| dc.description.abstract | Fungal biotechnology has served as a means for the transition to a bio-based economy. Being decomposers in the ecosystem, filamentous fungi are especially considered as a key in the biological cycle in the concept of a circular economy. To make better use of the natural resources, research related to sugar catabolism in fungi has been expanded. This project follows previous research about the pentose catabolic pathway (PCP) in Aspergillus niger, which is responsible for the conversion of D-xylose and L-arabinose. To date, most of the enzymes involved in this pathway have been identified. However, the understanding of L-xylulose conversion is still incomplete. In this study, we have analyzed the transcriptome data, gene sequencing and the enzyme activity of the putative new L-xylulose reductase(s). Ten genes encoding enzymes belonging to Pfam 00106 of A. niger NRRL3 were chose based on their higher expression values on sugars related to PCP. Although LxrA and LxrB also belong to the PF00106 family, all of them, including ten potential genes, are in separate places on the phylogenetic tree. Among them, NRRL3_09880 showed a similar pattern in the transcriptome data as LxrA, and were closer to LxrB on the phylogenetic tree, which indicates its potential in this complex mechanism of the fungal metabolic pathway. Furthermore, six of the candidate genes, NRRL3_00094, NRRL3_07289, NRRL3_07921, NRRL3_09880, NRRL3_09881, NRRL3_11712, were successfully cloned into pET28 a (+). The recombinant plasmids were then transformed into Escherichia coli expression strains: Arctic Express and BL21 Star to proceed heterologous protein production. Based on the results of the small-scale production, proteins were produced in the soluble form. Moreover, the initial enzyme activity assays showed the possibility of NAD dependence in the reaction of xylitol conversion, instead of NADP, which is required in all known Lxr catalyzed reactions in other filamentous fungi. This finding can respond to the assumption of the existence of NADH-dependent L-xylulose reductases in previous research. However, in big-scale protein production, the amount and concentration of proteins were an obstacle to gaining further characterization of potential enzymes. To improve this problem, multiple adjustments are recommended in further experiments of heterologous expression. For example, a softer induction with lactose as the inducer and a mixed feed system with lactose and glycerol. By understanding much more about the unknown L-xylulose reductases, advanced applications can be made within the cell factory and assist the manufacture of bio-based products. | |
