Kyoung Heon Kim

As a renewable energy from biomass, isobutanol is considered as a promising alternative to fossil fuels. To biotechnologically produce isobutanol, strain development using industrial microbial hosts, such as Escherichia coli , has been conducted by introducing a heterologous isobutanol synthetic pathway. However, the toxicity of produced isobutanol inhibits cell growth, thereby restricting improvements in isobutanol titer, yield, and productivity. Therefore, the development of robust microbial strains tolerant to isobutanol is required. In this study, isobutanol‐tolerant mutants were isolated from two E. coli parental strains, E. coli BL21(DE3) and MG1655(DE3), through adaptive laboratory evolution (ALE) under high isobutanol concentrations. Subsequently, 16 putative genes responsible for isobutanol tolerance were identified by transcriptomic analysis. When overexpressed in E. coli , four genes ( fadB, dppC, acs , and csiD ) conferred isobutanol tolerance. A fermentation study with a reverse engineered isobutanol‐producing E. coli JK209 strain showed that fadB or dppC overexpression improved isobutanol titers by 1.5 times, compared to the control strain. Through coupling adaptive evolution with transcriptomic analysis, new genetic targets utilizable were identified as the basis for the development of an isobutanol‐tolerant strain. Thus, these new findings will be helpful not only for a fundamental understanding of microbial isobutanol tolerance but also for facilitating industrially feasible isobutanol production.