Co-production of hydrogen and ethanol by pfkA-deficient Escherichia coli with activated pentose-phosphate pathway: Reduction of pyruvate accumulation

Abstract

Background: Fermentative hydrogen (H2) production suffers from low carbon-to-H2 yield, to which problem, co-production of ethanol and H2 has been proposed as a solution. For improved co-production of H2 and ethanol, we developed Escherichia coli BW25113 ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB Δpta-ackA ΔpfkA (SH8∗) and overexpressed Zwf and Gnd, the key enzymes in the pentose-phosphate (PP) pathway (SH8∗-ZG). However, the amount of accumulated pyruvate, which was significant (typically 0.20 mol mol-1 glucose), reduced the co-production yield. Results: In this study, as a means of reducing pyruvate accumulation and improving co-production of H2 and ethanol, we developed and studied E. coli SH9∗-ZG with functional acetate production pathway for conversion of acetyl-CoA to acetate (pta-ackA +). Our results indicated that the presence of the acetate pathway completely eliminated pyruvate accumulation and substantially improved the co-production of H2 and ethanol, enabling yields of 1.88 and 1.40 mol, respectively, from 1 mol glucose. These yields, significantly, are close to the theoretical maximums of 1.67 mol H2 and 1.67 mol ethanol. To better understand the glycolytic flux distribution, glycolytic flux prediction and RT-PCR analyses were performed. Conclusion: The presence of the acetate pathway along with activation of the PP pathway eliminated pyruvate accumulation, thereby significantly improving co-production of H2 and ethanol. Our strategy is applicable to anaerobic production of biofuels and biochemicals, both of which processes demand high NAD(P)H.