Native Carbonic Anhydrase Activity Provides a Critical and Sufficient CO2 Concentrating Mechanism for Escherichia coli Succinate Fermentation

While the biobased, fermentative production of succinate by Escherichia coli represents a sustainable alternative to its conventional synthesis from petroleum, this process requires substantial amounts of inorganic carbon (Ci) to support CO2-fixing reactions in the reductive branch of the tricarboxylic acid (rTCA) cycle. Accordingly, intracellular Ci availability represents a potential limiting factor during E. coli succinate fermentations. Here, we first investigate the role and importance of E. coli’s native CO2 concentrating mechanism (CCM)—comprising two carbonic anhydrases (CAs), Can and CynT—by comparing and contrasting the behaviors of wild-type E. coli and the engineered succinate-producing strain, KJ122. Deletion of can and cynT significantly impaired the aerobic growth of both strains under low CO2 atmosphere and/or low pH, outcomes that were further exacerbated under anaerobic conditions for KJ122. During bioreactor fermentations, KJ122 Δcan ΔcynT further exhibited a prolonged lag phase (~48 h) and 44% reduced succinate production relative to KJ122 by 96 h. Next, the relative functions and performance of mechanistically diverse, heterologous CCM components were investigated by characterizing their ability to restore growth and/or succinate production. While the cyanobacterial bicarbonate transporter SbtA and the Ci transporter DabAB from Halothiobacillus neapolitanus each complemented growth at 0.05% CO2 and pH 6.5–7.5, neither fully restored succinate production by KJ122 Δcan ΔcynT. Moreover, individual overexpression of sbtA, dabAB, or can in KJ122 rendered no additional improvements to succinate production. Collectively, while these results point to the critical importance of CA for supporting efficient fermentative succinate production by E. coli, they also suggest that this native CCM alone is sufficient for ensuring Ci acquisition at requisite levels under the conditions examined.