大肠杆菌aroG基因的克隆表达及与pheA、tyrB基因的串联表达

３－脱氧－２－阿拉伯庚酮糖－７－磷酸合成酶（ＤＡＨＰ）是苯丙氨酸合成途径中关键酶之一，在大肠杆菌中由ａｒｏＧ基因编码。本文用ＮＴＧ诱变得到对苯丙氨酸类似物间氟苯丙氨酸（ｍＦＰ）和对氟苯丙氨酸（ｐＦＰ）有抗性的大肠杆菌突变株，采用聚合酶链反应（ＰＣＲ）扩增得到了ａｒｏＧ基因，在大肠杆菌中进行了表达。结果表明，该基因能在λ噬菌体的ｐＲ启动子驱动下得到表达，在ＳＤＳ－聚丙烯酰胺凝胶电泳图上出现清晰的条带，酶的比活提高了１．７倍。在ｐｈｅＡ（编码分枝酸变位酶ＣＭ和预苯酸脱水酶ＰＤ）、ｔｙｒＢ（编码苯丙氨酸转氨酶ＰＡＴ）基因克隆、串联克隆和表达完成的基础上，将ａｒｏＧ基因和ｐｈｅＡ、ｔｙｒＢ基因以ａｒｏＧ－ｐｈｅＡ－ｔｙｒＢ的顺序三基因串联到表达载体进行表达，酶活测定结果表明，三个基因都能在λ噬菌体的ｐＲ启动子驱动下表达，与对照菌株相比，酶比活分别提高了１．７倍、１３．９／７．８倍和２．３倍。

Cloning and Expression of aro G Gene of E.coli and Its Co expression with phe A and tyr B Genes

Deoxy D arabino heptulonate 7 phosphate synthetase (DAHP) is one of the key enzymes in phenylalanine biosynthesis pathway. In E.coli , DAHP is encoded by aro G gene. In this work, aro G was cloned from an E.coli mutant strain resistant to m fluro L phenylalanine ( m PF) and p fluro L phenyalanine ( p PF) by polymerase chain reaction (PCR). The gene was expressed under the control of lambda phage promoter p R in P2392 strain of E.coli . Distint band was detected as the product of aro G on SDS polyacrylamide gel electrophoresis (SDS PAGE). The specific activity in crude extract of DAHP was raised to 1.7 fold. Based on the cloning and expression of phe A (encoding both chorsmate mutase CM and prephenate dehydratase PD) tyr B (encoding phenylalanine aminotransferase PAT)genes, aro G, phe A and tyr B genes were constructed and expressed in P2392, the result showed that the specific activities of DAPH, CM/PD and PAT in crude extracts were increased by 1.7, 13.9/7.8 and 2.3 fold, respectively.