Abstract
BackgroundSuccinate biosynthesis of Escherichia coli is reducing equivalent-dependent and the EMP pathway serves as the primary reducing equivalent source under anaerobic condition. Compared with EMP, pentose phosphate pathway (PPP) is reducing equivalent-conserving but suffers from low efficacy. In this study, the ribosome binding site library and modified multivariate modular metabolic engineering (MMME) approaches are employed to overcome the low efficacy of PPP and thus increase succinate production.ResultsAltering expression levels of different PPP enzymes have distinct effects on succinate production. Specifically, increased expression of five enzymes, i.e., Zwf, Pgl, Gnd, Tkt, and Tal, contributes to increased succinate production, while the increased expression of two enzymes, i.e., Rpe and Rpi, significantly decreases succinate production. Modular engineering strategy is employed to decompose PPP into three modules according to position and function. Engineering of Zwf/Pgl/Gnd and Tkt/Tal modules effectively increases succinate yield and production, while engineering of Rpe/Rpi module decreases. Imbalance of enzymatic reactions in PPP is alleviated using MMME approach. Finally, combinational utilization of engineered PPP and SthA transhydrogenase enables succinate yield up to 1.61 mol/mol glucose, which is 94% of theoretical maximum yield (1.71 mol/mol) and also the highest succinate yield in minimal medium to our knowledge.ConclusionsIn summary, we systematically engineered the PPP for improving the supply of reducing equivalents and thus succinate production. Besides succinate, these PPP engineering strategies and conclusions can also be applicable to the production of other reducing equivalent-dependent biorenewables.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0675-y) contains supplementary material, which is available to authorized users.
Highlights
Succinate biosynthesis of Escherichia coli is reducing equivalent-dependent and the EMP pathway serves as the primary reducing equivalent source under anaerobic condition
Embden–Meyerhof–Parnas (EMP) pathway is the predominant source of reducing equivalent: only 2 mol NADH was produced from glycolysis of 1 mol glucose, which is only enough for 1 mol succinate synthesis via the reductive TCA cycle [2]
Engineering effects of individual phosphate pathway (PPP) enzymes on succinate production We first measured the activities of all PPP enzymes within Suc-T110 under anaerobic condition
Summary
Succinate biosynthesis of Escherichia coli is reducing equivalent-dependent and the EMP pathway serves as the primary reducing equivalent source under anaerobic condition. We obtained a well-performing E. coli HX024 strain, which produces 813 mM succinate with a yield of 1.36 mol/mol glucose using AM1 12% glucose medium [8]. Embden–Meyerhof–Parnas (EMP) pathway is the predominant source of reducing equivalent: only 2 mol NADH was produced from glycolysis of 1 mol glucose, which is only enough for 1 mol succinate synthesis via the reductive TCA cycle [2]. Using EMP as the sole source of reducing equivalent, the theoretical succinate yield of 1 mol/mol glucose is only 58% of the maximal yield of 1.71 mol/mol [2]. To this end, recruitment of other reducing equivalent-conserving pathways is expected to further improve succinate production
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