TRIAD: a transposition-based approach for gene mutagenesis by random short in-frame insertions and deletions for directed protein evolution. TRIAD

Insertions and deletions (InDels) are among the most frequent changes observed in natural protein evolution, yet their potential has hardly been harnessed in directed evolution experiments. Here we describe TRIAD, a simple and efficient Mu transposon mutagenesis approach for generating libraries of single InDel variants with one, two or three triplet nucleotide insertions or deletions. The libraries give access to ~90% transposon insertion positions with larger coverage than previously estimated for the Mu transposon, so that >105 variants per insertion library can be achieved. Fitness analysis of InDel libraries of phosphotriesterase obtained via TRIAD revealed that InDels are 5- to 10-fold more deleterious than substitutions. However, directed evolution by screening these libraries for improved promiscuous arylesterase activity yielded over 80 distinct and substantially improved variants. Kinetic characterization of four hits revealed 10- to 20-fold increases in catalytic efficiency for arylesterase activity, while decreasing that for the native phosphotriesterase activity by 100-fold, in contrast to the weaker trade-off usually observed in evolution with substitution variants. Overall, our results suggest that InDel libraries created using TRIAD provide ready access to functionally distinct beneficial variants and expand the directed evolution toolbox toward functional trajectories that would not arise from substitution mutagenesis libraries.