Introduction of New Tools for Chemical Biology Research on Microbial Metabolites

Recently, high-throughput screening (HTS) has become the mainstream technique for drug discovery. Compounds that are synthesized by combinatorial chemistry might be more suitable than natural products to apply to HTS, because the purification procedure is a drawback of using natural products. Nevertheless, natural products remain an extremely important source of drugs. To overcome the demerits of natural products, we are constructing the RIKEN Natural Products Depository (NPDepo) that is focused primarily on microbial metabolites. In this review, I describe (i) engineering pathways for biosynthetic gene clusters of microbial metabolites, (ii) construction of fraction libraries of microbial metabolites, and (iii) the development of a new screening system using a chemical array and a protein library produced by GLORIA.     

Construction of a microbial natural product library for chemical biology studies

The RIKEN Natural Products Depository (NPDepo) is a public depository of small molecules. Currently, the NPDepo chemical library contains 39,200 pure compounds, half of which are natural products and their derivatives. In order to reinforce the uniqueness of our chemical library, we have improved our strategies for the collection of microbial natural products. Firstly, a microbial metabolite fraction library coupled with an MP (microbial products) plot database provides a powerful resource for the efficient isolation of microbial metabolites. Secondly, biosynthetic studies of microbial metabolites have enabled us to not only access ingenious biosynthetic machineries, but also obtain a variety of biosynthetic intermediates. Our chemical library contributes to the discovery of molecular probes for increasing our understanding of complex biological processes and for eventually developing new drug leads.     

Exploring anti-TB leads from natural products library originated from marine microbes and medicinal plants

Multidrug-resistant tuberculosis (MDR-TB) and TB–HIV co-infection have become a great threat to global health. However, the last truly novel drug that was approved for the treatment of TB was discovered 40?years ago. The search for new effective drugs against TB has never been more intensive. Natural products derived from microbes and medicinal plants have been an important source of TB therapeutics. Recent advances have been made to accelerate the discovery rate of novel TB drugs including diversifying strategies for environmental strains, high-throughput screening (HTS) assays, and chemical diversity. This review will discuss the challenges of finding novel natural products with anti-TB activity from marine microbes and plant medicines, including biodiversity- and taxonomy-guided microbial natural products library construction, target- and cell-based HTS, and bioassay-directed isolation of anti-TB substances from traditional medicines.     

Diversifying microbial natural products for drug discovery

Historically, nature has provided the source for the majority of the drugs in use today. More than 20,000 microbial secondary metabolites have been described, but only a small percentage of these have been carried forward as natural product drugs. Natural products are in tough competition with large chemical libraries and with combinatorial chemistries. Hence, each step of a natural product program has to be more efficient than ever, starting from the collection of environmental samples and the selection of strains, to metabolic expression, genetic exploitation, sample preparation and chemical dereplication. This review will focus on approaches for diversifying microbial natural product strains and extract libraries, while decreasing genetic and chemical redundancy.V. Knight and J.-J. Sanglier contributed equally to this work     

Bioenergetic and proteomic profiling to screen small molecule inhibitors that target cancer metabolisms

Cancer cells can reprogram their metabolic machinery to survive. This altered metabolism, which is distinct from the metabolism of normal cells, is thought to be a possible target for the development of new cancer therapies. In this study, we constructed a screening system that focuses on bioenergetic profiles (specifically oxygen consumption rate and extracellular acidification rate) and characteristic proteomic changes. Thus, small molecules that target cancer-specific metabolism were investigated. We screened the chemical library of RIKEN Natural Products Depository (NPDepo) and found that unantimycin A, which was recently isolated from the fraction library of microbial metabolites, and NPL40330, which is derived from a chemical library, inhibit mitochondrial respiration. Furthermore, we developed an in vitro reconstitution assay method for mitochondrial electron transport chain using semi-intact cells with specific substrates for each complex of the mitochondrial electron transport chain. Our findings revealed that NPL40330 and unantimycin A target mitochondrial complexes I and III, respectively.     

Informatic search strategies to discover analogues and variants of natural product archetypes

Natural products are a crucial source of antimicrobial agents, but reliance on low-resolution bioactivity-guided approaches has led to diminishing interest in discovery programmes. Here, we demonstrate that two in-house automated informatic platforms can be used to target classes of biologically active natural products, specifically, peptaibols. We demonstrate that mass spectrometry-based informatic approaches can be used to detect natural products with high sensitivity, identifying desired agents present in complex microbial extracts. Using our specialised software packages, we could elaborate specific branches of chemical space, uncovering new variants of trichopolyn and demonstrating a way forward in mining natural products as a valuable source of potential pharmaceutical agents.     

