Genome duplication and mutations in
            <i>ACE2</i>
            cause multicellular, fast-sedimenting phenotypes in evolved
            <i>Saccharomyces cerevisiae</i>

Oud, Bart; Guadalupe-Medina, Víctor; Nijkamp, Jurgen F.; Ridder, Dick de; Pronk, Jack T.; Maris, Antonius J. A. van; Daran, Jean‐Marc

doi:10.1073/pnas.1305949110

Cited by 101 publications

(95 citation statements)

References 74 publications

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“…These results are consistent with prior literature showing that loss-of-function mutations in this gene cause settling/clumping phenotypes in other experimental evolution scenarios (Voth et al 2005;Koschwanez et al 2013;Oud et al 2013;Ratcliff et al 2015). Furthermore, ace2 null mutants have the characteristic cell separation defect that we observed in our clones (Libby et al 2014;Ratcliff et al 2015).…”

Section: Majority Of Aggregating Clones Demonstrate Characteristics Osupporting

confidence: 93%

“…Using complementation with the wild-type allele, we verified that the ACE2 mutations were causative of the separation defect aggregation phenotype in these two clones (Table 1), with subtle modification to the cell morphology by BEM2-a gene involved in bud emergence that is also mutated in both clones ( Figure S2B in File S1) (Bender and Pringle 1991;Kim et al 1994). This type of separation defect in yeast is considered a type of multicellularity, and can be an adaptive trait when cells are challenged with local dispersal of nutrients (Koschwanez et al 2011(Koschwanez et al , 2013, settling (Ratcliff et al 2012(Ratcliff et al , 2013, or, as we show here, residence time in chemostats (Oud et al 2013). We would predict that floc formation could also be an adaptive strategy for cultures evolved under certain stress treatments.…”

Section: Majority Of Aggregating Clones Demonstrate Characteristics Omentioning

confidence: 60%

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Experimental evolution reveals favored adaptive routes to cell aggregation in yeast

Hope

Amorosi

Miller

et al. 2016

Preprint

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Yeast flocculation is a community-building cell aggregation trait that is an important mechanism of stress resistance and a useful phenotype for brewers; however, it is also a nuisance in many industrial processes, in clinical settings, and in the laboratory. Chemostat-based evolution experiments are impaired by inadvertent selection for aggregation, which we observe in 35% of populations. These populations provide a testing ground for understanding the breadth of genetic mechanisms Saccharomyces cerevisiae uses to flocculate, and which of those mechanisms provide the biggest adaptive advantages. In this study, we employed experimental evolution as a tool to ask whether one or many routes to flocculation are favored, and to engineer a strain with reduced flocculation potential. Using a combination of whole genome sequencing and bulk segregant analysis, we identified causal mutations in 23 independent clones that had evolved cell aggregation during hundreds of generations of chemostat growth. In 12 of those clones, we identified a transposable element insertion in the promoter region of known flocculation gene FLO1, and, in an additional five clones, we recovered loss-of-function mutations in transcriptional repressor TUP1, which regulates FLO1 and other related genes. Other causal mutations were found in genes that have not been previously connected to flocculation. Evolving a flo1 deletion strain revealed that this single deletion reduces flocculation occurrences to 3%, and demonstrated the efficacy of using experimental evolution as a tool to identify and eliminate the primary adaptive routes for undesirable traits.

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Section: Majority Of Aggregating Clones Demonstrate Characteristics Osupporting

confidence: 93%

Section: Majority Of Aggregating Clones Demonstrate Characteristics Omentioning

confidence: 60%

Experimental evolution reveals favored adaptive routes to cell aggregation in yeast

Hope

Amorosi

Miller

et al. 2016

Preprint

View full text Add to dashboard Cite

show abstract

“…It was demonstrated that these multicellular clusters are uniclonal and appeared as "snowflakes". This phenotype was also obtained in an ALE experiment with a haploid S. cerevisiae CEN.PK113-7D strain using long-term cultivation in sequential batch reactors [45]. It was shown that a frameshift mutation in ACE2-which encodes a transcriptional regulator involved in cell cycle control and mother-daughter cell separation (MDS)-caused the snowflake phenotype.…”

