Chemical screening method for the rapid identification of microbial sources of marine invertebrate-associated metabolites

Marine invertebrates have proven to be a rich source of secondary metabolites. The growing recognition that marine microorganisms associated with invertebrate hosts are involved in the biosynthesis of secondary metabolites offers new alternatives for the discovery and development of marine natural products. However, the discovery of microorganisms producing secondary metabolites previously attributed to an invertebrate host poses a significant challenge. This study describes an efficient chemical screening method utilizing a 96-well plate-based bacterial cultivation strategy to identify and isolate microbial producers of marine invertebrate-associated metabolites.     

Impact of Co-Culture on the Metabolism of Marine Microorganisms

Natural products from plants have been listed for hundreds of years as a source of biologically active molecules. In recent years, the marine environment has demonstrated its ability to provide new structural entities. More than 70% of our planet’s surface is covered by oceans, and with the technical advances in diving and remotely operated vehicles, it is becoming easier to collect samples. Although the risk of rediscovery is significant, the discovery of silent gene clusters and innovative analytical techniques has renewed interest in natural product research. Different strategies have been proposed to activate these silent genes, including co-culture, or mixed fermentation, a cultivation-based approach. This review highlights the potential of co-culture of marine microorganisms to induce the production of new metabolites as well as to increase the yields of respective target metabolites with pharmacological potential, and moreover to indirectly improve the biological activity of a crude extract.     

Recent Advances in the Heterologous Expression of Biosynthetic Gene Clusters for Marine Natural Products

Marine natural products (MNPs) are an important source of biologically active metabolites, particularly for therapeutic agent development after terrestrial plants and nonmarine microorganisms. Sequencing technologies have revealed that the number of biosynthetic gene clusters (BGCs) in marine microorganisms and the marine environment is much higher than expected. Unfortunately, the majority of them are silent or only weakly expressed under traditional laboratory culture conditions. Furthermore, the large proportion of marine microorganisms are either uncultivable or cannot be genetically manipulated. Efficient heterologous expression systems can activate cryptic BGCs and increase target compound yield, allowing researchers to explore more unknown MNPs. When developing heterologous expression of MNPs, it is critical to consider heterologous host selection as well as genetic manipulations for BGCs. In this review, we summarize current progress on the heterologous expression of MNPs as a reference for future research.     

Chemical Diversity and Biological Properties of Secondary Metabolites from Sea Hares of Aplysia Genus

The marine environment is an important source of structurally-diverse and biologically-active secondary metabolites. During the last two decades, thousands of compounds were discovered in marine organisms, several of them having inspired the development of new classes of therapeutic agents. Marine mollusks constitute a successful phyla in the discovery of new marine natural products (MNPs). Over a 50-year period from 1963, 116 genera of mollusks contributed innumerous compounds, Aplysia being the most studied genus by MNP chemists. This genus includes 36 valid species and should be distinguished from all mollusks as it yielded numerous new natural products. Aplysia sea hares are herbivorous mollusks, which have been proven to be a rich source of secondary metabolites, mostly of dietary origin. The majority of secondary metabolites isolated from sea hares of the genus Aplysia are halogenated terpenes; however, these animals are also a source of compounds from other chemical classes, such as macrolides, sterols and alkaloids, often exhibiting cytotoxic, antibacterial, antifungal, antiviral and/or antifeedant activities. This review focuses on the diverse structural classes of secondary metabolites found in Aplysia spp., including several compounds with pronounced biological properties.     

Investigation of Interspecies Interactions within Marine Micromonosporaceae Using an Improved Co-Culture Approach

With respect to bacterial natural products, a significant outcome of the genomic era was that the biosynthetic potential in many microorganisms surpassed the number of compounds isolated under standard laboratory growth conditions, particularly among certain members in the phylum Actinobacteria. Our group, as well as others, investigated interspecies interactions, via co-culture, as a technique to coax bacteria to produce novel natural products. While co-culture provides new opportunities, challenges exist and questions surrounding these methods remain unanswered. In marine bacteria, for example, how prevalent are interspecies interactions and how commonly do interactions result in novel natural products? In an attempt to begin to answer basic questions surrounding co-culture of marine microorganisms, we have tested both antibiotic activity-based and LC/MS-based methods to evaluate Micromonosporaceae secondary metabolite production in co-culture. Overall, our investigation of 65 Micromonosporaceae led to the identification of 12 Micromonosporaceae across three genera that produced unique metabolites in co-culture. Our results suggest that interspecies interactions were prevalent between marine Micromonosporaceae and marine mycolic acid-containing bacteria. Furthermore, our approach highlights a sensitive and rapid method for investigating interspecies interactions in search of novel antibiotics, secondary metabolites, and genes.     

