Marine Rare Actinomycetes: A Promising Source of Structurally Diverse and Unique Novel Natural Products

Rare actinomycetes are prolific in the marine environment; however, knowledge about their diversity, distribution and biochemistry is limited. Marine rare actinomycetes represent a rather untapped source of chemically diverse secondary metabolites and novel bioactive compounds. In this review, we aim to summarize the present knowledge on the isolation, diversity, distribution and natural product discovery of marine rare actinomycetes reported from mid-2013 to 2017. A total of 97 new species, representing 9 novel genera and belonging to 27 families of marine rare actinomycetes have been reported, with the highest numbers of novel isolates from the families Pseudonocardiaceae, Demequinaceae, Micromonosporaceae and Nocardioidaceae. Additionally, this study reviewed 167 new bioactive compounds produced by 58 different rare actinomycete species representing 24 genera. Most of the compounds produced by the marine rare actinomycetes present antibacterial, antifungal, antiparasitic, anticancer or antimalarial activities. The highest numbers of natural products were derived from the genera Nocardiopsis, Micromonospora, Salinispora and Pseudonocardia. Members of the genus Micromonospora were revealed to be the richest source of chemically diverse and unique bioactive natural products.


Introduction
Emerging infectious diseases and multidrug-resistant human pathogens are becoming a major threat to global health [1]. Therefore, there is an urgent need for new antibiotics to fight evolving bacterial infections. Despite the use of large synthetic combinatorial libraries of molecules to develop novel drugs, natural products and microbial metabolites, in particular, are a predominant source of bioactive scaffolds that represent the foundation for the development of life-saving antibiotics [2]. Nature encompasses millions of prokaryotes and eukaryotes with particularly high diversity in oceans and rainforests. However, so far only a small fraction (approximately 250,000-300,000 living species) of at least 1.5 million fungi, 0.5 million plant species and 10 11 -10 12 microbial species on Earth have been documented [3,4]. Moreover, even the known species have only been explored for bioactivity or for natural product discovery up to a limited extent. Therefore, natural resources are virtually unlimited for natural product discovery. The phylum Actinobacteria represents one of the largest phyla among the 30 major phyla currently recognized within the domain Bacteria. There are 6 classes, 18 orders, 14 suborders, 63 families and 374 genera recorded in this phylum until October 2016 (http://www.bacterio.net/-classifphyla.html#actinobacteria). In this review paper the term Marine habitats are also a rich source of diverse and largely uncharacterized microbial communities including actinomycetes [62]. This habitat shows extreme variations in ecological pressure, including competition for space, predation, available nutrients, light, oxygen concentration and pressure. Marine organisms including actinomycetes have developed a diverse range of secondary metabolites with unique structural elements to ensure their survival in these habitats [63]. Diverse new rare species including novel genera and novel families of actinomycetes have been isolated from marine habitats, such as coastal, tidal and deep-sea sediments, marine organisms (sponges, corals and ascidians), seawater and also mangrove forests [7]. Approximately 220 genera of rare actinomycetes were reported from marine sources until 2010 [64] and in the following sections we summarize new rare actinomycete isolates from these habitats since then [7]. For this review we've applied a conservative threshold on labelling a species as "novel" when sharing less than 97% similarity of the 16S rRNA gene to known species [65][66][67][68][69][70]. For the labelling of genera and families as "novel" we followed Silva taxonomy [71].

