William Lin B00554595 February 8, 2013 MICI.

4033 Summary Cell tolerance of physical and chemical stresses is an important research topic in biofuel production, which involves high temperatures and toxic compounds. Tolerance is governed by the holistic action of many proteins and genes in concert, not by a single enzyme. Mutating each specific gene in a metabolic pathway is laborious, and often the systems involved in resistance to a particular substance are not even completely characterized. Recent approaches like global transcriptional machinery engineering (gTME) circumvent this problem by altering transcription profiles to vary expression of downstream pathways and access new ones. Chen et al. describe a novel method of introducing irrE, an exogenous global transcription regulator associated with the extreme radiation tolerance of Deinococcus radiodurans, into Escherichia coli to elicit tolerance toward numerous stresses, including ethanol, butanol, osmotic, and heat stress tolerance. Although E. coli strains expressing wildtype irrE had improved tolerance of osmotic, heat and oxidative stress, mutation of the irrE protein was required in order for it to confer tolerance of ethanol and butanol, which are important biofuels. A mutant irrE library was constructed using error-prone PCR and placed under control of the GroESL promoter in plasmid pMD18T for constitutive expression. DH5 E. coli cells were transformed with the plasmid and were screened for growth in up to 5% ethanol. Cell growth was quantified by measuring the optical density of the culture (OD600). Five mutants were found to be significantly resistant to ethanol, one mutant strain having a 50-fold increase of growth in 5% ethanol compared to the control strain. An ethanol shock assay was also performed by growing cells and then exposing them to 12.5% ethanol for 1 hour. The best mutant displayed a 100-fold increase in surviving cells compared to the control.

Similar growth and shock experiments were performed using butanol and acetate, resulting in mutants that displayed a 5-fold increase of growth in 0.625% butanol and a 100-fold increase of surviving cells after shock with 2.1% butanol, and mutants that displayed 2-fold increase of growth in 0.05% acetate. Sequence alignment to D. desertis, whose irrE structure is solved, determined that mutations occurred in all three regions of the irrE protein, including the peptidase, DNA-binding, and small molecule-sensing domains. Reactive oxygen species (ROS) levels are increased in cells that are challenged with stress from oxidative or toxic substances. ROS levels were quantified by introducing H2DCFDA, which fluoresces when oxidized by ROS, into E. coli cells. Lower levels of ROS were found in mutant irrE cells compared to wild-type control cells, approximately a 15-fold decrease for cells grown in ethanol and a 10-fold decrease for cells grown in butanol. The authors describe a novel method of introducing an exogenous global regulator into E. coli to improve stress tolerance, supplementing existing methods of industrial strain improvement, like gTME. This study provides insight into cellular tolerance of toxic compounds and into extension of the perturbation range of transcriptional profiles, allowing for many more tolerance phenotypes to be discovered.

Chen T, Wang J, Yang R, Li J, Lin M, et al. (2011) Laboratory-Evolved Mutants of an Exogenous Global Regulator, IrrE from Deinococcus radiodurans, Enhance Stress Tolerances of Escherichia coli. PLoS ONE 6(1): e16228. doi:10.1371/journal.pone.0016228