Coexistence of the Entner–Doudoroff and Embden–Meyerhof–Parnas pathways enhances glucose consumption of ethanol-producing Corynebacterium glutamicum

Background It is interesting to modify sugar metabolic pathways to improve the productivity of biocatalysts that convert sugars to value-added products. However, this attempt often fails due to the tight control of the sugar metabolic pathways. Recently, activation of the Entner–Doudoroff (ED) pathway in Escherichia coli has been shown to enhance glucose consumption, though the mechanism underlying this phenomenon is poorly understood. In the present study, we investigated the effect of a functional ED pathway in metabolically engineered Corynebacterium glutamicum that metabolizes glucose via the Embden–Meyerhof–Parnas (EMP) pathway to produce ethanol under oxygen deprivation. This study aims to provide further information on metabolic engineering strategies that allow the Entner–Doudoroff and Embden–Meyerhof–Parnas pathways to coexist. Results Three genes (zwf, edd, and eda) encoding glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis were expressed in a genetically modified strain, C. glutamicum CRZ2e, which produces pyruvate decarboxylase and alcohol dehydrogenase from Z. mobilis. A 13C-labeling experiment using [1-13C] glucose indicated a distinctive 13C distribution of ethanol between the parental and the ED-introduced strains, which suggested an alteration of carbon flux as a consequence of ED pathway introduction. The ED-introduced strain, CRZ2e-ED, consumed glucose 1.5-fold faster than the parental strain. A pfkA deletion mutant of CRZ2e-ED (CRZ2e-EDΔpfkA) was also constructed to evaluate the effects of EMP pathway inactivation, which showed an almost identical rate of glucose consumption compared to that of the parental CRZ2e strain. The introduction of the ED pathway did not alter the intracellular NADH/NAD+ ratio, whereas it resulted in a slight increase in the ATP/ADP ratio. The recombinant strains with simultaneous overexpression of the genes for the EMP and ED pathways exhibited the highest ethanol productivity among all C. glutamicum strains ever constructed. Conclusions The increased sugar consumption observed in ED-introduced strains was not a consequence of cofactor balance alterations, but rather the crucial coexistence of two active glycolytic pathways for enhanced glucose consumption. Coexistence of the ED and EMP pathways is a good strategy for improving biocatalyst productivity even when NADPH supply is not a limiting factor for fermentation.

Enhancing control of cell-free metabolism through pH modulation

Engineering metabolism for the synthesis of bio-based products in non-model organisms can be challenging. One specific challenge is that biosynthetic pathways are often built from enzyme candidates sourced from diverse organisms, which can prove difficult to implement in recombinant hosts due to differences in their cellular environments (e.g. pH, cofactor balance). To address this problem, we report a cell-free synthetic biology approach for understanding metabolism in a range of environmental conditions, specifically under varied pH. The key idea is to control the pH of Escherichia coli-based cell-free systems for assessing pathway performance using enzymes sourced from organisms other than E. coli. As a model, we apply this approach to study the impact of pH on the n-butanol biosynthesis pathway derived from clostridia in E. coli lysates. Specifically, we exploit the open, cell-free reaction environment to explore pH outside the habitable range of E. coli, revealing insights into how chemical context impacts the interaction between native metabolism and heterologous enzymes. We find that the pH optimum for butanol production from acetyl-CoA is substantially lower than the optimal pH of glycolysis in E. coli-based crude lysates. In addition, pH is an essential factor to consider when activating metabolic pathways in the cell-free environment due to its effect on reaction yield or enzyme activity, the latter of which is demonstrated in this work for alcohol dehydrogenases from a range of extremophiles. Ultimately, altering metabolism through pH control will allow cell-free systems to be used in studying the metabolic state of organisms and identify suitable enzymes for pathway engineering.