Bioactive earthworm proteins and peptides from traditional Chinese medicine: structural diversity, biomedical applications, and emerging biomanufacturing strategies

Abstract

Dilong (earthworm) has been widely used in traditional Chinese medicine (TCM) for centuries to treat conditions such as thrombosis, inflammation, and hypertension. Increasing biochemical and pharmacological evidence indicates that these therapeutic effects are largely attributed to a diverse repertoire of bioactive proteins and peptides. These molecules exhibit a broad spectrum of biological activities, including fibrinolytic, anti-inflammatory, antimicrobial, antioxidant, and neuroprotective functions. With advances in omics technologies and protein engineering, growing attention has been directed toward the discovery, functional characterization, and biotechnological production of these macromolecules. This review summarizes recent progress in earthworm-derived proteins and peptides, with an emphasis on their structural diversity, pharmacological properties, and biomedical applications. We further discuss emerging recombinant production strategies and key challenges related to protein folding, secretion, and post-translational modifications. In addition, the integration of artificial intelligence (AI) with synthetic biology for protein design, structure prediction, and pathway optimization is highlighted. These advances collectively provide a foundation for accelerating the translation of earthworm-derived biomolecules into pharmaceutical, cosmetic, and industrial applications.

Graphical abstract

Keywords

Dilong / Earthworm proteins / Heterologous expression / Bioactive peptides / Synthetic biology / Artificial intelligence

References

[1] Adomako MO, Wu J, Yu F-H. Ecological and evolutionary responses of earthworm holobionts to environmental changes. ISME J, 2025

[2] Alotaiq N, Dermawan D, Elwali NE. Leveraging therapeutic proteins and peptides from Lumbricus earthworms: targeting SOCS2 E3 ligase for cardiovascular therapy through molecular dynamics simulations. Int J Mol Sci, 2024, 25(19): 10818

[3] Azmi N, Hashim P, Hashim D M, Halimoon N, Majid N M, Anti-elastase, anti-tyrosinase and matrix metalloproteinase-1 inhibitory activity of earthworm extracts as potential new anti-aging agent. Asian Pac. J. Trop. Biomed 4 (Suppl 1), 2014, S348–52. https://doi.org/10.12980/APJTB.4.2014C1166

[4] Bao Y, Hu S, Song Z, Shen H, Zhong W, Du S. Chinese medicine Di-long (Pheretima vulgaris) and its active fraction exhibit anti-rheumatoid arthritis effects by inhibiting CXCL10/CXCR3 chemotaxis in synovium. J Ethnopharmacol, 2024, 332: 118286

[5] Beygmoradi A, Homaei A, Hemmati R, Fernandes P. Recombinant protein expression: challenges in production and folding related matters. Int J Biol Macromol, 2023, 233: 123407

[6] Bhambri A, Dhaunta N, Patel SS, Hardikar M, Bhatt A, Srikakulam N, Shridhar S, Vellarikkal S, Pandey R, Jayarajan R, Verma A, Kumar V, Gautam P, Khanna Y, Khan JA, Fromm B, Peterson KJ, Scaria V, Sivasubbu S, Pillai B. Large scale changes in the transcriptome of Eisenia fetida during regeneration. PLoS ONE, 2018, 13(9): e0204234

[7] Blaxter M L, Spurgeon D, Kille P, The genome sequence of the common earthworm, Lumbricus terrestris (Linnaeus, 1758). Wellcome Open Res. 8, 2023; 500. https://doi.org/10.12688/wellcomeopenres.20178.1

[8] Braun M, Tripp A, Chakatok M, Kaltenbrunner S, Fischer C, Stoll D, Bijelic A, Elaily W, Totaro MG, Moser M, Hoch SY, Lechner H, Rossi F, Aleotti M, Hall M, Oberdorfer G. Computational enzyme design by catalytic motif scaffolding. Nature, 2026, 649(8095): 237-245

[9] Bui PT, Pham KT, Vo TDL. Earthworm (perionyx excavatus) protein hydrolysate: hypoglycemic activity and its stability for the hydrolysate and its peptide fractions. Processes, 2023, 11(8): 2490

[10] Cao Y, Zhang X, Wang W, Zhai W, Qian J, Wang J, Chen J, You N, Zhao Z, Wu Q, Xu Y, Yuan L, Li R, Liu C. Oral fibrinogen-depleting agent lumbrokinase for secondary ischemic stroke prevention: Results from a multicenter, randomized, parallel-group and controlled clinical trial. Chin Med J, 2013

[11] Carbonell P, Jervis AJ, Robinson CJ, Yan C, Dunstan M, Swainston N, Vinaixa M, Hollywood KA, Currin A, Rattray NJW, Taylor S, Spiess R, Sung R, Williams AR, Fellows D, Stanford NJ, Mulherin P, Le Feuvre R, Barran P, Goodacre R, Turner NJ, Goble C, Chen GG, Kell DB, Micklefield J, Breitling R, Takano E, Faulon J-L, Scrutton NS. An automated design-build-test-learn pipeline for enhanced microbial production of fine chemicals. Commun Biol, 2018, 1(1): 66

