Continuous Directed Evolution of a Feedback-Resistant Arabidopsis Arogenate Dehydratase in Plantized Escherichia coli.

ABSTRACT: Continuous directed evolution (CDE) is a powerful tool for enzyme engineering due to the depth and scale of evolutionary search that it enables. If suitably controlled and calibrated, CDE could be widely applied in plant breeding and biotechnology to improve plant enzymes ex planta. We tested this concept by evolving Arabidopsis arogenate dehydratase (AtADT2) for resistance to feedback inhibition. We used an Escherichia coli platform with a phenylalanine biosynthesis pathway reconfigured ("plantized") to mimic the plant pathway, a T7RNA polymerase-base deaminase hypermutation system (eMutaT7), and 4-fluorophenylalanine as selective agent. Selection schemes were prevalidated using a known feedback-resistant AtADT2 variant. We obtained variants that had 4-fluorophenylala

SUBMITTER: Leong BJ 

PROVIDER: S-EPMC9872817 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature