大肠杆菌苯丙氨酸合成途径关键酶基因pheA的克隆与表达

为了通过基因工程手段来增加苯丙氨酸的生物产量，利用ＰＣＲ方法从大肠杆菌中克隆了抗反馈抑制突变型及野生型的ｐｈｅＡ基因，进行了核苷酸序列分析，并利用高效的原核表达载体ＰＢＶ２２０对ｐｈｅＡ基因编码的突变型及野生型分支酸变位酶／预苯酸脱水酶（ＣＭ／ＰＤ）进行了表达。序列分析表明突变型基因碱基第５８０位由Ｔ变为Ｃ，相应氨基酸由Ｖａｌ变为Ａｌａ，ＳＤＳ－ＰＡＧＥ图谱扫描分析表明目的蛋白ＣＭ／ＰＤ的表达量占全菌体蛋白的４３％，占上清总蛋白的５７％。酶活性测定表明其ＣＭ和 ＰＤ活性分别提高了 １５．５和６．７倍，产酸量也有了一定的提高，为构建产苯丙氨酸的生物工程菌奠定了基础。

Molecular cloning and expression feedback inhibition-resistant pheA gene of Escherichia coli K-12

In order to improve the production of phenylalanine by bioengineering method, both the wild type and the feedback inhibition-resistant pheA gene were cloned. The result of DNA se- quencing indicated the T-580 in the wild type changed into C in the mutant, so the correspond amino acid changed from Val to Ala. The pheA gene of wild type and the feedback inhibition-re- sistant type encoding the double effect enzyme chorimate mutase/prephenate dehydratase (CM/ PD) were also high-level expressed in E. coli by PRPL promoter of pBV220. SDS-PAGE analysis indicated the yield of CM/PD is about 43% of total cellular proteins, and about 57% of total solvable protins. The specific activities of CM and PD were increased by 15. 5 and 6. 7 fold in strain harboring recombinant plasmid pBVpheA.