Optimized oxidoreductases for medium and large scale industrial biotransformations
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126
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[ 2016 ]
Kellner H, Pecyna MJ, Buchhaupt M, Ullrich R, Hofrichter M Draft Genome Sequence of the Chloroperoxidase-Producing Fungus Caldariomyces fumago Woronichin DSM1256
Genome Announc., 4
[ 2016 ]
Kracher D, Scheiblbrandner S, Felice AKG, Breslmayr E, Preims M, Ludwicka K, Haltrich D, Eijsink VG, Ludwig R Extracellular electron transfer systems fuel cellulose oxidative degradation
Science, 352: 1098-1101
[ 2016 ]
Linde D, Cañellas M, Coscolín C, Davó-Siguero I, Romero A, Lucas F, Ruiz-Dueñas FJ, Guallar V, Martínez AT Asymmetric sulfoxidation by engineering the heme pocket of a dye-decolorizing peroxidase: An experimental and computational study
Catal. Sci. Technol., 6: 6277-6285
[ 2016 ]
Lourenço A, Rencoret J, Chemetova C, Gominho J, Gutiérrez A, del Río JC, Pereira H Lignin Composition and Structure Differs between Xylem, Phloem and Phellem in Quercus suber L.
Front. Plant Sci., 7: 1612
[ 2016 ]
Lucas F, Babot ED, Cañellas M, del Río JC, Kalum L, Ullrich R, Hofrichter M, Guallar V, Martínez AT, Gutiérrez A Molecular determinants for selective C25-hydroxylation of vitamins D2 and D3 by fungal peroxygenases
Catal. Sci. Technol., 6: 288-295
[ 2016 ]
Martínez AT How to break down crystalline cellulose
Science, 352: 1050-1051
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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