Characterization of three glycerol transporters with broad sugar specificities from Aspergillus niger

Aspergillus niger is a versatile biotechnological workhorse whose generalist lifestyle enables the utilization of a wide range of sugar compounds, including hexoses, pentoses, and polyols. Yet most predicted A. niger sugar transporters remain uncharacterized. Here, we characterized three glycerol transporters-GlpA, GlpB, and GlpC-from A. niger. We generated single and double glp deletion strains for physiological analysis, among which the ΔglpA strain showed a clear phenotype with significantly reduced glycerol consumption, indicating that GlpA is essential for glycerol uptake. Furthermore, both growth on glycerol and its uptake were abolished in the ΔglpAΔglpB mutant, while the ΔglpAΔglpC mutant showed no growth phenotype on solid medium but lacked glycerol consumption in liquid cultures. This suggests that GlpB plays a supportive role in glycerol uptake, whereas GlpC affects it indirectly. Nevertheless, heterologous expression of the A. niger glp genes in a sugar uptake deficient Saccharomyces cerevisiae strain showed that all three transporters could transport not only glycerol but also D-mannitol, D-sorbitol, and D-xylitol. In addition, GlpC transported the hexose sugars D-fructose, D-glucose, and D-mannose. These results demonstrate the broad specificities of the A. niger Glp transporters, highlighting the need for thorough physiological and functional characterization of filamentous fungal sugar transporters. Our findings advance the understanding of polyol and sugar uptake in A. niger and provide candidate transporters for strain engineering towards biotechnological upcycling of glycerol. KEY POINTS: • GlpA is the main glycerol transporter in A. niger under the studied conditions • ΔglpAΔglpB and ΔglpAΔglpC double deletions abolished glycerol uptake in A. niger • GlpA, GlpB, and GlpC have broad polyol specificity, with GlpC also uptaking hexoses.