RESUMO
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.
Assuntos
Antifúngicos , Antifúngicos/química , Antifúngicos/farmacologia , Antifúngicos/metabolismo , Aspergillus oryzae/enzimologia , Aspergillus oryzae/metabolismo , Família Multigênica , Triterpenos/química , Triterpenos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismoRESUMO
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.
Assuntos
Fungos , Terpenos , Terpenos/metabolismo , Terpenos/química , Fungos/metabolismo , Fungos/química , Estrutura Molecular , Produtos Biológicos/metabolismo , Produtos Biológicos/química , Sistema Enzimático do Citocromo P-450/metabolismoRESUMO
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.
Assuntos
Polienos , Polienos/química , Polienos/farmacologia , Estrutura Molecular , Talaromyces/química , Antivirais/farmacologia , Antivirais/química , Vírus Sinciciais Respiratórios/efeitos dos fármacos , HumanosRESUMO
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 residueâ 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.
RESUMO
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.
Assuntos
Antifúngicos , Triterpenos , Antifúngicos/farmacologia , Triterpenos/farmacologia , Triterpenos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Triterpenos Pentacíclicos , Vias Biossintéticas/genéticaRESUMO
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.
Assuntos
Benzopiranos , Policetídeos , Compostos Radiofarmacêuticos , TerpenosRESUMO
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.
Assuntos
Hypocreales , Neuroblastoma , Humanos , Benzopiranos/química , Estrutura Molecular , FuranosRESUMO
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.
RESUMO
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 biscognienyneâ B based on heterologous expression, feeding experiments, and in vitro assay. This work shows that the alkyne moiety is constructed by an unprecedented cytochromeâ 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.
Assuntos
Alcinos/metabolismo , Ascomicetos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas Fúngicas/metabolismo , Hemiterpenos/biossíntese , Ascomicetos/enzimologia , Ascomicetos/genética , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Família Multigênica , Oxirredução , Especificidade por SubstratoRESUMO
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.
Assuntos
Antituberculosos/metabolismo , Antituberculosos/farmacologia , Aspergillus oryzae/metabolismo , Mycobacterium tuberculosis/enzimologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacologia , Aspergillus oryzae/enzimologia , Aspergillus oryzae/genética , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Liases/metabolismo , Família Multigênica , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológicoRESUMO
Lycibarbarspermidines are unusual phenolamide glycosides characterized by a dicaffeoylspermidine core with multiple glycosyl substitutions, and serve as a major class of bioactive ingredients in the wolfberry. So far, little is known about the enzymatic basis of the glycosylation of phenolamides including dicaffeoylspermidine. Here, we identify five lycibarbarspermidine glycosyltransferases, LbUGT1-5, which are the first phenolamide-type glycosyltransferases and catalyze regioselective glycosylation of dicaffeoylspermidines to form structurally diverse lycibarbarspermidines in wolfberry. Notably, LbUGT3 acts as a distinctive enzyme that catalyzes a tandem sugar transfer to the ortho-dihydroxy group on the caffeoyl moiety to form the unusual ortho-diglucosylated product, while LbUGT1 accurately discriminates caffeoyl and dihydrocaffeoyl groups to catalyze a site-selective sugar transfer. Crystal structure analysis of the complexes of LbUGT1 and LbUGT3 with UDP, combined with molecular dynamics simulations, revealed the structural basis of the difference in glycosylation selectivity between LbUGT1 and LbUGT3. Site-directed mutagenesis illuminates a conserved tyrosine residue (Y389 in LbUGT1 and Y390 in LbUGT3) in PSPG box that plays a crucial role in regulating the regioselectivity of LbUGT1 and LbUGT3. Our study thus sheds light on the enzymatic underpinnings of the chemical diversity of lycibarbarspermidines in wolfberry, and expands the repertoire of glycosyltransferases in nature.
Assuntos
Glicosiltransferases , Lycium , Glicosiltransferases/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosilação , Lycium/enzimologia , Lycium/metabolismo , Lycium/química , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/química , Glicosídeos/metabolismo , Glicosídeos/química , Cristalografia por Raios X , Piperidinas/metabolismo , Piperidinas/química , Especificidade por SubstratoRESUMO
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.
Assuntos
Técnicas de Cultura de Células/métodos , Glycyrrhiza/citologia , Glycyrrhiza/metabolismo , Linhagem Celular , Escuridão , Flavonoides/biossíntese , Glycyrrhiza/efeitos dos fármacos , Glycyrrhiza/efeitos da radiação , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Cefalosporinas/biossíntese , Sistema Enzimático do Citocromo P-450/metabolismo , Enzimas Multifuncionais/metabolismo , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Aspergillus oryzae/genética , Sequência de Bases , Carbonil Redutase (NADPH)/genética , Carbonil Redutase (NADPH)/metabolismo , Domínio Catalítico , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Ácido Fusídico/análogos & derivados , Ácido Fusídico/química , Regulação da Expressão Gênica , Hidroxilação , Estrutura Molecular , Enzimas Multifuncionais/genética , OxirreduçãoRESUMO
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.
Assuntos
Androstenos/metabolismo , Pregnanos/metabolismo , Xylariales/metabolismo , Androstenos/química , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Pregnanos/química , Xylariales/enzimologia , Xylariales/genéticaRESUMO
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.
Assuntos
Antibacterianos/química , Antibacterianos/metabolismo , Aspergillus oryzae/metabolismo , Ácido Fusídico/análogos & derivados , Antibacterianos/farmacologia , Aspergillus oryzae/química , Aspergillus oryzae/enzimologia , Aspergillus oryzae/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Desmetilação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ácido Fusídico/biossíntese , Ácido Fusídico/química , Ácido Fusídico/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Esteróis/biossíntese , Esteróis/químicaRESUMO
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.