Rare Actinomycetes from Marine Sediments, Seawater, Eukaryotic Hosts and Mangroves
Approximately 83% of marine sediments are more than 1000 m below sea level, so most marine sediments are located in a cold, lightless, high pressure habitat where food is supplied from distant surface waters [72]. Deep-sea environments are divided into three zones: the bathyal (depth range between 200 and 2000 m), the abyssal (depth from 2000 to 6000 m) and the hadal (depth below 6000 m) [73]. Especially the abyssal and hadal zones are largely unexplored. Highest biodiversity has been recorded at a depth of 3000 m and the heterogeneity of biomass is expanding to 5000 m [74,75]. Cold deep-sea muds have an astounding species richness and diversity compared to tropical rain forests [76]. The majority of these species has not been isolated in the laboratory and an estimated 95% of these species are unidentified and mostly considered as new species [74].
Actinomycetes, including rare actinomycetes, are abundant in diverse marine sediments. A total of 48 new rare actinomycete species belonging to 16 different actinomycete families were isolated from marine sediments in the period from mid 2013 to 2017 (Table 2). Among them, 5 novel genera: Flaviflexus, Halopolyspora, Mariniluteicoccus, Sediminivirga and Haloactinomyces were described. The actinomycete families reported from marine sediments to which the novel species belong are Pseudonocardiaceae Although earlier culture-dependent studies have described microbial population size to be only a few hundred cells per mL of seawater, the staining of cells using fluorescence microscopy studies demonstrated nominal cell densities of >10 5 cells per mL of seawater [77,78], which anticipates that the ocean harbors 3.6 × 10 29 microbial cells [79]. The microorganisms in the seawater play an important role in marine biogeochemical processes involved in cycling and decomposition of organic matter [80]. A total of 8 new rare actinomycete species were reported from seawater for the period mid-2013 to 2017 (Table 3), among which 2 novel genera, Pontimonas and Tamlicoccus. They belong to six actinomycete families: Nocardioidaceae (3 new species), Cellulomonadaceae (1 new species), Micrococcaceae (1 new species), Microbacteriaceae (1 new species), Dermacoccaceae (1 new species) and Dermabacteraceae (1 new species). From these studies, it is apparent that lower numbers of rare actinomycetes are isolated from seawater than from sediments. However, novel genera have been reported from seawater, which contribute to the extension of phylogenetic diversity of rare actinomycetes (Table 3) [7].
A substantial number of rare actinomycetes were reported to be associated to various members of marine benthic communities, such as sponges, corals, ascidians, sea anemones, sea cucumbers, sea urchins and seaweeds [7,62,81]. Five novel genera, 17 new rare actinomycete species belonging to 11 different actinomycete families were reported from marine plants and animals between 2007 and mid-2013 [7]. A total of 14 new species of rare actinomycetes belonging to 12 different families have been reported from various sponges, corals, algae, sea urchin, jelly fish and fish between mid-2013 and 2017 (Table 4) and Promicromonosporaceae (1 new species). Thus, marine organisms remain a rich source of novel rare actinomycetes (Table 4) and a substantial number of host-associated rare actinomycete genera have not been reported from other marine habitats (Labedella, Phycicola, Iamia, Euzebya and Koreibacter) [7]. Interestingly, Microbacterium aureliae was reported for the first time from Aurelia aurita, the moon jellyfish. Table 2. New species of rare actinomycetes from marine sediments reported during the period of mid 2013-2017.

Strain/Family
Nature of Sample Isolation Medium Ref.

Strain/Family Nature of Sample Isolation Medium
Ref.

Brachybacterium aquaticum/Dermabacteraceae
Seawater Tryptic soy agar medium (HiMedia) [127] Mangrove forests are highly dynamic ecosystems that cover and protect approximately 75% of the world's tropical and subtropical coastal areas [128] and harbor a rich diversity of marine, freshwater and terrestrial flora and fauna. The diversity of the microbial community in mangrove environments is still rather unexplored [60]. The large fluctuation of salinity and tidal gradients make the mangrove forests unique environments that favors the production of unusual metabolites among the residing microorganisms [60]. Novel actinomycetes reported from different mangrove habitats including sediments, mangrove plant rhizosphere soil and mangrove endophytes are classified into 25 genera, 11 families and 8 suborders [7,129]. A total of 27 new species of rare actinomycetes belonging to 13 different families have been reported from mangrove habitats for the period mid-2013-2017 (Table 5)  Marmoricola aquaticus/Nocardioidaceae marine sponge Glodia corticostylifera M1 agar (soluble starch 10 g L −1 , yeast extract 4 g L −1 , peptone 2 g L −1 , agar 15 g L −1 , 80% artificial seawater) [38] Arthrobacter echini/Micrococcaceae Purple sea urchin Heliocidaris crassispina Marine agar 2216 (Difco) [135] Ornithinimicrobium algicola/Intrasporangiaceae Marine green alga Ulva sp.