Metagenomic approaches to exploit the biotechnological potential of the microbial consortia of marine sponges

Natural products isolated from sponges are an important source of new biologically active compounds. However, the development of these compounds into drugs has been held back by the difficulties in achieving a sustainable supply of these often-complex molecules for pre-clinical and clinical development. Increasing evidence implicates microbial symbionts as the source of many of these biologically active compounds, but the vast majority of the sponge microbial community remain uncultured. Metagenomics offers a biotechnological solution to this supply problem. Metagenomes of sponge microbial communities have been shown to contain genes and gene clusters typical for the biosynthesis of biologically active natural products. Heterologous expression approaches have also led to the isolation of secondary metabolism gene clusters from uncultured microbial symbionts of marine invertebrates and from soil metagenomic libraries. Combining a metagenomic approach with heterologous expression holds much promise for the sustainable exploitation of the chemical diversity present in the sponge microbial community.     

Expression and isolation of antimicrobial small molecules from soil DNA libraries

Natural products have been a critically important source of clinically relevant small molecule therapeutics. However, the discovery rate of novel structural classes of antimicrobial molecules has declined. Recently, increasing evidence has shown that the number of species cultivated from soil represents less than 1% of the total population, opening up the exciting possibility that these uncultured species may provide a large untapped pool from which novel natural products can be discovered. We have constructed and expressed in E. coli a BAC (bacterial artificial chromosome) library containing genomic fragments of DNA (5-120kb) isolated directly from soil organisms (S-DNA). Screening of the library resulted in the identification of several antimicrobial activities expressed by different recombinant clones. One clone (mg1.1) has been partially characterized and found to express several small molecules related to and including indirubin. These results show that genes involved in natural product synthesis can be cloned directly from S-DNA and expressed in a heterologous host, supporting the idea that this technology has the potential to provide novel natural products from the wealth of environmental microbial diversity and is a potentially important new tool for drug discovery.     

Protein structure similarity clustering (PSSC) and natural product structure as inspiration sources for drug development and chemical genomics

Finding small molecules that modulate protein function is of primary importance in drug development and in the emerging field of chemical genomics. To facilitate the identification of such molecules, we developed a novel strategy making use of structural conservatism found in protein domain architecture and natural product inspired compound library design. Domains and proteins identified as being structurally similar in their ligand-sensing cores are grouped in a protein structure similarity cluster (PSSC). Natural products can be considered as evolutionary pre-validated ligands for multiple proteins and therefore natural products that are known to interact with one of the PSSC member proteins are selected as guiding structures for compound library synthesis. Application of this novel strategy for compound library design provided enhanced hit rates in small compound libraries for structurally similar proteins.     

Discovering new bioactive molecules from microbial sources

There is an increased need for new drug leads to treat diseases in humans, animals and plants. A dramatic example is represented by the need for novel and more effective antibiotics to combat multidrug-resistant microbial pathogens. Natural products represent a major source of approved drugs and still play an important role in supplying chemical diversity, despite a decreased interest by large pharmaceutical companies. Novel approaches must be implemented to decrease the chances of rediscovering the tens of thousands of known natural products. In this review, we present an overview of natural product screening, focusing particularly on microbial products. Different approaches can be implemented to increase the probability of finding new bioactive molecules. We thus present the rationale and selected examples of the use of hypersensitive assays; of accessing unexplored microorganisms, including the metagenome; and of genome mining. We then focus our attention on the technology platform that we are currently using, consisting of approximately 70 000 microbial strains, mostly actinomycetes and filamentous fungi, and discuss about high-quality screening in the search for bioactive molecules. Finally, two case studies are discussed, including the spark that arose interest in the compound: in the case of orthoformimycin, the novel mechanism of action predicted a novel structural class; in the case of NAI-112, structural similarity pointed out to a possible in vivo activity. Both predictions were then experimentally confirmed.     

微生物代谢产物库研究进展

微生物代谢物具有极大的化学结构多样性和复杂性,建立微生物代谢物库对发现新药有重要意义。对几种重要的微生物代谢物库及建库方法作一综述。     

Harnessing the potential of chemical defenses from antimicrobial activities

Resistance to the drugs used in the treatment of many infectious diseases is increasing, while microbial infections are being found to be responsible for more life-threatening diseases than previously thought. Despite a large investment in the invention and application of high-throughput screening techniques involving miniaturization and automation, and a diverse array of strategies for designing and constructing various chemical libraries, relatively few new drugs have resulted. Natural products, however, have been a major source of drugs for centuries. Since some of them are produced by organisms as a result of selection in favour of improved defense against competing deleterious microorganisms, in principle they would be less likely to incur resistance. Furthermore, the production of those defensive secondary metabolites is inducible because their original function is a response to environmental challenges. Moreover, symbioses, co-habitation associations between two or more different species of organisms, are universal in nature, and the production of secondary metabolites by symbiotic microbes may be an important adaptation allowing microbes to affect their hosts. Therefore, co-culture strategies, using combinations of plant cell-pathogenic microbes, plant cell-endophytes (or symbionts), and symbiont-pathogenic microbes, based on the principles of chemical defense and the known mechanisms of organism interactions, may be an efficient general approach in the search for new anti-microbial drugs.     

