Promoter Activation in Δhfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct Bioactivity Testing

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene‐sequence‐similarity‐based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non‐ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.     

Genome Mining and Molecular Networking-Based Metabolomics of the Marine Facultative Aspergillus sp. MEXU 27854

The marine-facultative Aspergillus sp. MEXU 27854, isolated from the Caleta Bay in Acapulco, Guerrero, Mexico, has provided an interesting diversity of secondary metabolites, including a series of rare dioxomorpholines, peptides, and butyrolactones. Here, we report on the genomic data, which consists of 11 contigs (N50~3.95 Mb) with a ~30.75 Mb total length of assembly. Genome annotation resulted in the prediction of 10,822 putative genes. Functional annotation was accomplished by BLAST searching protein sequences with different public databases. Of the predicted genes, 75% were assigned gene ontology terms. From the 67 BGCs identified, ~60% belong to the NRPS and NRPS-like classes. Putative BGCs for the dioxomorpholines and other metabolites were predicted by extensive genome mining. In addition, metabolomic molecular networking analysis allowed the annotation of all isolated compounds and revealed the biosynthetic potential of this fungus. This work represents the first report of whole-genome sequencing and annotation from a marine-facultative fungal strain isolated from Mexico.     

SYN-View: A Phylogeny-Based Synteny Exploration Tool for the Identification of Gene Clusters Linked to Antibiotic Resistance

The development of new antibacterial drugs has become one of the most important tasks of the century in order to overcome the posing threat of drug resistance in pathogenic bacteria. Many antibiotics originate from natural products produced by various microorganisms. Over the last decades, bioinformatical approaches have facilitated the discovery and characterization of these small compounds using genome mining methodologies. A key part of this process is the identification of the most promising biosynthetic gene clusters (BGCs), which encode novel natural products. In 2017, the Antibiotic Resistant Target Seeker (ARTS) was developed in order to enable an automated target-directed genome mining approach. ARTS identifies possible resistant target genes within antibiotic gene clusters, in order to detect promising BGCs encoding antibiotics with novel modes of action. Although ARTS can predict promising targets based on multiple criteria, it provides little information about the cluster structures of possible resistant genes. Here, we present SYN-view. Based on a phylogenetic approach, SYN-view allows for easy comparison of gene clusters of interest and distinguishing genes with regular housekeeping functions from genes functioning as antibiotic resistant targets. Our aim is to implement our proposed method into the ARTS web-server, further improving the target-directed genome mining strategy of the ARTS pipeline.     

γ-Ray induced catalytic properties of the silica nanoparticles dispersed in the aerated aqueous solution

Silica nanoparticles (NPs) dispersed in an aerated aqueous solution containing Ag⁺ were irradiated to a dose of 10 kGy using ⁶⁰Co γ-rays. The typical surface plasmon band of Ag NPs was observed around 400 nm, indicating that even in the presence of dissolved oxygen the reduction of Ag⁺ occurred by silica NPs. Transmission electron microscopy images indicated that Ag NPs formed on the surface of the silica NPs. The subtraction spectra showed broad absorption around 500 nm with the absorbance depending on the dose. The electrons generated by charge separation from silica NPs with a size of about 12 nm reduce Ag⁺ to Ag⁰ and form (Ag⁰) _n species on the silica NPs, and the type of (Ag⁰) _n species formed depended on the silica NP, and Ag⁺ contents, and the dose. In the co-presence of organic molecules on the silica NP such as rhodamine, the absorbance of the surface plasmon band of both Ag NPs and rhodamine decreased, indicating the electrons to participate in the reductive decomposition of rhodamine molecules adsorbed on the silica NP. Furthermore, in the case when the silica NPs contained fluorescein molecules, the fluorescein molecules were also decomposed, indicating that the fluorescein molecules adsorbed on the inner surface of the silica NPs. The addition of I₂ as an oxidative reagent prevented the decomposition of the fluorescein molecules, indicating that electrons are the main species emitted from irradiated silica NPs.     

