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Environmental Health : a Global Access... Jun 2024Risk assessment (RA) of microbial secondary metabolites (SM) is part of the EU approval process for microbial active substances (AS) used in plant protection products... (Review)
Review
Risk assessment (RA) of microbial secondary metabolites (SM) is part of the EU approval process for microbial active substances (AS) used in plant protection products (PPP). As the number of potentially produced microbial SM may be high for a certain microbial strain and existing information on the metabolites often are low, data gaps are frequently identified during the RA. Often, RA cannot conclusively clarify the toxicological relevance of the individual substances. This work presents data and RA conclusions on four metabolites, Beauvericin, 2,3-deepoxy-2,3-didehydro-rhizoxin (DDR), Leucinostatin A and Swainsonin in detail as examples for the challenging process of RA. To overcome the problem of incomplete assessment reports, RA of microbial AS for PPP is in need of new approaches. In view of the Next Generation Risk Assessment (NGRA), the combination of literature data, omic-methods, in vitro and in silico methods combined in adverse outcome pathways (AOPs) can be used for an efficient and targeted identification and assessment of metabolites of concern (MoC).
Topics: Risk Assessment; European Union; Secondary Metabolism; Depsipeptides; Humans
PubMed: 38835048
DOI: 10.1186/s12940-024-01092-0 -
Angewandte Chemie (International Ed. in... Feb 2024Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the...
Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the structural dynamics of these megasynthases, specifically the delivery of acyl carrier protein (ACP)-bound building blocks to the catalytic site of the ketosynthase (KS) domain, remains severely limited. Using a multipronged structural approach, we report details of the inter-domain interactions after C-C bond formation in a chain-branching module of the rhizoxin PKS. Mechanism-based crosslinking of an engineered module was achieved using a synthetic substrate surrogate that serves as a Michael acceptor. The crosslinked protein allowed us to identify an asymmetric state of the dimeric protein complex upon C-C bond formation by cryo-electron microscopy (cryo-EM). The possible existence of two ACP binding sites, one of them a potential "parking position" for substrate loading, was also indicated by AlphaFold2 predictions. NMR spectroscopy showed that a transient complex is formed in solution, independent of the linker domains, and photochemical crosslinking/mass spectrometry of the standalone domains allowed us to pinpoint the interdomain interaction sites. The structural insights into a branching PKS module arrested after C-C bond formation allows a better understanding of domain dynamics and provides valuable information for the rational design of modular assembly lines.
Topics: Polyketide Synthases; Cryoelectron Microscopy; Binding Sites; Catalytic Domain; Acyl Carrier Protein
PubMed: 38134222
DOI: 10.1002/anie.202315850 -
Environmental Microbiology Jan 2024Rhizopus microsporus often lives in association with bacterial and viral symbionts that alter its biology. This fungal model represents an example of the complex...
Rhizopus microsporus often lives in association with bacterial and viral symbionts that alter its biology. This fungal model represents an example of the complex interactions established among diverse organisms in functional holobionts. We constructed a Genome-Scale Model (GSM) of the fungal-bacterial-viral holobiont (iHol). We employed a constraint-based method to calculate the metabolic fluxes to decipher the metabolic interactions of the symbionts with their host. Our computational analyses of iHol simulate the holobiont's growth and the production of the toxin rhizoxin. Analyses of the calculated fluxes between R. microsporus in symbiotic (iHol) versus asymbiotic conditions suggest that changes in the lipid and nucleotide metabolism of the host are necessary for the functionality of the holobiont. Glycerol plays a pivotal role in the fungal-bacterial metabolic interaction, as its production does not compromise fungal growth, and Mycetohabitans bacteria can efficiently consume it. Narnavirus RmNV-20S and RmNV-23S affected the nucleotide metabolism without impacting the fungal-bacterial symbiosis. Our analyses highlighted the metabolic stability of Mycetohabitans throughout its co-evolution with the fungal host. We also predicted changes in reactions of the bacterial metabolism required for the active production of rhizoxin. This iHol is the first GSM of a fungal holobiont.
