28 research outputs found
The uptake of carbon sources by Aspergillus niger
No full textFungi have been used as food and in food fermentations long before written accounts were created and they have been used in folk medicine in ancient cultures. For centuries, species of the genus Aspergillus have been used for the preparation of traditional Asian foodstuffs or together with baker’s yeast in preparation of alcoholic beverages and have therefore been of great economical value. Later, Aspergillus niger has been used for large-scale production of organic acids, enzymes and other food-additives. Today, we aim to harness its saprophytic nature and extraordinary ability to degrade and utilize plant material that is naturally recalcitrant to degradation. To facilitate that ability, the range of sugar transporters employed by this fungus is large even among fungi. This makes it an excellent choice for the identification and characterization of a variety of proteins with different substrate specificities, with potential application in the design of newly engineered cell factories. This thesis was focused on the identification and characterization of previously unknown sugar uptake transporters. Aspergillus niger transporter proteins for the uptake of glucose, xylose, galacturonic acid and rhamnose were identified and characterized.</p
Overexpression of the Aspergillus niger GatA transporter leads to preferential use of D-galacturonic acid over D-xylose
No full textPectin is a structural heteropolysaccharide of the primary cell walls of plants and as such is a significant fraction of agricultural waste residues that is currently insufficiently used. Its main component, D-galacturonic acid, is an attractive substrate for bioconversion. The complete metabolic pathway is present in the genome of Aspergillus niger, that is used in this study. The objective was to identify the D-galacturonic acid transporter in A. niger and to use this transporter to study D-galacturonic acid metabolism. We have functionally characterized the gene An14g04280 that encodes the D-galacturonic acid transporter in A. niger. In a mixed sugar fermentation it was found that the An14g04280 overexpression strain, in contrast to the parent control strain, has a preference for D-galacturonic acid over D-xylose as substrate. Overexpression of this transporter in A. niger resulted in a strong increase of D-galacturonic acid uptake and induction of the D-galacturonic acid reductase activity, suggesting a metabolite controlled regulation of the endogenous D-galacturonic acid catabolic pathway
Identification of a Novel L-rhamnose Uptake Transporter in the Filamentous Fungus Aspergillus niger.
No full textThe 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
Time course transcriptional analysis of <i>rhtA</i> and <i>rhaB</i>.
No full textRelative transcription levels, measured by RT-qPCR, of rhtA (black bars) and rhaB (white bars) during A. niger 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
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>).
No full textA. niger 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.
No full textGrowth of strain EBY.VW4000 expressing the rhtA gene (rhtA+) 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
Classification of <i>A</i>. <i>niger</i> RhtA.
No full textSequences 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 [83]. A neighbour-joining tree was then generated with 1000 bootstrap replicates.</p
MOESM4 of Identification and functional characterization of novel xylose transporters from the cell factories Aspergillus niger and Trichoderma reesei
No full textAdditional file 4. HMMxylT, HMMgluT and HMMMFS scores of the top 15 proteins, from the A. niger and T. reesei predicted proteomes
Relative abundance of MFS transporter proteins detected in different <i>A</i>. <i>niger</i> growth conditions
No full textRelative abundance of MFS transporter proteins detected in different A. niger growth conditions</p