Discovery and characterisation of an amidine-containing ribosomally-synthesised peptide that is widely distributed in nature

Ribosomally synthesised and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural product with a wide range of bioactivities. Genome mining for RiPP biosynthetic gene clusters (BGCs) is often hampered by poor annotation of the short precursor peptides that are ultimately modified into the final molecule. Here, we utilise a previously described genome mining tool, RiPPER, to identify novel RiPP precursor peptides near YcaO-domain proteins, enzymes that catalyse various RiPP post-translational modifications including heterocyclisation and thioamidation. Using this dataset, we identified a novel and diverse family of RiPP BGCs spanning over 230 species of Actinobacteria and Firmicutes. A representative BGC from Streptomyces albidoflavus J1074 (formerly known as Streptomyces albus) was characterised, leading to the discovery of streptamidine, a novel amidine-containing RiPP. This new BGC family highlights the breadth of unexplored natural products with structurally rare features, even in model organisms.

Genome mining for pathways containing YcaO proteins revealed a widespread novel family of RiPP gene clusters. A model gene cluster was characterised through genetic and chemical analyses, which yielded streptamidine, a novel amidine-containing RiPP.     

Natural Enantiomers: Occurrence,Biogenesis and Biological Properties

The knowledge that natural products (NPs) are potent and selective modulators of important biomacromolecules (e.g., DNA and proteins) has inspired some of the world’s most successful pharmaceuticals and agrochemicals. Notwithstanding these successes and despite a growing number of reports on naturally occurring pairs of enantiomers, this area of NP science still remains largely unexplored, consistent with the adage “If you don’t seek, you don’t find”. Statistically, a rapidly growing number of enantiomeric NPs have been reported in the last several years. The current review provides a comprehensive overview of recent records on natural enantiomers, with the aim of advancing awareness and providing a better understanding of the chemical diversity and biogenetic context, as well as the biological properties and therapeutic (drug discovery) potential, of enantiomeric NPs.     

Natural product drug discovery in the artificial intelligence era

Natural products (NPs) are primarily recognized as privileged structures to interact with protein drug targets. Their unique characteristics and structural diversity continue to marvel scientists for developing NP-inspired medicines, even though the pharmaceutical industry has largely given up. High-performance computer hardware, extensive storage, accessible software and affordable online education have democratized the use of artificial intelligence (AI) in many sectors and research areas. The last decades have introduced natural language processing and machine learning algorithms, two subfields of AI, to tackle NP drug discovery challenges and open up opportunities. In this article, we review and discuss the rational applications of AI approaches developed to assist in discovering bioactive NPs and capturing the molecular “patterns” of these privileged structures for combinatorial design or target selectivity.

Natural products (NPs) are primarily recognized as privileged structures to interact with protein drug targets.     

Design,Synthesis, and Phenotypic Profiling of Pyrano‐Furo‐Pyridone Pseudo Natural Products

Natural products (NPs) inspire the design and synthesis of novel biologically relevant chemical matter, for instance through biology‐oriented synthesis (BIOS). However, BIOS is limited by the partial coverage of NP‐like chemical space by the guiding NPs. The design and synthesis of “pseudo NPs” overcomes these limitations by combining NP‐inspired strategies with fragment‐based compound design through de novo combination of NP‐derived fragments to unprecedented compound classes not accessible through biosynthesis. We describe the development and biological evaluation of pyrano‐furo‐pyridone (PFP) pseudo NPs, which combine pyridone‐ and dihydropyran NP fragments in three isomeric arrangements. Cheminformatic analysis indicates that the PFPs reside in an area of NP‐like chemical space not covered by existing NPs but rather by drugs and related compounds. Phenotypic profiling in a target‐agnostic “cell painting” assay revealed that PFPs induce formation of reactive oxygen species and are structurally novel inhibitors of mitochondrial complex I.     

