Tomer M. Salame

RNAi as a potential tool for biotechnological applications in fungi.

RNA interference (RNAi) is a post-transcriptional gene-silencing (PTGS) phenomenon in which double-stranded RNA (dsRNA) triggers the degradation of homologous mRNA species, thereby reducing gene expression. In fungi, the use of RNAi as a tool for reverse genetics, aimed at modification of gene expression, is constantly growing, with more than 40 species already proven to be responsive. This technology has the ability to co-down-regulate the expression of several genes; however, this trait also makes it susceptible to non-target effects, which can be addressed using both available and developing bioinformatic tools. Moreover, the functionality of absorbed exogenous RNAi molecules, and the various classes of small RNAs found in fungi, offer great versatility and flexibility in acquiring the desired effects on gene expression, even without the necessity to genetically modify the targeted strain. There is an emerging role for RNAi as a potential tool for biotechnological applications. This is evident from current investigations in fungi, demonstrating the contribution of RNAi to progress research and applications in biomaterials production, bioconversion, plant fungal interactions and virulence factors of human pathogens. Possible problems and prospects for the use of RNAi in fungal biotechnology are discussed.

Pleurotus ostreatus manganese-dependent peroxidase silencing impairs decolourization of Orange II.

Decolourization of azo dyes by Pleurotus ostreatus, a white‐rot fungus capable of lignin depolymerization and mineralization, is related to the ligninolytic activity of enzymes produced by this fungus. The capacity of P. ostreatus to decolourize the azo dye Orange II (OII) was dependent and positively co‐linear to Mn2+ concentration in the medium, and thus attributed to Mn2+‐dependent peroxidase (MnP) activity. Based on the ongoing P. ostreatus genome deciphering project we identified at least nine genes encoding for MnP gene family members (mnp1–9), of which only four (mnp1–4) were previously known. Relative real‐time PCR quantification analysis confirmed that all the nine genes are transcribed, and that Mn2+ amendment results in a drastic increase in the transcript levels of the predominantly expressed MnP genes (mnp3 and mnp9), while decreasing versatile peroxidase gene transcription (mnp4). A reverse genetics strategy based on silencing the P. ostreatus mnp3 gene by RNAi was implemented. Knock‐down of mnp3 resulted in the reduction of fungal OII decolourization capacity, which was co‐linear with marked silencing of the Mn2+‐dependent peroxidase genes mnp3 and mnp9. This is the first direct genetic proof of an association between MnP gene expression levels and azo dye decolourization capacity in P. ostreatus, which may have significant implication on understanding the mechanisms governing lignin biodegradation. Moreover, this study has proven the applicability of RNAi as a tool for gene function studies in Pleurotus research.

Predominance of a Versatile-Peroxidase-Encoding Gene, mnp4, as Demonstrated by Gene Replacement via a Gene Targeting System for Pleurotus ostreatus

ABSTRACT Pleurotus ostreatus (the oyster mushroom) and other white rot filamentous basidiomycetes are key players in the global carbon cycle. P. ostreatus is also a commercially important edible fungus with medicinal properties and is important for biotechnological and environmental applications. Efficient gene targeting via homologous recombination (HR) is a fundamental tool for facilitating comprehensive gene function studies. Since the natural HR frequency in Pleurotus transformations is low (2.3%), transformed DNA is predominantly integrated ectopically. To overcome this limitation, a general gene targeting system was developed by producing a P. ostreatus PC9 homokaryon Δku80 strain, using carboxin resistance complemented by the development of a protocol for hygromycin B resistance protoplast-based DNA transformation and homokaryon isolation. The Δku80 strain exhibited exclusive (100%) HR in the integration of transforming DNA, providing a high efficiency of gene targeting. Furthermore, the Δku80 strains produced showed a phenotype similar to that of the wild-type PC9 strain, with similar growth fitness, ligninolytic functionality, and capability of mating with the incompatible strain PC15 to produce a dikaryon which retained its resistance to the corresponding selection and was capable of producing typical fruiting bodies. The applicability of this system is demonstrated by inactivation of the versatile peroxidase (VP) encoded by mnp4. This enzyme is part of the ligninolytic system of P. ostreatus, being one of the nine members of the manganese-peroxidase (MnP) gene family, and is the predominantly expressed VP in Mn2+-deficient media. mnp4 inactivation provided a direct proof that mnp4 encodes a key VP responsible for the Mn2+-dependent and Mn2+-independent peroxidase activity under Mn2+-deficient culture conditions.

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 Mn2+ supplement to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds and lignin, we examined the effect of Mn2+ on the expression profile of the MnP gene family. In P. ostreatus (monokaryon PC9), mnp4 was found to be the predominantly expressed mnp in Mn2+-deficient media, whereas strongly repressed (to approximately 1%) in Mn2+-supplemented media. Accordingly, in-vitro Mn2+-independent activity was found to be negligible. We tested whether release of mnp4 from Mn2+ 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 Mn2+ 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 Mn2+-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. [14C]-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 Mn2+ suppression of VP4 by over-expression of the mnp4 gene in P. ostreatus improved its ligninolytic functionality.

Redundancy among Manganese Peroxidases in Pleurotus ostreatus

ABSTRACT Manganese peroxidases (MnPs) are key players in the ligninolytic system of white rot fungi. In Pleurotus ostreatus (the oyster mushroom) these enzymes are encoded by a gene family comprising nine members, mnp1 to -9 (mnp genes). Mn2+ amendment to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds (such as the azo dye orange II) and lignin. In Mn2+-amended glucose-peptone medium, mnp3, mnp4, and mnp9 were the most highly expressed mnp genes. After 7 days of incubation, the time point at which the greatest capacity for orange II decolorization was observed, mnp3 expression and the presence of MnP3 in the extracellular culture fluids were predominant. To determine the significance of MnP3 for ligninolytic functionality in Mn2+-sufficient cultures, mnp3 was inactivated via the Δku80 strain-based P. ostreatus gene-targeting system. In Mn2+-sufficient medium, inactivation of mnp3 did not significantly affect expression of nontargeted MnPs or their genes, nor did it considerably diminish the fungal Mn2+-mediated orange II decolorization capacity, despite the significant reduction in total MnP activity. Similarly, inactivation of either mnp4 or mnp9 did not affect orange II decolorization ability. These results indicate functional redundancy within the P. ostreatus MnP gene family, enabling compensation upon deficiency of one of its members.

