Multicopper oxidases from fungi that degrade lignocellulose: heterologous expression, characterization and engineering

[EN ] 1. Background Multicopper oxidases (MCOs) share a common catalytic mechanism of activation by oxygen and same cupredoxin-type fold, as well as some common structural determinants. Fungal laccases are the largest and widest distributed group of the MCO superfamily, with the greatest biotechnolo...

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Autor: Aza, Pablo
Tipo de recurso: tesis doctoral
Estado:Versión publicada
Fecha de publicación:2023
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/354503
Acceso en línea:http://hdl.handle.net/10261/354503
Access Level:acceso abierto
Palabra clave:Hongos
Oxidasas
Lignocelulosa
http://metadata.un.org/sdg/3
Ensure healthy lives and promote well-being for all at all ages
id ES_2441663cb53d4b34815d3534f8ab3b18
oai_identifier_str oai:digital.csic.es:10261/354503
network_acronym_str ES
network_name_str España
repository_id_str
dc.title.none.fl_str_mv Multicopper oxidases from fungi that degrade lignocellulose: heterologous expression, characterization and engineering
Oxidasas multicobre de hongos que degradan la lignocelulosa : expresión heteróloga, caracterización e ingeniería
title Multicopper oxidases from fungi that degrade lignocellulose: heterologous expression, characterization and engineering
spellingShingle Multicopper oxidases from fungi that degrade lignocellulose: heterologous expression, characterization and engineering
Aza, Pablo
Hongos
Oxidasas
Lignocelulosa
http://metadata.un.org/sdg/3
Ensure healthy lives and promote well-being for all at all ages
title_short Multicopper oxidases from fungi that degrade lignocellulose: heterologous expression, characterization and engineering
title_full Multicopper oxidases from fungi that degrade lignocellulose: heterologous expression, characterization and engineering
title_fullStr Multicopper oxidases from fungi that degrade lignocellulose: heterologous expression, characterization and engineering
title_full_unstemmed Multicopper oxidases from fungi that degrade lignocellulose: heterologous expression, characterization and engineering
title_sort Multicopper oxidases from fungi that degrade lignocellulose: heterologous expression, characterization and engineering
dc.creator.none.fl_str_mv Aza, Pablo
author Aza, Pablo
author_facet Aza, Pablo
author_role author
dc.contributor.none.fl_str_mv Camarero, Susana
Aza, Pablo [0000-0002-8703-8399 ]
Camarero, Susana [0000-0002-2812-895X ]
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Hongos
Oxidasas
Lignocelulosa
http://metadata.un.org/sdg/3
Ensure healthy lives and promote well-being for all at all ages
topic Hongos
Oxidasas
Lignocelulosa
http://metadata.un.org/sdg/3
Ensure healthy lives and promote well-being for all at all ages
description [EN ] 1. Background Multicopper oxidases (MCOs) share a common catalytic mechanism of activation by oxygen and same cupredoxin-type fold, as well as some common structural determinants. Fungal laccases are the largest and widest distributed group of the MCO superfamily, with the greatest biotechnological applicability due to their capability to oxidize a variety of aromatic compounds. However, their substrate versatility and low-sequence homology difficult their accurate classification. Many of the ever-increasing amount of MCO entries from fungal genomes analysis are automatically annotated as laccases, even though they await for experimental verification. In a recent comparative genomic study of 52 basidiomycete fungi from different orders with diverse lifestyles, the classification of MCOs was revised. The phylogenetic analysis revealed a total of 649 MCO enzymes assembled in different clusters according to their conserved structural motifs and theoretical activities, namely, Ascorbate Oxidases (AOs), Ferroxidases (FOXs), Laccase- Ferroxidases (LAC-FOXs) and Laccases sensu stricto. In addition, three novel welldefined clusters of laccase-like enzymes related to laccases sensu stricto were described as Novel Laccases (NLACs), Novel MCOs (NMCOs) and Novel Laccases with potential ferroxidase activity (NLAC-FOXs). 