Biosynthesis of Enfumafungin-type Antibiotic Reveals an Unusual Enzymatic Fusion Pattern and Unprecedented C-C Bond Cleavage.

Enfumafungin-type antibiotics, represented by enfumafungin and fuscoatroside, belong to a distinct group of triterpenoids derived from fungi. These compounds exhibit significant antifungal properties with ibrexafungerp, a semisynthetic derivative of enfumafungin, recently gaining FDA's approval as the first oral antifungal drug for treating invasive vulvar candidiasis. Enfumafungin-type antibiotics possess a cleaved E-ring with an oxidized carboxyl group and a reduced methyl group at the break site, suggesting unprecedented C-C bond cleavage chemistry involved in their biosynthesis. Here, we show that a 4-gene (fsoA, fsoD, fsoE, fsoF) biosynthetic gene cluster is sufficient to yield fuscoatroside by heterologous expression in Aspergillus oryzae. Notably, FsoA is an unheard-of terpene cyclase-glycosyltransferase fusion enzyme, affording a triterpene glycoside product that relies on enzymatic fusion. FsoE is a P450 enzyme that catalyzes successive oxidation reactions at C19 to facilitate a C-C bond cleavage, producing an oxidized carboxyl group and a reduced methyl group that have never been observed in known P450 enzymes. Our study thus sets the important foundation for the manufacture of enfumafungin-type antibiotics using biosynthetic approaches.

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.

Biosynthesis of fungal terpenoids.

Covering: up to August 2023Terpenoids, which are widely distributed in animals, plants, and microorganisms, are a large group of natural products with diverse structures and various biological activities. They have made great contributions to human health as therapeutic agents, such as the anti-cancer drug paclitaxel and anti-malarial agent artemisinin. Accordingly, the biosynthesis of this important class of natural products has been extensively studied, which generally involves two major steps: hydrocarbon skeleton construction by terpenoid cyclases and skeleton modification by tailoring enzymes. Additionally, fungi (Ascomycota and Basidiomycota) serve as an important source for the discovery of terpenoids. With the rapid development of sequencing technology and bioinformatics approaches, genome mining has emerged as one of the most effective strategies to discover novel terpenoids from fungi. To date, numerous terpenoid cyclases, including typical class I and class II terpenoid cyclases as well as emerging UbiA-type terpenoid cyclases, have been identified, together with a variety of tailoring enzymes, including cytochrome P450 enzymes, flavin-dependent monooxygenases, and acyltransferases. In this review, our aim is to comprehensively present all fungal terpenoid cyclases identified up to August 2023, with a focus on newly discovered terpenoid cyclases, especially the emerging UbiA-type terpenoid cyclases, and their related tailoring enzymes from 2015 to August 2023.

Identification of new bisabosqual-type meroterpenoids reveals non-enzymatic conversion of bisabosquals into seco-bisabosquals.

Bisabosqual-type meroterpenoids are fungi-derived polyketide-terpenoid hybrids bearing a 2,3,3a,3a1,9,9a-hexahydro-1H-benzofuro[4,3,2-cde]chromene skeleton (6/6/6/5 ring system) or its seco-C-ring structure, and exhibit diverse bioactivities. Their unique structural architecture and impressive biological activities have led to considerable interest in discovering new analogues. However, to date, only nine analogues have been identified. Herein, we reported the isolation and identification of six new bisabosqual-type meroterpenoids stachybisbins C-H (1-6), together with one known compound bisabosqual C (7), from Stachybotrys bisbyi PYH05-7. Intriguingly, we found that 7, which contains the intact tetracyclic skeleton, can be non-enzymatically converted into its seco derivative stachybisbin I (8), unveiling the biosynthetic relationship between bisabosquals and seco-bisabosquals. Moreover, based on CRISPR/Cas9-mediated gene disruption, we revealed that the three-gene cluster responsible for the formation of LL-Z1272ß is associated with the biosynthesis of bisabosqual-type meroterpenoids, and then proposed a plausible route to 1-8.

Isolation of new compounds related to xyloketals biosynthesis implies an alternative pathway for furan-fused-chromene formation.

