Natural products (NPs) with extensive structural diversity are widely applied in various fields, including drug discovery, food, cosmetics, and flavor development. However, the identification of novel NPs has become increasingly challenging with the growing number of discovered compounds. The rapid development of genome sequencing techniques and bioinformatics revealed the existence of abundant potentially novel and untapped NPs in microorganisms according to the analysis of biosynthetic gene clusters (BGCs) found in microbial genomes. The research on NP biosynthesis has led to the elucidation of the biosynthetic pathways, the discovery of novel NPs or new enzymatic mechanisms, and the engineering of NP biosynthesis.

Meroterpenoids are a class of hybrid NPs composed of terpenoid and non-terpenoid portions, showcasing remarkable structural diversity. Fungi serve as abundant sources of meroterpenoids, which exhibit a wide range of biological and pharmacological activities. These compounds have been explored as potential drug leads. For example, mycophenolate mofetil, a derivative of mycophenolic acid, is developed as an immunosuppressant, and ascofuranone is a promising lead for the treatment of African trypanosomiasis. The rich structural and biological diversity of fungal meroterpenoids has attracted significant attention from chemists. This research focused on the biosynthesis and genome mining-driven discovery of fungal meroterpenoids. The objectives encompass three key aspects. First, the research aims to comprehensively understand the biosynthesis of meroterpenoids in fungi and elucidate the intricate biosynthetic pathways. Secondly, the research endeavors to discover novel meroterpenoids and explore new biosynthesis mechanisms. Lastly, it is sought to engineer enzymes involved in the biosynthetic pathways of fungal meroterpenoids to facilitate the production of a diverse array of compounds.

The second chapter describes the elucidation of the biosynthesis of brevione E, a fungal meroterpenoid with unique oxepane and cycloheptenone moieties. The brv cluster was identified in a brevione E producer strain Penicillium bialowiezense CBS 227.28, using the set gene cluster involved in setosusin biosynthesis as a probe. Reconstitution of the brv cluster in a widely utilized and robust heterologous host Aspergillus oryzae NSAR1 successfully resulted in the production of brevione E. Structural determination of brevione E and reanalysis of the reported NMR data indicated the originally reported structure was incorrect. The revised structures for brevione E and related compounds were proposed. The biosynthetic pathway and mechanism of brevione E were elucidated through heterologous expression, in vitro enzymatic reactions, substrate feeding, and isotope-labeling experiments. Interestingly, the methyltransferase-like enzyme BrvO served as a decarboxylase. Further investigation of the earlier pathway of brevione E and setosusin biosynthesis revealed that two α-ketoglutarate-dependent dioxygenases, BrvJ and SetK, accepted the same substrate brevione B but produced distinct compounds for the downstream biosynthesis. The key factors that dictate the product selectivity were determined through mutational studies focusing on the active site amino acid residues in BrvJ.

The third chapter focuses on the genome mining-guided discovery of an uncharacterized gene cluster in Aspergillus funiculosus CBS 116.56. Reconstitution of the cluster in A. oryzae NSAR1 led to the identification of four new meroterpenoids with the polyketide portion derived from 5-methylorsellinic acid. The structures of these compounds, namely funiculolides A–D, were determined through NMR and X-ray crystallographic analyses. Furthermore, in vitro enzymatic reactions with FncG revealed it to be an Fe (Ⅱ) and α-ketoglutarate-dependent enzyme. Large-scale enzymatic reaction enabled the purification and structure determination of funiculolide D, which possessed an unusually rearranged spirocyclopentanone moiety.

The fourth chapter describes the exploration and characterization of unique meroterpenoid biosynthetic gene clusters. The mfm cluster, one of the gene clusters discovered in this study, exhibits some differences from the previously reported meroterpenoid BGCs. Specifically, the cluster lacks a gene encoding a flavin-dependent monooxygenase (FMO), which is typically responsible for the epoxidation reaction prior to cyclization in fungal meroterpenoid biosynthesis. In addition, a dimethylallyl tryptophan synthase (DMATS)-type prenyltransferase (MfmD), which is typically responsible for the transfer of a C₅ prenyl group, and a Pyr4-type terpenoid cyclase are involved in the pathway. These distinctive characteristics suggested the involvement of unusual reaction mechanisms. Heterologous expression of the mfm cluster led to the production of several new compounds with unstable properties. The reduction by NaBH₄ aided in the structure elucidation of these unstable compounds, and further X-ray study of the reduced final product determined the absolute configuration. The final product was characterized as a drimane–phthalide derivative. Although the drimane-like structure is widespread in nature, the biosynthetic mechanism represented a new category of meroterpenoid biosynthesis. Specifically, the pathway does not involve an FMO and adopts the DMATS-type prenyltransferase to introduce a farnesyl moiety. Replacement of the terpenoid cyclase with OcdTC from Colletotrichum orchidophilum IMI 309357 led to the production of a new monocyclic meroterpenoid. Furthermore, the introduction of another DMATS-type prenyltransferase encoded by a gene cluster from Aspergillus pseudotamarii CBS 117625 resulted in the generation of a meroterpenoid with a C₅ prenyl group. Mutational experiments on these two DMATS-type of prenyltransferases identified key amino acid residues responsible for the prenyl donor selectivity.

In summary, this research primarily focused on the biosynthesis and genome mining-driven discovery of fungal meroterpenoids. This study elucidated the biosynthetic pathways, discovered novel compounds and new meroterpenoid biosynthetic mechanisms, and characterized and engineered certain enzymes to modify their catalytic properties. The research contributes to a deeper understanding of fungal meroterpenoid biosynthesis, provides valuable insights into the enzymatic reactions involved therein, and expands the repertoire of meroterpenoids. In future investigations, structural biology studies and computational calculations would be employed to investigate the functions of selected enzymes, such as MfmD and BrvO. Additionally, extensive searches will be conducted to uncover unusual BGCs to identify new meroterpenoids. Furthermore, the biological activities of the obtained meroterpenoids will be assessed to evaluate their potential as drug leads.