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Upon consumption of contaminated food,\n                      <jats:italic>Listeria<\/jats:italic>\n                      is able to survive a number of gastrointestinal stressors, including competition with the host microbiota. The emergence of antibiotic-resistant clones of\n                      <jats:italic>L. monocytogene<\/jats:italic>\n                      s, together with the side effects of antibiotic treatment, highlights the need for alternatives or additives for its treatment and prevention.\n                      <jats:italic>Saccharomyces boulardii<\/jats:italic>\n                      is a probiotic yeast that is often used alongside antibiotics to minimize side effects since it is not affected by them as a result of its eukaryotic nature. Furthermore, it can be engineered to produce a wide range of molecules. We previously engineered\n                      <jats:italic>Saccharomyces cerevisiae<\/jats:italic>\n                      through CRISPR-Cas9 integration to produce Ply511, a bacteriophage endolysin active against\n                      <jats:italic>L. monocytogene<\/jats:italic>\n                      s, showing the potential of engineered yeast to produce endolysins for biocontrol. In this study, we extended this approach to the probiotic yeast\n                      <jats:italic>S. boulardii<\/jats:italic>\n                      and directly compared the two yeasts as secretion hosts for Ply511. Using a simulated human gastrointestinal environment, we evaluated their ability to retain endolysin activity and reduce\n                      <jats:italic>L. monocytogenes<\/jats:italic>\n                      levels. We then tested the cell extracts from both yeasts in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI), confirming a specificity for\n                      <jats:italic>Listeria<\/jats:italic>\n                      . Finally, we evaluated their activity in a simulated intestinal fermentation using fecal samples from human donors. Overall, this study demonstrates the potential of delivering endolysins to the gut via engineered probiotic\n                      <jats:italic>S. boulardii.<\/jats:italic>\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Key points<\/jats:title>\n                    <jats:p>\n                      <jats:list list-type=\"bullet\">\n                        <jats:list-item>\n                          <jats:p>\n                            <jats:italic>CRISPR-Cas9-engineered S. boulardii and S. cerevisiae were compared,\u00a0both allowing the expression and activity of endolysin Ply511 against L.\u00a0monocytogenes.<\/jats:italic>\n                          <\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>\n                            <jats:italic>Endolysin Ply511 retained its activity against L. monocytogenes in\u00a0simulated gastrointestinal digestion and was specific against Listeria in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI).<\/jats:italic>\n                          <\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>\n                            <jats:italic>Using fecal samples from human donors, the anti-Listeria effect was\u00a0reduced potentially due to the lower metabolic activity of S. boulardii and the\u00a0higher competition with the intestinal microbiome.<\/jats:italic>\n                          <\/jats:p>\n                        <\/jats:list-item>\n                      <\/jats:list>\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Graphical Abstract<\/jats:title>\n                  <\/jats:sec>","DOI":"10.1007\/s00253-026-13749-6","type":"journal-article","created":{"date-parts":[[2026,2,26]],"date-time":"2026-02-26T17:15:38Z","timestamp":1772126138000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Evaluation of the delivery of an anti-Listeria endolysin via CRISPR-Cas9 engineered probiotic Saccharomyces boulardii"],"prefix":"10.1007","volume":"110","author":[{"given":"David S\u00e1ez","family":"Moreno","sequence":"first","affiliation":[]},{"given":"Jo\u00e3o Paulo","family":"Carvalho","sequence":"additional","affiliation":[]},{"given":"Ellen","family":"Murray","sequence":"additional","affiliation":[]},{"given":"Natalia Soledad R\u00edos","family":"Colombo","sequence":"additional","affiliation":[]},{"given":"Alexandre","family":"Lamas","sequence":"additional","affiliation":[]},{"given":"Alejandra Cardelle","family":"Cobas","sequence":"additional","affiliation":[]},{"given":"Colin","family":"Hill","sequence":"additional","affiliation":[]},{"given":"Joana","family":"Azeredo","sequence":"additional","affiliation":[]},{"given":"Luc\u00edlia","family":"Domingues","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2026,2,26]]},"reference":[{"key":"13749_CR1","doi-asserted-by":"publisher","first-page":"853","DOI":"10.1021\/SB500372Z","volume":"4","author":"N Agmon","year":"2015","unstructured":"Agmon N, Mitchell LA, Cai Y, Ikushima S, Chuang J, Zheng A, Choi WJ, Martin JA, Caravelli K, Stracquadanio G, Boeke JD (2015) Yeast Golden Gate (yGG) for the efficient assembly of S. cerevisiae transcription units. 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