Anticancer Activities of Plant Secondary Metabolites: Rice Callus Suspension Culture as a New Paradigm

Plant natural products including alkaloids,polyphenols,terpenoids and flavonoids have been reported to exert anticancer activity by targeting various metabolic pathways.The biological pathways regulated by plant products can serve as novel drug targets.Plant natural compounds or their derivatives used for cancer treatment and some novel plant-based compounds which are used in clinical trials were discussed.Callus suspension culture with secondary metabolites can provide a continuous source of plant pharmaceuticals without time and space limitations.Previous research has shown that rice callus suspension culture can kill>95%cancer cells with no significant effect on the growth of normal cells.The role of candidate genes and metabolites which are likely to be involved in the process and their potential to serve as anticancer and anti-inflammatory agents were discussed.Large scale production of plant callus suspension culture and its constituents can be achieved using elicitors which enhance specific secondary metabolites combined with bioprocess technology.     

Culturable Microorganisms Associated with Sea Cucumbers and Microbial Natural Products

Sea cucumbers are a class of marine invertebrates and a source of food and drug. Numerous microorganisms are associated with sea cucumbers. Seventy-eight genera of bacteria belonging to 47 families in four phyla, and 29 genera of fungi belonging to 24 families in the phylum Ascomycota have been cultured from sea cucumbers. Sea-cucumber-associated microorganisms produce diverse secondary metabolites with various biological activities, including cytotoxic, antimicrobial, enzyme-inhibiting, and antiangiogenic activities. In this review, we present the current list of the 145 natural products from microorganisms associated with sea cucumbers, which include primarily polyketides, as well as alkaloids and terpenoids. These results indicate the potential of the microorganisms associated with sea cucumbers as sources of bioactive natural products.     

Comparative Metabologenomics Analysis of Polar Actinomycetes

Biosynthetic and chemical datasets are the two major pillars for microbial drug discovery in the omics era. Despite the advancement of analysis tools and platforms for multi-strain metabolomics and genomics, linking these information sources remains a considerable bottleneck in strain prioritisation and natural product discovery. In this study, molecular networking of the 100 metabolite extracts derived from applying the OSMAC approach to 25 Polar bacterial strains, showed growth media specificity and potential chemical novelty was suggested. Moreover, the metabolite extracts were screened for antibacterial activity and promising selective bioactivity against drug-persistent pathogens such as Klebsiella pneumoniae and Acinetobacter baumannii was observed. Genome sequencing data were combined with metabolomics experiments in the recently developed computational approach, NPLinker, which was used to link BGC and molecular features to prioritise strains for further investigation based on biosynthetic and chemical information. Herein, we putatively identified the known metabolites ectoine and chrloramphenicol which, through NPLinker, were linked to their associated BGCs. The metabologenomics approach followed in this study can potentially be applied to any large microbial datasets for accelerating the discovery of new (bioactive) specialised metabolites.     

Advances in Marine Microbial Symbionts in the China Sea and Related Pharmaceutical Metabolites

Marine animals and plants such as sponges, sea squirts, corals, worms and algae host diverse and abundant symbiotic microorganisms. Marine microbial symbionts are possible the true producers or take part in the biosynthesis of some bioactive marine natural products isolated from the marine organism hosts. Investigation of the pharmaceutical metabolites may reveal the biosynthesis mechanisms of related natural products and solve the current problem of supply limitation in marine drug development. This paper reviews the advances in diversity revelation, biological activity and related pharmaceutical metabolites, and functional genes of marine microbial symbionts from the China Sea.     

Structures and Biological Activities of Diketopiperazines from Marine Organisms: A Review

Diketopiperazines are potential structures with extensive biological functions, which have attracted much attention of natural product researchers for a long time. These compounds possess a stable six-membered ring, which is an important pharmacophore. The marine organisms have especially been proven to be a wide source for discovering diketopiperazine derivatives. In recent years, more and more interesting bioactive diketopiperazines had been found from various marine habitats. This review article is focused on the new 2,5-diketopiperazines derived from marine organisms (sponges and microorganisms) reported from the secondary half-year of 2014 to the first half of the year of 2021. We will comment their chemical structures, biological activities and sources. The objective is to assess the merit of these compounds for further study in the field of drug discovery.     

