Genome-wide Escherichia coli stress response and improved tolerance towards industrially relevant chemicals

Abstract

Background: Economically viable biobased production of bulk chemicals and biofuels typically requires high product titers. During microbial bioconversion this often leads to product toxicity, and tolerance is therefore a critical element in the engineering of production strains.

Results: Here, a systems biology approach was employed to understand the chemical stress response of Escherichia coli, including a genome-wide screen for mutants with increased fitness during chemical stress. Twelve chemicals with significant production potential were selected, consisting of organic solvent-like chemicals (butanol, hydroxy-γ-butyrolactone, 1,4-butanediol, furfural), organic acids (acetate, itaconic acid, levulinic acid, succinic acid), amino acids (serine, threonine) and membrane-intercalating chemicals (decanoic acid, geraniol). The transcriptional response towards these chemicals revealed large overlaps of transcription changes within and between chemical groups, with functions such as energy metabolism, stress response, membrane modification, transporters and iron metabolism being affected. Regulon enrichment analysis identified key regulators likely mediating the transcriptional response, including CRP, RpoS, OmpR, ArcA, Fur and GadX. These regulators, the genes within their regulons and the above mentioned cellular functions therefore constitute potential targets for increasing E. coli chemical tolerance. Fitness determination of genome-wide transposon mutants (Tn-seq) subjected to the same chemical stress identified 294 enriched and 336 depleted mutants and experimental validation revealed up to 60 % increase in mutant growth rates. Mutants enriched in several conditions contained, among others, insertions in genes of the Mar-Sox-Rob regulon as well as transcription and translation related gene functions.

Conclusions: The combination of the transcriptional response and mutant screening provides general targets that can increase tolerance towards not only single, but multiple chemicals.

Keywords: Biochemicals; Chemical stress; E. coli; Systems biology; Tn-seq; Tolerance; Transcription analysis.

Fig. 1

a Structures of the chemicals employed in this study. Twelve chemicals were included that based on properties can be divided into four groups. b Concentrations (mM) causing a 33 % growth rate reduction

Fig. 2

a Heatmap and hierarchical clustering of transcriptomic data for all conditions. Only significant genes are included. Red and blue signifies high and low relative expression, respectively. b Quantity of significantly upregulated (blue) and downregulated (red) genes for each condition. c Venn diagram displaying the overlap of significant genes within and between three of the chemical groups. Only significant genes changed in the same direction within or between groups are included

Fig. 3

Venn diagram displaying regulators with significantly enriched regulons within and between chemical groups. Regulons enriched in 3 out of 4 conditions for acids and organic solvents and 2 out of 2 conditions for amino acids and membrane-intercalating chemical groups are included as enriched in the respective chemical group

Fig. 4

Significantly enriched or depleted Tn-seq transposon insertion mutants. a Quantity of significantly enriched (blue) or depleted (red) gene transposon insertion mutants for each stress condition. b Fold changes of significant genes for which transposon mutants are either enriched (positive fold changes) or depleted (negative fold changes)

Fig. 5

Selected cellular functions with differential gene expression in multiple organic solvent and/or acid conditions. Blue highlighting signifies upregulation and red highlighting signifies downregulation of genes associated with the particular function. Colored squares denote gene expression changes present in at least 3 out of 4 organic solvent (green squares) or acid (orange squares) conditions. Gene regulators are depicted in grey letters with arrows indicating functions on which they exert a regulatory effect