In fungi, there is a rare group of natural products harboring the 2,3,3a,9a-tetrahydro-4H-furo[2,3-b]chromene skeleton, represented by xyloketal B, which display a wide range of biological activities and have drawn significant attention. In this work, four new analogues simpliketals A-D (1-4), as well as two other new compounds simplilactones A and B (5 and 6), were isolated from Simplicillium sp. AHK071-01. Their structures were elucidated by extensive NMR spectroscopic methods, 13C NMR calculation, single-crystal X-ray diffraction, and ECD calculation. In addition, five known compounds (7-11) including alboatrin (7) were also obtained. Based on the structural similarity of the above compounds, we inferred that compounds 5, 6, and 8-11 might be biosynthetically related with 1-4 and 7, which allowed us to propose an alternative biosynthetic route to generate the furan-fused chromene skeleton of this class of compounds, instead of a previously presumed polyketide-terpenoid hybrid pathway. Finally, cytotoxicity assays showed that 1-4 exhibited weak inhibitory activity on PANC-1 cells and that 2 and 3 possessed moderate activity against SH-SY5Y cells.

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.

Adversarial Caching Training: Unsupervised Inductive Network Representation Learning on Large-Scale Graphs.

Network representation learning (NRL) has far-reaching effects on data mining research, showing its importance in many real-world applications. NRL, also known as network embedding, aims at preserving graph structures in a low-dimensional space. These learned representations can be used for subsequent machine learning tasks, such as vertex classification, link prediction, and data visualization. Recently, graph convolutional network (GCN)-based models, e.g., GraphSAGE, have drawn a lot of attention for their success in inductive NRL. When conducting unsupervised learning on large-scale graphs, some of these models employ negative sampling (NS) for optimization, which encourages a target vertex to be close to its neighbors while being far from its negative samples. However, NS draws negative vertices through a random pattern or based on the degrees of vertices. Thus, the generated samples could be either highly relevant or completely unrelated to the target vertex. Moreover, as the training goes, the gradient of NS objective calculated with the inner product of the unrelated negative samples and the target vertex may become zero, which will lead to learning inferior representations. To address these problems, we propose an adversarial training method tailored for unsupervised inductive NRL on large networks. For efficiently keeping track of high-quality negative samples, we design a caching scheme with sampling and updating strategies that has a wide exploration of vertex proximity while considering training costs. Besides, the proposed method is adaptive to various existing GCN-based models without significantly complicating their optimization process. Extensive experiments show that our proposed method can achieve better performance compared with the state-of-the-art models.

Biosynthesis of alkyne-containing natural products.

Alkyne-containing natural products are important molecules that are widely distributed in microbes and plants. Inspired by the advantages of acetylenic products used in the fields of medicinal chemistry, organic synthesis and material science, great efforts have focused on discovering the biosynthetic enzymes and pathways for alkyne formation. Here, we summarize the biosyntheses of alkyne-containing natural products and introduce de novo biosynthetic strategies for alkyne-tagged compound production.

Extensive expansion of the chemical diversity of fusidane-type antibiotics using a stochastic combinational strategy.

Fusidane-type antibiotics, represented by helvolic acid, fusidic acid and cephalosporin P1, are fungi-derived antimicrobials with little cross-resistance to commonly used antibiotics. Generation of new fusidane-type derivatives is therefore of great value, but this is hindered by available approaches. Here, we developed a stochastic combinational strategy by random assembly of all the post-tailoring genes derived from helvolic acid, fusidic acid, and cephalosporin P1 biosynthetic pathways in a strain that produces their common intermediate. Among a total of 27 gene combinations, 24 combinations produce expected products and afford 58 fusidane-type analogues, of which 54 are new compounds. Moreover, random gene combination can induce unexpected activity of some post-tailoring enzymes, leading to a further increase in chemical diversity. These newly generated derivatives provide new insights into the structure‒activity relationship of fusidane-type antibiotics. The stochastic combinational strategy established in this study proves to be a powerful approach for expanding structural diversity of natural products.

Biosynthesis of Biscognienyne†B Involving a Cytochrome P450-Dependent Alkynylation.

The alkyne is a biologically significant moiety found in many natural products and a versatile functional group widely used in modern chemistry. Recent studies have revealed the biosynthesis of acetylenic bonds in fatty acids and amino acids. However, the molecular basis for the alkynyl moiety in acetylenic prenyl chains occurring in a number of meroterpenoids remains obscure. Here, we identify the biosynthetic gene cluster and characterize the biosynthetic pathway of an acetylenic meroterpenoid biscognienyne B based on heterologous expression, feeding experiments, and in vitro assay. This work shows that the alkyne moiety is constructed by an unprecedented cytochrome P450 enzyme BisI, which shows promiscuous activity towards C5 and C15 prenyl chains. This finding provides an opportunity for discovery of new compounds, featuring acetylenic prenyl chains, through genome mining, and it also expands the enzyme inventory for de novo biosynthesis of alkynes.

