Cloning and Random Mutagenesis of theErwinia herbicola tyrRGene for High-Level Expression of Tyrosine Phenol-Lyase

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 E201Gproteins were examined in vivo. Interestingly, as opposed to the wild-type TyrR protein, the mutant TyrR V67Aprotein had a repressive effect on the tyrP promoter in the presence of phenylalanine as the coeffector.Tyrosine phenol-lyase (Tpl) (EC 4.1.99.2) normally catalyzes the degradation of tyrosine into pyruvate, ammonia, and phenol (26-28, 56). However, this reaction is reversible, and if catechol is substituted for phenol, L-dihydroxyphenylalanine (L-DOPA) is produced (24, 57). L-DOPA is used in the treatment of Parkinson's disease, which afflicts 1 out of every 1,700 individuals. About 250 tons of L-DOPA is now supplied per year, and more than half of it is produced by an enzymatic method involving Tpl (24, 57).On an industrial scale, Erwinia herbicola cells with extremely high Tpl activity are prepared by cultivation in a medium containing L-tyrosine as an inducer of Tpl. The intact cells are then harvested by centrifugation and transferred to the reactor, as the catalyst, together with the substrate. This microbiological method is efficient; however, it actually has one serious drawback. Since Tpl is only synthesized under L-tyrosine-induced conditions (16, 49), the cells must be grown in medium supplemented with L-tyrosine. The extremely low solubility of L-tyrosine results in considerable carryover of it into the reactor, which severely complicates the separation of the final product, L-DOPA (hydroxyl derivative of L-tyrosine), from the remaining L-tyrosine. To avoid this drawback, the tpl genes of E. herbicola (17,20,50) and Citrobacter freundii (21) were cloned and expressed in Escherichia coli under the control of the tac promoter, respectively. In either case, Tpl was highly induced upon the addition of isopropyl-␤-D-thiogalactopyranoside (IPTG); however, the L-DOPA productivity of the cells was inferior to that of E. herbic...

Identification of the LIV-I/LS System as the Third Phenylalanine Transporter in Escherichia coli K-12

“…The same results were obtained for five independently constructed strains. This was surprising, because the expression of the aroP gene is known to be strongly repressed by tyrosine but not by tryptophan (9,22,36,(46)(47)(48)55) and the AroP system is known to exhibit almost equal affinity for the three aromatic amino acids (5,36). At present, the reason for this phenomenon is unclear.…”

Hyperproduction of 3,4-Dihydroxyphenyl-L-alanine (L-Dopa) UsingErwinia herbicolaCells Carrying a Mutant Transcriptional Regulator TyrR

“…One such mutant, TyrR V67A Y72C E201G , was found to increase Tpl expression effectively, 10) and when the mutant gene was introduced into E. herbicola, the resulting strain showed improved L-dopa productivity. 11) However, the addition of L-tyrosine to the medium was still required to achieve the maximum productivity of the strain (Fig.…”

Functional Analysis of the Erwinia herbicola tutB Gene and Its Product

“…In a preliminary experiment, we observed that the introduction of pACYC177 carrying the tutB gene into an E. coli strain with the ⌽(tplЈ-Јlac) gene (14) enhanced the tyrosine-induction ratio of tpl by twofold, but neither the phenylalanine-nor the tryptophan-mediated induction ratio was elevated. Since the expression of tpl is positively regulated by the TyrR protein (22) and its coeffectors (three aromatic amino acids), even though the magnitude of activation is quite different among the three ligands (13,28), the above observation implied that the tutB gene might encode a tyrosine transporter. In order to characterize the tutB gene product, an E. coli strain deficient in the uptake of all aromatic amino acids (5) was constructed (MG1655 ⌬aroP mtr24 ⌬pheP ⌬tna ⌬tyrP) and used for transport assays.…”

“…It has been shown that the F plasmid is stably maintained in Erwinia species (3). The Tpl contents of the cells grown under tyrosine-induced and non-induced conditions were determined by immunoblotting (13), and the results are presented in Fig. 4.…”

“…gene was cloned from an E. herbicola genomic library constructed as described previously (13), and the resulting plasmid was named pTK928. The 0.6-kb MfeI-HpaI fragment excised from pTK928 and the 3.3-kb SmaI-MfeI fragment cut off from pEBT were annealed at their MfeI-protruding ends, and the resulting 3.9-kb fragment containing the entire tpl and tutB genes in their native configurations was cloned into the SmaI site of pUC19 to generate pTK1016.…”