Identification of a Novel L-rhamnose Uptake Transporter in the Filamentous Fungus Aspergillus niger

The study of plant biomass utilization by fungi is a research field of great interest due to its many implications in ecology, agriculture and biotechnology. Most of the efforts done to increase the understanding of the use of plant cell walls by fungi have been focused on the degradation of cellulose and hemicellulose, and transport and metabolism of their constituent monosaccharides. Pectin is another important constituent of plant cell walls, but has received less attention. In relation to the uptake of pectic building blocks, fungal transporters for the uptake of galacturonic acid recently have been reported in Aspergillus niger and Neurospora crassa. However, not a single L-rhamnose (6-deoxy-L-mannose) transporter has been identified yet in fungi or in other eukaryotic organisms. L-rhamnose is a deoxy-sugar present in plant cell wall pectic polysaccharides (mainly rhamnogalacturonan I and rhamnogalacturonan II), but is also found in diverse plant secondary metabolites (e.g. anthocyanins, flavonoids and triterpenoids), in the green seaweed sulfated polysaccharide ulvan, and in glycan structures from viruses and bacteria. Here, a comparative plasmalemma proteomic analysis was used to identify candidate L-rhamnose transporters in A. niger. Further analysis was focused on protein ID 1119135 (RhtA) (JGI A. niger ATCC 1015 genome database). RhtA was classified as a Family 7 Fucose: H+ Symporter (FHS) within the Major Facilitator Superfamily. Family 7 currently includes exclusively bacterial transporters able to use different sugars. Strong indications for its role in L-rhamnose transport were obtained by functional complementation of the Saccharomyces cerevisiae EBY.VW.4000 strain in growth studies with a range of potential substrates. Biochemical analysis using L-[3H(G)]-rhamnose confirmed that RhtA is a L-rhamnose transporter. The RhtA gene is located in tandem with a hypothetical alpha-L-rhamnosidase gene (rhaB). Transcriptional analysis of rhtA and rhaB confirmed that both genes have a coordinated expression, being strongly and specifically induced by L-rhamnose, and controlled by RhaR, a transcriptional regulator involved in the release and catabolism of the methyl-pentose. RhtA is the first eukaryotic L-rhamnose transporter identified and functionally validated to date.
Author Summary The growth of filamentous fungi on plant biomass, which occurs through the utilization of its components (e.g. D-glucose, D-xylose, L-arabinose, L-rhamnose) as carbon sources, is a highly regulated event. L-rhamnose (6-deoxy-L-mannose) is a deoxy-sugar present in plant cell wall pectic polysaccharides (mainly rhamnogalacturonan I and rhamnogalacturonan II), but also in diverse plant secondary metabolites, ulvan from green seaweeds and glycan structures from virus and bacteria. The utilization, transformation or detoxification of this monosaccharide by fungi involves a first step of chemical hydrolysis, performed by alpha-L-rhamnosidases, and a second step of transport into the cell, prior to its metabolization. While many rhamnosidases have been identified, not a single eukaryotic plasma membrane L-rhamnose transporter is known to date. In this study we identified and characterized, for the first time, a fungal L-rhamnose transporter (RhtA), from the industrial workhorse Aspergillus niger. We also found that RhtA putative orthologs are conserved throughout different fungal orders, opening the possibility of identifying new transporters of its kind.