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Nature Communications Sep 2018Strobilurins from fungi are the inspiration for the creation of the β-methoxyacrylate class of agricultural fungicides. However, molecular details of the biosynthesis...
Strobilurins from fungi are the inspiration for the creation of the β-methoxyacrylate class of agricultural fungicides. However, molecular details of the biosynthesis of strobilurins have remained cryptic. Here we report the sequence of genomes of two fungi that produce strobilurins and show that each contains a biosynthetic gene cluster, which encodes a highly reducing polyketide synthase with very unusual C-terminal hydrolase and methyltransferase domains. Expression of stpks1 in Aspergillus oryzae leads to the production of prestrobilurin A when the fermentation is supplemented with a benzoyl coenzyme A (CoA) analogue. This enables the discovery of a previously unobserved route to benzoyl CoA. Reconstruction of the gene cluster in A. oryzae leads to the formation of prestrobilurin A, and addition of the gene str9 encoding an FAD-dependent oxygenase leads to the key oxidative rearrangement responsible for the creation of the β-methoxyacrylate toxophore. Finally, two methyltransferases are required to complete the synthesis.
Topics: Aspergillus oryzae; Basidiomycota; Multigene Family; Polyketide Synthases; Strobilurins
PubMed: 30258052
DOI: 10.1038/s41467-018-06202-4 -
Applied and Environmental Microbiology Dec 2022Angucyclines are a family of structurally diverse, aromatic polyketides with some members that exhibit potent bioactivity. Angucyclines have also attracted considerable...
Angucyclines are a family of structurally diverse, aromatic polyketides with some members that exhibit potent bioactivity. Angucyclines have also attracted considerable attention due to the intriguing biosynthetic origins that underlie their structural complexity and diversity. Balmoralmycin (compound 1) represents a unique group of angucyclines that contain an angular benz[]anthracene tetracyclic system, a characteristic C-glycosidic bond-linked deoxy-sugar (d-olivose), and an unsaturated fatty acid chain. In this study, we identified a strain that produces balmoralmycin and seven biosynthetically related coproducts (compounds 2-8). Four of the coproducts (compounds 5-8) are novel compounds that feature a highly oxygenated or fragmented lactone ring, and three of them (compounds 3-5) exhibited cytotoxicity against the human pancreatic cancer cell line MIA PaCa-2 with IC values ranging from 0.9 to 1.2 μg/mL. Genome sequencing and CRISPR/dCas9-assisted gene knockdown led to the identification of the ~43 kb balmoralmycin biosynthetic gene cluster ( BGC). The BGC encodes a type II polyketide synthase (PKS) system for assembling the angucycline aglycone, six enzymes for generating the deoxysugar d-olivose, and a hybrid type II/III PKS system for synthesizing the 2,4-decadienoic acid chain. Based on the genetic and chemical information, we propose a mechanism for the biosynthesis of balmoralmycin and the shunt products. The chemical and genetic studies yielded insights into the biosynthetic origin of the structural diversity of angucyclines. Angucyclines are structurally diverse aromatic polyketides that have attracted considerable attention due to their potent bioactivity and intriguing biosynthetic origin. Balmoralmycin is a representative of a small family of angucyclines with unique structural features and an unknown biosynthetic origin. We report a newly isolated strain that produces balmoralmycin in a high fermentation titer as well as several structurally related shunt products. Based on the chemical and genetic information, a biosynthetic pathway that involves a type II polyketide synthase (PKS) system, cyclases/aromatases, oxidoreductases, and other ancillary enzymes was established. The elucidation of the balmoralmycin pathway enriches our understanding of how structural diversity is generated in angucyclines and opens the door for the production of balmoralmycin derivatives via pathway engineering.
Topics: Humans; Biosynthetic Pathways; Multigene Family; Polyketide Synthases; Polyketides; Streptomyces; Cell Line, Tumor
PubMed: 36350133
DOI: 10.1128/aem.01208-22 -
Angewandte Chemie (International Ed. in... Dec 2014Following the biosynthesis of polyketide backbones by polyketide synthases (PKSs), post-PKS modifications result in a significantly elevated level of structural...
Following the biosynthesis of polyketide backbones by polyketide synthases (PKSs), post-PKS modifications result in a significantly elevated level of structural complexity that renders the chemical synthesis of these natural products challenging. We report herein a total synthesis of the widely used polyketide insecticide spinosyn A by exploiting the prowess of both chemical and enzymatic methods. As more polyketide biosynthetic pathways are characterized, this chemoenzymatic approach is expected to become readily adaptable to streamlining the synthesis of other complex polyketides with more elaborate post-PKS modifications.
Topics: Biosynthetic Pathways; Insecticides; Macrolides; Polyketide Synthases; Saccharopolyspora
PubMed: 25287333
DOI: 10.1002/anie.201407806 -
Complete Reconstitution and Deorphanization of the 3 MDa Nocardiosis-Associated Polyketide Synthase.Journal of the American Chemical Society Apr 2020Several strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly line polyketide synthase (PKS) that presumably...