[12] Cheng M, Yue T, Wang H, Jiang L, Huang Q, Li F. Biomimetic nanoparticles co-deliver hirudin and lumbrukinase to ameliorate thrombus and inflammation for atherosclerosis therapy. Asian J Pharm Sci, 2025, 20(1): 100990

[13] Cho I, Choi E, Lee H. Molecular cloning, sequencing, and expression of a fibrinolytic serine-protease gene from the earthworm Lumbricus rubellus. BMB Rep, 2004, 37: 574-581

[14] Chothia C, Lesk AM. The relation between the divergence of sequence and structure in proteins. EMBO J, 1986, 5(4): 823-826

[15] Chubb JJ, Boyle AL, Albanese KI. Rational protein design. Curr Opin Struct Biol, 2026, 97: 103224

[16] Cooper EL, Balamurugan M, Huang C, Tsao CR, Heredia J, Tommaseo-Ponzetta M, Paoletti MG. Earthworms Dilong: ancient, inexpensive, noncontroversial models may help clarify approaches to integrated medicine emphasizing neuroimmune systems. Evid Based Complement Alternat Med, 2012, 2012(1): 164152

[17] Dadkhahfar S, Farokh P, Demehri S, Nazarian RM. Radiation-induced skin injury: Mechanisms, clinical manifestations, and management. Int J Dermatol, 2025

[18] De Colibus L, Sonnen Andreas FP, Morris Keith J, Siebert CA, Abrusci P, Plitzko J, Hodnik V, Leippe M, Volpi E, Anderluh G, Gilbert Robert JC. Structures of lysenin reveal a shared evolutionary origin for pore-forming proteins and its mode of sphingomyelin recognition. Structure, 2012, 20(9): 1498-1507

[19] Dickey A, Wang N, Cooper E, Tull L, Breedlove D, Mason H, Liu D, Wang KY. Transient expression of lumbrokinase (PI239) in tobacco (nicotiana tabacum) using a geminivirus-based single replicon system dissolves fibrin and blood clots. Evid Based Complement Alternat Med, 2017, 2017(1): 6093017

[20] Dong G, Yuan X, Shan Y, Zhao Z, Chen J, Cong Y. Molecular cloning and characterization of cDNA encoding fibrinolytic enzyme-3 from earthworm Eisenia foetida. Acta Biochim Biophys Sin, 2004, 36(4): 303-308

[21] Du C, Li Y, Xia X, Du E, Lin Y, Lian J, Ren C, Li S, Wei W, Qin Y. Identification of a novel collagen-like peptide by high-throughput screening for effective wound-healing therapy. Int J Biol Macromol, 2021, 173: 541-553

[22] Feng Q, Wang X, Wang X, Xu M. A bibliometric analysis and literature review of earthworms used as bio-indicators for assessing soil heavy metal pollution. J Environ Sci, 2026, 162: 763-773

[23] Fierer N. Earthworms' place on earth. Science, 2019, 366(6464): 425-426

[24] Gao L, Zhou T, Yuan J, Zhang S, Hong K, Zhang Z, Hau LC, Cheang WS, Zhou C, Wu X. AI-powered de novo architecturing of marine-mimetic antioxidant peptides in yeast synbio factories. Chem Eng J, 2025, 524: 169604

[25] Ge T, Sun Z, Fu S, Liang G. Cloning of thrombolytic enzyme (lumbrokinase) from earthworm and its expression in the yeast Pichia pastoris. Protein Expression Purif, 2005, 42(1): 20-28

[26] Ge T, Fu S, Xu L, Tang Q, Wang H, Guan K, Liang G. High density fermentation and activity of a recombinant lumbrokinase (PI239) from Pichia pastoris. Protein Expression Purif, 2007, 52(1): 1-7

[27] Global Burden of Cardiovascular Diseases and Risks Collaborators. Global, regional, and national burden of cardiovascular diseases and risk factors in 204 countries and territories, 1990-2023. JACC 86 (22), 2025, 2167-2243 https://doi.org/10.1016/j.jacc.2025.08.015

[28] Gong X, Zhang J, Gan Q, Teng Y, Hou J, Lyu Y, Liu Z, Wu Z, Dai R, Zou Y, Wang X, Zhu D, Zhu H, Liu T, Yan Y. Advancing microbial production through artificial intelligence-aided biology. Biotechnol Adv, 2024, 74: 108399

[29] Guan C, Du X, Wang G, Ji J, Jin C, Li X. Expression of biologically active anti-thrombosis protein lumbrokinase in edible sunflower seed kernel. J Plant Biochem Biotechnol, 2014, 23(3): 257-265

[30] Hanahan D, Hallmarks of cancer-then and now, and beyond. Cell. https://doi.org/10.1016/j.cell.2025.12.049