2. Objectives The primary objective of this Doctoral Thesis was to carry out the heterologous expression of different types of MCOs from basidiomycete fungi with putative laccaselike activities to characterize them and improve their properties as biocatalysts by directed evolution and rational design. To attain this goal, we considered the following specific objectives that have been addressed in the chapters of this Doctoral Thesis: 1. Improvement of enzyme production in Saccharomyces cerevisiae by using several engineering approaches, and overexpression of selected enzyme variants in Aspergillus oryzae. 2. Optimization of the secretory potential of the α-factor preproleader of S. cerevisiae by protein engineering. 3. Biochemical, kinetical, and structural characterization of enzymes with putative laccase-like activity that belong to different families of MCOs, namely, laccases sensu stricto, LAC-FOXs and the recently described NLACs. 4. Assessment of the role and structure of the small proteins that form heterodimers with NLACs. 3. Results 3.1. Engineering approaches to enhance MCO production in S. cerevisiae The heterologous expression of fungal MCOs in S. cerevisiae was improved by using different approaches. First, we showed the remarkable secretory ability with fungal laccases of the α9H2 signal peptide, an engineered α-factor preproleader of S. cerevisiae obtained through successive laccase evolution campaigns. A second strategy was based on the consensus design of the NLAC member from Pleurotus eryngii (PeNL) to increase its difficult expression by the yeast. This was attained by introducing two conserved proline residues into the protein surface and one N-glycosylation site. 3.2. Design of an optimized signal peptide for enzyme production in S. cerevisiae The individual effects and possible epistatic interactions among the seven mutations accumulated in the fittest evolved α9H2 leader were analysed together with other reported mutations to obtain an optimized version of the signal peptide. The designing strategy was dual: a top-down design over the α9H2 leader and a bottom-up design over the native α-factor preproleader. The resulting αOPT leader (with simplified number of mutations) notably enhanced the secretion of different fungal enzymes. Additionally, we suggested a guideline to further enhance the heterologous production of a particular enzyme based on the combinatorial saturation mutagenesis of specific positions of the spacer region of the αOPT leader fused to the target protein. 3.3. Engineering and characterization of a laccase sensu stricto from Agrocybe pediades A laccase secreted by Agrocybe pediades under ligninolytic conditions (ApL), was heterologously expressed in S. cerevisiae. Through directed evolution of ApL we increased its expression by the yeast, enhanced its catalytic activity and shifted its optimal activity towards more neutral pH values. The evolved variant also showed tolerance to different inhibitors and ability to oxidize certain high-redox potential mediator compounds and recalcitrant organic dyes. We also studied the role of N-glycosylation in ApL by individually removing the three N-glycosylation sites through site-directed mutagenesis. Characterization of the resulting partially deglycosylated variants revealed the specific contribution of each N-glycosylation site to the heterologous expression or catalytic activity of the enzyme. 3.4. Laccase-Ferroxidase classification and study of a member from Heterobasidion annosum s. l. The phylogenetic study of the basidiomycete LAC-FOX family revealed two subgroups of enzymes, which seemed to correlate with the presence or absence of some of the three acidic residues responsible for ferroxidase activity in the canonical ferroxidase Fet3p from S. cerevisiae. Subgroup 1 harboured two acidic residues and reported efficient ferroxidase activity and some laccase activity, while LAC-FOXs grouped in subgroup 2, with only one acidic residue, were almost unexplored. We studied one member of subgroup 2, from Heterobasidion annosum s. l. (HaLF), after its heterologous expression in Aspergillus oryzae. HaLF showed good laccase activity similar to certain laccases sensu stricto, but no ferroxidase activity. Only after the full completion of the three acidic residues, equivalent to those of Fet3p, the enzyme oxidized Fe (II). The mutated variant also retained laccase activity although with poorer kinetic constants. 