Browsing by Subject "Xylose transporters"

Aspergillus niger is a well-studied filamentous ascomycete fungus and one of the most used fungal species in various biotechnological sectors. Recently the research focus on A. niger has shifted towards production of biochemicals and biomaterials from plant biomass residues. However, there are still many aspects of the plant biomass conversion process by A. niger that are not known in detail, including its sugar uptake systems. Sugar uptake in fungi relies on sugar transporter (ST) proteins that control the uptake of different sugar molecules. Fungal STs are abundant and diverse in their function, which is why many of them remain uncharacterized. Identification of fungal STs for substrates like xylose can improve the industrial production of lignocellulose-based bioproducts by e.g. improving the substrate uptake rate in fungal cell factories. In this thesis, four putative xylose STs from A. niger, XltD, XltE, XltG, and XltH, were characterized both physiologically and functionally in A. niger and Saccharomyces cerevisiae, respectively, together with two previously functionally characterized xylose transporters, XltA and XltB. In addition, a fifth putative xylose transporter XltF was characterized functionally in S. cerevisiae. For the physiological characterization, A. niger 6Δxlt strain was generated by deleting xltA, xltB, xltD, xltE, xltG, and xltH genes using CRISPR/Cas9 methodology. The physiological analysis of the 6Δxlt strain revealed the presence of additional xylose transporters in the A. niger genome, which still remain to be discovered. The functional characterization of the putative A. niger xylose STs was carried out by creating six recombinant S. cerevisiae IMK1010 strains producing the STs fused with a green fluorescent protein. Functional characterization confirmed the correct localization of the STs within plasma membrane, except for XltG which accumulated inside the yeast cells. This indicated potential localization of XltG within endoplasmic reticulum and a putative role in intracellular sugar transport. Growth assays of the recombinant yeast strains demonstrated variable affinities of the A. niger STs for hexoses. The results showed XltF not to be a xylose ST, but instead a hexose ST with low-affinity for fructose and dual-affinity for glucose. The STs were further tested for their substrate specificity and affinity for xylose in a competitive assay between xylose and glucose. XltE displayed a preference for xylose over glucose identifying it as a new low-affinity xylose transporter. Although further research is needed to elucidate the roles of the studied A. niger STs, XltE is a promising candidate for enhancing xylose uptake in fungal cell factories.