Abstract
BackgroundSuccinate is an important C4 building block chemical, and its production via fermentative processes in bacteria has many practical applications in the biotechnology field. One of the major goals of optimizing the bacterium-based succinate production process is to lower the culture pH from the current neutral conditions, as this would reduce total production costs. In our previous studies, we selected Enterobacter aerogenes, a rapid glucose assimilator at pH 5.0, in order to construct a metabolically engineered strain that could produce succinate under weakly acidic conditions. This engineered strain produced succinate from glucose with a 72.7% (g/g) yield at pH 5.7, with a volumetric productivity of 0.23 g/L/h. Although this demonstrates proof-of-concept that bacterium-based succinate fermentation can be improved under weakly acidic conditions, several parameters still required further optimization.ResultsIn this study, we genetically modified an E. aerogenes strain previously developed in our laboratory in order to increase the production of ATP during succinate synthesis, as we inferred that this would positively impact succinate biosynthesis. This led to the development of the ES08ΔptsG strain, which contains the following modifications: chromosomally expressed Actinobacillus succinogenes phosphoenolpyruvate carboxykinase, enhanced fumarate reductase, inactivated pyruvate formate lyase, pyruvate oxidase, and glucose-phosphotransferase permease (enzyme IIBCGlc). This strain produced 55.4 g/L succinate from glucose, with 1.8 g/L acetate as the major byproduct at pH 5.7 and anaerobic conditions. The succinate yield and volumetric productivity of this strain were 86.8% and 0.92 g/L/h, respectively.ConclusionsFocusing on increasing net ATP production during succinate synthesis leads to increased succinate yield and volumetric productivity in E. aerogenes. We propose that the metabolically engineered E. aerogenes ES08ΔptsG strain, which effectively produces succinate under weakly acidic and anaerobic conditions, has potential utility for economical succinate production.
Highlights
Succinate is an important C4 building block chemical, and its production via fermentative processes in bacteria has many practical applications in the biotechnology field
To construct an energy-conserving succinate synthesis pathway in E. aerogenes, we introduced adenosine triphosphate (ATP)-forming phosphoenolpyruvate carboxykinase (PCK) derived from Actinobacillus succinogenes [26], and disrupted the genes involved in the glucose phosphotransferase system
Strategy for increasing net ATP production during succinate synthesis in E. aerogenes To realize an energy conserving strategy that would increase net ATP production during succinate synthesis in E. aerogenes, we focused on modifications of carboxylation enzymes and the glucose uptake system (Figure 1)
Summary
Succinate is an important C4 building block chemical, and its production via fermentative processes in bacteria has many practical applications in the biotechnology field. For the last several decades, a number of groups have successfully achieved bacterium-based succinate fermentation using engineered strains of Escherichia coli, Corynebacterium glutamicum, Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens, and Mannheimia succiniciproducens [8,9,10,11,12]. All of these bacteria produced succinate effectively at neutral pH, this was not the case in acidic conditions [13,14,15]. There is a need to improve the economic and ecological impact of such bacteria in relation with the succinate production process
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