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.
