RESUMO
The fungus Parastagonospora nodorum causes septoria nodorum blotch on wheat. The role of the fungal Velvet-family transcription factor VeA in P. nodorum development and virulence was investigated here. Deletion of the P. nodorum VeA ortholog, PnVeA, resulted in growth abnormalities including pigmentation, abolished asexual sporulation and highly reduced virulence on wheat. Comparative RNA-Seq and RT-PCR analyses revealed that the deletion of PnVeA also decoupled the expression of major necrotrophic effector genes. In addition, the deletion of PnVeA resulted in an up-regulation of four predicted secondary metabolite (SM) gene clusters. Using liquid-chromatography mass-spectrometry, it was observed that one of the SM gene clusters led to an accumulation of the mycotoxin alternariol. PnVeA is essential for asexual sporulation, full virulence, secondary metabolism and necrotrophic effector regulation.
Assuntos
Ascomicetos , Proteínas Fúngicas , Doenças das Plantas , Metabolismo Secundário , Fatores de Transcrição , Triticum , Ascomicetos/genética , Ascomicetos/metabolismo , Ascomicetos/patogenicidade , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Lactonas , Família Multigênica , Micotoxinas/metabolismo , Micotoxinas/genética , Doenças das Plantas/microbiologia , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triticum/microbiologia , Virulência/genéticaRESUMO
Tertiary and allylic hydroxyl groups readily eliminate water during positive ion mode mass spectrometry and may show similar NMR spectra to their corresponding ethers. In a routine structure elucidation workflow, these factors can cause researchers to incorrectly assign diol moieties as ethers or vice versa, leading to inaccurate chemical structures. After facing this problem during our work on oxygenated sesquiterpenoids from a Fusarium sp. fungal strain, we became aware of this challenging issue. We examined the literature for oxygenated natural products bearing these functional groups, and with the aid of density functional calculations of NMR chemical shifts, we now report the structures of 15 natural products that should be revised. We further establish that derivatizing sub-micromolar amounts of alcohols to their sulfates can be used to distinguish these from their corresponding ethers using liquid chromatography negative ion mode mass spectrometry. Finally, we isolated lignoren/cyclonerodiol from the Fusarium sp. culture extract and supported its revised identity as cyclonerodiol using this sulfation approach. Our results suggest that ether-diol ambiguity could be a prevalent issue affecting the structure elucidation of oxygenated natural products and highlight the importance of using complementary techniques, such as sulfation with LC-(-)-ESI-MS or density functional calculations of NMR chemical shifts.
Assuntos
Produtos Biológicos , Fusarium , Produtos Biológicos/química , Estrutura Molecular , Fusarium/química , Éteres/química , Oxigênio/química , Espectroscopia de Ressonância MagnéticaRESUMO
BACKGROUND: Co-localized sets of genes that encode specialized functions are common across microbial genomes and occur in genomes of larger eukaryotes as well. Important examples include Biosynthetic Gene Clusters (BGCs) that produce specialized metabolites with medicinal, agricultural, and industrial value (e.g. antimicrobials). Comparative analysis of BGCs can aid in the discovery of novel metabolites by highlighting distribution and identifying variants in public genomes. Unfortunately, gene-cluster-level homology detection remains inaccessible, time-consuming and difficult to interpret. RESULTS: The comparative gene cluster analysis toolbox (CAGECAT) is a rapid and user-friendly platform to mitigate difficulties in comparative analysis of whole gene clusters. The software provides homology searches and downstream analyses without the need for command-line or programming expertise. By leveraging remote BLAST databases, which always provide up-to-date results, CAGECAT can yield relevant matches that aid in the comparison, taxonomic distribution, or evolution of an unknown query. The service is extensible and interoperable and implements the cblaster and clinker pipelines to perform homology search, filtering, gene neighbourhood estimation, and dynamic visualisation of resulting variant BGCs. With the visualisation module, publication-quality figures can be customized directly from a web-browser, which greatly accelerates their interpretation via informative overlays to identify conserved genes in a BGC query. CONCLUSION: Overall, CAGECAT is an extensible software that can be interfaced via a standard web-browser for whole region homology searches and comparison on continually updated genomes from NCBI. The public web server and installable docker image are open source and freely available without registration at: https://cagecat.bioinformatics.nl .
