Abstract
Fungal natural products include an important and diverse class of molecules with many valuable bioactivities. Among these, fungal terpenoids make up one of the largest groups. The biosynthesis of these compounds often involves key cyclization steps catalyzed by terpene cyclases (TCs). Although canonical TCs remain a major focus in terpenoid enzymology, recent advancements in genomic analysis and biosynthesis research have led to the identification and characterization of a growing number of non-canonical TCs. Pyr4, a representative non-canonical TC, is responsible for the production of pyripyropene A, which is a polyketide–sesquiterpenoid hybrid that potently inhibits acyl-coenzyme A:cholesterol acyl-transferase. Subsequent to the characterization of Pyr4, many Pyr4 homologues have been identified in the biosynthesis of diverse meroterpenoids, including polyketide–sesquiterpenoids, polyketide–diterpenoids, indole sesquiterpenoids, and indole diterpenoids. However, the structural foundations underlying the reactions catalyzed by these non-canonical Pyr4-family TCs remain largely unexplored, hindering the comprehensive characterization and rational engineering of these enzymes. This thesis focuses on genome mining and in-depth characterization of Pyr4-family TCs involved in the biosynthesis of fungal natural products. The research aims to (i) expand the enzymology of Pyr4-family TCs by discovering novel Pyr4 homologues, (ii) elucidate the catalytic mechanisms of Pyr4-family TCs, (iii) enhance the chemical diversity of fungal natural products by utilizing Pyr4-family TCs.Chapter two unveils the discovery of two new Pyr4-family TCs involved in branched meroterpenoid biosynthetic pathways. A prominent subgroup within fungal meroterpenoids originates from the aromatic precursor 3,5-dimethylorsellinic acid (DMOA). Through genome mining, a DMOA-derived meroterpenoid biosynthetic gene cluster (BGCs) was discovered in Aspergillus insuetus CBS 107.25, which encodes a Pyr4-like TC designated as InsA7. Intriguingly, InsA7 folds the substrate in a pre-boat-chair conformation, resulting in the formation of a new meroterpenoid compound with a C3-axial hydroxy group. Additionally, the A. insuetus strain possesses an extra BGC encoding another Pyr4-like TC, named InsB2, which cyclizes the same substrate as InsA7 with a distinct cyclization mode, leading to the branching of the biosynthetic pathway within the fungus. Further characterization of the tailoring enzymes encoded by the two BGCs resulted in the generation of 17 previously unidentified meroterpenoid compounds, thereby expanding the chemical landscape of fungal meroterpenoids.
Chapter three focuses on the functional analysis of Pyr4 homologues involved in fungal meroterpenoid biosynthesis. Although Pyr4-like enzymes are widely distributed among microorganisms, their three-dimensional structures remain experimentally uninvestigated. To bridge this knowledge gap, AlphaFold2-generated protein models of AdrI, a Pyr4-family TC for andrastin biosynthesis, and its homologues were utilized to investigate the mechanisms by which these enzymes govern cyclization reactions. Through a series of site-directed mutagenesis, amino acid residues that are essential for triggering the cyclization reactions and are important for product selectivity were identified. Importantly, multiple AdrI variants exhibiting shifted product selectivity were obtained, including one variant that predominantly produced a previously unreported meroterpenoid species. Furthermore, engineered metabolic routes were constructed in the fungus Aspergillus oryzae to enhance the molecular diversity of meroterpenoids, resulting in the production of six unnatural 5-methylorsellinate-derived meroterpenoids. These studies shed important light on the catalytic functions and characteristics of Pyr4-family TCs, which will facilitate the future engineering and optimization of these enzymes.
Chapter four describes the discovery of three new Pyr4-family TCs involved in fungal onoceroid triterpenoids. Although genomics-guided methodologies have significantly advanced the discovery of novel enzymes and natural products, currently available bioinformatic tools face challenges in recognizing certain biosynthetic proteins, particularly those lacking a detectable protein domain, as represented by Pyr4-family TCs. Additionally, these tools are unable to selectively extract BGCs with desired or specific characteristics. To overcome these challenges, our group has developed a widely applicable fungal genome mining tool, FunBGCeX, which enables selective extractions of BGCs encoding such domainless enzymes and those not recognized by existing bioinformatic tools. By utilizing FunBGCeX, three new Pyr4-family TCs, AlliB, FumiB, and HomoB, were discovered through a global genome mining approach. These enzymes are all involved in the biosynthesis of onoceroid triterpenoids, which had never been isolated from fungi. This study demonstrates the utility of global genome mining in discovering new forms of biosynthetic reactions.
Collectively, the genome mining and characterization of Pyr4-family TCs involved in fungal natural product biosynthesis have led to the identification of five new Pyr4-family TCs and enabled the successful generation of a diverse array of both natural and unnatural (mero)terpenoid molecules. Furthermore, the research provided the first comprehensive overview of the catalytic mechanisms of Pyr4-family TCs. Additionally, Pyr4-family TCs were first proven to participate in the biosynthesis of pure terpenoids, yielding the first example of fungal onoceroid triterpenoids. These findings significantly advanced our understanding of Pyr4-family TCs, opening new avenues for discovering and engineering these enzymes for the production of valuable molecules.
| Date of Award | 20 Dec 2024 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Yudai MATSUDA (Supervisor) |