{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T09:04:41Z","timestamp":1790240681820,"version":"4.1.0"},"reference-count":43,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T00:00:00Z","timestamp":1790208000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100014440","name":"Ministerio de Ciencia, Innovaci\u00f3n y Universidades","doi-asserted-by":"publisher","award":["CNS2023-144585"],"award-info":[{"award-number":["CNS2023-144585"]}],"id":[{"id":"10.13039\/100014440","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100014440","name":"Ministerio de Ciencia, Innovaci\u00f3n y Universidades","doi-asserted-by":"publisher","award":["PID2023-150939OB-I00"],"award-info":[{"award-number":["PID2023-150939OB-I00"]}],"id":[{"id":"10.13039\/100014440","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Bioeng. Biotechnol."],"abstract":"<jats:p>\n                    Bacteriocins are ribosomally synthesized antimicrobial peptides with promising applications in biotechnology, particularly in food preservation and animal and human health. Circular bacteriocins are especially attractive due to their head-to-tail cyclized structure, which confers enhanced stability and antimicrobial potency relative to linear peptides. Here, we report an\n                    <jats:italic>in vitro<\/jats:italic>\n                    cell-free protein synthesis system coupled with an enhanced Split Intein-Mediated Ligation platform (IV-CFPS\/SIML) for the efficient synthesis of circular bacteriocins through systematic evaluation of cyclization sites and alternative split inteins. Using enterocin AS-48 as a model, we systematically evaluated multiple serine-based cyclization sites in combination with three split inteins, NpuDnaE, Gp41-1, and SspGyrB, to identify configurations yielding high antimicrobial activity. Gp41-1 emerged as the most effective intein and was subsequently applied to the production of garvicin ML, amylocyclicin, and 27 naturally occurring sequence variants, demonstrating that cyclization site selection, intein identity, and minor sequence variations strongly influence antimicrobial potency and target range. Finally, SIML expression cassettes encoded in pUC-derived vectors enabled\n                    <jats:italic>in vivo<\/jats:italic>\n                    production and functional expression of selected circular bacteriocins in recombinant\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    . Collectively, these results establish SIML as a versatile platform for\n                    <jats:italic>in vitro<\/jats:italic>\n                    and\n                    <jats:italic>in vivo<\/jats:italic>\n                    production, screening, and functional characterization of known and putative circular bacteriocins.\n                  <\/jats:p>","DOI":"10.3389\/fbioe.2026.1950086","type":"journal-article","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T08:26:22Z","timestamp":1790238382000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["An enhanced split intein-mediated ligation (SIML) platform for rapid discovery and functional screening of circular bacteriocins"],"prefix":"10.3389","volume":"14","author":[{"given":"Ester","family":"Sevillano","sequence":"first","affiliation":[{"name":"Departamento de Nutrici\u00f3n y Ciencia de los Alimentos (NUTRYCIAL), Secci\u00f3n Departamental de Nutrici\u00f3n y Ciencia de los Alimentos (SD-NUTRYCIAL), Facultad de Veterinaria, Universidad Complutense de Madrid (UCM)","place":["Madrid, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohamed","family":"El Bakkoury","sequence":"additional","affiliation":[{"name":"Syngulon S.A","place":["Seraing, Belgium"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Irene","family":"Lafuente","sequence":"additional","affiliation":[{"name":"Departamento de Nutrici\u00f3n y Ciencia de los Alimentos (NUTRYCIAL), Secci\u00f3n Departamental de Nutrici\u00f3n y Ciencia de los Alimentos (SD-NUTRYCIAL), Facultad de Veterinaria, Universidad Complutense de Madrid (UCM)","place":["Madrid, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nuria","family":"Pe\u00f1a","sequence":"additional","affiliation":[{"name":"Departamento de Nutrici\u00f3n y Ciencia de los Alimentos (NUTRYCIAL), Secci\u00f3n Departamental de Nutrici\u00f3n y Ciencia de los Alimentos (SD-NUTRYCIAL), Facultad de Veterinaria, Universidad Complutense de Madrid (UCM)","place":["Madrid, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cleopatra","family":"Collado","sequence":"additional","affiliation":[{"name":"Departamento de Nutrici\u00f3n y Ciencia de los Alimentos (NUTRYCIAL), Secci\u00f3n Departamental de Nutrici\u00f3n y Ciencia de los Alimentos (SD-NUTRYCIAL), Facultad de Veterinaria, Universidad Complutense de Madrid (UCM)","place":["Madrid, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luis M.","family":"Cintas","sequence":"additional","affiliation":[{"name":"Departamento de Nutrici\u00f3n y Ciencia de los Alimentos (NUTRYCIAL), Secci\u00f3n Departamental de Nutrici\u00f3n y Ciencia de los Alimentos (SD-NUTRYCIAL), Facultad de Veterinaria, Universidad Complutense de Madrid (UCM)","place":["Madrid, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Estefan\u00eda","family":"Mu\u00f1oz-Atienza","sequence":"additional","affiliation":[{"name":"Departamento de Nutrici\u00f3n y Ciencia de los Alimentos (NUTRYCIAL), Secci\u00f3n Departamental de Nutrici\u00f3n y Ciencia de los Alimentos (SD-NUTRYCIAL), Facultad de Veterinaria, Universidad Complutense de Madrid (UCM)","place":["Madrid, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philippe","family":"Gabant","sequence":"additional","affiliation":[{"name":"Syngulon S.A","place":["Seraing, Belgium"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pablo E.","family":"Hern\u00e1ndez","sequence":"additional","affiliation":[{"name":"Departamento de Nutrici\u00f3n y Ciencia de los Alimentos (NUTRYCIAL), Secci\u00f3n Departamental de Nutrici\u00f3n y Ciencia de los Alimentos (SD-NUTRYCIAL), Facultad de Veterinaria, Universidad Complutense de Madrid (UCM)","place":["Madrid, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Borrero","sequence":"additional","affiliation":[{"name":"Departamento de Nutrici\u00f3n y Ciencia de los Alimentos (NUTRYCIAL), Secci\u00f3n Departamental de Nutrici\u00f3n y Ciencia de los Alimentos (SD-NUTRYCIAL), Facultad de Veterinaria, Universidad Complutense de Madrid (UCM)","place":["Madrid, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,24]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"403","DOI":"10.1016\/S0022-2836(05)80360-2","article-title":"Basic local alignment search tool","volume":"215","author":"Altschul","year":"1990","journal-title":"J. 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