A simple selection strategy for evolving highly efficient enzymes

Abstract

Combining tunable transcription with an enzyme-degradation tag affords an effective means to reduce intracellular enzyme concentrations from high to very low levels. Such fine-tuned control allows selection pressure to be systematically increased in directed-evolution experiments. This facilitates identification of mutants with wild-type activity, as shown here for an engineered chorismate mutase. Numerous selection formats and cell-based screening methodologies may benefit from the large dynamic range afforded by this easily implemented strategy.

Pseudomonas fluorescens WCS374r-Induced Systemic Resistance in Rice against Magnaporthe oryzae Is Based on Pseudobactin-Mediated Priming for a Salicylic Acid-Repressible Multifaceted Defense Response

“…Interestingly, recent evidence suggests that, in line with its closest homolog in P. aeruginosa, the catalytically promiscuous SA biosynthesis protein PchB, PmsB not only possesses isochorismate-pyruvate lyase but also chorismate mutase activity (Kunzler et al, 2005). Since chorismate mutase is located at the branch point of the shikimate pathway leading to the biosynthesis of Tyr and Phe, the enzyme constitutes a key point of regulation for maintaining the correct balance of aromatic amino acids in the cell (Neuenschwander et al, 2007). Hence, it can be envisaged that a mutation in such a regulatory enzyme might have a pleiotropic effect hampering the induction of ISR.…”

A general strategy for the evolution of bond-forming enzymes using yeast display

“…For example, screening enzyme libraries in a multiwell format has proven to be effective for enzymes that process chromogenic or fluorogenic substrates, and is typically limited to library sizes of approximately 10 2 -10 6 members, depending on the nature of the screen and on available infrastructure (10). Selections of cell-based libraries that couple product formation with auxotrophy complementation (11) or transcription of a reporter gene (12) enable larger library sizes to be processed, but also suffer from limited generality because they rely on specific properties of the substrate or product. Likewise, in vitro compartmentalization is a powerful genotype-phenotype colocalization platform that has been used to evolve protein enzymes with improved turnover, but also requires corresponding screening or selection methods that thus far have been substrate-or product-specific (13).…”

Design, selection, and characterization of a split chorismate mutase

“…The CM selection system is particularly well characterized, making the split CM a potentially useful addition to the toolkit of split sensors. The ability to adjust the stringency of the CM selection system with tightly regulatable promoters and specific degradation tags that control protein levels 40 could be advantageous, as it provides a simple means of fine-tuning the dynamic range of interactions that can be assayed. The relative orientation of possible interaction partners distinguishes individual split proteins.…”