Reprogramming a Hgt-family Kluyveromyces marxianus sugar transporter by site-directed mutagenesis to enable co-consumption of glucose and xylose.

Efficient utilization of lignocellulosic hydrolysates in yeast-based biorefineries requires simultaneous consumption of glucose and xylose, which is often limited by preferential glucose uptake. In Kluyveromyces marxianus, we kinetically characterized two native xylose transporters, KMAR_10 531 and KMAR_60 179, identifying medium- and low-affinity xylose transporters, respectively. KMAR_10 531 also mediated high-affinity glucose uptake (Km 0.28 ± 0.1 mM), limiting xylose utilization in mixed-sugar media. Guided by structural modelling, we engineered the KMAR_10 531 N325V variant, which reduced glucose affinity ∼20-fold while improving xylose affinity more than three-fold (Km reduced from 46.9 ± 9.5 to 14.9 ± 3.6 mM). Expression of KMAR_10 531 N325V in a pentose-transporter-deficient K. marxianus strain enabled simultaneous glucose-xylose co-consumption in flasks and bioreactors, overcoming the diauxic growth observed with the native transporter. In bioreactors, the engineered strain consumed ∼90% of available xylose within 45 h and produced increased biomass compared to native transporter. This study provides the first example of engineering an Hgt-like transporter for altered sugar specificity.