Connecting metabolome and phenotype: recent advances in functional metabolomics tools for the identification of bioactive natural products.

Covering: 1995 to 2023Advances in bioanalytical methods, particularly mass spectrometry, have provided valuable molecular insights into the mechanisms of life. Non-targeted metabolomics aims to detect and (relatively) quantify all observable small molecules present in a biological system. By comparing small molecule abundances between different conditions or timepoints in a biological system, researchers can generate new hypotheses and begin to understand causes of observed phenotypes. Functional metabolomics aims to investigate the functional roles of metabolites at the scale of the metabolome. However, most functional metabolomics studies rely on indirect measurements and correlation analyses, which leads to ambiguity in the precise definition of functional metabolomics. In contrast, the field of natural products has a history of identifying the structures and bioactivities of primary and specialized metabolites. Here, we propose to expand and reframe functional metabolomics by integrating concepts from the fields of natural products and chemical biology. We highlight emerging functional metabolomics approaches that shift the focus from correlation to physical interactions, and we discuss how this allows researchers to uncover causal relationships between molecules and phenotypes.

Enhanced Molecular Networking Shows Microbacterium sp. V1 as a Factory of Antioxidant Proline-Rich Peptides.

Two linear proline-rich peptides (1-2), bearing an N-terminal pyroglutamate, were isolated from the marine bacterium Microbacterium sp. V1, associated with the marine sponge Petrosia ficiformis, collected in the volcanic CO2 vents in Ischia Island (South Italy). Peptide production was triggered at low temperature following the one strain many compounds (OSMAC) method. Both peptides were detected together with other peptides (3-8) via an integrated, untargeted MS/MS-based molecular networking and cheminformatic approach. The planar structure of the peptides was determined by extensive 1D and 2D NMR and HR-MS analysis, and the stereochemistry of the aminoacyl residues was inferred by Marfey's analysis. Peptides 1-8 are likely to arise from Microbacterium V1 tailor-made proteolysis of tryptone. Peptides 1 and 2 were shown to display antioxidant properties in the ferric-reducing antioxidant power (FRAP) assay.

Novel Insights on Pyoverdine: From Biosynthesis to Biotechnological Application.

Pyoverdines (PVDs) are a class of siderophores produced mostly by members of the genus Pseudomonas. Their primary function is to accumulate, mobilize, and transport iron necessary for cell metabolism. Moreover, PVDs also play a crucial role in microbes' survival by mediating biofilm formation and virulence. In this review, we reorganize the information produced in recent years regarding PVDs biosynthesis and pathogenic mechanisms, since PVDs are extremely valuable compounds. Additionally, we summarize the therapeutic applications deriving from the PVDs' use and focus on their role as therapeutic target themselves. We assess the current biotechnological applications of different sectors and evaluate the state-of-the-art technology relating to the use of synthetic biology tools for pathway engineering. Finally, we review the most recent methods and techniques capable of identifying such molecules in complex matrices for drug-discovery purposes.

Mixotrophy in a Local Strain of Nannochloropsis granulata for Renewable High-Value Biomass Production on the West Coast of Sweden

A local strain of Nannochloropsis granulata (Ng) has been reported as the most productive microalgal strain in terms of both biomass yield and lipid content when cultivated in photobioreactors that simulate the light and temperature conditions during the summer on the west coast of Sweden. To further increase the biomass and the biotechnological potential of this strain in these conditions, mixotrophic growth (i.e., the simultaneous use of photosynthesis and respiration) with glycerol as an external carbon source was investigated in this study and compared with phototrophic growth that made use of air enriched with 1-2% CO2. The addition of either glycerol or CO2-enriched air stimulated the growth of Ng and theproduction of high-value long-chain polyunsaturated fatty acids (EPA) as well as the carotenoid canthaxanthin. Bioassays in human prostate cell lines indicated the highest antitumoral activity for Ng extracts and fractions from mixotrophic conditions. Metabolomics detected betaine lipids specifically in the bioactive fractions, suggesting their involvement in the observed antitumoral effect. Genes related to autophagy were found to be upregulated by the most bioactive fraction, suggesting a possible therapeutic target against prostate cancer progression. Taken together, our results suggest that the local Ng strain can be cultivated mixotrophically in summer conditions on the west coast of Sweden for the production of high-value biomass containing antiproliferative compounds, carotenoids, and EPA.