Discovery of daspyromycins A and B, 2-aminovinyl-cysteine containing lanthipeptides,through a genomics-based approach

Lanthipeptides are one of the largest groups of ribosomally synthesized and post-translationally modified peptides(RiPPs) and are characterized by the presence of lanthionine(Lan) or methyllanthionine residues(MeLan). Only very few lanthipeptides contain a C-terminal 2-aminovinyl-cysteine(AviCys) motif, but all of them show potent antibacterial activities. Recent advances of genome sequencing led to the rapid accumulation of new biosynthetic gene clusters(BGCs) for lanthipeptides. In this study,...     

Design,Synthesis, and Phenotypic Profiling of Pyrano‐Furo‐Pyridone Pseudo Natural Products

Natural products (NPs) inspire the design and synthesis of novel biologically relevant chemical matter, for instance through biology‐oriented synthesis (BIOS). However, BIOS is limited by the partial coverage of NP‐like chemical space by the guiding NPs. The design and synthesis of “pseudo NPs” overcomes these limitations by combining NP‐inspired strategies with fragment‐based compound design through de novo combination of NP‐derived fragments to unprecedented compound classes not accessible through biosynthesis. We describe the development and biological evaluation of pyrano‐furo‐pyridone (PFP) pseudo NPs, which combine pyridone‐ and dihydropyran NP fragments in three isomeric arrangements. Cheminformatic analysis indicates that the PFPs reside in an area of NP‐like chemical space not covered by existing NPs but rather by drugs and related compounds. Phenotypic profiling in a target‐agnostic “cell painting” assay revealed that PFPs induce formation of reactive oxygen species and are structurally novel inhibitors of mitochondrial complex I.     

Using SEC for Analyzing the Sizes of Au/Pt Core/Shell Nanoparticles

This paper describes the use of size-exclusion chromatography (SEC) to characterize the sizes of fabricated Au/Pt core/shell nanoparticles (NPs), which were monitored using a polymer-based SEC column (pore size: ca. 400 nm) and a mobile phase of 10 mM sodium dodecyl sulfate (SDS). The plot of the retention time with respect to the logarithm of the size of the Au NPs was very linear (R ² = 0.999) for NP diameters falling in the range from 5.3 to 59.9 nm. The relative standard deviation (n = 5) of these retention times was less than 0.20%. When using SEC to analyze synthetic products, we found that the sizes of the fabricated Au/Pt core/shell NPs could be controlled via modification of the volumes of the seed, SDS, and sodium hydroxide solutions. A good correlation existed between the sizes determined through SEC and those determined using transmission electron microscopy. Thus, polymer-based SEC appears to be a useful technique for monitoring the sizes of NPs—and nanomaterials in general.     

SEC Characterization of Au Nanoparticles Prepared through Seed-Assisted Synthesis

In this paper we report the use of size-exclusion chromatography (SEC) for rapid determination of the sizes and size distributions of Au nanoparticles (NPs) prepared by seed-assisted synthesis. Analytical separation of Au NPs was performed in a polymer-based column of pore size 400 nm. We characterized the sizes and size distributions of the Au NPs by using 10 mM sodium dodecyl sulfate (SDS) as mobile phase and obtained a linear relationship (R ² = 0.986) between retention time and size of Au NPs within the range 9.8–79.1 nm; the relative standard deviations of these retention times were less than 0.3%. These separation conditions were used to characterize the sizes and size distributions of Au NPs prepared by seed-assisted synthesis. In addition to observing the elution times of the Au NPs we also simultaneously characterized their size-dependent optical properties by spectral measurement of the eluting peaks by use of an on-line diode-array detector (DAD), i.e., monitoring of the stability of the Au NP products. By using this approach we found the presence of SDS was beneficial in stabilizing the synthesized Au NPs. We also found that the volume of Au metal ions used affected the sizes of the final products. SEC seems an efficient tool for characterizing the sizes of NPs fabricated by seed-assisted synthesis.     

