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Transmembrane segments 1, 5, 7 and 8 are required for high-affinity glucose transport by Saccharomyces cerevisiae Hxt2 transporter.

ABSTRACT: Hxt2 is a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae and belongs to the major facilitator superfamily. Hxt1 shares approximately 70% amino acid identity with Hxt2 in its transmembrane segments (TMs) and inter-TM loops, but transports D-glucose with an affinity about one-tenth of that of Hxt2. To determine which TMs of Hxt2 are important for high-affinity glucose transport, we constructed chimaeras of Hxt2 and Hxt1 by randomly replacing each of the 12 TMs of Hxt2 with the corresponding segment of Hxt1, for a total of 4096 different transporters. Among > 20000 yeast transformants screened, 39 different clones were selected by plate assays of high-affinity glucose-transport activity and sequenced. With only two exceptions, the selected chimaeras contained Hxt2 TMs 1, 5, 7 and 8. We then constructed chimaeras corresponding to all 16 possible combinations of Hxt2 TMs 1, 5, 7 and 8. Only one chimaera, namely that containing all four Hxt2 TMs, exhibited transport activity comparable with that of Hxt2. The K (m) and V (max) values for D-glucose transport, and the substrate specificity of this chimaera were almost identical with those of Hxt2. These results indicate that TMs 1, 5, 7 and 8 are necessary for exhibiting high-affinity glucose-transport activity of Hxt2.

SUBMITTER: Kasahara T

PROVIDER: S-EPMC1223383 | biostudies-other | 2003 May

REPOSITORIES: biostudies-other

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Project description: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-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.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.

Project description:L-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Especially at low concentrations of L-arabinose, its uptake via the Gal2 galactose transporter is an important rate-controlling step in the complete conversion of these feedstocks by engineered, pentose-metabolizing Saccharomyces cerevisiae strains. Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least three-fold higher in L-arabinose-limited cultures than in glucose-limited and ethanol-limited cultures. Of five genes that showed an over 30-fold higher transcript level in arabinose-grown cultures, only one (Pc20g01790) restored growth on L-arabinose upon expression in an engineered L-arabinose-fermenting S. cerevisiae strain in which GAL2 had been deleted. Sugar-transport assays indicated that Pc20g01790this transporter, designated as PcAraT, encodes functions as a high-affinity (Km = 0.13 mM) L-arabinose-proton symporter that does not transport xylose or glucose. An L-arabinose-metabolizing S. cerevisiae strain co-expressing Pc20g01790PcAraT and GAL2 showed lower residual substrate concentrations in L-arabinose-limited chemostat cultures (4 mg L-1) than a congenic strain in which L-arabinose import exclusively depended on Gal2 (1.8 g L-1). Inhibition of L-arabinose transport by these sugars was less pronounced than observed with Gal2. A hexose-phosphorylation-deficient, L-arabinose-metabolizing S. cerevisiae strain expressing PcAraT Pc20g0190 grew on 20 g L-1 L-arabinose in the presence of 20 g L-1 glucose, which completely inhibited growth on L-arabinose of a congenic strain dependent on L-arabinose transport via Gal2. Its high affinity and specificity for L-L-arabinose, combined with limited sensitivity to inhibition by glucose and D-D-xylose make PcAraT/ Pc20g01790 a valuable transporter gene for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.

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Transmembrane segments 1, 5, 7 and 8 are required for high-affinity glucose transport by Saccharomyces cerevisiae Hxt2 transporter.

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