Fernández-Fueyo Elena, Ruiz-Dueñas Francisco J, López-Lucendo María F, Pérez-Boada Marta, Rencoret Jorge, Gutiérrez Ana, Pisabarro Antonio G, Ramírez Lucía, Martínez Angel T
Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands.
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Biotechnol Biofuels. 2016 Feb 29;9:49. doi: 10.1186/s13068-016-0462-9. eCollection 2016.
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.
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.
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.
糙皮侧耳是全球第二大食用菌，也是用于脱木质素应用的模式真菌，具有能在木质和非木质原料上生长的优势。其基因组已测序，这使我们有机会开展蛋白质组学研究，以鉴定在木质纤维素培养物中过量产生的酶。
单核糙皮侧耳（PC9）以杨木或小麦秸秆作为唯一碳/氮源进行培养，并对细胞外蛋白质进行分析，同时分析了来自葡萄糖培养基的蛋白质。通过对全蛋白水解产物进行纳米液相色谱-串联质谱分析，鉴定出五百多种蛋白质。其中34%是秸秆培养物特有的，而葡萄糖和杨木培养物特有的分别仅为15%和6%（20%在三种条件下均产生，另外19%由两种木质纤维素培养物共有）。半定量分析表明，氧化还原酶是杨木（总丰度39%）和秸秆（31%）分泌蛋白组中的主要蛋白类型，而碳水化合物活性酶（CAZys）仅略有过量产生（14 - 16%）。漆酶10（LACC10）是两种木质纤维素分泌蛋白组中的主要蛋白（10 - 14%），并且与漆酶2、漆酶9、漆酶6、多功能过氧化物酶1（VP1）和锰过氧化物酶3（MnP3）一起在木质纤维素培养物中大量过量产生。七种CAZys也位列前50种蛋白质之中，但只有CE16乙酰酯酶在木质纤维素上过量产生。当比较木质和非木质分泌蛋白组时，GH1和GH3β-糖苷酶分别在杨木和秸秆上更为丰富，在丰度较低的蛋白质中，VP2在秸秆上过量产生，而VP3仅在杨木上被发现。通过二维核磁共振（2D NMR）对处理后的木质纤维素底物进行分析，观察到木质素相对于碳水化合物信号减少，同时一些次要木质素亚结构消失，以及糖还原端增加。
糙皮侧耳在木质和非木质木质纤维素底物上生长时，氧化还原酶被强烈诱导。一种漆酶在两种分泌蛋白组中均占据首位，另外三种漆酶与一种VP和一种MnP一起过量产生，表明其在木质纤维素降解中起重要作用。二维核磁共振显示木质素相对于碳水化合物优先被去除，这与上述分泌蛋白组学结果一致。
