Improved n-butanol production in Escherichia coli by combining strain development and flux optimization

Abstract

As the increasing demand from chemical and fuel markets, the interest in producing n-butanol using biological route has been rejuvenated to engineer an economical fermentation process, competing with the chemical synthesis. In addition to the traditional researches of understanding and engineering the n-butanol producer, Clostridium, to be more effective at producing solvents at the economically feasible level, one can transfer this synthesis pathway into more advanced hosts and optimize it with available engineering tools. It has been demonstrated that Escherichia coli is an appropriate alternative host for n-butanol pathway and it has become the second most effective host since the last 5 years of intensive study. Considering the feasibility to scaling up a fermentation process, beside the high efficiency of the synthetic pathway, how the host can be easily controlled and adapted to the industrial conditions is also very critical. The fact that n-butanol is very toxic to most of the commonly known microbial hosts, including Clostridium and E. coli is considered as a major drawback limiting the ability of this process to be expanded to the industrial scale. Having successfully improved the n-butanol tolerance of E. coli employing our Artificial Transcription Factor (ATF), we have sought other means to further increase the limit of E. coli tolerance and, at the same time, introduced and improved a heterologous n-butanol pathway in E. coli to justify the role of n-butanol tolerance with the whole process. Since the n-butanol tolerant ATF was based on a random approach, we applied rational strategies to complement it, including controlling the membrane fatty acid composition, overexpressions of molecular chaperones, efflux system, and iron uptake. On the other hand, a DNA scaffold system for creating an enzymatic cascade was constructed using our library of DNA-binding zinc finger proteins to facilitate the butanol synthesis pathway. Putting together, the use of ATF in combination with iron uptake protein and high membrane unsaturated fatty acid extended the ability of E. coli to tolerate up to 2% n-butanol, supporting the rapid enzymatic cascade generated by DNA scaffold system and improving the final n-butanol production.