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. 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.