Progress in Research on Bioactive Secondary Metabolites from Deep-Sea Derived Microorganisms

Deep sea has an extreme environment which leads to biodiversity of microorganisms and their unique physical and biochemical mechanisms. Deep-sea derived microorganisms are more likely to produce novel bioactive substances with special mechanism of action for drug discovery. This article reviews secondary metabolites with biological activities such as anti-tumor, anti-bacterial, anti-viral, and anti-inflammatory isolated from deep-sea fungi and bacteria during 2018–2020. Effective methods for screening and obtaining natural active compounds from deep-sea microorganisms are also summarized, including optimizing the culture conditions, using genome mining technology, biosynthesis and so on. The comprehensive application of these methods makes broader prospects for the development and application of deep sea microbial bioactive substances.     

Drugs from the Sea -Opportunities and Obstacles

The supply problem with regard to drug development and sustainable production lies in the limited amounts of biomass of most marine invertebrates available from wild stocks. Thus, most pharmacologically active marine natural products can only be isolated in minute yields. Total synthesis of pharmacologically active natural products has been successfully established but is in many cases economically not feasible due to the complexity of the molecular structures and the low yields. To solve the pressing supply issue in marine drug discovery, other strategies appear to be more promising. One of these is mariculture which has successfully been established with the bryozoan Bugula neritina (the source of the bryostatins) and the tunicate Ecteinascidia turbinata (the source of ET-743). Another strategy involves partial synthesis from precursors which are biotechnologically available. An example is ET-743 that can be partially synthesized from safracin B which is a metabolite of Pseudomonas fluorescens. There have been many examples of striking structural similarities between natural products obtained from marine invertebrates and those of microbial origin which suggests that microorganisms living in their invertebrate hosts could be the actual producers of these secondary metabolites. With regard to sustainable biotechnological production of pharmacologically important metabolites from marine invertebrates and their “endosymbionts”, a more advanced strategy is to focus on cloning and expression of the respective key biosynthetic gene clusters. This molecular biological approach will open up new avenues for biotechnological production of drugs or drug candidates from the sea.     

Genome Mining and Metabolic Profiling Uncover Polycyclic Tetramate Macrolactams from Streptomyces koyangensis SCSIO 5802

We have previously shown deep-sea-derived Streptomyces koyangensis SCSIO 5802 to produce two types of active secondary metabolites, abyssomicins and candicidins. Here, we report the complete genome sequence of S. koyangensis SCSIO 5802 employing bioinformatics to highlight its potential to produce at least 21 categories of natural products. In order to mine novel natural products, the production of two polycyclic tetramate macrolactams (PTMs), the known 10-epi-HSAF (1) and a new compound, koyanamide A (2), was stimulated via inactivation of the abyssomicin and candicidin biosynthetic machineries. Detailed bioinformatics analyses revealed a PKS/NRPS gene cluster, containing 6 open reading frames (ORFs) and spanning ~16 kb of contiguous genomic DNA, as the putative PTM biosynthetic gene cluster (BGC) (termed herein sko). We furthermore demonstrate, via gene disruption experiments, that the sko cluster encodes the biosynthesis of 10-epi-HSAF and koyanamide A. Finally, we propose a plausible biosynthetic pathway to 10-epi-HSAF and koyanamide A. In total, this study demonstrates an effective approach to cryptic BGC activation enabling the discovery of new bioactive metabolites; genome mining and metabolic profiling methods play key roles in this strategy.     

A Practical Strategy to Discover New Antitumor Compounds by Activating Silent Metabolite Production in Fungi by Diethyl Sulphate Mutagenesis

Many fungal biosynthetic pathways are silent in standard culture conditions, and activation of the silent pathways may enable access to new metabolites with antitumor activities. The aim of the present study was to develop a practical strategy for microbial chemists to access silent metabolites in fungi. We demonstrated this strategy using a marine-derived fungus Penicillium purpurogenum G59 and a modified diethyl sulphate mutagenesis procedure. Using this strategy, we discovered four new antitumor compounds named penicimutanolone (1), penicimutanin A (2), penicimutanin B (3), and penicimutatin (4). Structures of the new compounds were elucidated by spectroscopic methods, especially extensive 2D NMR analysis. Antitumor activities were assayed by the MTT method using human cancer cell lines. Bioassays and HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses were used to estimate the activated secondary metabolite production. Compounds 2 and 3 had novel structures, and 1 was a new compound belonging to a class of very rare natural products from which only four members are so far known. Compounds 1–3 inhibited several human cancer cell lines with IC₅₀ values lower than 20 μM, and 4 inhibited the cell lines to some extent. These results demonstrated the effectiveness of this strategy to discover new compounds by activating silent fungal metabolic pathways. These discoveries provide rationale for the increased use of chemical mutagenesis strategies in silent fungal metabolite studies.     

