Enhancing the 1-Butanol Tolerance in Escherichia coli through Repetitive Proton Beam Irradiation

The low butanol tolerance of most microorganisms severely limits the production of 1-butanol on an economical scale in alternative hosts other than the natural butanol producer Clostridium acetobutylicum, which is not amenable for genetic manipulation and requires demanding culture conditions. To generate butanol-tolerant E. coli, we devised a cyclic selection strategy that consists of an iterative application of proton irradiation at a dose of similar to 250 Gy by using 45-MeV protons and selection by daily serial transfer in a minimal medium containing increasing concentratioils of 1-butanol. Applying five rounds of the cyclic selection of E. colt ATCC 8739 (a C strain) over 61 days resulted in a mutant population that could tolerate 1.2% 1-butanol (v/v). However, without proton irradiation, the cells were unable to grow at >= 0.8% 1-butanol in a control experiment. Seven different mutations were identified within one clone from the endpoint population through 454 pyrosequencing of the genome. Tracing each mutation in terms of the prevalence in the population during the period of evolution suggested that proton beam irradiation-induced mutations were rapidly fixed during the early phase of the selection procedure. This approach, which is still being applied to increase butanol tolerance beyond 1.3%, can be considered useful for improving targeted traits whose corresponding genes are unknown.