Dihydroauroglaucin Isolated from the Mediterranean Sponge Grantia compressa Endophyte Marine Fungus Eurotium chevalieri Inhibits Migration of Human Neuroblastoma Cells.

Cancer cell migration is a hallmark of the aggressiveness and progression of malignancies such as high-risk neuroblastoma. Given the lack of effective therapeutic solutions to counteract cancer progression, basic research aims to identify novel bioactive molecules with inhibitory potential on cancer cell migration. In this context, this work investigated the role of members of the salicylaldehyde secondary metabolite set from the sponge endophyte fungus Eurotium chevalieri MUT 2316 as potential inhibitors of human neuroblastoma SH-SY5Y cell migration. Since tetrahydroauroglaucin (TAG) and dihydroauroglaucin (DAG) were isolated in large amounts, both were evaluated for their anticancer properties towards SH-SY5Y cells. Both molecules were found to be non-cytotoxic by MTT assay and cytofluorimetric analysis. Moreover, DAG showed efficacy in inhibiting the highly migratory phenotype of SH-SY5Y cells by wound healing assay; whereas TAG, although structurally similar to DAG, showed no anti-migratory effect. Therefore, this work provides good reasons to conduct further in vitro and in vivo studies focusing on DAG as a potentially useful migrastatic natural marine molecule.

Combining OSMAC Approach and Untargeted Metabolomics for the Identification of New Glycolipids with Potent Antiviral Activity Produced by a Marine Rhodococcus.

Natural products of microbial origin have inspired most of the commercial pharmaceuticals, especially those from Actinobacteria. However, the redundancy of molecules in the discovery process represents a serious issue. The untargeted approach, One Strain Many Compounds (OSMAC), is one of the most promising strategies to induce the expression of silent genes, especially when combined with genome mining and advanced metabolomics analysis. In this work, the whole genome of the marine isolate Rhodococcus sp. I2R was sequenced and analyzed by antiSMASH for the identification of biosynthetic gene clusters. The strain was cultivated in 22 different growth media and the generated extracts were subjected to metabolomic analysis and functional screening. Notably, only a single growth condition induced the production of unique compounds, which were partially purified and structurally characterized by liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). This strategy led to identifying a bioactive fraction containing >30 new glycolipids holding unusual functional groups. The active fraction showed a potent antiviral effect against enveloped viruses, such as herpes simplex virus and human coronaviruses, and high antiproliferative activity in PC3 prostate cancer cell line. The identified compounds belong to the biosurfactants class, amphiphilic molecules, which play a crucial role in the biotech and biomedical industry.

Assessment of the Degradation Potential and Genomic Insights towards Phenanthrene by Dietzia psychralcaliphila JI1D.

Extreme marine environments are potential sources of novel microbial isolations with dynamic metabolic activity. Dietzia psychralcaliphila J1ID was isolated from sediments originated from Deception Island, Antarctica, grown over phenanthrene. This strain was also assessed for its emulsifying activity. In liquid media, Dietzia psychralcaliphila J1ID showed 84.66% degradation of phenanthrene examined with HPLC-PDA. The identification of metabolites by GC-MS combined with its whole genome analysis provided the pathway involved in the degradation process. Whole genome sequencing indicated a genome size of 4,216,480 bp with 3961 annotated genes. The presence of a wide range of monooxygenase and dioxygenase, as well as dehydrogenase catabolic genes provided the genomic basis for the biodegradation. The strain possesses the genetic compartments for a wide range of toxic aromatic compounds, which includes the benABCD and catABC clusters. COG2146, COG4638, and COG0654 through COG analysis confirmed the genes involved in the oxygenation reaction of the hydrocarbons by the strain. Insights into assessing the depletion of phenanthrene throughout the incubation process and the genetic components involved were obtained. This study indicates the degradation potential of the strain, which can also be further expanded to other model polyaromatic hydrocarbons.

Antiviral Activity of Vitis vinifera Leaf Extract against SARS-CoV-2 and HSV-1.

