Improving Succinate Productivity by Engineering a Cyanobacterial CO2 Concentrating System (CCM) in Escherichia coli

Abstract

Biologically fixation of CO₂ has great potential as a significant carbon source for biosynthesis, which is also a major way to reduce CO₂ accumulation in atmosphere. Phosphoenolpyruvate (PEP) carboxylation is the key step of anaerobic succinate production in Escherichia coli. In this reaction, one mole CO₂ is assimilated with PEP to form oxaloacetate by PEP carboxykinase (PCK). The preferred substrate of PCK is CO₂ , which is very limited in cytoplasm. In this study, the carbon concentration mechanism (CCM) of cyanobacteria was introduced into Escherichia coli to enhance the intracellular inorganic carbon concentration for improving carboxylation velocity. Overexpression of the bicarbonate transporter (BT) or carbonic anhydrase (CA) gene from Synechococcus sp. PCC7002 led to a 22 or 35% increase in succinate titer at 36 h, respectively. The carboxylation rate of PCK increased from 2.46 to 3.92 µmol min^-1 mg^-1 protein by overexpression of the CA gene. In addition, co-overexpression of BT and CA genes had a synergetic effect, leading to a 44% increase in succinate titer at 36 h. This work is the first attempt to increase carbon fixation involved in microbial biosynthesis by engineering a biological CO₂ delivery system, which provides new direction and strategies for improving industrial fermentations based on biological CO₂ assimilation pathways.

Keywords: bicarbonate transporter; carbon concentration mechanism; carbonic anhydrase; phosphoenolpyruvate carboxykinase; succinate.