Release of Pleurotus ostreatus Versatile-Peroxidase from Mn 2+ Repression Enhances Anthropogenic and Natural Substrate Degradation Tomer M. Salame 1 , Doriv Knop 1 , Dana Levinson 1 , Sameer J. Mabjeesh 2 , Oded Yarden 1 , Yitzhak Hadar 1 * 1 Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Jerusalem, Israel, 2 Department of Animal Sciences, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Jerusalem, Israel 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 b- 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 b-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. Citation: Salame TM, Knop D, Levinson D, Mabjeesh SJ, Yarden O, et al. (2012) Release of Pleurotus ostreatus Versatile-Peroxidase from Mn 2+ Repression Enhances Anthropogenic and Natural Substrate Degradation. PLoS ONE 7(12): e52446. doi:10.1371/journal.pone.0052446 Editor: Fanis Missirlis, Queen Mary University of London, United Kingdom Received September 12, 2012; Accepted November 13, 2012; Published December 21, 2012 Copyright: ß 2012 Salame et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This research was partially supported by the Ministry of Science and Technology, Israel, and by grant No. 2011505 from the U.S.-Israel Binational Science Foundation (BSF). No additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hadar@agri.huji.ac.il Introduction Pleurotus ostreatus, the oyster mushroom, is a commercially important edible ligninolytic white-rot filamentous basidiomycete and a good model for the study of mechanisms involved in lignin biodegradation [1–3]. Mn 2+ supplements to P. ostreatus cultures have been shown to enhance degradation of anthropogenic aromatic compounds [4–5] and lignin [6–10]. Extracellular manganese peroxidases (MnPs) are considered to be key players in the ligninolytic system [4–18]. The MnP gene family (mnps) of P. ostreatus is comprised of five Mn 2+ -dependent peroxidases (mnp3, 6, 7, 8 and 9) and four versatile-peroxidases (mnp1, 2, 4 and 5; VPs), all having related gene and protein structure. Mn 2+ -dependent peroxidases (EC 1.11.1.13) exclusively oxidize Mn 2+ to Mn 3+ [4,15]. Mn 2+ is an obligatory co-substrate for these enzymes, as it is required to complete the catalytic cycle. VPs (EC 1.11.1.16) possess two catalytic sites, one for the direct oxidation of low- and high-redox- potential compounds, and the second for oxidation of Mn 2+ in a preferred manner [4,14,15,19,20]. This dual activity mode of action enables VPs to modify a wide range of substrates, and makes them attractive potential catalysts for a variety of bio- technological applications [15,20]. VPs have been isolated and thoroughly characterized in Pleurotus and Bjerkandera. The existence of VPs was also reported in Panus, Trametes, Dichomitus and Spongipellis [15,17]. Gene-expression analyses of P. ostreatus cultures have revealed that its nine mnps are transcribed in glucose-peptone medium (GP) and their expression is differentially affected by Mn 2+ supplements. This resulted in drastic up-regulation (200-fold increase) of the predominantly expressed Mn 2+ -dependent peroxidase-encoding genes mnp3 and mnp9, obtaining 0.2 level of expression relative to b-tubulin. In contrast, Mn 2+ supplement resulted in drastic down- regulation (0.03-fold level of expression relative to b-tubulin) of the VP4-encoding gene mnp4, which is by far the predominantly expressed gene in Mn 2+ -deficient medium, exhibiting 2.5-fold level of expression relative to b-tubulin. These findings provided an explanation for the lower activity levels detected in Mn 2+ - containing cultures [4,8–10]. Conclusive proof for the predomi- nance of VP4 under Mn 2+ deficiency was provided by inactivation of mnp4 via gene replacement [18]. Similar trends have been reported for Pleurotus eryngii and Trametes versicolor. Martı ´nez et al. PLOS ONE | www.plosone.org 1 December 2012 | Volume 7 | Issue 12 | e52446