Tailoring of global transcription sigma D factor by random mutagenesis to improve Escherichia coli tolerance towards low-pHs
•RAISE was first used to engineer the global regulator RpoD of E. coli to improve its acid tolerance.•Best mutant was obtained through multiwheel of acid-shock screening with significantly improved growth rate at pH 3.17.•Combined transcriptome and phenome analysis of E. coli was carried out to better understand the global effects of RpoD on the regulatory networks.•RpoD factor can regulate both the master regulators and gene/operon-specific transcription factors.
Bioconversion processes of organic acid or acid hydrolysis of raw material for microbial metabolism often suffer limitations as a result of microbial sensitivity in low-pH conditions. We adopted a three-step method called RAndom Insertional-deletional Strand Exchange mutagenesis (RAISE) to engineer the components of global regulator Sigma D factor (RpoD) of Escherichia coli to improve its acid tolerance. The best strain Mutant VII was identified from random mutagenesis libraries based on the growth performance, which exhibited much higher growth rate than the control (0.22 h−1 vs. 0.15 h−1) at pH as low as 3.17. Combined transcriptome and phenome analysis of E. coli was carried out to better understand the global effects of RpoD on the regulatory networks. Our analysis showed that 95 (2.1%) of all E. coli genes were induced and 178 (4.0%) genes were repressed, including those for trehalose biosynthesis, nucleotides biosynthesis, carbon metabolism, amino acid utilization, except for acid resistance. Also regulated were the master regulators (ArcA, EvgA, H-NS and RpoS) and gene/operon-specific transcription factors (GadX, GadW, AppY, YdeO, KdgR). These results demonstrated that RpoD acts as global regulator in the growth phase of E. coli and consequently improves acid tolerances.
Journal: Journal of Biotechnology - Volume 224, 20 April 2016, Pages 55–63