Actinomycetes as Sources of Antibiotics
Actinomycetes has been one of the most fertile sources for the discovery of new antibiotics since they were first discovered and a number of the antibiotics currently in use are natural products or analogs of natural products from actinomycetes [153]. Actinomycin was the first antibiotic discovered from actinomycetes in 1940 from a culture of Streptomyces antibioticus [154], followed by streptothricin from Streptomyces lavendulae in 1942 [155], and streptomycin from Streptomyces griseus in 1944 [156]. Streptomyces species have been the key source of clinical antibiotics, and more than 80% of all antibiotics of actinomycetes origin have been derived from this single genus [3,157]. Out of all microbially-derived antibiotic classes, 10 classes are exclusively produced by actinomycetes. Those are polyene macrolides, oligomycin-type large-membered macrolides, daunomycin-type anthracyclines, nigericin-type polyether antibiotics, nonactin-type cyclopolylactones, aminoglycosides, anthracyclines, streptothricins, actinomycins and quinoxaline-peptides [3]. The antibiotics production of different actinomycete strains can vary enormously as some actinomycete species produce a single antibiotic, whereas some produce a wide-range of different compounds and compound classes [5]. A total of 30 new antibiotics have been launched worldwide since 2000. Of the 30 new antibiotics, 2 were natural products (NP), 12 were NP-derived and 16 were synthetic antibiotics [158]. Out of these 30 new antibiotics, 12 were reported from members of actinomycetes, either as natural product or natural product-derivatives representing 7 different antibiotic classes (Table 7). Due to the decline in the number of new chemical scaffolds and rediscovery of known molecules, the innovation in antibiotic development has slowed down. The exploration of alternative taxa, which have not been previously cultivated, could alleviate urgent needs related to resistance against currently used antibiotics.

Rare Actinomycetes: A Target for Future Drugs
As a result, rare actinomycetes are becoming an increasingly important focus of investigation in the search for novel natural products because (1) they occupy a poorly explored taxonomic and environmental space, which reduces the likelihood of replication of discovery, and (2) the phylum Actinobacteria is a rich source of bioactive secondary metabolites [46] that can be expected to yield novel chemical scaffolds for the development of new antibiotics.

Marine Rare Actinomycetes Is a Source of Antibiotics
Approximately 100 new bioactive compounds were reported from 38 rare actinomycete strains belonging to 15 genera described between 2007 and mid-2013. Out of these 15 different genera, Salinispora (20 new compounds), Verrucosispora (18 new compounds), Nocardiopsis (12 new compounds), Actinoalloteichus (11 new compounds), Marinispora (10 new compounds) and Micromonospora (9 new compounds) were predominant for discovery of novel secondary metabolites from 2007 to mid-2013 [7]. A total of 4 compounds derived from marine actinomycetes are currently in clinical trials (Table 7) of which 3 were obtained from marine Salinispora spp. indicating that Streptomyces spp. are no longer the most important biological resource for new antibiotics.

