Collaborative regulation of CO(2) transport and fixation during succinate production in Escherichia coli

In Escherichia coli, succinic acid is synthesized by CO(2) fixation-based carboxylation of C3 metabolites. A two-step process is involved in CO(2) integration: CO(2) uptake into the cell and CO(2) fixation by carboxylation enzymes. The phosphoenolpyruvate (PEP) carboxylase (PPC) and carboxykinase (PCK) are two important carboxylation enzymes within the succinate synthetic pathway, while SbtA and BicA are two important bicarbonate transporters. In this study, we employed a dual expression system, in which genes regulating both CO(2) uptake and fixation were co-overexpressed, or overexpressed individually to improve succinate biosynthesis. Active CO(2) uptake was observed by the expression of SbtA or/and BicA, but the succinate biosynthesis was decreased. The succinate production was significantly increased only when a CO(2) fixation gene (ppc or pck) and a CO(2) transport gene (sbtA or bicA) were co-expressed. Co-expression of pck and sbtA provided the best succinate production among all the strains. The highest succinate production of 73.4 g L(−1) was 13.3%, 66.4% or 15.0% higher than that obtained with the expression of PCK, SbtA alone, or with empty plasmids, respectively. We believe that combined regulation of CO(2) transport and fixation is critical for succinate production. Imbalanced gene expression may disturb the cellular metabolism and succinate production.