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

From pseudo to real-time dynamics of T cell thymic differentiation

Summary: Numerous methods have recently emerged for ordering single cells along developmental trajectories. However, accurate depiction of developmental dynamics can only be achieved after rescaling the trajectory according to the relative time spent at each developmental point. We formulate a model which estimates local cell densities and fluxes, and incorporates cell division and apoptosis rates, to infer the real-time dimension of the developmental trajectory. We validate the model using mathematical simulations and apply it to experimental high dimensional cytometry data obtained from the mouse thymus to construct the true time profile of the thymocyte developmental process. Our method can easily be implemented in any of the existing tools for trajectory inference

Effects of cre1 modification in the white-rot fungus Pleurotus ostreatus PC9: altering substrate preference during biological pretreatment

Abstract Background During the process of bioethanol production, cellulose is hydrolyzed into its monomeric soluble units. For efficient hydrolysis, a chemical and/or mechanical pretreatment step is required. Such pretreatment is designed to increase enzymatic digestibility of the cellulose chains inter alia by de-crystallization of the cellulose chains and by removing barriers, such as lignin from the plant cell wall. Biological pretreatment, in which lignin is decomposed or modified by white-rot fungi, has also been considered. One disadvantage in biological pretreatment, however, is the consumption of the cellulose by the fungus. Thus, fungal species that attack lignin with only minimal cellulose loss are advantageous. The secretomes of white-rot fungi contain carbohydrate-active enzymes (CAZymes) including lignin-modifying enzymes. Thus, modification of secretome composition can alter the ratio of lignin/cellulose degradation. Results Pleurotus ostreatus PC9 was genetically modified to either overexpress or eliminate (by gene replacement) the transcriptional regulator CRE1, known to act as a repressor in the process of carbon catabolite repression. The cre1-overexpressing transformant demonstrated lower secreted cellulolytic activity and slightly increased selectivity (based on the chemical composition of pretreated wheat straw), whereas the knockout transformant demonstrated increased cellulolytic activity and significantly reduced residual cellulose, thereby displaying lower selectivity. Pretreatment of wheat straw using the wild-type PC9 resulted in 2.8-fold higher yields of soluble sugar compared to untreated wheat straw. The overexpression transformant showed similar yields (2.6-fold), but the knockout transformant exhibited lower yields (1.2-fold) of soluble sugar. Based on proteomic secretome analysis, production of numerous CAZymes was affected by modification of the expression level of cre1. Conclusions The gene cre1 functions as a regulator for expression of fungal CAZymes active against plant cell wall lignocelluloses, hence altering the substrate preference of the fungi tested. While the cre1 knockout resulted in a less efficient biological pretreatment, i.e., less saccharification of the treated biomass, the converse manipulation of cre1 (overexpression) failed to improve efficiency. Despite the inverse nature of the two genetic alterations, the expected “mirror image” (i.e., opposite regulatory response) was not observed, indicating that the secretion level of CAZymes, was not exclusively dependent on CRE1 activity

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

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

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

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

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