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Deep sequencing and fitness calculation for plasmids carrying the CREATE cassette from the enriched tolerant strains under different pressures by using Illumina protocol

ABSTRACT: We report using global regulator libraries based on the CRISPR-enabled trackable genome engineering (CREATE) method to engineer tolerance against multiple inhibitors in Escherichia coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target 34,340 mutations across 23 global regulators.These libraries were divided into G1, G2, G3, G4 and G5 sub-groups according to different gene functions (such as active sites, DNA binding sites, and predicted functional sites) . And we successively screened the libraries under different stress conditions (acetate, NaCl, furfural, high temperature and isobutanol), and enriched mutants able to tolerate multiple inhibitors. In order to determine the tolerance-conferring mutations screened under different stress conditions, deep sequencing and fitness analysis were used to detect the recombinant strains with good performance.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE200448 | GEO | 2024/04/01

REPOSITORIES: GEO

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Project description:BACKGROUND:Isobutanol is a promising next generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titers. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases on adaptation to exogenous isobutanol stress. RESULTS:The adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced rpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodelling the cell envelope and surprisingly, stress response attenuation. CONCLUSIONS:We have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for futurue global phenotype engineering. Two strains (WT strain and G3.2 mutant strain), each with two culture conditions (with and without isobutanol in medium). Three biological replicates for each strain/culture condition. Twelve samples in total.

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Deep sequencing and fitness calculation for plasmids carrying the CREATE cassette from the enriched tolerant strains under different pressures by using Illumina protocol

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