Biological and Chemical Diversity of Ascidian-Associated Microorganisms

Ascidians are a class of sessile filter-feeding invertebrates, that provide unique and fertile niches harboring various microorganisms, such as bacteria, actinobacteria, cyanobacteria and fungi. Over 1000 natural products, including alkaloids, cyclic peptides, and polyketides, have been isolated from them, which display diverse properties, such as antibacterial, antifungal, antitumor, and anti-inflammatory activities. Strikingly, direct evidence has confirmed that ~8% of natural products from ascidians are actually produced by symbiotic microorganisms. In this review, we present 150 natural products from microorganisms associated with ascidians that have been reported up to 2017.


Introduction
Ascidians are the most abundant and diverse class of the sub-phylum Tunicata, and more than 3000 species have been described. They have been found in diverse ecological niches, from shallow water to the deep sea [1]. Thousands of natural products have been isolated from ascidians; these include alkaloids, cyclic peptides, and polyketides [2,3]. Most of these secondary metabolites have diverse bioactivities, such as antibacterial, antifungal, antitumor and anti-inflammatory activities. In addition to the well-known molecules ecteinascidin (ET-743) and didemnin B, several other natural products or their derivatives (e.g., plitidepsin [4], midostaurin [5], lestaurtinib [6], edotecarin [7]) are also in clinical development. However, it has remained unclear whether these bioactive products were produced by ascidians themselves, or by ascidian-associated microorganisms [8,9].
Ascidians harbor rich microbial communities. The development of culture-independent methods has provided comprehensive information about ascidian microbial diversity [3]. In recent years, an increasing number of microorganisms associated with ascidians (including fungi, bacteria, actinobacteria, and cyanobacteria) have been isolated [10]. In this review, we mainly focus on ascidian-associated microorganisms that were isolated by culture-dependent methods.
Microorganisms associated with ascidians represent a potential source of natural products [11]. Many compounds isolated from ascidian-associated microorganisms are extremely potent [12][13][14]. Ecteinascidin 743 (or ET-743, or the trade name Yondelis) was originally isolated from the Ecteinascidia turbinata [15]. In 2007, it was approved for the treatment of advanced soft tissue sarcoma by EMEA. In 2011, with the help of metagenomic methods, it was proven that Candidatus Endoecteinascidia frumentensis was the actual producer of ET-743 [16]. Didemnin B, originally isolated from the Caribbean ascidian Trididemnum solidum [17] was the first marine natural product used in clinical
Ascidians can be divided into colonial and solitary classes. Colonial ascidians consist of many small individuals, called zooids, and the whole ascidians were used as the samples for the isolation of microorganisms. Solitary ascidians live as separate individuals with larger bodies, and the corresponding microbial diversity within these diverse ascidian tissues is different [3]. Thus, microorganisms have been isolated from different ascidian tissues, such as the tunic, gonads, gut and pharynx.
To date, diverse microorganisms, such as fungi, bacteria, actinobacteria and cyanobacteria have been isolated from ascidians. Bacteria represent the most abundant class of ascidian-associated microorganisms, and exhibit a high degree of diversity. On the other hand, cyanobacteria have also been widely used to study the symbiosis between microorganisms and their ascidian counterparts.

Bacteria
Ascidians are associated with diverse bacterial populations, and there is species-selective pairing of ascidians and bacteria [21]. For example, bacteria Acinetobacter sp. were isolated from the surface of Stomozoa murrayi [12], and bacteria Candidatus Endoecteinascidia frumentensis was found in symbiosis with Ecteinascidia turbinate [16], whereas Trididemnum solidum harbours the bacteria Tistrella mobilis and Tistrella bauzanensis [18,19]. To date, 21 genera belonging to 16 families in four phyla have been cultured from ascidians ( genus Exiguobacterium belonging to unclassified family in phylum Firmicutes; genus Rubritalea belonging to family Rubritaleaceae in phylum Verrucomicrobia; genus Labilibacter belonging to family Marinilabiliaceae and genus Tenacibaculum belonging to family Flavobacteriaceae in phylum Bacteroidetes. The dominant phylum of Proteobacteria is represented by 13 genera, which belong to 9 families. Ascidian genus Didemnum showed high bacterial diversity, and nearly half of the bacterial genera mentioned in this paper (Acinetobacter, Bacillus, Endozoicomonas, Exiguobacterium, Paenibacillus, Paucisalibacillus, Pseudomonas, Pseudovibrio, Ruegeria, Staphylococus, Stappia and Vibrio) were isolated from them. Surprisingly, culture-dependent and -independent approaches have not often been used to study the symbiosis between bacteria and ascidians, and further work is required in this area [22].

