Cloning and random mutagenesis of the Erwinia herbicola tyrR gene for high-level expression of tyrosine phenol-lyase
Cloning and random mutagenesis of the Erwinia herbicola tyrR gene for high-level expression of tyrosine phenol-lyase
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DOI：
10.1128/aem.66.11.4764-4771.2000
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发表时间：
2000-11-01
影响因子：
4.4
通讯作者：
Kumagai, H
中科院分区：
文献类型：
--
作者：
Katayama, T;Suzuki, H;Kumagai, H
酪氨酸苯酚裂解酶（Tyrosine phenol-lyase, Tpl）是一种酪氨酸诱导酶，能以丙酮酸、氨和儿茶酚为原料合成3,4-二羟基苯丙氨酸。以往的研究表明，除草剂欧文氏菌的tpl启动子可被大肠杆菌的TyrR蛋白激活。为了创造一个高tpl表达的菌株，我们克隆了tyrR基因。除草剂，然后随机诱变。利用lac报告系统在大肠杆菌中筛选了具有增强tpl激活能力的TyrR突变蛋白。突变tyrR等位基因的菌株tpl转录增加最多，其中缬氨酸-67、酪氨酸-72和谷氨酸-201分别被丙氨酸、半胱氨酸和甘氨酸取代。构建了一株缺乏tyrR的除草剂E. herola衍生物，并用携带突变tyrR等位基因（V67A Y72C E201G取代）的质粒进行转化。所得菌株在培养基中不添加酪氨酸的情况下表达Tpl，其产量与在酪氨酸诱导条件下生长的野生型菌株一样多。在体内检测突变体TyrR（V67A）、TyrR（V72C）、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，突变体TyrRV67A蛋白在苯丙氨酸作为系数存在时对tyrP启动子具有抑制作用。
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 off. 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(V72C), TyrR(E201G), and TyrR(V67A Y72C E201G) proteins were examined in vivo. Interestingly, as opposed to the wild-type TyrR protein, the mutant TyrRV67A protein had a repressive effect on the tyrP promoter in the presence of phenylalanine as the coeffector.