Metabolic engineering of a genetic selection system with tunable stringency

Abstract

The biosynthesis of small molecules can be fine-tuned by (re)engineering metabolic flux within cells. We have adapted this approach to optimize an in vivo selection system for the conversion of prephenate to phenylpyruvate, a key step in the production of the essential aromatic amino acid phenylalanine. Careful control of prephenate concentration in a bacterial host lacking prephenate dehydratase, achieved through provision of a regulable enzyme that diverts it down a parallel biosynthetic pathway, provides the means to systematically increase selection pressure on replacements of the missing catalyst. Successful differentiation of dehydratases whose activities vary over a >50,000-fold range and the isolation of mechanistically informative prephenate dehydratase variants from large protein libraries illustrate the potential of the engineered selection strain for characterizing and evolving enzymes. Our approach complements other common methods for adjusting selection pressure and should be generally applicable to any selection system that is based on the conversion of an endogenous metabolite. directed evolution ͉ enzyme design ͉ genetic complementation ͉ prephenate dehydratase ͉ tetracycline promoter

Salmonella Co-opts Host Cell Chaperone-mediated Autophagy for Intracellular Growth

“…As the SCV has been reported to acidify within a short time postinfection (23,24), the strain shown in Fig. 1D also harbors a plasmid encoding an inducible cyclohexadienyl dehydrogenase (CDH) to eliminate residual growth of pheA mutants under acidic conditions (25). Although the intracellular growth of the mutant was similar to that of the wild-type strain regardless of the presence or absence of the CDH plasmid (data not shown), the studies shown were performed with strains harboring the CDH plasmid.…”

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

“…Due to this property, this enzyme was used recently for the development of a tunable selection system for mutant prephenate dehydratases. In this system, TyrC diverted PPA into the L-Tyr pathway to avoid the nonenzymatic conversion of PPA into phenylpyruvate in L-Phe biosynthesis (26). On the other hand, CM-prephenate dehydratase (P-protein) from E. coli is involved in L-Phe biosynthesis.…”

13C isotope effect on the reaction catalyzed by prephenate dehydratase

“…In one report, 15 residues were found to be conserved among 12 different PDT-containing proteins [8], including a strictly conserved TRF motif. Mutation of these residues revealed the importance of Thr172 and Phe174 (MjPDT numbering) for catalysis, whereas Arg 173 was surprisingly tolerant to substitution [8,10]. Another mutational study using the PDT domain of the E. coli P-Protein provided independent evidence for the importance of Thr172 in catalysis [7].…”

“…Located at the bottom of the cleft is the conserved TRF motif with the side chains of Thr172 and Phe174 pointing into the cleft. Although many of the other conserved residues were located within or lining the active site cleft, Arg173 was oriented away from the pocket, precluding a role in substrate recognition or catalysis [10]. In this structure, Thr172 had hydrogen-bonding interactions with a solvent molecule as well as the backbone oxygen of Asn57.…”