Actinobacteria
The bacterial phylum of Actinobacteria is widely known for the ability to produce bioactive compounds. Marine actinobacteria are widely distributed across different marine ecosystems, such as sediments, water, mangrove, algae, and animals [23][24][25][26]. As is the case with marine invertebrate sponges and corals, ascidians are associated with rich and diverse actinobacteria communities.

Cyanobacteria
Cyanobacteria is a phylum of bacteria that produce oxygen during photosynthesis. In 1982, Kott discovered the symbiotic relationship between cyanobacteria and 20 ascidian species. Of these, 17 ascidian species are obligate associates with the symbiotic cyanobacteria genus Prochloron, and the other three species (Trididemnum solidum, T. Cyanophorum and Didemnum viride) are associated with

Structure and Bioactivities of Natural Products
To date, 150 natural products have been isolated from ascidian-associated microorganisms. These compounds include polyketides, terpenoids, peptides, and alkaloids. These natural products have diverse properties, such as antimicrobial, antitumor and anti-inflammatory activities.

Polyketides
Polyketides, including macrolides, anthraquinones and polyethers, are derived from the polymerization of acetyl and propionyl groups, and are biosynthesized by three types of polyketide synthases (PKSs). Type I PKSs are multifunctional enzymes, type II PKSs are multienzyme complexes, and type III PKSs are homodimeric enzymes, which are also referred to as 'chalcone synthase-like PKSs' [88]. Thirty-seven of the compounds under review here (24.7%) are polyketide-based, and many of them have biological and pharmacological activities.
The antimelanoma drug palmerolide A (1) (Figure 2), a new enamide-bearing polyketide, was isolated from Synoicum adareanum, and was possibly of bacterial origin [89]. It has potent cytotoxicity against melanoma cells (UACC-62, MI14, SK-MEL-5, LOX IMVI), colon cancer cell line HCC-2998 and renal cancer cell line RXF 393. It was also found to be V-ATPase inhibitor [90,91]. Another ascidian, Lissoclinum patella, produces patellazoles A-C (2-4); these natural compounds have strong cytotoxicity against HCT-116 tumour cells [92]. Chemical and biological evidence suggested that the bacterium Candidatus Endolissoclinum faulkneri synthesizes patellazoles [34]. Further studies indicate that these products were the foundation of the symbiotic relationship between ascidians and bacteria, and were conserved even during the drive of genome reduction over millions of years [93].
The ascidian-associated bacterium, Streptomyces sp. PTY087I2, exhibited enhanced production of three naphthoquinone derivatives, granaticin (5), granatomycin D (6), and dihydrogranaticin B (7), and increased antibacterial activity when co-cultured with the human pathogens Bacillus subtilis, methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa) [63]. The isolation of Streptomyces sp. #N1-78-1 from Ecteinascidia turbinata in Puerto Rico led to the purification of bisanthraquinones 1 and 2 (8,9), and derivative 3 (10), the dehydration product of bisanthraquinone 1. Bisanthraquinones 1 and 2 showed potent antimicrobial activities against MRSA (methicillin-resistant Staphylococcus aureus) and VRE (vancomycin-resistant Enterococcus faecalis), and these three compounds displayed cytotoxic activity against HCT-116 cells [13]. Two novel chlorinated pyrones, halomadurones A and B (11,12), and two novel brominated analogues, halomadurones C and D (13,14) were isolated from Actinomadura sp. strain WMMB499 associated with Ecteinascidia turbinata in the Florida Keys. Halomadurones C and D showed potent nuclear factor E2-related factor antioxidant response element (Nrf2-ARE) activation, but were toxic at high concentrations [46]. Arenimycin (15) was the first report of the benzo[α] naphthacene quinone class of antibiotic isolated from marine actinobacteria Salinispora arenicola strain CNR-647, which is associated with Ecteinascidia turbinate. Arenimycin exhibited potent antimicrobial activities against drug-resistant Staphylococci, some other Gram-positive microorganisms and one Mycobacterium strain [14]. Ubiquinone Q9 (16), which was determined as 2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone by NMR spectroscopy and mass spectrometry, has been isolated from Nocardia sp. strain KMM 3749, a bacterium associated with an unidentified ascidian. This compound inhibited the development of fertilized eggs from the sea urchin Strongylocentrotus intermedius and caused haemolysis of mouse erythrocytes [52]. Griseorhodin A (17), a member of the rubromycin family, is an inhibitor of human telomerase [94]. The biosynthesis gene cluster for griseorhodin A was isolated from Streptomyces sp. JP95, which is associated with Aplidium lenticulum collected at Heron Island, Queensland, Australia [60]. In order to find the actual producer of namenamicin, a potent antitumour compound isolated from Polysyncraton lithostrotum, a number of actinobacteria were isolated from the inner core of the host ascidian. Among them, the actinobacteria Salinispora pacifica (originally proposed to be Micromonospora lomaivitiensis), strain LL-37I366 produced two novel lomaiviticin compounds, A and B (18, 19). These natural products, which are members of the angucycline family of aromatic polyketides, contain a distinctive diazotetrahydrobenzo[b]fluorene scaffold also found in the kinamycins [56]. Both compounds were demonstrated to be potent DNA damaging agents by biochemical induction assay (BIA), and have antimicrobial activities against Staphylococcus aureus and Enterococcus faecium. Lomaiviticin A also showed cytotoxicity against a number of cancer cell lines [55]. The actinobacteria Streptomyces coelicoflavus strain HQA809, which is associated with Styela clava, produced two natural compounds, germicidin (20) and 6-isopropyl group-3-ethyl-4-hydroxy-2-pyrone (21). Both of these compounds were lethal to Artemia salina [53]. The isolation of Actinomadura sp. from Ecteinascidia turbinata led to the purification of ecteinamycin (22). It showed potent antimicrobial activity against Clostridium difficile NAP1/B1/027 [47].

