Genomic Analysis Enlightens Agaricales Lifestyle Evolution and Increasing Peroxidase Diversity

Abstract

As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay. Based on the gene families with the predicted highest evolutionary rates −namely cellulose-binding CBM1, glycoside hydrolase GH43, lytic polysaccharide monooxygenase AA9, class-II peroxidases, glucose-methanol-choline oxidase/dehydrogenases, laccases, and unspecific peroxygenases− we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. The changes in the enzymatic toolkit of ancestral Agaricales are correlated with the evolution of their ability to grow not only on wood but also on leaf-litter and decayed wood, with grass-litter decomposers as the most recent eco-physiological group. In this context, the above families were analyzed in detail in connection with lifestyle diversity. Peroxidases appear as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes, consistent with their essential role in lignin degradation and high evolutionary rates. This includes not only expansions/losses in peroxidase genes common to other basidiomycetes, but also the widespread presence in Agaricales (and Russulales) of new peroxidases types not found in wood-rotting Polyporales, and other Agaricomycetes orders. Therefore, we analyzed the peroxidase evolution in Agaricomycetes by ancestral-sequence reconstruction revealing several major evolutionary pathways, and mapped the appearance of the different enzyme types in a time-calibrated species tree.

Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae

“…Two laccases from Agaricales fungi, Agrocybe pediades , ApL (ID 823,363 JGI), and Pleurotus eryngii , PeL (ID 152,153 JGI), were assayed. PeL belongs to the recently classified NLAC that constitute a separate cluster of laccase-like enzymes that are not found in Polyporales [ 38 ], whereas ApL is a sensu stricto laccase. The CDS of both laccases were synthesized de novo for expression in S. cerevisiae , replacing the predicted native signal peptides by the ⍺-factor preproleader, in particular by ⍺ nat and ⍺ 9H2 leaders.…”

“…Three putative N -glycosylation sites were found in PeL: N89, N256 and N436, respectively located in domains D1, D2 and D3. We analysed the N -glycosylation sites in the 25 NLAC sequences found in the 52 fungal genomes previously studied [ 38 ]. We observed that N89 and N436 were largely conserved in NLAC sequences, as well as in basidiomycete laccases sensu stricto, whereas N256 was much less frequent.…”

“…The N220 site introduced in PeL is analogous to the N215 site of the domain-swap laccase, and it is conserved in 40% of the Polyporales laccases studied [ 38 ], supporting the possible contribution of glycosylation in this site for proper laccase production. This N site is not present in Agrocybe pediades laccase (ApL).…”

“…The introduction of the N220 site in PeL is part of the consensus design we followed in an attempt to facilitate the difficult heterologous expression of the enzyme by exploiting the evolutionary information encapsulated in homologous NLAC sequences [ 38 ]. Consensus protein design is based on the hypothesis that, at a given position, the respective consensus amino acid contributes more than the average to the stability of the protein than non-conserved ones [ 46 ].…”

“…The mature sequence of PeL was compared with the consensus sequence obtained from the multiple alignment of the 25 NLAC sequences found in 52 Agaricomycotina genomes [ 38 ]. We searched for putatively conserved N -glycosylation sites and proline residues that were absent in PeL.…”