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Methods in Enzymology 2024Magnesium ions (Mg) are crucial in class II terpene cyclases that utilize substrates with diphosphate groups. Interestingly, these enzymes catalyze reactions without...
Magnesium ions (Mg) are crucial in class II terpene cyclases that utilize substrates with diphosphate groups. Interestingly, these enzymes catalyze reactions without cleaving the diphosphate group, instead initiating the reaction through protonation. In our recent research, we discovered a novel class II sesquiterpene cyclase in Streptomyces showdoensis. Notably, we determined its crystal structure and identified Mg within its active site. This finding has shed light on the previously elusive question of Mg binding in class II terpene cyclases. In this chapter, we outline our methods for discovering this novel enzyme, including steps for its purification, crystallization, and kinetic analysis.
Topics: Magnesium; Sesquiterpenes; Streptomyces; Binding Sites; Kinetics; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Structure-Activity Relationship; Crystallization; Carbon-Carbon Lyases
PubMed: 38942506
DOI: 10.1016/bs.mie.2024.02.018 -
Journal of Natural Products Jun 2024The marine tunicate-derived RKJM0023 was cultured in the presence of a rhamnolipid mixture in an effort to elicit the production of silent natural products. MS/MS-based...
The marine tunicate-derived RKJM0023 was cultured in the presence of a rhamnolipid mixture in an effort to elicit the production of silent natural products. MS/MS-based molecular networking analysis enhanced with nonparametric statistics highlighted the upregulation of a molecular cluster (Kruskal-Wallis = 1.6 e for ) in which no MS/MS features had library matches. Targeted isolation of these features resulted in the discovery of nine new acylated lipopeptides, albubactins A-H (-) each containing a unique glutamine tripeptide and a -terminal ethyl ester moiety. Three related albubactin acids A-C (-) lacking the ethyl ester were also identified. NMR spectroscopy and UPLC-HR-ESI-MS/MS demonstrated that the albubactins were obtained as mixtures that shared a common / and differed only in their acylated terminal groups. Due to the complex spectroscopic elucidation with many overlapping shifts, a total synthesis of albubactin A () was completed and used to determine the absolute configuration of the new albubactins.
PubMed: 38940698
DOI: 10.1021/acs.jnatprod.3c01234 -
Journal of Agricultural and Food... Jun 2024A study targeting novel antifungal metabolites identified potent antifungal activity against key plant pathogens in acetone extracts of sp. strain CA-296093....
A study targeting novel antifungal metabolites identified potent antifungal activity against key plant pathogens in acetone extracts of sp. strain CA-296093. Feature-based molecular networking revealed the presence in this extract of antimycin-related compounds, leading to the isolation of four new compounds: escuzarmycins A-D (-). Extensive structural elucidation, employing 1D and 2D NMR, high-resolution mass spectrometry, Marfey's analysis, and NOESY correlations, confirmed their structures. The bioactivity of these compounds was tested against six fungal phytopathogens, and compounds and demonstrated strong efficacy, particularly against , with compound exhibiting the highest potency (EC: 11 nM). Both compounds also displayed significant antifungal activity against and , with compound proving to be the most potent. Despite moderate cytotoxicity against the human cancer cell line HepG2, compounds and emerge as promising fungicides for combating blotch, anthracnose, and gray mold.
PubMed: 38935555
DOI: 10.1021/acs.jafc.4c01303 -
Organic Letters Jun 2024Halogenated phenazine meroterpenoids are a structurally unusual family of marine actinobacterial natural products that exhibit antibiotic, antibiofilm, and cytotoxic...
Halogenated phenazine meroterpenoids are a structurally unusual family of marine actinobacterial natural products that exhibit antibiotic, antibiofilm, and cytotoxic bioactivities. Despite a lack of established phenazine halogenation biochemistry, genomic analysis of sp. CNZ-289, a prolific lavanducyanin and C2-halogenated derivative producer, suggested the involvement of vanadium-dependent haloperoxidases. We subsequently discovered lavanducyanin halogenase (LvcH), characterized it as a regioselective vanadium-dependent chloroperoxidase, and applied it in late-stage chemoenzymatic synthesis.
PubMed: 38934639
DOI: 10.1021/acs.orglett.4c01869 -
Pharmaceuticals (Basel, Switzerland) Jun 2024A tremendous increase in the green synthesis of metallic nanoparticles has been noticed in the last decades, which is due to their unique properties at the nano...
Exploring the Potential of Halotolerant Actinomycetes from Rann of Kutch, India: A Study on the Synthesis, Characterization, and Biomedical Applications of Silver Nanoparticles.
