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

Background: l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via theSaccharomyces cerevisiaeGal2 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-metabolizingS. cerevisiaestrains.

Results: Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungusPenicillium chrysogenumwhose 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-fermentingS. cerevisiaestrain in which the endogenous l-arabinose transporter,GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated asPcAraT, is a high-affinity (Km = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizingS. cerevisiaestrain in whichGAL2was replaced byPcaraTshowed 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 × 10-3and 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 ofPcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizingS. cerevisiaestrain enabled growth in media supplemented with both 20 g L-1l-arabinose and 20 g L-1d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2.

Conclusion: Its high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, makePcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineeringS. cerevisiaestrains for efficient conversion of lignocellulosic hydrolysates.