Abstract
Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst, and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. 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. In this study, we 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. Accordingly, this study opens new avenues for UPO design by bringing together neutral genetic drift and DNA recombination in vivoIMPORTANCE Fungal peroxygenases resemble the peroxide shunt pathway of cytochrome P450 monoxygenases, performing selective oxyfunctionalizations of unactivated C-H bonds in a broad range of organic compounds. In this study, we combined neutral genetic drift and in vivo DNA shuffling to generate highly functional peroxygenase mutant libraries. The panel of neutrally evolved peroxygenases showed different activity profiles for peroxygenative substrates and improved stability with respect to temperature and the presence of organic cosolvents, making the enzymes valuable blueprints for emerging evolution campaigns. This association of DNA recombination and neutral drift is paving the way for future work in peroxygenase engineering and, from a more general perspective, to any other enzyme system heterologously expressed in S. cerevisiae.
Highlights
Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst, and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications
Taking advantage of the peroxide shunt pathway of classic cytochrome P450 monooxygenases, UPO can behave as a self-sufficient monooxygenase triggered by H2O2, which acts as the final electron acceptor and main oxygen source [3]
In which screening efforts focus on the selection of the fittest, directed evolution by neutral genetic drift pursues the gradual accumulation of neutral mutations within a population of variants with similar phenotypes but different genotypes [16]
Summary
Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst, and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. The panel of neutrally evolved peroxygenases showed different activity profiles for peroxygenative substrates and improved stability with respect to temperature and the presence of organic cosolvents, making the enzymes valuable blueprints for emerging evolution campaigns. This association of DNA recombination and neutral drift is paving the way for future work in peroxygenase engineering and, from a more general perspective, to any other enzyme system heterologously expressed in S. cerevisiae. The potential to use DNA recombination in genetic drift experiments has long been suggested, all of the studies reported to date have lacked an efficient recombination system that enables neutral mutations to be simultaneously recombined between homologues in each round of neutral evolution
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