【摘要】 目的：蛋白质赖氨酸乙酰化(KAc)修饰广泛存在于糖酵解、TCA循环等中心代谢途径的关键酶中，这些在特定位点进行可逆的KAc修饰作用能通过影响酶活来调控胞内各种代谢路径；cg3035基因编码N-乙酰谷氨酸合酶(NAGS),负责催化谷氨酸乙酰化以提供前体。探究KAc修饰及提高前体L-谷氨酸利用对钝齿棒杆菌产L-精氨酸的影响。方法：利用无痕技术敲除cobB基因(编码去乙酰化酶),提高胞内KAc水平；过表达cg3035基因，增强对前体L-谷氨酸的利用。通过Western blot和HPLC/MS/MS初步分析敲除cobB基因对蛋白质KAc水平的影响，并鉴定特定蛋白质的乙酰化位点变化；通过ArgF点突变验证乙酰化修饰对酶活的影响。结果：成功构建了一株提高L-精氨酸产量菌株，其产量提升53.8%。cobB敲除显著改变蛋白质KAc修饰动态规律，通过分析CCM01和CCM02中可溶性蛋白的Western blot结果，切取84 h位于约48 kDa处两个样的蛋白质条带进行HPLC/MS/MS分析，显示ArgC、ArgF等精氨酸合成途径酶及TCA循环关键酶的乙酰化水平显著上调。ArgF的K281Q和K294Q突变使其酶活分别降低5.14%和13.34%,表明乙酰化水平提升不一定能提高酶活。结论：通过提高蛋白质KAc水平和优化前体供应，可显著促进钝齿棒杆菌产L-精氨酸。cobB基因敲除通过改变精氨酸途径酶和TCA循环关键酶的乙酰化水平调控α-精氨酸的合成效率，这为工业菌株的代谢工程改造提供了新的分子育种策略。更多还原

【Abstract】 Objective: To explore how protein lysine acetylation(KAc) modification and the increased utilization of the amino acid precursor L-glutamic acid affect the production of L-arginine by Corynebacterium crenatum. Methods: The cobB gene was knocked out using scarless technology to increase the intracellular KAc level, while the cg3035 gene was overexpressed to enhance the utilization of the precursor L-glutamic acid. The influence of knocking out the cobB gene on the protein KAc level was preliminarily analyzed by Western blot and HPLC/MS/MS, and the changes in the acetylation sites of specific proteins were identified. Additionally, the effect of acetylation modification on enzyme activity was verified by performing an ArgF point mutation. Results: A strain that increases L-arginine yield was successfully constructed, increasing its yield by 53.8%. Knocking out cobB significantly altered the dynamic pattern of protein KAc modification. By HPLC/MS/MS, identification of the 48 kDa band at 84 h showed that the acetylation levels of enzymes in the arginine synthesis pathway, such as ArgC and ArgF, and key enzymes in the TCA cycle, were significantly upregulated. The K281Q and K294Q mutations in the ArgF enzyme reduced its activity by 5.14% and 13.34%, respectively, indicating that an increase in acetylation level does not necessarily enhance enzyme activity. Conclusion: By increasing the protein KAc level and optimizing the supply of precursors, the production of L-arginine by Corynebacterium crenatum can be significantly promoted. Knocking out the cobB gene regulates the efficiency with which arginine is synthesized by altering the levels at which arginine pathway enzymes and key enzymes in the TCA cycle are acetylated. This provides a new molecular breeding strategy for the metabolic engineering modification of industrial strains.更多还原