Overexpression of the Aspergillus niger GatA transporter leads to preferential use of D-galacturonic acid over D-xylose

Abstract

Pectin 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.

Keywords: Aspergillus niger; D-galacturonic acid; Pectin; Sustainable resources; Transmembrane transport.

Figure 1

Schematic representation of Funbrick expression vector. Restriction sites flank functional modules. Capital P and T represent promoter and terminator. Bacterial marker: amR. Fungal uridine auxotrophy complementation marker: pyrA.

Figure 2

Concentration of GalA in the medium during growth on sorbitol and GalA. Induction of expression system with D-xylose at T = 0. Overexpression strains of gatA (number of replicates =4) in comparison to the control NW185 PYR A⁺ (number of replicates =2).

Figure 3

Percentage remaining in medium of the three most important substrates from the synthetic cell-wall hydrolysate for gatA overexpression strain (a) (number of replicates =2) and the parent (NW185 PYR A ⁺ strain ) (b) (number of replicates = 2).

Figure 4

Carbon consumed (a) and produced (b), expressed in Cmoles, for control strain (NW185 PYR A ⁺ strain) and transformant JS013 after 72 h of growth on SCH medium. Samples taken from direct cultures, D-xylose in the mixed substrate medium serves as an inducer of the xlnD promoter. Totals represent 0.1 Cmoles.

Figure 5

Substrate remaining in culture medium and pH during growth in fermentors of the control, (NW185 PYR A ⁺ strain) (a) and overexpression strain, JS013 (b). The strains were pre-grown and mycelium was transferred at T = 0 to fermentors containing D-xylose and D-galacturonic acid as substrates.

Figure 6

Galacturonic acid reductase activity (Bars; left Y axis) and CO ₂ in offgas (Diamonds; right Y axis). Samples were taken during growth on 25mM of D-xylose and 25mM of D-galacturonic acid as a carbon source in fermentors.

Figure 7

Neighbor-joining tree of GatA and 17 putative orthologs, constructed with 1000 bootstrap replicates using MEGA6.06 (Tamura et al.[2013]), and a characterized high-affinity glucose transporter protein ( mstA_A.niger) as an outgroup (vanKuyk et al.[2004]). Stars indicate the three currently characterized D-galacturonic acid transporters. Bootstrap values are indicated at the branch-points. Labels indicate Protein Accession numbers followed by species abbreviations. Abbreviations:Aspergillus clavatus; A. clavatus, Aspergillus flavus; A. flavus, Aspergillus fumigatus; A. fumigatus, Aspergillus nidulans; A. nidulans, Aspergillus niger; A. niger, Aspergillus oryzae; A. oryzae, Aspergillus terreus; A. terreus, Botrytis cinerea; B. cinerea, Chaetomium globosum; C. globosum, Fusarium verticilliodes; F. verticilliodes, Neosartorya fischeri; N. fischeri, Neurospora crassa; N. crassa, Penicillium chrysogenum; P. chrysogenum, Phaeosphaeria nodorum; Ph. nodorum, Sclerotinia sclerotiorum; S. sclerotiorum, Trichoderma reesei; T. reesei.

Figure 8

Transmembrane domain prediction and indication of the two interacting domains (1, 3) and the putative substrate binding domain (2) used for comparison of the putative GatA orthologs (Viklund and Elofsson[2004]).