title: Release of Pleurotus ostreatus versatile-peroxidase from Mn2+ repression enhances anthropogenic and natural substrate degradation.
abstract: 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.
authors.affiliation: Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Tomer M
authors.initials: TM
authors.lastname: Salame
authors.forename: Doriv
authors.initials: D
authors.lastname: Knop
authors.forename: Dana
authors.initials: D
authors.lastname: Levinson
authors.forename: Sameer J
authors.initials: SJ
authors.lastname: Mabjeesh
authors.forename: Oded
authors.initials: O
authors.lastname: Yarden
authors.forename: Yitzhak
authors.initials: Y
authors.lastname: Hadar
mesh_terms: D001391:Azo Compounds / Q000378:metabolism; D001673:Biodegradation, Environmental / Q000187:drug effects; D002247:Carbon Isotopes; D004396:Coloring Agents / Q000378:metabolism; D005285:Fermentation / Q000187:drug effects / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005800:Genes, Fungal / Q000235:genetics; D005818:Genetic Engineering; D006801:Humans; D008031:Lignin / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology*; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism*; D020076:Pleurotus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000187:drug effects; D013997:Time Factors
chemical_list: D001391:Azo Compounds; D002247:Carbon Isotopes; D004396:Coloring Agents; C036909:lignocellulose; D008345:Manganese; D008031:Lignin; D010544:Peroxidases; C051129:manganese peroxidase