Access to 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 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 are distinct from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more deleterious, a larger proportion has successfully adapted for the new activity, exhibiting different functional profiles: ( i ) both strong and weak trade-off in original vs promiscuous activity are observed; ( ii ) trade-off is more severe (10- to 20-fold increased k cat/ K M in arylesterase with ∼100-fold decreases in the original phosphotriesterase activity) and ( iii ) improvements show up in k cat rather than KM, suggesting novel adaptive solution. These distinct features make TRIAD an alternative to widely used point mutagenesis, providing access to functional innovations and traversing unexplored fitness landscape regions.