The Construction Of Recombinant Escherichia Coli By A Global Transcription Regulator IrrE And Its Performance Analysis

IrrE, a global regulator of Deinococcus radiodurans, show good effect on directed evolution of microbial complex phenotype. This paper used the heterologous expression of irrE gene, from D. radiodurans R1, in model microorganism Escherichia coli and investigated the influence of different stress tolerance for the expression of IrrE in E.coli.The heterologous expression of irrE in E. coli BL21 (DE3) was carried out using the vector pET-28a(+). The optimal protein expression conditions of IrrE were confirmed as follows:2 mmol/L IPTG (the final concentration), and the induction temperature was 37℃ for 6 h. The expression of IrrE in E.coli was confirmed by SDS-PAGE and purified by Ni-NTA His bind resins. The influence of the wild-type IrrE on cell growths BioLog and stress tolerance were further investigated. The results indicated that the wild-type IrrE could enhance the growth rate of E. coli under no stress condition, and improve the cell viability under stress conditions (3 mol/L sorbitol, pH=2, pH=12,10% methanol,12% ethanol and 1%H2O2) to different extent, especially the tolerance to osmotic shock. The cell growth and the final biomass of recombinant strain were significantly higher than the control strain under stress conditions(4% ethanol,5% methanol and 1mol/L sorbitol). The carbon source utilization profile of E. coli expressing IrrE was determined using Biolog.The result shows the wild-type IrrE could improved the substrate utilization of 18 types carbon sourcesA mutant (M4) can significantly improved the tolerance to 8% ethanol was obtained by error-prone PCR. The OD600 value of mutant strain (M4) reached 1.6 after 28 h. The cell viability of mutant M4 under pH=5, pH=10, high osmotic and 15% methanol shock as well as the growth rate and the final biomass under stress were improved obviously.The sequence of irrE gene was analyzed, and the results showed that the mutation were C24T and G530A. The DNA sequencing of mutant irrE revealed 177 glycine mutated into glutamic. The homology modeling of IrrE was build based on the solved structure of . IrrE protein from Deinoeoccus deserti. The results showed the mutational amino acid was in HTH motif of IrrE. The work laid a foundation for further research on the role of global transcription factor IrrE in microbial tolerance phenotypes directed evolution.