Several strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly line polyketide synthase (PKS) that presumably synthesizes an unknown polyketide. Here, we report the discovery and structure elucidation of the NOCAP (nocardiosis-associated polyketide) aglycone by first fully reconstituting the NOCAP synthase from purified protein components followed by heterologous expression in and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all- trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a -acyltransferase assembly line PKS and of using these approaches to "deorphanize" a complete assembly line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human infections.
Topics: Bacterial Proteins; Humans; Multigene Family; Nocardia; Nocardia Infections; Polyketide Synthases; Polyketides
PubMed: 32182063
DOI: 10.1021/jacs.0c00904 -
Nature Communications Oct 2022Biosynthesis of the flavonoid naringenin in plants and bacteria is commonly catalysed by a type III polyketide synthase (PKS) using one p-coumaroyl-CoA and three...
Biosynthesis of the flavonoid naringenin in plants and bacteria is commonly catalysed by a type III polyketide synthase (PKS) using one p-coumaroyl-CoA and three malonyl-CoA molecules as substrates. Here, we report a fungal non-ribosomal peptide synthetase -polyketide synthase (NRPS-PKS) hybrid FnsA for the naringenin formation. Feeding experiments with isotope-labelled precursors demonstrate that FnsA accepts not only p-coumaric acid (p-CA), but also p-hydroxybenzoic acid (p-HBA) as starter units, with three or four malonyl-CoA molecules for elongation, respectively. In vitro assays and MS/MS analysis prove that both p-CA and p-HBA are firstly activated by the adenylation domain of FnsA. Phylogenetic analysis reveals that the PKS portion of FnsA shares high sequence homology with type I PKSs. Refactoring the biosynthetic pathway in yeast with the involvement of fnsA provides an alternative approach for the production of flavonoids such as isorhamnetin and acacetin.
Topics: Polyketide Synthases; Phylogeny; Flavonoids; Tandem Mass Spectrometry; Peptide Synthases; Malonyl Coenzyme A; Catalysis
PubMed: 36289208
DOI: 10.1038/s41467-022-34150-7 -
Fungal Genetics and Biology : FG & B Sep 2019Filamentous fungi produce a vast number of bioactive secondary metabolites (SMs), some of which have found applications in the pharmaceutical industry including as... (Review)
Review
Filamentous fungi produce a vast number of bioactive secondary metabolites (SMs), some of which have found applications in the pharmaceutical industry including as antibiotics and immunosuppressants. As more and more species are whole genome sequenced the number of predicted clusters of genes for SM biosynthesis is ever increasing - holding a promise of novel useful bioactive SMs. To be able to fully utilize the potential of novel SMs, it is necessary to link the SM and the genes responsible for producing it. This can be challenging, but many strategies and tools have been developed for this purpose. Here we provide an overview of the methods used to establish the link between SM and biosynthetic gene cluster (BGC) and vice versa, along with the challenges and advantages of each of the methods. Part I of the review, associating BCG with SM, is divided into gene manipulations native strain and heterologous expression strategies, depending on the fungal species. Part II, associating SM with BGC, is divided into three main approaches: (1) homology search (2) retro-biosynthesis and (3) comparative genomics.
Topics: Biosynthetic Pathways; Fungal Proteins; Fungi; Gene Expression Regulation, Fungal; Genome, Fungal; Genomics; Multigene Family; Peptide Synthases; Polyketide Synthases; Secondary Metabolism
PubMed: 31195124
DOI: 10.1016/j.fgb.2019.06.001 -
International Journal of Molecular... Jan 2023Adaptation to a wide variety of habitats allows fungi to develop unique abilities to produce diverse secondary metabolites with diverse bioactivities. In this study, 30...
Adaptation to a wide variety of habitats allows fungi to develop unique abilities to produce diverse secondary metabolites with diverse bioactivities. In this study, 30 fungi isolated from St. John's Island, Singapore were investigated for their general biosynthetic potential and their ability to produce antimicrobial secondary metabolites (SMs). All the 30 fungal isolates belong to the Phylum and are distributed into 6 orders and 18 genera with Order having the highest number of representative (37%). Screening for polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes using degenerate PCR led to the identification of 23 polyketide synthases (PKSs) and 5 nonribosomal peptide synthetases (NRPSs) grouped into nine distinct clades based on their reduction capabilities. Some of the identified PKSs genes share high similarities between species and known reference genes, suggesting the possibility of conserved biosynthesis of closely related compounds from different fungi. Fungal extracts were tested for their antimicrobial activity against , Methicillin-resistant (MRSA), and . Bioassay-guided fractionation of the active constituents from two promising isolates resulted in the isolation of seven compounds: Penilumamides A, D, and E from strain F4335 and xanthomegnin, viomellein, pretrichodermamide C and vioxanthin from strain F7180. Vioxanthin exhibited the best antibacterial activity with IC values of 3.0 μM and 1.6 μM against and MRSA respectively. Viomellein revealed weak antiproliferative activity against A549 cells with an IC of 42 μM. The results from this study give valuable insights into the diversity and biosynthetic potential of fungi from this unique habitat and forms a background for an in-depth analysis of the biosynthetic capability of selected strains of interest with the aim of discovering novel fungal natural products.