3.5. Role and structure of the heterodimers formed by NLACs with small proteins The NLAC from the fungus P. eryngii (PeNL) was expressed in A. oryzae alone or together with a small protein of unknown function found in the P. eryngii genome, to study the putative formation of the PeNL-ss complex, previously reported for this type of enzymes. The complex was formed both in vitro and in vivo. After purification of monomeric PeNL and PeNL-ss heterodimer, it was proved the role of the small subunit in enhancing the stability of the heterodimer towards temperature, acidic pH and the presence of co-solvents. Moreover, the heterodimeric form also improved the catalytic activity of the NLAC. Finally, we solved the crystallographic structure of the small protein of P. eryngii expressed in Escherichia coli (at 1.6 Å resolution), which is the first structure of a small protein obtained. 4. Conclusions In this Doctoral Thesis, we use a tandem approach based on the heterologous expression and engineering of the MCO enzymes under study in S. cerevisiae and the overproduction of the most interesting enzyme variants in A. oryzae. To facilitate the heterologous production of the enzymes by the yeast, we took advantage of the best evolved α-factor preproleader available in our lab, that has been further optimized here, as well as of other approaches that included consensus mutations and addition of Nglycosylation sites. The three fungal MCO enzymes studied covered three families with laccase-like activity: i) ApL is a member of laccases sensu stricto from the Agaricales order; ii) PeNL is a NLAC also from Agaricales; and iii) HaLF is a LAC-FOX from the Russulales order. We demonstrated the three share certain laccase activities. The evolved DM variant of ApL is a laccase with versatile activity towards phenolic compounds, aryl amines, ABTS, HBT, violuric acid and different organic dyes, some of them with high-redox potential. The enzyme HaLF, from the unexplored subgroup 2 of LAC-FOXs, is able to oxidize ABTS, phenols and aryl amines, as well as recalcitrant organic dyes, though its catalytic efficiencies are remarkably lower than laccases sensu stricto. It also exhibits null Fe (II) oxidation, unless the full completion of the three acidic residues equivalent to those of Fet3p is attained in HaLF A227E, F344D variant. As for the NLAC PeNL, the small protein confers higher stability and enhanced catalytic activity to the enzyme in the heterodimeric form. Still, the activity of the heterodimeric complex NLAC PeNL-ss towards phenolic and aryl amine substrates is extremely poor compared to those of ApL laccase sensu stricto or HaLF LAC-FOX.
publishDate 2023
dc.date.none.fl_str_mv 2023
2024
2024
dc.type.none.fl_str_mv info:eu-repo/semantics/doctoralThesis
http://purl.org/coar/resource_type/c_db06
Publisher's version
info:eu-repo/semantics/publishedVersion
format doctoralThesis
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/354503
url http://hdl.handle.net/10261/354503
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Universidad Complutense de Madrid
CSIC - Centro de Investigaciones Biológicas Margarita Salas (CIB)
publisher.none.fl_str_mv Universidad Complutense de Madrid
CSIC - Centro de Investigaciones Biológicas Margarita Salas (CIB)
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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spelling Multicopper oxidases from fungi that degrade lignocellulose: heterologous expression, characterization and engineeringOxidasas multicobre de hongos que degradan la lignocelulosa : expresión heteróloga, caracterización e ingenieríaAza, PabloHongosOxidasasLignocelulosahttp://metadata.un.org/sdg/3Ensure healthy lives and promote well-being for all at all ages[EN ] 1. Background Multicopper oxidases (MCOs) share a common catalytic mechanism of activation by oxygen and same cupredoxin-type fold, as well as some common structural determinants. Fungal laccases are the largest and widest distributed group of the MCO superfamily, with the greatest biotechnological applicability due to their capability to oxidize a variety of aromatic compounds. However, their substrate versatility and low-sequence homology difficult their accurate classification. Many of the ever-increasing amount of MCO entries from fungal genomes analysis are automatically annotated as laccases, even though they await for experimental verification. In a recent comparative genomic study of 52 basidiomycete fungi from different orders with diverse lifestyles, the classification of MCOs was revised. The phylogenetic analysis revealed a total of 649 MCO enzymes assembled in different clusters according to their conserved structural motifs and theoretical activities, namely, Ascorbate Oxidases (AOs), Ferroxidases (FOXs), Laccase- Ferroxidases (LAC-FOXs) and Laccases sensu stricto. In addition, three novel welldefined clusters of laccase-like enzymes related to laccases sensu stricto were described as Novel Laccases (NLACs), Novel MCOs (NMCOs) and Novel Laccases with potential ferroxidase activity (NLAC-FOXs). 