Abstract

Aspergillus niger is a filamentous ascomycete fungus commonly found in soil and decomposing plant matter. It possesses numerous genes for primary and secondary metabolism and efficiently utilizes plant polysaccharides via a broad set of extracellular enzymes. Recently, sequenced fungal genomes, including A. niger, reveal gene models with amino acid sequences similar to bacterial compounds-degrading genes. Despite advances in genome sequencing, there remains a significant gap in the fundamental understanding of catabolic pathways for plant-derived aromatic compounds in fungi. Many of the genes encoding aromatic compound-degrading enzymes in fungi have not been identified, and in some cases, some of these pathway intermediates remain unknown in filamentous fungi. As part of an ongoing effort to understand the genes involved in the degradation of aromatics in A. niger. I have investigated in vivo the genes encoding enzymes in the pathway of two aromatic compounds, namely gentisate and resveratrol. In the first research chapter, I investigated the role of genes involved in the catabolism of gentisate in A. niger using multiple approaches of sequence homology to functionally characterize gentisate pathway genes in bacteria, as well as transcriptome analysis. This initially identified two gene clusters (NRRL3_05234–NRRL3_05237 and NRRL3_10244–NRRL3_10250) encoding enzymes with potentially similar functions. Further investigation through gene knock-out studies, functional complementation test, and the detection of pathway intermediates using LC-MS identified genes in the first gene cluster (ΔNRRL3_05234–NRRL3_05237) as the genes that catalyze gentisate degradation in A. niger. Of note is the identification of a gene (miaA) with no homology to known bacteria gentisate pathway gene but which was found to be indispensable in the catabolism of gentisate through the conversion of maleylpyruvate to fumarylpyruvate. With these approaches, I was able to confirm and refine the assignment of these genes and their roles in the catabolism of gentisate in A. niger. The second research chapter is the result of the attempt to assign roles to the second gene cluster (NRRL3_10244- 10250). Using similar approaches as stated above, excluding the functional complementation tests and transcriptome analysis, I identified the genes and their role in the resveratrol catabolic pathway in A. niger.