A Fungal secretome tailored to enable a radical (oxidative) wood decay mechanism in brown rot fungi

Abstract

Wood is one of the most important carbon sinks on earth, and it is composed mainly of cellulose, hemicellulose, and lignin, which together form what is known as the lignocellulosic complex. The lignin component in wood is highly recalcitrant and prevents wood from being degraded by most organisms. Wood degrading fungi have evolved mechanisms to handle the lignin barrier effectively and harness the carbon present in wood, which makes them also plausible biological templates for industry-related applications such as the production of biofuels. However, there are still several knowledge gaps about how fungi orchestrate the complex mechanisms behind wood decay, which prevents further usage of these fungi in biotechnological fields. Wood degrading fungi were initially classified by the physical properties of the rotted wood as white, brown, and soft rot fungi – with brown and white rot fungi being the most efficient. These properties are correlated with important genetic and regulatory differences that distinguish the different modes of decay. Recently, it was shown that brown rot fungi evolved from white rot fungi losing an important number of carbohydrate-degrading enzymes (CAZy) and oxidoreductases. This loss was accompanied by regulatory changes that included the overexpression of retained CAZys, and the development of a two-step mechanism that segregated a Fenton-based oxidative phase from a hydrolytic phase in early and late wood decay, respectively. However, although some main differences between white and brown rot fungi have been identified, several details remain obscure. For example, it is unknown how brown rot fungi manage to produce highly oxidative and unspecific hydroxyl radicals while producing some glycosyl hydrolases necessary for the depolymerization of pectin and hemicellulose at early decay stages. It seems reasonable, that brown rot fungi could protect their enzymes by making them more naturally tolerant of reactive oxygen species (ROS) radicals than other wood degraders associated with a different rot type. Also, although transcriptomics and proteomics data suggest brown rot fungi use some Fenton chemistry at early decay, there is little information about how these fungi regulate the concentration of the chemical species that enable this chemistry. This dissertation contributes to widen the knowledge about fungal wood decay mechanisms, especially those used by brown rot fungi. For starters, by studying the wood decay progression with the brown rot fungus Rhodonia placenta, we found that this fungus displays different mechanisms to harness the use of ROS during wood decay without inflicting damage on itself. First, R. placenta controls the extracellular production of ROS by regulating the concentrations of H2O2 and Fe2+ in the media (avoidance mechanism). Second, this fungus presents a high antioxidant capacity as decay progresses, potentially to quench any possible leaks of ROS from earlier decay stages (suppression mechanism). Thirdly, several R. placenta secreted CAZys, important for early wood decay, displayed tolerance of high concentrations of ROS compared to the soft rot fungus Trichoderma reesei (an industrially relevant cellulase producer), which enables these enzymes to work under harsh operating conditions (tolerance mechanism). Collectively, this indicates that R. placenta uses avoidance, suppression and tolerance mechanisms, extracellularly, to complement intracellular differential expression, enabling this brown rot fungus to use ROS to degrade wood. After finding these results, we decided to incorporate a white rot fungus (Trametes versicolor) in the tolerance comparison and observed similar results, with tolerance of ROS only present in the side-chain hemicellulases of R. placenta. Proteomics analysis, meant to examine the presence of oxidative modifications induced after an in-vitro oxidative treatment, revealed that not only side-chain hemicellulases but also several other enzymes such as laccases, glutathione-S-transferases, and proteases were differentially tolerant of ROS compared to white and soft rot fungi. This suggest that the fungal secretome in brown rot fungi has been tailored as a whole to better endure the presence of ROS. Also, we found that several of the oxidative modifications that occurred in the glycosyl hydrolases of T. reesei and T. versicolor happened in amino acid residues in the vicinity of the active site, which can be linked to the loss of enzyme activity after the oxidative treatment. In a follow-up study addressing the effects of these modifications, we used molecular dynamics to understand the effect of some of the oxidative modifications in an α-L-arabinofuranosidase of T. reesei. Even though the number of oxidative modifications that we could include in the modeled protein was limited due to the lack of force field parameters, the simulations still showed some of the negative potential outcomes when a number of amino acid residues become oxidized. For instance, there were significant alterations of the conformational stability of the protein when oxidized, as evidenced by changes in root mean square deviation (RMSD) and principal component analyses (PCA) trajectories. Likewise, enzyme-ligand interactions such as hydrogen bonds were greatly reduced in quantity and quality in the oxidized protein. In addition, free energy landscape plots showed that there was a more rugged energy surface in the oxidized protein, implying a less favorable reaction pathway. Collectively, these results revealed the basis for the loss of function in the α-L-arabinofuranosidase of the commercially-relevant fungus T. reesei. Finally, metabolomics experiments were carried to find out whether the different modes of decay translated into signature metabolite profiles that could be assigned to either brown or white rot fungi. For this purpose, we cultured two brown rot fungi (R. placenta and Gloephylum trabeum) and two white rot fungi (Pleurotus ostreatus and T. versicolor). The results showed that brown rot fungi have a distinct metabolite pattern at late decay stages that clearly distinguish them from white rot fungi. Different metabolites such as organic acids, sugars, pyranones, and furanones contributed to this result. The finding of several pyranones and furanones being differentially more abundant in brown rot fungi was interesting since it agrees with the expansion of polyketide synthase genes in brown rot fungi. In contrast to brown rot fungi, we could not find a lot of similarities in white rot fungi as deduced by the PCA plots and heatmaps. However, some commonalities were evident such as the presence of galactitol as a potential biomarker, and the higher efficiency of these fungi at removing phenolic compounds originally found in undecayed wood. When focusing on both types of decay, we found that wood degrading fungi tend to accumulate sugars and carboxylic acids at late decay stages. Also, as fungal decay progresses, we observed an accumulation of different furans such as furfural or 5-methylfurfural in all fungi.