Phase control and its mechanism of CuInS2 nanoparticles

CuInS₂ nanoparticles (NPs) usually take chalcopyrite-(CP) structure. Recently, CuInS₂ NPs with pseudo-wurtzite (WZ) structure, which is thermodynamically less favored, have been synthesized. However, the formation mechanism of this metastable-phase has not been understood yet. In this report, the key issue of phase selectivity of CuInS₂ (CIS) NPs has been investigated using various metal sources and ligands. Experimental results suggested that the crystalline structure and morphology of CIS NPs were decided by the stability of indium ligand complex; the active ligand reduces the precipitation rate of In₂S₃, resulting in pre-generation of Cu₂S seed NPs. Crystallographic analogy and superionic conductivity of Cu₂S remind us that the formation of WZ CIS NPs is attributed to the pre-generation of Cu₂S seed NPs and the following cation exchange reaction. In order to confirm this hypothesis, Cu_2-xS seed NPs with various structures have been annealed in indium-ligand solution. This experiment revealed that the crystalline structure of CIS NP was determined by that of pre-generation Cu_2-xS NPs. Our results provide the important information for the phase control and synthesis of ternary chalcogenide NPs with a novel crystalline structure.     

On the Hopping Efficiency of Nanoparticles in the Electron Transfer across Self‐Assembled Monolayers

Redox reactions of solvated molecular species at gold‐electrode surfaces modified by electrochemically inactive self‐assembled molecular monolayers (SAMs) are found to be activated by introducing Au nanoparticles (NPs) covalently bound to the SAM to form a reactive Au–alkanedithiol–NP–molecule hybrid entity. The NP appears to relay long‐range electron transfer (ET) so that the rate of the redox reaction may be as efficient as directly on a bare Au electrode, even though the ET distance is increased by several nanometers. In this study, we have employed a fast redox reaction of surface‐confined 6‐(ferrocenyl) hexanethiol molecules and NPs of Au, Pt and Pd to address the dependence of the rate of ET through the hybrid on the particular NP metal. Cyclic voltammograms show an increasing difference in the peak‐to‐peak separation for NPs in the order Au<Pt<Pd, especially when the length of the alkanedithiol increases from octanedithiol to decanedithiol. The corresponding apparent rate constants, k_app, for decanedithiol are 1170, 360 and 14 s^?1 for NPs of Au, Pt and Pd, respectively, indicating that the efficiency of NP mediation of the ET clearly depends on the nature of the NP. Based on a preliminary analysis rooted in interfacial electrochemical ET theory, combined with a simplified two‐step view of the NP coupling to the electrode and the molecule, this observation is referred to the density of electronic states of the NPs, reflected in a broadening of the molecular electron/NP bridge group levels and energy‐gap differences between the Fermi levels of the different metals.     

Synthesis of open-mouthed,yolk–shell Au@AgPd nanoparticles with access to interior surfaces for enhanced electrocatalysis

We have successfully produced open-mouthed, yolk–shell (OM-YS) Au@AgPd nanoparticles (NPs) via galvanic replacement reaction at room temperature; each NP has a large opening on its AgPd shells. Owing to the openings on the AgPd shells, the inner surfaces of the AgPd shells of as-prepared OM-YS Au@AgPd NPs become accessible to the surrounding media. These new structural characters make the present OM-YS Au@AgPd NPs excellent catalysts for electrochemical oxidation of ethanol in alkaline media. Their electrochemical active surface area is 87.8 m² g^–1 and the mass activity is 1.25 A mg_Pd^–1. Moreover, the openings on the AgPd shells also make the surfaces of the Au cores in OM-YS Au@AgPd NPs accessible to the reaction media, which significantly facilitates the removal of CO and other carbonaceous intermediate species, thus leading to substantially enhanced durability and stability. This superior electrocatalytic performance cannot be implemented by using conventional YS Au@AgPd NPs or commercially available Pd/C catalysts.     

