Temporal alterations in the secretome of the selective ligninolytic fungus Ceriporiopsis subvermispora during growth on aspen wood reveal this organism's strategy for degrading lignocellulose

Abstract

The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such as Ceriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue, C. subvermispora was grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis by C. subvermispora.

FIG 1

Distribution of C. subvermispora proteins identified by LC-MS/MS in media containing BMA on days 3, 5, and 7 (A) and the aggregate number of proteins within AA and CAZy families identified during cultivation (B). “Other” proteins are largely without recognizable secretion signals and likely intracellular proteins released by hyphal lysis during incubation. Low-molecular-weight proteins, those without accessible trypsin cleavage sites, those bound to the lignocellulose substrate, and rapidly degraded proteins are likely to be underestimated or missed entirely by shotgun LC-MS/MS.

FIG 2

Time course of emPAI values of enzymes associated with lignocellulose degradation secreted by C. subvermispora grown in media containing BMA. (A) AA1; (B) AA2; (C) AA5; (D) AA3-2; (E) AA3-1; (F) GH6 and GH7; (G) GH12 and GH45; (H) AA9/GH61; (I) GH5; (J) GH10; (K) GH51; (L) CE1 and CE15.

FIG 3

(A) Hierarchical analysis of protein expression patterns of 248 detected genes based on normalized emPAI values. (B) Thirty-four genes with significant changes were categorized into six clusters.

FIG 4

Manganese peroxidase (A), laccase (B), aryl alcohol oxidase (C), and cellobiose dehydrogenase (D) activities of culture filtrates produced by C. subvermispora grown in BMA cultures. Error bars indicate standard deviations from three replicate cultivations. Consistent with LC-MS/MS analysis, no LiP or GLOX activity was detected.