Unlocking the biosynthetic machinery for bioactive natural products in marine extremophiles: metabologenomic approaches to biotechnological production

Marine habitats are among the largest and most biodiverse ecosystems on the planet, constituting an almost untapped reservoir of bioactive compounds with enormous application potential in various fields, including medicine, biotechnology, and industry. The chemical diversity of marine natural products (MNPs), often referred to as secondary metabolites, has attracted increasing scientific interest due to their structural uniqueness and potent biological activities. Indeed, many classes of natural products have been designed by nature which placed every single atom at the right position to interact selectively with key cellular targets and play specific roles in cell processes. These compounds include alkaloids, peptides, lipopeptides, and polysaccharides, which have demonstrated significant therapeutic activities, including antimicrobial, anticancer, antiviral, anti-inflammatory, and antioxidant properties. It has become a sort of paradigm that many MNPs are synthesized by marine bacteria, which may exist as free-living organisms or form symbiotic or pathogenic relationships with eukaryotic hosts (sponges, tunicates, soft corals) or other prokaryotes. Therefore, marine environments are a compelling arena where each microbial strain had to develop chemical signaling systems and secondary-metabolite biosynthetic machineries to (a) communicate with the host and other microbial species, (b) gain advantage over competitors, (c) defend itself or the host against predators, and (d) retrieve nutrients from the environment. This makes marine bacteria, and particularly those inhabiting extremophilic niches (e.g., deep seas and oceans, hydrothermal vents, contaminated waters) a cornucopia for the discovery of new chemical entities of potential efficacy for clinical and industrial uses. However, the exploitation of marine resources brings challenges related to biodiversity conservation, the difficulty of cultivating extremophilic microorganisms under controlled conditions, and the supply of such compounds. In this context, the integration of traditional and modern biotechnological approaches appears to be a promising strategy to sustainably explore and enhance marine biodiversity while minimizing ecological impact. The advancement of knowledge about extremophilic bacteria and their metabolic capabilities is a key contribution to an expanding field of research that highlights the vast untapped potential of marine ecosystems as sources of biotechnological solutions. In this scenario, this doctoral thesis focuses on the discovery, characterization and biotechnological application of novel MNPs derived from extremophilic bacteria. Using an approach that integrates genomic analysis, metabolomics, and bioactivity assays, this study aims to address the main challenges related to marine bioprospecting and to discover novel bioactive compounds with promising applications in pharmaceutical and industrial fields. One of the bacteria studied in this work is Shewanella aquimarina CIP 108633T, belonging to a genus known for its extraordinary ecological versatility, which allows it to thrive in extreme environments, from deep marine sediments to sea ice and freshwater ecosystems. S. aquimarina is a Gram-negative rod-shaped bacterium that exhibits remarkable metabolic and physiological adaptability, allowing it to utilize a wide range of substrates. This bacterium is also capable of producing numerous bioactive metabolites of great biotechnological interest. S. aquimarina CIP 108633T turned out to be a factory of imidazolium alkaloids, namely shewazoles, which have attracted attention for their structural novelty and potent biological activities. These compounds were identified using advanced dereplication techniques, i.e. molecular networking, and bioactivity-guided fractionation. The alkaloid mixture was shown to (a) exert antimicrobial and antibiofilm activity against antibiotic-resistant Staphylococcus aureus clinical isolates, (b) act synergistically with antibiotics currently included in clinical settings such as tigecycline and linezolid, (c) restore methicillin-resistant Staphylococcus aureus (MRSA) sensitivity to fosfomycin, and (d) have anthelminthic effects against Caenorhabditis elegans. In addition, the mixture exhibited antiviral properties against viruses with and without envelopes, such as coronavirus HcoV-229E, herpes simplex viruses, and poliovirus PV-1. The biosurfactant nature of shewazoles partially explains their broad-spectrum activities, thus making them promising candidates for breaking down drug-resistant infectious diseases. The second aim of my research work was exploring the metabolome of Rhodococcus sp. I2R, isolated from deep-sea sediments collected in the Southern Tyrrhenian Sea. The genus Rhodococcus is known for its ability to degrade a wide range of organic compounds, including hydrocarbons and solvents, thereby being used in environmental biotechnology for bioremediation. In a previous work, Rhodococcus sp. I2R has been identified as a producer of unprecedented succinoyl trehalolipids, glycolipid biosurfactants possessing antiviral properties. However, genomic analysis of Rhodococcus sp. I2R revealed a still unexplored biosynthetic repertoire, with numerous biosynthetic gene clusters responsible for the production of compounds of industrial and pharmaceutical interest, such as biosurfactants, carotenoids, and lipopeptides. In particular, lipopeptides are low molecular weight metabolites known for their antimicrobial and anticancer activities. These compounds are mainly synthesized by non-ribosomal peptide synthetases (NRPS), large multimodular enzymes that assemble complex molecules through sequential condensation of amino acids. Genome mining revealed several NRPS gene clusters, two of them being associated with the biosynthesis of lipopeptides. Using a metabologenomic approach, two families of novel lipopeptides were identified and designated as rhodoheptins and rhodamides. Rhodoheptins are cyclic lipoheptapeptides and rhodamides are linear 14‐mer glycolipopeptides, which demonstrated significant tensioactive activity for potential applications in bioremediation and pharmaceutics. These findings highlighted the utility of the genome-driven metabolite discovery to disclose bioactive molecules and elucidate their stereostructure. The last bacterium investigated during my PhD research, was Novosphingobium sp. PP1Y, isolated from hydrocarbon-contaminated waters in the harbour of Pozzuoli (Napoli, Italy). Bacterial genome mining led to the discovery of new lassopeptides, i. e. novosphingocins, a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) bearing a stable macrolactam ring. Novosphingocins were shown to inhibit the entry of herpes simplex virus type I into human cells. Heterologous production of novosphingocins in Escherichia coli allowed to overcome the low-yield production in the native host, paving the way for industrial large-scale fermentation to obtain these promising lead compounds.