双功能尿苷酰转移/去除酶GlnD在谷氨酸棒杆菌JNR中的功能

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

摘要:

[目的] 通过改造谷氨酸棒杆菌JNR中双功能尿苷酰转移/去除酶GlnD，减弱尿苷酰去除酶的活性，增强NH₄⁺的转运和利用，提高L-精氨酸的合成。[方法] 本文对来源于谷氨酸棒杆菌的突变菌株JNR中的双功能尿苷酰转移/去除酶GlnD进行整合突变，采用同源重组的方法将H₄₁₄和D₄₁₅位点突变为两个丙氨酸AA，在此菌株的基础上过量表达PII蛋白GlnK，并对其进行尿苷酰化研究，离子色谱检测摇瓶发酵过程中NH₄⁺的浓度，并对最终的改造菌株进行连续流加发酵分析。[结果] 该双功能尿苷酰转移/去除酶在谷氨酸棒杆菌中成功进行整合突变，有效减弱了尿苷酰去除酶的活性；同时过表达PII蛋白GlnK，其酰基化程度明显增强。摇瓶发酵结果表明菌株L4消耗NH₄⁺增加，L-精氨酸产量为36.2±1.2 g/L，比对照菌株L3高出22.7%。5-L发酵罐实验结果显示改造菌株L4的L-精氨酸的产量为52.2 g/L，较野生型菌株L0提高了25.3%。[结论] 谷氨酸棒杆菌合成L-精氨酸的过程中氮源是必不可少的。减弱GlnD尿苷酰去除酶的活性后，胞内尿苷酰化的GlnK-UMP增加，GlnK-UMP与氮转录调控因子AmtR结合，转运至胞内的NH₄⁺浓度提高，促使L-精氨酸产量显著提高。

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.