外源基因pheA、aroG和tyrB在苯丙氨酸合成途径中的共表达

Co-expression of Heterologous Genes pheA,aroG and tyrB for Biosynthesis Pathway of L-phenylalanine

利用基因工程技术提高了短杆菌的苯丙氨酸合成途径中关键酶活性 ,大幅度地增加了生物合成苯丙氨酸的产量。首先采用聚合酶链反应 (PCR)从大肠杆菌的氟代苯丙氨酸抗性变异菌株基因组中扩增到与苯丙氨酸合成相关的aroG ,pheA和tyrB 3个基因。其中aroG编码 3 脱氧 2 阿拉伯庚酮糖 7 磷酸合成酶 (DS) ,pheA编码双功能酶蛋白 分枝酸变位酶 (CM)和预苯酸脱水酶 (PD) ,tyrB编码转氨酶 (AT)。设计不同的酶切位点 ,利用质粒pUC118的多克隆位点 ,将 3个基因按不同的顺序组合串联 ,然后插入穿梭质粒pCZ 10 ,导入短杆菌中表达。结果表明 3个大肠杆菌基因在短杆菌中能够表达。其中以aroG pheA tyrB顺序串联而构建的黄色短杆菌工程菌株1311 GAB中的DS酶活力提高 4 5倍 ,CM提高 4 2倍 ,PD提高 2 7倍 ,AT提高 3 2倍 ;它的苯丙氨酸合成量提高2 35倍。

Three genes related to the biosynthesis of phenylalanine, pheA,aroG, and tyrB , encode key enzymes: a bifunctional protein\|chorismate mutase (CM)/prephenate dehydratase (PD).3\|deoxy\|D\|arabino\|heptulonate\|7\|phosphate synthetase (DS), and aminotransferase (AT),respectively.In this work,these genes were amplified from Escherichia coli mutants resistant to fluro\|L\|phenylalanine (FP) by polymerase chain reaction (PCR).The genes were expressed either separately or linked tandem as operons in the plasmids pUC118 and pCZ10.To study the effect of each gene,the recombinant plasmids of pUC and pCZ involving different linkage order of the genes were constructed.When three genes linking tandem by aroG\|pheA\|tyrB order on the plasmid pCZ\|gab transfered into Brevibacterium flavum, the specific activities of DS,CM,PD and AT were increased by 4.5, 4.2, 2.7 and 3.2 folds,repectively.As the result,the amount of phenylalanine biosynthesis was increased by 2.35 folds compared with that of host strain.