Over-expression of NAD kinase in Corynebacterium crenatum and its Impact on L-Arginine Biosynthesis

Purpose: To improve the biosynthesis of L-arginine by overexpressing homologous NAD kinase (ppnk) in Corynebacterium crenatum SYPA5-5 and to study its impact in presence of high (HOS) and low oxygen supply (LOS). Methods: A recombinant plasmid (pJC1-tac-ppnK) harboring homologous NAD kinase (ppnk) was constructed in a shuttle vector pJC1 and transferred in L-arginine producing strain Corynebacterium crenatum SYPA5-5. Furthermore, fermentation was performed by shake flask method with consecutive determination of cell growth and glucose concentration. NAD(+) kinase activity was studied by stop method and NADP(H) concentrations were determined by spectrophotometric enzymatic cycling method. To check the biosynthesis of amino acids, HPLC method was used to determine extracellular amino acid concentrations. Results: In HOS condition, NAD(+) kinase activity increased by 116 %, while intracellular concentrations of NADP(+) and NADPH increased by 7.3 and 36.8 %, respectively. Whereas, in LOS condition, NAD(+) kinase activity increased 49 %, with intracellular 14.67 and 15 % increases in NADP(+) and NADPH respectively. More importantly, recombinant strain could produce 26.47 and 11.36 g/L L-arginine in HOS and LOS respectively, which is higher than control strain value of 24.29 and 7.58 g/L respectively. Conclusion: These results suggest that altering the concentration of co-enzymes by NAD kinase in Corynebacterium crenatum is an effective way to increase NADP(+) with concurrent production of NADPH for further enhanced L-arginine biosynthesis in Corynebacterium crenatum in both conditions of high and low oxygen supply.