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

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 arylesterase with 60-400-fold decreases in phosphotriesterase activity) and (iii) improvements are present in k cat rather than just in K M , suggesting adaptive solutions. These distinct features make TRIAD an alternative to widely used point mutagenesis, accessing functional innovations and traversing unexplored fitness landscape regions. Insertions/Deletions (InDels) remain an untapped source of protein diversity in laboratory evolution. The method TRIAD generates libraries of random variants with short in-frame InDels using transposons, allowing a comparison of their evolutionary potential with widely-used point mutant libraries.