[31] Haque S, Hussain A, Almalki AH, Aldawsari MF, Lal B, Rai AK, Srivastava M, Fiołka M. Prospects of earthworm coelomic fluid as a potential therapeutic agent to treat cancer. Cancer Metastasis Rev, 2024, 43(2): 621-637

[32] He P, Zhang Y, Zhang Y, Zhang L, Lin Z, Sun C, Wu H, Zhang M. Isolation, identification of antioxidant peptides from earthworm proteins and analysis of the structure-activity relationship of the peptides based on quantum chemical calculations. Food Chem, 2024, 431: 137137

[33] Hirano Y, Tsukamoto K, Ariki S, Naka Y, Ueda M, Tamada T. X-ray crystallographic structural studies of α-amylase I from Eisenia fetida. Acta Cryst D, 2020, 76(9): 834-844

[34] Hong K, Rong Y, Jiang Y, Yuan J, Wu F, Gao L, Wu X. Different strategies for the biosynthesis of bioactive peptide using bioengineering technology in Pichia pastoris: a review. Food Bioprocess Technol, 2025, 18(5): 4326-4344

[35] Hossack EJ, Hardy FJ, Green AP. Building enzymes through design and evolution. ACS Catal, 2023, 13(19): 12436-12444

[36] Hu R, Zhang S, Liang H, Li N, Tu C. Codon optimization, expression, and characterization of recombinant lumbrokinase in goat milk. Protein Expression Purif, 2004, 37(1): 83-88

[37] Hu R, Zhang S, Xu Q, Tu C. A convenient method for the identification and expression of eukaryotic genes. Biotechnol Appl Biochem, 2004, 39(3): 307-312

[38] Hu Y, Meng X, Xu J, Lu W, Wang J. Cloning and expression of earthworm fibrinolytic enzyme PM₂₄₆ in Pichia pastoris. Protein Expression Purif, 2005, 43(1): 18-25

[39] Hua C, Guo Z, Dai M, Zhou J, Ge H, Xue G, Xu F, Ru L, Lv K, Zhang G, Zheng L, Wang M, Teng Y, Yu W, Guo W. Lumbrokinase extracted from earthworms synergizes with bevacizumab and chemotherapeutics in treating non-small cell lung cancer by targeted inactivation of BPTF/VEGF and NF-κB/COX-2 signaling. Biomolecules, 2024, 14(7): 741

[40] Huang C, Li W, Wu B, Chen W, Chen L, Mo G, Zhang Q, Gong L, Li J, Zhang H, Zhu H, Zeng Q. Pheretima aspergillum decoction suppresses inflammation and relieves asthma in a mouse model of bronchial asthma by NF-κB inhibition. J Ethnopharmacol, 2016, 189: 22-30

[41] Huang M, Zhu Y, Xin G, Wang Y, Li F, Li S, Dong Y, Zhang K, Feng L, Tang L, Zhang B, Huang W. Multi-enzyme mimetic iridium nanozymes-based thrombus microenvironment-modulated nanoplatform for enhanced thrombolytic therapy. Chem Eng J, 2023, 470: 144156

[42] Hussain N, Chatterjee SK, Maiti TK, Goswami L, Das S, Deb U, Bhattacharya SS. Metal induced non-metallothionein protein in earthworm: a new pathway for cadmium detoxification in chloragogenous tissue. J Hazard Mater, 2021, 401: 123357

[43] Jafari M, Shoeibi A, Khodatars M, Ghassemi N, Moridian P, Alizadehsani R, Khosravi A, Ling SH, Delfan N, Zhang Y, Wang S, Gorriz JM, AlinejadRokny H, Acharya UR. Automated diagnosis of cardiovascular diseases from cardiac magnetic resonance imaging using deep learning models: a review. Comput Biol Med, 2023, 160: 106998

[44] Ji M, Chen X, Luo J, Wan Y. Improved blood compatibility of polysulfone membrane by anticoagulant protein immobilization. Colloids Surf B, 2019, 175: 586-595

[45] Jiang P, Tang B. The expression of lumbrokinase gene in Pichia pastoris and the optimizationof high density fermentation conditions. Food Ferment Ind, 2018, 44(10): 79-83

[46] Jiang R, Yuan S, Zhou Y, Wei Y, Li F, Wang M, Chen B, Yu H. Strategies to overcome the challenges of low or no expression of heterologous proteins in Escherichia coli. Biotechnol Adv, 2024, 75: 108417

[47] Jin M, Chen W, Huang W, Rong L, Gao Z. Preparation of pegylated lumbrokinase and an evaluation of its thrombolytic activity both in vitro and in vivo. Acta Pharm Sin B, 2013, 3(2): 123-129

[48] Jin F, Zhou ZL, Guo Q, Liang ZW, Yang RY, Jiang JB, He YL, Zhao Q, Zhao Q. High-quality genome assembly of Metaphire vulgaris. PeerJ, 2020

[49] Karbalaei M, Rezaee SA, Farsiani H. Pichia pastoris: a highly successful expression system for optimal synthesis of heterologous proteins. J Cell Physiol, 2020, 235(9): 5867-5881

