Metabolic Engineering Of Escherichia Coli For C4-dicarboxylic Acids Production From Glycerol

Glycerol has become an abundant and inexpensive feedstock for bio-based production of fuels and valuable chemicals as the major by-product of biodiesel. Dicarboxylic acids, which include succinate, fumaric acid, malate, have significant applications in agricultural, food, and pharmaceutical and chemical industries. In this study, Escherichia coli was engineered through metabolic and evolutionary strategies for dicarboxylic acids production from glycerol.Elementary mode analysis was applied to systematically analyze the aerobic, microaerobic, and anaerobic metabolic network model of E. coli for succinate production from glycerol.Based on elementary mode analysis, E. coli strain capable of producing succinate aerobically was developed, and succinate yield was 0.22 mol/mol glycerol. However, α-ketoglutarate was produced as the main product. Via the directed pathway evolution, the glyoxylate shunt was recruited as the primary anaplerotic pathway in a ppc mutant. As a result, the amount of α-ketoglutarate was reduced by 46% and succinate production was increased by 30%. Overexpression of α-ketoglutarate dehydrogenase complex further reduced the amount of byproduct significantly and improved succinate production by 20%. In addition, a G583 T mutation in gene icdA was identified in the evolved strain as the main mutation responsible for the observed phenotype. The final strain E2-Δsdh-ppc-sucAB produced 43.2 g/L succinate from 120 g/L glycerol in minimal medium in fed-batch fermentation.The evolved mutant developed for succinate production from glycerol was further engineered for fumaric acid production under aerobic conditions. Through deletion of three fumarases, fumaric acid was produced as the main product from glycerol, and meanwhile a large amount of acetate was accumulated. In order to reduce acetate production several strategies were attempted, among which increasing the flux of the anaplerotic pathways through overexpression of phosphoenolpyruvate carboxylase gene ppc or the glyoxylate shunt operon aceBA effectively reduced acetate and improved fumaric acid production. In fed-batch culture, the resulting strain EF02(pSCppc) produced 41.5 g/L fumaric acid from 94 g/L glycerol with 70% of the maximum theoretical yield.In addition, E. coli was engineered for succinate production from glycerol under microaerobic conditions, based on which malate producing strains were also developed. The effects of multiple pathways on malate production were studied. Finally, the resulting strain could produce 11.5 g/L succinate with a yield of 0.64 mol/mol glycerol, and the malate producing strain could produce 7.5 g/L malate with a yield of 0.57 mol/mol glycerol.