Improving acetate tolerance of Escherichia coli by rewiring Its global regulator cAMP receptor protein (CRP)
Date of Issue2013
School of Chemical and Biomedical Engineering
The presence of acetate exceeding 5 g/L is a major concern during E. coli fermentation due to its inhibitory effect on cell growth, thereby limiting high-density cell culture and recombinant protein production. Hence, engineered E. coli strains with enhanced acetate tolerance would be valuable for these bioprocesses. In this work, the acetate tolerance of E. coli was much improved by rewiring its global regulator cAMP receptor protein (CRP), which is reported to regulate 444 genes. Error-prone PCR method was employed to modify crp and the mutagenesis libraries (~3×106) were subjected to M9 minimal medium supplemented with 5–10 g/L sodium acetate for selection. Mutant A2 (D138Y) was isolated and its growth rate in 15 g/L sodium acetate was found to be 0.083 h-1, much higher than that of the control (0.016 h-1). Real-time PCR analysis via OpenArray® system revealed that over 400 CRP-regulated genes were differentially expressed in A2 with or without acetate stress, including those involved in the TCA cycle, phosphotransferase system, etc. Eight genes were chosen for overexpression and the overexpression of uxaB was found to lead to E. coli acetate sensitivity.
DRNTU::Engineering::Chemical engineering::Biochemical engineering
© 2013 The Author(s). This paper was published in PLoS ONE and is made available as an electronic reprint (preprint) with permission of The Author(s). The paper can be found at the following official DOI: http://dx.doi.org/10.1371/journal.pone.0077422. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper is prohibited and is subject to penalties under law.