苯丙氨酸合成的关键酶基因aroG与pheA串联表达

EXPRESSION OF GENES aroG AND pheA IN PHENYLALANINE BIOSYNTHESIS

ａｒｏＧ 和ｐｈｅＡ 是与苯丙氨酸合成有关的两个重要基因。在大肠杆菌（ Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ） 中，ａｒｏＧ 基因编码脱氧阿拉伯糖型庚酮糖磷酸合酶（ＤＳ） ，该酶催化由糖代谢中心途径分流出来的磷酸烯醇丙酮酸（ＰＥＰ） 和赤鲜糖４磷酸（Ｅ４Ｐ） 缩合形成脱氧阿拉伯糖型庚酮糖磷酸（ＤＡＨＰ） 的反应；ｐｈｅＡ 基因编码一个双功能酶蛋白，它同时催化两步关键反应，即具有分枝酸变位酶（ＣＭ） 和预苯酸脱水（ＰＤ） 的两种功能。采用ＰＣＲ 技术分别从两个不同品系的大肠杆菌染色体ＤＮＡ 中扩增到ａｒｏＧ 和ｐｈｅＡ。当这两个基因串联在一个质粒上导入大肠杆菌Ｐ２３９２ 中进行表达时，它们编码的酶ＤＳ、ＣＭ 和ＰＤ 活性分别提高４－３ 、４－４ 和２－２ 倍；导入短杆菌（ Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ）２７３１ 中表达时，相应的酶活性分别提高１２－３ 、２－３ 和５－６ 倍。两基因的串联表达能大幅度地提高工程菌株的苯丙氨酸发酵产量。

aroGand pheAgenes,encoding 3 Deoxy D arabinoheptulonate 7 phosphatesyn thase(DS) and Chorismate mutase (CM) prephenate dehydratase(PD) in the pathway of phenylalnine biosynthesisrespectively,were amplified by polymerase chain reaction(PCR) .The genes were assembled on the multicopy vectors and expressed in both Escherichia coli and Brevibacterium .The productsoftwogene weredetected by SDS PAGE.Theactivities ofrelevantenzymes were measuredinthecrudeextractofthe hoststrain. When aroG pheA genes wereintroducedinto E.coli p2392 ,the activities of DS,CMand PDwereincreased by 4 .3 fold,4 .4 fold and 2.2 fold respectively. Whereas in the case of Brevibacterium flavum 2732,the activitiesof DS,CMand PDwereincreased 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 geneticengineeringstrain of B.flavum .