Improved Co-Production of Hydrogen and Ethanol in Escherichia coli By the Deletion of Phosphofructokinase-1 and Activating Pentose-Phosphate Pathway

Co-production of H₂ and ethanol has been suggested to overcome the low yield limitation in fermentative biohydrogen production and co-production of these valuable biofuels in a single process is beneficial [1]. We have suggested the activation of pentose phosphate pathway for improving the reduced co-factors needed for ethanol production. We tried to upregulate the carbon flux through PP pathway by down-regulation of Embden-Meyerhof-Parnas pathway and overexpressing Zwf and Gnd, the key enzymes in PP pathway. The overexpression of Zwf and Gnd could activate PP pathway and down-regulation of EMP pathway (ΔpfkA) additionally improved the flux through PP pathway. Further, acetate production pathway was deleted to divert more acetyl-CoA towards ethanol production. The deletion of acetate production pathway significantly reduced the co-production yield by excreting pyruvate. We also attempted to eliminate acetate/pyruvate production by increasing the expression levels of Zwf and Gnd. The co-production yield of 1.55 mol/mol H₂ and 1.41 mol/mol ethanol could be obtained with a slight accumulation of acetate. The production levels of NAD(P)H suggested that ~70% of carbon was consumed through PP pathway. The carbon distribution analysis of the mutant strain and enzyme activities of Zwf and Gnd were studied. The gene expression analysis was also performed and the effect of Zwf and Gnd overexpression was characterized. Our strategy should be very useful for the production of biochemicals requiring reduced co-factors.

References     

1. Seol E, Ainala SK, Sekar BS, Park S. Metabolic engineering of Escherichia coli strains for co-production of hydrogen and ethanol from glucose. Int J Hydrogen Energ. 2014; 39:19323-19330.
2. Seol E, Sekar BS, Raj SM, Park S. Co‐production of hydrogen and ethanol from glucose modification of glycolytic pathways in Escherichia coli–from Embden‐Meyerhof‐Parnas pathway to pentose phosphate pathway. Biotechnol J. 2015; 11:249-256.