Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis.

ABSTRACT: Insertions and deletions (InDels) are frequently observed in natural protein evolution, yet their potential remains untapped in laboratory evolution. Here we introduce a transposon-based mutagenesis approach (TRIAD) to generate libraries of random variants with short in-frame InDels, and screen TRIAD libraries to evolve a promiscuous arylesterase activity in a phosphotriesterase. The evolution exhibits features that differ from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more compromised, a larger proportion has successfully adapted for the activity. Different functional profiles emerge: (i) both strong and weak trade-off between activities are observed; (ii) trade-off is more severe (20- to 35-fold increased k_cat/K_M in aryles

SUBMITTER: Emond S 

PROVIDER: S-EPMC7351745 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature