Journal of the Korean Physical Society, Vol. 61, No. 2, July 2012, pp. 227∼233 Changes in Membrane Fatty Acid Composition through Proton-induced fabF Mutation Enhancing 1-butanol Tolerance in E. coli Haeyoung Jeong, ∗ Sun Hong Kim, Sang Soo Han and Myung Hee Kim Systems and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea Keun Chul Lee Korean Collection for Type Cultures, Biological Resource Center, KRIBB, Daejeon 305-806, Korea (Received 24 November 2011) While a rational approach based on genomic data has become the preferred method for microbial strain development, radiation-induced random mutagenesis is still a robust method for organisms such as plants whose genome or target gene information is unavailable. We previously reported on a combined approach that consists of proton irradiation and a long-term experimental evolution to enhance 1-butanol tolerance of the E. coli C strain so that it can be used as a basal strain for the production of 1-butanol, a potential biofuel along with ethanol. Genome sequencing of one randomly chosen clone (PKH5000) from the endpoint population revealed eleven mutations occurring in the coding regions, and we found that a mutation (F74C) in fabF gene encoding β- ketoacyl-ACP synthases II is associated with a twofold increase in the major unsaturated fatty acid, cis -vaccenic acid. The increase of cis -vaccenic acid by wild-type FabF, which is more active at low temperatures or in the presence of organic compounds, is considered to be a protective mechanism against cold stress. A structural analysis of the FabF protein suggests that the F74C mutation may affect the enzyme activity through a change in flexibility around the catalytic site. The expression of a plasmid that harbors mutant fabF gene in the fabF knockout strain enhanced growth in a medium containing butanol with a concomitant elevation of the cis -vaccenic acid level. Among the eight available Keio knockout strains for genes that have amino acid substitution in the PKH5000 strain, the fabF mutant showed the slowest growth in the presence of 0.7% butanol. We propose that fabF, as probably the gene most responsible for butanol tolerance in wild-type form, contributes further when converted into a F74C missense mutation, which is beneficial as it increases the level of cis -vaccenic acid. PACS numbers: 87.50.Gi Keywords: Proton beam, Mutation, Escherichia coli, 1-butanol, Tolerance, fabF, cis -vaccenic acid DOI: 10.3938/jkps.61.227 I. INTRODUCTION Until now, radiation breeding has been widely used to improve biological resources, particularly plants, as it can facilitate the process of mutation induction, which usually requires considerable time and resources [1]. Con- temporary recombinant DNA technology, followed by genome-scale metabolic engineering in the post-genome or ‘omics’ era, has drawn more interest from researchers for its potential application to strain development with desirable characteristics [2,3]. However, mutation breed- ing through radiation is still a valuable tool for crops because the simplicity of its application does not require * E-mail: hyjeong@kribb.re.kr; Tel: +82-42-860-4237; Fax: +82- 42-879-8595 much omics information for the biological sample and be- cause it can simultaneously accommodate a large quan- tity of samples. Moreover, mutation breeding mimics a spontaneous mutagenic process during the evolutionary pathway in nature, much like the generation of cisgenic plants, and thus has an advantage over the genetically- modified organisms (GMOs), which has created world- wide concern [4]. In addition to traditional electromagnetic radiation sources such as UV lights or γ -rays, ion beams produced from large-scale particle accelerators have become avail- able at national-level research facilities supporting both basic sciences and industrial applications. Ion beam ra- diation is regarded as being superior to conventional ra- diation sources as it can generate more diverse mutations with minimal damage to the biological samples [5–7]. We previously reported the enhancement of 1-butanol -227-