pepc和pck介导的草酰乙酸回补途径对谷氨酸棒杆菌v1氨基酸代谢 ....doc

磷酸烯醇式丙酮酸羧化酶和磷酸烯醇式丙酮酸羧激酶介导的草酰乙酸回补途径对谷氨酸棒杆菌V1氨基酸代谢的影响张旦旦1 仇爱梅1 鲍勇1 窦文芳1 许正宏2江南大学药学院制药工程研究室 江苏 无锡 214122) 江南大学粮食发酵工艺与技术国家工程实验室 江苏 无锡 214122) 摘 要【目的】谷氨酸棒杆菌是工业生产氨基酸的主要菌株，以缬氨酸高产菌株谷氨酸棒杆菌V1为研究对象，探讨磷酸烯醇式丙酮酸羧化酶(PEPC)和磷酸烯醇式丙酮酸羧激酶(PCK)介导的草酰乙酸回补途径对菌株生理特性以及主要氨基酸代谢流量的影响。【方法】通过基因工程手段，在谷氨酸棒杆菌V1中过表达pepc (编码PEPC)和pck (编码PCK)，比较重组菌与出发菌关键酶活性、发酵特性、以及主要氨基酸积累量变化。【结果】成功构建两株重组菌V1-pepc (强化草酰乙酸回补途径)和V1-pck (弱化草酰乙酸回补途径)，重组菌生长均较出发菌延缓，总生物量、葡萄糖和硫酸铵消耗基本不变；过表达pck，PCK活性提高22.8%，丙氨酸、缬氨酸、谷氨酸、精氨酸积累量分别提高了11.8%、17.2%、27.8%和19.5%；过表达pepc，PEPC活性提高27.5%，同时PC活性降低12.9%，天冬氨酸族和谷氨酸族氨基酸的整体流量变化不大，丙氨酸族氨基酸的整体流量降低了14.7%。【结论】丙氨酸族氨基酸受此回补途径影响较大，天冬氨酸族氨基酸受此影响较小。 关键词谷氨酸棒杆菌V1，磷酸烯醇式丙酮酸羧化酶，磷酸烯醇式丙酮酸羧激酶，草酰乙酸回补途径，缬氨酸Effects of oxaloacetate anaplerotic reaction mediated by phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PCK) on amino acid etabolism of Corynebacterium glutamicum V1 Zhang Dan-Dan1 QIU Ai-Mei1 BAO Yong1 DOU Wen-Fang1 XU Zheng-Hong2* (1. Laboratory of Phamaceutical Engineering, School of Medicine, Jiangnan University, Wuxi, Jiangsu 214122, China) (2. National Engineering Laboratory for Cereal Fermention Technology, Jiangnan University, Wuxi, Jiangsu 214122, China) Abstract: [Objective] Corynebacterium glutamicum is the main industry strain for production of amino acids, to explore the effects of oxaloacetate anaplerotic reaction mediated by phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PCK) on physiological characteristics and amino acid metabolism, the valine producing strain Corynebacterium glutamicum V1 was researched. [Methods] Overexpressing of pepc encoding PEPC and pck encoding PCK in Corynebacterium glutamicum V1 by means of genetic engineering, the changes of key enzyme activities, fermentation characteristics and main amino acids accumulation were discussed compared with the original strain. [Results] Two recombinant strains V1-pepc (enhanced oxaloacetate anaplerotic pathway) and V1-pck (weakened oxaloacetate anaplerotic pathway) were