Modular design of butanol production pathway from red sea-weed in Escherichia coli using the synthetic expression controllers

Refactoring microorganisms for robust production from alternative biomass containing a mixture of sugars of variable composition should be an essential goal of strain development. In this study, we engineered Escherichia coli to produce n-butanol from red seaweed containing variable sugar mixtures, a promising alternative biomass. Since red seaweed contained non-preferred sugars such as galactose whose uptake rate is significantly low and regulated by glucose. To redesign entire pathway from sugar uptake to butanol production, a synthetic biology approach called “modular design” is known as the most efficient approach. In this study, two modules, i.e., galactose utilization module and butanol production module, were individually designed by UTR Designer. Both modules were connected at pyruvate node. NADH was optimally rebalanced by supplementing Saccharomyces cerevisiae originated formate dehydrogenase gene with synthetic UTR libraries designed by UTR Library Designer. The engineered strain produced 6.2 g/L of n-butanol, with a productivity of 0.13 g/L/h after 48 h fermentation in galatose supplemented medium and utilized galactose and glucose simultaneously. The strain also showed the ability to maintain the productivity for robust production of n-butanol regardless of sugar composition in red sea-weed media. The strategy demonstrated in this study can be used to engineer efficient and robust producers of biofuels and value-added chemicals from alternative biomass containing variable sugar composition.