2. Objectives The primary objective of this Doctoral Thesis was to carry out the heterologous expression of different types of MCOs from basidiomycete fungi with putative laccaselike activities to characterize them and improve their properties as biocatalysts by directed evolution and rational design. To attain this goal, we considered the following specific objectives that have been addressed in the chapters of this Doctoral Thesis: 1. Improvement of enzyme production in Saccharomyces cerevisiae by using several engineering approaches, and overexpression of selected enzyme variants in Aspergillus oryzae. 2. Optimization of the secretory potential of the α-factor preproleader of S. cerevisiae by protein engineering. 3. Biochemical, kinetical, and structural characterization of enzymes with putative laccase-like activity that belong to different families of MCOs, namely, laccases sensu stricto, LAC-FOXs and the recently described NLACs. 4. Assessment of the role and structure of the small proteins that form heterodimers with NLACs. 3. Results 3.1. Engineering approaches to enhance MCO production in S. cerevisiae The heterologous expression of fungal MCOs in S. cerevisiae was improved by using different approaches. First, we showed the remarkable secretory ability with fungal laccases of the α9H2 signal peptide, an engineered α-factor preproleader of S. cerevisiae obtained through successive laccase evolution campaigns. A second strategy was based on the consensus design of the NLAC member from Pleurotus eryngii (PeNL) to increase its difficult expression by the yeast. This was attained by introducing two conserved proline residues into the protein surface and one N-glycosylation site. 3.2. Design of an optimized signal peptide for enzyme production in S. cerevisiae The individual effects and possible epistatic interactions among the seven mutations accumulated in the fittest evolved α9H2 leader were analysed together with other reported mutations to obtain an optimized version of the signal peptide. The designing strategy was dual: a top-down design over the α9H2 leader and a bottom-up design over the native α-factor preproleader. The resulting αOPT leader (with simplified number of mutations) notably enhanced the secretion of different fungal enzymes. Additionally, we suggested a guideline to further enhance the heterologous production of a particular enzyme based on the combinatorial saturation mutagenesis of specific positions of the spacer region of the αOPT leader fused to the target protein. 3.3. Engineering and characterization of a laccase sensu stricto from Agrocybe pediades A laccase secreted by Agrocybe pediades under ligninolytic conditions (ApL), was heterologously expressed in S. cerevisiae. Through directed evolution of ApL we increased its expression by the yeast, enhanced its catalytic activity and shifted its optimal activity towards more neutral pH values. The evolved variant also showed tolerance to different inhibitors and ability to oxidize certain high-redox potential mediator compounds and recalcitrant organic dyes. We also studied the role of N-glycosylation in ApL by individually removing the three N-glycosylation sites through site-directed mutagenesis. Characterization of the resulting partially deglycosylated variants revealed the specific contribution of each N-glycosylation site to the heterologous expression or catalytic activity of the enzyme. 3.4. Laccase-Ferroxidase classification and study of a member from Heterobasidion annosum s. l. The phylogenetic study of the basidiomycete LAC-FOX family revealed two subgroups of enzymes, which seemed to correlate with the presence or absence of some of the three acidic residues responsible for ferroxidase activity in the canonical ferroxidase Fet3p from S. cerevisiae. Subgroup 1 harboured two acidic residues and reported efficient ferroxidase activity and some laccase activity, while LAC-FOXs grouped in subgroup 2, with only one acidic residue, were almost unexplored. We studied one member of subgroup 2, from Heterobasidion annosum s. l. (HaLF), after its heterologous expression in Aspergillus oryzae. HaLF showed good laccase activity similar to certain laccases sensu stricto, but no ferroxidase activity. Only after the full completion of the three acidic residues, equivalent to those of Fet3p, the enzyme oxidized Fe (II). The mutated variant also retained laccase activity although with poorer kinetic constants. 