Assuntos
Computadores , Software , Família Multigênica , Genoma , Análise por ConglomeradosRESUMO
Covering: up to May 2022Fungal genetics has transformed natural product research by enabling the elucidation of cryptic metabolites and biosynthetic steps. The enhanced capability to add, subtract, modulate, and rewrite genes via CRISPR/Cas technologies has opened up avenues for the manipulation of biosynthetic gene clusters across diverse filamentous fungi. This review discusses the innovative and diverse strategies for fungal natural product discovery and engineering made possible by CRISPR/Cas-based tools. We also provide a guide into multiple angles of CRISPR/Cas experiment design, and discuss current gaps in genetic tool development for filamentous fungi and the promising opportunities for natural product research.
Assuntos
Produtos Biológicos , Edição de Genes , Sistemas CRISPR-Cas/genética , Produtos Biológicos/metabolismo , Fungos/genética , Fungos/metabolismoRESUMO
Covering: up to the end of July, 2022Fungi are prolific producers of piperazine alkaloids, which have been shown to exhibit an array of remarkable biological activities. Since the first fungal piperazine, herquline A, was reported from Penicillium herquei Fg-372 in 1979, a plethora of structurally diverse piperazines have been isolated and characterised from various fungal strains. Significant advancements have been made in recent years towards unravelling the biosynthesis of fungal piperazines and numerous synthetic routes have been proposed. This review provides a comprehensive summary of the current knowledge of the discovery, classification, bioactivity and biosynthesis of piperazine alkaloids reported from fungi, and discusses the perspectives for exploring the structural diversity of fungal piperazines via genome mining of the untapped piperazine biosynthetic pathways.
Assuntos
Alcaloides , Piperazinas , Piperazina/metabolismo , Alcaloides/química , Fungos/metabolismoRESUMO
Fourteen-membered macrolides are a class of compounds with significant clinical value as antibacterial agents. As part of our ongoing investigation into the metabolites of Streptomyces sp. MST-91080, we report the discovery of resorculins A and B, unprecedented 3,5-dihydroxybenzoic acid (α-resorcylic acid)-containing 14-membered macrolides. We sequenced the genome of MST-91080 and identified the putative resorculin biosynthetic gene cluster (rsn BGC). The rsn BGC is hybrid of type I and type III polyketide synthases. Bioinformatic analysis revealed that the resorculins are relatives of known hybrid polyketides: kendomycin and venemycin. Resorculin A exhibited antibacterial activity against Bacillus subtilis (MIC 19.8 µg mL-1), while resorculin B showed cytotoxic activity against the NS-1 mouse myeloma cell line (IC50 3.6 µg mL-1).
Assuntos
Mieloma Múltiplo , Policetídeos , Streptomyces , Animais , Camundongos , Policetídeos/farmacologia , Policetídeos/metabolismo , Macrolídeos/farmacologia , Macrolídeos/metabolismo , Linhagem Celular Tumoral , Streptomyces/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Família MultigênicaRESUMO
Penicillium turbatum has previously been reported to produce A26771B, a 16-membered macrocyclic polyketide with activity against Gram-positive bacteria, mycoplasma, and fungi, as well as the structurally related compounds berkeleylactone E and berkeleylactones I-O. In this work, large-scale cultivation of P. turbatum NRRL 5630 on rice yielded seven new berkeleylactone analogues, berkeleylactone E methyl ester, 14-epi-berkeleylactone F, berkeleylactones P-R, 12-epi-berkeleylactone Q, and 13-epi-berkeleylactone R, and six previously reported analogues, A26771B and berkeleylactones E-G and J-K. The structures of the berkeleylactones were elucidated by detailed analysis of spectroscopic data, molecular modeling, and comparison with literature values. Interestingly, six of the berkeleylactone analogues were isolated as pairs of hydroxy epimers, highlighting how Nature can exploit stereodivergence in biosynthetic pathways to increase chemical diversity. The genome of P. turbatum was sequenced, and a putative gene cluster (bekl) responsible for the biosynthesis of the berkeleylactones was identified. The new berkeleylactone analogues exhibited no significant biological activity against a panel of bacteria, fungi, the parasite Giardia duodenalis, or NS-1 murine myeloma cells, suggesting a hitherto undiscovered biological role.
