Production of terpenes in Aspergillus nidulans: Engineering of the secondary metabolite gene cluster: Dissertation

Secondary metabolites, or natural products, are produced by a variety of microorganisms and plants but are particularly abundant in filamentous fungi. Secondary metabolites can be toxic or have pharmaceutical applications as antibiotics, anticancer, antiparasitic and antifungal agents. Terpenes are a large group of secondary metabolites with wide array of bioactivities. Filamentous fungus, Aspergillus nidulans, is a fungal model organism and a close relative to other industrially relevant Aspergillus species. A. nidulans is a known producer of many bioactive metabolites, including polyketides, nonribosomal peptides and sesquiterpenes. Sequencing of A. nidulans genome has revealed numerous secondary metabolite gene clusters, yet products of many of these biosynthetic pathways are unknown since the expression of the clustered genes usually remains silent in laboratory conditions. Finding ways to induce the gene expression of silent biosynthetic clusters can lead to discovery of novel metabolites with unknown bioactivities. Furthermore, identifying key biosynthetic genes in interesting metabolite pathways is a prerequisite for genetic engineering of heterologous production systems. In this study, we wanted to explore the terpene producing ability of A. nidulans. We discovered a novel diterpene, ent-pimara-8(14),15-diene (PD), in A. nidulans by predicting a biosynthetic gene cluster with genomic mining. We identified a Zn(II)₂Cys₆-type transcription factor, PbcR, and demonstrated its role as a positive regulator for the predicted gene cluster. The transcriptional profile of the pbcR overexpression strain (oe:PbcR) was analyzed by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and DNA microarray. Overexpression of pbcR led to upregulation of the predicted biosynthetic cluster genes and subsequent PD production in A. nidulans. In addition to PD cluster genes, transcriptional changes were observed for a number of secondary metabolite genes in oe:PbcR. Microscopy of the plate cultures revealed changes in the oe:PbcR morphology. To study the bioactivity of PD, we developed and optimized a PD extraction method for the engineered A. nidulans production strain. An accelerated solvent extraction (ASE) with a combined High-Performance-Liquid-Chromatography (HPLC) purification enabled us to collect highly pure PD for the Nuclear Magnetic Resonance (NMR) analysis and bioactivity testing. While PD did not exhibit inhibitory function against Staphylococcus aureus or Leishmania donovani, we observed an antioxidant activity for PD in a radical scavenging assay. We also characterized A. nidulans PD synthase (PbcA) with overexpression and deletion assays and showed that it is indispensable for PD production in A. nidulans. Furthermore, we extended our genetic engineering to study the potential of A. nidulans as a heterologous host for mono- and diterpene production. Overexpression of Fusarium fujikuroi ent-kaurene synthase (cps/ks) and Citrus unshiu gamma-terpinene synthase resulted in ent-kaurene and gamma-terpinene production, respectively. The results reported in this thesis highlight the advantage of using genomic mining in the search for novel biosynthetic pathways. Additionally, this thesis presents a proof-of-concept study for using A. nidulans as heterologous terpene production host.