Evolution of Escherichia coli to 42 °C and Subsequent Genetic Engineering Reveals Adaptive Mechanisms and Novel Mutations
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ABSTRACT:
ORGANISM(S): Escherichia coli str. K-12 substr. MG1655
PROVIDER: GSE132442 | GEO | 2019/06/11
REPOSITORIES: GEO
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Project description:We carried out adaptive laboratory evolution of an E. coli strain lacking four genes (adhE, pta, ldhA, frdA) involved in acetyl-CoA consumption, allowing the efficient utilization of acetate as its sole carbon and energy source. The transcriptomes according to the medium status (M9 aceate, M9 glucose) of the evolved strain (SBA01) and its parent strain (DSM01) were compared using RNA-seq.
2020-09-15 | GSE157942 | GEO
Project description:Adaptive laboratory evolution (ALE) has emerged as a valuable method by which to investigate microbial adaptation to a desired environment. Here, we performed ALE to 42 °C of ten parallel populations of Escherichia coli K-12 MG1655 grown in glucose minimal media. Tightly controlled experimental conditions allowed selection based on exponential-phase growth rate, yielding strains that uniformly converged toward a similar phenotype along distinct genetic paths. Adapted strains possessed as few as 6 and as many as 55 mutations, and of the 144 genes that mutated in total, 14 arose independently across two or more strains. This mutational recurrence pointed to the key genetic targets underlying the evolved fitness increase. Genome engineering was used to introduce the novel ALE-acquired alleles in random combinations into the ancestral strain, and competition between these engineered strains reaffirmed the impact of the key mutations on the growth rate at 42 °C. Interestingly, most of the identified key gene targets differed significantly from those found in similar temperature adaptation studies, highlighting the sensitivity of genetic evolution to experimental conditions and ancestral genotype. Additionally, transcriptomic analysis of the ancestral and evolved strains revealed a general trend for restoration of the global expression state back toward preheat stressed levels. This restorative effect was previously documented following evolution to metabolic perturbations, and thus may represent a general feature of ALE experiments. The widespread evolved expression shifts were enabled by a comparatively scant number of regulatory mutations, providing a net fitness benefit but causing suboptimal expression levels for certain genes, such as those governing flagellar formation, which then became targets for additional ameliorating mutations. Overall, the results of this study provide insight into the adaptation process and yield lessons important for the future implementation of ALE as a tool for scientific research and engineering.
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Project description:Laboratory adaptive evolution experiments were conducted using serial passage of E. coli in M9 minimal medium supplemented with either 2 g/L of lactate for 60 days or 2 g/L of glycerol for 44 days. 7 parallel evolution strains were generated for growth on lactate and 7 parallel evolution strains were generated for growth on glycerol. Affymetrix arrays were used to study the time-course change in gene expression from unevolved E. coli (day 0) to a midpoint evolved strain (day 20) and evolutionary endpoints
2011-10-22 | GSE33147 | GEO
Project description:Laboratory adaptive evolution experiments were conducted using serial passage of E. coli in M9 minimal medium supplemented with either 2 g/L of lactate for 60 days or 2 g/L of glycerol for 44 days. 7 parallel evolution strains were generated for growth on lactate and 7 parallel evolution strains were generated for growth on glycerol. Affymetrix arrays were used to study the time-course change in gene expression from unevolved E. coli (day 0) to a midpoint evolved strain (day 20) and evolutionary endpoints Biological replicate arrays were conducted for each of the time points tested for the different evolution strains
2011-10-21 | E-GEOD-33147 | biostudies-arrayexpress
Project description:Evolution of Escherichia coli to 42 °C and Subsequent Genetic Engineering Reveals Adaptive Mechanisms and Novel Mutations
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Project description:mRNA sequencing was performed on E. coli evolved at 37C on M9 Minimal Media with Glucose
2014-12-03 | GSE61327 | GEO
Project description:Transcriptional profiling of E. coli MG1655 to Trimethoprim in i) LB media, ii) M9 minimal media and iii) M9 minimal media with supplements to study the association of gene expression with corresponding lethal and non-lethal growth conditions. Supplementation conditions in M9 media included addition of 50 microgram/ml of adenine, methionine, glycine or thymine in various combinations.
2011-10-04 | GSE32562 | GEO
Project description:We performed genomic sequencing of whole-genome amplified DNA and native DNA isolated during growth in one of five conditions. We sequenced the DNA using Oxford Nanopore and compared the signals from the whole genome amplified DNA to the native DNA to infer sites at which the native DNA was methylated. The file names here are denoted via the strain name (SC419, SC452, or SC469), the growth condition (37C M9, 42C M9, 25C M9, rich media LB, 96 hours of growth), and in two cases, the replicate culture (M9_rep1 and M9_rep2)
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Project description:The strategy of anaerobic biosynthesis of β-alanine by Escherichia coli (E. coli) has been reported. However, the low energy production under anaerobic condition limited cell growth and then affected the production efficiency of β-alanine. Here, the adaptive laboratory evolution was carried out to improve energy production of E. coli lacking phosphoenolpyruvate carboxylase under anaerobic condition. Five mutants were isolated and analyzed. Sequence analysis showed that most of the consistent genetic mutations among the mutants were related with pyruvate accumulation, indicating that pyruvate accumulation enabled the growth of the lethal parent. It is possible that the accumulated pyruvate provides sufficient precursors for energy generation and CO2 fixing reaction catalyzed by phosphoenolpyruvate carboxykinase. B0016-100BB (B0016-090BB, recE::FRT, mhpF::FRT, ykgF::FRT, mhpB:: mhpB *, mhpD:: mhpD *, rcsA:: rcsA *) was engineered based on the analysis of the genetic mutations among the mutants for the biosynthesis of β-alanine. Along with the recruitment of glycerol as the sole carbon source, 1.07 g/L β-alanine was generated by B0016-200BB (B0016-100BB, aspA::FRT) harboring pET24a-panD-AspDH, which was used for overexpression of two key enzymes in β-alanine fermentation process. Compared with the starting strain, which can hardly generate β-alanine under anaerobic condition, the production efficiency of β-alanine of the engineered cell factory was significantly improved.
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Project description:Efforts are underway to construct several recoded genomes anticipated to exhibit multivirus resistance, enhanced nonstandard amino acid (nsAA) incorporation, and capability for synthetic biocontainment. Although our laboratory pioneered the first genomically recoded organism (Escherichia coli strain C321.∆A), its fitness is far lower than that of its nonrecoded ancestor, particularly in defined media. This fitness deficit severely limits its utility for nsAA-linked applications requiring defined media, such as live cell imaging, metabolic engineering, and industrial-scale protein production. Here, we report adaptive evolution of C321.∆A for more than 1,000 generations in independent replicate populations grown in glucose minimal media. Evolved recoded populations significantly exceeded the growth rates of both the ancestral C321.∆A and nonrecoded strains. We used next-generation sequencing to identify genes mutated in multiple independent populations, and we reconstructed individual alleles in ancestral strains via multiplex automatable genome engineering (MAGE) to quantify their effects on fitness. Several selective mutations occurred only in recoded evolved populations, some of which are associated with altering the translation apparatus in response to recoding, whereas others are not apparently associated with recoding, but instead correct for off-target mutations that occurred during initial genome engineering. This report demonstrates that laboratory evolution can be applied after engineering of recoded genomes to streamline fitness recovery compared with application of additional targeted engineering strategies that may introduce further unintended mutations. In doing so, we provide the most comprehensive insight to date into the physiology of the commonly used C321.∆A strain.
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