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/K in arylesterase with 60-400-fold decreases in phosphotriesterase activity) and (iii) improvements are present in k rather than just in K, suggesting adaptive solutions. These distinct features make TRIAD an alternative to widely used point mutagenesis, accessing functional innovations and traversing unexplored fitness landscape regions.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351745 | PMC |
http://dx.doi.org/10.1038/s41467-020-17061-3 | DOI Listing |
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