Release of Pleurotus ostreatus versatile-peroxidase from Mn2+ repression enhances anthropogenic and natural substrate degradation.

The versatile-peroxidase (VP) encoded by mnp4 is one of the nine members of the manganese-peroxidase (MnP) gene family that constitutes part of the ligninolytic system of the white-rot basidiomycete Pleurotus ostreatus (oyster mushroom). VP enzymes exhibit dual activity on a wide range of substrates. As Mn(2+) supplement to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds and lignin, we examined the effect of Mn(2+) on the expression profile of the MnP gene family. In P. ostreatus (monokaryon PC9), mnp4 was found to be the predominantly expressed mnp in Mn(2+)-deficient media, whereas strongly repressed (to approximately 1%) in Mn(2+)-supplemented media. Accordingly, in-vitro Mn(2+)-independent activity was found to be negligible. We tested whether release of mnp4 from Mn(2+) repression alters the activity of the ligninolytic system. A transformant over-expressing mnp4 (designated OEmnp4) under the control of the β-tubulin promoter was produced. Now, despite the presence of Mn(2+) in the medium, OEmnp4 produced mnp4 transcript as well as VP activity as early as 4 days after inoculation. The level of expression was constant throughout 10 days of incubation (about 0.4-fold relative to β-tubulin) and the activity was comparable to the typical activity of PC9 in Mn(2+)-deficient media. In-vivo decolorization of the azo dyes Orange II, Reactive Black 5, and Amaranth by OEmnp4 preceded that of PC9. OEmnp4 and PC9 were grown for 2 weeks under solid-state fermentation conditions on cotton stalks as a lignocellulosic substrate. [(14)C]-lignin mineralization, in-vitro dry matter digestibility, and neutral detergent fiber digestibility were found to be significantly higher (about 25%) in OEmnp4-fermented substrate, relative to PC9. We conclude that releasing Mn(2+) suppression of VP4 by over-expression of the mnp4 gene in P. ostreatus improved its ligninolytic functionality

Gene-expression, lignin degradation and lignocellulose digestibility under solid-state fermentation.

<p>(A) <i>mnp4</i> expression level, (B) [¹⁴C]-lignin mineralization (percentage of ¹⁴CO2 emitted from the total initial radiolabelled [¹⁴C]-lignin is presented), (C) <i>in-vitro</i> dry matter digestibility (IVDMD) and (D) neutral detergent fiber digestibility (NDFD) by <i>P. ostreatus</i>. The wild-type strain (PC9) and <i>mnp4</i> over-expressing strain (OE<i>mnp4</i>) were incubated for 14 days under solid-state fermentation conditions, using a lignocellulosic substrate of cotton stalks containing either 8.0 (basal Mn²⁺ concentration) or 23.3 (concentration in Mn²⁺ supplemented stalks) mg/kg dry matter (DM) of Mn²⁺. Non-inoculated substrate was used as a control. Data represent the average of three biological replicates. Bars denote SD. Statistical analysis was performed by analysis of variance with Tukey-Kramer HSD test (significance accepted at <i>P</i><0.05).</p

Time-course assay of peroxidase activity.

<p>Mn²⁺-dependent (+Mn²⁺) and Mn²⁺-independent (−Mn²⁺) peroxidase activities of <i>P. ostreatus</i> wild-type strain (PC9) and <i>mnp4</i> over-expressing strain (OE<i>mnp4</i>). The fungi were grown in GP medium containing 27 µM Mn²⁺ supplemented with 100 mg/l Orange II for 10 days. Data represent the average of three biological replicates. Bars denote SD.</p

Strategy for producing a <i>mnp4</i> over-expressing <i>P. ostreatus</i>

<p><b>strain.</b> (A) Map of the VP4 (encoded by <i>mnp4</i>) over-expression and carboxin-resistance-conferring (<i>Cbx</i>^R) cassette, TMS12. Small arrows indicate the location of primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052446#pone-0052446-t001" target="_blank">Table 1</a>) used for construction and detection of the construct. (B) PCR screening of <i>P. ostreatus</i> genomic DNA targeting TMS12, using primers R4 and btubTR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052446#pone-0052446-g001" target="_blank">Figure 1A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052446#pone-0052446-t001" target="_blank">Table 1</a>). The arrow indicates the expected 6866 bp amplicon. M – DNA size marker (GeneRuler DNA Ladder Mix, Fermentas); PC9– wild-type; 1, 13, 51, 61, 68, 69, 70, 76– carboxin-resistant transformant strains.</p

Expression of the <i>P. ostreatus manganese peroxidase</i> genes in PC9 and OE<i>mnp4</i> strains.

<p>Total RNA was extracted from <i>P. ostreatus</i> wild-type strain (PC9) and <i>mnp4</i> over-expressing strain (OE<i>mnp4</i>) cultures at (A) 4 days, (B) 7 days and (C) 10 days of incubation. The fungi were grown in GP medium containing 27 µM Mn²⁺ supplemented with 100 mg/l Orange II. Data represent the average of three biological replicates. Bars denote SD.</p

Time-course assay of <i>in-vivo</i> azo dyes decolorization.

<p><i>P. ostreatus</i> wild-type strain (PC9) and <i>mnp4</i> over-expressing strain (OE<i>mnp4</i>) were grown for 10 days in GP medium containing 27 µM Mn²⁺ supplemented with 100 mg/l of (A) Orange II, (B) Reactive Black 5 and (C) Amaranth. Data represent the average of three biological replicates. Bars denote SD. The chemical structure of each dye is shown.</p

MOESM1 of Transcriptomic characterization of Caecomyces churrovis: a novel, non-rhizoid-forming lignocellulolytic anaerobic fungus

Additional file 1: Table S1. Alignment results for transcriptomes of A. robustus, N. californiae, and P. finnis [5]. Shown are # of transcripts (% of transcriptome) in the transcriptome of the fungus listed on the left side successfully aligned to the transcriptome of the fungus listed across the top row using blastn analysis. Table S2. Alignment of scaffoldin amino acid sequences from P. finnis [13] to the transcriptome of C. churrovis by tblastn identifies scaffoldin transcripts. Only results with an alignment E value of 0 are shown. Figure S1. ITS1 Phylogeny of Caecomyces strains shows C. churrovis is significantly different compared to other strains. ITS1 phylogeny of only Caecomyces fungal strains identified a clear separation of C. churrovis from other isolated strains. Figure S2. Full ITS Phylogeny confirms observations about C. churrovis. Phylogeny of ITS1-5.8S-ITS2 regions confirmed the observations that C. churrovis represents a new species in the Caecomyces genus. Figure S3. Catabolic pathways for biomass derived sugars were reconstructed using transcriptome annotations. Enzyme commission numbers and BLAST alignments were used to identify complete sugar pathways present in the transcriptome of C. churrovis. This analysis revealed catabolic routes for glucose, xylose, and fructose, but not mannose, sucrose, and arabinose. Catabolism of ι-d-galactose was identified using BLAST annotations, but not EC numbers. Figure S4. The secretomes of anaerobic gut fungi display free enzymes and multi enzymes complexes (cellulosomes). The same amount of secreted proteins (determined by BCA assay) of P. finnis (F), N. californiae (G1), A. robustus (S4) and C. churrovis (C) were loaded on Native (A) and SDS (B) PAGE. While the Native PAGE (stained by silver staining) shows strong bands indicative of cellulosomes around 1200 kDa, the SDS PAGE (stained by SYPRO Ruby) shows many bands in dissociated cellulosome complexes