Shared Multi-View Data Representation for Multi-Domain Event Detection.

Internet platforms provide new ways for people to share experiences, generating massive amounts of data related to various real-world concepts. In this paper, we present an event detection framework to discover real-world events from multiple data domains, including online news media and social media. As multi-domain data possess multiple data views that are heterogeneous, initial dictionaries consisting of labeled data samples are exploited to align the multi-view data. Furthermore, a shared multi-view data representation (SMDR) model is devised, which learns underlying and intrinsic structures shared among the data views by considering the structures underlying the data, data variations, and informativeness of dictionaries. SMDR incorpvarious constraints in the objective function, including shared representation, low-rank, local invariance, reconstruction error, and dictionary independence constraints. Given the data representations achieved by SMDR, class-wise residual models are designed to discover the events underlying the data based on the reconstruction residuals. Extensive experiments conducted on two real-world event detection datasets, i.e., Multi-domain and Multi-modality Event Detection dataset, and MediaEval Social Event Detection 2014 dataset, indicating the effectiveness of the proposed approaches.

Biosynthetic Study of Cephalosporin P1 Reveals a Multifunctional P450 Enzyme and a Site-Selective Acetyltransferase.

Fusidane-type antibiotics are a group of triterpenoid antibiotics. They include helvolic acid, fusidic acid, and cephalosporin P1, among which fusidic acid has been used clinically. We have recently elucidated the biosynthesis of helvolic acid and fusidic acid, which share an early biosynthetic route involving six conserved enzymes. Here, we report two separate gene clusters for cephalosporin P1 biosynthesis. One consists of the six conserved genes, and the other contains three genes encoding a P450 enzyme (CepB4), an acetyltransferase (CepD2), and a short-chain dehydrogenase/reductase (CepC2). Introduction of these three genes into Aspergillus oryzae, which harbors the six conserved genes, produced cephalosporin P1. Stepwise introduction revealed that CepB4 not only catalyzes stereoselective dual oxidation of C6 and C7, but also monooxygenation of C6 or C7. This led to the generation of five new analogues. Using monohydroxylated products as substrates, we demonstrated that CepD2 specifically acetylates C6-OH, although both C6-OH and C7-OH acetylated analogues have been identified in nature.

Biosynthesis of clinically used antibiotic fusidic acid and identification of two short-chain dehydrogenase/reductases with converse stereoselectivity.

Fusidic acid is the only fusidane-type antibiotic that has been clinically used. However, biosynthesis of this important molecule in fungi is poorly understood. We have recently elucidated the biosynthesis of fusidane-type antibiotic helvolic acid, which provides us with clues to identify a possible gene cluster for fusidic acid (fus cluster). This gene cluster consists of eight genes, among which six are conserved in the helvolic acid gene cluster except fusC1 and fusB1. Introduction of the two genes into the Aspergillus oryzae NSAR1 expressing the conserved six genes led to the production of fusidic acid. A stepwise introduction of fusC1 and fusB1 revealed that the two genes worked independently without a strict reaction order. Notably, we identified two short-chain dehydrogenase/reductase genes fusC1 and fusC2 in the fus cluster, which showed converse stereoselectivity in 3-ketoreduction. This is the first report on the biosynthesis and heterologous expression of fusidic acid.

Biosynthesis of an anti-tuberculosis sesterterpenoid asperterpenoid A.

A putative three-gene cluster for asperterpenoid A was identified. Step-wise reconstitution of this gene cluster in Aspergillus oryzae reveals that astC encodes a sesterterpene cyclase to synthesize preasperterpenoid A, which is dually oxidized by a P450 enzyme AstB to give asperterpenoid A along with a minor product asperterpenoid B, and asperterpenoid A is further oxidized by another P450 eznyme AstA to afford a new sesterterpenoid asperterpenoid C. Unexpectedly, asperterpenoids A and B, but not the final product asperterpenoid C, exhibit potent inhibitory activity against Mycobacterium tuberculosis protein tyrosine phosphatase B with IC50 values of 3-6 µM.

