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

Abstract

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.

Combinatorial control of gene expression in Aspergillus niger grown on sugar beet pectin

“…Release, uptake and metabolism of another building block of pectin, L-rhamnose, is regulated by RhaR 15 , 22 . Gruben et al .…”

In vivo functional analysis of L-rhamnose metabolic pathway in Aspergillus niger: a tool to identify the potential inducer of RhaR

“…An L-rhamnose transporter (RhtA) has been characterized recently in A. niger [ 6 ]. Interestingly, it has been found that rhtA is co-localized with the α-L-rhamnosidase gene rhaB .…”

“…Interestingly, it has been found that rhtA is co-localized with the α-L-rhamnosidase gene rhaB . The transcriptional profile of rhtA and rhaB genes during a time-course growth experiment of A. niger on L-rhamnose suggests not only that there is a coordinated role in the release and the transport of L-rhamnose during consumption but also that strong activation of these two genes do not require high concentrations of L-rhamnose but only low levels [ 6 ].…”

Organic acid production in Aspergillus niger and other filamentous fungi

“…the standard Blast algorithm) are in many cases not precise enough (Mishra et al, 2014;Sloothaak et al, 2016a). The use of profile hidden Markov models (HMM) to segregate sugar transporter proteins based on their substrate has been shown as an effective approach, however their precision largely depends on the availability of a consistent training set of biochemically characterized proteins with the function of interest (Sloothaak et al, 2015(Sloothaak et al, , 2016a. In this study we aimed, for the first time, to identify a eukaryotic L-rhamnose transporter and consequently, no eukaryotic examples of L-rhamnose transporters were available in the literature.…”

“…In this experiment, the L-rhamnose uptake ability of the EBY.VW4000_RhtA strain was determined, using as a negative control the EBY.VW4000 strain expressing the A. niger specific D-xylose transporter XltB (Sloothaak et al, 2016a (Tate et al, 1992), however, the experimental approach used by Tate et al, which performed overnight cultivations of different E. coli strains (WT and L-rhamnose negative strains) for that purpose, make a comparison difficult. The L-rhamnose uptake rate determined for RhtA was, however, comparable to the maximum sugar uptake rate determined for other fungal MFS transporters, like the D-xylose transporters GXS1 from Candida intermedia (Young et al, 2012) or XylH from Debaryomyces hansenii (Ferreira et al, 2013).…”