A multilayered exploration of Aspergillus niger sugar metabolic enzymes

Filamentous fungi are one of the pillars of sustainable production. This kingdom contains species able to degrade plant biomass, which is characterized by high structural complexity and is therefore difficult to decompose. Aspergillus niger can produce extracellular enzymes that degrade plant polymers into monomers and short oligomers, which are subsequently transported into the cell and metabolized through a complex network of metabolic pathways. The sugar metabolic model of A. niger is based on data obtained from transcriptome, proteome, and metabolome analyses together with growth profiling. These datasets are essential for the construction of a metabolic model but do not provide complete information on the reactions involved. Biochemical analysis provides additional insight into the efficiency and specificity of metabolic enzymes. Sugar metabolism in A. niger is characterized by a high degree of redundancy; therefore, incorporating kinetic data into metabolic models is necessary to obtain a comprehensive overview of fungal carbon metabolism. The aim of this thesis was to improve understanding of the roles of selected A. niger reductases and dehydrogenases by combining biochemical analysis with transcriptome data and phylogenetic analysis. The research focused on the following enzymes: D-xylose reductase XyrB, L-xylulose reductases LxrA and LxrB, L-rhamnose dehydrogenase LraA, and D-sorbitol dehydrogenase SbdA, formerly known as SdhA. Chapter 2 describes the role of the second D-xylose reductase, XyrB, which is involved in D-xylose and L-arabinose conversion through the pentose catabolic pathway. Phylogenetic analysis revealed that XyrB evolved distantly from the primary D-xylose reductase XyrA. XyrB is an NADP(H)-dependent reductase with exceptionally high affinity for D-xylose and L-arabinose. Although XyrB displays activity toward a wide range of substrates, expression analysis showed that xyrB is not expressed in the presence of several sugars it can convert in vitro. High expression on D-xylose and L-arabinose and regulation by XlnR and AraR indicate a primary role in pentose conversion. Genetic analysis showed that XyrA, XyrB, and LarA all contribute to pentose metabolism at different levels, providing metabolic flexibility. Chapter 3 describes the L-xylulose reductases LxrA and LxrB, which catalyze the conversion of L-xylulose to xylitol. LxrB exhibits broad substrate specificity and activity in multiple pathways, while LxrA is more specific and has higher affinity for L-xylulose. Expression analysis supported LxrA as the primary L-xylulose reductase in the pentose catabolic pathway. Chapter 4 describes LraA, a highly specific L-rhamnose dehydrogenase catalyzing the first step of the L-rhamnose catabolic pathway. LraA shows near exclusive specificity for L-rhamnose, matching its highly specific expression profile. This lack of redundancy likely reflects the low preference of A. niger for L-rhamnose. Chapter 5 demonstrates that SbdA is a general zinc-binding polyol dehydrogenase with low to medium affinity for D-sorbitol and xylitol. Combined biochemical, expression, and genetic data indicate that SbdA likely contributes to multiple pathways and may play a role in stress-related metabolism.