Importance of microbial natural products and the need to revitalize their discovery

Microbes are the leading producers of useful natural products. Natural products from microbes and plants make excellent drugs. Significant portions of the microbial genomes are devoted to production of these useful secondary metabolites. A single microbe can make a number of secondary metabolites, as high as 50 compounds. The most useful products include antibiotics, anticancer agents, immunosuppressants, but products for many other applications, e.g., antivirals, anthelmintics, enzyme inhibitors, nutraceuticals, polymers, surfactants, bioherbicides, and vaccines have been commercialized. Unfortunately, due to the decrease in natural product discovery efforts, drug discovery has decreased in the past 20 years. The reasons include excessive costs for clinical trials, too short a window before the products become generics, difficulty in discovery of antibiotics against resistant organisms, and short treatment times by patients for products such as antibiotics. Despite these difficulties, technology to discover new drugs has advanced, e.g., combinatorial chemistry of natural product scaffolds, discoveries in biodiversity, genome mining, and systems biology. Of great help would be government extension of the time before products become generic.     

Advances in microbial biotechnology of bile acids

The recent advances in microbial biotechnology of production of bile acid metabolites helped to identify a number of neutral and acidic steroidal compounds useful as drugs and drug intermediates on a scale which would not have been possible by classical chemical transformations. Microbial transformations viz., hydroxylation, dehydroxylation, reduction of the carbonyl moieties, epimerization, side-chain metabolism, introduction of carbon-carbon double bonds into the steroid nucleus, deconjugation of bile acid conjugates carried out by various microorganisms for production of useful metabolites with special reference to newer techniques including cell immobilization and transposon mutagenesis for selective transformations are reviewed. The different pathways of microbial degradation of bile acids leading to the formation of various products are discussed. A compilation of the metabolites formed by various microorganisms from the bile acids or their conjugates and reported during the period 1979-1992 is also provided.     

Microbial metabolism of methyl protodioscin by Aspergillus niger culture--a new androstenedione producing way from steroid

Methyl protodioscin (1), a natural furostanol biglycoside steroid, was a preclinical anticancer drug, which showed potent activity against most cell lines from leukemia and solid tumors in the National Cancer Institute's (NCI) human cancer panel. Metabolism of methyl protodioscin by Aspergillus niger was investigated. Seven metabolites were isolated and identified. Two main metabolites were pregnane glycosides and four were furostanol glycosides, together with the aglycone. It was found that steroidal saponin skeleton could be converted to pregnenolone skeleton only using microbial methods, which must have chemical procedures in the reported literatures. The proposed biosynthetic pathways of the microbial conversion products of methyl protodioscin were drawn. The found enriched the reaction types of microbial bioconversion and provided a new producing way of androstenedione from steroid. Most metabolites showed strong cytotoxic activities against HepG2, NCI-H460, HeLa, and MCF-7 cell lines.     

All natural

Natural products research focuses on the chemical properties, biosynthesis and biological functions of secondary metabolites. As our scientific understanding of all things 'natural' is rapidly expanding, we should also make time to communicate the subtleties of chemical distinctions to the public.     

Natural products potential of Dictyoceratida sponges-associated micro-organisms

The marine environment represents one of the most underexplored environments in the world. Marine sponges have a higher taxonomic diversity according to definite environmental conditions. They have been considered interesting sources for bioactive compounds. Dictyoceratida sponges are divided into five families which are widely distributed and habituating different types of micro-organisms. However, some secondary metabolites are probably not produced by the sponges themselves, but rather by their associated micro-organisms. These secondary metabolites are characterized by different chemical structures and consequently different biological activities. This review outlines the reported secondary metabolites from micro-organisms associated with Dictyoceratida sponges and their investigated biological activities from 1991 to 2019. The increasing research studies in this field can play a major role in marine microbial natural products drug discovery in the future.     

Role of natural product diversity in chemical biology

Through the natural selection process, natural products possess a unique and vast chemical diversity and have been evolved for optimal interactions with biological macromolecules. Owing to their diversity, target affinity, and specificity, natural products have demonstrated enormous potential as modulators of biomolecular function, been an essential source for drug discovery, and provided design principles for combinatorial library development.     

Fate of 14C-labeled microbial products derived from nitrifying bacteria in autotrophic nitrifying biofilms

The cross-feeding of microbial products derived from 14C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [14C]bicarbonate, biofilm samples were incubated with and without NH4+ as a sole energy source for 10 days. The transfer of 14C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the beta-Proteobacteria showed significant uptake of 14C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH4+ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized 14C-labeled products in the culture with NH4+ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of 14C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

Mixed fermentation for natural product drug discovery

Natural products continue to play a major role in drug discovery and development. However, chemical redundancy is an ongoing problem. Genomic studies indicate that certain groups of bacteria and fungi have dozens of secondary metabolite pathways that are not expressed under standard laboratory growth conditions. One approach to more fully access the metabolic potential of cultivatable microbes is mixed fermentation, where the presence of neighboring microbes may induce secondary metabolite synthesis. Research to date indicates that mixed fermentation can result in increased antibiotic activity in crude extracts, increased yields of previously described metabolites, increased yields of previously undetected metabolites, analogues of known metabolites resulting from combined pathways and, importantly, induction of previously unexpressed pathways for bioactive constituents.