重排NAD^+合成途径提高磷酸烯醇式丙酮酸羧化酶活性

Enhanced phosphoenolpyruvate carboxylase activity by rewiring NAD^+ biosynthesis in E. coli

为提高磷酸烯醇式丙酮酸羧化酶的催化活性,构建了两个载体分别表达:(1)磷酸烯醇式丙酮酸羧化酶(PEPC);(2)与NAD+合成相关的关键酶,包括烟酸转磷酸核糖激酶(pnc B)、烟酸单核苷酸腺苷酰转移酶(nad D)和NAD+合成酶(nad E)。将这两个载体共转化大肠杆菌,获得重组菌E.coli(p ET-pepc&p UC-pnc B-nad D-nad E)。聚丙烯酰胺凝胶电泳显示这些酶均实现了高表达。摇瓶发酵显示,与单独过表达PEPC的重组菌相比,双质粒工程菌的PEPC酶活提高了4.36倍;与野生型大肠杆菌相比,双质粒工程菌的琥珀酸产量提高了4.23倍,达到0.9 g/L,富马酸产量提高了5.23倍,达到5.4 mg/L。上述结果表明加强NAD+合成途径提高了PEPC活性,并增加了还原三羧酸循环的代谢流量。

In order to enhance the catalytic activity of phosphoenolpyruvate carboxylase( PEPC),we have constructed two expression vectors which respectively overexpressed the following genes:( i) the PEPC gene from Synechocystis sp. PCC6803 and( ii) the NAD+synthesis genes pnc B,nad D and nad E,which encode nicotinate phosphoribosyltransferase,nicotinic acid mononucleotide adenylyltransferase and NAD+synthetase,respectively. After the two vectors were co-transformed into E. coli BL21,a recombinant strain named E. coli( p ET-pepc & p ET-pnc Bnad D-nad E) was acquired. SDS-PAGE analysis showed high expression of these enzymes. Under shake-flask conditions,this recombinant E. coli strain exhibited a 4. 36-fold increase in PEPC catalytic activity compared with the control strain that only harbored the vector p ET-pepc. Moreover,this strain produced 0. 9 g / L of succinic acid and5. 4 mg / L of fumaric acid,which are 4. 23- and 5. 23-times the corresponding values produced by wild-type E. coli BL21. Overall these results indicate that NAD+augmentation enhanced PEPC activity and increased the carbon flux in r TCA.