Genomic Analyses and Enzyme Characterization Provide Insights into the Catabolism of Lignin-Related Aromatic Compounds in White-Rot Fungi
Teeratas Kijpornyongpan,1 Eugene Kuatsjah,1 Alexa Schwartz,1 Allison Yaguchi,1 Sean Woodworth,1 Michael Zahn,2 and Davinia Salvachúa1* (firstname.lastname@example.org)
1National Renewable Energy Laboratory; and 2University of Portsmouth, United Kingdom
The overall goal of this project is to test the hypothesis that white-rot fungi (WRF) can simultaneously depolymerize lignin extracellularly and catabolize depolymerization products intracellularly as carbon and energy sources. The results from this project will lead to improved understanding of lignin utilization by WRF and enable identification of promising fungal strains and enzymes for lignin catabolism and valorization. To date, researchers have confirmed the utilization of lignin-related compounds as carbon sources by two WRF species and have initiated pathway elucidation via systems biology approaches. To continue this effort, the team is using enzymology approaches and comparative genomic and phylogenetic analyses to validate and broaden the knowledge on the catabolism of lignin-related compounds by WRF.
Plant-derived biomass is the most abundant biogenic carbon source on Earth. Despite this abundance of lignocellulose, only a small clade of organisms known as WRF can efficiently break down both the polysaccharide and lignin components of plant cell walls. This unique ability imparts a key role for WRF in global carbon cycling. To date, research on WRF has almost universally and intensely focused on their extracellular enzymes that depolymerize plant polymers, whereas knowledge of their intracellular metabolism remains underexplored (Kijpornyongpan et al. 2022). This project aims to elucidate intracellular pathways in WRF with a particular focus on aromatic catabolic pathways. Recently, the team confirmed the utilization of two lignin-related compounds (e.g., 4-hydroxybenzoic acid and vanillic acid) as carbon sources in two species of WRF (Trametes versicolor and Gelatoporia subvermispora). Additionally, the team proposed a catabolic route via the hydroxyquinol pathway from 4-hydroxybenzoic acid to central carbon metabolism, which was informed by differential transcriptomic, proteomic, and metabolomic analyses (del Cerro et al. 2021). This pathway has been considerably less studied than the b-ketoadipate pathway, which is well known in aromatic catabolic bacteria to convert 4-hydroxybenzoic acid to the tricarboxylic acid cycle but which includes different catabolic intermediates. Based on this discovery, researchers were motivated to conduct mechanistic pathway validation and to seek better understanding of the distribution and prevalence of these pathways in WRF and the fungal kingdom generally.
For the enzyme validation work, team members focused on three main proposed steps in the catabolism of 4-hydroxybenzoic acid: oxidative decarboxylation, hydroxylation, and ring-cleavage (del Cerro et al. 2021). To date, five enzymes have been validated via in vitro approaches, and two additional enzymes have also been validated in vivo in a bacterial host. The former five enzymes have been purified, and enzymology analyses have been conducted to understand substrate and cofactor preference as well as inhibitory mechanisms in the presence of various lignin-related compounds. Structural biology efforts are also underway.
In parallel to its enzyme validation efforts, the team sought to understand the distribution of these catabolic activities in WRF and other microbes. For that purpose, researchers performed a large-scale comparative genomic and phylogenetic study across the bacterial and fungal kingdoms. Team members selected protein domains related to specific catabolic activities and sampled 255 bacterial genomes and 317 fungal genomes. Researchers have shown that some of these enzymes are highly conserved in certain fungal lineages and that substrate specificity of aromatic ring-cleavage enzymes has expanded during fungal evolution. In addition, the abilities to depolymerize lignin and catabolize lignin-related aromatic compounds seem to be independent. Lastly, these analyses have also revealed a series of unique enzymes from WRF that may broaden the spatial location of some catabolic reactions with aromatic compounds. These new enzymes are undergoing biochemical characterization. Overall, these studies will provide a deeper understanding of carbon turnover during wood decay and discover enzymes and pathways that could be exploited to convert the undervalued biopolymer lignin into value-added compounds.
Kijpornyongpan, T., et al. 2022. “Systems Biology–Guided Understanding of White-Rot Fungi for Biotechnological Applications: A Review,” iScience 25, 104640.
del Cerro, C., et al. 2021. “Intracellular Pathways for Lignin Catabolism in White-Rot Fungi,” PNAS 118, e2017381118.
This research is supported by the U.S. Department of Energy (DOE) Office of Science, Office of Biological and Environmental Research under the Early Career Award Program. This work was authored by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36- 08GO28308.