Collaborative Research: EDGE FGT: Establishing functional genomics in anaerobic fungi for applications in agriculture, sustainability, and carbon cycling

厌氧真菌是一种强大的微生物，原产于反刍动物和后肠发酵罐的消化道，包括牛、绵羊和山羊等重要牲畜。在它们的宿主体内，这些真菌提供必需的营养和化合物，驱动动物的生长和行为。具体来说，它们促进富含纤维的原料的消化，分泌维持其他对消化至关重要的微生物生长的代谢物，产生增强宿主健康的化合物，并控制甲烷的产生。该项目开发并优化了厌氧真菌的工程方法，使科学家能够研究和控制这些分子过程，用于各种应用。本科生和研究生、博士后、K-12学生和公众将通过课程作业、实验室研究、YouTube视频和南加州和大西洋中部社区的当地外展活动参与到这些研究中。还将编写培训材料，以便向科学界广泛传播项目发现，加速对这些不寻常微生物的研究。该项目对植物生物量分解、经济生物燃料、可再生化学品生产、气候变化、动物营养和健康以及药物发现具有深远的影响。本项目开发基础遗传工具来研究厌氧肠道真菌。到目前为止，只有少数关于这些物种的短暂转化的报道。然而，CRISPR-Cas技术的出现和最近获得的这些物种的完整基因组为永久性基因改造提供了新的策略。本项目以Neocallimastigomycota门中几个属的代表性分离物为研究对象，优化了将遗传物质传递到真菌游动孢子（该门的幼年生命阶段）的方法，并将通过选择和荧光激活细胞分选对其进行量化。接下来，该项目开发了两种引入或敲除基因的策略。在第一个策略中，基于crispr的工具将被部署和优化，以设计新的细胞表型。在互补的第二种方法中，该项目利用分布在厌氧真菌基因组中的LTR反转录转座子作为平台，提高DNA整合频率并识别真菌启动子。研究成果将被提炼成培训材料，通过国际和跨学科研究团体，如瘤胃微生物基因组学（RMG）网络和厌氧真菌网络（AFN）传播，以促进一些相关领域的科学进步。同样，公众将通过与圣巴巴拉动物园等组织的合作伙伴关系参与进来。这项研究将产生第一个测试厌氧真菌基因功能的实验工具，使人们能够深入了解它们的生活方式，并为微生物工程和假设检验提供途径。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Anaerobic fungi are powerful microorganisms native to the digestive tracts of ruminants and hindgut fermenters, including important livestock such as cattle, sheep, and goats. Within their hosts, these fungi provide essential nutrients and compounds that drive animal growth and behavior. Specifically, they promote the digestion of fiber-rich feedstocks, secrete metabolites that sustain the growth of other microbes critical for digestion, produce compounds that enhance host health, and control the production of methane. This project develops and optimizes methods to engineer anaerobic fungi allowing scientists to study and control these molecular processes for diverse applications. Undergraduate and graduate students, postdoctoral fellows, K-12 students, and the public will be integrated into the studies through coursework, laboratory research, YouTube videos and local outreach to communities in Southern California and the Mid Atlantic. Training materials will also be developed to broadly disseminate project findings to the scientific community, accelerating research into these unusual microbes. This project has far reaching impacts on plant biomass breakdown, economical biofuels, renewable chemical production, climate change, animal nutrition and health, and drug discovery.This project develops foundational genetic tools to study anaerobic gut fungi. To date, only a handful of reports exist for the transient transformation of these species. However, the advent of CRISPR-Cas technology and the recent acquisition of complete genomes for these species enable new strategies for permanent genetic modification. Focusing on representative isolates from several genera within the Neocallimastigomycota phylum, this project optimizes methods to deliver genetic materials to fungal zoospores, the juvenile life stage of this phylum, which will be quantified through selection and fluorescence-activated cell sorting. Next, the project develops two strategies l to introduce or knock down/out genes. In the first strategy, CRISPR-based tools will be deployed and optimized to engineer novel cellular phenotypes. In the complementary second approach, the project leverages LTR retrotransposons distributed throughout anaerobic fungal genomes as platforms to enhance DNA integration frequency and identify fungal promoters. Research findings will be distilled into training materials that will be disseminated through international and interdisciplinary research communities such as the Rumen Microbial Genomics (RMG) Network and the Anaerobic Fungi Network (AFN) to catalyze scientific advancement in a number of allied fields. Similarly, the public will be engaged through partnerships with organizations such as the Santa Barbara Zoo. This research will result in the first experimental tools to test gene function in the anaerobic fungi, enabling insight into their lifestyle and providing a path to microbial engineering and hypothesis testing.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.