Co‐production of hydrogen and ethanol from glucose by modification of glycolytic pathways in Escherichia coli – from Embden‐Meyerhof‐Parnas pathway to pentose phosphate pathway

Abstract

Hydrogen (H2) production from glucose by dark fermentation suffers from the low yield. As a solution to this problem, co-production of H2 and ethanol, both of which are good biofuels, has been suggested. To this end, using Escherichia coli, activation of pentose phosphate (PP) pathway, which can generate more NADPH than the Embden-Meyhof-Parnas (EMP) pathway, was attempted. Overexpression of two key enzymes in the branch nodes of the glycolytic pathway, Zwf and Gnd, significantly improved the co-production of H2 and ethanol with concomitant reduction of pyruvate secretion. Gene expression analysis and metabolic flux analysis (MFA) showed that, upon overexpression of Zwf and Gnd, glucose assimilation through the PP pathway, compared with that of the EMP or Entner-Doudoroff (ED) pathway, was greatly enhanced. The maximum co-production yields were 1.32 mol H2 mol(-1) glucose and 1.38 mol ethanol mol(-1) glucose, respectively. It is noteworthy that the glycolysis and the amount of NAD(P)H formed under anaerobic conditions could be altered by modifying (the activity of) several key enzymes. Our strategy could be applied for the development of industrial strains for biological production of reduced chemicals and biofuels which suffers from lack of reduced co-factors.

EColiCore2: a reference network model of the central metabolism of Escherichia coli and relationships to its genome-scale parent model

“…For a product of the central metabolism such as ethanol, we expect that a large number of MCS found in ECC2 are sufficient to reach the desired production phenotype. In fact, successful E. coli strains for ethanol overproduction reported in the literature contained only deletions in the central metabolism343536 and the example of the suggested deletion of the acetaldehyde dehydrogenase reaction R_ACALD in the ECGS model shows that “exotic” intervention strategies (that redirect the flux to alternative product synthesis pathways) might be found in the genome-scale model whose relevance is unclear but cannot be excluded a priori . Generally, if calculated (smallest) MCS in ECGS require a huge and unrealistic number of knockouts or if a calculation of (large sets of) MCS for a given intervention problem is not feasible at all in the ECGS model, it would thus be practical to start with a MCS calculated in the core network ECC2.…”

Co-production of hydrogen and ethanol from glucose in Escherichia coli by activation of pentose-phosphate pathway through deletion of phosphoglucose isomerase (pgi) and overexpression of glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd)

“…Accordingly, while acetate production decreased, ethanol production and the CO 2 /H 2 ratio gradually increased. This indicated that when Gnd activity is enhanced, the flux through the PP pathway becomes more prominent than the ED pathway [16]. However, the ethanol yield increase, or the decrease in acetate production, almost halted at ~0.1 mM IPTG; moreover, even at the highest IPTG concentration tested in this study, 0.3 mM, about 0.2 mol acetate mol −1 glucose was produced.…”

“…In response, for co-production of H 2 and ethanol, alternative, phosphofructokinase-1 (PfkA) deletion mutants have been developed and studied (Fig. 1) [14, 16]. The Δ pfkA strains could grow well after long adaptation to anaerobic conditions [14] and produced significant amounts of H 2 and ethanol (~1.8 mol H 2  mol −1 and ~1.40 mol ethanol mol −1 ) when Zwf and Gnd, the key enzymes of the PP pathway, were overexpressed.…”

Engineering Corynebacterium glutamicum for fast production of l-lysine and l-pipecolic acid

“…b Means and standard deviations of three replicates are given control by global regulators such as ArcA (Perrenoud and Sauer 2005). Overexpression of genes encoding glycolytic enzymes as applied in E. coli (Xie et al 2014;Seol et al 2015), B. subtilis ), or S. coelicolor (Borodina et al 2008) was also used for C. glutamicum, e.g., to improve the production of high value compounds like amino acids (Yamamoto et al 2012;Reddy and Wendisch 2014), alcohols (Jojima et al 2015;Yamamoto et al 2013), or the organic acid D-lactate (Tsuge et al 2015). Under these conditions or when a repressor gene of glycolysis genes was deleted, i.e., that of the DeoR-type transcriptional regulator SugR, high titers of L-lactate were formed as side effect of faster glucose utilization (Engels and Wendisch 2007;Engels et al 2008;Teramoto et al 2011).…”