The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae

l -Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d -glucose. Especially at low concentrations of l -arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains. Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l -arabinose-limited cultures than in d -glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l -arabinose-grown cultures compared to d -glucose-grown cultures, only one (Pc20g01790) restored growth on l -arabinose upon expression in an engineered l -arabinose-fermenting S. cerevisiae strain in which the endogenous l -arabinose transporter, GAL2 , had been deleted. Sugar transport assays indicated that this fungal transporter, designated as Pc AraT, is a high-affinity ( K m = 0.13 mM), high-specificity l -arabinose-proton symporter that does not transport d -xylose or d -glucose. An l -arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l -arabinose-limited chemostat cultures than a congenic strain in which l -arabinose import depended on Gal2 (4.2 × 10 −3 and 1.8 g L −1 , respectively). Inhibition of l -arabinose transport by the most abundant sugars in hydrolysates, d -glucose and d -xylose was far less pronounced than observed with Gal2. Expression of Pc AraT in a hexose-phosphorylation-deficient, l -arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L −1 l -arabinose and 20 g L −1 d -glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l -arabinose transport via Gal2. Its high affinity and specificity for l -arabinose, combined with limited sensitivity to inhibition by d -glucose and d -xylose, make Pc AraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.