3.5. Role and structure of the heterodimers formed by NLACs with small proteins The NLAC from the fungus P. eryngii (PeNL) was expressed in A. oryzae alone or together with a small protein of unknown function found in the P. eryngii genome, to study the putative formation of the PeNL-ss complex, previously reported for this type of enzymes. The complex was formed both in vitro and in vivo. After purification of monomeric PeNL and PeNL-ss heterodimer, it was proved the role of the small subunit in enhancing the stability of the heterodimer towards temperature, acidic pH and the presence of co-solvents. Moreover, the heterodimeric form also improved the catalytic activity of the NLAC. Finally, we solved the crystallographic structure of the small protein of P. eryngii expressed in Escherichia coli (at 1.6 Å resolution), which is the first structure of a small protein obtained. 4. Conclusions In this Doctoral Thesis, we use a tandem approach based on the heterologous expression and engineering of the MCO enzymes under study in S. cerevisiae and the overproduction of the most interesting enzyme variants in A. oryzae. To facilitate the heterologous production of the enzymes by the yeast, we took advantage of the best evolved α-factor preproleader available in our lab, that has been further optimized here, as well as of other approaches that included consensus mutations and addition of Nglycosylation sites. The three fungal MCO enzymes studied covered three families with laccase-like activity: i) ApL is a member of laccases sensu stricto from the Agaricales order; ii) PeNL is a NLAC also from Agaricales; and iii) HaLF is a LAC-FOX from the Russulales order. We demonstrated the three share certain laccase activities. The evolved DM variant of ApL is a laccase with versatile activity towards phenolic compounds, aryl amines, ABTS, HBT, violuric acid and different organic dyes, some of them with high-redox potential. The enzyme HaLF, from the unexplored subgroup 2 of LAC-FOXs, is able to oxidize ABTS, phenols and aryl amines, as well as recalcitrant organic dyes, though its catalytic efficiencies are remarkably lower than laccases sensu stricto. It also exhibits null Fe (II) oxidation, unless the full completion of the three acidic residues equivalent to those of Fet3p is attained in HaLF A227E, F344D variant. As for the NLAC PeNL, the small protein confers higher stability and enhanced catalytic activity to the enzyme in the heterodimeric form. Still, the activity of the heterodimeric complex NLAC PeNL-ss towards phenolic and aryl amine substrates is extremely poor compared to those of ApL laccase sensu stricto or HaLF LAC-FOX.[ES] 1. Antecedentes Las oxidasas multicobre (MCO) comparten un mecanismo catalítico común de activación por oxígeno y similar plegamiento proteico de tipo cupredoxina, así como algunos determinantes estructurales. De esta superfamilia de enzimas las lacasas fúngicas constituyen el grupo más numeroso y ampliamente distribuido, con la mayor aplicabilidad biotecnológica debido a su capacidad para oxidar una gran variedad de compuestos aromáticos. Sin embargo, esta amplia versatilidad a la hora de oxidar diferentes tipos de sustratos y la escasa homología de secuencia entre las lacasas siguen dificultando enormemente su clasificación. Los continuos análisis de genomas fúngicos aportan un número masivo de secuencias de MCOs que son anotadas automáticamente como lacasas, pero que requieren una verificación experimental. En un reciente estudio donde se comparan 52 genomas de hongos basidiomicetos pertenecientes a diferentes órdenes y diversos estilos de vida, se revisó la clasificación de las MCOs. El análisis filogenético reveló un total de 649 enzimas MCO agrupadas en diferentes familias según algunos motivos conservados y actividades teóricas. Entre estas familias de MCOs se encontraban las Ascorbato Oxidasas (AOs), Ferroxidasas (FOXs), Lacasa-Ferroxidasas (LAC-FOXs) y Lacasas sensu stricto. Además, se describieron tres nuevos grupos de enzimas similares a las lacasas y relacionadas con las de tipo sensu stricto: Nuevas Lacasas (NLACs), Nuevas MCOs (NMCOs) y Nuevas Lacasa con potencial actividad ferroxidasa (NLACFOXs). 