Novel/New Compounds from Marine Rare Actinomycetes between mid-2013 and 2017
A total of 167 different new bioactive compounds were reported from 58 rare actinomycete strains belonging to 24 genera from mid-2013 to 2017 (Table 8). Among them, genera such as Nocardiopsis (40 new compounds), Micromonospora (37 new compounds), Salinispora (21 new compounds) and Pseudonocardia (14 new compounds) are leading with respect to the number of novel secondary metabolites (Table 8). Among them, there are new/novel pyrones, structurally diverse natural products and unique chemical moieties (Figures 2-5).    A total of 7 different chemical classes of natural products were reported from marine Nocardiopsis spp. between mid-2013 and 2017 of which, α-pyrones (18 out of 40 compounds) were predominant (Table 9; Figure 2). These molecules have a wide range of biological activities, such as pro-inflammatory activity (enhancing and stimulating the inflammatory response), anti-inflammatory activity, antibacterial and cytotoxic activities (Table 9). In addition, the genera Streptomonospora and Saccharomonospora also produce a substantial number of α-pyrones. Besides, nocarimidazoles from Nocardiopsis sp. possess a 4-aminoimidazole ring rarely found in microbial secondary metabolites [162] and rare prolinyl-macrolactam polyketides were isolated from Nocardiopsis sp. [163]. Sterol O-acyltransferase [SOAT, also known as acyl-CoA: cholesterol acyltransferase (ACAT)], an endoplasmic reticulum membrane protein, catalyzes the synthesis of cholesteryl ester from free cholesterol and long-chain fatty acyl-CoA. SOAT has been postulated as a target for modulation by a new type of antiatherosclerotic agent. Interestingly, a diketopiperazine derived from marine Nocardiopsis sp. was found to be an effective SOAT inhibitor [164].  A marine sponge-derived Actinokineospora sp. produces actinosporins with selective activity against the parasite Trypanosoma brucei brucei, the causative agent of sleeping sickness [182]. Fungal infections, particularly candidiasis, is one of the serious diseases worldwide. A novel antifungal polyketide, forazoline A isolated from Actinomadura sp. showing significant activity against Candida albicans works through a new mechanism of action by disrupting membrane integrity [186]. Another way of controlling candidiasis is by capping enzyme repressors. Inhibitors of the enzyme RNA 5'triphosphatase in yeast may be used against pathogenic yeasts such as Candida. Interestingly, novel kribellosides from a marine Kribbella sp. inhibit activity of Cet1p (RNA 5'-triphosphatase) from Saccharomyces cerevisiae in vitro [208]. Another interesting biological activity is anti-allergic activity shown by nesterenkoniane, a novel cyclic ether isolated from the deep-sea-derived Nesterenkonia flava. Nesterenkoniane is the first report on secondary metabolites from the genus of Nesterenkonia [202]. Furthermore, discovery of anti-malarial drugs is one of the targets of research in pharma industries. Salinipostin A, isolated from the marine genus Salinispora shows potent antimalarial activity against Plasmodium falciparum growth (EC50 = 50 nM) and a high selectivity index (SI > 10 3 ) [192] (Figure 5).  (Table 9; Figure 3). In total, 13 chemical classes, including macrolides, polyene macrocyclic lactams, polycyclic tetramic acid macrolactams, aromatic tautomers, hydroxamates, diterpenoids, diterpenes, angucyclines, quinolone alkaloids, dioxanes, glycosylated paulomycins, glycosides and aglycone spirotetrorates were identified in Micromonospora spp. during this period. Polyene macrolactams are an underexplored group of natural products that have only been found in actinomycetes. Micromonolactam is a new polyene macrocyclic lactam isolated from a marine Micromonospora sp. (Figure 3). However, micromonolactam did not show antibacterial activities against test pathogens [165]. Another interesting group of natural products, paulomycins, are glycosylated molecules containing a pauloate residue that are of pharmacological interest due to their strong antibiotic properties [166]. Paulomycin G is structurally unique because it is the smallest bioactive paulomycin in the paulomycin family of antibiotics, lacking the paulomycose moiety ( Figure 3). Furthermore, a number of novel chemical skeletal structures are reported from marine Micromonospora spp. For example, polycyclic tetramic acid macrolactams of butremycin [167], halimane-type diterpenoids of micromonohalimanes [168] and a novel pimarane diterpene in isopimara-2-one-3-ol-8,15-diene [169] ( Figure 3).

