{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T09:24:39Z","timestamp":1787217879733,"version":"build-2736575974"},"reference-count":34,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:00:00Z","timestamp":1787184000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100009590","name":"Gansu Province University Teacher Innovation Fund Project","doi-asserted-by":"publisher","award":["2024B-034"],"award-info":[{"award-number":["2024B-034"]}],"id":[{"id":"10.13039\/501100009590","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100017619","name":"Northwest Minzu University","doi-asserted-by":"publisher","award":["00400-Z2200603"],"award-info":[{"award-number":["00400-Z2200603"]}],"id":[{"id":"10.13039\/501100017619","id-type":"DOI","asserted-by":"publisher"}]},{"award":["00400-Z2200603"],"award-info":[{"award-number":["00400-Z2200603"]}],"id":[{"id":"https:\/\/ror.org\/04cyy9943","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100017619","name":"Fundamental Research Funds for the Central Universities","doi-asserted-by":"publisher","award":["31920240123"],"award-info":[{"award-number":["31920240123"]}],"id":[{"id":"10.13039\/501100017619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100017619","name":"Fundamental Research Funds for the Central Universities","doi-asserted-by":"publisher","award":["31920260001-91"],"award-info":[{"award-number":["31920260001-91"]}],"id":[{"id":"10.13039\/501100017619","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Functional annotation gaps limit the interpretation of carbohydrate metabolism in silage microbiomes. We developed Context-Aware Annotation Completion (CAAC), a framework integrating ESM2 embeddings, genomic-neighborhood features, three-class classification, confidence-tiered neighbor voting, and Enzyme Commission (EC)-to-KEGG Orthology (KO) mapping. CAAC was applied to 21 metagenomes from uninoculated and Lacticaseibacillus paracasei-inoculated silages sampled before ensiling and at 7 and 90 days. Five-fold cross-validation yielded an F1-macro of 84.64% for negative, positive, and hard-sequence classification. Among 800,000 selected annotation-poor sequences, 545,671 Tier 1 or Tier 2 predictions passed the annotation-validity and EC-to-KO mapping criteria, of which 524,814 were eligible for sample-level annotation supplementation. After silage-focused filtering and KO\u2013EC summarization, these predictions yielded 102 KO\u2013EC features repeatedly detected across the silage metagenomes and increased coverage in 25 of 47 carbohydrate-metabolism pathways, mainly by recovering enzyme-level components related to starch and sucrose, cellulose and cellobiose, xylan and hemicellulose, and pectin and glucuronate metabolism. Taxon-linked analyses further revealed treatment- and stage-associated patterns in the taxonomic sources of the supplemented annotations. A database-derived temporal benchmark using the July 2025 CAZy release showed 94.94% Tier 1 family-level annotation-transfer consistency. CAAC extends the enzyme-level interpretation of under-annotated silage metagenomes, while the inferred assignments remain computational predictions requiring experimental validation.<\/jats:p>","DOI":"10.3390\/microorganisms14081848","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T08:10:20Z","timestamp":1787213420000},"page":"1848","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["ESM2-Guided Context-Aware Annotation Completion Supplements Carbohydrate Metabolism Coverage in Silage Microbial Metagenomes"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-6624-9260","authenticated-orcid":false,"given":"Jiewei","family":"Zhang","sequence":"first","affiliation":[{"name":"School of Electrical Engineering, Northwest Minzu University, Lanzhou 730030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-0586-8187","authenticated-orcid":false,"given":"Xinyu","family":"Du","sequence":"additional","affiliation":[{"name":"School of Electrical Engineering, Northwest Minzu University, Lanzhou 730030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-5231-2629","authenticated-orcid":false,"given":"Jinbiao","family":"Tang","sequence":"additional","affiliation":[{"name":"School of Electrical Engineering, Northwest Minzu University, Lanzhou 730030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-9888-614X","authenticated-orcid":false,"given":"Xiaoning","family":"Dong","sequence":"additional","affiliation":[{"name":"School of Electrical Engineering, Northwest Minzu University, Lanzhou 730030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5587-3920","authenticated-orcid":false,"given":"Xusheng","family":"Guo","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Lanzhou University, Lanzhou 730000, China"},{"name":"Probiotics and Life Health Institute, Lanzhou University, Lanzhou 730000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7666-1954","authenticated-orcid":false,"given":"Mingxue","family":"Li","sequence":"additional","affiliation":[{"name":"School of Electrical Engineering, Northwest Minzu University, Lanzhou 730030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3359-9702","authenticated-orcid":false,"given":"Dongmei","family":"Xu","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Lanzhou