2. Objetivos El objetivo general de esta Tesis Doctoral ha sido estudiar y mejorar la expresión heteróloga de las MCOs de hongos basidiomicetos para caracterizar diferentes tipos de estas enzimas con actividad lacasa. A su vez, se pretende mejorar sus propiedades como biocatalizadores mediante su ingeniería por evolución dirigida y diseño racional. Para alcanzar este objetivo nos planteamos los siguientes objetivos específicos que han sido abordados en los capítulos de esta tesis: 1. Mejorar la producción de enzimas en Saccharomyces cerevisiae mediante el uso de varios enfoques de ingeniería de proteínas, y la sobreexpresión de variantes enzimáticas en Aspergillus oryzae. 2. Optimización del potencial de secreción del péptido señal del factor α de S. cerevisiae mediante ingeniería de proteínas. 3. Caracterización bioquímica, cinética y estructural de enzimas con actividad de tipo lacasa que pertenecen a diferentes familias de MCOs: lacasas sensu stricto, LACFOXs y las recientemente descritas NLACs. 4. Evaluación del papel y la estructura de las proteínas de pequeño tamaño que forman heterodímeros con las NLACs. 3. Resultados 3.1. Ingeniería para mejorar la producción de MCOs en S. cerevisiae La expresión heteróloga de MCOs fúngicas en S. cerevisiae se mejoró mediante diferentes estrategias. En primer lugar, demostramos la notable capacidad secretora sobre lacasas fúngicas del péptido señal α9H2, una secuencia del preprolíder del factor α de S. cerevisiae obtenida tras varias campañas evolutivas. En segundo lugar, se llevó a cabo el diseño por consenso de una NLAC de Pleurotus eryngii (PeNL), para así aumentar su difícil expresión en la levadura. Esto se consiguió introduciendo dos residuos conservados de prolina en la superficie de la proteína y un sitio de N-glicosilación. 3.2. Diseño de un péptido señal optimizado para la producción de enzimas en S. cerevisiae Se analizaron los efectos individuales y las posibles epistasis entre las siete mutaciones acumuladas en el péptido señal α9H2, junto con otras mutaciones publicadas, para obtener una versión optimizada de la secuencia líder. La estrategia de diseño fue doble: un diseño “Bottom-up” sobre el preprolíder nativo del factor α y un diseño “Topdown” sobre α9H2, previamente confirmado como el mejor péptido señal para la producción heteróloga de lacasas en levadura. El péptido señal resultante, αOPT, fue una versión simplificada con un número menor de mutaciones respecto al α9H2, y que mejoró notablemente la secreción de diferentes enzimas fúngicas. Además, sugerimos una guía para mejorar aún más, y de forma personalizada, la producción heteróloga de una enzima concreta. Esta se basa en la mutagénesis saturada combinatorial de posiciones específicas de la región espaciadora del péptido señal αOPT, fusionado a la proteína cuya producción se desea mejorar. 3.3. Ingeniería y caracterización de una lacasa sensu stricto de Agrocybe pediades Se expresó de forma heteróloga en S. cerevisiae una lacasa de Agaricales secretada por Agrocybe pediades en condiciones ligninolíticas (ApL). Mediante la evolución dirigida de ApL aumentamos su expresión en levadura, mejoramos su actividad catalítica y modificamos su pH óptimo hacia valores más neutros. La variante evolucionada también mostró tolerancia a diferentes inhibidores y capacidad oxidativa sobre ciertos compuestos mediadores de alto potencial redox y colorantes orgánicos recalcitrantes. También estudiamos el papel de la N-glicosilación en ApL eliminando individualmente sus tres sitios de N-glicosilación mediante mutagénesis sitio dirigida. La caracterización de las variantes parcialmente desglicosiladas demostró como la N-glicosilación en cada sitio contribuye de forma específica a la expresión heteróloga de la enzima y a su actividad catalítica. 