Topics: Macrolides; Rhizopus; Bacteria; Nucleotides; Symbiosis
PubMed: 38072824
DOI: 10.1111/1462-2920.16551 -
Access Microbiology 2023Several species of soil-dwelling nematodes are used in the biocontrol of crop pests, due to their natural capacity to kill diverse lepidopteran species. Although this...
Several species of soil-dwelling nematodes are used in the biocontrol of crop pests, due to their natural capacity to kill diverse lepidopteran species. Although this insect-killing trait is known to be augmented by the nematodes' endosymbionts, the role of other steinernematid-associated bacterial genera in the nematode lifecycle remains unclear. This genomic study aimed to determine the potential of to contribute to the entomopathogenicity of its host. Insect larvae were infected with three separate cultures. From each of the three treatments, the prevalent bacteria in the haemocoel of cadavers, four days post-infection, were isolated. These three bacterial isolates were morphologically characterised. DNA was extracted from each of the three bacterial isolates and used for long-read genome sequencing and assembly. Assemblies were used to delineate species and identify genes that encode insect toxins, antimicrobials, and confer antibiotic resistance. We assembled three complete genomes. Through digital DNA-DNA hybridisation analyses, we ascertained that the haemocoels of insect cadavers previously infected with sp. Kalro, sp. 75, and sp. 97 were dominated by Kalro, 75, and 97, respectively. Kalro and 97 formed a subspecies with other symbionts of steinernematids from Kenya. 75 phylogenetically clustered with pseudomonads that are characterised by high insecticidal activity. The 75 genome encoded the production pathway of insect toxins such as orfamides and rhizoxins, antifungals such as pyrrolnitrin and pyoluteorin, and the broad-spectrum antimicrobial 2,4-diacetylphloroglucinol. The 75 genome encoded resistance to over ten classes of antibiotics, including cationic lipopeptides. Steinernematid-associated bacteria hence have the biosynthetic potential to contribute to nematode entomopathogenicity.
PubMed: 37970093
DOI: 10.1099/acmi.0.000659.v3 -
Frontiers in Fungal Biology 2022Mucoralean fungi from the genus are common inhabitants of terrestrial ecosystems, being some pathogens of animals and plants. In this study, we analyzed the symbiotic...
Mucoralean fungi from the genus are common inhabitants of terrestrial ecosystems, being some pathogens of animals and plants. In this study, we analyzed the symbiotic and toxinogenic potential of species derived from agricultural soils dedicated to the production of papaya ( L.) in Mexico. Four representative strains of soil-derived spp. were analyzed employing molecular, microscopic, and metabolic methods. The ITS phylogenies identified the fungi as HP499, HP475 and HP479, and HP487. We discovered that HP499 and HP475 harbor similar endofungal bacterial symbionts that belong to the genus ( sensu lato) and that none of the four fungi were associated with RmNV-20S and RmNV-23S. Intriguingly, the interaction between - showed different phenotypes from known - symbioses. Elimination of bacteria in HP475 did not cause a detrimental effect on fungal growth or asexual reproduction. Moreover, metabolic and molecular analyses confirmed that, unlike symbiotic HP499, HP475 does not produce rhizoxin, one of the best-characterized toxins produced by spp. The rhizoxin () biosynthetic gene cluster seems absent in this symbiotic bacterium. Our study highlights that the symbioses between and are more diverse than anticipated. Our findings contribute to expanding our understanding of the role bacterial symbionts have in the pathogenicity, biology and evolution of Mucorales.
PubMed: 37746220
DOI: 10.3389/ffunb.2022.893700 -
ACS Synthetic Biology Oct 2023Bacterial natural products (NPs) are an indispensable source of drugs and biopesticides. Heterologous expression is an essential method for discovering bacterial NPs and...
