钝齿棒杆菌黄素血红蛋白Hmp在L-精氨酸合成中的作用

Role of Corynebacterium crenatum flavohemoglobin Hmp in the synthesis of L-arginine

钝齿棒杆菌(Corynebacterium crenatum)SYPA5-5是本实验室选育的一株L-精氨酸高产菌,基于前期转录组和蛋白组数据分析,发现黄素血红蛋白(flavohemoglobin,Hmp)在高氮源相对于低氮源条件下表达水平上调,进一步研究Hmp的抗氧化胁迫功能和在L-精氨酸合成中的作用.构建了hmp过表达和敲除菌株,首先以Hmp蛋白为研究对象,分析Hmp的圆二色谱结构,用SWISS-MODEL建模分析,测其NAD(P)H氧化酶酶活,验证了Hmp的结构与功能.接下来研究Hmp与氧化应激之间的关系,首先测定发酵过程胞内活性氧(reactive oxygen species,ROS)水平以及超氧化物歧化酶(SOD)和过氧化氢酶(CAT)酶活,最后通过5-L发酵罐扩大发酵体系来探究Hmp在L-精氨酸合成中的作用.过表达hmp在发酵过程中降低了活性氧水平,分别提高了9.2%和8.7%的SOD和CAT酶活,促进了菌体生长代谢和提高目标代谢产物的积累.通过检测发酵过程的细胞干重、葡萄糖含量和L-精氨酸产量,发现hmp过表达菌株Cc-hmp的L-精氨酸产量和得率相比于原始菌株Cc5-5分别提高了19.7%和29.6%,hmp敲除菌株Cc-Δhmp的L-精氨酸产量和得率相比于原始菌株Cc5-5分别减少37.9%和14.8%.本研究表明高氮高溶氧条件下,使用发酵法生产L-精氨酸的过程中,氧化应激的产生及带来的氧化损伤不可避免,Hmp可以有效抵抗胞内氧化应激,改善胞内的氧化还原状态,维持一个良好的胞内环境,从而提高L-精氨酸的产量.(图10表3参31)

Transcriptome and proteome analyses of the high L-arginine producing Corynebacterium crenatum SYPA5-5 strain previously characterized in our laboratory revealed that under high nitrogen concentrations,at the transcription level,the amount of hmp increased compared to that at low nitrogen concentrations.This study found Hmp functions as a stress antioxidant and further explored its role in the synthesis of L-arginine.Therefore,we first constructed the hmp overexpression and knockout strains.Next,we analyzed the circular dichroism structure of Hmp,modeled the protein structure using SWISS-MODEL,and measured its NAD(P)H oxidase activity,before verifying its structure and function.Next,we studied the relationship between Hmp and oxidative stress by measuring reactive oxygen species(ROS)levels and superoxide dismutase and catalase enzyme activities during fermentation.Finally,we expanded the fermentation system through a 5-L fermenter to explore its effects on the synthesis of L-arginine.hmp overexpression reduced the level of ROS during the fermentation process,improved SOD and CAT activities by 9.2%and 8.7%,respectively,and increased the yield of target substances.Detection of cell density,glucose content,and L-arginine production during the fermentation process,indicated that compared to Cc5-5,the production and yield of Cc-hmp L-arginine were increased by 19.7%and 29.6%,and those of Cc-Δhmp L-arginine were reduced by 37.9%and 14.8%,respectively.Hence,this study shows that in the process of L-arginine fermentation with high nitrogen and dissolved oxygen,oxidative stress and oxidative damage are inevitable.Hmp effectively resisted intracellular oxidative stress and improved the redox state of cells.This strategy could be applied to maintain a good cellular environment during fermentation,thereby increasing the production of L-arginine.