[50] Karwal M, Kaushik A. Co-composting and vermicomposting of coal fly-ash with press mud: changes in nutrients, micro-nutrients and enzyme activities. Environ Technol Innov, 2020, 18: 100708

[51] Khlebodarova TM, Bogacheva NV, Zadorozhny AV, Bryanskaya AV, Vasilieva AR, Chesnokov DO, Pavlova EI, Peltek SE. Komagataella phaffii as a platform for heterologous expression of enzymes used for industry. Microorganisms, 2024, 12(2): 346

[52] Koh HY, Zheng Y, Yang M, Arora R, Webb GI, Pan S, Li L, Church GM. AI-driven protein design. Nat Rev Bioeng, 2025, 3(12): 1034-1056

[53] Kowald GR, Stürzenbaum SR, Blindauer CA. Earthworm Lumbricus rubellus MT-2: Metal binding and protein folding of a true cadmium-MT. Int J Mol Sci, 2016, 17(1): 65

[54] Kuroki C, Hirano Y, Nakazawa M, Sakamoto T, Tamada T, Ueda M, (2023) A single mutation Asp43Arg was increased 2.5-fold the catalytic activity and maintained the stability of cold-adapted endo-1,4-beta glucanase (Ef-EG2) from Eisenia fetida. Curr Res Biotechnol 2023; 5: 100126 https://doi.org/10.1016/j.crbiot.2023.100126

[55] Lauko A, Pellock S J, Sumida K H, Anishchenko I, Juergens D, Ahern W, Jeung J, Shida A F, Hunt A, Kalvet I, Norn C, Humphreys I R, Jamieson C, Krishna R, Kipnis Y, Kang A, Brackenbrough E, Bera A K, Sankaran B, Houk K N, Baker D, (2025) Computational design of serine hydrolases. Science 388(6744): eadu2454 https://doi.org/10.1126/science.adu2454

[56] Li D, Tong W, Yang Y. Functional expression of an earthworm fibrinolytic enzyme in Escherichia coli. World J Microbiol Biotechnol, 2008, 24(5): 613-618

[57] Li W, Wang C, Sun Z. Vermipharmaceuticals and active proteins isolated from earthworms. Pedobiologia, 2011, 54: S49-S56

[58] Li G, Wang KY, Li D, Wang N, Liu D. Cloning, expression and characterization of a gene from earthworm Eisenia fetida encoding a blood-clot dissolving protein. PLoS ONE, 2012, 7(12): e53110

[59] Li C, Chen M, Li X, Yang M, Wang Y, Yang X. Purification and function of two analgesic and anti-inflammatory peptides from coelomic fluid of the earthworm, Eisenia foetida. Peptides, 2017, 89: 71-81

[60] Li S, Yang Q, Chen F, Tian L, Huo J, Meng Y, Tang Q, Wang W. The antifibrotic effect of pheretima protein is mediated by the tgf-β1/smad2/3 pathway and attenuates inflammation in bleomycin-induced idiopathic pulmonary fibrosis. J Ethnopharmacol, 2022, 286: 114901

[61] Lin Z, Wu H, Zhang M. Isolation, identification, and structure-activity relationship of novel ace inhibitory peptides from earthworm protein in vitro gastrointestinal digestion product. Food Biosci, 2023, 55: 103010

[62] Liu J, Wang X, Xu L, Zhang J, Liang D, Chang W. cDNA cloning and expression of earthworm fibrinolytic enzyme component A. Chin Sci Bull, 2003, 48(1): 68-71

[63] Liu Y, Jia K, Chen H, Wang Z, Zhao W, Zhu L. Cold-adapted enzymes: mechanisms, engineering and biotechnological application. Bioprocess Biosyst Eng, 2023, 46(10): 1399-1410

[64] Liu M, Svirskis D, Proft T, Loh J, Yin N, Li H, Li D, Zhou Y, Chen S, Song L, Chen G, Lu W-Y, Zhang Z, Zhou Z, Li L, Huang Y, Bunt C, Sun G, Harris PWR, Brimble MA, Wen J. Progress in peptide and protein therapeutics: challenges and strategies. Acta Pharm Sin B, 2025, 15(12): 6342-6381

[65] Longfei L, Bin T, Song L, Peng J. Heterologous expression of lumbrokinase from and primary research on the recombinant enzyme characteristics. Chin Pharm J, 2017, 48(12): 1744-1750

[66] Luitjens A, van Corven E, Production and purification of recombinant proteins. Pharmaceutical biotechnology: Fundamentals and applications, Crommelin, D. J. A., Sindelar, R. D., Meibohm, B., Eds. Springer International Publishing: Cham, 2024, pp 69–94. https://doi.org/10.1007/978-3-031-30023-3_4

[67] Luo X, Bi Q, Huang D, Li Y, Yao C, Zhang J, Wei W, Li J, Li Z, Zhang J, Ji S, Wang Y. Guo D-a, Characterization of natural peptides in pheretima by integrating proteogenomics and label-free peptidomics. J Pharm Anal, 2023, 13(9): 1070-1079

