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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| 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 |
| Sumario: | [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. |
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