Shuffling the Neutral Drift of Unspecific Peroxygenase in Saccharomyces cerevisiae

Applied and Environmental Microbiology
Javier Martin-DiazMiguel Alcalde

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 pero...Continue Reading

References

Jan 22, 2004·Bioinformatics·Emmanuel ParadisKorbinian Strimmer
Aug 6, 2004·Applied and Environmental Microbiology·René UllrichMartin Hofrichter
Jul 3, 2007·Applied and Environmental Microbiology·Dau Hung AnhMartin Hofrichter
Jul 21, 2007·BMC Biology·Jesse D BloomFrances H Arnold
Dec 1, 2007·Nature Methods·Sergio G Peisajovich, Dan S Tawfik
May 23, 2008·Journal of Molecular Biology·Shimon BershteinDan S Tawfik
Aug 9, 2008·Molecular Biology and Evolution·Shimon Bershtein, Dan S Tawfik
Oct 22, 2008·Nature Methods·Rinkoo D Gupta, Dan S Tawfik
Jun 17, 2009·Proceedings of the National Academy of Sciences of the United States of America·Jesse D Bloom, Frances H Arnold
Dec 20, 2011·Bioengineered Bugs·Susan BullmanRoy D Sleator
Dec 6, 2012·Nature Communications·Nobuhiko TokurikiDan S Tawfik
Feb 21, 2013·Methods in Enzymology·Moshe Goldsmith, Dan S Tawfik
Oct 16, 2013·The Journal of Biological Chemistry·Klaus PiontekDietmar A Plattner
Mar 13, 2014·Current Opinion in Chemical Biology·Martin Hofrichter, René Ullrich
Apr 1, 2014·Applied and Environmental Microbiology·Patricia Molina-EspejaMiguel Alcalde
Apr 15, 2014·Nature Chemical Biology·Timothy G KeysRita Gerardy-Schahn
Jun 7, 2014·Biotechnology and Bioengineering·Jing ZhaoUlrich Schwaneberg
Jul 25, 2014·Methods in Molecular Biology·Miriam Kaltenbach, Nobuhiko Tokuriki
May 24, 2015·Enzyme and Microbial Technology·Patricia Molina-EspejaMiguel Alcalde
May 24, 2015·Advances in Experimental Medicine and Biology·Martin HofrichterRené Ullrich
Dec 19, 2015·Chembiochem : a European Journal of Chemical Biology·Patricia Molina-EspejaMiguel Alcalde
May 20, 2016·Chembiochem : a European Journal of Chemical Biology·Dirk Holtmann, Frank Hollmann
Dec 20, 2016·Current Opinion in Chemical Biology·Yonghua WangFrank Hollmann
Jan 20, 2017·Chembiochem : a European Journal of Chemical Biology·Jan KiebistKatrin Scheibner

❮ Previous
Next ❯

Citations

Feb 6, 2021·Biotechnology Advances·Carmen ArandaAna Gutiérrez
Jan 6, 2019·Current Opinion in Biotechnology·Devin L Trudeau, Dan S Tawfik
May 9, 2021·Biotechnology and Bioengineering·Javier Martin-DiazMiguel Alcalde
Jul 27, 2021·Frontiers in Bioengineering and Biotechnology·Alina KinnerStephan Lütz

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR

Software Mentioned

Python
PyMOL
SigmaPlot
R
ape
ClustalX
Mega
studio

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

© 2022 Meta ULC. All rights reserved