Coexistence of the Entner\u2013Doudoroff and Embden\u2013Meyerhof\u2013Parnas pathways enhances glucose consumption of ethanol-producing Corynebacterium glutamicum

Toru Jojima,Akira Watanabe,Masako Suda,Masayuki Inui,Takafumi Igari,Ryoji Noburyu

doi:10.1186/s13068-021-01876-3

Abstract

BackgroundIt 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.ResultsThree 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.ConclusionsThe 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.

Highlights

It is interesting to modify sugar metabolic pathways to improve the productivity of biocatalysts that convert sugars to value-added products
Our findings suggested that coexistence of the ED and EMP pathways was needed to enhance the rate of glucose consumption; cofactors such as NADH and ATP were unlikely to be involved in enhanced glycolysis by the ED pathway
Pathway design The ED pathway was introduced into the genome of the ethanol-producing C. glutamicum CRZ2e that was previously constructed by expressing the heterologous genes for Z. mobilis pyruvate decarboxylase and alcohol dehydrogenase from pCRA723 and disrupting the chromosomal genes for lactate dehydrogenase and phosphoenolpyruvate carboxylase to suppress the formation of lactate and succinate [21]

Summary

Introduction

It is interesting to modify sugar metabolic pathways to improve the productivity of biocatalysts that convert sugars to value-added products. Modifying cell metabolism to produce valuable molecules is of substantial economic and scientific interest Most of these efforts are directed toward the expression of heterologous biosynthetic pathways, inactivation of enzymes to repress by-product formation, and enhancement of the expression of enzymes related to product biosynthesis [1]. All these approaches have produced valuable information on the metabolic engineering of microorganisms. The processes for amino acid production by C. glutamicum are performed under aerobic conditions; this microorganism can metabolize glucose and produce mixed organic acids under oxygen-deprived conditions, its growth is suppressed under the circumstances [8]. These properties of C. glutamicum have been exploited to develop unique bioprocesses that decouple cell growth from biofuel and biochemical production [9]

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