S. eubayanus maltose genes (the three identical SeAGT1 copies being undistinguishable) were transcriptionally induced under these conditions (Figure 6), confirming that the hierarchical regulatory role of glucose catabolite repression (45, 48) also takes place in S. eubayanus . During growth on maltose, all nine genes were significantly upregulated relative to glucose-grown cultures but large variations in expression level were observed.…”

Section: Resultsmentioning

confidence: 73%

“…In S. cerevisiae transcriptional regulation of MALx2 and MALx1 genes is tightly controlled by a transcription factor encoded by MALx3 genes. Malx3 binds an activating site located in the bidirectional promoters that control expression of MALx2 and MALx1 genes (45, 46). To test whether absence of maltose consumption in Himalayan S. eubayanus strains was caused by a lack transcriptional upregulation of SeMALT and SeMALS , the S. cerevisiae ScMAL13 gene was integrated at the SeSGA1 locus in S. eubayanus CDFM21L.1, under the control of a constitutive ScPGK1 promoter and ScTEF2 terminator.…”

Section: Resultsmentioning

confidence: 99%

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Maltotriose consumption by hybridSaccharomyces pastorianusis heterotic and results from regulatory cross-talk between parental sub-genomes

Brouwers

Brickwedde

et al. 2019

Preprint

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S. pastorianus strains are hybrids of S. cerevisiae and S. eubayanus that have been domesticated for several centuries in lager-beer brewing environments. As sequences and structures of S. pastorianus genomes are being resolved, molecular mechanisms and evolutionary origin of several industrially relevant phenotypes remain unknown. This study investigates how maltotriose metabolism, a key feature in brewing, may have arisen in early S. eubayanus × S. cerevisiae hybrids. To address this question, we generated a near-complete genome assembly of Himalayan S. eubayanus strains of the Holarctic subclade. This group of strains have been proposed to be the origin of the S. eubayanus subgenome of current S. pastorianus strains. The Himalayan S. eubayanus genomes harbored several copies of a SeAGT1 α-oligoglucoside transporter gene with high sequence identity to genes encountered in S. pastorianus. Although Himalayan S. eubayanus strains are unable to grown on maltose and maltotriose, their maltose-hydrolase and SeMALT1 and SeAGT1 maltose-transporter genes complemented the corresponding null mutants of S. cerevisiae. Expression, in a Himalayan S. eubayanus strain, of a functional S. cerevisiae maltose-metabolism regulator gene (MALx3) enabled growth on oligoglucosides. The hypothesis that the maltotriose-positive phenotype in S. pastorianus is a result of heterosis was experimentally tested by constructing a S. cerevisiae × S. eubayanus laboratory hybrid with a complement of maltose-metabolism genes that resembles that of current S. pastorianus strains. The ability of this hybrid to consume maltotriose in brewer’s wort demonstrated regulatory cross talk between sub-genomes and thereby validated this hypothesis. These results provide experimental evidence of the evolutionary origin of an essential phenotype of lager-brewing strains and valuable knowledge for industrial exploitation of laboratory-made S. pastorianus-like hybrids.ImportanceS.pastorianus, a S.cerevisiae X S.eubayanus hybrid, is used for production of lager beer, the most produced alcoholic beverage worldwide It emerged by spontaneous hybridization and have colonized early lager-brewing processes. Despite accumulation and analysis of genome sequencing data of S.pastorianus parental genomes, the genetic blueprint of industrially relevant phenotypes remains unknown. Assimilation of wort abundant sugar maltotriose has been postulated to be inherited from S.cerevisiae parent. Here, we demonstrate that although Asian S.eubayanus isolates harbor a functional maltotriose transporter SeAGT1 gene, they are unable to grow on α-oligoglucosides, but expression of S. cerevisae regulator ScMAL13 was sufficient to restore growth on trisaccharides. We hypothesized that S. pastorianus maltotriose phenotype results from regulatory interaction between S.cerevisae maltose transcription activator and the promoter of SeAGT1. We experimentally confirmed the heterotic nature of the phenotype and thus this results provide experimental evidence of the evolutionary origin of an essential phenotype of lager-brewing strains.

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Section: Resultsmentioning

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Maltotriose consumption by hybridSaccharomyces pastorianusis heterotic and results from regulatory cross-talk between parental sub-genomes

Brouwers

Brickwedde

et al. 2019

Preprint

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“…The p GAL promoter is inducible by galactose but also repressible by glucose, which together could result in a merely moderate increase in p GAL -driven expression at high (2%) glucose concentration. 24 Therefore, we also assayed the effect of TUP1 and CYC8 induction on cell adhesion in GM and YD, using strains BRF-p CUP -TUP1 and BRF-p CUP -CYC8, inducible by copper. The results ( Supplementary Fig.…”

Section: Adhesion To Plastic Varies In Different S Cerevisiae Strainsmentioning

confidence: 99%

Glucose, Cyc8p and Tup1p regulate biofilm formation and dispersal in wild Saccharomyces cerevisiae

