Characterization and directed evolution of propionyl-CoA carboxylase and its application in succinate biosynthetic pathway with two CO2 fixation reactions.

Propionyl-CoA carboxylase (PCC) is a promising enzyme in the fields of biological CO 2 utilization, synthesis of natrual products, and so on. The activity and substrate specificity of PCC are dependent on its key subunit carboxyltransferase (CT). To obtain PCC with high enzyme activity, seven pccB genes encoding CT subunit from diverse microorganisms were expressed in recombinant E. coli , and PccB from Bacillus subtilis showed the highest activity in vitro. To further optimize this protein using directed evolution, a genetic screening system based on oxaloacetate availability was designed to enrich the active variants of PccB Bs. Four amino acid substitutions (D46G, L97Q, N220I and I391T) proved of great assistance in PccB Bs activity improvement, and a double mutant of PccB Bs (N220I/I391T) showed a 94-fold increase of overall catalytic efficiency indicated by k cat / K m. Moreover, this PccB Bs double mutant was applied in construction of new succinate biosynthetic pathway. This new pathway produces succinate from acetyl-CoA with fixation of two CO 2 molecules, which was confirmed by isotope labeling experiment with NaH13CO 3. Compared with previous succinate production based on carboxylation of phosphoenolpyruvate or pyruvate, this new pathway showed some advantages including higher CO 2 fixation potentiality and availability under aerobic conditions. In summary, this study developed a PCC with high enzyme activity which can be widely used in biotechnology field, and also demonstrated the feasibility of new succinate biosynthetic pathway with two CO 2 fixation reactions. • Several propionyl-CoA carboxylases (PCCs) were cloned and assayed in vitro. • Activity of B. subtilis PCC were notable improved by directed evolution. • The improved PCC was applied in a novel succinate biosynthetic pathway. • Succinate is produced from acetyl-CoA with fixation of two CO 2 molecules. [ABSTRACT FROM AUTHOR]