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.