钝齿棒杆菌GlnK在氮代谢调控及L-精氨酸合成中的功能

Role of GlnK in regulating nitrogen metabolism and L-arginine synthesis in Corynebacterium crenatum

【目的】钝齿棒杆菌是重要的氨基酸生产菌株,本研究针对氮代谢PⅡ信号转导蛋白GlnK展开相关功能研究,分析其在钝齿棒杆菌氮代谢调控及L-精氨酸合成中的作用。【方法】以GlnK蛋白为研究对象,通过基因敲除等遗传方法获得过表达、敲除及敲弱glnK的重组钝齿棒杆菌,研究GlnK对NH4+吸收的影响,通过RT-qPCR和酶活测定,从转录水平和蛋白水平上揭示GlnK对氮代谢和L-精氨酸合成相关基因表达水平及酶活的影响,通过5-L发酵罐发酵产L-精氨酸研究GlnK对L-精氨酸合成的影响。【结果】过表达glnK能明显促进NH4+的吸收,而敲除glnK后则会抑制NH4+的摄取;RT-qPCR和酶活测定发现,相比于野生型菌株Cc5-5,glnK过表达菌株Cc-glnK中与铵吸收相关的基因,表达量平均上调约4.58倍,L-精氨酸合成基因簇中基因的表达水平平均上调1.50倍。Cc-glnK中氮代谢相关蛋白的酶活平均提高46.97%;L-精氨酸合成途径上7个关键酶的酶活平均提高30.00%;5-L发酵罐发酵各重组菌株结果表明,Cc-glnK菌株的产量可达49.53 g/L,产率为0.516 g/(L·h),相比于出发菌株Cc5-5,其L-精氨酸产量提高了28.65%。【结论】过表达GlnK能促进NH4+的吸收及利用,并通过影响L-精氨酸合成途径上关键基因的表达水平,提高关键酶的酶活,最终提高L-精氨酸的产量。本研究为后续探索钝齿棒杆菌氮代谢调控机制及代谢工程改造钝齿棒杆菌生产L-精氨酸提供了一种新的策略。

[Objective] In this study, we analyzed the roles of PⅡ signal transduction protein GlnK in nitrogen metabolism regulation and L-arginine biosynthesis in Corynebacterium crenatum. [Methods] The glnK overexpressed, glnK deletion and glnK knock-down strains were constructed. RT-qPCR and determination of enzyme activities were carried out to reveal the effects of GlnK on the expression levels and enzyme activities of nitrogen metabolism-related and L-arginine biosynthesis-related genes and enzymes. The changes of various parameters during the fermentation of recombinant strains were also investigated. [Results] Overexpression of GlnK protein had a significant effect on the absorption of NH4+. The expression levels and enzyme activities of nitrogen metabolism-related and L-arginine biosynthesis-related genes and proteins have generally been up-regulated in Cc-glnK strain. Among them, genes encoding ammonium absorption-related enzymes, such as glnA, gltD and gdh, were significantly up-regulated with an average increase about 4.58 times. The L-arginine yield and productivity of Cc-glnK reached 49.53 g/L and 0.516 g/(L·h), respectively, at the end of fermentation. [Conclusion] Overexpression of GlnK could promote the absorption of NH4+, increase the expression levels of genes and enzymes activities on the L-arginine biosynthesis pathway, ultimately increase the yield of L-arginine. It provided guidance for the subsequent exploration of the nitrogen regulation mechanism and metabolic modification of C. crenatum in the production of nitrogen-containing compounds.