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)

Abstract

BackgroundBiologically, hydrogen (H2) can be produced through dark fermentation and photofermentation. Dark fermentation is fast in rate and simple in reactor design, but H2 production yield is unsatisfactorily low as <4 mol H2/mol glucose. To address this challenge, simultaneous production of H2 and ethanol has been suggested. Co-production of ethanol and H2 requires enhanced formation of NAD(P)H during catabolism of glucose, which can be accomplished by diversion of glycolytic flux from the Embden–Meyerhof–Parnas (EMP) pathway to the pentose-phosphate (PP) pathway in Escherichia coli. However, the disruption of pgi (phosphoglucose isomerase) for complete diversion of carbon flux to the PP pathway made E. coli unable to grow on glucose under anaerobic condition.ResultsHere, we demonstrate that, when glucose-6-phosphate dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd), two major enzymes of the PP pathway, are homologously overexpressed, E. coli Δpgi can recover its anaerobic growth capability on glucose. Further, with additional deletions of ΔhycA, ΔhyaAB, ΔhybBC, ΔldhA, and ΔfrdAB, the recombinant Δpgi mutant could produce 1.69 mol H2 and 1.50 mol ethanol from 1 mol glucose. However, acetate was produced at 0.18 mol mol−1 glucose, indicating that some carbon is metabolized through the Entner–Doudoroff (ED) pathway. To further improve the flux via the PP pathway, heterologous zwf and gnd from Leuconostoc mesenteroides and Gluconobacter oxydans, respectively, which are less inhibited by NADPH, were overexpressed. The new recombinant produced more ethanol at 1.62 mol mol−1 glucose along with 1.74 mol H2 mol−1 glucose, which are close to the theoretically maximal yields, 1.67 mol mol−1 each for ethanol and H2. However, the attempt to delete the ED pathway in the Δpgi mutant to operate the PP pathway as the sole glycolytic route, was unsuccessful.ConclusionsBy deletion of pgi and overexpression of heterologous zwf and gnd in E. coli ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB, two important biofuels, ethanol and H2, could be successfully co-produced at high yields close to their theoretical maximums. The strains developed in this study should be applicable for the production of other biofuels and biochemicals, which requires supply of excessive reducing power under anaerobic conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0768-2) contains supplementary material, which is available to authorized users.

Two‐stage carbon distribution and cofactor generation for improving l‐threonine production of Escherichia coli

“…Thus, GDH was very likely to compete for NADPH with them, which might have a detrimental effect on l ‐threonine production. The commonly used NADPH generating enzymes to increase NADPH availability included glucose‐6‐phosphate dehydrogenase (Sundara Sekar, Seol, & Park, ), malic enzyme (L. Liu et al, ), and glyceraldehyde 3‐phosphate dehydrogenase (Martínez, Zhu, Lin, Bennett, & San, ). However, since these enzymes were related to central carbon metabolism, enhancing their expression might affect cell growth.…”

Computer-Aided Rational Design of Efficient NADPH Production System by Escherichia coli pgi Mutant Using a Mixture of Glucose and Xylose

“…The typical approach for the overproduction of NADPH is, therefore, to increase the flux through the OPP pathway by the disruption of pgi gene encoding phosphoglucose isomerase (Pgi) (and also pfkA encoding phosphofructokinase (Pfk) but to a lesser extent), which forces the flux of the imported glucose toward the OPP pathway at the glucose 6posphate (G6P) node, which resulted in increased NADPH. In fact, this strategy has been employed to produce several chemicals in practice (Kabir and Shimizu, 2003;Marx et al, 2003;Blombach et al, 2008;Wang et al, 2013;Park et al, 2014;Seol et al, 2014;Kim et al, 2015;Ng et al, 2015;Aslan et al, 2017;Sundara Sekar et al, 2017). Since excess NADPH allosterically inhibits the activity of G6PDH, G6P accumulates and the glucose uptake rate (GUR) reduces through the posttranscriptional regulation, causing instability of mRNA of ptsG encoding EIIBC of the phosphotransferase system (PTS) (Morita et al, 2003).…”

Metabolic engineering of Escherichia coli for biosynthesis of β‐nicotinamide mononucleotide from nicotinamide

“…The precursor of PRPP, ribose‐5‐phosphate (R5P) is mainly synthesized through the pentose phosphate (PP) pathway in E. coli . Enhancing the expression of 6‐phosphate gluconate dehydrogenase (encoded by gnd ) or glucose 6‐phosphate dehydrogenase (encoded by zwf ) could increase the carbon flux through the PP pathway (Sekar et al ., 2017 ; Wu et al ., 2020 ). In order to increase PRPP supply by means of increasing the carbon flux of the PP pathway, genes zwf and gnd were additionally integrated into genome at the site of yee P and yji V, generating strains NMN05 and NMN06, respectively.…”