Differential Expression in Phanerochaete chrysosporium of Membrane-Associated Proteins Relevant to Lignin Degradation

Abstract

Fungal lignin-degrading systems likely include membrane-associated proteins that participate in diverse processes such as uptake and oxidation of lignin fragments, production of ligninolytic secondary metabolites, and defense of the mycelium against ligninolytic oxidants. Little is known about the nature or regulation of these membrane-associated components. We grew the white rot basidiomycete Phanerochaete chrysosporium on cellulose or glucose as the carbon source and monitored the mineralization of a 14 C-labeled synthetic lignin by these cultures to assess their ligninolytic competence. The results showed that the cellulose-grown cultures were ligninolytic, whereas the glucose-grown ones were not. We isolated microsomal membrane fractions from both types of culture and analyzed tryptic digests of their proteins by shotgun liquid chromatography-tandem mass spectrometry. Comparison of the results against the predicted P. chrysosporium proteome showed that a catalase (Joint Genome Institute P. chrysosporium protein identification number [I.D.] 124398), an alcohol oxidase (126879), two transporters (137220 and 132234), and two cytochrome P450s (5011 and 8912) were upregulated under ligninolytic conditions. Quantitative reverse transcription-PCR assays showed that RNA transcripts encoding all of these proteins were also more abundant in ligninolytic cultures. Catalase 124398, alcohol oxidase 126879, and transporter 137220 were found in a proteomic analysis of partially purified plasma membranes from ligninolytic P. chrysosporium and are therefore most likely associated with the outer envelope of the fungus.White rot basidiomycetes make an essential contribution to global carbon cycling by efficiently degrading the recalcitrant aromatic biopolymer lignin, which encases the cellulose and hemicelluloses of vascular plants and is second only to these polysaccharides as a repository of terrestrial biomass. It is generally thought that the lignin is first oxidatively depolymerized outside the fungal hyphae by the combined action of lignin peroxidases, manganese peroxidases, reactive oxygen species, and secreted secondary metabolites, after which the resulting lignin fragments are taken up and mineralized intracellularly (7,9,11,14). Proteins associated with fungal membranes probably have a major role in many steps of this process, including biosynthesis and secretion of secondary metabolites, uptake and intracellular oxidation of lignin fragments, and protection of the mycelium against ligninolytic oxidants. Now that the genome of Phanerochaete chrysosporium has been sequenced and annotated (20, 34), proteomic work has begun on this intensively researched white rot fungus. Recent studies have shown variation in the expression patterns of many extracellular and cytoplasmic enzymes relevant to ligninolysis (1,21,26,28,30,35) but have so far revealed little about the regulation of membrane-associated components. In part, this lack of information likely reflects the poor behavior of some membrane proteins during the electro...

Aromatic Metabolism of Filamentous Fungi in Relation to the Presence of Aromatic Compounds in Plant Biomass

“…Although fungal manganese peroxidases are able to mineralize polymeric lignin to carbon dioxide in vitro ( (Hatakka, 2001;Kapich, Hofrichter, Vares, & Hatakka, 1999) and references therein), it is likely that at least part of aromatic degradation products are taken inside the fungal cell and metabolized by intracellular enzymes. However, almost nothing is known about the transport of aromatic compounds from the growth medium to fungal cell (Shary et al, 2008). The best characterized of these enzymes are oxidoreductases such as class II heme-containing peroxidases from the CAZy family AA2 and laccases (CAZy family AA1_1) (Martinez, Ruiz-Dueñas, Guillen, & Martinez, 1996;Mester et al, 2001;Youn, Hah, & Kang, 1995).…”

Transcriptome and Secretome Analyses ofPhanerochaete chrysosporiumReveal Complex Patterns of Gene Expression

“…Since demethoxylation of lignin is common to white and brown rot, oxidation of methanol may be a crucial reaction. Catalase activity has been identified in the periplasmic space of P. chrysosporium grown under ligninolytic conditions (6), and recent MS studies associated the model Pchr124398 protein with membrane fractions (42).…”

Time-Dependent Profiles of Transcripts Encoding Lignocellulose-Modifying Enzymes of the White Rot Fungus Phanerochaete carnosa Grown on Multiple Wood Substrates

“…Transcripts for actin and GAPDH have traditionally been used as standards in studies of P. chrysosporium (6,14,17). However, previous transcriptomic analysis of P. carnosa suggests that the abundance of these transcripts varies during the growth of this organism on different substrates, and that the level of transcripts encoding a chitin synthase (Chs-257626) is more stable (Fig.…”