Tyrosine phenol-lyase (Tpl), which can synthesize 3, 4-dihydroxyphenylalanine from pyruvate, ammonia, and catechol, is a tyrosine-inducible enzyme. Previous studies demonstrated that the tpl promoter of Erwinia herbicola is activated by the TyrR protein of Escherichia coli. In an attempt to create a high-Tpl-expressing strain, we cloned the tyrR gene of E. herbicola and then randomly mutagenized it. Mutant TyrR proteins with enhanced ability to activate tpl were screened for by use of the lac reporter system in E. coli. The most increased transcription of tpl was observed for the strain with the mutant tyrR allele involving amino acid substitutions of alanine, cysteine, and glycine for valine-67, tyrosine-72, and glutamate-201, respectively. A tyrR-deficient derivative of E. herbicola was constructed and transformed with a plasmid carrying the mutant tyrR allele (V67A Y72C E201G substitutions). The resultant strain expressed Tpl without the addition of tyrosine to the medium and produced as much of it as was produced by the wild-type strain grown under tyrosine-induced conditions. The regulatory properties of the mutant TyrR(V67A), TyrR(Y72C), TyrR(E201G), and TyrR(V67A Y72C E201G) proteins were examined in vivo. Interestingly, as opposed to the wild-type TyrR protein, the mutant TyrR(V67A) protein had a repressive effect on the tyrP promoter in the presence of phenylalanine as the coeffector.

译文

酪氨酸酚裂解酶(Tpl)可以从酪氨酸,氨水和邻苯二酚合成3,4-二羟基苯丙氨酸,是一种酪氨酸诱导酶。先前的研究表明,草食欧文氏菌的tpl启动子被大肠杆菌的TyrR蛋白激活。为了创建高表达Tpl的菌株,我们克隆了E. herbicola的tyrR基因,然后对其进行了随机诱变。通过使用大肠杆菌中的lac报告系统筛选具有增强的tpl激活能力的突变TyrR蛋白。对于具有突变tyrR等位基因的菌株,观察到tpl的转录增加最多,所述突变体分别涉及丙氨酸,半胱氨酸和甘氨酸的氨基酸取代缬氨酸67,酪氨酸72和谷氨酸201。构建了E. herbicola的tyrR缺陷型衍生物,并用携带突变型tyrR等位基因(V67A Y72C E201G替代)的质粒转化。所得菌株在不向培养基中添加酪氨酸的情况下表达Tpl,并产生与在酪氨酸诱导的条件下生长的野生型菌株所产生的一样多的菌株。在体内检查了突变体TyrR(V67A),TyrR(Y72C),TyrR(E201G)和TyrR(V67A Y72C E201G)蛋白的调控特性。有趣的是,与野生型TyrR蛋白相反,突变型TyrR(V67A)蛋白在存在苯丙氨酸作为协同增效剂的情况下对tyrP启动子具有抑制作用。

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