Cloning and Random Mutagenesis of the Erwinia herbicola tyrR Gene for High-Level Expression of Tyrosine Phenol-Lyase

Abstract

ABSTRACT 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.