van Erven Gijs, Kleijn Anne F, Patyshakuliyeva Aleksandrina, Di Falco Marcos, Tsang Adrian, de Vries Ronald P, van Berkel Willem J H, Kabel Mirjam A
1Laboratory of Food Chemistry, Wageningen University and Research, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands.
2Fungal Physiology, Westerdijk Fungal Biodiversity Institute and Fungal Molecular Physiology, Utrecht University, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
Biotechnol Biofuels. 2020 Apr 16;13:75. doi: 10.1186/s13068-020-01713-z. eCollection 2020.
The ascomycete fungus has been appreciated for its targeted carbohydrate-active enzymatic arsenal. As a late colonizer of herbivorous dung, the fungus acts specifically on the more recalcitrant fraction of lignocellulose and this lignin-rich biotope might have resulted in the evolution of ligninolytic activities. However, the lignin-degrading abilities of the fungus have not been demonstrated by chemical analyses at the molecular level and are, thus far, solely based on genome and secretome predictions. To evaluate whether might provide a novel source of lignin-active enzymes to tap into for potential biotechnological applications, we comprehensively mapped wheat straw lignin during fungal growth and characterized the fungal secretome.
Quantitative C lignin internal standard py-GC-MS analysis showed substantial lignin removal during the 7 days of fungal growth (24% w/w), though carbohydrates were preferably targeted (58% w/w removal). Structural characterization of residual lignin by using py-GC-MS and HSQC NMR analyses demonstrated that C-oxidized substructures significantly increased through fungal action, while intact β--4' aryl ether linkages, -coumarate and ferulate moieties decreased, albeit to lesser extents than observed for the action of basidiomycetes. Proteomic analysis indicated that the presence of lignin induced considerable changes in the secretome of . This was particularly reflected in a strong reduction of cellulases and galactomannanases, while HO-producing enzymes clearly increased. The latter enzymes, together with laccases, were likely involved in the observed ligninolysis.
For the first time, we provide unambiguous evidence for the ligninolytic activity of the ascomycete fungus and expand the view on its enzymatic repertoire beyond carbohydrate degradation. Our results can be of significance for the development of biological lignin conversion technologies by contributing to the quest for novel lignin-active enzymes and organisms.
子囊菌因其靶向性的碳水化合物活性酶库而受到关注。作为食草动物粪便的晚期定殖者，该真菌专门作用于木质纤维素中更难降解的部分，而这种富含木质素的生物群落可能导致了木质素分解活性的进化。然而，该真菌的木质素降解能力尚未通过分子水平的化学分析得到证实，迄今为止，仅基于基因组和分泌组预测。为了评估该真菌是否能为潜在的生物技术应用提供一种新的木质素活性酶来源，我们全面绘制了真菌生长过程中小麦秸秆木质素的图谱，并对真菌分泌组进行了表征。
定量C木质素内标py-GC-MS分析表明，在真菌生长的7天内，木质素大量去除（24% w/w），尽管碳水化合物是更优先的作用靶点（去除率为58% w/w）。通过py-GC-MS和HSQC NMR分析对残留木质素进行结构表征表明，通过真菌作用，C-氧化亚结构显著增加，而完整的β-O-4'芳基醚键、对香豆酸和阿魏酸部分减少，尽管减少程度小于担子菌作用所观察到的。蛋白质组学分析表明，木质素的存在诱导了该真菌分泌组的显著变化。这尤其体现在纤维素酶和半乳甘露聚糖酶的强烈减少，而产H2O2的酶明显增加。后一类酶与漆酶一起，可能参与了观察到的木质素分解。
我们首次为子囊菌的木质素分解活性提供了明确的证据，并将其酶库的观点扩展到碳水化合物降解之外。我们的结果对于生物木质素转化技术的发展可能具有重要意义，有助于寻找新型木质素活性酶和生物体。
