From 13C-lignin to 13C-mycelium: Agaricus bisporus uses polymeric lignin as a carbon source

Abstract

Plant biomass conversion by saprotrophic fungi plays a pivotal role in terrestrial carbon (C) cycling. The general consensus is that fungi metabolize carbohydrates, while lignin is only degraded and mineralized to CO₂. Recent research, however, demonstrated fungal conversion of ¹³C-monoaromatic compounds into proteinogenic amino acids. To unambiguously prove that polymeric lignin is not merely degraded, but also metabolized, carefully isolated ¹³C-labeled lignin served as substrate for Agaricus bisporus, the world's most consumed mushroom. The fungus formed a dense mycelial network, secreted lignin-active enzymes, depolymerized, and removed lignin. With a lignin carbon use efficiency of 0.14 (g/g) and fungal biomass enrichment in ¹³C, we demonstrate that A. bisporus assimilated and further metabolized lignin when offered as C-source. Amino acids were high in ¹³C-enrichment, while fungal-derived carbohydrates, fatty acids, and ergosterol showed traces of ¹³C. These results hint at lignin conversion via aromatic ring-cleaved intermediates to central metabolites, underlining lignin's metabolic value for fungi.

Fig. 1.. Overview of approach to assess whether A. bisporus structurally modifies and delignifies, assimilates and metabolizes ¹³C-isotope labeled lignin.

Main research aims (A). Experimental setup and sample codes of lignin treatments by A. bisporus (B). C, carbon; LG, lignin; G, glucose; AX, arabinose and xylose mix [50/50 (w/w %)]; Ab, A. bisporus; CUE, carbon use efficiency.

Fig. 2.. A. bisporus metabolizes lignin for fungal biomass formation.

Removal of lignin by A. bisporus grown on nonlabeled lignin spiked with ¹³C-carbohydrates and lipids (¹²C*_LG, striped bars) and ¹³C-labeled lignin (¹³C_LG, gray bars). Delignification was calculated by mass recoveries of residual insoluble (LR), soluble (LS), and fungal biomass (FBM) fractions and lignin contents thereof analyzed by quantitative pyrolysis–GC-MS. Lignin removal was corrected for sample-handling losses, based on recoveries of uninoculated lignin controls (A). FBM per substrate (CUE); g/g) of A. bisporus cultivated on glucose (¹²C_G, blue bar), arabinose-xylose [50:50 (w/w)] (¹²C_AX, yellow bar), nonlabeled lignin spiked with ¹³C-carbohydrates and lipids (¹²C*_LG, striped bar), and ¹³C-lignin (¹³C_LG, gray bar). Values have been corrected for inoculation seed and lignin embedded in FBM (B). FBM formed (mg) extrapolated to 100 mg of substrate (C-source). Theoretically possible FBM (mg) based on C-containing impurities in the lignin isolate (black bar) (using a CUE of 0.5) and total measured FBM (mg, green plus black bars) generated on lignin media (C). Scanning electron microscopy (SEM) pictures of A. bisporus grown on glucose (D), grown on lignin (E), and of untreated lignin (uninoculated) (F).

Fig. 3.. ¹H-¹³C HSQC NMR spectra of fungal treated lignin and controls from soluble lignin fraction (LS).

Aliphatic regions (A), aromatic regions (B), region of HPV/S (C), and aldehyde regions (D) of ¹H-¹³C HSQC NMR spectra of SPE-purified supernatant of untreated ¹³C-lignin control (LS¹³C_Lg) and of A. bisporus–treated ¹³C-lignin (S¹³C_Lg + Ab) (right). Semiquantitative analysis of volume integrals based on normalized signal intensity of interunit linkages, hydroxycinnamic acids, flavonoids and end groups (E), and aromatic subunits and signal ratios (F) of SPE-purified ¹³C-lignin supernatants (black bars, LS¹³C_Lg; gray bars, LS¹³C_Lg + Ab). Averages, including SDs, of LS¹³C_Lg + Ab are of biological duplicates. Color codes in the spectra correspond to structures in (G), and gray represents unassigned spectra. See Fig. 1 for explanation of sample codes.

Fig. 4.. Secretome profiling of A. bisporus lignin treatments.

Protein IDs and log2-transformed IBAQ intensities of enzymes active on lignin and aromatics in secretomes of biological duplicates (coded A15^I and A15^II) in analytical duplicates of A. bisporus cultivated on nonlabeled lignin in minimal medium (secretomes of ¹²CLg + Ab; data S1). CAZyme family and subfamily, protein ID, and putative functions are provided if available [according to Joint Genome Institute identifier “jgi|Agabi_varbisH97_2” and Morin et al. (11)]. MCO, multicopper oxidase; UPO, unspecific peroxygenase; ODC, oxalate decarboxylase; MnP, manganese peroxidase; GLOX, glyoxal oxidase; AAO, aryl alcohol oxidase; GDH, glucose dehydrogenase; GST, glutathione S-transferase.

Fig. 5.. ¹³C-fractional labeling of FBM compounds.

Fatty acids and ergosterol (A), amino acids (B), carbohydrates (C), of A. bisporus (Ab) FBM from ¹²C*_Lg + Ab (orange bars; ¹²C-lignin “spiked” used as carbon source), and from ¹³C_Lg + Ab (turquoise bars; ¹³C-lignin used as carbon source). All results are the average of biological duplicates, and error bars represent the SDs. The graphs on the right side indicate maximal labeling possible, calculated from FBM increase and mycelium seed used, for ¹³C_Lg + Ab (turquoise), and labeled spike used for ¹²C*_Lg + Ab (orange).

Fig. 6.. Extracellular lignin active enzymes and intracellular pathways and conversion of assimilated lipids, arabinose/xylose, glucose, and lignin degradation products in a fungal cell.

Metabolic pathways show key intermediates where they branch off for biosynthesis of amino acids, hexoses/riboses, and lipids. Amino acids are shown in their one-letter codes (magenta). Key-branching points for biosynthesis of riboses, β-glucan, and chitin are glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P; green). These fungal components/fibers can be hydrolyzed and result in hexoses and riboses (green) for determining the ¹³C-enrichment. Lipids and steroids are marked in yellow and ¹³C-fractional enrichment was tested in fatty acids and ergosterol. Extracellularly, lignin is depolymerized by lignin active enzymes (dark blue), which have been investigated in A. bisporus secretome cultivated on nonlabeled lignin. MCO, multicopper oxidase; PPP, pentose phosphate pathway; EMP, Embden-Meyerhof-Parnas pathway/glycolysis; GNG, gluconeogenesis; TCA cycle, tricarboxylic acid cycle; G3P, glycerol-3-phosphate; 3PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate; X5P, xylose-5-phosphate; R5P, ribose-5-phosphate; αKG, α-ketoglutarate; Oa, oxaloacetate; PW, pathway. The figure was created with BioRender (BioRender.com). Metabolic pathways were based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (33, 34), and proposed end products of aromatic-ring cleavage pathways are based on the schemes proposed by Holesova et al. (31), Lubbers et al. (29), del Cerro et al. (10), Kijpornyongpan et al. (5), and Patyshakuliyeva et al. (47).