Phenotypic engineering of Escherichia coli using novel artificial transcription factors = 새로운 인공 전사 인자를 이용한 대장균의 phenotypic engineering 연구

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Now that many genomes have been sequenced and the products of newly identified genes have been annotated, the next goal is to engineer the desired phenotypes in organisms of interest. For the phenotypic engineering of microorganisms, we have developed novel artificial transcription factors (ATFs) capable of reprogramming innate gene expression circuits in Escherichia coli. These ATFs are composed of zinc finger (ZF) DNA-binding proteins, with distinct specificities, fused to an $\It{E. coli}$ cyclic AMP receptor protein (CRP). By randomly assembling 40 different types of ZFs, we have constructed more than $6.4\times10^4$ ATFs that consist of 3 ZF DNA-binding domains and a CRP effector domain. Using these ATFs, we induced various phenotypic changes in $\It{E. coli}$ and selected for industrially important traits, such as resistance to heat shock, osmotic pressure, and cold shock. The genes of $\It{E. coli}$ that are associated with the thermotolerance were then identified and characterized. We have also observed that our ATF can generate various levels of phenotypic improvements, such as the different degree of thermotolerance, when they were expressed at various levels with different expression vectors. In addition, with these ATFs, we selected a butanol-tolerant $\It{E. coli}$ which can tolerate up to 1.5% (vol/vol) butanol, ith a concomitant increase in heat resistance, and then engineered a butanol production pathway into the selected butanol-tolerant $\It{E. coli}$. This engineered butanol-tolerant $\It{E. coli}$ shows an $\sim 13%$ increase in butanol production compared to the wild-type $\It{E. coli}$. Furthermore, we showed the application of these ATFs to the butanol-producing $\It{Clostridium acetobutylicum}$ for improved butanol tolerance and selected a butanol-tolerant $\It{C. acetobutylicum}$, which shows an $\sim 110%$ increase in butanol production compared to the wild-type $\It{C. acetobutylicum}$. These results and the general applic...
Kim, Sun-Changresearcher김선창researcher
한국과학기술원 : 생명과학과,
Issue Date
418831/325007  / 020055868

학위논문(박사) - 한국과학기술원 : 생명과학과, 2010.2, [ vii, 140 p. ]


Heat resistance; Phenotype engineering; Artificial transcription factor; E. coli; Biofuel butanol; 바이오 에너지 부탄올; 열저항성; 균주 특성 개발; 인공 전사 인자; 대장균

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