Improving E.coli performance under stress by rewiring its global regulator Camp Receptor Protein (CRP)
Date of Issue2013
School of Chemical and Biomedical Engineering
BioMedical Engineering Research Centre
Strain engineering tools are usually adopted to improve microorganism tolerance towards stressful environments in bioindustries. However, classical strain engineering techniques of using UV/chemical mutagens are often both labor- and time- intensive. In recent years, transcriptional engineering approach has started to attract attention in strain engineering. The better efficiency of this method has made it more preferable over the classical methods. Here in this thesis, I would adopt error-prone PCR technique to engineer global transcription factor cAMP receptor protein (CRP), which can regulate more than 400 genes in Escherichia coli, to enhance its tolerance against organic solvents, oxidative stress, and low pH. E. coli DH5α-∆crp strain was transformed with the plasmid-containing crp mutants, which were generated from error-prone PCR, followed by a selection under various stresses. All selected mutants exhibited much better tolerance than the wild type (WT) against respective stress. For example, the best toluene tolerant mutant showed growth even in 0.23% (v/v) toluene 0.51 h-1 whereas the growth of WT was completely inhibited. The best oxidative stress mutant demonstrated viability in 12 mM H2O2, while WT growth was halted at 6 mM H2O2. The best mutant identified under various stresses were then subjected to various characterizations, including cross-tolerance check, DNA microarray analysis, quantitative real time PCR and enzyme assay. For instance, toluene-tolerant mutant was also able to have improved growth against n-hexane, cyclohexane and p-xylene while oxidative-stress-tolerant mutant also exhibits thermotolerance. DNA microarray analysis and qRT-PCR results have demonstrated that the modifications to CRP would not only bring differential expression in CRP-regulated genes but also those non-CRP-regulated genes. In conclusion, random mutagenesis of CRP can provide an efficient alternative for E. coli strain engineering.