Abstract

Secondary metabolites (SMs) from fungi have become an integral part of various scientific and medical fields as well as providing economic benefits. The genes involved in the biosynthesis of fungal and bacterial SMs are often co-localized in closely linked clusters that are generally dormant under laboratory conditions. Genomics technologies are being intensively used to identify SM gene clusters, and suggest methods to promote their expression. These efforts have already been used to discover new natural products, reveal the biosynthetic underpinnings of known products, and establish methods to eliminate toxic substances from industrially relevant organisms. An important aspect of SM gene cluster characterization is the development of methods to overproduce known compounds which are difficult to synthesize in the lab. This thesis presents the annotation of SM gene clusters in the sequenced genome of Aspergillus niger NRRL3. Using our cluster data as a guide to induce production of secondary metabolites through the overproduction of clustered transcription factors, we confirm the identity of the regulators that are involved in the biosynthesis of the fumonisins, TAN-1612/BMS-192548, and the azanigerones which were also used as positive controls. Additionally, we discovered three previously uncharacterized regulators of compounds whose masses correspond to the malformins, pyrophen, and the alkyl citric acids hexylitaconic acid, 2-carboxymethyl-3-hexylmaleic acid and the tensyuic acids. Selecting the alkyl citric acids for further investigation, we obtained a transcriptomic profile to define the genes of the alkyl citric acid gene cluster in A. niger. Using the identified compounds from mass spectrometry and NMR as well as the functional annotations of our defined cluster, I reconstruct a biosynthetic pathway for the alkyl citrates I identified. I then tested the robustness of my reconstruction by successfully predicting the accumulation of two early precursors, 2-hexylcitric acid (log2 fold change = 2.982 ± 0.15, p-value < 0.01) and its anhydride (log2 fold change = 3.792 ± 0.26, p-value < 0.01), as well as the elimination of all downstream compounds.