University, Lanzhou 730000, China"},{"name":"Probiotics and Life Health Institute, Lanzhou University, Lanzhou 730000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Xu, S., Yin, G., and Yu, X. (2025). Dynamics of Microorganisms and Metabolites in the Mixed Silage of Oats and Vetch in Alpine Pastures, and Their Regulatory Mechanisms Under Low Temperatures. Microorganisms, 13.","DOI":"10.3390\/microorganisms13071535"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Ren, J., Han, L., Ma, X., Liu, X., Hao, Y., Yuan, J., Ding, Z., Li, X., Wang, J., and Sun, J. (2026). Integrated Bacterial Community and Differential Metabolites Reveal the Impact of Growth Stage on the Quality of Oat Silage. Microorganisms, 14.","DOI":"10.3390\/microorganisms14030516"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"skad163","DOI":"10.1093\/jas\/skad163","article-title":"Metagenomics Analysis Reveals the Performance of Homo- and Heterofermentative Lactic Acid Bacteria in Alfalfa Silage Fermentation, Bacterial Community, and Functional Profiles","volume":"101","author":"Okoye","year":"2023","journal-title":"J. Anim. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Chen, L., Chen, Y., Peng, Q., Zhou, D., and Feng, S. (2026). Metagenomics and Metagenome-Assembled Genomes Analysis of Highland Barley Baijiu Daqu. Microorganisms, 14.","DOI":"10.3390\/microorganisms14040877"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"D571","DOI":"10.1093\/nar\/gkab1045","article-title":"The Carbohydrate-Active Enzyme Database: Functions and Literature","volume":"50","author":"Drula","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"D672","DOI":"10.1093\/nar\/gkae909","article-title":"KEGG: Biological Systems Database as a Model of the Real World","volume":"53","author":"Kanehisa","year":"2025","journal-title":"Nucleic Acids Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1236","DOI":"10.1093\/bioinformatics\/btu031","article-title":"InterProScan 5: Genome-Scale Protein Function Classification","volume":"30","author":"Jones","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e67667","DOI":"10.7554\/eLife.67667","article-title":"Unifying the Known and Unknown Microbial Coding Sequence Space","volume":"11","author":"Vanni","year":"2022","journal-title":"eLife"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e2016239118","DOI":"10.1073\/pnas.2016239118","article-title":"Biological Structure and Function Emerge from Scaling Unsupervised Learning to 250 Million Protein Sequences","volume":"118","author":"Rives","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1123","DOI":"10.1126\/science.ade2574","article-title":"Evolutionary-Scale Prediction of Atomic-Level Protein Structure with a Language Model","volume":"379","author":"Lin","year":"2023","journal-title":"Science"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2896","DOI":"10.1073\/pnas.96.6.2896","article-title":"The Use of Gene Clusters to Infer Functional Coupling","volume":"96","author":"Overbeek","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1204","DOI":"10.1101\/gr.10.8.1204","article-title":"Predicting Protein Function by Genomic Context: Quantitative Evaluation and Qualitative Inferences","volume":"10","author":"Huynen","year":"2000","journal-title":"Genome Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"R0020","DOI":"10.1186\/gb-2001-2-6-research0020","article-title":"Evolution of Gene Order Conservation in Prokaryotes","volume":"2","author":"Tamames","year":"2001","journal-title":"Genome Biol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Abramova, A., Karkman, A., and Bengtsson-Palme, J. (2024). Metagenomic Assemblies Tend to Break Around Antibiotic Resistance Genes. BMC Genom., 25.","DOI":"10.1186\/s12864-024-10876-0"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"The UniProt Consortium (2023). UniProt: The Universal Protein Knowledgebase in 2023. Nucleic Acids Res., 51, D523\u2013D531.","DOI":"10.1093\/nar\/gkac1052"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"The Gene Ontology Consortium, Aleksander, S.A., Balhoff, J., Carbon, S., Cherry, J.M., Drabkin, H.J., Ebert, D., Feuermann, M., Gaudet, P., and Harris, N.L. (2023). The Gene Ontology Knowledgebase in 2023. Genetics, 224, iyad031.","DOI":"10.1093\/genetics\/iyad031"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1658","DOI":"10.1093\/bioinformatics\/btl158","article-title":"Cd-Hit: A Fast Program for Clustering and Comparing Large Sets of Protein or Nucleotide Sequences","volume":"22","author":"Li","year":"2006","journal-title":"Bioinformatics"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Bao, J., Wang, L., and Yu, Z. (2025). Effect of Lactic Acid Bacteria and Propionic Acid on Fermentation Characteristics, Chemical Composition, and Aerobic Stability of High-Moisture Corn Grain Silage. Microorganisms, 13.","DOI":"10.3390\/microorganisms13010033"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Wei, L., Zhao, M., Song, J., Gao, D., Li, X., Sun, J., Yu, Z., and Bai, C. (2025). Effect of Cordyceps militaris Residue and Lactiplantibacillus plantarum on Fermentation Quality and Bacterial Community of Alfalfa Silage. Microorganisms, 