Data_Sheet_1_The Secretome of Phanerochaete chrysosporium and Trametes versicolor Grown in Microcrystalline Cellulose and Use of the Enzymes for Hydrolysis of Lignocellulosic Materials.pdf

The ability of white-rot fungi to degrade polysaccharides in lignified plant cell walls makes them a suitable reservoir for CAZyme prospects. However, to date, CAZymes from these species are barely studied, which limits their use in the set of choices for biomass conversion in modern biorefineries. The current work joined secretome studies of two representative white-rot fungi, Phanerochaete chrysosporium and Trametes versicolor, with expression analysis of cellobiohydrolase (CBH) genes, and use of the secretomes to evaluate enzymatic conversion of simple and complex sugarcane-derived substrates. Avicel was used to induce secretion of high levels of CBHs in the extracellular medium. A total of 56 and 58 proteins were identified in cultures of P. chrysosporium and T. versicolor, respectively, with 78–86% of these proteins corresponding to plant cell wall degrading enzymes (cellulolytic, hemicellulolytic, pectinolytic, esterase, and auxiliary activity). CBHI predominated among the plant cell wall degrading enzymes, corresponding to 47 and 34% of the detected proteins in P. chrysosporium and T. versicolor, respectively, which confirms that Avicel is an efficient CBH inducer in white-rot fungi. The induction by Avicel of genes encoding CBHs (cel) was supported by high expression levels of cel7D and cel7C in P. chrysosporium and T. versicolor, respectively. Both white-rot fungi secretomes enabled hydrolysis experiments at 10 FPU/g substrate, despite the varied proportions of CBHs and other enzymes present in each case. When low recalcitrance sugarcane pith was used as a substrate, P. chrysosporium and T. versicolor secretomes performed similarly to Cellic® CTec2. However, the white-rot fungi secretomes were less efficient than Cellic® CTec2 during hydrolysis of more recalcitrant substrates, such as acid or alkaline sulfite-pretreated sugarcane bagasse, likely because Cellic® CTec2 contains an excess of CBHs compared with the white-rot fungi secretomes. General comparison of the white-rot fungi secretomes highlighted T. versicolor enzymes for providing high glucan conversions, even at lower proportion of CBHs, probably because the other enzymes present in this secretome and CBHs lacking carbohydrate-binding modules compensate for problems associated with unproductive binding to lignin.

白腐真菌降解木质化植物细胞壁中多糖的能力，使其成为挖掘碳水化合物活性酶（CAZyme）潜力的优质资源库。然而迄今为止，针对这类真菌所产CAZyme的研究仍较为匮乏，这限制了其在现代生物炼制生物质转化工艺中的应用潜力。本研究结合了两种典型白腐真菌——黄孢原毛平革菌（Phanerochaete chrysosporium）与彩绒革盖菌（Trametes versicolor）的分泌组研究、纤维二糖水解酶（cellobiohydrolase, CBH）基因表达分析，并利用分泌组评估了简单及复杂甘蔗来源底物的酶解转化效果。研究采用微晶纤维素（Avicel）诱导胞外培养基分泌高水平CBH。分别在黄孢原毛平革菌和彩绒革盖菌的培养物中鉴定出56种和58种蛋白质，其中78%~86%为植物细胞壁降解酶，包括纤维素酶、半纤维素酶、果胶酶、酯酶及辅助活性酶类。纤维二糖水解酶I（CBHI）在植物细胞壁降解酶中占主导地位，分别占黄孢原毛平革菌和彩绒革盖菌检测到的总蛋白的47%和34%，这证实了Avicel是白腐真菌中高效的CBH诱导剂。黄孢原毛平革菌的cel7D基因与彩绒革盖菌的cel7C基因均呈现高表达水平，佐证了Avicel可诱导CBH编码基因（cel）的表达。尽管两种白腐真菌分泌组中CBH与其他酶的比例存在差异，但二者均可在10滤纸酶活力单位（FPU）每克底物的条件下开展酶解实验。当以低抗解性甘蔗髓为底物时，黄孢原毛平革菌和彩绒革盖菌的分泌组酶解效果与Cellic® CTec2相当。然而在处理更高抗解性底物（如酸性或碱性亚硫酸盐预处理的甘蔗渣）的酶解实验中，白腐真菌分泌组的酶解效率低于Cellic® CTec2，这可能是因为相较于白腐真菌分泌组，Cellic® CTec2含有过量的CBH。对两种白腐真菌分泌组的综合对比显示，即便CBH占比更低，彩绒革盖菌的酶系仍可实现更高的葡聚糖转化率，这可能得益于该分泌组中其他酶类以及不含碳水化合物结合模块（carbohydrate-binding modules, CBM）的CBH，可弥补非生产性结合木质素所带来的问题。