Microbial Natural Products in Drug Discovery

: Over a long period of time, humans have explored many natural resources looking for remedies of various ailments. Traditional medicines have played an intrinsic role in human life for thousands of years, with people depending on medicinal plants and their products as dietary supplements as well as using them therapeutically for treatment of chronic disorders, such as cancer, malaria, diabetes, arthritis, inﬂammation, and liver and cardiac disorders. However, plant resources are not su ﬃ cient for treatment of recently emerging diseases. In addition, the seasonal availability and other political factors put constrains on some rare plant species. The actual breakthrough in drug discovery came concurrently with the discovery of penicillin from Penicillium notatum in 1929. This discovery dramatically changed the research of natural products and positioned microbial natural products as one of the most important clues in drug discovery due to availability, variability, great biodiversity, unique structures, and the bioactivities produced. The number of commercially available therapeutically active compounds from microbial sources to date exceeds those discovered from other sources. In this review, we introduce a short history of microbial drug discovery as well as certain features and recent research approaches, specifying the microbial origin, their featured molecules, and the diversity of the producing species. Moreover, we discuss some bioactivities as well as new approaches and trends in research in this ﬁeld.


Historical Overview of Natural Products in Drug Discovery
Nature sustains unlimited resources of novel bioactive molecules, and the study of these resources is very useful in the process of drug discovery [1]. These bioactive molecules are called natural products (NPs). Natural products are metabolites and/or by-products derived from living organisms, such as plants, animals, and microorganisms [2]. Natural products have played an intrinsic role in human life for thousands of years. Due to their low cost and availability, natural products have been used as a source of medicine, especially in developing countries. Moreover, they are chemically diverse with various bioactivities and are the most valuable sources of drug discovery and development [3]. Today, many microbial-originated antibiotics are available in the market, and more than 120 of the most important medicines in use are obtained from terrestrial microorganisms [36]. A large number of bioactive metabolites are used in medicine, agriculture, and industry, but about 100 of them are used for therapeutic purposes, herbicidal activity, growth-promoting agents, or tools for biochemistry [37].
Recently, there has been great interest in natural products from unexplored microbial sources, especially actinomycetes [38], marine ecosystems [39], and microorganisms associated with plants [40], mammals [41], and invertebrates [42] from marine and terrestrial habitats. Despite the most important antibiotics currently in use being derived from cultivable microorganisms, only a tiny fraction of microorganisms can be cultivated in routine lab cultures [43]. The majority of microorganisms in biosamples cannot be cultivated under normal laboratory conditions and are called uncultivated microorganisms. This kind of microorganisms can be cultivated using systems developed specifically for the organisms, such as synthetic medium mimicking the biosystem conditions and several other in situ cultivation strategies [44].

Natural Products from Fungal Sources
Fungi are distributed in nature, and these eukaryotic, heterotrophic microorganisms often live symbiotically. Fungi have been used for a long time by humankind for many purposes, including food production (beer, wine, leavened bread, soy foods), treatments, and in everyday life. Thousands of years ago, fungi were used to treat intestinal diseases by the Mayans. Since the discovery of penicillin, which was isolated from the fungus Penicillium notatum, fungi have been a rich source of many therapeutic agents [45]. Fungi are a rich source of biologically active secondary metabolites ( Figure 2). Today, many microbial-originated antibiotics are available in the market, and more than 120 of the most important medicines in use are obtained from terrestrial microorganisms [36]. A large number of bioactive metabolites are used in medicine, agriculture, and industry, but about 100 of them are used for therapeutic purposes, herbicidal activity, growth-promoting agents, or tools for biochemistry [37].
Recently, there has been great interest in natural products from unexplored microbial sources, especially actinomycetes [38], marine ecosystems [39], and microorganisms associated with plants [40], mammals [41], and invertebrates [42] from marine and terrestrial habitats. Despite the most important antibiotics currently in use being derived from cultivable microorganisms, only a tiny fraction of microorganisms can be cultivated in routine lab cultures [43]. The majority of microorganisms in biosamples cannot be cultivated under normal laboratory conditions and are called uncultivated microorganisms. This kind of microorganisms can be cultivated using systems developed specifically for the organisms, such as synthetic medium mimicking the biosystem conditions and several other in situ cultivation strategies [44].

