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Here, we systematically evaluated 1,3-propanediol dinitrate (1,3-PDN), a more hydrophobic derivative of 3-NOP, for its antimethanogenic potential and underlying mode of action using ruminal fermentation, pure-culture assays, multi-omics analyses, and molecular docking. Intracellular accumulation assays indicated greater cellular accumulation of 1,3-PDN than 3-NOP in rumen-derived methanogen Methanobrevibacter olleyae. Ruminal fermentation assays showed that 1,3-PDN reduced CH4 production by ~55%, and altered hydrogen (H2) metabolism, leading to 11-fold increase in H2 accumulation. Metatranscriptomic profiling revealed that 1,3-PDN significantly altered the active archaeal community, with a notable reduction in Methanobrevibacter_A and suppression of hydrogenotrophic methanogenesis. Molecular docking suggested that 1,3-PDN may bind to the conserved active site of methyl-coenzyme M reductase (MCR), potentially contributing to MCR-associated inhibition. Proteomic analysis further indicated that 1,3-PDN supplementation downregulated key MCR subunits and simultaneously affected other redox-sensitive methanogenesis-related processes, including tetrahydromethanopterin S-methyltransferase subunits and proteins involved in the biosynthesis of cofactors F430 and cobalamin. Nitrogen-equivalent assay suggested partial contribution of nitrite to the methanogenesis inhibition and oxidative effects induced by 1,3-PDN. Together, these findings identify 1,3-PDN as an effective inhibitor of ruminal methanogenesis with enhanced cellular enrichment, providing mechanistic insights for the rational development of new CH4 inhibitors.<\/jats:p>","DOI":"10.1093\/ismejo\/wrag200","type":"journal-article","created":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T11:14:35Z","timestamp":1785237275000},"source":"Crossref","is-referenced-by-count":0,"title":["Intracellular accumulation of hydrophobic 1,3-PDN inhibits methanogenesis in rumen methanogens"],"prefix":"10.1093","volume":"20","author":[{"given":"Bingzi","family":"Ouyang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Forage Breeding-by-Design and Utilization, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences , Changsha, Hunan 410125 ,","place":["China"]},{"name":"College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences , Beijing 100049 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yurong","family":"Cao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Forage Breeding-by-Design and Utilization, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences , Changsha, Hunan 410125 ,","place":["China"]},{"name":"College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences , Beijing 100049 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiushuang","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Forage Breeding-by-Design and Utilization, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences , Changsha, Hunan 410125 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7742-0685","authenticated-orcid":false,"given":"Shizhe","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Forage Breeding-by-Design and Utilization, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences , Changsha, Hunan 410125 ,","place":["China"]},{"name":"College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences , Beijing 100049 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiang","family":"Zhou","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Forage Breeding-by-Design and Utilization, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences , Changsha, Hunan 410125 ,","place":["China"]},{"name":"College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences , Beijing 100049 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiwu","family":"Wu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Forage Breeding-by-Design and Utilization, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences , Changsha, Hunan 410125 ,","place":["China"]},{"name":"College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences , Beijing 100049 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyi","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Forage Breeding-by-Design and Utilization, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences , Changsha, Hunan 410125 ,","place":["China"]},{"name":"College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences , Beijing 100049 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cancan","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Forage Breeding-by-Design and Utilization, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences , Changsha, Hunan 410125 ,","place":["China"]},{"name":"College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences , Beijing 100049 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,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaojia","family":"Liu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University , Beijing 100193 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lixiang","family":"Yin","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University , Beijing 100193 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiliang","family":"Tan","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Forage Breeding-by-Design and Utilization, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences , Changsha, Hunan 410125 ,","place":["China"]},{"name":"College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences , Beijing 100049 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kui","family":"Zhu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University , Beijing 100193 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Le Luo","family":"Guan","sequence":"additional","affiliation":[{"name":"Faculty of Land and Food Systems, The University of British Columbia , Vancouver, BC V6T 1Z4 ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7818-7659","authenticated-orcid":false,"given":"Xiumin","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Forage Breeding-by-Design and Utilization, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences , Changsha, Hunan 410125 ,","place":["China"]},{"name":"College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences , Beijing 100049 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Diversity and Innovative Utilization, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3645-7315","authenticated-orcid":false,"given":"Min","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Forage Breeding-by-Design and Utilization, National Engineering Laboratory for Pollution Control 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