Signal Transducers and Activators of Transcription (STAT) Regulatory Networks in Marine Organisms: From Physiological Observations towards Marine Drug Discovery

Part of our ocean’s richness comes from its extensive history of supporting life, resulting in a highly diverse ecological system. To date, over 250,000 species of marine organisms have been identified, but it is speculated that the actual number of marine species exceeds one million, including several hundreds of millions of species of marine microorganisms. Past studies suggest that approximately 70% of all deep-sea microorganisms, gorgonians, and sea sponges produce secondary metabolites with anti-cancer activities. Recently, novel FDA-approved drugs derived from marine sponges have been shown to reduce metastatic breast cancer, malignant lymphoma, and Hodgkin’s disease. Despite the fact that many marine natural products have been shown to possess a good inhibition potential against most of the cancer-related cell signaling pathways, only a few marine natural products have been shown to target JAK/STAT signaling. In the present paper, we describe the JAK/STAT signaling pathways found in marine organisms, before elaborating on the recent advances in the field of STAT inhibition by marine natural products and the potential application in anti-cancer drug discovery.     

Marketed Marine Natural Products in the Pharmaceutical and Cosmeceutical Industries: Tips for Success

The marine environment harbors a number of macro and micro organisms that have developed unique metabolic abilities to ensure their survival in diverse and hostile habitats, resulting in the biosynthesis of an array of secondary metabolites with specific activities. Several of these metabolites are high-value commercial products for the pharmaceutical and cosmeceutical industries. The aim of this review is to outline the paths of marine natural products discovery and development, with a special focus on the compounds that successfully reached the market and particularly looking at the approaches tackled by the pharmaceutical and cosmetic companies that succeeded in marketing those products. The main challenges faced during marine bioactives discovery and development programs were analyzed and grouped in three categories: biodiversity (accessibility to marine resources and efficient screening), supply and technical (sustainable production of the bioactives and knowledge of the mechanism of action) and market (processes, costs, partnerships and marketing). Tips to surpass these challenges are given in order to improve the market entry success rates of highly promising marine bioactives in the current pipelines, highlighting what can be learned from the successful and unsuccessful stories that can be applied to novel and/or ongoing marine natural products discovery and development programs.     

Quality not Quantity: The Role of Marine Natural Products in Drug Discovery and Reverse Chemical Proteomics

Reverse chemical proteomics combines affinity chromatography with phage display and promises to be a powerful new platform technology for the isolation of natural product receptors, facilitating the drug discovery process by rapidly linking biologically active small molecules to their cellular receptors and the receptors’ genes. In this paper we review chemical proteomics and reverse chemical proteomics and show how these techniques can add value to natural products research. We also report on techniques for the derivatisation of polystyrene microtitre plates with cleavable linkers and marine natural products that can be used in chemical proteomics or reverse chemical proteomics. Specifically, we have derivatised polystyrene with palau’amine and used reverse chemical proteomics to try and isolate the human receptors for this potent anticancer marine drug.     

Biomaterials and Bioactive Natural Products from Marine Invertebrates: From Basic Research to Innovative Applications

Aquatic invertebrates are a major source of biomaterials and bioactive natural products that can find applications as pharmaceutics, nutraceutics, cosmetics, antibiotics, antifouling products and biomaterials. Symbiotic microorganisms are often the real producers of many secondary metabolites initially isolated from marine invertebrates; however, a certain number of them are actually synthesized by the macro-organisms. In this review, we analysed the literature of the years 2010–2019 on natural products (bioactive molecules and biomaterials) from the main phyla of marine invertebrates explored so far, including sponges, cnidarians, molluscs, echinoderms and ascidians, and present relevant examples of natural products of interest to public and private stakeholders. We also describe omics tools that have been more relevant in identifying and understanding mechanisms and processes underlying the biosynthesis of secondary metabolites in marine invertebrates. Since there is increasing attention on finding new solutions for a sustainable large-scale supply of bioactive compounds, we propose that a possible improvement in the biodiscovery pipeline might also come from the study and utilization of aquatic invertebrate stem cells.