Natural Products from Fungal Sources
Fungi are distributed in nature, and these eukaryotic, heterotrophic microorganisms often live symbiotically. Fungi have been used for a long time by humankind for many purposes, including food production (beer, wine, leavened bread, soy foods), treatments, and in everyday life. Thousands of years ago, fungi were used to treat intestinal diseases by the Mayans. Since the discovery of penicillin, which was isolated from the fungus Penicillium notatum, fungi have been a rich source of many therapeutic agents [45]. Fungi are a rich source of biologically active secondary metabolites ( Figure 2). Many therapeutic agents, such as cyclosporine and mycophenolic acid (immunosuppressive activity), fusidic acid and griseofulvin (antimicrobial activity), and other novel semisynthetic antifungal drugs, such as anidulafungin and caspafungin, have been derived from fungal metabolites [45]. Recently, cyclosporine was used to develop Debio 025, which was clinically proven to have potent antiviral activity [46].
One of the most important drugs are statins, including mevastatin from Penicillium citrinum [47] and lovastatin from Aspergillus terreus [48]. Statins, an important class of antilipidemic drugs for the treatment of cardiovascular diseases [49], are also derived from microbial sources. Fungal metabolites are not only important for medicine but also for plant protection. For instance, the discovery of strobilurins, which were first isolated from Strobilurus species, led to compounds for synthetic fungicides, such as trifloxystrobin [50].
Plant endophytes have been defined in several ways. The most common definition is "all organisms inhabiting plant organs that at some time in their life can colonize internal plant tissues without causing apparent harm to the host" [51]. Fungi are more frequently observed as endophytes than bacteria [52]. An endophytic fungus is a fungus that can colonize healthy tissues of the host plant, typically causing no apparent symptoms of disease. There are symbiotic relationships between the host plants and their endophytes by which the host can support and provide nutrients to the fungus and later produce metabolites that are important to the host. This symbiotic relationship may be suddenly reversed to opportunistic if the host plant is weakened [52].
Endophytes are a polyphyletic group of primarily ascomycetous fungi, whereas basidiomycetes, deuteromycetes, and oomycetes rarely exist [53]. There is no host specificity, but it has been noticed that some families frequently colonize certain hosts. The great diversity and ecological roles of endophytes produce a variety of pharmaceutically and agrochemically promising secondary metabolites [54,55].
About 140 new bioactive compounds were isolated from endophytic fungi in the period between 1987 and 2000. Between 2000 and 2006, a similar number of compounds were isolated [56]. The ability to produce pharmacologically important natural products is not only restricted to plant sources but is also inherent to associated endophytes [57,58]. Amongst the isolated products, cryptocin, from the Many therapeutic agents, such as cyclosporine and mycophenolic acid (immunosuppressive activity), fusidic acid and griseofulvin (antimicrobial activity), and other novel semisynthetic antifungal drugs, such as anidulafungin and caspafungin, have been derived from fungal metabolites [45]. Recently, cyclosporine was used to develop Debio 025, which was clinically proven to have potent antiviral activity [46].
One of the most important drugs are statins, including mevastatin from Penicillium citrinum [47] and lovastatin from Aspergillus terreus [48]. Statins, an important class of antilipidemic drugs for the treatment of cardiovascular diseases [49], are also derived from microbial sources. Fungal metabolites are not only important for medicine but also for plant protection. For instance, the discovery of strobilurins, which were first isolated from Strobilurus species, led to compounds for synthetic fungicides, such as trifloxystrobin [50].
Plant endophytes have been defined in several ways. The most common definition is "all organisms inhabiting plant organs that at some time in their life can colonize internal plant tissues without causing apparent harm to the host" [51]. Fungi are more frequently observed as endophytes than bacteria [52]. An endophytic fungus is a fungus that can colonize healthy tissues of the host plant, typically causing no apparent symptoms of disease. There are symbiotic relationships between the host plants and their endophytes by which the host can support and provide nutrients to the fungus and later produce metabolites that are important to the host. This symbiotic relationship may be suddenly reversed to opportunistic if the host plant is weakened [52].
Endophytes are a polyphyletic group of primarily ascomycetous fungi, whereas basidiomycetes, deuteromycetes, and oomycetes rarely exist [53]. There is no host specificity, but it has been noticed that some families frequently colonize certain hosts. The great diversity and ecological roles of endophytes produce a variety of pharmaceutically and agrochemically promising secondary metabolites [54,55].
About 140 new bioactive compounds were isolated from endophytic fungi in the period between 1987 and 2000. Between 2000 and 2006, a similar number of compounds were isolated [56]. The ability to produce pharmacologically important natural products is not only restricted to plant sources but is also inherent to associated endophytes [57,58]. Amongst the isolated products, cryptocin, from the endophytic fungus Cryptosporiopsis quercina, an endophyte of Tripterigeum wilfordii, has shown potent activity against the world's worst plant pest, Pyricularia oryzae, and other plant pathogenic fungi [57]. Phomol, an active polyketide lactone that is isolated from the endophyte Phomopsis sp., an endophyte of the medicinal plant Erythrina crista-galli, exhibits anti-inflammatory as well as antimicrobial activity [59]. Some of the US Food and Drug Administration (FDA)-approved drugs from fungi are presented in Table 1.