Inactivation of a Pleurotus ostreatus versatile peroxidase-encoding gene (mnp2) results in reduced lignin degradation.

Lignin biodegradation by white-rot fungi is pivotal to the earths carbon cycle. Manganese peroxidases (MnPs), the most common extracellular ligninolytic peroxidases produced by white-rot fungi, are considered key in ligninolysis. Pleurotus ostreatus, the oyster mushroom, is a preferential lignin degrader occupying niches rich in lignocellulose such as decaying trees. Here, we provide direct, genetically based proof for the functional significance of MnP to P. ostreatus ligninolytic capacity under conditions mimicking its natural habitat. When grown on a natural lignocellulosic substrate of cotton stalks under solid-state culture conditions, gene and isoenzyme expression profiles of its short MnP and versatile peroxidase (VP)-encoding gene family revealed that mnp2 was predominately expressed. mnp2, encoding the versatile short MnP isoenzyme 2 was disrupted. Inactivation of mnp2 resulted in three interrelated phenotypes, relative to the wild-type strain: (i) reduction of 14% and 36% in lignin mineralization of stalks non-amended and amended with Mn(2+), respectively; (ii) marked reduction of the bioconverted lignocellulose sensitivity to subsequent bacterial hydrolyses; and (iii) decrease in fungal respiration rate. These results may serve as the basis to clarify the roles of the various types of fungal MnPs and VPs in their contribution to white-rot decay of wood and lignocellulose in various ecosystems.

Mn2+-deficiency reveals a key role for the Pleurotus ostreatus versatile peroxidase (VP4) in oxidation of aromatic compounds

The manganese peroxidase gene family (mnps) is a part of the ligninolytic system of Pleurotus ostreatus. This gene family is comprised of nine members, mnp1–9, encoding short manganese peroxidases (short-MnPs) or versatile peroxidases (VPs). We show that unlike in Mn2+-amended glucose–peptone (GP) medium, where redundancy among mnps was reported, in Mn2+-deficient GP medium mnp4 [encoding versatile peroxidase isoenzyme 4 (VP4)] has a key and nonredundant function. The abundance of mnps transcripts at time points corresponding to the tropophase (active growth), early idiophase, and idiophase indicates that mnp4 is the predominantly expressed mnp gene and that its relative predominance is dependent on the age of the culture. In this medium, azo dye, Orange II (OII) decolorization occurs only during the idiophase and a Δmnp4 strain showed a drastic reduction in this decolorization. Three degradation metabolites were identified by liquid chromatography-mass spectroscopy (LC-MS), indicating both asymmetric and symmetric enzymatic cleavage of the azo-bond. In addition, the culture filtrate of Δmnp4 showed negligible values of oxidation capability of four typical VP substrates: Mn2+, 2,6-dimethoxyphenol, phenol red, and Reactive Black 5 (RB5), compared to the wild-type strain PC9. We concluded that under Mn2+-deficient GP culture, VP4 (encoded by mnp4) is the main active ligninolytic enzyme able to oxidize Mn2+ as well as high and low redox potential aromatic substrate, including dyes. Furthermore, other VPs/MnPs do not compensate for the lack of VP4 activity.

Sensitive detection and identification of DNA and RNA using a patterned capillary tube.

We report on a new ultrasensitive and fast technique for the detection and identification of both DNA and RNA with sensitivity of a few molecules. The new method is based on a patterned capillary tube (PCT) in which the internal surface of a glass tube is patterned with rings of different single-stranded DNA probes. A solution containing single-stranded analyte flows through the tube. Upon hybridization of appropriate DNA and RNA from the solution, DNA polymerase and reverse transcriptase (RT) are employed to synthesize the complementary nucleic acids with deoxynucleoside triphosphate (dNTP) labeled with fluorophores. The sample-analyte hybrids are detected by their fluorescence signal. We show that the new method is sensitive, is specific, can detect simultaneously both DNA and RNA from the same sample, and allows detection of analytes in serum.

A recombinant fungal compound induces anti-proliferative and pro-apoptotic effects on colon cancer cells

Finding intracellular pathways and molecules that can prevent the proliferation of colon cancer cells can provide significant bases for developing treatments for this disease. Ostreolysin (Oly) is a protein found in the mushroom Pleurotus ostreatus, and we have produced a recombinant version of this protein (rOly). We measured the viability of several colon cancer cells treated with rOly. Xenografts and syngeneic colon cancer cells were injected into in vivo mouse models, which were then treated with this recombinant protein. rOly treatment induced a significant reduction in viability of human and mouse colon cancer cells. In contrast, there was no reduction in the viability of normal epithelial cells from the small intestine. In the search for cellular targets of rOly, we showed that it enhances the anti-proliferative activity of drugs targeting cellular tubulin. This was accompanied by a reduction in the weight and volume of tumours in mice injected with rOly as compared to their respective control mice in two in vivo models. Our results advance the functional understanding of rOly as a potential anti-cancer treatment associated with pro-apoptotic activities preferentially targeting colon cancer cells.

Lignin-degrading fungi

Fungi that are able to degrade lignin (a polymer that together with cellulose forms the woody cell …