P104: Native pathway engineering in E. coli for production of bioethanol from pentose and hexose sugars

Bioethanol is a form of renewable energy that can be produced from agricultural feedstocks. Traditionally bioethanol is produced from sugarcane fermentation by Saccharomyces cerevisiae but its inability to utilize pentose sugars necessiates the search for other bioethanol producing microorganisms. Escherichia coli is one such organism that has the property to metabolize variety of sugars to produce basal level of ethanol under anaerobic conditions. We report an engineered Escherichia coli strain for increased ethanol production through native pathway engineering. The reducing equivalence needed for homoethanol production was made available by expressing the native pyruvate dehydrogenase (PDH) operon under anaerobic condition by replacing its promoter with five different promoters used by anaerobically expressed genes. The strain where it was expressed under gapA promoter, yielded highest PDH mRNA expression, enzyme activity and improvement in ethanol yield as compared to the wild type strain. Deletion of genes responsible for competing products led to minimal side product formation with ethanol being the major product. However, the gene deletion also led to slower growth rate of the engineered strain. Introduction of basal level acetate kinase activity regained cell growth rate and improved ethanol productivity significantly. Further bioprocess optimization resulted in improved glucose and xylose consumption and ethanol productivity. Taken together, we report an engineered E. coli strain for its maximum ethanol yield and should be considered for further study to evaluate ethanol production from hydrolysate of lignocellulosic biomass.