Print Email Facebook Twitter The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae Title The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae Author Bracher, J.M. (TU Delft BT/Industriele Microbiologie) Verhoeven, M.D. (TU Delft BT/Industriele Microbiologie) Wisselink, H. Wouter (Isobionics) Crimi, B. (TU Delft BT/Industriele Microbiologie; UMR9002-CNRS-UM) Nijland, Jeroen G. (Rijksuniversiteit Groningen) Driessen, Arnold J.M. (Rijksuniversiteit Groningen) Klaassen, Paul (DSM) van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie; AlbaNova University Center) Daran, J.G. (TU Delft BT/Industriele Microbiologie) Pronk, J.T. (TU Delft BT/Industriele Microbiologie) Date 2018 Abstract Background: 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. Results: 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 PcAraT, 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 PcAraT 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. Conclusion: Its high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates. Subject l-Arabinose transporterMetabolic engineeringPenicilliumProton symportSecond-generation bioethanolSugar transportTranscriptomeYeast To reference this document use: http://resolver.tudelft.nl/uuid:113d8e73-d8f4-4825-92f0-6950a582af0b DOI https://doi.org/10.1186/s13068-018-1047-6 ISSN 1754-6834 Source Biotechnology for Biofuels, 11 (1) Part of collection Institutional Repository Document type journal article Rights © 2018 J.M. Bracher, M.D. Verhoeven, H. Wouter Wisselink, B. Crimi, Jeroen G. Nijland, Arnold J.M. Driessen, Paul Klaassen, A.J.A. van Maris, J.G. Daran, J.T. Pronk Files PDF s13068_018_1047_6.pdf 1.43 MB Close viewer /islandora/object/uuid:113d8e73-d8f4-4825-92f0-6950a582af0b/datastream/OBJ/view