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Early-branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes

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Science  11 Mar 2016:
Vol. 351, Issue 6278, pp. 1192-1195
DOI: 10.1126/science.aad1431
  1. Fig. 1 Anaerobic fungi degrade crude biomass.

    (A) Relative growth of gut fungal isolates on crystalline cellulose and crude C3 and C4 bioenergy crops (see table S3 for specific growth rates). (B) Relative xylan activity of cellulose-precipitated gut fungal secretions and commercial Trichoderma [Celluclast (Sigma-Aldrich)] and Aspergillus [Viscozyme (Sigma-Aldrich)]. (C) Relative hemicellulose:cellulose [xylan versus carboxymethylcellulose (CMC)] activity of cellulose-precipitated gut fungal secretions and commercial preparations. For all panels, data represent mean ± SEM (error bars) of more than three samples.

  2. Fig. 2 Anaerobic fungi contain a wealth of biomass-degrading machinery.

    (A) Distribution of cellulolytic carbohydrate-active enzyme (CAZy) transcripts and their regulatory antisense transcripts in Piromyces. CAZymes are shown in bold, whereas antisense transcripts are indicated in parentheses and plotted in a lighter shade. In the key, “Other” refers to pectinases and accessory enzymes that separate cellulose and hemicellulose from other cell wall constituents. PD, polysaccharide deacetylase (acetylxylan esterase); CE, carbohydrate esterase (excluding pectinesterases); RL, rhamnogalacturonate lyase. (B) Proposed model for an extracellular catalytic complex for cellulose degradation. (C) CAZyme composition of the putative extracellular complex. Each square represents a single enzyme that encodes a CAZyme fused to at least one dockerin domain. (D) Identity of predominant secreted gut fungal CAZymes in the cellulose-precipitated fraction. Bands were excised and mapped to the transcriptome by tandem MS (fig. S4).

  3. Fig. 3 Anaerobic fungal biomass-degrading machinery is catabolically repressed.

    (A) Glucose consumption in fungal cultures. Exponential cultures of Piromyces were pulsed with 5 mg of glucose, and mRNA and secretome samples were collected during glucose depletion (yellow region). Blue diamonds, accumulated pressure; brown triangles, glucose concentration. Error bars indicate SEM. (B) Cluster analysis of genes strongly regulated by glucose. Transcript abundance data were compared to uninduced samples at time t = 0 to calculate the log2 fold change in expression. Transcripts with large, significant regulation are displayed (P ≤ 0.01, negative binomial distribution, ≥twofold change). Clusters were manually annotated based on the most common protein domains and/or BLAST (Basic Local Alignment Search Tool) hits. (C) Relative expression [fragments per kilobase of transcript per million mapped reads (FPKM)] of biomass-degrading enzymes (table S1) and their corresponding activity (cellulosome fraction) on CMC (21). Data represent the mean ± SEM (error bars) of at least two replicates.

  4. Fig. 4 Anaerobic fungi degrade complex substrates with increasingly diverse enzymes.

    (A) Relative expression (FPKM) of biomass-degrading enzymes (table S1) and their activity (cellulosome fraction) on CMC. Data represent the mean ± SEM (error bars) of at least two replicates. (B) Normalized enrichment scores of positively enriched specified gene sets relative to growth on glucose. Gene sets containing genes that are expressed more highly in a given substrate are indicated (FDR ≤ 10%; Kolmogorov-Smirnov distribution). Enrichment scores are directly proportional to expression level. Gene sets shown in bold were analyzed in aggregate and in subsets (unbolded sets below). asRNA, antisense RNA that targets CAZy domains (Fig. 2A); Cellulosome, dockerin tagged transcripts. Numbers in the “Glucose responsive” subset indicate clusters.

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