大肠杆菌色氨酸操纵子基因突变株的构建与表达

Construction and Expression of Trp Operon Gene Mutant of E.coli

目的构建大肠杆菌色氨酸操纵子基因突变株,提高邻氨基苯甲酸合成酶和色氨酸合成酶的产量。方法利用依赖于DpnⅠ酶的PCR方法突变表达载体pET-22b(+)-Trp Operon上的关键位点,PCR扩增Trp OperonM基因,构建pET-22b(+)-Trp OperonM重组表达质粒,经酶切及测序鉴定正确后,转化大肠杆菌BL21(DE3),IPTG诱导表达。制备粗酶液,经比色法测定邻氨基苯甲酸合成酶和色氨酸合成酶的活性。结果 PCR扩增产物可见约7000bp的Trp OperonM条带;所构建的重组表达质粒经酶切及测序鉴定正确;邻氨基苯甲酸合成酶和色氨酸合成酶的活性比大肠杆菌BL21(DE3)空菌分别提高了4.5倍和5.2倍。结论已成功构建了大肠杆菌色氨酸操纵子基因突变株BL21(DE3)/pET-22b(+)-Trp OperonM,邻氨基苯甲酸合成酶和色氨酸合成酶的活性均有提高,为高产色氨酸基因工程菌的构建奠定了基础。

Objective To construct the Trp operon gene mutant of E.coli and increase the yields of anthranilic acid synthase and tryptophan synthase.Methods The mutant of Trp Operon gene（Trp OperonM）was amplified by DpnⅠ-dependent PCR,based on which recombinant plasmid pET-22b（＋）-Trp OperonM was constructed,then identified by restriction analysis and sequencing and transformed to E.coli BL21（DE3）for expression under induction of IPTG.The crude extract of the recombinant E.coli was determined for the activities of anthranilic acid synthase and tryptophan synthase by colorimetry.Results The PCR product showed a Trp OperonM band at a length of about 7 000 bp on electrophoretic profile.Both restriction analysis and sequencing proved that recombinant plasmid pET-22b（＋）-Trp OperonM was constructed correctly.As compared with those of empty E.coli BL21（DE3）,the anthranilic acid synthase and tryptophan synthase activities of recombinant E.coli BL21（DE3）/pET-22b（＋）-Trp OperonM increased by 4.5 and 5.2 folds respectively.Conclusion The Trp operon gene mutant of E.coli was successfully constructed,with increased activities of anthranilic acid synthase and tryptophan synthase,which laid a foundation of construction of recombinant E.coli highly producing tryptophan.