Mechanisms of manganese(II) oxidation by filamentous Ascomycete fungi vary with species, time, and composition of the secretome
Biogenic manganese (Mn) oxide minerals are ubiquitous in the environment, and their high reactivity can profoundly impact carbon cycling and climate dynamics. Basidiomycete fungi, such as white-rot wood degraders, utilize extracellular enzymes such as laccases and Mn peroxidases to breakdown lignocellulose in plant material. These enzymes oxidize Mn(II) to the reactive intermediate Mn(III), in addition to generating reactive oxygen species (ROS) and organic radicals, all of which can attack recalcitrant compounds. Mn(III/IV) oxide minerals may form as a result of these initial enzymatic processes, but more often Mn(III) is reduced back to Mn(II) during lignocellulose oxidation. In contrast, many filamentous Ascomycete fungi have recently been isolated that can oxidize Mn(II) to Mn(III/IV) oxides, but the pathways utilized by these ubiquitous yet understudied organisms remain largely unknown.
Here, we explore the mechanisms of Mn(II) oxidation by a phylogenetically diverse group of filamentous Ascomycetes. Fungi were grown in liquid culture, and their secretomes, which encompass the extracellular proteins and metabolites produced during growth, were harvested at various time points. These secretomes were subjected to a combination of chemical assays, bulk mass spectrometry, and iTRAQ proteomics to identify enzymes and reactive metabolites involved in extracellular Mn oxide formation.
We show that the mechanisms of Mn(II) oxidation vary with species and over time as a function of secretome composition. Specifically, our work reveals a dynamic transition in Mn(II) oxidation pathways that varies between species. In particular, while ROS produced via transmembrane NADPH oxidases are involved in hyphal-associated Mn(II) oxidation, secreted enzymes are important mediators of Mn(II) oxidation in the bulk secretome. Proteomic analysis of the secretomes reveals a variety of redox-active enzymes involved in Mn(II) oxidation, including several multicopper oxidases and ROS-generating enzymes such as GMC oxidoreductases and glyoxal oxidases. Interestingly, while many of these enzymes have not been previously linked to Mn(II) oxidation, some have been implicated in cellulose degradation by brown-rot Basidiomycetes, suggesting a link between Mn(II) and carbon oxidation mechanisms.
Overall, our work identifies a suite of secreted oxidative enzymes not previously implicated in Mn(II) oxidation by fungi or bacteria. Future work will explore the connection between Ascomycete Mn(II) oxidation and the ability to degrade cellulose, a key carbon reservoir for biofuel production. This work will highlight the contribution of filamentous Ascomycetes to the oxidative capacity of lignocellulose-degrading communities in the environment.