Abstract
Backgroundl-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.ResultsChemostat-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−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.ConclusionIts 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.
Highlights
At an annual production of 100 Mton [1], bioethanol produced by the yeast Saccharomyces cerevisiae is by volume the largest fermentation product in industrial biotechnology
synthetic medium (SM), synthetic medium with urea (SM-urea) and YP media were further supplemented with 20 g L−1 d-glucose or l-arabinose, by adding concentrated solutions autoclaved at 110 °C for 20 min, yielding SMD or SMA, SMD-urea or SMA-urea and YPD or YPA, respectively
To identify candidate structural genes for l-arabinose transporters in P. chrysogenum, carbon-limited chemostat cultures of strain DS17690 were grown at a dilution rate of 0.03 h−1 on different carbon sources
Summary
At an annual production of 100 Mton [1], bioethanol produced by the yeast Saccharomyces cerevisiae is by volume the largest fermentation product in industrial biotechnology. Cane sugar and corn starch, which are still the predominant feedstocks for bioethanol production, almost exclusively yield sucrose and d-glucose as fermentable sugars. Alternative lignocellulosic feedstocks, derived from agricultural residues or energy crops, contain cellulose, hemicellulose, and in some cases, pectin [2]. The pentoses d-xylose and l-arabinose typically represent 10–25 and 2–3%, respectively, of the monomeric sugars in lignocellulosic hydrolysates [3]. In hydrolysates of corn fibre and sugar beet pulp, l-arabinose represents 16 and 26% of the total sugar content, respectively [4, 5]
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