Physico-Chemical Properties of Inorganic NPs Influence the Absorption Rate of Aquatic Mosses Reducing Cytotoxicity on Intestinal Epithelial Barrier Model

Noble metals nanoparticles (NPs) and metal oxide NPs are widely used in different fields of application and commercial products, exposing living organisms to their potential adverse effects. Recent evidences suggest their presence in the aquifers water and consequently in drinking water. In this work, we have carefully synthesized four types of NPs, namely, silver and gold NPs (Ag NPs and Au NPs) and silica and titanium dioxide NPs (SiO₂ NPs and TiO₂ NPs) having a similar size and negatively charged surfaces. The synthesis of Ag NPs and Au NPs was carried out by colloidal route using silver nitrate (AgNO₃) and tetrachloroauric (III) acid (HAuCl₄) while SiO₂ NPs and TiO₂ NPs were achieved by ternary microemulsion and sol-gel routes, respectively. Once the characterization of NPs was carried out in order to assess their physico-chemical properties, their impact on living cells was studied. We used the human colorectal adenocarcinoma cells (Caco-2), known as the best representative intestinal epithelial barrier model to understand the effects triggered by NPs through ingestion. Then, we moved to explore how water contamination caused by NPs can be lowered by the ability of three species of aquatic moss, namely, Leptodictyum riparium, Vesicularia ferriei, and Taxiphyllum barbieri, to absorb them. The experiments were conducted using two concentrations of NPs (100 μM and 500 Μm as metal content) and two time points (24 h and 48 h), showing a capture rate dependent on the moss species and NPs type. Then, the selected moss species, able to actively capture NPs, appear as a powerful tool capable to purify water from nanostructured materials, and then, to reduce the toxicity associated to the ingestion of contaminated drinking water.     

In Silico Analysis of P450s and Their Role in Secondary Metabolism in the Bacterial Class Gammaproteobacteria

The impact of lifestyle on shaping the genome content of an organism is a well-known phenomenon and cytochrome P450 enzymes (CYPs/P450s), heme-thiolate proteins that are ubiquitously present in organisms, are no exception. Recent studies focusing on a few bacterial species such as Streptomyces, Mycobacterium, Cyanobacteria and Firmicutes revealed that the impact of lifestyle affected the P450 repertoire in these species. However, this phenomenon needs to be understood in other bacterial species. We therefore performed genome data mining, annotation, phylogenetic analysis of P450s and their role in secondary metabolism in the bacterial class Gammaproteobacteria. Genome-wide data mining for P450s in 1261 Gammaproteobacterial species belonging to 161 genera revealed that only 169 species belonging to 41 genera have P450s. A total of 277 P450s found in 169 species grouped into 84 P450 families and 105 P450 subfamilies, where 38 new P450 families were found. Only 18% of P450s were found to be involved in secondary metabolism in Gammaproteobacterial species, as observed in Firmicutes as well. The pathogenic or commensal lifestyle of Gammaproteobacterial species influences them to such an extent that they have the lowest number of P450s compared to other bacterial species, indicating the impact of lifestyle on shaping the P450 repertoire. This study is the first report on comprehensive analysis of P450s in Gammaproteobacteria.     

Integration of microfiltration and anion-exchange nanoparticles-based magnetic separation with MALDI mass spectrometry for bacterial analysis

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is powerful in characterizing and identifying bacterial isolates. However, sufficient quantities of bacterial cells are required for generating MALDI mass spectra and a procedure to isolate and enrich target bacteria from sample matrix prior to MALDI-MS analysis is often necessary. In this paper, anion-exchange superparamagnetic nanoparticles (NPs), i.e., fluidMAG-DEAE and fluidMAG-Q, were employed to capture Aeromonas, Salmonella, Pseudomonas, Enterococcus, Bacillus, Staphylococcus and Escherichia coli from aqueous solutions and fresh water. The magnetically isolated bacteria were then characterized by whole cell MALDI-MS. The capture efficiency was found to be dependent on bacterial species, medium pH, the functional group and concentration of the NPs. The experimental results demonstrated that fluidMAG-DEAE and fluidMAG-Q were broad spectrum probes for bacteria. Furthermore, both dead and live bacteria could be captured by the NPs, and the live bacteria captured remained viable. Membrane filtration prior to the magnetic isolation could increase enrichment factor and eliminate potential matrix interference. A detection limit of 1 × 10³ cfu/ml was achieved for the bacteria spiked in tap water and reservoir water, and the analytical time was around 2 h.