title: A secretomic view of woody and nonwoody lignocellulose degradation by Pleurotus ostreatus.
abstract: BACKGROUND
  Pleurotus ostreatus is the second edible mushroom worldwide, and a model fungus for delignification applications, with the advantage of growing on woody and nonwoody feedstocks. Its sequenced genome is available, and this gave us the opportunity to perform proteomic studies to identify the enzymes overproduced in lignocellulose cultures.
  
  
  RESULTS
  Monokaryotic P. ostreatus (PC9) was grown with poplar wood or wheat straw as the sole C/N source and the extracellular proteins were analyzed, together with those from glucose medium. Using nano-liquid chromatography coupled to tandem mass spectrometry of whole-protein hydrolyzate, over five-hundred proteins were identified. Thirty-four percent were unique of the straw cultures, while only 15 and 6 % were unique of the glucose and poplar cultures, respectively (20 % were produced under the three conditions, and additional 19 % were shared by the two lignocellulose cultures). Semi-quantitative analysis showed oxidoreductases as the main protein type both in the poplar (39 % total abundance) and straw (31 %) secretomes, while carbohydrate-active enzymes (CAZys) were only slightly overproduced (14-16 %). Laccase 10 (LACC10) was the main protein in the two lignocellulose secretomes (10-14 %) and, together with LACC2, LACC9, LACC6, versatile peroxidase 1 (VP1), and manganese peroxidase 3 (MnP3), were strongly overproduced in the lignocellulose cultures. Seven CAZys were also among the top-50 proteins, but only CE16 acetylesterase was overproduced on lignocellulose. When the woody and nonwoody secretomes were compared, GH1 and GH3 β-glycosidases were more abundant on poplar and straw, respectively and, among less abundant proteins, VP2 was overproduced on straw, while VP3 was only found on poplar. The treated lignocellulosic substrates were analyzed by two-dimensional nuclear magnetic resonance (2D NMR), and a decrease of lignin relative to carbohydrate signals was observed, together with the disappearance of some minor lignin substructures, and an increase of sugar reducing ends.
  
  
  CONCLUSIONS
  Oxidoreductases are strongly induced when P. ostreatus grows on woody and nonwoody lignocellulosic substrates. One laccase occupied the first position in both secretomes, and three more were overproduced together with one VP and one MnP, suggesting an important role in lignocellulose degradation. Preferential removal of lignin vs carbohydrates was shown by 2D NMR, in agreement with the above secretomic results.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands.
authors.forename: Elena
authors.initials: E
authors.lastname: Fernández-Fueyo
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Ruiz-Dueñas
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: María F
authors.initials: MF
authors.lastname: López-Lucendo
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Pérez-Boada
authors.affiliation: Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, PO Box 1052, 41080 Seville, Spain.
authors.forename: Jorge
authors.initials: J
authors.lastname: Rencoret
authors.affiliation: Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, PO Box 1052, 41080 Seville, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Gutiérrez
authors.affiliation: Department of Agrarian Production, Universidad Pública de Navarra, 31006, Pamplona, Spain.
authors.forename: Antonio G
authors.initials: AG
authors.lastname: Pisabarro
authors.affiliation: Department of Agrarian Production, Universidad Pública de Navarra, 31006, Pamplona, Spain.
authors.forename: Lucía
authors.initials: L
authors.lastname: Ramírez
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Angel T
authors.initials: AT
authors.lastname: Martínez
keywords: 2D NMR; Carbohydrate-active enzymes; LC–MS/MS; Laccases; Lignin-modifying enzymes; Pleurotus ostreatus; Poplar wood; Secreted proteins; Wheat straw