Intracellular pathways for lignin catabolism in white-rot fungi

Abstract

Lignin is a biopolymer found in plant cell walls that accounts for 30% of the organic carbon in the biosphere. White-rot fungi (WRF) are considered the most efficient organisms at degrading lignin in nature. While lignin depolymerization by WRF has been extensively studied, the possibility that WRF are able to utilize lignin as a carbon source is still a matter of controversy. Here, we employ ¹³C-isotope labeling, systems biology approaches, and in vitro enzyme assays to demonstrate that two WRF, Trametes versicolor and Gelatoporia subvermispora, funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways. These results provide insights into global carbon cycling in soil ecosystems and furthermore establish a foundation for employing WRF in simultaneous lignin depolymerization and bioconversion to bioproducts-a key step toward enabling a sustainable bioeconomy.

Keywords: Gelatoporia subvermispora; Trametes versicolor; aromatic compounds; carbon cycling; metabolism.

Fig. 1.

Potential routes for lignin mineralization to CO₂ by WRF and analyses conducted in the current study. Metabolic pathways that are unclear or have not been described before in WRF are designated by red-dashed lines. Typical syringyl (S), hydroxyphenyl (H), and coniferyl (G) lignin-derived motifs from poplar are also depicted. See SI Appendix, Fig. S1 for additional details on aromatic catabolic pathways.

Fig. 2.

¹³C-labeling experiments demonstrate carbon flux of lignin-derived compounds to central metabolism. (A) Abbreviated map showing central carbon metabolic pathways and amino acid biosynthesis in WRF based on the current KEGG model for T. versicolor. (B–E) Substrate level (molar percent) of extracellular metabolites (cellobiose and 4-HBA) in (B) 6-d and (C) 17-d T. versicolor cultivations and (D) 8-d and (E) 16-d G. subvermispora cultivations. A value of 100% corresponds to the initial concentration (7.5 mM in all cases, excluding cellobiose concentration in the 17-d T. versicolor cultivations, which was 3.75 mM). Arrows indicate the sample collection time for ¹³C intracellular analysis. (F–G) Fractional labeling in detected proteinogenic amino acid fragments and other metabolites (acetate and succinate) in (F) T. versicolor and (G) G. subvermispora cultivations when providing unlabeled 4-HBA [negative control, CTL(-)] and ¹³C-ring–labeled 4-HBA. Amino acids fragments (i.e., [15], [57], [85], [159], and [302]) are the result of the derivatization and analysis as thoroughly detailed by Nalsen et al. (33). Individual points are connected with discontinuous lines to facilitate visualization. All results are the average of biological triplicates, and error bars represent the SD. Statistical significance (t test) is presented in SI Appendix, Figs. S4 and S5.

Fig. 3.

Proposed metabolic pathway in T. versicolor and G. subvermispora for the conversion of 4-HBA. Intracellular and extracellular metabolites detected in cellobiose, 4-HBA, lignin, and poplar cultivations (see Dataset S3 and SI Appendix, Figs. S7–S9 for quantitative and qualitative information for each metabolite and SI Appendix, Table S2 for the complete list of metabolites analyzed). As expected, based on previously described syringic acid catabolic pathways (SI Appendix, Fig. S1), none of these metabolites were detected in syringic acid cultivations and neither were the predicted metabolites included as standards in metabolomic analyses (i.e., 3-O-methylgallic acid, gallic acid, pyrogallol, and 2-pyrone-4,6-dicarboxylic acid; SI Appendix, Table S2). Fungal cultivations were conducted in triplicate. A metabolite is considered to be present in biological cultivations if it is detected in at least in two replicates. Media not inoculated with fungi (noninoculated) was also used as control for extracellular metabolomic analyses and intracellular when applicable (i.e., for pellets from lignin and poplar cultivations). Molecules without boxes next to the structure do not have commercially available standards. Continuous gray arrows indicate potential transport through the cell membrane. Continuous and discontinuous black lines correspond to validated (in this work) and proposed enzymatic steps, respectively. The steps are as follows: 1) oxidative decarboxylases GS_120062 and GS_90429, 2) hydroxylase TV_58730, 3) hydroxylation by cytochrome P450, 4) hydroxylases TV_58730 and GS_82057, 5) oxidative decarboxylases GS_120062 and TV_32834 and GS_90429, 6) dioxygenase, 7) ring-cleaving dioxygenases TV_28066 and GS_116134, 8) 4-hydroxymuconic semialdehyde dehydrogenase, 9) maleylacetate reductase, 10) ketoacid CoA transferase, 11) thiolase, 12) CARs, 13) aldehyde dehydrogenase,14) alcohol dehydrogenase, 15) alcohol oxidase, 16) aldehyde oxidase, 17) 4-O-methyl transferase, and 18) demethylase. Additional information for each of these putative enzymatic reactions is detailed in SI Appendix, Text S2.

Fig. 4.

Proteomic and trancriptomic analyses and in vitro biochemical validation. (A) Phylogenetic relationships with putative oxidative decarboxylases, hydroxylases, and dioxygenases selected from in silico analyses in T. versicolor (TV) and G. subvermispora (GS). The white/black matrix indicates the enzymes that were selected for an initial “screening” and for further in vitro activity “validation” with purified enzymes. The heat map shows proteomic (P) and transcriptomic (T) results for protein expression and gene regulation levels, respectively, in each growth media compared to the inoculated control (cellobiose-containing media) from biological triplicates. (B, C) Apparent specific activity in µmol NADH (Left) or NAD(P)H (Right) turnover per minute per milligram of enzyme of selected (B) oxidative decarboxylase and (C) hydroxylase candidates on diverse substrates. (D) Apparent specific activity in µmol O₂ consumed per minute per milligram enzyme of selected dioxygenase enzymes on diverse substrates. Substrates and products from these enzymatic reactions are depicted in SI Appendix, Fig. S17. BZT = 1,2,4-benzenetriol; CAT = catechol; HQ = hydroquinone; NS = nonsignificant differential expression compared to the control; CTL(-) = negative control, no substrate; PCA = protocatechuate; SA = syringic acid; U = unique. Enzymes assays were conducted in triplicate and error bars show the SD.