A global deep-sea small protein atlas reveals a reservoir of noncanonical antimicrobial peptides

Abstract Small proteins encoded by small open reading frames (smORFs; ≤ 100 aa) represent a largely unexplored dimension of microbial diversity, especially in the deep sea. By analyzing 708 metagenomes from five major deep-sea biomes (hadal trenches, cold seeps, hydrothermal vents, abyssal plains, and seamounts), we constructed the Deep-Sea Small Protein Atlas, comprising 88.7 million smORFs with exceptional novelty and strong habitat specificity. Deep-learning predictions identified 5.47 million candidate antimicrobial peptides (c_AMPs), revealing a peptide space far larger and structurally distinct from known AMPs. Deep-sea c_AMPs are longer, enriched in nonpolar and acidic residues, and exhibit low charge and high intrinsic disorder, suggesting non-membranolytic modes of action. We synthesized 131 representative peptides, of which 87% were antimicrobial, with MICs as low as 1.25 μM, broad-spectrum antibacterial and even antifungal efficacy, and minimal mammalian cytotoxicity. Transcriptomics, TEM imaging, and peptide–protein modeling showed that representative peptides preserve membrane integrity while disrupting intracellular processes such as translation and metabolism, supporting intracellular, non-lytic mechanisms. This work uncovers a vast reservoir of previously unrecognized deep-sea small proteins and structurally unconventional AMPs, providing a foundational resource for discovering next-generation peptide therapeutics. Competing Interest Statement The authors have declared no competing interest. Footnotes In this revised version of the manuscript, the author affiliation of Yiqian Duan has been corrected. This update ensures that the institutional information accurately reflects the author's correct affiliation. No other changes have been made to the manuscript, and the scientific content, results, and conclusions remain unchanged.