Vitis vinifera represents an important and renowned source of compounds with significant biological activity. Wines and winery bioproducts, such as grape pomace, skins, and seeds, are rich in bioactive compounds against a wide range of human pathogens, including bacteria, fungi, and viruses. However, little is known about the biological properties of vine leaves. The aim of this study was the evaluation of phenolic composition and antiviral activity of Vitis vinifera leaf extract against two human viruses: the Herpes simplex virus type 1 (HSV-1) and the pandemic and currently widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). About 40 phenolic compounds were identified in the extract by HPLC-MS/MS analysis: most of them were quercetin derivatives, others included derivatives of luteolin, kaempferol, apigenin, isorhamnetin, myricetin, chrysoeriol, biochanin, isookanin, and scutellarein. Leaf extract was able to inhibit both HSV-1 and SARS-CoV-2 replication in the early stages of infection by directly blocking the proteins enriched on the viral surface, at a very low concentration of 10 µg/mL. These results are very promising and highlight how natural extracts could be used in the design of antiviral drugs and the development of future vaccines.

Isolation and Characterization of Strain Exiguobacterium sp. KRL4, a Producer of Bioactive Secondary Metabolites from a Tibetan Glacier.

Extremophilic microorganisms represent a unique source of novel natural products. Among them, cold adapted bacteria and particularly alpine microorganisms are still underexplored. Here, we describe the isolation and characterization of a novel Gram-positive, aerobic rod-shaped alpine bacterium (KRL4), isolated from sediments from the Karuola glacier in Tibet, China. Complete phenotypic analysis was performed revealing the great adaptability of the strain to a wide range of temperatures (5-40 °C), pHs (5.5-8.5), and salinities (0-15% w/v NaCl). Genome sequencing identified KRL4 as a member of the placeholder genus Exiguobacterium_A and annotation revealed that only half of the protein-encoding genes (1522 of 3079) could be assigned a putative function. An analysis of the secondary metabolite clusters revealed the presence of two uncharacterized phytoene synthase containing pathways and a novel siderophore pathway. Biological assays confirmed that the strain produces molecules with antioxidant and siderophore activities. Furthermore, intracellular extracts showed nematocidal activity towards C. elegans, suggesting that strain KRL4 is a source of anthelmintic compounds.

Antioxidant Molecules from Marine Fungi: Methodologies and Perspectives.

The marine environment represents a prosperous existing resource for bioprospecting, covering 70% of the planet earth, and hosting a huge biodiversity. Advances in the research are progressively uncovering the presence of unknown microorganisms, which have evolved unique metabolic and genetic pathways for the production of uncommon secondary metabolites. Fungi have a leading role in marine bioprospecting since they represent a prolific source of structurally diverse bioactive metabolites. Several bioactive compounds from marine fungi have already been characterized including antibiotics, anticancer, antioxidants and antivirals. Nowadays, the search for natural antioxidant molecules capable of replacing those synthetic currently used, is an aspect that is receiving significant attention. Antioxidants can inactivate reactive oxygen and nitrogen species, preventing the insurgence of several degenerative diseases including cancer, autoimmune disorders, cardiovascular and neurodegenerative diseases. Moreover, they also find applications in different fields, including food preservation, healthcare and cosmetics. This review focuses on the production of antioxidants from marine fungi. We begin by proposing a survey of the available tools suitable for the evaluation of antioxidants, followed by the description of various classes of marine fungi antioxidants together with their extraction strategies. In addition, a view of the future perspectives and trends of these natural products within the "blue economy" is also presented.

Molecular Network and Culture Media Variation Reveal a Complex Metabolic Profile in Pantoea cf. eucrina D2 Associated with an Acidified Marine Sponge.

The Gram-negative Pantoea eucrina D2 was isolated from the marine sponge Chondrosia reniformis. Sponges were collected in a shallow volcanic vents system in Ischia island (South Italy), influenced by CO2 emissions and lowered pH. The chemical diversity of the secondary metabolites produced by this strain, under different culture conditions, was explored by a combined approach including molecular networking, pure compound isolation and NMR spectroscopy. The metabolome of Pantoea cf. eucrina D2 yielded a very complex molecular network, allowing the annotation of several metabolites, among them two biosurfactant clusters: lipoamino acids and surfactins. The production of each class of metabolites was highly dependent on the culture conditions, in particular, the production of unusual surfactins derivatives was reported for the first time from this genus; interestingly the production of these metabolites only arises by utilizing inorganic nitrogen as a sole nitrogen source. Major components of the extract obtained under standard medium culture conditions were isolated and identified as N-lipoamino acids by a combination of 1D and 2D NMR spectroscopy and HRESI-MS analysis. Assessment of the antimicrobial activity of the pure compounds towards some human pathogens, indicated a moderate activity of leucine containing N-lipoamino acids towards Staphylococcus aureus, Staphylococcus epidermidis and a clinical isolate of the emerging food pathogen Listeria monocytogenes.

