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Bio-based succinate production from renewable resources has prospective economic and environmental benefits that caused heightened interest towards the study of succinate-producing microorganisms. The pathways of succinate formation have been well studied, and microorganisms that are capable of biomass convertion into the target substance (bacteria of the genera Actinobacillus, Anaerobiospirillum, and Mannheimia) have been isolated and characterized; however, the realization of economically feasible industrial processes using native producers still remains a challenge. Traditionally, the Escherichia coli bacterium has been used as a workhouse to develop new processes for the biosynthesis of many valuable chemicals due to the extensive knowledge of its metabolism, available genetic tools, and good growth characteristics, combined with low nutrient requirements. This review is focused on modern rational approaches to the construction of recombinant E. coli strains that efficiently produce succinic acid from glucose.

Two different approaches to activate the glyoxylate bypass in model Escherichia coli K-12 strains for succinate biosynthesis during dual-phase fermentation in minimal glucose media were examined. Inactivation of IclR and FadR, the transcriptional regulators of the aceBAK operon, were insufficient for the involvement of the glyoxylate bypass in anaerobic succinate biosynthesis by strains grown aerobically under glucose-abundant conditions. In contrast, the strains that constitutively expressed the aceEF-lpdA operon coding for the pyruvate dehydrogenase complex could partially synthesise succinate anaerobically via the glyoxylate bypass, even in the presence of intact regulators. The results suggest that the intensive acetyl-CoA formation in the strains constitutively expressing pyruvate dehydrogenase matches the physiological conditions that favour the activation of the glyoxylate bypass.

Efficient succinate production in Escherichia coli is attained during anaerobic glucose fermentation in biosynthetic processes combining the reductive branch of the TCA cycle and the glyoxylate bypass. Pyruvate dehydrogenase (PDH) or pyruvate formate lyase (PFL) serves in E. coli as a source of acetyl-CoA, a substrate for the glyoxylate bypass. Depending on enzymes responsible for acetyl-CoA generation, the contribution of the glyoxylate bypass to the anaerobic succinate biosynthesis may vary to support redox balance resulting in diverse maximum achievable yield values. Anaerobic succinate biosynthesis from glucose was studied using E. coli strains with altered expression of genes encoding PFL and PDH. For acetyl-CoA formation by PFL, the yield of 1.32 mol succinate per mole of glucose was achieved with the theoretical value of 1.6 mol/mol. Involvement of PDH in anaerobic acetyl-CoA synthesis increased succinate yield up to 1.49 mol/mol, which is 89.8% of the predicted maximum (1.6(6) mol/mol). The maximum yield of 1.69 mol succinate per mol glucose, amounting to 98.8% of the stoichiometric maximum (1.71 mol/mol), was achieved with the strain possessing PDH as the primary anaerobic source of acetyl-CoA. During high cell density fermentation, the best engineered strain produced high amounts of succinate (570.7 mM) and only small quantities of acetate (11.9 mM).

Effect of constitutive expression of the aceEF-lpdA operon genes coding for the enzymes of NAD+ reducing pyruvate dehydrogenase complex on the anaerobic production of succinic acids from glucose by recombinant Escherichia coli strains was studied. Basic producer strains were obtained by inactivation of the main pathways for synthesis of acetic and lactic acids by deletion of the genes ackA, pta, poxB, and ldhA (SGMO.1) in E. coli strain MG 1655 cells and additional introduction of the Bacillus subtilis pyruvate carboxylase (SG M0.1 [pPYC]). A constitutive expression of the genes aceEF-lpdA in derivatives of the basic strains SGM0.1 PL-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] was provided by replacing the native regulatory region of the operon with the lambda phage PL promoter. Molar yields of succinic acid in anaerobic glucose fermentation by strains SGM0.1 P(L)-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] exceeded the corresponding yields displayed by several control strains (exceeded considerably in the case of the strains with a pyruvate carboxylase activity). It is concluded that an increase in the succinic acid production by strain SGM0.1 PL-aceEF-lpdA [pPYC] as compared with the strains SGM0.1 and SGM0.1 [pPYC], which synthesize this substance in the reductive tricarboxylic acid cycle, is determined by activation of the glyoxylate shunt.