Functional characterization of a xylose transporter in Aspergillus nidulans

Abstract

BackgroundThe production of bioethanol from lignocellulosic feedstocks will only become economically feasible when the majority of cellulosic and hemicellulosic biopolymers can be efficiently converted into bioethanol. The main component of cellulose is glucose, whereas hemicelluloses mainly consist of pentose sugars such as D-xylose and L-arabinose. The genomes of filamentous fungi such as A. nidulans encode a multiplicity of sugar transporters with broad affinities for hexose and pentose sugars. Saccharomyces cerevisiae, which has a long history of use in industrial fermentation processes, is not able to efficiently transport or metabolize pentose sugars (e.g. xylose). Subsequently, the aim of this study was to identify xylose-transporters from A. nidulans, as potential candidates for introduction into S. cerevisiae in order to improve xylose utilization.ResultsIn this study, we identified the A. nidulans xtrD (xylose transporter) gene, which encodes a Major Facilitator Superfamily (MFS) transporter, and which was specifically induced at the transcriptional level by xylose in a XlnR-dependent manner, while being partially repressed by glucose in a CreA-dependent manner. We evaluated the ability of xtrD to functionally complement the S. cerevisiae EBY.VW4000 strain which is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae, XtrD was targeted to the plasma membrane and its expression was able to restore growth on xylose, glucose, galactose, and mannose as single carbon sources, indicating that this transporter accepts multiple sugars as a substrate. XtrD has a high affinity for xylose, and may be a high affinity xylose transporter. We were able to select a S. cerevisiae mutant strain that had increased xylose transport when expressing the xtrD gene.ConclusionsThis study characterized the regulation and substrate specificity of an A. nidulans transporter that represents a good candidate for further directed mutagenesis. Investigation into the area of sugar transport in fungi presents a crucial step for improving the S. cerevisiae xylose metabolism. Moreover, we have demonstrated that the introduction of adaptive mutations beyond the introduced xylose utilization genes is able to improve S. cerevisiae xylose metabolism.

Transcriptional Regulation and Responses in Filamentous Fungi Exposed to Lignocellulose

“…In A. niger, the gene encoding the high affinity sugar transporter MstA is regulated by CreA-mediated CCR ensuring that it is expressed under conditions when concentrations of monomers are low [128]. The xtrD gene from A. nidulans described above is also regulated in a CreA-dependent manner [127].…”

“…The CLR-1 transcription factor -a regulator of cellulase genes -was the primary regulator of cdt-1, which encodes the transporter of breakdown products of cellulose (although CLR-1 also has a role in regulating cdt-2) [126]. In A. nidulans, a gene encoding a high affinity xylose transporter, xtrD, was induced by xylose in a XlnR-dependent manner [127]. When fungi are exposed to lignocellulose, high affinity transporters are induced because the concentrations of free sugars are generally low [120].…”

Organic acid production in Aspergillus niger and other filamentous fungi

“…Native industrially relevant xylose transporters that show high affinity and capacity for xylose uptake have been identified from Escherichia coli, Pichia stipitis and Candida intermedia (Leandro et al, 2006;Weierstall et al, 1999). Additionally several transporters from filamentous fungi have been reported to transport xylose, such as An25 and An29-2 from Neurospora crassa, XtrD from A. nidulans, MstA from A. niger and Xlt1 and Str1 from T. reesei (Colabardini et al, 2014;Du et al, 2010;Huang et al, 2015;Saloheimo et al, 2007). Of this list only An25, and MstA have been biochemically characterized.…”

“…Poor growth of xylose utilizing yeast transformant strains has been described before (Colabardini et al, 2014;Huang et al, 2015;Saloheimo et al, 2007), and this could be due to metabolic imbalances as a result of different cofactor specificities of the heterologous genes XYL1 and XYL2 present in the modified S. cerevisiae strain (Hector et al, 2011). To obtain a better growing xylose-utilizing host for routine identification of xylose transporters, the ability of the EBY.XP strain to metabolize xylose was improved through laboratory-evolution.…”

“…These yeast mutant strains, unable to grow on glucose, fructose, mannose and galactose as a single carbon source, have also subsequently been used as tools for the functional characterisation of sugar transporters from other fungal species (Colabardini et al, 2014;dos Reis et al, 2013;Du et al, 2010;Leandro et al, 2013;Polidori et al, 2007;Saloheimo et al, 2007;Vankuyk PA et al, 2004;Wahl et al, 2010) In contrast to S. cerevisiae, the only functionally validated sugar transporters in A. niger are the recently identified D-galacturonic acid transporter GatA (Martens-Uzunova and Schaap, 2008;Sloothaak et al, 2014), two fructose transporters (Coelho et al, 2013) and the high-affinity sugar/H + symporter MstA (Vankuyk PA et al, 2004). Furthermore, transcriptional data for the A. niger mstC gene suggests that it encodes a low-affinity glucose transporter (Jørgensen et al, 2007), but no experimental data supporting its role as a functional sugar transporter is publicly available.…”