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器的消化道，包括重要的牲畜，如牛、绵羊和山羊。在宿主体内，这些真菌提供必要的营养和化合物，推动动物的生长和行为。具体地说，它们促进富含纤维的饲料的消化，分泌维持其他消化关键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。该项目开发和优化了设计厌氧真菌的方法，使科学家能够研究和控制这些不同应用的分子过程。本科生和研究生、博士后研究员、K-12学生和公众将通过课程作业、实验室研究、YouTube视频和对南加州和大西洋中部社区的当地宣传融入到研究中。还将开发培训材料，向科学界广泛传播项目成果，加快对这些不同寻常的微生物的研究。该项目在植物生物量分解、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等方面产生了深远的影响。该项目开发了研究厌氧肠道真菌的基础基因工具。到目前为止，关于这些物种的瞬时转化的报道寥寥无几。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使永久遗传修改的新策略成为可能。该项目专注于新马尾菌门中几个属的代表性菌株，优化了向真菌游动孢子传递遗传物质的方法，游动孢子是该门的幼年生命阶段，将通过选择和荧光激活的细胞分类来量化。接下来，该项目开发了L引入或敲除/敲除基因的两种策略。在第一个战略中，将部署和优化基于CRISPR的工具来设计新的细胞表型。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，通过瘤胃微生物基因组学(RMG)网络和厌氧真菌网络(AFN)等国际和跨学科研究社区传播，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伙伴关系参与进来。这项研究将产生第一批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，并提供一条通往微生物工程和假设检验的途径。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.