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Title

Shuffling the neutral drift of unspecific peroxygenase in Saccharomyces cerevisiae

AuthorsMartín-Díaz, Javier ; Paret, Carmen; García-Ruiz, Eva CSIC ORCID ; Molina-Espeja, Patricia CSIC ORCID ; Alcalde Galeote, Miguel CSIC ORCID
KeywordsPeroxygenases
Neutral genetic drift
In vivo DNA shuffling
Saccharomyces cerevisiae
Directed evolution
Issue DateAug-2018
PublisherAmerican Society for Microbiology
CitationApplied and Environmental Microbiology 84(15): 00808-18 (2018)
AbstractUnspecific 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 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 vivo.
Publisher version (URL)http://dx.doi.org/10.1128/AEM.00808-18
URIhttp://hdl.handle.net/10261/165826
DOI10.1128/AEM.00808-18
ISSN0099-2240
E-ISSN1098-5336
Appears in Collections:(ICP) Artículos




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