Natural Products from Bacterial Sources
Nearly three-quarters of microbial-produced bioactive compounds are from actinomycete bacteria. Streptomycetes are the most widely identified group, producing a wide range of biologically active compounds. They are Gram-positive aerobic filamentous (often soil) bacteria [68]. Euzeby (2008) [69] described more than 500 species of streptomycetes. They mostly produce spores and are characterized by the production of geosmin, a volatile metabolite that give them "earthy" odor. The spore germination process depends on the environmental conditions. In normal conditions, the germination of streptomycete spores starts by arthrospore (substrate mycelium), but in the case of nutrient depletion, the growth starts with aerial mycelium. In other words, under favorable conditions, a fully matured mycelia is produced. Under drastic conditions, on the other hand, the aerial mycelium is subdivided by septa, then into spores, which in turn can, under certain conditions, germinate into mycelium [70].
The production of secondary metabolites in actinomycetes is greatly affected by various fermentation parameters, such as nutrients availability, pH, aeration, temperature, mineral salts, heavy metals, precursors, inducers, and inhibitors, which often vary from organism to organism [85,86].
Streptomycetes are good soil inhabitants and are considered to be valuable sources of many enzymes, such as lipases and cellulases [87]. In addition, some genes from these bacteria may be applied to plants to produce genetically modified plants with improved characteristics [88]. The genes for the production of secondary metabolites are considered to be nonessential and are often found near the ends of linear chromosomes; the chromosomes of streptomycetes, in general, are linear [89].
Streptomycetes are a rich source of many bioactive compounds. Most antifungals derived from