[68] Lv F, Zhang J, You S, Qi W. From traditional to AI-driven: the evolution of intelligent enzyme engineering for biocatalysis. Biotechnol Adv, 2026, 87: 108788

[69] Mazurenko S, Prokop Z, Damborsky J. Machine learning in enzyme engineering. ACS Catal, 2020, 10(2): 1210-1223

[70] Mihara H, Sumi H, Akazawa K, Yoneda T, Mizumoto H. Fibrinolytic enzyme extracted from the earthworm. Thromb Haemost, 1983, 50(1): 258-263

[71] Mihara H, Sumi H, Yoneta T, Mizumoto H, Ikeda R, Seiki M, Maruyama M. A novel fibrinolytic enzyme extracted from the earthworm. Lumbricus Rubellus Jpn J Physiol, 1991, 41(3): 461-472

[72] Min KH, Kim KH, Ki M-R, Pack SP. Antimicrobial peptides and their biomedical applications: a review. Antibiotics, 2024, 13(9): 794

[73] Nakajima N, Sugimoto M, Ishihara K. Earthworm-serine protease: characterization, molecular cloning, and application of the catalytic functions. J Mol Catal B: Enzym, 2003, 23(2): 191-212

[74] Ozkan AD, Eskiler GG, Sarihan M, Kazan N, Aksoy O, Yuksel B, Betts Z. Anticancer properties of Eisenia Foetida proteins in prostate cancer cells in vitro. Int J Pept Res Ther, 2022, 28(4): 119

[75] Park YD, Kim JW, Min BG, Seo JW, Jeong JM. Rapid purification and biochemical characteristics of lumbrokinase iii from earthworm for use as a fibrinolytic agent. Biotechnol Lett, 1998, 20(2): 169-172

[76] Park IY, Cha JR, Ok S, Shin C, Kim J, Kwak H, Yu Y, Kim Y, Medina B, Cho S, Park SC. A new earthworm cellulase and its possible role in the innate immunity. Dev Comp Immunol, 2017, 67: 476-480

[77] Peng G, Qin Y, Yan Z, He M, Zhao Y, Jiang N, Wei C. The high-quality telomere-to-telomere genome assembly of the earthworm (Amynthas aspergillum). Sci Data, 2025, 12(1): 931

[78] Phillips H R P, Guerra C A, Bartz M L C, Briones M J I, Brown G, Crowther T W, Ferlian O, Gongalsky K B, van den Hoogen J, Krebs J, Orgiazzi A, Routh D, Schwarz B, Bach E M, Bennett J M, Brose U, Decaëns T, König-Ries B, Loreau M, Mathieu J, Mulder C, van der Putten W H, Ramirez K S, Rillig M C, Russell D, Rutgers M, Thakur M P, de Vries F T, Wall D H, Wardle D A, Arai M, Ayuke F O, Baker G H, Beauséjour R, Bedano J C, Birkhofer K, Blanchart E, Blossey B, Bolger T, Bradley R L, Callaham M A, Capowiez Y, Caulfield M E, Choi A, Crotty F V, Crumsey J M, Dávalos A, Diaz Cosin D J, Dominguez A, Duhour A E, van Eekeren N, Emmerling C, Falco L B, Fernández R, Fonte S J, Fragoso C, Franco A L C, Fugère M, Fusilero A T, Gholami S, Gundale M J, López M G, Hackenberger D K, Hernández L M, Hishi T, Holdsworth A R, Holmstrup M, Hopfensperger K N, Lwanga E H, Huhta V, Hurisso T T, Iannone B V, Iordache M, Joschko M, Kaneko N, Kanianska R, Keith A M, Kelly C A, Kernecker M L, Klaminder J, Koné A W, Kooch Y, Kukkonen S T, Lalthanzara H, Lammel D R, Lebedev I M, Li Y, Jesus Lidon J B, Lincoln N K, Loss S R, Marichal R, Matula R, Moos J H, Moreno G, Morón-Ríos A, Muys B, Neirynck J, Norgrove L, Novo M, Nuutinen V, Nuzzo V, P95 M R, Pansu J, Paudel S, Pérès G, Pérez-Camacho L, Piñeiro R, Ponge J-F, Rashid M I, Rebollo S, Rodeiro-Iglesias J, Rodríguez M Á, Roth A M, Rousseau G X, Rozen A, Sayad E, van Schaik L, Scharenbroch B C, Schirrmann M, Schmidt O, Schröder B, Seeber J, Shashkov M P, Singh J, Smith S M, Steinwandter M, Talavera J A, Trigo D, Tsukamoto J, de Valença A W, Vanek S J, Virto I, Wackett A A, Warren M W, Wehr N H, Whalen J K, Wironen M B, Wolters V, Zenkova I V, Zhang W, Cameron E K, Eisenhauer N, Global distribution of earthworm diversity. Science 366 (6464), 2019, 480-485. https://doi.org/10.1126/science.aax4851

