Omics analyses and biochemical study of Phlebiopsis gigantea elucidate its degradation strategy of wood extractives

Abstract

Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor impeding or excluding fungal colonization on the freshly harvested conifers. Among wood decay fungi, the basidiomycete Phlebiopsis gigantea has evolved a unique enzyme system to efficiently transform or degrade conifer extractives but little is known about the mechanism(s). In this study, to clarify the mechanism(s) of softwood degradation, we examined the transcriptome, proteome, and metabolome of P. gigantea when grown on defined media containing microcrystalline cellulose and pine sapwood extractives. Beyond the conventional enzymes often associated with cellulose, hemicellulose and lignin degradation, an array of enzymes implicated in the metabolism of softwood lipophilic extractives such as fatty and resin acids, steroids and glycerides was significantly up-regulated. Among these, a highly expressed and inducible lipase is likely responsible for lipophilic extractive degradation, based on its extracellular location and our characterization of the recombinant enzyme. Our results provide insight into physiological roles of extractives in the interaction between wood and fungi.

Figure 1

Chemical composition of lipophilic compounds in lyophilized Avicel microcrystalline cellulose medium containing none (AV0X) or increasing amounts of loblolly pine extract (AV1X, AV2X, AV4X) without and with P. gigantea inoculation by GC–MS analyses (n = 1). The culture media were harvested five days after the inoculation (AV0X Phlgi, AV1X Phlgi, AV2X Phlgi, AV4X Phlgi) (n = 1). Numbers in parentheses correspond to peaks shown in GC–MS chromatograms (Figure S1). *The fatty acids present in control samples (AV0X and AV0X Phlgi) were thought to be contaminants arisen during sample preparation.

Figure 2

(A) Heat maps showing expression values of all 11,891 P. gigantea genes (left panel) and a subset of 161 significantly (P < 0.02) regulated genes (right panel) when cultured in microcrystalline cellulose medium containing none (AV0X) compared to increasing amounts of loblolly pine extract (AV1X, AV2X, AV4X). DNAStar’s hierarchical clustering program with euclidean distance and centroid linkage methods were used. (Gene tree not shown). (B) Functional classification of 161 P. gigantea genes whose transcripts levels increased (upper panel) or decreased (lower panel) relative to AV0X. Only those exhibiting > twofold change, P < 0.02 and RPKM values > 10 are shown. See Supplemental data file S2 for complete listing of Gene Ontology (GO) and related terms. (C) Line graph plot of signals for each of the 161 regulated transcripts observed in the four media. Highlighted transcripts corresponding to four cytochrome P450s and lipase protein model #19028 exhibiting significant accumulation (> fourfold) even at relatively low extract addition (AV1X).

Figure 3

Glyoxylate shunt and proposed relationship to lipid metabolism when P. gigantea is cultivated on extracts-containing media (AV1X, AV2X, AV4X) relative to Avicel microcrystalline cellulose medium (AV0X). Enzymes encoded by upregulated genes are red highlighted and associated with thick arrows. Heatmap includes transcriptional expression amount (RPKM) in AV0X, AV1X, AV2X, AV4X and LPAS media and ratio with respect to AV0X. ADH/AO acyl-CoA dehydrogenase/oxidase, AH aconitate hydratase, CoA ligase long fatty acid-CoA ligase, DLAT dihydrolipoyllysine-residue acetyltransferase, DLST dihydrolipoyllysine-residue succinyltransferase, EH enoyl-CoA hydratase, FDH formate dehydrogenase, FH fumarate hydratase, GO glyoxylate oxidase, KT ketothiolase (acetyl-CoA C-acyltransferase), HAD 3-hydroxyacyl-CoA dehydrogenase, ICL isocitrate lyase, IDH isocitrate dehydrogenase, MDH malate dehydrogenase, MS malate synthase, ODH oxoglutarate dehydrogenase, OXA oxaloacetase, OXDC oxalate decarboxylase, OXO oxalate oxidase, PC pyruvate carboxylase, PDH pyruvate dehydrogenase, PEP phosphoenolpyruvate, PEPCK phosphoenolpyruvate carboxykinase, PEPK phosphoenolpyruvate kinase, SDH succinate dehydrogenase.

Figure 4

Time course of lipase activities in the secretomes of Phlebiopsis gigantea grown on AV0X, AV1X, AV2X and AV4X (A). Student’s t-tests were performed; **P value < 0.05, *P value < 0.1. The venn diagram of secretomes of Phlebiopsis gigantea grown on AV0X and AV4X based on LC–MS/MS identifications (B).

Figure 5

Phylogenetic tree of amino acids of nine lipases encoded in P. gigantea genome with gene expression (A). ClustalW alignments and RaxML with bootstrap was used for tree construction. Signal, “○” indicates secretion signal peptide at N-terminal, while “x” indicates lack of secretion signal; *secreted protein detected by LC–MS/MS; G-X-S-X-G motif conserved in lipases. (GQSAG and GESAG, found in other Basidiomycetes such as PleoLip369, PleoLip241 and lip2 from Pleurotus. Note: Protein model Phlgi_99110 contains an extended intervening sequence. Lipase activities of recombinant PgLip19028 and vector control (B). Lipase activities of were measured using pNPD as a substrate in acetate buffer (pH 5.0) for 10-min incubation at 26.5 °C. Optimal pH (C) and temperature (D) activities of recombinant PgLip19028. pNPD was used as a substrate. Reaction temperature for optimal pH was 25 °C, and reaction pH for optimal temperature was acetate buffer (pH 4.5). Square, tartrate buffer; circle, acetate buffer; diamond, phosphate buffer. Activities of vector control were subtracted.

Figure 6

GC–MS analysis of the released products from triolein (A) and wood extractives (B) after 17 h-incubation with the recombinant lipase Phlgi_19028 (PgLip19028) or vector control (Ctl) expressed by P. pastoris in acetate buffer (pH4.5) and at 25 °C.