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

• Published in: PloS one 2012-12, Vol.7 (12), p.e52446
• Main Authors: Salame, Tomer M, Knop, Doriv, Levinson, Dana, Mabjeesh, Sameer J, Yarden, Oded, Hadar, Yitzhak
• Format: Article
• Language: English
• Subjects: Agriculture; Amaranth; Anthropogenic factors; Azo Compounds - metabolism; Azo dyes; BASIC BIOLOGICAL SCIENCES; Basidiocarps; Biodegradation, Environmental - drug effects; Biology; Carbon Isotopes; Cloning; Coloring Agents - metabolism; Cotton; Decoloring; Decolorization; Degradation; Digestibility; Dry matter; Fermentation; Fermentation - drug effects; Fermentation - genetics; Fermented food; Food; Fungi; Gene expression; Gene Expression Regulation, Fungal - drug effects; Genes, Fungal - genetics; Genetic Engineering; Glucose; Humans; Incubation; Inoculation; Laboratories; Lignin; Lignin - metabolism; Lignocellulose; liquids; Manganese; Manganese - pharmacology; Mineralization; MnP gene; Orange II; Overexpression; Oxidation; Peroxidase; peroxidases; Peroxidases - genetics; Peroxidases - metabolism; Plant pathology; Pleurotus - drug effects; Pleurotus - enzymology; Pleurotus - genetics; Pleurotus ostreatus; polymerase chain reaction; Solid state fermentation; Substrate Specificity - drug effects; Substrates; Time Factors; Transcription; Tubulin; White rot
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0052446

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.