Metabolic engineering of Escherichia coli for production of n-butanol from crude glycerol.

BACKGROUND: Crude glycerol in the waste stream of the biodiesel production process is an abundant and renewable resource. However, the glycerol-based industry is usually afflicted by the cost for refinement of crude glycerol. This issue can be addressed by developing a microbial process to convert crude glycerol to value-added chemicals. In this study, Escherichia coli was implemented for the production of n-butanol based on the reduced nature of glycerol. RESULTS: The central metabolism of E. coli was rewired to improve the efficiency of glycerol metabolism and provide the reductive need for n-butanol in E. coli. This was carried out in several steps by (1) forcing the glycolytic flux through the oxidation pathway of pyruvate, (2) directing the gluconeogenic flux into the oxidative pentose phosphate pathway, (3) enhancing the anaerobic catabolism for glycerol, and (4) moderately suppressing the tricarboxylic acid cycle. Under the microaerobic condition, the engineered strain enabled the production of 6.9 g/L n-butanol from 20 g/L crude glycerol. The conversion yield and the productivity reach 87% of the theoretical yield and 0.18 g/L/h, respectively. CONCLUSIONS: The approach by rational rewiring of metabolic pathways enables E. coli to synthesize n-butanol from glycerol in an efficient way. Our proposed strategies illustrate the feasibility of manipulating key metabolic nodes at the junction of the central catabolism. As a result, it renders the intracellular redox state adjustable for various purposes. Overall, the developed technology platform may be useful for the economic viability of the glycerol-related industry.

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

BACKGROUND: 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. Microbial production of n-butanol apparently represents one typical example. RESULTS: In 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. CONCLUSIONS: The 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.