Topics: Singapore; Methicillin-Resistant Staphylococcus aureus; Staphylococcus aureus; Polyketide Synthases; Ascomycota; Peptide Synthases; Fungi; Phylogeny
PubMed: 36674548
DOI: 10.3390/ijms24021033 -
ACS Chemical Biology Aug 2022Piscibactins and photoxenobactins are metallophores and virulence factors, whose biosynthetic gene cluster, termed , is the most prevalent polyketide...
Piscibactins and photoxenobactins are metallophores and virulence factors, whose biosynthetic gene cluster, termed , is the most prevalent polyketide synthase/non-ribosomal peptide synthetase hybrid cluster across entomopathogenic bacteria. They are structurally similar to yersiniabactin, which contributes to the virulence of the human pathogen . However, the -derived products feature various chain lengths and unusual carboxamide, thiocarboxylic acid, and dithioperoxoate termini, which are rarely found in thiotemplated biosyntheses. Here, we characterize the biosynthetic logic by gene deletions, site-directed mutagenesis, and isotope labeling experiments. Notably, we propose that it involves (1) heterocyclization domains with various catalytic efficiencies catalyzing thiazoline and amide/thioester bond formation and (2) putative C-N and C-S bond cleavage off-loading manners, which lead to products with different chain lengths and usual termini. Additionally, the post-assembly-line spontaneous conversions of the biosynthetic end product contribute to production titers of the other products in the culture medium. This study broadens our knowledge of thiotemplated biosynthesis and how bacterial host generate a chemical arsenal.
Topics: Bacteria; Humans; Multigene Family; Polyketide Synthases; Virulence
PubMed: 35860925
DOI: 10.1021/acschembio.2c00367 -
Nature Communications Aug 2021Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid systems typically use complex protein-protein interactions to facilitate direct transfer of...
Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between these multimodular megaenzymes. In the canal-associated neurons (CANs) of Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides biosynthesized by animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional enzymes expressed in the CANs that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes and discover a mechanism for trafficking intermediates between a PKS and an NRPS. Specifically, the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking mechanism provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.
Topics: Animals; Animals, Genetically Modified; Biosynthetic Pathways; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Chromatography, Liquid; Enzymes; Gene Expression; Mass Spectrometry; Molecular Structure; Mutation; Neurons; Peptide Synthases; Peptides; Polyketide Synthases; Polyketides
PubMed: 34389721
DOI: 10.1038/s41467-021-24682-9 -
PloS One 2021Pseudocercospora fijiensis is the causal agent of the highly destructive black Sigatoka disease of banana. Previous research has focused on polyketide synthase gene...
Pseudocercospora fijiensis is the causal agent of the highly destructive black Sigatoka disease of banana. Previous research has focused on polyketide synthase gene clusters in the fungus, given the importance of polyketide pathways in related plant pathogenic fungi. A time course study of expression of the previously identified PKS7-1, PKS8-2, and PKS10-2 gene clusters showed high expression of all three PKS genes and the associated clustered genes in infected banana plants from 2 weeks post-inoculation through 9 weeks. Engineered transformants silenced for PKS8-2 and PKS10-2 were developed and tested for pathogenicity. Inoculation of banana plants with silencing transformants for PKS10-2 showed significant reduction in disease symptoms and severity that correlated with the degree of silencing in the conidia used for inoculation, supporting a critical role for PKS10-2 in disease development. Unlike PKS10-2, a clear role for PKS8-2 could not be determined. Two of four PKS8-2 silencing transformants showed reduced disease development, but disease did not correlate with the degree of PKS8-2 silencing in the transformants. Overall, the degree of silencing obtained for the PKS8-2 transformants was less than that obtained for the PKS10-2 transformants, which may have limited the utility of the silencing strategy to identify a role for PKS8-2 in disease. Orthologous PKS10-2 clusters had previously been identified in the related banana pathogens Pseudocercospora musae and Pseudocercospora eumusae. Genome analysis identified orthologous gene clusters to that of PKS10-2 in the newly sequenced genomes of Pseudocercospora fuligena and Pseudocercospora cruenta, pathogens of tomato and cowpea, respectively. Our results support an important role for the PKS10-2 polyketide pathway in pathogenicity of Pseudocercospora fijiensis, and suggest a possible role for this pathway in disease development by other Pseudocercospora species.
Topics: Ascomycota; Fungal Proteins; Mycoses; Plant Diseases; Polyketide Synthases
PubMed: 34705882
DOI: 10.1371/journal.pone.0258981