Fan C, Zeng X, Chai Y, Jiang P, Huang W
Department of Microbiology, Fudan University, Shanghai, China 200433.
Wei Sheng Wu Xue Bao. 1999 Oct;39(5):430-5.
aroG and pheA genes, encoding 3-Deoxy-D-arabinoheptulonate-7-phosphate synthase(DS) and Chorismate mutase (CM)-prephenate dehydratase(PD) in the pathway of phenylalanine biosynthesis respectively, were amplified by polymerase chain reaction(PCR). The genes were assembled on the multicopy vectors and expressed in both Escherichia coli and Brevibacterium. The products of two gene were detected by SDS-PAGE. The activities of relevant enzymes were measured in the crude extract of the host strain. When aroG-pheA genes were introduced into E. coli p2392, the activities of DS, CM and PD were increased by 4.3-fold, 4.4-fold and 2.2-fold respectively. Whereas in the case of Brevibacterium flavum 2732, the activities of DS, CM and PD were increased by 12.3-fold, 2.3-fold and 5.6-fold, respectively. As the results, the overproduction of phenylalanine was brought about by using the genetic engineering strain of B. flavum.
分别编码苯丙氨酸生物合成途径中的3-脱氧-D-阿拉伯庚酮糖-7-磷酸合酶(DS)和分支酸变位酶(CM)-预苯酸脱水酶(PD)的aroG和pheA基因,通过聚合酶链反应(PCR)进行扩增。这些基因被组装到多拷贝载体上,并在大肠杆菌和短杆菌中表达。通过SDS-PAGE检测两个基因的产物。在宿主菌株的粗提取物中测定相关酶的活性。当aroG-pheA基因导入大肠杆菌p2392时,DS、CM和PD的活性分别提高了4.3倍、4.4倍和2.2倍。而在黄色短杆菌2732中,DS、CM和PD的活性分别提高了12.3倍、2.3倍和5.6倍。结果,使用黄色短杆菌的基因工程菌株实现了苯丙氨酸的过量生产。