Function of uridylyltransferase and uridylyl-removing enzyme GlnD in Corynebacterium glutamicum JNR

Abstract:

[Objective] We modified the bifunctional uridylyltransferase and uridylyl-removing enzyme GlnD to reduce the activity of uridylyl-removing enzyme, thus, increasing the NH₄⁺ transition and application, to improve L-arginine production.[Methods] PII protein GlnK was overexpressed and its uridylation was studied. A glnD_AA(including H₄₁₄A and D₄₁₅A) mutant was generated from C. glutamicum JNR using the double-crossover chromosome replacement technique. The concentration of NH₄⁺ in the fermentation medium was measured by ion chromatography. Then the resulting strain was cultivated in a 5-L stirring bioreactor to performed a fed-batch fermentation.[Results] The bifunctional uridylyltransferase and uridylyl-removing enzyme was successfully mutated in C. glutamicum JNR and the resulting strain L4 showed a weakened activity of uridylyl-removing enzyme. The shaking flask fermentation showed that the L4 strain consumed more NH₄⁺, and the L-arginine yield was 36.2±1.2 g/L, 22.7% higher than the control strain. The production of L-arginine of L4 strain was 52.2 g/L, which was 25.3% higher than that of L0 strain in 5-L fermentation.[Conclusion] In C. glutamicum, nitrogen is necessary for the L-arginine biosynthesis. We conclude that reducing uridylyl-removing activity resulted in more intracellular GlnK-UMP. The GlnK-UMP interacts with the nitrogen regulator AmtR and enhances the intracellular consumption of NH₄⁺. Subsequently, the increased uptake of NH₄⁺ could promote the L-arginine production in C. glutamicum.