Development of 1-Butanol Tolerant Escherichia coli using Synthetic Systems Biology Approach
Date Issued
2008
Date
2008
Author(s)
Chang, Hsin-Yi
Abstract
1-Butanol with energy content similar to gasoline is an important industrial solvent and potentially a better fuel substitution than ethanol. However, 1-butanol causes serious product inhibition on the cells themselves while producing the solvents. The aim of this study was to develop 1-butanol tolerant Escherichia coli for applying it in biofuel production. A systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection) was used to screen the butanol-tolerant bacteria. Of the 3,985 nonessential gene mutants, eighty-five were found to exhibit 1-butanol tolerant phenotype, and two of them were displayed reliable 1-butanol tolerance. Four strains were characterized by a novel high-throughput technique, Phenotype MicroArrays (PMs)-Biolog, which can test up to 2000 cellular phenotypes simultaneously. Furthermore, we used proteomics approach to reveal potential proteins involved in 1-butanol tolerance and to understand the physiological response induced by 1-butanol stimulus. After E. coli cells were treated with 0 or 2 % (v/v) 1-butanol, thirty-three differential expressed proteins were separated and identified by 2DE and Q-TOF MS/MS. Our results showed that the anti-oxidative enzymes, chaperones, membrane transporters were the major group increased and proteins participated glycolysis, arginine degradation, tryptophan degradation, ATP synthesis, ATP transport and membrane signaling transduction were down-regulated. Thirteen of them were independently cloned into an IPTG-inducible vector; furthermore, their 1-butanol tolerance was evaluated. Among these overexpressed strains, six had higher 1-butanol tolerance than non-induced control. By introducing the protective proteins, PhoH, MdoG, YdfG, Hmp, YqhD, and TolB, into the ydhF- and yheT- knockout mutants, twelve novel E coli mutants were created. Ultimately, ydhF- mutant overexpressed PhoH demonstrated high 1-butanol tolerance up to 5.5 folds of the control and can tolerate up to 5 % 1-butanol. From this study, we not only obtain the 1-butanol tolerant E. coli mutants, but also have some impact on the development of alternative energy by synthetic systems biology knowledge. Our serial enhancement approach provided a more integrated observation of the process under the 1-butanol treatment. Further characterization of the genes and proteins identified in this study will likely improve our understanding of the complicated nature of solvent tolerance.
Subjects
1-Butanol
Biofuel
Tolerance
E. coli
Systems biology
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