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

合作研究：EDGE FGT：建立厌氧真菌的功能基因组学，用于农业、可持续发展和碳循环

• 批准号: 2128272
• 负责人: Kevin Solomon
• 金额: $ 52万
• 依托单位: University of Delaware
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2128272&HistoricalAwards=false
• 关键词: Collaborative; Research; EDGE; FGT; Establishing

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.

厌氧菌真菌是反刍动物和后肠发酵罐的消化道的强大微生物，包括重要的牲畜，例如牛，绵羊和山羊。这些真菌在宿主中提供了必需的营养和化合物，可促进动物的生长和行为。具体来说，它们促进了富含纤维的原料的消化，分泌的代谢产物维持对消化至关重要的其他微生物的生长，产生可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工程厌氧菌真菌的方法，使科学家可以研究和控制这些分子过程以进行多种应用。本科生和研究生，博士后研究员，K-12学生以及公众将通过课程工作，实验室研究，YouTube视频以及向南加州和中大西洋中部的社区进行融入研究。还将开发培训材料，以将项目发现广泛传播给科学界，从而加速对这些异常微生物的研究。该项目对植物生物量分解，经济生物燃料，可再生化学生产，气候变化，动物营养和健康以及药物发现具有很大的影响。迄今为止，仅存在少数报告这些物种的瞬时转化。但是，CRISPR-CAS技术的出现以及最近对这些物种的完整基因组的获得，为永久遗传修饰提供了新的策略。该项目专注于新植物菌属中的几个属中的代表性分离物，该项目优化了将遗传材料传递到真菌动物孢子的方法，真菌生物孢子是该门的少年生命阶段，将通过选择和荧光激活的细胞分类来量化。接下来，该项目制定了两种策略，以引入或击倒/消除基因。在第一个策略中，将基于CRISPR的工具进行部署并进行优化，以设计新型的细胞表型。在互补的第二种方法中，该项目利用LTR逆转座子分布在厌氧真菌基因组中，作为平台，以增强DNA整合频率并识别真菌启动子。研究发现将被蒸馏成培训材料，这些材料将通过国际和跨学科研究社区（例如瘤胃微生物基因组学（RMG）网络）和厌氧菌真菌网络（AFN）进行分发，以催化许多盟军领域的科学进步。同样，公众将通过与圣塔芭芭拉动物园（Santa Barbara Zoo）等组织的合作伙伴关系。这项研究将为厌氧真菌中测试基因功能的第一个实验工具，从而深入了解其生活方式，并为微生物工程和假设测试提供了一条途径。该奖项反映了NSF的法定任务，并通过评估该基金会的知识功能和广泛的影响来评估NSF的法定任务。

项目成果

A Genetic Engineering Toolbox for the Lignocellulolytic Anaerobic Gut Fungus Neocallimastix frontalis.

• DOI: 10.1021/acssynbio.2c00502
• 发表时间: 2023-03
• 期刊: ACS synthetic biology
• 影响因子: 4.7
• 作者: Casey A. Hooker;Radwa A. Hanafy;Ethan T. Hillman;Javier Muñoz Briones;Kevin V. Solomon

Characterization and rank assignment criteria for the anaerobic fungi (Neocallimastigomycota)

厌氧真菌（Neocallimastigomycota）的特征和等级划分标准

• DOI: 10.1099/ijsem.0.005449
• 发表时间: 2022-01-01
• 期刊: INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY
• 影响因子: 2.8
• 作者: Elshahed, Mostafa S.;Hanafy, Radwa A.;Youssef, Noha H.
• 通讯作者: Youssef, Noha H.