Natural Products from Bacterial Sources
Nearly three-quarters of microbial-produced bioactive compounds are from actinomycete bacteria. Streptomycetes are the most widely identified group, producing a wide range of biologically active compounds. They are Gram-positive aerobic filamentous (often soil) bacteria [68]. Euzeby (2008) [69] described more than 500 species of streptomycetes. They mostly produce spores and are characterized by the production of geosmin, a volatile metabolite that give them "earthy" odor. The spore germination process depends on the environmental conditions. In normal conditions, the germination of streptomycete spores starts by arthrospore (substrate mycelium), but in the case of nutrient depletion, the growth starts with aerial mycelium. In other words, under favorable conditions, a fully matured mycelia is produced. Under drastic conditions, on the other hand, the aerial mycelium is subdivided by septa, then into spores, which in turn can, under certain conditions, germinate into mycelium [70].
The production of secondary metabolites in actinomycetes is greatly affected by various fermentation parameters, such as nutrients availability, pH, aeration, temperature, mineral salts, heavy metals, precursors, inducers, and inhibitors, which often vary from organism to organism [85,86].
Streptomycetes are good soil inhabitants and are considered to be valuable sources of many enzymes, such as lipases and cellulases [87]. In addition, some genes from these bacteria may be applied to plants to produce genetically modified plants with improved characteristics [88]. The genes for the production of secondary metabolites are considered to be nonessential and are often found near the ends of linear chromosomes; the chromosomes of streptomycetes, in general, are linear [89].
Streptomycetes are a rich source of many bioactive compounds. Most antifungals derived from streptomycetes tend to be macrolide polyene, such as nystatin, produced by streptomyces. noursei [90]; amphotericin B, produced from streptomyces nodosus, and natamycin, produced by streptomyces natalensis [91]. A huge number of streptomyces-derived antibiotics are used as antibacterial agents. Starting with aminoglycosides, a large number show antibacterial activity, such as streptomycin, produced by streptomyces griseus [92]; neomycin, produced by streptomyces fradiae [93]; and kanamycin, produced by streptomyces kanamyceticus [94]. Other antibacterial antibiotics from streptomycetes include erythromycin, produced by streptomyces erythraea; tetracycline produced by streptomyces rimosus [95]; chloramphenicol produced by streptomyces venezuelae [96]; vancomycin, produced by streptomyces orientalis; and thienamycin, produced by Streptomyces cattleya [97]. Some chemical alterations could be useful for producing novel structures with new properties in so-called "semisynthetic drugs" [91].
About two-thirds of bioactive compounds are produced by this group, and they have many clinical efficacies against different kinds of organisms, such as bacteria, fungi, and parasites. In addition, other drugs in this category exhibit antitumor activities, such as aclacinomycin A, actinomycin D, bleomycin, daunorubicin, mithramycin, mitomycin C, and nogalamycin (produced by Streptomyces glalilaeus, Streptomyces antibioticus, Streptoverticillium verticillium, Streptomyces paecetius, Streptomyces argillaceus, Streptomyces lavendulae, and Streptomyces nogalater, respectively) [101]. These drugs can act on DNA by altering its function via different mechanisms, such as intercalation, cross-linking, DNA strand breakage, or interacting with DNA non-intercalatively [102]. Approximately 3% of all antibacterial have been synthesized by streptomycetes [103], which serves as a promising source for discovering novel drugs.
Accordingly, actinobacteria have played a significant role in human health in the last decades throughout the world. Like fungi, there are many actinobacteria that can be associated with and colonize the inner tissues of higher plants but do not visibly harm the plants. These are called endophytic actinobacteria and represent an important source of many bioactive compounds. These microbes inhabit different plant organs inter-and intracellularly. It is worth mentioning that there are about 300,000 plant species on Earth and that each individual plant is considered to host one or more type of endophytes, creating huge biodiversity of compounds and functions [104]. Endophytic actinobacteria associated with traditionally used medicinal plants, especially of the tropics, could be a rich source of promising compounds. Many endophytic actinobacteria, especially those from medicinal plants, possess the ability to inhibit or kill a wide variety of harmful microorganisms like pathogenic bacteria, fungi, and viruses [44].
The most promising value of endophytes is to produce many new antitumor and anti-inflammatory agents. Considering that endophytic actinobacteria are closely associated with their host plant, it is possible for horizontal gene transfer (HGT) to occur, resulting in the production of plant-derived compounds by a microbe, such as the paclitaxel-producing Kitasatospora sp. isolated from Taxus baccata in Italy [105]. Maytansinoids are extraordinarily potent antitumor agents that were originally isolated from members of the higher plant families Celastraceae, Rhamnaceae, and Euphorbiaceae [106,107] as well as some mosses [108] and, remarkably, from plant-associated actinomycete Actino-synnema pretiosum [109].
Endophytic actinobacteria have sparked great interest because they possess many properties that could be beneficial for plant growth. For example, several endophytic actinobacteria isolated from winter rye produced indolyl-3-acetic acid, which enhanced seedling germination [113]. In most cases, natural compounds from bacteria need some modifications to optimize their properties. These alterations may be controlled during synthesis by metabolic engineering or by changing synthesis technology, as studied in several bacteria [114]. Such methods may produce novel compounds by expressing a newly identified pathway or using gene combination to create a new synthetic pathway. However, the enzymes involved in biosynthetic pathway are like fatty acid synthases, which are conserved in eukaryotes and prokaryotes [115].
Due to the drug-resistance phenomena, new approaches have been employed to find new drugs from microorganisms by studying well-known productive strains, developing new screening methods [116], carrying out chemical modifications of biosynthesized precursors and combinatorial biosynthesis [117], and doing intensive studies to select and discover new strains from new sources. Thus, much effort has to be made to compensate for the emerging resistance as no novel compounds have been discovered during the period between the introduction of quinolone nalidixic acid (1962) and linezolid (2000) [118].
One of the factors that enhanced the resistance problem is the use of about 50% of existing antimicrobials for purposes other than therapeutic use [119], such as food additives in livestock breeding. The study of metabolic pathways and the genetics of microbes are beneficial in production strategies and in regulatory mechanisms employed by the productive strain, such as in the Streptomyces coelicolor [120] and Streptomyces avirmitilis genome projects [121].
Recently, marine actinomycetes have been considered to be a promising and unique resource for novel bioactive secondary metabolites [122] because environmental conditions of the sea are extremely different from terrestrial conditions and they are widely distributed within the marine ecosystem and found in intertidal zones, seawater, animals, plants, sponges, and in ocean sediments [123][124][125].
In addition, these actinomycetes have the ability to form stable populations in different habitats and produce many compounds with various activities [126]. This explains the importance of this group as a source of novel compounds. Many novel pharmaceutically important compounds have been produced from marine actinomycetes, such as the anticancer salinosporamide A, which is produced by Salinispora tropica [127]; salinipyrones A and B, produced by Salinispora pacifica [128]; iodopyridone, produced by marine Saccharomonospora sp. [128]; and srenimycin, produced by Salinispora arenicola [129] ( Figure 4). Table 2 lists some of FDA-approved drugs from actinomycetes.

