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

Abstract

BackgroundEconomically 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.ResultsHere, 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.ConclusionsThe combination of the transcriptional response and mutant screening provides general targets that can increase tolerance towards not only single, but multiple chemicals.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0577-5) contains supplementary material, which is available to authorized users.

Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida

“…The role of these genes, of which 40% have unknown function, and their involvement in a general stress response are an interesting area for future investigation. In addition, the insights gained here could be combined with transposon-insertion sequencing experiments under similar conditions to identify target genes that affect fitness during stress, with the aim of generating more robust strains as in several studies in E. coli (84,85). Moreover, the transcriptomic data collected here combined with proteomic studies may yield important insights into regulation at the posttranscriptional level, including the involvement of sRNAs.…”

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

“…We therefore decided to screen P. putida KT2440 for its tolerance toward a number of chemical compounds that could be relevant for biobased production, and that are known to be relatively toxic to E. coli (Rau, Calero, Lennen, Long, & Nielsen, 2016). Six of the compounds, hydroxy‐ γ ‐butyrolactone, furfural, itaconic acid, levulinic acid, succinic acid, and L‐threonine, have previously been identified as being among the top 30 predicted chemical building blocks in a report from the U.S Department of Energy (Werpy & Petersen, 2004).…”

“…P. putida KT2440 tolerance to these chemical compounds was tested by assaying their effect on the growth rate using different concentrations, using conditions identical to those used previously for E. coli K‐12 MG1655 (Rau et al, 2016). In order to compare the effect the different compounds have on P. putida KT2440 and E. coli K‐12 MG1655 growth, we calculated the concentration of each compound required to reduce the growth rate by 33%, as described previously (Rau et al, 2016).…”

“…In order to compare the effect the different compounds have on P. putida KT2440 and E. coli K‐12 MG1655 growth, we calculated the concentration of each compound required to reduce the growth rate by 33%, as described previously (Rau et al, 2016). The concentrations of compounds needed to reduce the growth rate in P. putida KT2440 varied significantly.…”

Investigation of monoterpenoid resistance mechanisms in Pseudomonas putida and their consequences for biotransformations

“…For the development of microbial monoterpenoid production strains, a deep molecular understanding of the tolerance mechanisms for such compounds represents a prerequisite. As P. putida shows strong monoterpenoid tolerance (Inoue and Horikoshi 1991;Speelmans et al 1998;Mi et al 2014;Rau et al 2016) and its organic solvent resistance mechanisms have been extensively studied (Isken and de Bont 1998;Ramos et al 2015), we chose the P. putida strain GS1 (Speelmans et al 1998) to explore its behavior after exposure to a number of different monoterpenoids (Fig. 1).…”