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

【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。

[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 gln DAA(including H414 A and D415 A)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.