精氨酸合成途径关键酶基因的过量表达及其对L-精氨酸合成的影响

Overexpression of Key Enzymes in Arginine Biosynthetic Pathway and their Effects on L-arginine Production

摘要:

由CarAB、ArgD、ArgJ三个基因分别编码的氨基甲酰磷酸合成酶、乙酰鸟氨酸转氨酶、鸟氨酸乙酰基转移酶是L-精氨酸合成途径的关键酶。以谷氨酸棒杆菌ATCC14067为出发菌株，利用大肠杆菌-谷氨酸棒杆菌的穿梭质粒pEC-XK99E-p’、PEC-T18mob2，分别对精氨酸生物合成途径的关键酶基因CarAB、ArgD、ArgJ进行单个基因表达、对ArgD、ArgJ基因进行串联表达以及对CarAB、ArgD、ArgJ三个基因进行共表达，分别得到过量表达菌株14067-pEC-CarAB、14067-T18-ArgD、14067-T18-ArgJ、14067-T18-ArgDJ、14067-pEC-CarAB-T18-ArgDJ。检测上述三个基因单独表达，串联表达及共表达对L-精氨酸产量的影响。摇瓶发酵实验证明，这五株菌产精氨酸的能力较出发菌株ATCC14067分别提高了1.61、1.14、1.19、1.28、2.91倍。产量最高的共表达菌株14067-pEC-CarAB-T18-ArgDJ，通过实时荧光定量PCR检测，表明CarA、CarB、ArgD、ArgJ基因的表达量分别是ATCC14067的2.50、5.60、3.59、4.70倍。这表明过量表达基因CarAB、ArgD、ArgJ可以有效提高精氨酸的产量。

Abstract:

Carbamoyl phosphate synthase, Acetyl ornithine amino transferase and ornithine acetyl transferase encoded by CarAB, ArgD and ArgJ were the key enzymes in L-arginine biosynthetic pathway. In Corynebacterium glutamicum ATCC14067, with the plasmids pEC- XK99E-p and PEC-T18mob2, the genes of CarAB, ArgD and ArgJ in the arginine biosynthetic pathway were expressed individually, ArgD and ArgJ were overexpressed in series and the CarAB, ArgD and ArgJ were co-expressed. The ability of producing L-arginine of the recombination strains, 14067-pEC-CarAB, 14067-T18-ArgD, 14067-T18-ArgJ, 14067-T18-ArgDJ and 14067-pEC-CarAB-T18-ArgDJ, increased 1.61, 1.14, 1.19, 1.28 and 2.19 times, respectively, in shake flask which compared with the starting strain ATCC14067. The real-time quantitative PCR showed that the relative expression of genes CarA, CarB, ArgD and ArgJ in strain 14067-pEC-CarAB-T18-ArgDJ were 2.50, 5.60, 3.59 and 4.70 times, respectively, than ATCC14067. Therefore, overexpression of CarAB, ArgD and ArgJ genes can effectively improve the production of L-arginine.