Biosynthetic pathway for furanosteroid demethoxyviridin and identification of an unusual pregnane side-chain cleavage.

Furanosteroids, represented by wortmannin, viridin, and demethoxyviridin, are a special group of fungal-derived, highly oxygenated steroids featured by an extra furan ring. They are well-known nanomolar-potency inhibitors of phosphatidylinositol 3-kinase and widely used in biological studies. Despite their importance, the biosyntheses of these molecules are poorly understood. Here, we report the identification of the biosynthetic gene cluster for demethoxyviridin, consisting of 19 genes, and among them 15 biosynthetic genes, including six cytochrome P450 monooxygenase genes, are deleted. As a result, 14 biosynthetic intermediates are isolated, and the biosynthetic pathway for demethoxyviridin is elucidated. Notably, the pregnane side-chain cleavage requires three enzymes: flavin-dependent Baeyer-Villiger monooxygenase, esterase, and dehydrogenase, in sharp contrast to the single cytochrome P450-mediated process in mammalian cells. Structure-activity analyses of these obtained biosynthetic intermediates reveal that the 3-keto group, the C1ß-OH, and the aromatic ring C are important for the inhibition of phosphatidylinositol 3-kinase.

Biosynthesis of helvolic acid and identification of an unusual C-4-demethylation process distinct from sterol biosynthesis.

Fusidane-type antibiotics represented by helvolic acid, fusidic acid and cephalosporin P1 are a class of bacteriostatic agents, which have drawn renewed attention because they have no cross-resistance to commonly used antibiotics. However, their biosynthesis is poorly understood. Here, we perform a stepwise introduction of the nine genes from the proposed gene cluster for helvolic acid into Aspergillus oryzae NSAR1, which enables us to isolate helvolic acid (~20 mg L-1) and its 21 derivatives. Anti-Staphylococcus aureus assay reveals that the antibacterial activity of three intermediates is even stronger than that of helvolic acid. Notably, we observe an unusual C-4 demethylation process mediated by a promiscuous short-chain dehydrogenase/reductase (HelC) and a cytochrome P450 enzyme (HelB1), which is distinct from the common sterol biosynthesis. These studies have set the stage for using biosynthetic approaches to expand chemical diversity of fusidane-type antibiotics.

[Significant impact of different induction conditions on metabolic diversity of callus cell lines of Glycyrrhiza sp].

The purpose of this study was to evaluate the impact of callus induction and culture conditions on secondary metabolic diversity of the callus cell lines of traditional Chinese medicinal plant Glycyrrhiza sp. (Glycyrrhiza) by combined chemical analysis and HPLC fingerprint. These callus induction conditions included two Glycyrrhiza species, two types of explants, light and dark conditions, and two combinations of hormones. The evaluation was firstly based on the contents of total flavonoids in the callus by chemical analysis and one way ANOVA. The content of total flavonoids in callus was significantly (P < 0.05) influenced by Glycyrrhiza species, light condition, and the combination of hormones. The callus was further evaluated using diversity factor based on the comparison of HPLC fingerprints of these callus cell lines. Diversity factor varies significantly for calli induced under different conditions, with the highest being at 0.45 under light condition and combination of hormones. These results provide important knowledge for the selection of suitable callus cell lines for the production of pharmacologically important secondary metabolites or bioactive fractions by in vitro culture of Glycyrrhiza sp.

Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions.

To increase the lipid productivity and thus to reduce the production cost of microalgal biodiesel, effects of cultivation conditions including KNO(3)-level, CO(2) concentration and irradiance on the cell growth, chlorophyll a content and lipid accumulation of Chlorella vulgaris were systematically investigated in a membrane sparged photobioreactor. The biochemical compositions including carbohydrates, proteins and lipids were analyzed simultaneously by the FT-IR spectroscopy. The results showed that the largest biomass productivity and the highest lipid content were obtained at different cultivation conditions. The algae should be harvested at a point that optimized the biomass productivity and lipid content. When the cultivation conditions were controlled at 1.0mM KNO(3), 1.0% CO(2) and 60 micromol photons m(-2)s(-1) at 25 degrees C, the highest lipid productivity obtained was 40 mg L(-1)d(-1), which was about 2.5-fold that had been reported by Illman et al. (2000). The influences of cultivation conditions on the cell growth, lipid accumulation, and other biochemical compositions of cells were further discussed and illustrated by a schematic which was also useful for other microalgal species.