13.","DOI":"10.3390\/microorganisms13081919"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1674","DOI":"10.1093\/bioinformatics\/btv033","article-title":"MEGAHIT: An Ultra-Fast Single-Node Solution for Large and Complex Metagenomics Assembly via Succinct de Bruijn Graph","volume":"31","author":"Li","year":"2015","journal-title":"Bioinformatics"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Hyatt, D., Chen, G.L., Locascio, P.F., Land, M.L., Larimer, F.W., and Hauser, L.J. (2010). Prodigal: Prokaryotic Gene Recognition and Translation Initiation Site Identification. BMC Bioinform., 11.","DOI":"10.1186\/1471-2105-11-119"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Sui, J., Wang, X., Su, Y., Gao, M., Huang, H., Liu, H., Zhang, J., and Tang, Y. (2026). ProMoHGT: A Heterogeneous Graph Transformer with Graph Contrastive Learning for Robust Microbial Protein Function Prediction. BMC Genom., 27.","DOI":"10.1186\/s12864-025-12383-2"},{"key":"ref_23","unstructured":"Vaswani, A., Shazeer, N., Parmar, N., Uszkoreit, J., Jones, L., Gomez, A.N., Kaiser, \u0141., and Polosukhin, I. (2017). Attention Is All You Need. Proceedings of the 31st Annual Conference on Neural Information Processing Systems, Long Beach, CA, USA, 4\u20139 December 2017, Curran Associates Inc."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Kurtz, Z.D., M\u00fcller, C.L., Miraldi, E.R., Littman, D.R., Blaser, M.J., and Bonneau, R.A. (2015). Sparse and Compositionally Robust Inference of Microbial Ecological Networks. PLoS Comput. Biol., 11.","DOI":"10.1371\/journal.pcbi.1004226"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1111\/j.2517-6161.1995.tb02031.x","article-title":"Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing","volume":"57","author":"Benjamini","year":"1995","journal-title":"J. R. Stat. Soc. Ser. B"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Yang, B., Na, N., Wu, N., Sun, L., Li, Z., Qili, M., Han, H., and Xue, Y. (2024). Impact of Additives and Packing Density on Fermentation Weight Loss, Microbial Diversity, and Fermentation Quality of Rape Straw Silage. Microorganisms, 12.","DOI":"10.3390\/microorganisms12101985"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Chen, X., Qiu, S., Huang, L., Yang, Y., Huang, X., Huang, X., and Feng, D. (2024). Effects of Selenium Application on Fermentation Quality, Chemical Composition, and Bacterial Community of Hybrid Pennisetum Silage. Microorganisms, 12.","DOI":"10.3390\/microorganisms12112144"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Bai, B., Qiu, R., Wang, Z., Liu, Y., Bao, J., Sun, L., Liu, T., Ge, G., and Jia, Y. (2023). Effects of Cellulase and Lactic Acid Bacteria on Ensiling Performance and Bacterial Community of Caragana korshinskii Silage. Microorganisms, 11.","DOI":"10.3390\/microorganisms11020337"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"D625","DOI":"10.1093\/nar\/gkae1045","article-title":"CAZac: An Activity Descriptor for Carbohydrate-Active Enzymes","volume":"53","author":"Lombard","year":"2025","journal-title":"Nucleic Acids Res."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Jia, H., Wu, C., Liu, Z., Sun, Y., He, Y., Chen, H., Zu, X., Wang, L., Gao, Y., and Wang, M. (2025). Improving the Quality of Ensiling High-Moisture Alfalfa with Peanut Vine in Different Additives: Fermentation, Nutritional Quality, and Microbial Communities. Microorganisms, 13.","DOI":"10.3390\/microorganisms13102228"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Liu, X., Zhao, G., Bai, J., Qu, X., Chai, J., and Lin, D. (2025). Pediococcus pentosaceus OL77 Enhances Oat (Avena sativa) Silage Fermentation Under Cold Conditions. Microorganisms, 13.","DOI":"10.3390\/microorganisms13102248"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Zhang, L., Zeng, Y., Fu, L., Zhou, Y., Chen, J., Wang, G., Ran, Q., Hu, L., Hu, R., and Zhou, J. (2025). Dynamic Changes in Microorganisms and Metabolites During Silage Fermentation of Whole Winter Wheat. Vet. Sci., 12.","DOI":"10.3390\/vetsci12080708"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Niu, Y., Ma, X., Wang, C., Zhang, P., Lu, Q., Long, R., Wu, Y., and Zhang, W. (2025). Microbial and Metabolomic Insights into Lactic Acid Bacteria Co-Inoculation for Dough-Stage Triticale Fermentation. Microorganisms, 13.","DOI":"10.3390\/microorganisms13081723"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Zhang, Y., Feng, D., Huo, J., Xu, J., Wang, Y., Liu, Q., Bai, W., Liu, Q., and Zhang, Y. (2025). Cultivar-Dependent Differences in Agronomic Characteristics, Nutritional Value, Fermentation Quality, and Bacteriome Profile of Whole-Plant Sorghum Silage. Microorganisms, 13.","DOI":"10.3390\/microorganisms13112634"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/8\/1848\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T08:36:59Z","timestamp":1787215019000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/8\/1848"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,20]]},"references-count":34,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["microorganisms14081848"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms14081848","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,20]]}}}