ArticleJournal of visualized experiments : JoVE2016
Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening.
Article in Journal of visualized experiments : JoVE, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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Who cites it
4 citing papers in PubMed, 10 citations in OpenAlex.
- Improvements of the productivity and saccharification efficiency of the cellulolytic β-glucosidase D2-BGL in Pichia pastoris via directed evolution.Biotechnology for biofuels · 2021Article
- Next Generation Winemakers: Genetic Engineering inBioengineering (Basel, Switzerland) · 2020Review
- Directed evolution of the aryl-alcohol oxidase: Beyond the lab bench.Computational and structural biotechnology journal · 2020Review
- Shuffling the Neutral Drift of Unspecific Peroxygenase in Saccharomyces cerevisiae.Applied and environmental microbiology · 2018Article
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Authors and funding
3 authors at 1 institution in 1 country.
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Abstract
Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.
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