A tremendous increase in the green synthesis of metallic nanoparticles has been noticed in the last decades, which is due to their unique properties at the nano dimension. The present research work deals with synthesis mediated by the actinomycete of silver nanoparticles (AgNPs), isolated from Little and Greater Rann of Kutch, India. The confirmation of the formation of AgNPs by the actinomycetes was carried out by using a UV-Vis spectrophotometer where an absorbance peak was obtained at 420 nm. The X-ray diffraction pattern demonstrated five characteristic diffraction peaks indexed at the lattice plane (111), (200), (231), (222), and (220). Fourier transform infrared showed typical bands at 531 to 1635, 2111, and 3328 cm. Scanning electron microscopy shows that the spherical-shaped AgNPs particles have diameters in the range of 40 to 90 nm. The particle size analysis displayed the mean particle size of AgNPs in aqueous medium, which was about 55 nm (±27 nm), bearing a negative charge on their surfaces. The potential of the -mediated synthesized AgNPs was evaluated for their antimicrobial, anti-methicillin-resistant (MRSA), anti-biofilm, and anti-oxidant activity. The maximum inhibitory effect was observed against at (8 µg/mL), followed by and at (32 µg/mL), and against (64 µg/mL), whereas (128 µg/mL) and (256 µg/mL) were much less sensitive to AgNPs. The biosynthesized AgNPs displayed activity against MRSA, and the free radical scavenging activity was observed with an increase in the dosage of AgNPs from 25 to 200 µg/mL. AgNPs in combination with ampicillin displayed inhibition of the development of biofilm in and at 98% and 83%, respectively. AgNPs were also successfully coated on the surface of cotton to prepare antimicrobial surgical cotton, which demonstrated inhibitory action against (15 mm) and (12 mm). The present research integrates microbiology, nanotechnology, and biomedical science to formulate environmentally friendly antimicrobial materials using halotolerant actinomycetes, evolving green nanotechnology in the biomedical field. Moreover, this study broadens the understanding of halotolerant actinomycetes and their potential and opens possibilities for formulating new antimicrobial products and therapies.
PubMed: 38931410
DOI: 10.3390/ph17060743 -
Plants (Basel, Switzerland) Jun 2024The use of rhizospheric SynComs can be a new and sustainable strategy in the agrobiotechnology sector. The objective of this study was to create the most appropriate...
The use of rhizospheric SynComs can be a new and sustainable strategy in the agrobiotechnology sector. The objective of this study was to create the most appropriate SynCom composition; examine the ability to dissolve natural rock phosphate (RP) from Morocco in liquid-modified NBRIP medium; determine organic acids, and phytohormones; and verify plant growth promoting and nutrition uptake effect in the pot experiments of winter wheat (). A total of nine different microorganisms were isolated, which belonged to three different genera: , , and . Out of the 21 treatments tested, four SynComs had the best phosphate-dissolving properties: IJAK-27+44+91 (129.17 mg L), IIBEI-32+40 (90.95 µg mL), IIIDEG-45+41 (122.78 mg L), and IIIDEG-45+41+72 (120.78 mg L). We demonstrate that these SynComs are capable of producing lactic, acetic, gluconic, malic, oxalic, citric acids, and phytohormones such as indole-3-acetic acid, zeatin, gibberellic acid, and abscisic acid. In pot experiments with winter wheat, we also demonstrated that the designed SynComs were able to effectively colonize the plant root rhizosphere and contributed to more abundant plant growth characteristics and nutrient uptake as uninoculated treatment or uninoculated treatment with superphosphate (NPK 0-19-0). The obtained results show that the SynCom compositions of IJAK-27+44+91, IIBEI-32+40, IIIDEG-45+41, and IIIDEG-45+41+72 can be considered as promising candidates for developing biofertilizers to facilitate P absorption and increase plant nutrition.
PubMed: 38931148
DOI: 10.3390/plants13121716 -
Microorganisms Jun 2024Sigma factors are transcriptional regulators that are part of complex regulatory networks for major cellular processes, as well as for growth phase-dependent regulation...
Sigma factors are transcriptional regulators that are part of complex regulatory networks for major cellular processes, as well as for growth phase-dependent regulation and stress response. sp. SE50/110 is the natural producer of acarbose, an α-glucosidase inhibitor that is used in diabetes type 2 treatment. Acarbose biosynthesis is dependent on growth, making sigma factor engineering a promising tool for metabolic engineering. ACSP50_0507 is a homolog of the developmental and osmotic-stress-regulating σH. Therefore, the protein encoded by was named σH. Here, an sp. SE50/110 expression strain for the alternative sigma factor gene () achieved a two-fold increased acarbose yield with acarbose production extending into the stationary growth phase. Transcriptome sequencing revealed upregulation of acarbose biosynthesis genes during growth and at the late stationary growth phase. Genes that are transcriptionally activated by σH frequently code for secreted or membrane-associated proteins. This is also mirrored by the severely affected cell morphology, with hyperbranching, deformed and compartmentalized hyphae. The dehydrated cell morphology and upregulation of further genes point to a putative involvement in osmotic stress response, similar to its homolog. The DNA-binding motif of σH was determined based on transcriptome sequencing data and shows high motif similarity to that of its homolog. The motif was confirmed by in vitro binding of recombinantly expressed σH to the upstream sequence of a strongly upregulated gene. Autoregulation of σH was observed, and binding to its own gene promoter region was also confirmed.
