Bridging the gap between genes and fungal natural products: Functional heterologous expression of the genes from the lichen Cladonia uncialis

Abstract

Fungi, with an estimated 5 million species, constitutes the second most diverse kingdom on Earth. However, only about 10% of these species have been identified, highlighting the vast, untapped potential for discovering bioactive natural products. This thesis focuses on two main objectives: (1) exploring the chemical diversity of fungal natural products and (2) heterologous expression of biosynthetic gene clusters (BGCs) from the lichenized fungus Cladonia uncialis. The first objective involved the isolation, characterization, and bioactivity profiling of secondary metabolites (SMs) from Aspergillus fumigatus and Penicillium sanguifluum. Two compounds were characterized from the A. fumigatus: N-formyl-4-hydroxyphenyl-acetamide (compound 1) and atraric acid (compound 2), with the latter being the first instance of its production from a non-lichenized fungus. In addition, two compounds were identified from P. sanguifluum: dehydrocurvularin (compound 3) and 11-hydroxycurvularin (compound 4). Despite the identification of over 1,000 SMs, establishing definitive connections between these SMs and their BGCs remains elusive. Our group identified and annotated 48 secondary metabolite BGCs from the fungal partner of C. uncialis. This thesis investigates the heterologous expression of two BGCs from this collection: the orsellinic acid (OA) BGC (cu-nr-pks-5) and the halogenated isocoumarin BGC (cu-pks-4) in A. oryzae NSAR1. The OA gene cluster contains two genes, one encodes for the PKS enzyme and the other for a post-PKS tailoring enzyme, decarboxylase. The heterologous expression of PKSs revealed that A. oryzae can successfully produce de-novo OA from non-lichen fungi (Fusarium spp.), but not from lichen fungus C. uncialis. The expression of the decarboxylase gene demonstrated reversible decarboxylation of OA, marking the first instance of a native lichen gene expression in a heterologous host. Additionally, halogenated isocoumarin BGC was explored. The core PKS (Cu-A) showed similarity to TerA in Aspergillus terreus, suggesting it catalyzes similar condensation reactions to form 2,3-dihydro-6-hydroxymellein. Unique genes within the cluster, Cu-halo and Cu-OMT, are proposed to catalyze halogenation and O-methylation, respectively, to produce halogenated isocoumarin. The heterologous expression of the cluster, both at the individual gene level and as a complete BGC, resulting in the production of various compounds, confirming its functional activity and potential for novel metabolite synthesis.