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.

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.

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.

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.

Spider venom-derived peptide JZTX-14 prevents migration and invasion of breast cancer cells via inhibition of sodium channels.

Nav1.5 channel is crucial for the proliferation and migration of breast cancer cells. In this study, we investigated the anticancer effect of JZTX-14, a natural peptide considered an effective antagonist of Nav1.5. First, we successfully isolated and purified the 31 amino acid peptide JZTX-14 containing three pairs of disulfide bonds from spider venom and synthesised JZTX-14 by solid phase synthesis. We then predicted their physiochemical properties and structures in the peptide database. Further, we investigated the effects of natural and synthetic JZTX-14 on the proliferation and migration of MDA-MB-231 breast cancer cells via modulation of sodium current through the Nav1.5 channel. The results showed that both synthetic and natural JZTX-14 inhibited Nav1.5 currents, indicating the successful synthesis of JZTX-14. However, JZTX-14 did not affect MDA-MB-231 cell proliferation but inhibited its migration. Transcriptome analysis revealed that JZTX-14 downregulated S100A4 and FBXO2 and upregulated SERPINB2 in MDA-MB-231 cells. Western blot analysis demonstrated an increased level of the epithelial marker, E-cadherin, and decreased levels of the mesenchymal markers, N-cadherin and vimentin, and matrix metalloproteinase (MMP2), indicating the possible underlying mechanism of the inhibition of MDA-MB-231 cell migration by JZTX-14. This study provides a new target for inhibiting breast cancer metastasis and identifies a potent natural peptide for treating Triple-negative breast cancer.

Automated rock mass condition assessment during TBM tunnel excavation using deep learning.

Rock mass condition assessment during tunnel excavation is a critical step for the intelligent control of tunnel boring machine (TBM). To address this and achieve automatic detection, a visual assessment system is installed to the TBM and a lager in-situ rock mass image dataset is collected from the water conveyance channel project. The rock mass condition assessment task is transformed into a fine-grain classification task. To fulfill the task, a self-convolution based attention fusion network (SAFN) is designed in this paper. The core of our method is the discovery and fusion of the object attention map within a deep neural network. The network consists of two novel modules, the self-convolution based attention extractor (SAE) module and the self-convolution based attention pooling algorithm (SAP) module. The former is designed to detect the intact rock regions generating the attention map, and the latter is designed to improve the performance of classifier by fusing the attention map that focuses on the intact rock regions. The results of SAFN are evaluated from aspects of interpretability, ablation, accuracy and cross-validation, and it outperforms state-of-the-art models in the rock mass assessment dataset. Furthermore, the dynamic filed test show that our assessment system based on the SAFN model is accurate and efficient for automated classification of rock mass.

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.