Effect of modification of phosphoenolpyruvate carboxylase on the production of L-ornithine

Phosphoenolpyruvate carboxylase (PEPC) is an enzyme in the glycolysis pathway of C. glutamicum and plays an important role in the regulation of the TCA cycle (Fig.1). Phosphoenolpyruvate and carbon dioxide synthesize oxaloacetic acid under the catalysis of PEPC, and then oxaloacetic acid enters the TCA cycle for further metabolism. The expression and activity of PEPC affect the synthesis of glutamic acid inC. glutamicum (Nagano-Shoji et al., 2017; Wada et al., 2015). Lysine at position 653 (K653) is essential for the regulation of PEPC acetylation. Megumi ed al. found that acetylation of PEPC at K653 decreased enzymatic activity and reduced glutamate production, this means K653-acetylation regulates PEPC activity negatively. Mutated K653 into arginine can lower the level of acetylation on PEPC, which correspondingly improve its activity (Nagano-Shoji et al., 2017). In addition, PEPC will be feedback suppressed by aspartic acid when it is overexpressed in C. glutamicum , mutated aspartic acid at position 299 of PEPC to asparagine with a similar structure can effectively reduce the inhibitory effect (Wada et al., 2015). In our study, we attempt to combine the weakening acetylation by K653R and the attenuating feedback inhibition by D299N of PEPC to enhance the activity of this enzyme, leading to promote the glutamate synthesis and the L-ornithine yield. We selected the more preferred CGC and AAC codons inC. glutamicum S9114 to mutate K653 and D299. At the same time, the pepc gene was overexpressed by adding a strong constitutive promoter H36 (Yim et al., 2013). Multiple gene fragments are fused into a complete fragment by PCR overlapping amplification technology. After two point mutations in pepc , strain XAB03 was constructed. The shaking flask fermentation of strain XAB03 showed that the production of L-ornithine was 27.1 ± 0.32 g/L, which was 11.5% higher than that of strain XAB01 (Fig.4b). Moreover, it has a positive effect on the growth of C. glutamicum (Fig.4a). These results indicate the importance of PEPC in L-ornithine synthesis, and this modification strategy shows value for the synthesis of L-glutamate, L-citrulline and L-arginine.