3.4. Clasificación de las LAC-FOXs y estudio de un miembro de Heterobasidion annosum s. l. El estudio filogenético de la familia LAC-FOX de basidiomicetos reveló dos subgrupos, cuya división parecía correlacionarse con la presencia o ausencia de algunos de los tres residuos ácidos responsables de la oxidación de Fe (II) en la ferroxidasa canónica Fet3p de S. cerevisiae. El subgrupo 1 albergaba dos de estos residuos ácidos y, además, las enzimas que habían sido caracterizadas mostraron una eficiente actividad híbrida lacasa-ferroxidasa. Por el contrario, las secuencias agrupadas en el subgrupo 2 sólo tenían un residuo ácido y ningún miembro había sido caracterizado de forma exhaustiva. Estudiamos una LAC-FOX de este subgrupo 2 perteneciente al hongo Heterobasidion annosum s. l. (HaLF). Tras su expresión heteróloga en Aspergillus oryzae HaLF mostró una buena actividad lacasa, similar a la de ciertas lacasas sensu stricto, pero la actividad ferroxidasa fue nula. La enzima solo oxidó el Fe (II) después de restaurar los tres residuos ácidos equivalentes a los de Fet3p. La variante mutada también conservaba la actividad lacasa, aunque con constantes cinéticas más pobres. 3.5. Función y estructura de los heterodímeros formados por enzimas NLAC con proteínas pequeñas La NLAC del hongo P. eryngii (PeNL) se expresó en A. oryzae individualmente o junto con una pequeña proteína de función desconocida encontrada en el genoma de P. eryngii (subunidad pequeña, ss), para estudiar la posible formación del complejo PeNLss entre ambas. Este heterodímero ya había sido previamente descrito para este tipo de enzimas, y durante este trabajo se confirmó que se podía formar tanto in vivo como in vitro. Tras la purificación de la enzima PeNL monomérica y del heterodímero PeNL-ss se comprobó que la proteína pequeña incrementaba la estabilidad de la enzima frente a la temperatura, el pH ácido y la presencia de co-solventes, así como aumentaba la actividad catalítica de la NLAC. Por último, resolvimos la estructura cristalográfica de la proteína pequeña de P. eryngii expresada en Escherichia coli (a 1,6 Å de resolución), siendo la primera estructura obtenida de una proteína pequeña de este tipo. 4. Conclusiones En esta Tesis Doctoral utilizamos una estrategia de tipo tándem basada en la expresión heteróloga e ingeniería de las enzimas en S. cerevisiae y la sobreproducción de las variantes enzimáticas más interesantes en A. oryzae. Para facilitar la producción heteróloga de las enzimas por la levadura aprovechamos la mejor secuencia evolucionada del preprolíder del factor α, denominada α9H2, disponible en nuestro laboratorio, y que se ha optimizado aún más durante esta Tesis Doctoral. Del mismo modo, se incluyen otros enfoques como mutaciones consenso y la adición de sitios de N-glicosilación para mejorar la expresión heteróloga de las MCOs. Las tres enzimas MCO fúngicas estudiadas abarcan tres familias con actividad de tipo lacasa: i) ApL es un miembro de las lacasas sensu stricto del orden Agaricales; ii), PeNL es una NLAC también de Agaricales; y iii) HaLF es una LAC-FOX del orden de los Russulales. Hemos demostrado que las tres comparten cierta actividad lacasa. La variante DM evolucionada de ApL es una lacasa con actividad versátil frente a compuestos fenólicos, aril aminas, ABTS, HBT, ácido violúrico y diferentes colorantes orgánicos, algunos de ellos con alto potencial redox. La enzima HaLF, del apenas explorado subgrupo 2 de las LAC-FOXs, es capaz de oxidar ABTS, fenoles y aril aminas, así como colorantes orgánicos recalcitrantes, aunque sus eficiencias catalíticas son notablemente inferiores a las de las lacasas sensu stricto. Aunque HaLF no fue capaz de oxidar Fe (II), la actividad ferroxidasa fue obtenida tras completar los tres residuos ácidos equivalentes a los de Fet3p en la variante HaLF A227E, F344D. En cuanto a la NLAC PeNL, la proteína pequeña confiere una mayor estabilidad y actividad catalítica a la enzima en forma de heterodímero. Aun así, la actividad del complejo heterodimérico PeNL-ss es extremadamente pobre frente a sustratos fenólicos y aril aminas en comparación con la lacasa sensu stricto ApL o la LAC-FOX HaLF.Peer reviewedUniversidad Complutense de MadridCSIC - Centro de Investigaciones Biológicas Margarita Salas (CIB)Camarero, SusanaAza, Pablo [0000-0002-8703-8399 ]Camarero, Susana [0000-0002-2812-895X ]Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202420242023info:eu-repo/semantics/doctoralThesishttp://purl.org/coar/resource_type/c_db06Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/354503reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)InglésSíinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/3545032026-05-22T06:33:51Z
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