Improving Tolerance of Escherichia Coli Strain KO11 to Bagasse Hydrolysate and Ethanol by Screening Libraries of Mutated Transcriptional Machinery

In pursuit of ethanol or higher alcohol fuel production from cellulosic feedstocks, the ideal industrial strain has a number of characteristics that require improved tolerance to stresses such as high sugar or high ethanol tolerance that elicit a complex phenotypic response. The engineering of these complex responses require the use of combinatorial or random based strain engineering approaches along with appropriately designed screens to search for improved strains. Our lab has shown the utility of using global transcriptional machinery engineering (gTME) to search for mutant factors that improve complex phenotypes such as solvent tolerance and metabolite overproduction in bacteria and yeast. We have also recently developed a high- throughput microfluidic droplet system for use in screening libraries of clones. We will discuss the use of the microfluidic droplet system and other screening methods to screen two gTME libraries in E. coli strain KO11 to find strains with improved tolerance to high sugars and high ethanol in sugarcane bagasse hydrolysate.