Abstract
In Escherichia coli, succinic acid is synthesized by CO2 fixation-based carboxylation of C3 metabolites. A two-step process is involved in CO2 integration: CO2 uptake into the cell and CO2 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 CO2 uptake and fixation were co-overexpressed, or overexpressed individually to improve succinate biosynthesis. Active CO2 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 CO2 fixation gene (ppc or pck) and a CO2 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 CO2 transport and fixation is critical for succinate production. Imbalanced gene expression may disturb the cellular metabolism and succinate production.
Highlights
In E. coli, succinic acid is synthesized by CO2 fixation-based carboxylation of C3 metabolites
Our results showed that the best succinate production was attained only when one CO2 transport and one CO2 fixation gene were co-expressed
The succinate production obtained with Tang1501, Tang1502, Tang1503, and Tang1504 was between 62.6 and 67.3 g L−1
Summary
In E. coli, succinic acid is synthesized by CO2 fixation-based carboxylation of C3 metabolites. Overexpression of ppc gene from Sorghum vulgare in E. coli strain SB2020 increased succinate production by 1.5 folds[13]. Another critical step in succinic acid production is the CO2 uptake by cells. The Na+-dependent SbtA transporter was originally identified in the cyanobacterium, Synechocystis PCC6803. It is a single gene transporter with relatively high affinity for HCO3−, requiring Na+ for maximal HCO3− uptake activity[18]. In an attempt to further enhance succinic acid production, we employed a dual expression system, in which genes regulating both PEP carboxylation and CO2 uptake were overexpressed individually or co-overexpressed. This work provides useful information for metabolic regulation of CO2 to improve succinate production
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