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In Kluyveromyces marxianus, we kinetically characterized two native xylose transporters, KMAR_10\u2009531 and KMAR_60\u2009179, identifying medium- and low-affinity xylose transporters, respectively. KMAR_10\u2009531 also mediated high-affinity glucose uptake (Km 0.28 \u00b1 0.1 mM), limiting xylose utilization in mixed-sugar media. Guided by structural modelling, we engineered the KMAR_10\u2009531 N325V variant, which reduced glucose affinity \u223c20-fold while improving xylose affinity more than three-fold (Km reduced from 46.9 \u00b1 9.5 to 14.9 \u00b1 3.6 mM). Expression of KMAR_10\u2009531 N325V in a pentose-transporter-deficient K. marxianus strain enabled simultaneous glucose\u2013xylose co-consumption in flasks and bioreactors, overcoming the diauxic growth observed with the native transporter. In bioreactors, the engineered strain consumed \u223c90% of available xylose within 45\u00a0h and produced increased biomass compared to native transporter. This study provides the first example of engineering an Hgt-like transporter for altered sugar specificity.<\/jats:p>","DOI":"10.1093\/femsyr\/foag035","type":"journal-article","created":{"date-parts":[[2026,8,8]],"date-time":"2026-08-08T11:52:21Z","timestamp":1786189941000},"source":"Crossref","is-referenced-by-count":1,"title":["Reprogramming a Hgt-family\n                    <i>Kluyveromyces marxianus<\/i>\n                    sugar transporter by site-directed mutagenesis to enable co-consumption of glucose and xylose"],"prefix":"10.1093","volume":"26","author":[{"given":"Lorena","family":"Donzella","sequence":"first","affiliation":[{"name":"School of Microbiology, University College Cork , T12 K8AF Cork ,","place":["Ireland"]},{"name":"CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho , 4710-057 Braga 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