Catalytic Residue Reprogramming Enhances Enzyme Activity at Alkaline pH via Phenolate-Mediated Proton Transfer.

Achieving efficient enzyme catalysis under extreme pH conditions remains a major challenge in biocatalysis and synthetic biology. To address this, we present an enzyme engineering strategy that integrates rational redesign of catalytic residues with directed evolution to enable robust enzyme function at alkaline pH. The core principle involves replacing the conserved general base with an ionizable residue of higher intrinsic p, shifting the proton transfer mechanism from carboxylate- to phenolate-mediated catalysis.

An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes.

In vivo selection systems are powerful tools for directed evolution of enzymes. The selection pressure of the systems can be tuned by regulating the expression levels of the catalysts. In this work, we engineered a selection system for laboratory evolution of highly active enzymes by incorporating a translationally suppressing cis repressor as well as an inducible promoter to impart stringent and tunable selection pressure.