Assuntos
Penicillium , Camundongos , Animais , Estrutura Molecular , Hidroxilação , Penicillium/químicaRESUMO
Turonicin A (1) was isolated from Streptomyces sp. MST-123921, which was recovered from soil collected on the banks of the Turon River in New South Wales, Australia. Turonicin A (1) is an amphoteric linear polyene polyketide featuring independent pentaene and tetraenone chromophores and is structurally related to linearmycins A-C (2-4). The structure of 1 was determined by detailed spectroscopic analysis and comparison to literature data. Bioinformatic analysis of the linearmycin biosynthetic gene cluster also allowed the previously unresolved absolute stereostructures of 2-4 to be elucidated. Turonicin A (1) exhibited very potent activity against the fungi Candida albicans (MIC 0.0031 µg/mL, 2.7 nM) and Saccharomyces cerevisiae (MIC 0.0008 µg/mL, 0.7 nM), moderate activity against the bacteria Bacillus subtilis (MIC 0.097 µg/mL, 85 nM) and Staphylococcus aureus (MIC 0.39 µg/mL, 340 nM), and no cytotoxicity against human fibroblasts, making it an attractive candidate for further development as a potential next-generation antibiotic scaffold.
Assuntos
Policetídeos , Streptomyces , Humanos , Antifúngicos/farmacologia , Policetídeos/farmacologia , Streptomyces/química , Austrália , Antibacterianos/química , Polienos/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
The necrotrophic fungal pathogen Cochliobolus victoriae produces victorin, a host-selective toxin (HST) essential for pathogenicity to certain oat cultivars with resistance against crown rust. Victorin is a mixture of highly modified heterodetic cyclic hexapeptides, previously assumed to be synthesized by a nonribosomal peptide synthetase. Herein, we demonstrate that victorin is a member of the ribosomally synthesized and posttranslationally modified peptide (RiPP) family of natural products. Analysis of a newly generated long-read assembly of the C. victoriae genome revealed three copies of precursor peptide genes (vicA1-3) with variable numbers of "GLKLAF" core peptide repeats corresponding to the victorin peptide backbone. vicA1-3 are located in repeat-rich gene-sparse regions of the genome and are loosely clustered with putative victorin biosynthetic genes, which are supported by the discovery of compact gene clusters harboring corresponding homologs in two distantly related plant-associated Sordariomycete fungi. Deletion of at least one copy of vicA resulted in strongly diminished victorin production. Deletion of a gene encoding a DUF3328 protein (VicYb) abolished the production altogether, supporting its predicted role in oxidative cyclization of the core peptide. In addition, we uncovered a copper amine oxidase (CAO) encoded by vicK, in which its deletion led to the accumulation of new glycine-containing victorin derivatives. The role of VicK in oxidative deamination of the N-terminal glycyl moiety of the hexapeptides to the active glyoxylate forms was confirmed in vitro. This study finally unraveled the genetic and molecular bases for biosynthesis of one of the first discovered HSTs and expanded our understanding of underexplored fungal RiPPs.
Assuntos
Ascomicetos/metabolismo , Proteínas Fúngicas/metabolismo , Micotoxinas/metabolismo , Ascomicetos/genética , Desaminação , Proteínas Fúngicas/genética , Proteínas Fúngicas/toxicidade , Deleção de Genes , Família Multigênica , Micotoxinas/genética , Micotoxinas/toxicidade , Biossíntese de Proteínas , Processamento de Proteína Pós-TraducionalRESUMO
Covering up to June 2021Ribosomally synthesized and post-translationally modified peptides (RiPPs) from fungi are an underexplored class of natural products, despite their propensity for diverse bioactivities and unique structural features. Surveys of fungal genomes for biosynthetic gene clusters encoding RiPPs have been limited in their scope due to our incomplete understanding of fungal RiPP biosynthesis. Through recent discoveries, along with earlier research, a clearer picture has been emerging of the biosynthetic principles that underpin fungal RiPP pathways. In this Highlight, we trace the approaches that have been used for discovering currently known fungal RiPPs and show that all of them can be assigned to one of three distinct families based on hallmarks of their biosynthesis, which are in turn imprinted on their corresponding gene clusters. We hope that our systematic exposition of fungal RiPP structural and gene cluster features will facilitate more comprehensive approaches to genome mining efforts in the future.
