大肠杆菌苯丙氨酸生物合成关键酶的串联表达

ａｒｏＧ基因编码的 ３－脱氧－２－阿拉伯庚酮糖－７－磷酸合成酶（ＤＡＨＰ　Ｓｙｎｔｈｅｔａｓｅ　ＤＳ）和 ｐｈｅＡ基因编码的分支酸变位酶／预苯酸脱水酶（Ｃｈｏｒｉｍａｔｅ ｍｕｔａｓｅ／ Ｐｒｅｐｈｅｎａｔｅ　ｄｅｈｙｄｒａｔａｓｅ，ＣＷ／ＰＤ）都是本丙氨酸合成途径中的关键酶，为了通过基因工程手段来增加本丙氨酸生物的产量，在利用高效的原核表达载体ｐＢＶ２２ ０对ｐｈｅＡ基因编码的ＣＭ／ ＰＤ 酶进行了表达的基础上，采用ＰＣＲ方法扩增了抗反馈抑制的ａｒｃＧ基因，进行克隆表达，并与ｐｈｅＡ基因串联，以ＰＲＰＬ－ａｒｏＧ－ＰＬ－ｐｈｅＡ的形式，实现了２种酶基因在大肠杆菌中的表达， ＳＤＳＰＡＧＥ 图谱显示了新增的４３ｋｕ及３５ｋｕ蛋白带，经酶活性测定ＤＳ、ＣＭ／ＰＤ酶的比活分别提高了 ４．６７倍、８０５／１０．７１倍。

CO-EXPRESSION OF THE KEY ENZYMES FOR PHENYLALANINE PRODUCTION IN E. coli

Deoxy-D-arabino-heptulonate-7- phosphate synthetase (DAHP synthetase ) (DS ) and chorismate mutase/ prephenate dehydratase (CM/PD) are key enzymes in phenylalanine biosynthesis pathway. They are encoded by aroG and pheA respectively. In order to improve bio-production of phenylalanine through bioengineering means, a feedback inhibition-re- sistant aroG gene was cloned by PCR and co-expressed with gene pheA,which was cloned and expressed before. In the recom- binant,the two genes were clustered in the form of PRPL-aroG-PL-pheA,SDS-PAGE analysis shows two target protein prod- ucts of 35 KD and 43 KD. The enzyme activity analysis indicated the specific activities of DS, CM/PD were increased by 4. 67, 8. 05/10. 71 folds respectively.