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PROVIDER: PRJNA566398 | ENA |
REPOSITORIES: ENA
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Project description:Background: Evolutionary engineering is a powerful approach to isolate suppressor mutants and industrially relevant genotypes. Until recently, DNA microarray analysis was the only affordable genome-wide approach to identify the responsible mutations. This situation has changed due to the rapidly decreasing costs of whole genome (re)sequencing. DNA microarray-based mRNA expression analysis and whole genome resequencing were combined in a study on lactate transport in Saccharomyces cerevisiae. Jen1p is the only S. cerevisiae lactate transporter reported in literature. To identify alternative lactate transporters, a jen1Δ strain was evolved for growth on lactate. Results: Two independent evolution experiments yielded Jen1p-independent growth on lactate (μmax 0.14 and 0.18 h-1 for single-cell lines IMW004 and IMW005, respectively). Whereas mRNA expression analysis did not provide leads, whole-genome resequencing showed different single nucleotide changes (C755G/Leu219Val and C655G/Ala252Gly) in the acetate transporter gene ADY2. Analysis of mRNA levels and depth of coverage of DNA sequencing combined with karyotyping, gene deletions and diagnostic PCR showed that in IMW004 an isochromosome III (~475 kb), which contains two additional copies of ADY2C755G, was formed via crossover between YCLWΔ15 and YCRCΔ6. Introduction of the ADY2 alleles in a jen1 ady2 strain resulted in growth on lactate (μmax 0.14 h-1 for Ady2pLeu219Val and 0.12 h-1 for Ady2pAla252Gly). Conclusions: Whole-genome resequencing of yeast strains obtained from independent evolution experiments enabled rapid identification of a key gene that was not identified by mRNA expression analysis of the same strains. Reverse metabolic engineering showed that mutated alleles of ADY2 (C655G and C755G) encode efficient lactate transporters. The goal of the present study was to investigate whether evolutionary engineering and subsequent elucidation of the underlying mutations can lead to the identification of alternative lactate transporters in S. cerevisiae. Transport of carboxylic acids plays a key role in weak organic acids stress and the responsible membrane transporters are often poorly studied and encoded by multiple redundant genes . Additionally, import of lactate is an essential step in the reutilization of lactate produced during intense exercise in mammals [8]. In S. cerevisiae, Jen1p was previously identified as the only efficient lactate importer by generation of a UV-mutant unable to grow on lactate and subsequent functional complementation with a genomic library . The lactate uptake rate of the resulting jen1 knockout strain was close to the detection limit. Interestingly, export of lactate in engineered lactate producing S. cerevisiae is unaffected by deletion of JEN1 (our unpublished results). These observations suggest the presence of at least one alternative lactate transporter. To test this, a jen1 knockout strain was constructed and evolved for faster growth on lactate as the sole carbon and energy source. The evolved strains were subjected to a combination of mRNA expression analysis and whole genome DNA (re)sequencing to identify the relevant mutations. The resulting leads were tested for lactate transport activity via knockout studies in the evolved strains and reverse engineering of the portable genetic elements into non-evolved strains.
2011-12-21 | E-GEOD-31634 | biostudies-arrayexpress
Project description:Metabolomic analysis of Wildtype, crp mutant and its five adaptively evolved populations evolved in glucose minimal media with 40 mM MOPS during its exponential phase of growth. Three biological and two technical replicate samples (n=6) were harvested for each of the strains while growing in a bioreactor aerobically at 37 degree Celsius and 700 rpm. This study aims to characterize and compare the metabolic profile of all these strains.
2020-06-06 | ST001394 | MetabolomicsWorkbench
Project description:Neurospora crassa D27 Resequencing
| PRJNA696601 | ENA
Project description:Extremely thermoacidophilic Crenarchaeota belonging to the family Sulfolobales flourish in hot acidic habitats that are strongly oxidizing. However, the pH extremes of these habitats often exceed the acid tolerance of type species and strains. Here, experimental evolution was used to test whether such organisms harbor additional thermoacidophilic capacity. Three distinct cell lines derived from a single type species were subjected to high temperature serial passage while culture acidity was gradually increased. A 178-fold increase in thermoacidophily was achieved after 29 increments of shifted culture pH resulting in growth at pH 0.8 and 80°C. These strains are named super acid resistant crenarchaeota (SARC). Mathematical modeling using growth parameters predicted the limits of acid resistance while genome and transcriptome resequencing provided insights into the underlying mechanisms responsible for evolved thermoacidophily. Transcriptomics of the evolved strains indicates that their unique phenotype may be due to an increased rate of membrane turnover under strong acid conditions.