Assuntos
Produtos Biológicos , Biologia Computacional , Produtos Biológicos/metabolismo , Fungos/genética , Fungos/metabolismo , Humanos , Peptídeos/química , Processamento de Proteína Pós-Traducional , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Species from the Metarhizium genus are the causal agents of the green muscardine disease of insects. These fungi have been successfully employed for the biological control of pests over decades. Besides the biocontrol applications, recent efforts for genome sequencing of species in this genus have revealed a great diversity of biosynthetic gene clusters potentially associated with secondary metabolite synthesis. Amongst such molecules are the pseurotins, compounds with several activities, as chitin synthase inhibitors, and immunoglobulin E suppressors. Here, we report, for the first time, the isolation of pseurotin A from the culture broth of M. anisopliae, as well as the characterization of the effects of this compound over the model-arthropod Galleria mellonella. Pseurotin A displayed dose-dependent reversible paralysis effects when injected into the larvae hemocoel. However, the posterior challenge of the treated insects with M. anisopliae conidia did not lead to increased mortality, suggesting that pseurotin A treatment did not increase larvae susceptibility to the green muscardine disease. Although apparent insecticidal effects were not observed for pseurotin A, the paralysis effect observed can be important in M. anisopliae infection development.
Assuntos
Metarhizium , Mariposas , Animais , Larva , Metarhizium/genética , PirrolidinonasRESUMO
SUMMARY: Genes involved in biological pathways are often collocalised in gene clusters, the comparison of which can give valuable insights into their function and evolutionary history. However, comparison and visualization of gene cluster similarity is a tedious process, particularly when many clusters are being compared. Here, we present clinker, a Python based tool and clustermap.js, a companion JavaScript visualization library, which used together can automatically generate accurate, interactive, publication-quality gene cluster comparison figures directly from sequence files. AVAILABILITY AND IMPLEMENTATION: Source code and documentation for clinker and clustermap.js is available on GitHub (github.com/gamcil/clinker and github.com/gamcil/clustermap.js, respectively) under the MIT license. clinker can be installed directly from the Python Package Index via pip. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
RESUMO
Metarhizium anisopliae is an important entomopathogenic species and model for arthropod-fungus interaction studies. This fungus harbors a diverse arsenal of unexplored secondary metabolite biosynthetic gene clusters, which are suggested to perform diverse roles during host interaction and soil subsistence as a saprophytic species. Here we explored an unusual carnitine acyltransferase domain-containing highly reducing polyketide synthase found in the genome of M. anisopliae. Employing heterologous expression in Aspergillus nidulans, two new polyketides were obtained, named BAA and BAB, as well as one known polyketide [(2Z,4E,6E)-octa-2,4,6-trienedioic acid]. Intra-hemocoel injection of the most abundant compound (BAA) in the model-arthropod Galleria mellonella larvae did not induce mortality or noticeable alterations, suggesting that this compound may not harbor insecticidal activity. Also, the potential role of such molecules in polymicrobial interactions was evaluated. Determination of minimum inhibitory concentration assays using distinct fungal species revealed that BAA and BAB did not alter Cryptococcus neoformans growth, while BAA exhibited weak antifungal activity against Saccharomyces cerevisiae. Unexpectedly, these compounds increased Candida albicans growth compared to control conditions. Furthermore, BAA can mitigate the fungicidal effects of fluconazole over C. albicans. Although the exact role of these compounds on the M. anisopliae life cycle is elusive, the described results add up to the complexity of secondary metabolites produced by Metarhizium spp. Moreover, up to our knowledge, these are the first polyketides isolated from filamentous fungi that can boost the growth of another fungal species.
Assuntos
Vias Biossintéticas/genética , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Metarhizium/genética , Metarhizium/metabolismo , Policetídeos/metabolismo , Policetídeos/farmacologia , Animais , Antifúngicos , Aspergillus nidulans/genética , Fungos/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica , Genoma Fúngico/genética , Larva/microbiologia , Interações Microbianas/fisiologia , Testes de Sensibilidade Microbiana , Mariposas , Família Multigênica , Policetídeos/química , Policetídeos/isolamento & purificação , Metabolismo Secundário/genéticaRESUMO
Chemical exploration of the recently described Australian fungus, Aspergillus burnettii, uncovered a new metabolite, burnettiene A. Here, we characterise the structure of burnettiene A as a polyene-decalin polyketide. Bioinformatic analysis of the genome of A. burnettii identified a putative biosynthetic gene cluster for burnettiene A (bue), consisting of eight genes and sharing similarity to the fusarielin gene cluster. Introduction of the reassembled bue gene cluster into Aspergillus nidulans for heterologous expression resulted in the production of burnettiene A under native promoters. Omission of bueE encoding a cytochrome P450 led to the production of preburnettiene A, confirming that BueE is responsible for catalysing the regiospecific multi-oxidation of terminal methyl groups to carboxylic acids. Similarly, bueF was shown to encode an ester-forming methyltransferase, with its omission resulting in the production of the tricarboxylic acid, preburnettiene B. Introduction of an additional copy of the transcription factor bueR under the regulation of the gpdA promoter significantly improved the heterologous production of the burnettienes. Burnettiene A displayed strong in vitro cytotoxicity against mouse myeloma NS-1 cells (MIC 0.8 µg mL-1).
