Abstract
Natural products have been crucial to drug development, with approximately 70% of modern small-molecule drugs derived from them, directly or indirectly. Fungi, the second-largest category of organisms in nature, significantly contribute to the production of natural products. The biosynthesis of these fungal secondary metabolites is governed by biosynthetic gene clusters (BGCs). Advances in genome sequencing have unveiled various unexplored BGCs, underscoring the substantial potential for the development of fungal-derived bioactive natural products. Molecular tools, such as genome editing and heterologous expression, aid in elucidating biosynthetic pathways of natural products of interest and unraveling the molecular bases of critical reactions. Beyond the discovery of novel molecules, studying the biosynthesis of natural products, particularly novel enzymatic synthetic mechanism for molecules with unique structures, offers biocatalysts and inspiration for organic synthesis. This, in turn, fosters the development of drug leads, agrochemicals, and other valuable molecules. After two centuries of effort, there is no good estimation of the inventory of undiscovered natural products, and the biosynthetic mechanisms of numerous known molecules remain mysterious. This project has delved into several natural products that are representative in terms of both chemical structures and biosynthetic mechanisms, aiming to provide valuable clues and insights toward unraveling the vast puzzle of natural products.Xanthones, widely present in fungi, have attracted significant research attention in recent decades due to their diverse structures and a wide range of biological activities. However, an investigation a single xanthone natural product is lacking. To deeply understand the mechanism of tetrahydroxanthone (THX) backbone formation and the enzymes involved therein, the biosynthesis of blennolides A and C was reconstituted through heterologous expression and in vitro enzymatic reaction. The detailed model of the THX biosynthesis encompasses three crucial enzymes: the isomerase NsrQ, the flavin-dependent monooxygenase NsrK, and the short-chain dehydrogenase/reductase NsrO. NsrQ plays a central role in the formation of the THX skeleton, in which the keto group was installed by 1,2- hydride shift, followed by epimerization and heterocyclization to give the final architecture. Based on our results, the biosynthesis of secalonic acid D, a xanthone homodimer with the potential to be developed as a novel antitumor agent, was investigated. AacuH catalyzes the Baeyer–Villiger oxidation, selectively preparing the substrate of the THX monomer for the biosynthesis of secalonic acid D, a process different from its homologous enzyme. Simultaneously, the dimerizing enzyme AacuE, with its broad substrate recognition, facilitates the synthesis of various THX dimers, including the target molecule. The elucidation of the biosynthesis and key enzymes involved in this process can facilitate the biosynthetic research on other THXs and the synthesis of novel THX analogues for future drug development.
Nonribosomal peptides (NRPs), exemplified by penicillin antibiotics, are the main class of natural products synthesized by nonribosomal peptide synthetases (NRPSs), which utilize a broad range of building blocks, including γ-aminobutyric acid (GABA). Several GABA-containing cyclic peptides have demonstrated potential as drug carriers and antitumor agents. Unguisin A is one of the GABA-containing cyclic peptides and was reported to serve as an anion receptor; however, its biosynthesis has never been investigated. Gene deletion experiments and in vitro enzymatic reactions confirmed that UngC acts as an alanine racemase, playing a pivotal role in supplying D-alanine during unguisin biosynthesis. The hydrolase UngD transforms unguisins into previously unreported linear peptides. The reconstitution of the unguisins and linear peptides was successfully completed by heterologous expression of ungA, ungC, and ungD. Consequently, a novel methodology for introducing a sizable gene into an A. oryzae host was established. In addition, two new unguisin congeners with a (2R,3R)-β-methylphenylalanine residue were discovered by genome mining. This work offers insights into the mechanisms through which fungi synthesize a variety of natural peptide products and provides a foundation for the future engineering of NRPS.
Meroterpenoids are hybrid molecules biosynthesized partially through the terpenoid pathway, conferring high levels of structural diversity and a wide range of biological activities. Setosusin is a fungal meroditerpenoid featuring a unique spiro-fused 3(2H)-furanone moiety, recognized as a pharmacophore present in numerous biologically active natural products. Investigating setosusin biosynthesis could unveil a novel mechanism for spirofuranone formation, discover unknown enzymes, and expand the current enzymatic repertoires. In the heterologous reconstitution of setosusin, the cytochrome P450 enzyme SetF was identified as responsible for 3(2H)-furanone formation. Isotope-labeling experiments, computational calculations, and mutational studies were conducted to elucidate the in-depth reaction mechanism of SetF. This novel bifunctional enzyme first oxidizes olefin to epoxide, followed by protonation-initiated skeletal rearrangement to generate the spirofuranone. The comprehensive characterization of the biosynthesis of setosusin presents a rare example in which a P450 enzyme catalyzes a complex rearrangement to yield 3(2H)-furanone, serving as inspiration for both biosynthesis and chemical synthesis, fostering innovative pathways in drug development.
Collectively, key enzymes in the biosynthesis of three types of molecules have been successfully identified and well characterized using multidisciplinary methodologies. The studies described above include the total biosynthesis of known compounds and the discovery and determination of new compounds. This may offer further support for the subsequent exploration and biosynthesis of natural products from fungi.
| Date of Award | 23 Sept 2024 |
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| Original language | English |
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| Supervisor | Yudai MATSUDA (Supervisor) |