[79] Ren M, Yang L, He L, Wang J, Zhao W, Yang C, Yang S, Cheng H, Huang M, Gou M. Non-viral gene therapy for melanoma using lysenin from Eisenia Foetida. Adv Sci, 2024, 11(17): e2306076

[80] Rong Y, Jensen SI, Lindorff-Larsen K, Nielsen AT. Folding of heterologous proteins in bacterial cell factories: Cellular mechanisms and engineering strategies. Biotechnol Adv, 2023, 63: 108079

[81] Rowaiye A B, Bur D, Agbalalah T, Ogugua J A, Ibeanu G C, Garcia J H F, Molecules of therapeutic and prophylactic value in the earthworm. Vermitechnology: economic, environmental and agricultural sustainability, Aransiola, S. A., Maddela, N. R., Eds. Springer Nature Switzerland: Cham, 2025, pp 83–110. https://doi.org/10.1007/978-3-031-96212-7_5

[82] Royer WE, Strand K, van Heel M, Hendrickson WA. Structural hierarchy in erythrocruorin, the giant respiratory assemblage of annelids. Proc Natl Acad Sci USA, 2000, 97(13): 7107-7111

[83] Rybicka M, Czaplewska P, Rzymowska J, Sofińska-Chmiel W, Wójcik-Mieszawska S, Lewtak K, Węgrzyn K, Jurczak P, Szpiech A, Nowak J, Musiał N, Fiołka MJ. Novel Venetin-1 nanoparticle from earthworm coelomic fluid as a promising agent for the treatment of non-small cell lung cancer. Sci Rep, 2022, 12(1): 18497

[84] Scherlach K, Hertweck C. Mining and unearthing hidden biosynthetic potential. Nat Commun, 2021, 12(1): 3864

[85] Seo M, Lee JH, Baek M, Kim MA, Ahn MY, Kim SH, Yun EY, Hwang JS. A novel role for earthworm peptide lumbricusin as a regulator of neuroinflammation. Biochem Biophys Res Commun, 2017, 490(3): 1004-1010

[86] Shao Y, Wang X, Zhang J, Li M, Wu S, Ma X, Wang X, Zhao H, Li Y, Zhu HH, Irwin DM, Wang D, Zhang G, Ruan J, Wu D. Genome and single-cell RNA-sequencing of the earthworm Eisenia andrei identifies cellular mechanisms underlying regeneration. Nat Commun, 2020, 11(1): 2656

[87] Slathia P S, Sagrika, Sharma E, Khan I A, Thakur R S, Sharma P, Recombinant production of therapeutic proteins. Protein-based therapeutics, Singh, D. B., Tripathi, T., Eds. Springer, 2023, pp 101–129. https://doi.org/10.1007/978-981-19-8249-1_4

[88] Shemesh P, Fishman A. Optimal fermentation conditions for growth and recombinant protein production in Pichia pastoris: Strain selection, ploidy level and carbon source. Curr Res Food Sci, 2024, 9: 100840

[89] Shi G, Ji M, Manar S, Chen J, Jiang G. Liposomes functionalized with cyclic RGD for targeted chemo-photothermal-combined thrombus therapy. J Drug Delivery Sci Technol, 2026, 117: 108068

[90] Short S, Green Etxabe A, Robinson A, Spurgeon D, Kille P, The genome sequence of the red compost earthworm, Lumbricus rubellus (Hoffmeister, 1843). Wellcome Open Res. 8, 2023; 354. https://doi.org/10.12688/wellcomeopenres.19834.1

[91] Stephani L, Rahayu P, Retnoningrum D, Suhartono MT, Rachmawati H, Tjandrawinata RR. Purification and proteomic analysis of potent fibrinolytic enzymes extracted from Lumbricus rubellus. Proteome Sci, 2023, 21(1): 8

[92] Stourac J, Dubrava J, Musil M, Horackova J, Damborsky J, Mazurenko S, Bednar D. Fireprotdb: database of manually curated protein stability data. Nucleic Acids Res, 2020, 49(D1): D319-D324

[93] Sugimoto M, Nakajima N. Molecular cloning, sequencing, and expression of cDNA encoding serine protease with fibrinolytic activity from earthworm. Biosci Biotechnol Biochem, 2001, 65(7): 1575-1580

[94] Tsukamoto K, Ariki S, Nakazawa M, Sakamoto T, Ueda M. Novel cold-adapted raw-starch digesting α-amylases from Eisenia fetida: Gene cloning, expression, and characterization. Biotechnol Rep, 2021, 31: e00662

[95] Ueda M, Ito A, Nakazawa M, Miyatake K, Sakaguchi M, Inouye K. Cloning and expression of the cold-adapted endo-1,4-β-glucanase gene from Eisenia fetida. Carbohydr Polym, 2014, 101: 511-516

[96] Ueda M, Hirano Y, Fukuhara H, Naka Y, Nakazawa M, Sakamoto T, Ogata Y, Tamada T. Gene cloning, expression, and X-ray crystallographic analysis of a β-mannanase from Eisenia fetida. Enzyme Microb Technol, 2018, 117: 15-22

