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.
- sugar transport
- proton symport
- l-arabinose transporter
- second-generation bioethanol
- metabolic engineering
- YEAST HEXOSE TRANSPORTERS
- ALCOHOLIC FERMENTATION
- GALACTOSE TRANSPORT
MOESM2 of The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae
Bracher, J. M. (Contributor), Verhoeven, M. D. (Contributor), Wisselink, H. W. (Contributor), Crimi, B. (Contributor), Nijland, J. (Contributor), Driessen, A. (Contributor), Klaassen, P. (Contributor), Van Maris, A. J. A. (Contributor), Daran, J. G. (Contributor) & Pronk, J. T. (Contributor), University of Groningen, 13-Mar-2018