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

【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】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。

Effects of oxaloacetate anaplerotic reaction mediated by phosphoenolpyruvate carboxylase (PEPC) and phosphoenolpyruvate carboxykinase (PCK) on amino acid etabolism of Corynebacterium glutamicum V1

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 studied. 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 constructed, both the growth trends were delayed, the total biomass and the consumption of sugar and ammonium basically unchanged; Overexpression of pck, the activity of PCK has 22.8% increased, the yields of alanine, valine, glutamic and arginine were increased by 11.8%, 17.2%, 27.8% and 19.5% respectively; Over expression of pepc, the activity of PEPC increased by 27.5%, while the activity of PC decreased 12.9%, the overall flow of aspartic and glutamic-family amino acids changes little, while the flow of alanine-family reduced by 14.7%. Conclusion] Alanine-family amino acids are greatly influenced by this pathway while aspartate-family not.