Terpenoids and Meroterpenoids
The terpenoids are derived from five-carbon isoprene units assembled and modified in thousands of ways, as well as their oxygen-containing derivatives. Terpenes are generally considered to be plant metabolites, although more and more terpenoids are isolated from marine microorganism [97]. The number of terpenoids reported from ascidian-associated microorganisms is very small and most of them are sesquiterpenoids, and these compounds showed diverse bioactivities. The meroterpenoids are natural products of mixed biosynthetic origin, which are partially derived from terpenoids.
Two new terpenoids gifhornenolones A (38) and B (39), together with a known sesquiterpene compound cyperusol C (40) were isolated from actinobacterial strain Verrucosispora gifhornensis YM28-088 associated with ascidian. However, only gifhornenolone A was reported to have potent inhibitory activity against the androgen receptor [98].
Didemnum molle was the source of fungus Penicillium sp. strain SS080624SCf1, and this strain produced two novel sesquiterpenoids JBIR-27 (41) and JBIR-28 (42), together with two known compounds sporogen-AO1 (43) and phomenone (44). They showed cytotoxicity against HeLa expect for JBIR-27 [80]. The fungus Humicola fuscoatra strain KMM 4629 associated with ascidian produced a new sesquiterpene of the caryophyllene series, fuscoatrol A (45), and a known compound 11-epiter-pestacin (46). This is the first report of fuscoatrol A, but its acetyl form, pestalotiopsin B, has been isolated from the endophytic fungus associated with the bark and the leaves of Taxus brevifolia. These two compounds both showed antimicrobial activities against Staphylococcus aureus and Bacillus subtilis, and fuscoatrol A also exhibited cytotoxic action on the developing eggs of sea urchin Strongylocentrotus intermedius [76].

