Optimized oxidoreductases for medium and large scale industrial biotransformations
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[ 2016 ]
van Kuijk SJA, del Río JC, Rencoret J, Gutiérrez A, Sonnenberg ASM, Baars JJP, Hendriks WH, Cone JW Selective ligninolysis of wheat straw and wood chips by the white-rot fungus Lentinula edodes and its influence on in vitro rumen degradability
J. Anim. Sci. Biotechnol., 7: 55
[ 2016 ]
Viña-Gonzalez J, González-Pérez D, Alcalde M Directed evolution method in Saccharomyces cerevisiae: Mutant library creation and screening
J. Vis. Exp., doi: 10.3791/53761
[ 2015 ]
Alcalde M Engineering the ligninolytic enzyme consortium
Trends Biotechnol., 33: 155-162
[ 2015 ]
Babot ED, del Río JC, Cañellas M, Sancho F, Lucas F, Guallar V, Kalum L, Lund H, Gröbe G, Scheibner K, Ullrich R, Hofrichter M, Martínez AT, Gutiérrez A Steroid hydroxylation by basidiomycete peroxygenases: A combined experimental and computational study
Appl. Environ. Microbiol., doi: 10.1128/AEM.00660-15
[ 2015 ]
Babot ED, del Río JC, Kalum L, Martínez AT, Gutiérrez A Regioselective Hydroxylation in the Production of 25-Hydroxyvitamin D by Coprinopsis cinerea Peroxygenase
ChemCatChem, 7: 283-290
[ 2015 ]
Baratto MC, Sinicropi A, Linde D, Saez-Jimenez V, Sorace L, Ruiz-Dueñas FJ, Martínez AT, Basosi R, Pogni R Redox-Active Sites in Auricularia auricula-judae Dye-Decolorizing Peroxidase and Several Directed Variants: A Multifrequency EPR Study
J. Phys. Chem. B, 119: 13583-13592
year2016
Directed evolution method in Saccharomyces cerevisiae: Mutant library creation and screening
Viña-Gonzalez J, González-Pérez D, Alcalde M
J. Vis. Exp., doi: 10.3791/53761
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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[ industrialoxidoreductases ]. Optimized oxidoreductases for medium and large scale industrial biotransformations. This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under Grant Agreement nº: FP7-KBBE-2013-7-613549. © indox 2013. Developed by
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