Department of Chemical Engineering, University of California, Santa Barbara, Santa Barbara, California, USA.
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington, USA.
mBio. 2021 Jun 29;12(3):e0083221. doi: 10.1128/mBio.00832-21. Epub 2021 Jun 1.
Anaerobic fungi () isolated from the guts of herbivores are powerful biomass-degrading organisms that enhance their degradative ability through the formation of cellulosomes, multienzyme complexes that synergistically colocalize enzymes to extract sugars from recalcitrant plant matter. However, a functional understanding of how fungal cellulosomes are deployed to orchestrate plant matter degradation is lacking, as is knowledge of how cellulosome production and function vary throughout the morphologically diverse life cycle of anaerobic fungi. In this work, we generated antibodies against three major fungal cellulosome protein domains, a dockerin, scaffoldin, and glycoside hydrolase (GH) 48 protein, and used them in conjunction with helium ion and immunofluorescence microscopy to characterize cellulosome localization patterns throughout the life cycle of Piromyces finnis when grown on simple sugars and complex cellulosic carbon sources. Our analyses reveal that fungal cellulosomes are cell-localized entities specifically targeted to the rhizoids of mature fungal cells and bodies of zoospores. Examination of cellulosome localization patterns across life stages also revealed that cellulosome production is independent of growth substrate in zoospores but repressed by simple sugars in mature cells. This suggests that further exploration of gene regulation patterns in zoospores is needed and can inform potential strategies for derepressing cellulosome expression and boosting hydrolytic enzyme yields from fungal cultures. Collectively, these findings underscore how life cycle-dependent cell morphology and regulation of cellulosome production impact biomass degradation by anaerobic fungi, insights that will benefit ongoing efforts to develop these organisms and their cellulosomes into platforms for converting waste biomass into valuable bioproducts. Anaerobic fungi () isolated from the guts of herbivores excel at degrading ingested plant matter, making them attractive potential platform organisms for converting waste biomass into valuable products, such as chemicals and fuels. Major contributors to their biomass-hydrolyzing power are the multienzyme cellulosome complexes that anaerobic fungi produce, but knowledge gaps in how cellulosome production is controlled by the cellular life cycle and how cells spatially deploy cellulosomes complicate the use of anaerobic fungi and their cellulosomes in industrial bioprocesses. We developed and used imaging tools to observe cellulosome spatial localization patterns across life stages of the anaerobic fungus under different environmental conditions. The resulting spatial details of how anaerobic fungi orchestrate biomass degradation and uncovered relationships between life cycle progression and regulation of cellulosome production will benefit ongoing efforts to develop anaerobic fungi and their cellulosomes into useful biomass-upgrading platforms.
从食草动物肠道中分离出的厌氧真菌是强大的生物量降解生物，它们通过形成纤维素酶多酶复合物来增强其降解能力，该复合物协同定位酶以从植物物质中提取糖。然而，对于真菌纤维素酶如何协调植物物质降解的功能理解还很缺乏，对于纤维素酶的产生和功能如何在厌氧真菌形态多样的生命周期中变化的了解也很缺乏。在这项工作中，我们针对三种主要的真菌纤维素酶蛋白结构域（一个 dockerin、一个 scaffoldin 和一个糖苷水解酶（GH）48 蛋白）产生了抗体，并将其与氦离子和免疫荧光显微镜结合使用，以在简单糖和复杂纤维素碳源上生长时，描述 Piromyces finnis 生命周期中的纤维素酶定位模式。我们的分析表明，真菌纤维素酶是针对成熟真菌细胞的根和游动孢子体的细胞内实体，专门靶向这些细胞。对生命阶段的纤维素酶定位模式的检查还表明，纤维素酶的产生与游动孢子中的生长底物无关，但在成熟细胞中被简单糖抑制。这表明需要进一步探索游动孢子中的基因调控模式，并可以为从真菌培养物中释放纤维素酶表达和提高水解酶产量提供潜在策略。总的来说，这些发现强调了生命周期依赖性细胞形态和纤维素酶产生的调控如何影响厌氧真菌对生物量的降解，这些见解将有助于正在努力将这些生物及其纤维素酶开发为将废生物质转化为有价值的生物制品的平台的工作。从食草动物肠道中分离出的厌氧真菌擅长降解摄入的植物物质，因此它们成为将废生物质转化为有价值产品（如化学品和燃料）的有吸引力的潜在平台生物。厌氧真菌产生的多酶纤维素酶复合物是其生物质水解能力的主要贡献者，但细胞周期对纤维素酶产生的控制以及细胞如何空间部署纤维素酶的知识空白，使厌氧真菌及其纤维素酶在工业生物过程中的应用变得复杂。我们开发并使用成像工具来观察在不同环境条件下厌氧真菌生命周期不同阶段的纤维素酶空间定位模式。这些关于厌氧真菌如何协调生物量降解的空间细节以及生命周期进展与纤维素酶产生的调控之间的关系，将有助于正在努力将厌氧真菌及其纤维素酶开发为有用的生物质升级平台的工作。
