"Site and Mutation"-Specific Predictions Enable Minimal Directed Evolution Libraries

ACS Synthetic Biology
Jeffrey C MooreMatthew D Truppo

Abstract

Directed evolution experiments designed to improve the activity of a biocatalyst have increased in sophistication from the early days of completely random mutagenesis. Sequence-based and structure-based methods have been developed to identify "hotspot" positions that when randomized provide a higher frequency of beneficial mutations that improve activity. These focused mutagenesis methods reduce library sizes and therefore reduce screening burden, accelerating the rate of finding improved enzymes. Looking for further acceleration in finding improved enzymes, we investigated whether two existing methods, one sequence-based (Protein GPS) and one structure-based (using Bioluminate and MOE), were sufficiently predictive to provide not just the hotspot position, but also the amino acid substitution that improved activity at that position. By limiting the libraries to variants that contained only specific amino acid substitutions, library sizes were kept to less than 100 variants. For an initial round of ATA-117 R-selective transaminase evolution, we found that the methods used produced libraries where 9% and 18% of the amino acid substitutions chosen were amino acids that improved reaction performance in lysates. The ability to crea...Continue Reading

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Jul 26, 2018·Chembiochem : a European Journal of Chemical Biology·Aitao LiManfred T Reetz
Sep 11, 2019·International Journal of Biological Macromolecules·Felipe de SalasSusana Camarero
Dec 29, 2019·Trends in Genetics : TIG·Phillip A ClevesDebashish Bhattacharya
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Jan 2, 2021·Current Opinion in Chemical Biology·Serena Gargiulo, Patrice Soumillion
Feb 9, 2019·Accounts of Chemical Research·Alexandria Deliz LiangThomas R Ward
Nov 27, 2021·Journal of Molecular Biology·Guido ScarabelliAgustina Rodriguez-Granillo

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