Evolución dirigida de la peroxigenasa inespecífica de agrocybe aegerita: tolerancia a disolventes orgánicos mediante deriva genética neutral y evolución adaptativa

Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst, and its selective mono(per)oxygenase activity is attracting the attention of the biotechnology community. This highly selective and stable biocatalyst inserts oxygen into unactivated C-H bonds at room temperature and at atmospheric pressure, which represented a mere pipedream only a few years ago. Synthetic chemists are demanding a new generation of UPOs with improved activity in organic solvents whereby the most challenging oxyfunctionalization reactions can be explored. In this Thesis we have applied a palette of different laboratory evolution methods (neutral genetic drift, adaptive evolution, site directed recombination) to tailor UPO variants active and stable in organic solvents. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be accumulated gradually within a polymorphic network of variants. We first conducted a campaign of genetic drift with UPO in Saccharomyces cerevisiae, so that neutral mutations were simply added and recombined in vivo. With low mutational loading and an activity threshold of 45% of the parent’s native function, mutant libraries enriched in folded active UPO variants were generated. After only eight rounds of genetic drift and DNA shuffling, we identified an ensemble of 25 neutrally evolved variants with changes in peroxidative and peroxygenative activities, kinetic thermostability, and enhanced tolerance to organic solvents. With an average of 4.6 substitutions introduced per clone, neutral mutations covered approximately 10% of the protein sequence. In a parallel approach, we run adaptive evolution in the presence of increasing concentrations of different cosolvents. The best variant of this process was further subjected to site directed recombination in vivo aimed at unveiling epistatic interactions with neutral mutations that emerged from the genetic drift campaign. The final variant, WamPa, brought together a combination of 9 neutral and adaptive/beneficial mutations that conferred a broad resistance to organic solvents of different chemical nature and polarity, becoming a potential departure point for a new generation of oxyfunctionalization biocatalysts