2015-12-31 | GSE76423 | GEO
Project description:Extremely thermoacidophilic Crenarchaeota belonging to the family Sulfolobales flourish in hot acidic habitats that are strongly oxidizing. However, the pH extremes of these habitats often exceed the acid tolerance of type species and strains. Here, experimental evolution was used to test whether such organisms harbor additional thermoacidophilic capacity. Three distinct cell lines derived from a single type species were subjected to high temperature serial passage while culture acidity was gradually increased. A 178-fold increase in thermoacidophily was achieved after 29 increments of shifted culture pH resulting in growth at pH 0.8 and 80°C. These strains are named super acid resistant crenarchaeota (SARC). Mathematical modeling using growth parameters predicted the limits of acid resistance while genome and transcriptome resequencing provided insights into the underlying mechanisms responsible for evolved thermoacidophily. Transcriptomics of the evolved strains indicates that their unique phenotype may be due to an increased rate of membrane turnover under strong acid conditions. 6 Samples were analyzed: 2 replicate control samples [SULA] and 2 replicate experimental samples [SULC and SULB]
2015-12-31 | E-GEOD-76423 | biostudies-arrayexpress
Project description:Background: Evolutionary engineering is a powerful approach to isolate suppressor mutants and industrially relevant genotypes. Until recently, DNA microarray analysis was the only affordable genome-wide approach to identify the responsible mutations. This situation has changed due to the rapidly decreasing costs of whole genome (re)sequencing. DNA microarray-based mRNA expression analysis and whole genome resequencing were combined in a study on lactate transport in Saccharomyces cerevisiae. Jen1p is the only S. cerevisiae lactate transporter reported in literature. To identify alternative lactate transporters, a jen1Δ strain was evolved for growth on lactate. Results: Two independent evolution experiments yielded Jen1p-independent growth on lactate (μmax 0.14 and 0.18 h-1 for single-cell lines IMW004 and IMW005, respectively). Whereas mRNA expression analysis did not provide leads, whole-genome resequencing showed different single nucleotide changes (C755G/Leu219Val and C655G/Ala252Gly) in the acetate transporter gene ADY2. Analysis of mRNA levels and depth of coverage of DNA sequencing combined with karyotyping, gene deletions and diagnostic PCR showed that in IMW004 an isochromosome III (~475 kb), which contains two additional copies of ADY2C755G, was formed via crossover between YCLWΔ15 and YCRCΔ6. Introduction of the ADY2 alleles in a jen1 ady2 strain resulted in growth on lactate (μmax 0.14 h-1 for Ady2pLeu219Val and 0.12 h-1 for Ady2pAla252Gly). Conclusions: Whole-genome resequencing of yeast strains obtained from independent evolution experiments enabled rapid identification of a key gene that was not identified by mRNA expression analysis of the same strains. Reverse metabolic engineering showed that mutated alleles of ADY2 (C655G and C755G) encode efficient lactate transporters.
2011-12-21 | GSE31634 | GEO
Project description:Saccharomyces cerevisiae has been used as a secretion host for production of various products, including pharmaceuticals. However, few antibody molecules have been functionally expressed in S. cerevisiae due to the incompatible surface glycosylation. Our laboratory previously isolated a group of yeast mutant strains with different α-amylase secretory capacities, and these evolved strains have showed advantages for production of some heterologous proteins. However, it is not known whether these secretory strains are generally suitable for pharmaceutical protein production. Here, three non-glycosylated antibody fragments with different configurations (Ran-Fab fragment Ranibizumab, Pex-the scFv peptide Pexelizumab, and Nan-a single V-type domain) were successfully expressed and secreted in three background strains with different secretory capacities, including HA (wild type), MA (evolved strain), and LA (evolved strain). However, the secretion of Ran and Nan were positively correlated with the strains’ secretory capacity, while Pex was most efficiently secreted in the parental strain. Therefore, transcriptional analysis was performed to explore the fundamental changes triggered by the expression of the different pharmaceutical proteins in these selected yeast strains.
2021-07-06 | GSE179391 | GEO
Project description:Salmonella enterica serovar Typhimurium genome sequencing of hns mutant evolved strains
| PRJNA213246 | ENA
Project description:Analysis of copy number variation in evolved haploid, diploid, tetraploid strains.
2015-02-28 | GSE51017 | GEO
Project description:C. glutamicum strains adapted to higher growth temperatures were obtained through an adaptive laboratory evolution experiment. To elucidate molecular basis for thermotolerance acquired by the evolved strains, we examined transcriptional responses of the evolved and parental strains to thermal stress using microarray technology.
2015-01-06 | GSE64654 | GEO