Assuntos
PolicetídeosRESUMO
LCMS-guided screening of a library of biosynthetically talented bacteria and fungi identified Streptomyces sp. MST- as a prolific producer of chlorinated metabolites. We isolated and characterised six new and nine reported compounds from MST-, belonging to three discrete classes - the depsipeptide svetamycins, the indolocarbazole borregomycins and the aromatic polyketide anthrabenzoxocinones. Following genome sequencing of MST-, we describe, for the first time, the svetamycin biosynthetic gene cluster (sve), its mosaic structure and its relationship to several distantly related gene clusters. Our analysis of the sve cluster suggested that the reported stereostructures of the svetamycins may be incorrect. This was confirmed by single-crystal X-ray diffraction analysis, allowing us to formally revise the absolute configurations of svetamycins A-G. We also show that the borregomycins and anthrabenzoxocinones are encoded by a single supercluster (bab) implicating superclusters as potential nucleation points for the evolution of biosynthetic gene clusters. These clusters highlight how individual enzymes and functional subclusters can be co-opted during the formation of biosynthetic gene clusters, providing a rare insight into the poorly understood mechanisms underpinning the evolution of chemical diversity.
Assuntos
StreptomycesRESUMO
The hancockiamides are an unusual new family of N-cinnamoylated piperazines from the Australian soil fungus Aspergillus hancockii. Genomic analyses of A. hancockii identified a biosynthetic gene cluster (hkm) of 12 genes, including two single-module nonribosomal peptide synthetase (NRPS) genes. Heterologous expression of the hkm cluster in A. nidulans confirmed its role in the biosynthesis of the hancockiamides. We further demonstrated that a novel cytochrome P450, Hkm5, catalyses the methylenedioxy bridge formation, and that the PAL gene hkm12 is dispensable, but contributes to increased production of the cinnamoylated hancockiamides. In vitro enzymatic assays and substrate feeding studies demonstrated that NRPS Hkm11 activates and transfers trans-cinnamate to the piperazine scaffold and has flexibility to accept bioisosteric thienyl and furyl analogues. This is the first reported cinnamate-activating fungal NRPS. Expression of a truncated cluster lacking the acetyltransferase gene led to seven additional congeners, including an unexpected family of 2,5-dibenzylpiperazines. These pleiotropic effects highlight the plasticity of the pathway and the power of this approach for accessing novel natural product scaffolds.
Assuntos
Aspergillus/metabolismo , Peptídeo Sintases/metabolismo , Piperazinas/química , Piperazinas/metabolismo , Aspergillus/genética , Cinética , Família Multigênica/genéticaRESUMO
1-Benzazepine is a pharmaceutically important scaffold but is rare among natural products. Nanangelenin A (1), containing an unprecedented 3,4-dihydro-1-benzazepine-2,5-dione-N-prenyl-N-acetoxy-anthranilamide scaffold, was isolated from a novel species of Australian fungus, Aspergillus nanangensis. Genomic and retrobiosynthetic analyses identified a putative nonribosomal peptide synthetase (NRPS) gene cluster (nan). The detailed biosynthetic pathway to 1 was established by heterologous pathway reconstitution in A. nidulans, which led to biosynthesis of intermediates nanagelenin B-F (2-5 and 7). We demonstrated that the NRPS NanA incorporates anthranilic acid (Ant) and l-kynurenine (l-Kyn), which is supplied by a dedicated indoleamine-2,3-dioxygenase NanC encoded in the gene cluster. Using heterologous in vivo assays and mutagenesis, we demonstrated that the C-terminal condensation (CT) and thiolation (T3) domains of NanA are responsible for the regioselective cyclization of the tethered Ant-l-Kyn dipeptide to form the unusual benzazepine scaffold in 1. We also showed that NanA-CT catalyzes the regioselective cyclization of a surrogate synthetic substrate, Ant-l-Kyn-N-acetylcysteamine, to give the benzazepine scaffold, while spontaneous cyclization of the dipeptide yielded the alternative kinetically favored benzodiazepine scaffold. The discovery of 1 and the characterization of NanA have expanded the chemical and functional diversities of fungal NRPSs.
