(593aa) Engineering Global Regulator cAMP Receptor Protein (CRP) of E. Coli to Improve Strain Performance Under Stress

Strain engineering has long been an intensively studied topic in both industry and academic. In general, there are two major categories in strain engineering: 1) “random” approach by using mutagens (UV, N-methyl-N’-nitro-N-nitrosoguanidine, and etc.), which is often time and resource intensive; 2) “rational”approach that uses metabolic engineering tools. Since most microorganisms lack of detailed metabolism knowledge and genotype-phenotype information, the use of metabolic engineering tools is often limited. Even engineering the simplest microbes like E. coli can be very complicated.

Here, we want to demonstrate that engineering global regulator cAMP receptor protein (CRP) of E. coli can improve cell performance under stressful conditions. CRP is one of the global regulatory proteins that can regulate the transcription of over 400 genes in E. coli. Our first target phenotype was strain osmotolerance.  Amino acid mutations were introduced to CRP by error-prone PCR and DNA shuffling, and the random mutagenesis libraries were subjected to enrichment selection under NaCl stress. Five CRP mutants (MT1− MT5) were selected from the error-prone PCR libraries with enhanced osmotolerance. DNA shuffling technique was employed to generate mutant MT6 with MT1 – MT5 as templates. All of these variants showed much better growth in the presence of NaCl compared to wild type, and MT6 presented the best tolerance towards NaCl. In the presence of 0.9M NaCl, the growth rate of MT6 was 0.113 h^-1, while that of WT is 0.077h^-1. MT6 also exhibited resistance to other osmotic stressors, such as KCl, glucose and sucrose. Microarray analysis demonstrated that mutations in CRP could influence the expression level of genes not regulated by CRP. Scanning electron microscopy images confirmed the elongation of both WT and MT6 when exposed to NaCl but the cell surface of MT6 was relatively smooth.¹

We have also applied the same technique to improve strain biofuel (1-butanol) tolerance², tolerance towards oxidative stress, and organic solvent tolerance. All selected mutant E. coli strains have shown much better performance under stress. In conclusion, transcriptional engineering via CRP can provide an efficient alternative for E. coli strain engineering.

1. Zhang, H.; Chong, H.; Ching, C.; Jiang, R. Biotech. Bioeng. 2012, 109(5), 1165-1172
2. Zhang, H.; Chong, H.; Ching, C.; Song, H.; Jiang, R. Appl. Microb. Biotech. 2012, 94 (4), 1107-1117