INRAE, Aix Marseille University, UMR1163 Biodiversité et Biotechnologie Fongiques, Marseille, France.
IFP Energies nouvelles, Rueil-Malmaison, France.
Appl Environ Microbiol. 2022 Dec 13;88(23):e0158122. doi: 10.1128/aem.01581-22. Epub 2022 Nov 10.
Filamentous fungi are keystone microorganisms in the regulation of many processes occurring on Earth, such as plant biomass decay and pathogenesis as well as symbiotic associations. In many of these processes, fungi secrete carbohydrate-active enzymes (CAZymes) to modify and/or degrade carbohydrates. Ten years ago, while evaluating the potential of a secretome from the maize pathogen Ustilago maydis to supplement lignocellulolytic cocktails, we noticed it contained many unknown or poorly characterized CAZymes. Here, and after reannotation of this data set and detailed phylogenetic analyses, we observed that several CAZymes (including glycoside hydrolases and carbohydrate oxidases) are predicted to act on the fungal cell wall (FCW), notably on β-1,3-glucans. We heterologously produced and biochemically characterized two new CAZymes, called GH16_1-A and AA3_2-A. We show that GH16_1-A displays β-1,3-glucanase activity, with a preference for β-1,3-glucans with short β-1,6 substitutions, and AA3_2-A is a dehydrogenase catalyzing the oxidation of β-1,3- and β-1,6-gluco-oligosaccharides into the corresponding aldonic acids. Working on model β-1,3-glucans, we show that the linear oligosaccharide products released by GH16_1-A are further oxidized by AA3_2-A, bringing to light a putative biocatalytic cascade. Interestingly, analysis of available transcriptomics data indicates that both GH16_1-A and AA3_2-A are coexpressed, only during early stages of U. maydis infection cycle. Altogether, our results suggest that both enzymes are connected and that additional accessory activities still need to be uncovered to fully understand the biocatalytic cascade at play and its physiological role. Filamentous fungi play a central regulatory role on Earth, notably in the global carbon cycle. Regardless of their lifestyle, filamentous fungi need to remodel their own cell wall (mostly composed of polysaccharides) to grow and proliferate. To do so, they must secrete a large arsenal of enzymes, most notably carbohydrate-active enzymes (CAZymes). However, research on fungal CAZymes over past decades has mainly focused on finding efficient plant biomass conversion processes while CAZymes directed at the fungus itself have remained little explored. In the present study, using the maize pathogen Ustilago maydis as model, we set off to evaluate the prevalence of CAZymes directed toward the fungal cell wall during growth of the fungus on plant biomass and characterized two new CAZymes active on fungal cell wall components. Our results suggest the existence of a biocatalytic cascade that remains to be fully understood.
丝状真菌是调节地球上许多过程的关键微生物,例如植物生物质的分解和发病机制以及共生关系。在许多这些过程中,真菌分泌碳水化合物活性酶(CAZymes)来修饰和/或降解碳水化合物。十年前,在评估玉米病原体玉米黑粉菌的分泌组以补充木质纤维素分解鸡尾酒时,我们注意到其中包含许多未知或特征描述不佳的 CAZymes。在这里,并且在重新注释该数据集和详细的系统发育分析之后,我们观察到几种 CAZymes(包括糖苷水解酶和碳水化合物氧化酶)被预测作用于真菌细胞壁(FCW),特别是β-1,3-葡聚糖。我们异源产生并对两种新的 CAZymes 进行了生化表征,分别称为 GH16_1-A 和 AA3_2-A。我们表明 GH16_1-A 具有β-1,3-葡聚糖酶活性,对具有短β-1,6 取代的β-1,3-葡聚糖具有偏好性,而 AA3_2-A 是一种脱氢酶,可催化β-1,3-和β-1,6-葡糖低聚糖氧化成相应的醛酸。在模型β-1,3-葡聚糖上进行工作时,我们表明 GH16_1-A 释放的线性寡糖产物被 AA3_2-A 进一步氧化,揭示了潜在的生物催化级联。有趣的是,对可用转录组学数据的分析表明,GH16_1-A 和 AA3_2-A 仅在玉米黑粉菌感染周期的早期阶段共同表达。总的来说,我们的结果表明这两种酶是相关的,并且还需要发现其他辅助活性,才能完全了解起作用的生物催化级联及其生理作用。丝状真菌在地球上起着核心调节作用,特别是在全球碳循环中。无论其生活方式如何,丝状真菌都需要重塑自己的细胞壁(主要由多糖组成)才能生长和增殖。为此,它们必须分泌大量的酶,尤其是碳水化合物活性酶(CAZymes)。然而,过去几十年对真菌 CAZymes 的研究主要集中在寻找有效的植物生物质转化过程上,而针对真菌本身的 CAZymes 则研究甚少。在本研究中,我们以玉米病原体玉米黑粉菌为模型,评估了真菌在植物生物质上生长时针对真菌细胞壁的 CAZymes 的普遍性,并对两种针对真菌细胞壁成分的新型 CAZymes 进行了表征。我们的结果表明存在一个生物催化级联,仍有待充分理解。