Bacterial natural products (NPs) are an indispensable source of drugs and biopesticides. Heterologous expression is an essential method for discovering bacterial NPs and the efficient biosynthesis of valuable NPs, but the chassis for Gram-negative bacterial NPs remains inadequate. In this study, we built a mutant Δ::attB by introducing an integrated site () to inactivate the native gladiolin () biosynthetic gene cluster, which stabilizes large foreign gene clusters and reduces the native metabolite profile. The growth and successful heterologous production of high-value NPs such as phylogenetically close -derived antitumor polyketides (PKs) rhizoxins, phylogenetically distant -derived anti-MRSA (methicillin-resistant ) antibiotics WAP-8294As, and -derived antitumor PKs disorazols demonstrate that this strain is a potential chassis for Gram-negative bacterial NPs. We further improved the yields of WAP-8294As through promoter insertions and precursor pathway overexpression based on heterologous expression in this strain. This study provides a robust bacterial chassis for genome mining, efficient production, and molecular engineering of bacterial NPs.
Topics: Biological Products; Burkholderia gladioli; Methicillin-Resistant Staphylococcus aureus; Anti-Bacterial Agents; Biological Control Agents; Polyketides; Multigene Family
PubMed: 37708405
DOI: 10.1021/acssynbio.3c00389 -
Frontiers in Microbiology 2022Heterologous expression is an indispensable approach to exploiting natural products from phylogenetically diverse microbial communities. In this study, we constructed a...
Heterologous expression is an indispensable approach to exploiting natural products from phylogenetically diverse microbial communities. In this study, we constructed a heterologous expression system based on strain E264 by deleting efflux pump genes and screening constitutive strong promoters. The biosynthetic gene cluster (BGC) of disorazol from So ce12 was expressed successfully with this host, and the yield of its product, disorazol F, rather than A, was improved to 38.3 mg/L by promoter substitution and insertion. In addition to the disorazol gene cluster, the BGC of rhizoxin from was also expressed efficiently, whereas no specific peak was detected when shuangdaolide BGC from sp. B59 was transformed into the host. This system provides another option to explore natural products from different phylogenetic taxa.
PubMed: 36466684
DOI: 10.3389/fmicb.2022.1073243 -
Comprehensive Reviews in Food Science... Nov 2022Fermented soybean products are widely consumed worldwide, and their popularity is increasing. Filamentous fungi, such as Actinomucor, Aspergillus, Monascus, Mucor,... (Review)
Review
Fermented soybean products are widely consumed worldwide, and their popularity is increasing. Filamentous fungi, such as Actinomucor, Aspergillus, Monascus, Mucor, Penicillium, Rhizopus, and Zymomonas, play critical roles in the fermentation processes of many soybean foods. However, besides producing essential enzymes for food fermentation, filamentous fungi can release undesirable or even toxic metabolites into the food. Mycotoxins are toxic secondary metabolites produced by certain filamentous fungi and may be detected during the food production process. Without effective prevention strategies, mycotoxin contamination in fermented soybean products poses a risk to human health. This review focused on the changes in mycotoxigenic fungal abundance and mycotoxin contamination at different stages during the production of soybean-based fermented foods, as well as effective strategies for preventing mycotoxin contamination in such products. Data from relevant studies demonstrated a tendency of change in the genera of mycotoxigenic fungi and types of mycotoxins (aflatoxins, alternariol, alternariol monomethyl ether, deoxynivalenol, fumonisins, ochratoxin A, rhizoxins, T-2 toxin, and zearalenone) present in the raw materials and the middle and final products. The applicability of traditional chemical and physical mitigation strategies and novel eco-friendly biocontrol approaches to prevent mycotoxin contamination in soybean-based fermented foods were discussed. The present review highlights the risks of mycotoxin contamination during the production of fermented soybean products and recommends promising strategies for eliminating mycotoxin contamination risk in soybean-based fermented foods.
Topics: Humans; Mycotoxins; Glycine max; Food Contamination; Fumonisins; Fungi
PubMed: 36084140
DOI: 10.1111/1541-4337.13032 -
MBio Oct 2022The fungus Rhizopus microsporus harbors a bacterial endosymbiont () for the production of the antimitotic toxin rhizoxin. Although rhizoxin is the causative agent of...
