Optimized oxidoreductases for medium and large scale industrial biotransformations
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Project Secretariat
Dr Marta Pérez-Boada
E-mail: MPBoada@cib.csic.es
Consejo Superior de Investigaciones Científicas (CSIC)
Biological Research Centre (CIB)
Calle Ramiro de Maeztu 9, E-28040 Madrid, Spain
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publications
Total records: 126
Pages:    1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21  

[ 2015 ] Poraj-Kobielska M, Peter S, Leonhardt S, Ullrich R, Scheibner K, Hofrichter M Immobilization of unspecific peroxygenases (EC 1.11.2.1) in PVA/PEG gel and hollow fiber modules Biochem. Eng. J., 98: 144-150
[ 2015 ] Rico A, Rencoret J, del Río JC, Martínez AT, Gutiérrez A In-Depth 2D NMR Study of Lignin Modification During Pretreatment of Eucalyptus Wood with Laccase and Mediators Bioenerg. Res., 8: 211-230
[ 2015 ] Saez-Jimenez V, Acebes S, Guallar V, Martínez AT, Ruiz-Dueñas FJ Improving the oxidative stability of a high redox potential fungal peroxidase by rational design PlosOne, 10-4
[ 2015 ] Saez-Jimenez V, Baratto MC, Pogni R, Rencoret J, Gutiérrez A, Santos JI, Martínez AT, Ruiz-Dueñas FJ Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A Transient-state Kinetics, Directed Mutagenesis, EPR and NMR Study J. Biol. Chem., 290: 23201-23213
[ 2015 ] Saez-Jimenez V, Fernandez-Fueyo E, Medrano FJ, Romero A, Martínez AT, Ruiz-Dueñas FJ Improving the pH-stability of Versatile Peroxidase by Comparative Structural Analysis with a Naturally-Stable Manganese Peroxidase PlosOne, doi: 10.1371/journal.pone.0140984
[ 2015 ] Tan TC, Kracher D, Gandini R, Sygmund C, Kittl R, Haltrich D, Hällberg BM, Ludwig R, Divine C Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation Nat. Commun., 6: 7542
year2015
Focused directed evolution of aryl-alcohol oxidase in yeast using chimeric signal peptides
Viña-Gonzalez J, González-Pérez D, Ferreira P, Martínez AT, Alcalde M
Appl. Environ. Microbiol., 81: 6451-6462

Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H2O2 during natural wood decay. With a broad substrate specificity and highly stereoselective reaction mechanism, AAO is an attractive candidate for studies into organic synthesis and synthetic biology, yet the lack of suitable heterologous expression systems has precluded its engineering by directed evolution. In this study, the native signal sequence of AAO from Pleurotus eryngii was replaced by those of the mating α-factor and the K1 Killer toxin, as well as different chimeras of both prepro-leaders in order to drive secretion in Saccharomyces cerevisiae. The secretion of these AAO constructs increased in the order preproα-AAO>preαproK-AAO>preKproα-AAO>preproK-AAO. The chimeric preαproK-AAO was subjected to focused-directed evolution with the aid of a dual screening assay based on the Fenton reaction. Random mutagenesis and DNA recombination was concentrated on two protein segments (Met[α1]-Val109, Phe392-Gln566) and an array of improved variants was identified, among which the FX7 mutant (harboring the H91N mutation) showed a dramatic 96-fold improvement in total activity with secretion levels of 2 mg/L. Analysis of the N-terminal sequence of the FX7 variant confirmed the correct processing of the preαproK hybrid peptide by the KEX2 protease. FX7 showed higher stability in terms of pH and temperature whereas the pH activity profiles and the kinetic parameters were maintained. The Asn91 lies in the flavin attachment loop motif, and it is a highly conserved residue in all members of the GMC superfamily, except P. eryngii and P. pulmonarius AAO. The in vitro involution of the enzyme by restoring the consensus ancestor Asn91 promoted AAO expression and stability.

Official webpage of indox [ 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 garcíarincón