Model-driven Optimal Redox-Rebalancing for n-Butanol Synthetic Pathway in Escherichia coli

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Jae Hyung Lim, Sang Woo Seo, Gyoo Yeol Jung

POSTECH, South Korea

Advances in metabolic engineering and synthetic biology could fulfill a global demand for the production of commercially valuable chemicals such as petroleum-derived chemicals, fuels, and pharmaceuticals. To achieve the successful design of the biological systems, however, one of the important issues to be solved is balancing the intracellular redox state that plays a governing factor for the continuation of both catabolism and anabolism. The concept of the rebalancing redox state is also important to make cells efficiently utilize various feedstocks, because typically different catalytic amounts of reducing equivalent are required depending on the carbon flux of the different substrates. Here, we show that how the changes of intracellular redox state affect the pathway performance of n-butanol production from glucose and galactose in Escherichia coli as a model system. We built the synthetic n-butanol production pathway by implementing synthetic constitutive promoters and designing synthetic 5’-untranslated regions (5’-UTR) based on our predictive model (UTR Designer), which we term “UTR engineering”, for each gene. The redox rebalancing was achieved by anaerobically activating pyruvate dehydrogenase (PDH) complex and additionally tuning expression level of NAD+-dependent formate dehydrogenase (fdh1 from Saccharomyces cerevisiae) through UTR engineering. As a result, we found that the optimal expression levels of fdh1 were dramatically different to efficiently produce n-butanol from glucose or galactose. This work provides intriguing insight that genetic contexts dependent-engineering is promising for strain improvement, even with the same genetic contents, by rebalancing of the intracellular redox state depending on the target products and substrates.