The fungus Rhizopus microsporus harbors a bacterial endosymbiont () for the production of the antimitotic toxin rhizoxin. Although rhizoxin is the causative agent of rice seedling blight, the toxinogenic bacterial-fungal alliance is, not restricted to the plant disease. It has been detected in numerous environmental isolates from geographically distinct sites covering all five continents, thus raising questions regarding the ecological role of rhizoxin beyond rice seedling blight. Here, we show that rhizoxin serves the fungal host in fending off protozoan and metazoan predators. Fluorescence microscopy and coculture experiments with the fungivorous amoeba revealed that ingestion of R. microsporus spores is toxic to . This amoebicidal effect is caused by the dominant bacterial rhizoxin congener rhizoxin S2, which is also lethal toward the model nematode Caenorhabditis elegans. By combining stereomicroscopy, automated image analysis, and quantification of nematode movement, we show that the fungivorous nematode Aphelenchus avenae actively feeds on R. microsporus that is lacking endosymbionts, whereas worms coincubated with symbiotic R. microsporus are significantly less lively. This study uncovers an unexpected ecological role of rhizoxin as shield against micropredators. This finding suggests that predators may function as an evolutionary driving force to maintain toxin-producing endosymbionts in nonpathogenic fungi. The soil community is a complex system characterized by predator-prey interactions. Fungi have developed effective strategies to defend themselves against predators. Understanding these strategies is of critical importance for ecology, medicine, and biotechnology. In this study, we shed light on the defense mechanisms of the phytopathogenic - symbiosis that has spread worldwide. We report an unexpected role of rhizoxin, a secondary metabolite produced by the bacterium residing within the hyphae of R. microsporus. We show that this bacterial secondary metabolite is utilized by the fungal host to successfully fend off fungivorous protozoan and metazoan predators and thus identified a fundamentally new function of this infamous cytotoxic compound. This endosymbiont-dependent predator defense illustrates an unusual strategy employed by fungi that has broader implications, since it may serve as a model for understanding how animal predation acts as an evolutionary driving force to maintain endosymbionts in nonpathogenic fungi.
Topics: Animals; Burkholderia; Antimitotic Agents; Macrolides; Symbiosis; Oryza; Toxins, Biological; Seedlings; Soil
PubMed: 36005392
DOI: 10.1128/mbio.01440-22 -
Archives of Biochemistry and Biophysics Sep 2022A tritiated derivative of the sponge-derived natural product spongistatin 1 was prepared, and its interactions with tubulin were examined. [H]Spongistatin 1 was found to...
A tritiated derivative of the sponge-derived natural product spongistatin 1 was prepared, and its interactions with tubulin were examined. [H]Spongistatin 1 was found to bind rapidly to tubulin at a single site (the low specific activity of the [H]spongistatin 1, 0.75 Ci/mmol, prevented our defining an association rate), and the inability of spongistatin 1 to cause an aberrant assembly reaction was confirmed. Spongistatin 1 bound to tubulin very tightly, and we could detect no significant dissociation reaction from tubulin. The tubulin-[H]spongistatin 1 complex did dissociate in 8 M urea, so there was no evidence for covalent bond formation. Apparent K values were obtained by Scatchard analysis of binding data and by Hummel-Dreyer chromatography (3.5 and 1.1 μM, respectively). The effects of a large cohort of vinca domain drugs on the binding of [H]spongistatin 1 to tubulin were evaluated. Compounds that did not cause aberrant assembly reactions (halichondrin B, eribulin, maytansine, and rhizoxin) caused little inhibition of [H]spongistatin 1 binding. Little inhibition also occurred with the peptides dolastatin 15, its active pentapeptide derivative, vitilevuamide, or diazonamide A, nor with the vinca alkaloid vinblastine. Strong inhibition was observed with dolastatin 10, hemiasterlin, and cryptophycin 1, all of which cause aberrant assembly reactions that might actually mask the spongistatin 1 binding site. Spongistatin 5 was found to be a competitive inhibitor of [H]spongistatin 1 binding, with an apparent K of 2.2 μM. We propose that the strong picomolar cytotoxicity of spongistatin 1 probably derives from its extremely tight binding to tubulin.
Topics: Antineoplastic Agents; Binding Sites; Macrolides; Microtubules; Tubulin; Vinblastine
PubMed: 35594923
DOI: 10.1016/j.abb.2022.109296