000061354 001__ 61354
000061354 005__ 20210121114547.0
000061354 0247_ $$2doi$$a10.1128/AEM.01966-15
000061354 0248_ $$2sideral$$a92983
000061354 037__ $$aART-2015-92983
000061354 041__ $$aeng
000061354 100__ $$aViña-Gonzalez, J.
000061354 245__ $$aFocused directed evolution of aryl-alcohol oxidase in Saccharomyces cerevisiae by using chimeric signal peptides
000061354 260__ $$c2015
000061354 5060_ $$aAccess copy available to the general public$$fUnrestricted
000061354 5203_ $$aAryl-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, and 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 a-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 following order: preproa-AAO> preaproK-AAO>preKproa-AAO>preproK-AAO. The chimeric preaproK-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 (Meta1]-Val109 and 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/liter. Analysis of the N-terminal sequence of the FX7 variant confirmed the correct processing of the preaproK 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 for P. eryngii and P. pulmonarius AAO. The in vitro involution of the enzyme by restoring the consensus ancestor Asn91 promoted AAO expression and stability.
000061354 536__ $$9info:eu-repo/grantAgreement/ES/MINECO/RTC-2014-1777-3$$9info:eu-repo/grantAgreement/ES/MINECO/BIO2013-43407-R$$9info:eu-repo/grantAgreement/EC/FP7/613549/EU/Optimized oxidoreductases for medium and large scale industrial biotransformations/INDOX
000061354 540__ $$9info:eu-repo/semantics/openAccess$$aAll rights reserved$$uhttp://www.europeana.eu/rights/rr-f/
000061354 590__ $$a3.823$$b2015
000061354 591__ $$aBIOTECHNOLOGY & APPLIED MICROBIOLOGY$$b33 / 161 = 0.205$$c2015$$dQ1$$eT1
000061354 591__ $$aMICROBIOLOGY$$b31 / 123 = 0.252$$c2015$$dQ2$$eT1
000061354 592__ $$a1.877$$b2015
000061354 593__ $$aApplied Microbiology and Biotechnology$$c2015$$dQ1
000061354 593__ $$aFood Science$$c2015$$dQ1
000061354 593__ $$aEcology$$c2015$$dQ1
000061354 593__ $$aBiotechnology$$c2015$$dQ1
000061354 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion
000061354 700__ $$aGonzalez-Perez, D.
000061354 700__ $$0(orcid)0000-0003-4076-6118$$aFerreira, P.$$uUniversidad de Zaragoza
000061354 700__ $$aMartinez, A.T.
000061354 700__ $$aAlcalde, M.
000061354 7102_ $$11002$$2060$$aUniversidad de Zaragoza$$bDpto. Bioq.Biolog.Mol. Celular$$cÁrea Bioquímica y Biolog.Mole.
000061354 773__ $$g81, 18 (2015), 6451-6462$$pAppl. environ. microbiol.$$tApplied and environmental microbiology$$x1098-5336
000061354 8564_ $$s1702031$$uhttps://zaguan.unizar.es/record/61354/files/texto_completo.pdf$$yVersión publicada
000061354 8564_ $$s137633$$uhttps://zaguan.unizar.es/record/61354/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada
000061354 909CO $$ooai:zaguan.unizar.es:61354$$particulos$$pdriver
000061354 951__ $$a2021-01-21-11:19:43
000061354 980__ $$aARTICLE