[97] Wan F, Wong F, Collins JJ, de la Fuente-Nunez C. Machine learning for antimicrobial peptide identification and design. Nat Rev Bioeng, 2024, 2(5): 392-407

[98] Wang X, Wang X, Zhang Y, Qu X, Yang S. An antimicrobial peptide of the earthworm Pheretima tschiliensis: cDNA cloning, expression and immunolocalization. Biotechnol Lett, 2003, 25(16): 1317-1323

[99] Wang F, Wang C, Li M, Zhang J, Gui L, An X, Chang W. Crystal structure of earthworm fibrinolytic enzyme component b: a novel, glycosylated two-chained trypsin. J Mol Biol, 2005, 348(3): 671-685

[100] Wang K, Tull L, Cooper E, Wang N, Liu D. Recombinant protein production of earthworm lumbrokinase for potential antithrombotic application. Evid Based Complement Alternat Med, 2013, 2013(1): 783971

[101] Wang Q, Duan L, Xu Z, Wang J, Xi S. The protective effect of the earthworm active ingredients on hepatocellular injury induced by endoplasmic reticulum stress. Biomed Pharmacother, 2016, 82: 304-311

[102] Wang X, Zhang Y, Zhang Y, Kang M, Li Y, James SW, Yang Y, Bi Y, Jiang H, Zhao Y, Sun Z. Amynthas corticis genome reveals molecular mechanisms behind global distribution. Commun Biol, 2021, 4(1): 135

[103] Wang W, Hsu Y, Lin M, Chen S, Lai Y-H, Huang C, Yao C. Ex vivo model to evaluate the antibacterial and anti-inflammatory effects of gelatin–tricalcium phosphate composite incorporated with emodin and lumbrokinase for bone regeneration. Bioengineering, 2023, 10(8): 906

[104] Wang W, Lai Y, Huang C, Lai J, Yao C. Lumbrokinase-containing gelatin nanofibers with multiple bioactivities for effective skin wound healing. Mater Today Bio, 2025, 32: 101713

[105] Wei H, Shao J, Liu B. Biological sequence analysis: advances, medical applications, and challenges. Fundam Res, 2026, 6(1): 6-10

[106] Weinstein JJ, Goldenzweig A, Hoch S, Fleishman SJ. Pross 2: a new server for the design of stable and highly expressed protein variants. Bioinformatics, 2020, 37(1): 123-125

[107] Weisel JW, Litvinov RI. Exploring the thrombus niche: lessons learned and potential therapeutic opportunities. Blood, 2025, 146(12): 1389-1399

[108] Wijma HJ, Fürst M, Janssen DB. A computational library design protocol for rapid improvement of protein stability: fresco. Methods Mol Biol, 2018, 1685: 69-85

[109] Wojtaszek J, Kolaczkowska A, Kowalska J, Nowak K, Wilusz T, LTCI, a novel chymotrypsin inhibitor of the potato I family from the earthworm Lumbricus terrestris. Purification, cDNA cloning, and expression. Comp Biochem Physiol B Biochem Mol Biol 2006; 143(4): 465–72. https://doi.org/10.1016/j.cbpb.2005.12.023

[110] Wu Y, Hu S, Ma Y, Zhao B, Yang W, Lu Y, Li P, Du S. Novel Pheretima guillelmi-derived antithrombotic protein DPf3: identification, characterization, in vitro evaluation and antithrombotic mechanisms investigation. Int J Biol Macromol, 2020, 154: 545-556

[111] Wu Z, Xia F, Lin R. Global burden of cancer and associated risk factors in 204 countries and territories, 1980–2021: a systematic analysis for the GBD 2021. J Hematol Oncol, 2024, 17(1): 119

[112] Wu W, Cui C, Zhu Y, Chen J, Zhuang Q, Wang Y, Liu Z, Gao H, Ou G, Liu C, Tao M, Chen Y, Pan R, Zhang G, Cai H, Yang J, Chen X, Zhou X, Wang S, Shen X. Structural genomics sheds light on protein functions and remote homologs across the insect tree of life. Cell Res, 2026

[113] Xiao N, Ge F, Edwards CA. The regeneration capacity of an earthworm, Eisenia fetida, in relation to the site of amputation along the body. Acta Ecol Sin, 2011, 31(4): 197-204

[114] Xing Z, Gao H, Wang D, Shang Y, Tuliebieke T, Jiang J, Li C, Wang H, Li Z, Jia L, Wu Y, Wang D, Yang W, Chang Y, Zhang X, Xu L, Jiang C, Huang L, Tian X, A novel biological sources consistency evaluation method reveals high level of biodiversity within wild natural medicine: a case study of amynthas earthworms as “guang Dilong”. Acta Pharm Sin B 2023; 13 (4): 1755–1770. https://doi.org/10.1016/j.apsb.2022.10.024

