Mechanistic characterisation of a fungal fusicoccane-type diterpene synthase involved in the biosynthesis of talaro-7,13-diene.

The cyclisation mechanism of the fungal fusicoccane (FC)-type diterpene synthase (DTS) TadA was investigated by extensive isotopic labelling experiments, and the pH-dependency of the product selectivity of this enzyme was explored. These studies provide new insights into the cyclisation mechanisms of FC-type DTSs.

Identification of three novel P450 enzymes involved in the oxidative modification of a newly discovered fusicoccane diterpene.

Fusicoccane (FC)-type diterpenoids are a class of diterpenoids characterized by a unique 5-8-5 ring system and exhibit diverse biological activities. Recently, we identified a novel FC-type diterpene synthase MgMS, which produces a myrothec-15(17)-en-7-ol (1) hydrocarbon skeleton, however, its tailoring congeners have not been elucidated. Here, we discovered two additional gene clusters Bn and Np, each encoding a highly homologous terpene synthase to MgMS but distinct tailoring enzymes. Heterologous expression of the terpene synthases BnMS and NpMS yielded the same product as MgMS. Subsequent introduction of three P450 enzymes MgP450, BnP450 and NpP450 from individual gene clusters resulted in four new FC-type diterpenoids 2-5. Notably, MgP450 serves as the first enzyme responsible for hydroxylation of the C19 methyl group, whereas NpP450 functions as a multifunctional P450 enzyme involved in the oxidations at C5, C6, and C19 positions of the 5-8-5 tricyclic skeleton. C5 oxidation of the hydrocarbon skeleton 1 led to broadening of the NMR signals and incomplete spectra, which was resolved by high-temperature NMR spectral analysis.

A Functional Switch Between Asperfumene and Fusicoccadiene Synthase and Entrance to Asperfumene Biosynthesis through a Vicinal Deprotonation-Reprotonation Process.

The diterpene synthase AfAS was identified from Aspergillus fumigatiaffinis. Its amino acid sequence and-according to a structural model-active site architecture are highly similar to those of the fusicocca-2,10(14)-diene synthase PaFS, but AfAS produces a structurally much more complex diterpene with a novel 6-5-5-5 tetracyclic skeleton called asperfumene. The cyclisation mechanism of AfAS was elucidated through isotopic labelling experiments and DFT calculations. The reaction cascade proceeds in its initial steps through similar intermediates as for the PaFS cascade, but then diverges through an unusual vicinal deprotonation-reprotonation process that triggers a skeletal rearrangement at the entrance to the steps leading to the unique asperfumene skeleton. The structural model revealed only one major difference between the active sites: The PaFS residue F65 is substituted by I65 in AfAS. Intriguingly, site-directed mutagenesis experiments with both diterpene synthases revealed that position 65 serves as a bidirectional functional switch for the biosynthesis of tetracyclic asperfumene versus structurally less complex diterpenes.

Absolute Configuration of Oxabornyl Polyenes Prugosenes A1-A3 and Structural Revision of Prugosene A2.

Oxabornyl polyenes represent a unique group of polyketides characterized by a central polyene core flanked by a conserved oxabornyl moiety and a structurally diverse oxygen heterocyclic ring. They are widely distributed in fungi and possess a variety of biological activities. Due to the significant spatial separation between the two stereogenic ring systems, it is difficult to establish their overall relative configurations. Here, we isolated three oxabornyl polyenes, prugosenes A1-A3 (1-3), from Talaromyces sp. JNU18266-01. Although these compounds were first reported from Penicillium rugulosum, their overall relative and absolute configurations remained unassigned. By employing ozonolysis in combination with ECD calculations, we were able to establish their absolute configurations, and additionally obtained seven new chemical derivatives (4-10). Notably, through NMR data analysis and quantum chemical calculations, we achieved the structural revision of prugosene A2. Furthermore, prugosenes A1-A3 exhibited potent antiviral activity against the respiratory syncytial virus, with compound 1 displaying an IC50 value of 6.3 µM. Our study thus provides a valuable reference for absolute configuration assignment of oxabornyl polyene compounds.

Biosynthetic characterization of the antifungal fernane-type triterpenoid polytolypin for generation of new analogues via combinatorial biosynthesis.

Fernane-type triterpenoids are a small group of natural products mainly found in plants and fungi with a wide range of biological activities. Polytolypin is a representative fernane-type triterpenoid from fungi and possesses potent antifungal activity. So far, biosynthesis of fungal-derived fernane-type triterpenoids has not been characterized, which hinders the expansion of their structural diversity using biosynthetic approaches. Herein, we identified the biosynthetic gene cluster of polytolypin and elucidated its biosynthetic pathway through heterologous expression in Aspergillus oryzae NSAR1, which involves a new triterpene cyclase for the biosynthesis of the hydrocarbon skeleton motiol, followed by multiple oxidations via three P450 enzymes. Moreover, two new triterpene cyclases for the biosynthesis of two other fernane-type skeletons isomotiol and fernenol were identified from fungi, and were individually co-expressed with the three P450 enzymes involved in polytolypin biosynthesis. These studies led to the generation of 13 fernane-type triterpenoids including eight new compounds, and two of them showed stronger antifungal activity towards Candida albicans FIM709 than polytolypin.

Characterization of a new fusicoccane-type diterpene synthase and an associated P450 enzyme.

Fusicoccane-type terpenoids are a subgroup of diterpenoids featured with a unique 5-8-5 ring system. They are widely distributed in nature and possess a variety of biological activities. Up to date, only five fusicoccane-type diterpene synthases have been identified. Here, we identify a two-gene biosynthetic gene cluster containing a new fusicoccane-type diterpene synthase gene tadA and an associated cytochrome P450 gene tadB from Talaromyces wortmannii ATCC 26942. Heterologous expression reveals that TadA catalyzes the formation of a new fusicoccane-type diterpene talaro-7,13-diene. D2O isotope labeling combined with site-directed mutagenesis indicates that TadA might employ a different C2,6 cyclization strategy from the known fusicoccane-type diterpene synthases, in which a neutral intermediate is firstly formed and then protonated by an environmental proton. In addition, we demonstrate that the associated cytochrome P450 enzyme TadB is able to catalyze multiple oxidation of talaro-7,13-diene to yield talaro-6,13-dien-5,8-dione.

Development of a versatile and conventional technique for gene disruption in filamentous fungi based on CRISPR-Cas9 technology.

Filamentous fungi represent an invaluable source of pharmaceutically active compounds. The development of versatile methods to genetically manipulate filamentous fungi is of great value for improving the low yields of bioactive metabolites and expanding chemical diversity. The CRISPR-Cas9-based system has become a common platform for genome editing in a variety of organisms. However, recent application of this technology in filamentous fungi is limited to model strains, a versatile method for efficient gene disruption in different fungi is lacking. Here, we investigated the utility of the CRISPR-Cas9 system in a less-studied fungus Nodulisporium sp. (No. 65-12-7-1), and we have developed an efficient CRISPR-Cas9-based gene disruption strategy by simultaneous transformation of in vitro transcriptional gRNA and the linear maker gene cassette into the Cas9-expressing fungi. We found that the linear marker gene cassette could not only allow for selection of transformants, but also significantly enhance the gene disruption efficiency by inserting itself into the Cas9 cut site. Moreover, the above approach also demonstrated its efficiency in two other phylogenetically distinct strains Aspergillus oryzae NSAR1 and Sporormiella minima (No. 40-1-4-1) from two different classes of Ascomycota. These results suggested that a versatile CRISPR-Cas9-based gene disruption method in filamentous fungi was established.