Genome Mining of Marine Rare Actinomycetes
The rapid development of genome and metagenome sequencing methods including identification of secondary metabolite gene clusters has lead to the discovery of the genetic machinery encoding for novel natural products from microorganisms that have not yet been chemically identified [211]. The majority of these gene clusters encode for polyketides (PK), non-ribosomally synthesized peptides (NRP), ribosomally and post-translationally modified peptides (RiPPs) and aminoglycosides [211]. The bioinformatic analysis of genomes can also reveal silent secondary metabolite gene clusters, which are not expressed under standard laboratory conditions [212]. More than 23,000 PK and NRP have been reported so far, many of them found in actinomycetes, and they are being widely tested for pharmaceutical applications [213,214]. This approach has also been used for the identification of new antibiotic scaffolds from rare genera of actinomycetes from marine sediments [16]. Recently, Schorn and colleagues [215] have shown that rare marine actinomycetesderived genomes demonstrated a high degree of novelty and diversity, with Corynebacterium, Gordonia, Nocardiopsis, Saccharomonospora and Pseudonocardia as genera representing the highest biosynthetic gene cluster diversity. A total of 13 new bioactive compounds have been derived from marine rare actinomycetes, such as Saccharomonospora sp., Salinispora spp., Micromonospora spp. and Streptosporangium sp. using genome-based approaches between mid 2013 and 2017 (Table 10).
These numbers of new biosynthetic gene clusters and corresponding compounds will undoubtly increase in the near future due to revolutionary developments in the genome-and metagenomebased approaches for drug discovery [215] and it likely that omics-based screening for novel bioactive compounds will prevail over microbial isolation as the most efficient method for first identification of bioactivity potential of strains and environmental samples [216].

Conclusions
In the last decade (2007-2017), a great range of diverse, new and rare actinomycetes have been isolated from the marine environment. Employment of heat-treatment of marine sediment samples, Additionally, other rare actinomycete genera have yielded a number of unique chemical moieties, which were not previously reported from microbially-derived natural products ( Figure 4). For example, thiasporine A is the first natural product with a 5-hydroxy-4H-1,3-thiazin-4-one moiety, along with two new thiazole derivatives and were reported from Actinomycetospora chlora [170] (Figure 4). Other unusual structures include a curvularin macrolide with a rare α-D-glucopyranose substituent from Pseudonocardia sp. [171]; a butrepyrazinone, from Verrucosispora sp. that possesses an unusual methylation pattern on the pyrazinone ring [172], a novel indole microindolinone A from Microbacterium sp. [173], new dimeric indole derivatives with acetylcholinesterase (AchE) inhibitory activity from Rubrobacter radiotolerans [174] and a structurally new amycofuran bearing a rhamnose sugar from Amycolatopsis sp. [175].
Actinomadura sp. derived halomadurones ( Figure 5) demonstrated potent nuclear factor E2-related factor antioxidant response element (Nrf2-ARE) activation, which is an important therapeutic approach for treatment of neurodegenerative diseases [176]. Cyanogramide obtained from Actinoalloteichus cyanogriseus showed efficient anticancer activity by reversing the adriamycin-induced resistance of K562/A02 and MCF-7/Adr cells, and the vincristine-induced resistance of KB/VCR cells [177]. Table 9. Novel/new bioactive compounds produced by marine rare actinomycetes between mid 2013 and 2017.
No cytotoxicity [210] A marine sponge-derived Actinokineospora sp. produces actinosporins with selective activity against the parasite Trypanosoma brucei brucei, the causative agent of sleeping sickness [182]. Fungal infections, particularly candidiasis, is one of the serious diseases worldwide. A novel antifungal polyketide, forazoline A isolated from Actinomadura sp. showing significant activity against Candida albicans works through a new mechanism of action by disrupting membrane integrity [186]. Another way of controlling candidiasis is by capping enzyme repressors. Inhibitors of the enzyme RNA 5'-triphosphatase in yeast may be used against pathogenic yeasts such as Candida. Interestingly, novel kribellosides from a marine Kribbella sp. inhibit activity of Cet1p (RNA 5'-triphosphatase) from Saccharomyces cerevisiae in vitro [208]. Another interesting biological activity is anti-allergic activity shown by nesterenkoniane, a novel cyclic ether isolated from the deep-sea-derived Nesterenkonia flava. Nesterenkoniane is the first report on secondary metabolites from the genus of Nesterenkonia [202]. Furthermore, discovery of anti-malarial drugs is one of the targets of research in pharma industries. Salinipostin A, isolated from the marine genus Salinispora shows potent antimalarial activity against Plasmodium falciparum growth (EC 50 = 50 nM) and a high selectivity index (SI > 10 3 ) [192] (Figure 5).

