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

Abstract

Background: Fermentative hydrogen (H-2) production suffers from low carbon-to-H-2 yield, to which problem, co-production of ethanol and H-2 has been proposed as a solution. For improved co-production of H-2 and ethanol, we developed Escherichia coli BW25113 Delta hycA Delta hyaAB Delta hybBC Delta ldhA Delta frdAB Delta pta-ackA Delta pfkA (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 H-2 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 H-2 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 H-2 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 H-2 and ethanol. Our strategy is applicable to anaerobic production of biofuels and biochemicals, both of which processes demand high NAD(P)H.