Assuntos
Alcaloides/metabolismo , Aspergillus/patogenicidade , Benzazepinas/síntese química , Cinurenina/metabolismo , Família Multigênica/genética , Benzazepinas/química , Catálise , CiclizaçãoRESUMO
Aspergillus burnettii is a new species belonging to the A. alliaceus clade in Aspergillus subgenus Circumdati section Flavi isolated from peanut-growing properties in southern Queensland, Australia. A. burnettii is a fast-growing, floccose fungus with distinctive brown conidia and is a talented producer of biomass-degrading enzymes and secondary metabolites. Chemical profiling of A. burnettii revealed the metabolites ochratoxin A, kotanins, isokotanins, asperlicin E, anominine and paspalinine, which are common to subgenus Circumdati, together with burnettiene A, burnettramic acids, burnettides, and high levels of 14α-hydroxypaspalinine and hirsutide. The genome of A. burnettii was sequenced and an annotated draft genome is presented. A. burnettii is rich in secondary metabolite biosynthetic gene clusters, containing 51 polyketide synthases, 28 non-ribosomal peptide synthetases and 19 genes related to terpene biosynthesis. Functional annotation of digestive enzymes of A. burnettii and A. alliaceus revealed overlapping carbon utilisation profiles, consistent with a close phylogenetic relationship.
Assuntos
Aspergillus/genética , Vias Biossintéticas/genética , Peptídeo Sintases/genética , Filogenia , Aspergillus/classificação , Aspergillus/metabolismo , Classificação , Genômica , Família Multigênica/genética , Policetídeo Sintases/genética , Análise de Sequência de DNARESUMO
The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits diverse biological and pharmacologic activities. Its biosynthetic origin has remained obscure for more than 60 y, however. Here we report the identification of the biosynthetic gene cluster (BGC) of anisomycin in Streptomyces hygrospinosus var. beijingensis by a bioactivity-guided high-throughput screening method. Using a combination of bioinformatic analysis, reverse genetics, chemical analysis, and in vitro biochemical assays, we have identified a core four-gene ensemble responsible for the synthesis of the pyrrolidine system in anisomycin: aniQ, encoding a aminotransferase that catalyzes an initial deamination and a later reamination steps; aniP, encoding a transketolase implicated to bring together an glycolysis intermediate with 4-hydroxyphenylpyruvic acid to form the anisomycin molecular backbone; aniO, encoding a glycosyltransferase that catalyzes a cryptic glycosylation crucial for downstream enzyme processing; and aniN, encoding a bifunctional dehydrogenase that mediates multistep pyrrolidine formation. The results reveal a BGC for pyrrolidine alkaloid biosynthesis that is distinct from known bacterial alkaloid pathways, and provide the signature sequences that will facilitate the discovery of BGCs encoding novel pyrrolidine alkaloids in bacterial genomes. The biosynthetic insights from this study further set the foundation for biosynthetic engineering of pyrrolidine antibiotics.
Assuntos
Anisomicina/biossíntese , Antibacterianos/biossíntese , Vias Biossintéticas , Família Multigênica , Streptomyces/efeitos dos fármacos , Anisomicina/farmacologia , Antibacterianos/farmacologia , Biologia Computacional , Genoma Bacteriano , Ensaios de Triagem em Larga EscalaRESUMO
Paecilomyces variotii produces the antibacterial and cytotoxic ( M)-viriditoxin (1) together with a trace amount of its atropisomer ( P)-viriditoxin 1'. Elucidation of the biosynthesis by heterologous pathway reconstruction in Aspergillus nidulans identified the multicopper oxidase (MCO) VdtB responsible for the regioselective 6,6'-coupling of semiviriditoxin (10), which yielded 1 and 1' at a ratio of 1:2. We further uncovered that VdtD, an α/ß hydrolase-like protein lacking the catalytic serine, directs the axial chirality of the products. Using recombinant VdtB and VdtD as cell-free extracts from A. nidulans, we demonstrated that VdtD acts like a dirigent protein to control the stereoselectivity of the coupling catalyzed by VdtB to yield 1 and 1' at a ratio of 20:1. Furthermore, we uncovered a unique Baeyer-Villiger monooxygenase (BVMO) VdtE that could transform the alkyl methylketone side chain to methyl ester against the migratory aptitude.