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

Author:
Bracher, Jasmine M., Verhoeven, Maarten D., Wisselink, H. Wouter, Crimi, Barbara, Nijland, Jeroen G., Driessen, Arnold J. M., Klaassen, Paul, van Maris, Antonius J. A., Daran, Jean-Marc G., Pronk, Jack T.
Source:
Biotechnology for biofuels 2018 v.11 no.1 pp. 63
ISSN:
1754-6834
Subject:
Penicillium chrysogenum, Saccharomyces cerevisiae, arabinose, feedstocks, fungi, galactose, genes, glucose, growth retardation, hydrolysates, lignocellulose, metabolic engineering, symporters, transcriptomics, xylose
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 (Kₘ = 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.
Agid:
5911339