Genome Mining of Marine Rare Actinomycetes
The rapid development of genome and metagenome sequencing methods including identification of secondary metabolite gene clusters has lead to the discovery of the genetic machinery encoding for novel natural products from microorganisms that have not yet been chemically identified [211]. The majority of these gene clusters encode for polyketides (PK), non-ribosomally synthesized peptides (NRP), ribosomally and post-translationally modified peptides (RiPPs) and aminoglycosides [211]. The bioinformatic analysis of genomes can also reveal silent secondary metabolite gene clusters, which are not expressed under standard laboratory conditions [212]. More than 23,000 PK and NRP have been reported so far, many of them found in actinomycetes, and they are being widely tested for pharmaceutical applications [213,214]. This approach has also been used for the identification of new antibiotic scaffolds from rare genera of actinomycetes from marine sediments [16]. Recently, Schorn and colleagues [215] have shown that rare marine actinomycetes-derived genomes demonstrated a high degree of novelty and diversity, with Corynebacterium, Gordonia, Nocardiopsis, Saccharomonospora and Pseudonocardia as genera representing the highest biosynthetic gene cluster diversity. A total of 13 new bioactive compounds have been derived from marine rare actinomycetes, such as Saccharomonospora sp., Salinispora spp., Micromonospora spp. and Streptosporangium sp. using genome-based approaches between mid 2013 and 2017 (Table 10).
These numbers of new biosynthetic gene clusters and corresponding compounds will undoubtly increase in the near future due to revolutionary developments in the genome-and metagenome-based approaches for drug discovery [215] and it likely that omics-based screening for novel bioactive compounds will prevail over microbial isolation as the most efficient method for first identification of bioactivity potential of strains and environmental samples [216]. Lobosamides A-C Polyene macrolactams Micromonospora sp. Anti-protozoan parasite, Trypanosoma brucei [220] Hexaricins A-C Pentangular polyphenols Streptosporangium sp. Not specified [221] Tetrocarcin N and O Glycosidic spirotetronates Micromonospora sp. Modest antibacterial activity [222] Rifsaliniketal Saliniketal Salinispora sp. Not specified [223] Nenestatin A Benzofluorene Micromonospora echinospora Antibacterial activity [224]

Conclusions
In the last decade (2007-2017), a great range of diverse, new and rare actinomycetes have been isolated from the marine environment. Employment of heat-treatment of marine sediment samples, the use of low-nutrient agar medium (seawater agar) or a growth medium with natural seawater along with the use of antifungal agents, favor the isolation of marine rare actinomycetes. At least 177 new species, which represent 29 novel genera and 3 novel families, were obtained as pure cultures. Micromonosporaceae, Nocardioidaceae, Pseudonocardiaceae, Microbacteriaceae, Micrococcaceae, Demequinaceae, Nocardiopsaceae, Propionibacteriaceae and Intrasporangiaceae were the families most frequently isolated from the marine environment.
In total, 267 new natural products derived from 96 different marine rare actinomycete strains belonging to 28 genera have been reported from 2007 to 2017. Out of these 28 marine genera, Nocardiopsis, Micromonospora, Salinispora, Verrucosispora, Pseudonocardia and Actinoalloteichus are topmost producers of novel new secondary metabolites.
The rare actinomycetes isolated and biomolecules discovered represent most likely only the low-hanging fruits and the immense diversity of microorganisms in marine habitats as shown from large cultivation-independent studies [225,226] are the proof for the presence of an even larger diversity of currently uncultivable rare actinomycetes and putative secondary metabolites. This uncultured majority should be the target of future selective isolation strategies and procedures. In addition, genetic engineering of whole biosynthetic gene clusters is finally gaining ground [216] and may be the key to access hidden gene clusters from rare actinomycetes. A breakthrough in heterologous expression would herald 'another golden age' of novel bioactive natural product discovery, for which marine rare actinomycetes may be one of the important sources.
Author Contributions: R.S. conceived, designed the work and wrote the manuscript; D.S. conceived, critically analyzed the data, revised and corrected the manuscript.