Fig. 1

Production of succinate anaerobically in E. coli. Through EMP pathway, 14.3 mol glucose produces 28.6 mol PEP and 28.6 mol NADH (catalyzed by gapA). Through PPP, 85.7 mol glucose produces 142.8 mol PEP, 171.4 mol NADPH (catalyzed by zwf and gnd), 142.8 mol NADH (catalyzed by gapA), and 85.7 mol CO₂. Through combinational utilization of EMP and PPP, 100 mol glucose produces 171.4 mol PEP, 171.4 mol NADH, 171.4 mol NADPH, and 85.7 mol CO₂. The formed 171.4 mol NADPH can be converted into 171.4 mol NADH by increased expression of SthA. In Suc-T110, phosphoenolpyruvate carboxykinase (pck) is the predominant PEP carboxylase and most of PEP flux goes into the reductive TCA cycle (mdh-fumB-frd) for succinate biosynthesis [25]. Finally, 100 mol glucose coupled with 85.7 mol CO₂ can produce 171.4 mol succinate, by which it achieves the theoretical maximum yield of 1.71 mol/mol glucose. EMP, Embden–Meyerhof–Parnas (glycolysis) pathway; PPP, pentose phosphate pathway; 6-P-Glc, glucose-6-phosphate; 6-P-Fru, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; 1,6-2P-Fru, 1,6-bisphosphate fructose; G3P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; 1,3-BPG, 1,3-bisphosphoglycerate; PEP, phosphoenolpyruvate; Pyr, pyruvate; OAA, oxaloacetate; 6-P-Glc-I, 6-phosphoglucono-δ-lactone; Glc6P, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; X5P, xylulose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; Mal, malate; Fum, fumarate; pgi, phosphoglucose isomerase; zwf, glucose-6-phosphate dehydrogenase; pgl, 6-phosphogluconolactonase; gnd, 6-phosphogluconate dehydrogenase; rpiA, ribose-5-phosphate isomerase; rpe, ribulose-5-phosphate-3-epimerase; tktA, transketolase; talB, transaldolase; gapA, glyceraldehyde 3-phosphate dehydrogenase; ppc, phosphoenolpyruvate carboxylase; pck, phosphoenolpyruvate carboxykinase; maeA, NAD⁺-dependent malic enzyme; maeB, NADP⁺-dependent malic enzyme; mdh, malate dehydrogenase; fumB, fumarase; frd, fumarate reductase; sthA, soluble pyridine nucleotide transhydrogenase; ADP, adenosine diphosphate; ATP, adenosine triphosphate; NAD⁺, oxidized nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; NADP⁺, oxidized nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate