Shuffling the neutral drift of unspecific peroxygenase in Saccharomyces cerevisiae.

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 and active UPO variants were generated. After only 8 rounds of genetic drift and DNA shuffling, we identified an ensemble of 25 neutrally evolved variants with modifications in peroxidative and peroxygenative activities, kinetic thermostability and enhanced tolerance to organic solvents. With an average 4.6 substitutions introduced per clone, neutral mutations covered roughly 10% of the protein sequence. As such, this study opens new avenues of UPO design by bringing together neutral genetic drift and DNA recombination in vivoIMPORTANCE Fungal unspecific peroxygenase (UPO) resembles the peroxide shunt pathway of P450s to perform selective oxyfunctionalizations of unactivated C-H bonds with a broad range of organic compounds. In this study, we have combined neutral genetic drift and in vivo DNA shuffling to generate highly functional UPO mutant libraries. The panel of neutrally evolved UPOs showed different activity profiles for peroxygenative substrates and improved stability vs temperature and the presence of organic co-solvents making them valuable blueprints for emergent evolution campaigns. This association of DNA recombination and neutral drift is paving the way for future works in UPO ...
Source: Applied and Environmental Microbiology - Category: Microbiology Authors: Tags: Appl Environ Microbiol Source Type: research