Characterization of a New Mixture of Mono-Rhamnolipids Produced by Pseudomonas gessardii Isolated from Edmonson Point (Antarctica).

Rhamnolipids (RLs) are surface-active molecules mainly produced by Pseudomonas spp. Antarctica is one of the less explored places on Earth and bioprospecting for novel RL producer strains represents a promising strategy for the discovery of novel structures. In the present study, 34 cultivable bacteria isolated from Edmonson Point Lake, Ross Sea, Antarctica were subjected to preliminary screening for the biosurfactant activity. The positive strains were identified by 16S rRNA gene sequencing and the produced RLs were characterized by liquid chromatography coupled to high resolution mass spectrometry (LC-HRESIMS) and liquid chromatography coupled with tandem spectrometry (LC-MS/MS), resulting in a new mixture of 17 different RL congeners, with six previously undescribed RLs. We explored the influence of the carbon source on the RL composition using 12 different raw materials, such as monosaccharides, polysaccharides and petroleum industry derivatives, reporting for the first time the production of RLs using, as sole carbon source, anthracene and benzene. Moreover, we investigated the antimicrobial potential of the RL mixture, towards a panel of both Gram-positive and Gram-negative pathogens, reporting very interesting results towards Listeria monocytogenes with a minimum inhibitory concentration (MIC) value of 3.13 µg/mL. Finally, we report for the first time the antimicrobial activity of RLs towards three strains of the emerging multidrug resistant Stenotrophomonas maltophilia with MIC values of 12.5 µg/ml.

Genomics-Metabolomics Profiling Disclosed Marine Vibrio spartinae 3.6 as a Producer of a New Branched Side Chain Prodigiosin.

A wide range of prescreening tests for antimicrobial activity of 59 bacterial isolates from sediments of Ria Formosa Lagoon (Algarve, Portugal) disclosed Vibrio spartinae 3.6 as the most active antibacterial producing strain. This bacterial strain, which has not previously been submitted for chemical profiling, was subjected to de novo whole genome sequencing, which aided in the discovery and elucidation of a prodigiosin biosynthetic gene cluster that was predicted by the bioinformatic tool KEGG BlastKoala. Comparative genomics led to the identification of a new membrane di-iron oxygenase-like enzyme, annotated as Vspart_02107, which is likely to be involved in the biosynthesis of cycloprodigiosin and analogues. The combined genomics-metabolomics profiling of the strain led to the isolation and identification of one new branched-chain prodigiosin (5) and to the detection of two new cyclic forms. Furthermore, the evaluation of the minimum inhibitory concentrations disclosed the major prodigiosin as very effective against multi-drug-resistant pathogens including Stenotrophomonas maltophilia, a clinical isolate of Listeria monocytogenes, as well as some human pathogens reported by the World Health Organization as prioritized targets.

Marine Collagen from Alternative and Sustainable Sources: Extraction, Processing and Applications.

Due to its unique properties, collagen is used in the growing fields of pharmaceutical and biomedical devices, as well as in the fields of nutraceuticals, cosmeceuticals, food and beverages. Collagen also represents a valid resource for bioplastics and biomaterials, to be used in the emerging health sectors. Recently, marine organisms have been considered as promising sources of collagen, because they do not harbor transmissible disease. In particular, fish biomass as well as by-catch organisms, such as undersized fish, jellyfish, sharks, starfish, and sponges, possess a very high collagen content. The use of discarded and underused biomass could contribute to the development of a sustainable process for collagen extraction, with a significantly reduced environmental impact. This addresses the European zero-waste strategy, which supports all three generally accepted goals of sustainability: sustainable economic well-being, environmental protection, and social well-being. A zero-waste strategy would use far fewer new raw materials and send no waste materials to landfills. In this review, we present an overview of the studies carried out on collagen obtained from by-catch organisms and fish wastes. Additionally, we discuss novel technologies based on thermoplastic processes that could be applied, likewise, as marine collagen treatment.