Natural Products from Algae
Algae are a prolific source in natural product chemistry and include prokaryotic (cyanobacteria) and eukaryotic species. They are represented by approximately 30,000 species that have a function of supplying oxygen to the biosphere [137]. They are also a very good nutritional source for fish and humans. Moreover, they can be used in medicine and fertilizers. The most important group of compounds produced by algae are terpenoids, which comprise many classes, including brominated derivatives, phenazine derivatives, oxygen and nitrogen heterocycles, amino acids, and guanidine derivatives [138].
Investigation of natural products from algae started in 1970 [139]. Among the important compounds produced by algae are polycavernoside A from the red alga Polycaverosa tsudai [128];

Natural Products from Algae
Algae are a prolific source in natural product chemistry and include prokaryotic (cyanobacteria) and eukaryotic species. They are represented by approximately 30,000 species that have a function of supplying oxygen to the biosphere [137]. They are also a very good nutritional source for fish and humans. Moreover, they can be used in medicine and fertilizers. The most important group of compounds produced by algae are terpenoids, which comprise many classes, including brominated derivatives, phenazine derivatives, oxygen and nitrogen heterocycles, amino acids, and guanidine derivatives [138].

Natural Products from Microbial Community Interactions
Among the many recent approaches for the discovery of new drugs is cocultivation (mixed cultures), by which we can cultivate together two or more organisms from different species, mimicking the natural microbial community interactions. Recent investigations have indicated that microbial interactions induce the production of new specialized metabolites through the activation of some cryptic genes, providing a very promising tool for drug discovery [145,146].

Natural Products from Microbial Community Interactions
Among the many recent approaches for the discovery of new drugs is cocultivation (mixed cultures), by which we can cultivate together two or more organisms from different species, mimicking the natural microbial community interactions. Recent investigations have indicated that microbial interactions induce the production of new specialized metabolites through the activation of some cryptic genes, providing a very promising tool for drug discovery [145,146].

Conclusions
In summary, natural products play a significant role in human life. Microbial natural products are the most versatile and are of interest because of their unique structures and functions, due to which they are considered the cornerstone of drug discovery. Despite the use of microorganisms as a source of drugs being a recent discovery, the most important and commercially available antibiotics and many other anti-infectives are obtained from them. Recent research initiatives have been directed toward endophytic microorganisms due to their importance as a source of novel compounds.
Furthermore, marine macro-and microorganisms provide endless resources for novel bioactive compounds as they cover about 70% of the Earth's area. Even marine macroorganisms (plants and animals) are interesting sources of novel bioactive compounds, mostly due to their inhabitant microbiota, which are often responsible for the production of their secondary metabolites. However, only a small number of microorganisms can be used for defense mechanisms of their hosts (plants and animals) and for other ecological interactions within their microbiota, such as commensalism and symbiosis, which produce very useful products for them and their hosts. Because only a small number of microorganisms can be cultivated under laboratory conditions, more of such systems need to be developed using in situ cultivation strategies.
One of the most recent approaches in drug discovery from microorganisms is cocultivation. In the cocultivation strategy, we can cultivate two or more organism from different species. Through this, their physiology can be changed to produce cryptic compounds that cannot otherwise be produced in routine cultivation media.
Overall, the present review highlighted the role of biodiversity in providing a vital link for expanding the molecular diversity needed for successful drug discovery attempts in the future, with microbial inhabitants being a unique source of bioactive secondary metabolites and their therapeutic applications. This review therefore attempted to place emphasis on many important concepts in the field of microbial natural products that use cost-effective techniques utilizing recent cultivation ideas mimicking the natural ecological conditions where microorganisms always co-exist within complex microbial communities, such as cocultivation and in situ cultivation. We will further discuss the recombinant DNA technology and other probable molecular tools in future studies to address the most applicable approaches.