Feedback Inhibition of Chorismate Mutase/Prephenate Dehydrogenase (TyrA) ofEscherichia coli: Generation and Characterization of Tyrosine-Insensitive Mutants

Abstract

In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/ prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA fbr mutants were selected by virtue of their resistance toward m-fluoro-D,L-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His 6 -tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrA WT , but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrA fbr mutants were similar to those of the TyrA WT protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA fbr contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.Chorismate is a central intermediate of the shikimate pathway and branch point for five different metabolic pathways in microorganisms (10). The aromatic amino acids L-phenylalanine and L-tyrosine are formed from chorismate via prephenate, which undergoes either decarboxylation/dehydration or decarboxylation/dehydrogenation, followed by a transamination to form the respective amino acids (Fig. 1).Chorismate mutase/prephenate dehydrogenase (CM/PDH, TyrA) is a bifunctional enzyme occurring as a homodimer with a molecular weight of approximately 78,000 and is involved in tyrosine biosynthesis in Escherichia coli (13). The rearrangement of chorismate to prephenate is catalyzed by the N-terminal CM domain of TyrA, whereas the C-terminal PDH domain catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate. Analogously, the PDT domain of the bifunctional CM/PDT (PheA) yields phenylpyruvate, which is subsequently transaminated to phenylalanine.Aromatic amino acid biosynthesis is tightly regulated, in particular, at the chorismate branch point and at the first committed step, i.e., at 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, which catalyzes the condensation of phosphoenolpyruvate and erythrose-4-phosphate. The DAHP synthase occurs as three isoforms, each feedback regulated by either phenylalanine, tyrosine, or tryptophan (20). Furthermore, the TyrR protein, binding any one of the three aromatic amino acids, provides transcriptional control of eight different promoters related to aromatic amino acid biosynthesis (21).Due to the industrial importance of L-phenylalanine, for example, as a precursor for the sweetener aspartame (L-aspartyl-L-phenylalanine methyl ester), much more effort has been devoted to the investigation of the phenylalanine biosynthesis pathway than to the tyrosine branch (1). For the biotechnological production of phenylalanine, engineered E. coli strains were employed, exhibiting, among other characteristics, alleviated feedback inhibition by the end product (23, 24). As a consequence, different feedback-inhibition-...

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“…This concentration would have to be optimized by increasing the flux through the phenylalanine biosynthetic pathway. To do so, strategies have already been applied in other organisms (i.e., Escherichia coli [43] and S. cerevisiae [44]). In S. cerevisiae, the simultaneous replacement of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (ARO4) and the chorismate mutase (ARO7) with tyrosine feedback-insensitive alleles (ARO4 K229L and ARO7 G141S ) led to a 4.5-fold increase of the in vivo flux toward phenylalanine (44).…”

Metabolic Engineering of Escherichia coli for l -Tyrosine Production by Expression of Genes Coding for the Chorismate Mutase Domain of the Native Chorismate Mutase-Prephenate Dehydratase and a Cyclohexadienyl Dehydrogenase from Zymomonas mobilis

“…In these studies, feedback inhibition-resistant variants of CM-TyrA p were obtained by error-prone PCR of the encoding gene tyrA and were selected using the toxic analog 3-fluoro-DL-tyrosine. Using E. coli as a host strain, Takai et al (U.S. patent application 20050277179) and Lütke-Eversloh and Stephanopoulos (32) constructed E. coli L-Tyr producers by overexpression of feedback inhibition-resistant variants of DAHPS and CM-TyrA p (13,29,31,32; Takai et al, U.S. patent application 20050277179). One of the E. coli L-Tyr producers, which had additional central metabolism genetic modifications, produced 9.7 g/liter of L-Tyr, with an L-Tyr yield on glucose (Y L-Tyr/Glc ) of 0.10 g/g and an L-Tyr-specific production rate (q L-Tyr ) of 73 mg/g (dry weight)/h in a fed-batch fermentation (32).…”

Heterologous Production of Curcuminoids

“…Also, the sequences of the repressed genes were modified so that their products were no longer sensitive to feedback inhibition. The overexpression of feedback inhibition-resistant derivatives of the aroG (aroG fbr ) and tyrA (tyrA fbr ) genes proved to help in amino acid overexpression (145)(146)(147)(148)(149).…”