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Adaptation to High Ethanol Reveals Complex Evolutionary Pathways.

Voordeckers K, Kominek J, Das A, Espinosa-Cantú A, De Maeyer D, Arslan A, Van Pee M, van der Zande E, Meert W, Yang Y, Zhu B, Marchal K, DeLuna A, Van Noort V, Jelier R, Verstrepen KJ - PLoS Genet. (2015)

Bottom Line: Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance.Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration.Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67.

View Article: PubMed Central - PubMed

Affiliation: VIB Laboratory for Systems Biology, Leuven, Belgium.

ABSTRACT
Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts.

No MeSH data available.


Related in: MedlinePlus

Evolved populations are more ethanol tolerant.Evolved populations from different reactors show increased fitness in EtOH. Fitness was determined for population samples of each reactor after 40 generations (blue) and 200 generations (red). Fitness is expressed relative to the ancestral strain of each reactor (haploid for reactor 1–2; diploid for reactor 3–4 and tetraploid for reactor 5–6). Data represent the average of three independent measurements, error bars represent standard deviation. Populations with mutator phenotypes showed higher standard deviations. This might be due to the large supply of mutations generated by these mutators, which could affect mutation-selection balance.
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pgen.1005635.g002: Evolved populations are more ethanol tolerant.Evolved populations from different reactors show increased fitness in EtOH. Fitness was determined for population samples of each reactor after 40 generations (blue) and 200 generations (red). Fitness is expressed relative to the ancestral strain of each reactor (haploid for reactor 1–2; diploid for reactor 3–4 and tetraploid for reactor 5–6). Data represent the average of three independent measurements, error bars represent standard deviation. Populations with mutator phenotypes showed higher standard deviations. This might be due to the large supply of mutations generated by these mutators, which could affect mutation-selection balance.

Mentions: We determined the relative fitness of the evolved populations, isolated at 40 and 200 generations, and generally observed increases in fitness in high (9% v/v) EtOH (Fig 2 & Materials and Methods). In general, clones taken from the same population at the same time point had similar fitness; with some notable exceptions suggesting considerable heterogeneity within populations (S2 Fig). These fitness measurements were performed in 9% ethanol because the ancestral reference strains did not grow at higher ethanol levels. It is important to note that the actual increase in fitness of our evolved strains in higher ethanol levels (above 9% (v/v)) is much larger than what can be appreciated from Fig 2. Spotting of our evolved lineages on agar plates with increasing levels of ethanol indicates that these adapted strains can grow on concentrations up to 11% (v/v) EtOH, with the haploid control ancestral strain showing almost no growth under these conditions (i.e. an infinite increase in fitness compared to the ancestral haploid strain) (S3 Fig). Ancestral diploid and tetraploid strains show growth on 11% EtOH, although they are still outperformed by their evolved clones under these conditions.


Adaptation to High Ethanol Reveals Complex Evolutionary Pathways.

Voordeckers K, Kominek J, Das A, Espinosa-Cantú A, De Maeyer D, Arslan A, Van Pee M, van der Zande E, Meert W, Yang Y, Zhu B, Marchal K, DeLuna A, Van Noort V, Jelier R, Verstrepen KJ - PLoS Genet. (2015)

Evolved populations are more ethanol tolerant.Evolved populations from different reactors show increased fitness in EtOH. Fitness was determined for population samples of each reactor after 40 generations (blue) and 200 generations (red). Fitness is expressed relative to the ancestral strain of each reactor (haploid for reactor 1–2; diploid for reactor 3–4 and tetraploid for reactor 5–6). Data represent the average of three independent measurements, error bars represent standard deviation. Populations with mutator phenotypes showed higher standard deviations. This might be due to the large supply of mutations generated by these mutators, which could affect mutation-selection balance.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4636377&req=5

pgen.1005635.g002: Evolved populations are more ethanol tolerant.Evolved populations from different reactors show increased fitness in EtOH. Fitness was determined for population samples of each reactor after 40 generations (blue) and 200 generations (red). Fitness is expressed relative to the ancestral strain of each reactor (haploid for reactor 1–2; diploid for reactor 3–4 and tetraploid for reactor 5–6). Data represent the average of three independent measurements, error bars represent standard deviation. Populations with mutator phenotypes showed higher standard deviations. This might be due to the large supply of mutations generated by these mutators, which could affect mutation-selection balance.
Mentions: We determined the relative fitness of the evolved populations, isolated at 40 and 200 generations, and generally observed increases in fitness in high (9% v/v) EtOH (Fig 2 & Materials and Methods). In general, clones taken from the same population at the same time point had similar fitness; with some notable exceptions suggesting considerable heterogeneity within populations (S2 Fig). These fitness measurements were performed in 9% ethanol because the ancestral reference strains did not grow at higher ethanol levels. It is important to note that the actual increase in fitness of our evolved strains in higher ethanol levels (above 9% (v/v)) is much larger than what can be appreciated from Fig 2. Spotting of our evolved lineages on agar plates with increasing levels of ethanol indicates that these adapted strains can grow on concentrations up to 11% (v/v) EtOH, with the haploid control ancestral strain showing almost no growth under these conditions (i.e. an infinite increase in fitness compared to the ancestral haploid strain) (S3 Fig). Ancestral diploid and tetraploid strains show growth on 11% EtOH, although they are still outperformed by their evolved clones under these conditions.

Bottom Line: Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance.Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration.Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67.

View Article: PubMed Central - PubMed

Affiliation: VIB Laboratory for Systems Biology, Leuven, Belgium.

ABSTRACT
Tolerance to high levels of ethanol is an ecologically and industrially relevant phenotype of microbes, but the molecular mechanisms underlying this complex trait remain largely unknown. Here, we use long-term experimental evolution of isogenic yeast populations of different initial ploidy to study adaptation to increasing levels of ethanol. Whole-genome sequencing of more than 30 evolved populations and over 100 adapted clones isolated throughout this two-year evolution experiment revealed how a complex interplay of de novo single nucleotide mutations, copy number variation, ploidy changes, mutator phenotypes, and clonal interference led to a significant increase in ethanol tolerance. Although the specific mutations differ between different evolved lineages, application of a novel computational pipeline, PheNetic, revealed that many mutations target functional modules involved in stress response, cell cycle regulation, DNA repair and respiration. Measuring the fitness effects of selected mutations introduced in non-evolved ethanol-sensitive cells revealed several adaptive mutations that had previously not been implicated in ethanol tolerance, including mutations in PRT1, VPS70 and MEX67. Interestingly, variation in VPS70 was recently identified as a QTL for ethanol tolerance in an industrial bio-ethanol strain. Taken together, our results show how, in contrast to adaptation to some other stresses, adaptation to a continuous complex and severe stress involves interplay of different evolutionary mechanisms. In addition, our study reveals functional modules involved in ethanol resistance and identifies several mutations that could help to improve the ethanol tolerance of industrial yeasts.

No MeSH data available.


Related in: MedlinePlus