Systematic engineering of the central metabolism in Escherichia coli for effective production of n-butanol.

Mukesh Saini,Yun-Peng Chao,Chung-Jen Chiang,Si-Yu Li,Ze Win Wang

doi:10.1186/s13068-016-0467-4

Abstract

BackgroundMicrobes have been extensively explored for production of environment-friendly fuels and chemicals. The microbial fermentation pathways leading to these commodities usually involve many redox reactions. This makes the fermentative production of highly reduced products challenging, because there is a limited NADH output from glucose catabolism. Microbial production of n-butanol apparently represents one typical example.ResultsIn this study, we addressed the issue by adjustment of the intracellular redox state in Escherichia coli. This was initiated with strain BuT-8 which carries the clostridial CoA-dependent synthetic pathway. Three metabolite nodes in the central metabolism of the strain were targeted for engineering. First, the pyruvate node was manipulated by enhancement of pyruvate decarboxylation in the oxidative pathway. Subsequently, the pentose phosphate (PP) pathway was amplified at the glucose-6-phosphate (G6P) node. The pathway for G6P isomerization was further blocked to force the glycolytic flux through the PP pathway. It resulted in a growth defect, and the cell growth was later recovered by limiting the tricarboxylic acid cycle at the acetyl-CoA node. Finally, the resulting strain exhibited a high NADH level and enabled production of 6.1 g/L n-butanol with a yield of 0.31 g/g-glucose and a productivity of 0.21 g/L/h.ConclusionsThe production efficiency of fermentative products in microbes strongly depends on the intracellular redox state. This work illustrates the flexibility of pyruvate, G6P, and acetyl-CoA nodes at the junction of the central metabolism for engineering. In principle, high production of reduced products of interest can be achieved by individual or coordinated modulation of these metabolite nodes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0467-4) contains supplementary material, which is available to authorized users.

Highlights

Microbes have been extensively explored for production of environment-friendly fuels and chemicals
In E. coli, pyruvate is oxidized to acetyl-CoA by a reaction mediated by pyruvate dehydrogenase (PDH) under the aerobic growth and by pyruvate formate-lyase
formate dehydrogenase (FDH) such as Candida boidinii fdh and Saccharomyces cerevisiae fdh1 catalyzes oxidation of formate to CO2 associated with NADH generation [20]

Summary

Introduction

Microbes have been extensively explored for production of environment-friendly fuels and chemicals. The microbial fermentation pathways leading to these commodities usually involve many redox reactions. This makes the fermentative production of highly reduced products challenging, because there is a limited NADH output from glucose catabolism. The rising price, the insecure supply, and the environmental concern of fossil fuels have currently overshadowed these industries. It provokes the demand for renewable and environmentfriendly fuels and chemicals [1]. The microbial fermentation pathways involve many redox reactions, which usually require NADH and NAD+ as cofactors. The result of the reductive reactions usually leads to production of ethanol, lactate, and succinate as exemplified in fermentative Escherichia coli [4]. Maintaining the redox balance of NADH and NAD+ is a key to ensure the continued operation of cellular metabolism under the fermentative condition

Methods

Results

Conclusion