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Applied and Environmental Microbiology, November 2000, p. 4764-4771, Vol. 66, No. 11
Applied Molecular Microbiology, Division of
Applied Life Sciences, Graduate School of
Agriculture,1 and Applied Molecular
Microbiology, Division of Integrated Life Science, Graduate School
of Biostudies,2 Kyoto University, Kyoto, Japan
Received 4 May 2000/Accepted 4 August 2000
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 TyrRV67A, TyrRY72C,
TyrRE201G, and TyrRV67A 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.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cloning and Random Mutagenesis of the Erwinia
herbicola tyrR Gene for High-Level Expression of Tyrosine
Phenol-Lyase
*
Corresponding author. Mailing address: Applied
Molecular Microbiology, Division of Integrated Life Science, Graduate
School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. Phone: 81-75-753-6276. Fax: 81-75-753-6275. E-mail: hidekuma{at}kais.kyoto-u.ac.jp.
Applied and Environmental Microbiology, November 2000, p. 4764-4771, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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