A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations

Abstract

BackgroundAcetic acid, released during hydrolysis of lignocellulosic feedstocks for second generation bioethanol production, inhibits yeast growth and alcoholic fermentation. Yeast biomass generated in a propagation step that precedes ethanol production should therefore express a high and constitutive level of acetic acid tolerance before introduction into lignocellulosic hydrolysates. However, earlier laboratory evolution strategies for increasing acetic acid tolerance of Saccharomyces cerevisiae, based on prolonged cultivation in the presence of acetic acid, selected for inducible rather than constitutive tolerance to this inhibitor.ResultsPreadaptation in the presence of acetic acid was shown to strongly increase the fraction of yeast cells that could initiate growth in the presence of this inhibitor. Serial microaerobic batch cultivation, with alternating transfers to fresh medium with and without acetic acid, yielded evolved S. cerevisiae cultures with constitutive acetic acid tolerance. Single-cell lines isolated from five such evolution experiments after 50–55 transfers were selected for further study. An additional constitutively acetic acid tolerant mutant was selected after UV-mutagenesis. All six mutants showed an increased fraction of growing cells upon a transfer from a non-stressed condition to a medium containing acetic acid. Whole-genome sequencing identified six genes that contained (different) mutations in multiple acetic acid-tolerant mutants. Haploid segregation studies and expression of the mutant alleles in the unevolved ancestor strain identified causal mutations for the acquired acetic acid tolerance in four genes (ASG1, ADH3, SKS1 and GIS4). Effects of the mutations in ASG1, ADH3 and SKS1 on acetic acid tolerance were additive.ConclusionsA novel laboratory evolution strategy based on alternating cultivation cycles in the presence and absence of acetic acid conferred a selective advantage to constitutively acetic acid-tolerant mutants and may be applicable for selection of constitutive tolerance to other stressors. Mutations in four genes (ASG1, ADH3, SKS1 and GIS4) were identified as causative for acetic acid tolerance. The laboratory evolution strategy as well as the identified mutations can contribute to improving acetic acid tolerance in industrial yeast strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0583-1) contains supplementary material, which is available to authorized users.

The sole introduction of two single-point mutations establishes glycerol utilization in Saccharomyces cerevisiae CEN.PK derivatives

“…Several previously published adaptive evolution approaches of S.   cerevisiae (such as for acetic acid and n-butanol tolerance) have shown that strains evolved in parallel experiments can have mutations affecting different sets of genes [44, 45]. Therefore, the results of the current study strongly suggest that UBR2 and GUT1 are absolutely crucial in the establishment of glycerol utilization capability in CEN.PK strains.…”

Evolutionary adaptation of Aspergillus niger for increased ferulic acid tolerance

“…Adaptive evolution is a powerful tool, which uses the ability of micro‐organisms to adapt to environmental changes, in order to improve strains for various biotechnological applications (Wallace‐Salinas and Gorwa‐Grauslund ; Culleton et al ; González‐Ramos et al ; Patyshakuliyeva et al ). Most of the current filamentous fungi obtained through adaptive evolution were created for increased enzymatic activity.…”

“…For example, a Saccharomyces cerevisiae strain was created with improved tolerance to higher temperature and hydrolysate‐derived inhibitors for second generation ethanol production (Wallace‐Salinas and Gorwa‐Grauslund ). Another example was the S. cerevisiae strains that were created with increased acetic acid tolerance to withstand the negative effects of acetic acid released during hydrolysis of lignocellulosic feedstocks for bio‐ethanol production (González‐Ramos et al ). Evolutionary adaptation depends on spontaneous mutation(s) that is beneficial under the selection condition (Schoustra et al ; Schoustra and Punzalan ).…”

Nanopore sequencing enables near-complete de novo assembly of Saccharomyces cerevisiae reference strain CEN.PK113-7D

“…CEN.PK113-7D is a haploid strain used as a model organism in biotechnology-related research and systems biology because of its convenient growth characteristics, its robustness under industrially relevant conditions and its excellent genetic accessibility (Canelas et al 2010;Nijkamp et al 2012;González-Ramos et al 2016;Papapetridis et al 2017). CEN.PK113-7D was sequenced using a combination of 454 and Illumina short-read libraries, and a draft genome was assembled consisting of over 700 contigs (Nijkamp et al 2012).…”