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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 -
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 -
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 -
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 -
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