PubMed: 38930623
DOI: 10.3390/microorganisms12061241 -
Microorganisms Jun 2024species are attractive sources of secondary metabolites that serve as major sources of antibiotics and other drugs. In this study, genome mining was used to determine...
species are attractive sources of secondary metabolites that serve as major sources of antibiotics and other drugs. In this study, genome mining was used to determine the biosynthetic potential of sp. 21So2-11 isolated from Antarctic soil. 16S rRNA gene sequencing revealed that this strain is most closely related to NBRC 101007, with a similarity of 98.02%. Genome comparisons based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) showed that strain 21So2-11 represents a novel species of the genus . In addition to a large number of genes related to environmental adaptation and ecological function, a total of 28 putative biosynthetic gene clusters (BGCs) responsible for the biosynthesis of known and/or novel secondary metabolites, including terpenes, lantipeptides, polyketides, nonribosomal peptides, RiPPs and siderophores, were detected in the genome of strain 21So2-11. In addition, a total of 1456 BGCs were predicted to contribute to the biosynthesis of more than 300 secondary metabolites based on the genomes of 47 strains originating from polar regions. The results indicate the potential of sp. 21So2-11 for bioactive secondary metabolite production and are helpful for understanding bacterial adaptability and ecological function in cold terrestrial environments.
PubMed: 38930610
DOI: 10.3390/microorganisms12061228 -
Microorganisms May 2024is an emerging human pathogen that has a high rate of incidence in immunocompromised individuals. We have found a putative secondary metabolite pathway within , which...
is an emerging human pathogen that has a high rate of incidence in immunocompromised individuals. We have found a putative secondary metabolite pathway within , which may be a key factor in its pathogenesis. This novel pathway is encoded in a gene cluster spanning MAB_0284c to 0305 and is related to pathways, producing the secondary metabolites streptonigrin and nybomycin. We constructed an in-frame deletion of the MAB_0295 () gene and tested it in our animal model. We have previously shown that tadpoles, which have functional lungs and T cells, can serve as a reliable comparative model for persistent infection and pathogenesis. Here, we report that tadpoles intraperitoneally infected with the ∆ mutant exhibit early decreased bacterial loads and significantly increased survival compared with those infected with WT . ∆ mutant also induced lower transcript levels of several pro-inflammatory cytokines (, , , ) than those of WT in the liver and lungs. In addition, there was impaired macrophage recruitment and decreased macrophage infection in tadpoles infected with the ∆ mutant, by tail wound inoculation, compared to those infected with the WT bacteria, as assayed by intravital confocal microscopy. These data underline the relevance and usefulness of tadpoles as a novel comparative animal model to identify genetic determinants of immunopathogenesis, suggesting a role for this novel and uncharacterized pathway in pathogenesis and macrophage recruitment.
PubMed: 38930501
DOI: 10.3390/microorganisms12061120 -
Biology May 2024This work aimed to study the genome organization and the metabolic potential of strain SCPM-O-B-9993, a promising plant-protecting and plant-stimulating strain isolated...
This work aimed to study the genome organization and the metabolic potential of strain SCPM-O-B-9993, a promising plant-protecting and plant-stimulating strain isolated from brown semi-desert soils with very high salinity. The strain genome contains a linear chromosome 5,968,715 bp long and has no plasmids. The genome contains 5331 coding sequences among which 2139 (40.1%) are functionally annotated. Biosynthetic gene clusters (BGCs) of secondary metabolites exhibiting antimicrobial properties (ohmyungsamycin, pellasoren, naringenin, and ansamycin) were identified in the genome. The most efficient period of SCPM-O-B-9993 strain cultivation was 72 h: during this period, the culture went from the exponential to the stationary growth phase as well as exhibited excellent phytostimulatory properties and antiviral activity against the cucumber mosaic virus in tomatoes under laboratory conditions. The SCPM-OB-9993 strain is a biotechnologically promising producer of secondary metabolites exhibiting antiviral and phytostimulatory properties.
PubMed: 38927268
DOI: 10.3390/biology13060388