Section: Discussionmentioning

confidence: 75%

“…The morphology of the flocs can be described as "snowflake" aggregates. Yeast cell clusters with a comparable morphology have been recently described [44][45][46]. The evolved cells were plated out on WL nutrient agar, which can be used to detect morphological differences between strains [36].…”

Section: Adaptive Evolution During Continuous Operationmentioning

confidence: 99%

Gravity-Driven Adaptive Evolution of an Industrial Brewer’s Yeast Strain towards a Snowflake Phenotype in a 3D-Printed Mini Tower Fermentor

Conjaerts

Willaert

2017

Fermentation

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Abstract:We designed a mini tower fermentor that is suitable to perform adaptive laboratory evolution (ALE) with gravity imposed as selective pressure, and suitable to evolve a weak flocculating industrial brewers' strain towards a strain with a more extended aggregation phenotype. This phenotype is of particular interest in the brewing industry, since it simplifies yeast removal at the end of the fermentation, and many industrial strains are still not sufficiently flocculent. The flow of particles (yeast cells and flocs) was simulated, and the theoretical retainment advantage of aggregating cells over single cells in the tower fermentor was demonstrated. A desktop stereolithography (SLA) printer was used to construct the mini reactor from transparent methacrylic acid esters resin. The printed structures were biocompatible for yeast growth, and could be sterilised by autoclaving. The flexibility of 3D printing allowed the design to be optimized quickly. During the ALE experiment, yeast flocs were observed within two weeks after the start of the continuous cultivation. The flocs showed a "snowflake" morphology, and were not the result of flocculin interactions, but probably the result of (a) mutation(s) in gene(s) that are involved in the mother/daughter separation process.

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“…Single cell lines were isolated at the end of each of the accelerostat experiments and named IMS0478, IMS0480, and IMS0481 (Table 1). The SBR experiment was terminated when, after 47 days and 11 cycles, multicellular aggregates appeared in the culture due to the SBR setup-inherent empty-refill selection procedure, which is prone to unintended selection of cells developing a clumping phenotype, enabling their persistence in the bioreactor without further improvement of their growth rate under selective conditions (29). At this point, the specific growth rate, as estimated from CO 2 production profiles, had reached 0.22 h Ϫ1 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Laboratory Evolution of a Biotin-Requiring Saccharomyces cerevisiae Strain for Full Biotin Prototrophy and Identification of Causal Mutations

Bracher

Hulster

Koster

et al. 2017

Appl Environ Microbiol

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Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [] Ϸ 0.01 h Ϫ1 ). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates ( Յ 0.36 h Ϫ1 ) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium ( ϭ 0.15 h Ϫ1 ). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h Ϫ1 . The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast. IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are insufficient to sustain fast growth and product formation. Consequently, this expensive vitamin is routinely added to baker's yeast cultures. In this study, laboratory evolution in biotin-free growth medium yielded new strains that grew as fast in the absence of biotin as in its presence. By analyzing the DNA sequences of evolved biotin-independent strains, mutations were identified that contributed to this ability. This work demonstrates full biotin independence of an industrially relevant yeast and identifies mutations whose introduction into other yeast strains may reduce or eliminate their biotin requirements.KEYWORDS Saccharomyces cerevisiae, adaptive laboratory evolution, biotin, prototrophy, reverse metabolic engineering, vitamin biosynthesis, whole-genome sequencing

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Genome duplication and mutations in ACE2 cause multicellular, fast-sedimenting phenotypes in evolved Saccharomyces cerevisiae

Cited by 101 publications

References 74 publications

Experimental evolution reveals favored adaptive routes to cell aggregation in yeast

Experimental evolution reveals favored adaptive routes to cell aggregation in yeast

Gravity-Driven Adaptive Evolution of an Industrial Brewer’s Yeast Strain towards a Snowflake Phenotype in a 3D-Printed Mini Tower Fermentor

Laboratory Evolution of a Biotin-Requiring Saccharomyces cerevisiae Strain for Full Biotin Prototrophy and Identification of Causal Mutations

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