[115] Xu Y, Liang G, Sun Z, Chen F, Fu S, Chai Y, Hou Y. Cloning and expression of the novel gene-PV₂₄₂ of earthworm fibrinolytic enzyme. Prog Biochem Biophys, 2002, 29(4): 610-614

[116] Xu Z, Yang Y, Gui Q, Zhang L, Hu L. Expression, purification, and characterization of recombinant lumbrokinase PI239 in Escherichia coli. Protein Expression Purif, 2010, 69(2): 198-203

[117] Xu L, Bai X, Joong OhE. Strategic approaches for designing yeast strains as protein secretion and display platforms. Crit Rev Biotechnol, 2025, 45(3): 491-508

[118] Yang Y, Hu H, Wang W, Duan X, Luo S, Wang X, Sun Y. The identification of functional proteins from amputated lumbricus Eisenia fetida on the wound healing process. Biomed Pharmacother, 2017, 95: 1469-1478

[119] Yang W, Wang W, Ma Y, Yang Q, Li P, Du S. Bioevaluation of Pheretima vulgaris antithrombotic extract, PvQ, and isolation, identification of six novel PvQ-derived fibrinolytic proteases. Molecules, 2021, 26(16): 4946

[120] Yang R, Roshani D, Gao B, Li P, Shang N. Metallothionein: a comprehensive review of its classification, structure, biological functions, and applications. Antioxidants, 2024, 13(7): 825

[121] Yang J, Lal RG, Bowden JC, Astudillo R, Hameedi MA, Kaur S, Hill M, Yue Y, Arnold FH. Active learning-assisted directed evolution. Nat Commun, 2025, 16(1): 714

[122] Yang Y, Ni P, Ma L, Tang H, Yang J, Zhou X, Sun Y, Xu W. Therapeutic effects of G90′-ES2 protein complex from regenerated Eisenia fetida on radiation-induced skin injury. J Ethnopharmacol, 2026, 355: 120583

[123] Yuan X, Cao C, Shan Y, Zhao Z, Chen J, Cong Y, Expression and characterization of earthworm Eisenia foetida lumbrokinase-3 in Pichia pastoris. Prep. Biochem Biotechnol 36 (3): 273–9 https://doi.org/10.1080/10826060600716703

[124] Zhang L, Wang J, Yu M, Ru B. Expression and characterization of ARSP1 from Eisenia fetida. Comp Biochem Physiol C: Toxicol Pharmacol, 2004, 137(2): 115-122

[125] Zhang G, Liu C, Lu J, Zhang S, Zhu L. The role of AI-driven de novo protein design in the exploration of the protein functional universe. Biology, 2025, 14(9): 1268

[126] Zhang Y, Zhao S, Ma Y, Pan Y, Yan X. Lumbrokinase-loaded GelMA hydrogels with inflammatory regulatory capacity promote vascularized bone regeneration in critical-sized cranial defects. RSC Adv, 2025, 15(35): 28689-28702

[127] Zhao MM, Li M, Han ZL, Wang M, Du LX. Cloning and expression of lumbrokinase gene in Pichia pastoris. Acta Microbiol Sin, 2006, 46(4): 581-585

[128] Zhao X, Gao S, Cui D, Geng F. In silico cloning of EfP-0, a novel earthworm fibrinolytic enzyme gene, and verification of its coding region by RT-PCR. Chin J Biotechnol, 2006, 22(6): 897-901

[129] Zhao Z, Liu Z, Li X, Wang B. Expression of lumbrokinase protein genes in Escherichia coli. J Chromatogr B, 2025, 1263: 124675

[130] Zhao L, Cao J, Liu X, Li Y, Wu J, Su L. Optimizing protein folding in prokaryotes: strategies to enhance soluble expression of recombinant proteins. Bioresour Technol, 2026, 439: 133266

[131] Zhong WL, Yang JQ, Liu H, Wu YL, Shen HJ, Li PY, Du SY. Antithrombotic effect in zebrafish of a fibrinolytic protein EPF3 from Dilong (Pheretima vulgaris Chen) and its transport mechanism in Caco-2 monolayer through cell bypass pathway. J Integrat Med, 2025, 23(4): 415-428

[132] Zhou Q, Yang J, Zhang W, Jiang W, Chen J, Chen L, Jiang Y, Xin F. Komagataella phaffii: a versatile platform for the production of value-added chemicals. Green Carbon, 2025, 3(4): 407-418

[133] Zhu Z, Deng X, Xie W, Li H, Li Y, Deng Z. Pharmacological effects of bioactive agents in earthworm extract: a comprehensive review. Anim Models Exp Med, 2024, 7(5): 653-672

Funding

China Postdoctoral Science Foundation(2025M772527)

Postdoctoral Fellowship Program of CPSF(GZC20251638)

National Key Research and Development Program of China(2024YFF1106300)

National Natural Science Foundation of China(U24A20368)

Fundamental Research Funds for the Central Universities(JUSRP622003)

Guangdong S&T Program(2024B1111160002)

Jiangsu Basic Research Center for Synthetic Biology(BK20233003)

RIGHTS & PERMISSIONS

Jiangnan University