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In the present study, publicly available metagenomic and metatranscriptomic datasets of rumen microbiota from different herbivorous mammals were used to prospect novel XIs sequences. Seven putative XIs from moose, camel, cow, and sheep were cloned into a strain modified for xylose metabolism. Out of those, five XIs demonstrated activity and efficiently converted xylose into xylulose, resulting in ethanol as the final product. A XI from camel rumen microbiota exhibited a K<jats:sub>M<\/jats:sub> of 16.25\u00a0mM, indicating high substrate affinity. The strains expressing enzymes XI11 and XI12, obtained from sheep rumen microbiota, were able to deplete 40\u00a0g\/L of xylose within 72 and 96\u00a0h, achieving theoretical ethanol yields of 90% and 88%, respectively. These results are comparable to those obtained with <jats:italic>Orpinomyces sp<\/jats:italic>. ukk1 XI, a benchmark enzyme previously reported as highly efficient in <jats:italic>S. cerevisiae<\/jats:italic>. This study also provides the first report on the successful expression of XIs mined from the ruminal microbiotas of sheep and camels in <jats:italic>S. cerevisiae<\/jats:italic>, expanding the perspectives for the optimization of fermentation processes and the production of lignocellulosic biofuels from xylose.<\/jats:p>","DOI":"10.1002\/biot.70050","type":"journal-article","created":{"date-parts":[[2025,6,10]],"date-time":"2025-06-10T00:43:31Z","timestamp":1749516211000},"source":"Crossref","is-referenced-by-count":5,"title":["Engineering <i>Saccharomyces Cerevisiae<\/i> With Novel Functional Xylose Isomerases From Rumen Microbiota for Enhanced Biofuel Production"],"prefix":"10.1002","volume":"20","author":[{"given":"Beatriz de Oliveira","family":"Vargas","sequence":"first","affiliation":[{"name":"Departamento de Gen\u00e9tica Evolu\u00e7\u00e3o Microbiologia e Imunologia UNICAMP  Campinas S\u00e3o Paulo Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marcelo 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