Identification and functional characterization of novel xylose transporters from the cell factories Aspergillus niger and Trichoderma reesei

Background: Global climate change and fossil fuels limitations have boosted the demand for robust and efficient microbial factories for the manufacturing of bio-based products from renewable feedstocks. In this regard, efforts have been done to enhance the enzyme-secreting ability of lignocellulose-degrading fungi, aiming to improve protein yields while taking advantage of their ability to use lignocellulosic feedstocks. Access to sugars in complex polysaccharides depends not only on their release by specific hydrolytic enzymes, but also on the presence of transporters capable of effectively transporting the constituent sugars into the cell. This study aims to identify and characterize xylose transporters from Aspergillus Niger and Trichoderma reesei, two fungi that have been industrially exploited for decades for the production of lignocellulose-degrading hydrolytic enzymes. Results: A hidden Markov model for the identification of xylose transporters was developed and used to analyze the A. Niger and T. reesei in silico proteomes, yielding a list of candidate xylose transporters. From this list, three A. Niger (XltA, XltB and XltC) and three T. reesei (Str1, Str2 and Str3) transporters were selected, functionally validated and biochemically characterized through their expression in a Saccharomyces cerevisiae hexose transport null mutant, engineered to be able to metabolize xylose but unable to transport this sugar. All six transporters were able to support growth of the engineered yeast on xylose but varied in affinities and efficiencies in the uptake of the pentose. Amino acid sequence analysis of the selected transporters showed the presence of specific residues and motifs recently associated to xylose transporters. Transcriptional analysis of A. Niger and T. reesei showed that XltA and Str1 were specifically induced by xylose and dependent on the XlnR/Xyr1 regulators, signifying a biological role for these transporters in xylose utilization. Conclusions: This study revealed the existence of a variety of xylose transporters in the cell factories A. Niger and T. reesei. The particular substrate specificity and biochemical properties displayed by A. Niger XltA and XltB suggested a possible biological role for these transporters in xylose uptake. New insights were also gained into the molecular mechanisms regulating the pentose utilization, at inducer uptake level, in these fungi. Analysis of the A. Niger and T. reesei predicted transportome with the newly developed hidden Markov model showed to be an efficient approach for the identification of new xylose transporting proteins.</p

Identification of a Novel L-rhamnose Uptake Transporter in the Filamentous Fungus <i>Aspergillus niger</i>

<div><p>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 <i>Aspergillus niger</i> and <i>Neurospora crassa</i>. 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 <i>A</i>. <i>niger</i>. Further analysis was focused on protein ID 1119135 (RhtA) (JGI <i>A</i>. <i>niger</i> 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 <i>Saccharomyces cerevisiae</i> 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 (<i>rhaB</i>). Transcriptional analysis of <i>rhtA</i> and <i>rhaB</i> 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.</p></div

Phenotype analysis of <i>A</i>. <i>niger</i> strains N402 (WT), JS14 (Δ<i>rhaR</i>), JS16 (Δ<i>rhtA</i>) and JS19 (Δ<i>rhaB</i>).

<p><i>A</i>. <i>niger</i> strains were plated on minimal media supplemented either with D-glucose (1%; w/v), L-rhamnose (1%; w/v) or rhamnogalacturonan I (RG1) (1%; w/v) as sole carbon source, and cultured for 144 hours. Mutants with the same gene deleted showed the same growth pattern; the figure depicts only one representative knockout strain per gene.</p

RhtA functional analysis in yeast.

<p>Growth of strain EBY.VW4000 expressing the <i>rhtA</i> gene (<i>rhtA</i>⁺) or harbouring the empty expression vector p426HXT7-6His (EV) in minimal medium agar plates containing maltose (M; 29 mM), D-glucose (G; 56 mM), D-fructose (F; 56 mM) or D-mannose (Mn; 56 mM) as sole carbon sources. Agar plates were incubated at 30°C for 96 h. Transformants expressing RhtA showed the same growth pattern; the figure depicts only one representative transformant.</p

Time course transcriptional analysis of <i>rhtA</i> and <i>rhaB</i>.

<p>Relative transcription levels, measured by RT-qPCR, of <i>rhtA</i> (black bars) and <i>rhaB</i> (white bars) during <i>A</i>. <i>niger</i> N400 fermentations in minimal medium with an initial concentration of L-rhamnose 1 mM. Concentration of L-rhamnose over time is represented by grey line with triangles (concentration at t = 4h is equal to 0). Transcript levels of both genes always refer to the reference sample (D-sorbitol 100 mM; t = 1h). Results are given as relative transcript ratios in logarithmic scale (lg(10)). The values provided in the figures correspond to two biological replicates per culture condition. Error bars are means of three technical replicates.</p

Relative abundance of MFS transporter proteins detected in different <i>A</i>. <i>niger</i> growth conditions

<p>Relative abundance of MFS transporter proteins detected in different <i>A</i>. <i>niger</i> growth conditions</p

Role of the CreA and RhaR transcriptional regulators on the expression of <i>rhtA</i> and <i>rhaB</i>.

<p>Strains N402 (WT; black bars), NW283 (<i>ΔcreA;</i> grey bars), and JS014 (<i>ΔrhaR</i>; open bars) were used. Relative transcription levels were measured by RT-qPCR in samples obtained 2 hours after mycelium transfer to 5 mM L-rhamnose or 5 mM L-rhamnose plus 50 mM D-glucose. Relative transcript levels of <i>rhtA</i> and <i>rhaB</i> were calculated using the pre-culture condition of each strain (D-sorbitol 100 mM; t = 18h), sampled prior to the mycelium transfer to inducing and inducing/repressing conditions, as reference (*). Results are given as relative transcript ratios in logarithmic scale (lg(10)). The values provided in the figures correspond to two biological replicates per culture condition. Error bars are means of three technical replicates.</p

Transcriptional analysis of <i>rhtA</i>.

<p>Relative transcription levels were measured by RT-qPCR, in <i>A</i>. <i>niger</i> N400 sampled 2 hours after mycelium transfer to minimal medium with 100 mM D-sorbitol (reference), 5 mM L-rhamnose or 5 mM D-fructose. Transcript levels are relative to reference sample (D-sorbitol 100 mM), indicated with an asterisk. Results are given as relative transcript ratios in logarithmic scale (lg(10)). The values provided in the figures correspond to two biological replicates per culture condition. Error bars are means of three technical replicates.</p

Classification of <i>A</i>. <i>niger</i> RhtA.

<p>Sequences of biochemically characterized sugar transporters were collected and a multiple sequence alignment was created using Praline alignment suite, which takes secondary structure predictions into account [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006468#pgen.1006468.ref083" target="_blank">83</a>]. A neighbour-joining tree was then generated with 1000 bootstrap replicates.</p

Functional characterization of <i>A</i>. <i>niger</i> RhtA sugar transporter.

<p>Uptake at high cell density of radiolabeled substrates by <i>S</i>. <i>cerevisiae</i> EBY.VW4000 expressing the <i>Aspergillus niger</i> L-rhamnose transporter gene <i>rhtA</i> (black bars) or D-xylose transporter gene <i>xltB</i> (open bars). Radiolabeled L-rhamnose, D-xylose or D-fructose were added at a final concentration of 20 μM.</p