Error-prone PCR of global transcription factor cyclic AMP receptor protein for enhanced organic solvent (toluene) tolerance

Abstract

Organic solvents are often applied in biphasic biocatalysis or involved in bioremediation, but native microorganisms usually have low tolerance toward these organic solvents and cannot survive in the stressful environment. Here, we aimed to improve the organic solvent tolerance of Escherichia coli by engineering its global transcription factor cAMP receptor protein (CRP). Toluene was chosen as model organic solvent. The mutated crp genes were generated via error-prone PCR and three toluene-tolerant mutants M1–M3 were isolated from random mutagenesis libraries by enrichment selection. All mutants exhibited much better growth than the wild type upon exposure to 0.2–0.23% (v/v) toluene, with M2 the best. When toluene concentration was at 0.2%, M1–M3 shared similar growth rate at 0.68 h−1 despite the elongated 20-h lag phase, with WT exhibiting null growth. In 0.23% toluene, the growth rate of M2 was found to be 0.51 h−1 while the growth of WT was completely inhibited. M2 also demonstrated excellent growth in other organic solvents such as n-hexane, cyclohexane and p-xylene as compared to the wild type. Our quantitative real-time reverse transcription PCR analysis with organic-solvent stress associated genes indicated that amino acid mutations in CRP would lead to changes in the expression level of genes that are not regulated by CRP. The field emission scanning electron microscopy images revealed that both wild type and M2, though combating toluene stress, did not have any significant changes in their cellular size and shape.