Identification , functional expression in Saccharomyces cerevisiae and characterization of a Penicillium chrysogenum high-affinity L-arabinose transporter. Identification , functional expression in Saccharomyces cerevisiae and characterization of a Penicillium chrysogenum high-affinity L-arabinose transporter

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. Overall design: The goal of this study was to explore the P. chrysogenum genome for L-arabinose transporters that can be functionally expressed in S. cerevisiae and support glucose- and D-xylose insensitive, high-affinity transport of L-arabinose. To this end, transcriptomes of L-arabinose-, ethanol- and glucose-limited chemostat cultures of P. chrysogenum were compared, and putative L-arabinose transporter genes were tested for their ability to support L-arabinose transport upon expression in an S. cerevisiae strain engineered for L-arabinose fermentation in which GAL2 had been deleted. A P. chrysogenum transporter identified in this screen, PcAraTPc20g01790, was subjected to more detailed analysis, including kinetic sugar-uptake studies with radiolabelled substrates, in vivo studies on uptake inhibition, and physiological studies with engineered S. cerevisiae strains in L-arabinose-limited chemostat cultures.