Nguyen

Plocek

Váchová

et al. 2020

npj Biofilms Microbiomes

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Saccharomyces cerevisiae is a mainly beneficial yeast, widely used in the food industry. However, there is growing evidence of its potential pathogenicity, leading to fungemia and invasive infections. The medical impact of yeast pathogens depends on formation of biofilms: multicellular structures, protected from the environment. Cell adhesion is a prerequisite of biofilm formation. We investigated the adherence of wild and genetically modified S. cerevisiae strains, formation of solid-liquid interface biofilms and associated regulation. Planktonic and static cells of wild strain BRF adhered and formed biofilms in glucose-free medium. Tup1p and Cyc8p were key positive and negative regulators, respectively. Glucose caused increased Cyc8p levels and blocked cell adhesion. Even low glucose levels, comparable with levels in the blood, allowed biofilm dispersal and release of planktonic cells. Cyc8p could thus modulate cell adhesion in different niches, dependently on environmental glucose level, e.g., high-glucose blood versus low-glucose tissues in host organisms.

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“…Many existing inducible gene expression systems show considerable variation in expression of the controlled gene, making it difficult to achieve homogenous pheno-types at the population level. 13,19,20 In S. cerevisiae and C. elegans, comparison of signals from strains with different constellations of reporter genes allows quantification of different sources of variation in protein dosage. 31,39,40 Here, we quantified overall variation in protein dosage by measuring the Coefficient of Variation (CoV) in fluorescent output from a single reporter ( Figure S2), and we developed a second measure (explained in SI) that normalized variation in dosage with respect to a key confounding variable, cell volume, to correct for its effect on protein concentration.…”

Section: Complex Autorepressing (Car) Controller Architecture Expandsmentioning

confidence: 99%

“…16 These methods suffer from a number of drawbacks, including basal expression when not induced,, 8,13,17 deleterious effects on cell growth due to sequestration of cellular components by the activation domain 18 induction of genes in addition to the controlled gene, 14 and high cell-to-cell variability in expression of the controlled genes. 13,19,20 These inducible systems rely on "activation by recruitment", 21 the activator binds a site on DNA upstream of a yeast gene and recruits general transcription factors and regulators of the Pre-Initiation Complex (PIC). These assemble downstream at the "core promoter", and recruit RNA polymerase II to induce transcription.…”

Section: Introductionmentioning

confidence: 99%

A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery

Azizoğlu

Brent

Rudolf

2019

Preprint

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For more than 65 years, means to express genes conditionally into phenotype have remained central to biological experimentation and discovery. Current experimental methods typically enable either on/o↵ gene expression or growth-condition-specific, imprecisely controlled graded expression in response to exogenous inducers. Here we describe a "well-tempered" controller, one that allows precise and graded conditional expression of genes in Saccharomyces cerevisiae. This system, WTC 846 , appropriates genetic and design elements from bacterial, eukaryotic, and engineered systems. Its main autorepressing circuitry relies on two identical instances of a strong, growth rate regulated promoter P TDH3 engineered to be repressible by the TetR protein. This allows for titration of inducer concentration to drive variation-reduced expression of regulated genes across the entire range of the proteome. To reduce basal expression to zero, a TetR-Tup1 fusion protein is expressed at a low, constitutive level. We showed that strains carrying conditional, WTC 846 allelic forms of genes encoding stable low abundance proteins (eg. Cdc42), unstable low abundance proteins (eg. Ipl1), and highly expressed proteins including the glycolytic enzyme Tpi1 recapitulated known knockout and overexpression phenotypes. Strains bearing WTC 846:Cdc20 alleles allowed inducer controlled cell cycle synchronization of batch cultures and release. Chemical titration of WTC 846 allelic strains of CDC28, TOR1, PBR1 and PMA1 brought about distinct, gene dosage dependent controlled growth rates, and highly penetrant morphological phenotypes expected for the di↵erent gene doses. The ability to generate WTC 846 controlled, "expression-clamped" genes may operationally define a new kind of conditional allele whose cell-to-cell variation in expression is minimized and whose e↵ective dosage is under the experimenter's control. We expect Well-tempered Controller 846 (WTC 846 ) strains to find use in assessment of phenotypes now incompletely penetrant due to variable dosage of the causative gene product, in targeted cell biological experimentation, and in genome-wide studies such as gene by gene epistasis screens. In higher cells, we hope that implementation of titratable, expression clamped control logic via mammalian specific transcription elements will enable experiments now impossible due to cell-to-cell variation and imprecise control.

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The regulatableMAL32promoter inS. cerevisiae: characteristics and tools to facilitate its use

Cited by 5 publications

References 28 publications

Maltotriose consumption by hybridSaccharomyces pastorianusis heterotic and results from regulatory cross-talk between parental sub-genomes

Maltotriose consumption by hybridSaccharomyces pastorianusis heterotic and results from regulatory cross-talk between parental sub-genomes

Glucose, Cyc8p and Tup1p regulate biofilm formation and dispersal in wild Saccharomyces cerevisiae

A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery

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