The Improved Stress Tolerance of Escherichia coli by Directed Evolution of IrrE

Abstract

IrrE, a global regulator of Deinococcus radiodurans, has proven to be effective in enhancing microbial stress tolerance. In this paper, IrrE from D. radiodurans R1, was introduced into Escherichia coli and directed evolved by error-prone PCR. The influence of mutation of IrrE on the cell growth and tolerance to various stresses was further investigated. First, one of the mutations, designated M4 with higher ethanol tolerance was obtained by error-prone PCR using the pET-28a(+)-irrE(W) as a template. The OD₆₀₀ value of M4 reached 1.6 after 28 h cultivation under 8 % ethanol, while no obvious cell growth was observed in the recombinant strain harboring plasmid pET-28a(+)-irrE(W) and the control strain harboring plasmid pET-28a(+). The cell viability of M4 under different stress shock conditions (such as pH = 5.0, pH = 10.0, 3 mmol/L sorbitol and 15 % methanol), the cell growth rate, and the final biomass were improved obviously. The sequence comparison of irrE revealed that there were two sense substitutions (C24T and G530A). The substitution of G530A caused one amino acid change Gly177Glu. The homology modeling of IrrE was built according to the known structure of IrrE protein from D. deserti, which showed that the amino acid mutation located in HTH motif of IrrE. The work laid a foundation for further research on the relationship of IrrE structure and host cell tolerance to stress.