Peptides
Peptides isolated from ascidian-associated microorganisms are mainly cyclic. They are nonribosomal peptides (NRPs) synthesized by huge protein complexes called nonribosomal peptide synthetases (NRPSs), and NRPs contain a high proportion of cyclic or branched nonproteogenic amino acids. Most of these cyclopeptides have biological and pharmacological properties, such as antibiotic and antitumor activities [99].
Didemnins A, B, and C, a class of cyclic depsipeptides, were first isolated from the Caribbean ascidian Trididemnum solidum in 1981 [17]. These compounds showed significant in vitrocytotoxicity and in vivoantitumor activity [18], and were also active against both DNA and RNA viruses [100]. Didemnin B (57) was the first marine compound to enter clinical trials as an antineoplastic agent, and exhibited anticancer activity in phase II clinical trials; however, it ultimately failed as a drug, because of its significant toxicity. Didemnin B now was confirmed to be produced by the marine α-proteobacteria Tistrella mobilis [18,19]. Complete genome sequence analysis of the T. mobilis strain KA081020-065 discovered the didemnin biosynthetic gene clusters; this lead to the hypothesis that didemnin X and Y precursors may be converted to didemnin B in this organism, which is an unusual post-synthetase activation mechanism [19].
The patellamides are cyclic peptides that exemplify both the unique structural features and potent bioactivities of natural products isolated from ascidians of the Didemnidae family [64]. For example, in 1982 Lissoclinum patella was reported to produce cyclic peptide patellamides A-C, all of which contained an unusual fused oxazoline-thiazole unit. Subsequently patellamide D (1993), patellamide E (1992) and patellamide F (1995) were also isolated from L. patella. Patellamides A-C have cytotoxic activity against L1210 murine leukaemia cells, whereas patellamide D is a selective resistance-modifying agent [101][102][103][104]. Cyanobacteria of the genus Prochloron are obligate symbionts of many didemnid ascidians, and have been identified as the real producers of cyclic peptides of the patellamide class. For example, genetic evidence has shown that Prochloron didemni (associated with Lissoclinum patella, Republic of Palau) is the source of cytotoxic compounds patellamide A (65) and C (66) [64,68]. The patellamide biosynthesis gene from Prochloron sp. (associated with Lissoclinum patella, Great Barrier Reef, Australia) has been expressed in Escherichia coli, leading to the production of patellamide D (67) and ascidiacyclamide (68); both of these molecules are highly cytotoxic [66].
Trichoderma virens, a fungus isolated from Didemnum molle, produces two modified dipeptide trichodermamides, A (69) and B (70). The trichodermamides possess a rare cyclic O-alkyl-oxime functionality incorporated into a six-membered ring. Trichodermamide B displayed cytotoxicity against HCT-116 and antimicrobial activity against amphoterocin resistant Candida albicans, methacillin resistant Staphylococcus aureus and vancomycin resistant Enterococcus faecium [86]. Depsipeptide JBIR-113 (71) was isolated from the fungus Meyerozyma sp., which is associated with the ascidian Ciona intestinalis in China. This compound was reported to have lethality against brine shrimp Artemia salina [77]. This compound was previously isolated from a marine sponge-derived Penicillium sp. This compound was previously isolated from a marine sponge-derived Penicillium sp. fS36 in Japan, together with JBIR-114 and JBIR-115. These peptides are all of marine origin and contain pipecolic acid, which is very rarely found in natural products [105].

Alkaloids
Alkaloids are structurally diverse compounds generally classified as such, due to the basic character of the molecule, and the presence of at least one nitrogen atom, preferably in a heterocycle [106]. Alkaloids have been isolated from diverse natural organisms, including ascidians and microorganisms.
Sesbanimides A-C were previously isolated from the seeds of the leguminous plant Sesbania drummondii [107,108]. Later, sesbanimide A (72) was isolated from the bacteria Agrobacterium PH-130, which is associated with Ecteinascidia turbinata from the Florida peninsula, and sesbanimide C (73) was isolated from the bacteria Agrobacterium PH-A034C (associated with Polycitonidae sp.) along the Turkish coast [31]. Sesbanimide A is one of the most active sesbania alkaloids, with excellent in vitro cytotoxicity against KB cellsand potent in vivo activity against P-388 murine leukaemia [109]. Isolated from bacteria LL-14I352 (associated with an unidentified orange ascidian, Pacific Ocean, Fiji), phenazine compounds LL-14I352 α (or pelagiomicin) (74) and β (75) have diverse properties, such as antimicrobial activity, and the ability to inhibit DNA, RNA and protein synthesis, DNA-damaging activity; and cytotoxic activity [44]. 6-bromoindole-3-carbaldehyde (76) and its debromo analogue indole-3-carbaldehyde (77) were isolated from Acinetobacter sp. (associated with Stomozoa murrayi). Both compounds inhibit the settlement of cyprid larvae from the barnacle, Balanus amphitrite. Compound 76 also presented antimicrobial activity against strain SM-S2, strain SM-Z, Bacillus marinus and Vibrio campbellii [12]. In 1990, the structures of six newly isolated bioactive compounds (ecteinascidins 729, 743, 745, 759A, 759B, and 770) were assigned. The most abundant compound ET-743 showed excellentin vitro cytotoxicity against L1210 leukaemia cells and potentin vivoactivity against P388 murine leukaemia [15]. However, its clinical utility was hampered by inefficient methodologies for isolation of the compound. This led to the development of (semi)-synthetic methods for its large-scale production, which resulted in a novel anticancer agent sold under the brand name Yondelis (Trabectedin) [110]. Trabectedin is the first marine-derived anticancer drug to be approved by the European Union (2007), and is currently approved in more than 70 countries for the treatment of soft tissue sarcoma [111]. In recent years, using metagenomic sequencing of total DNA from the ascidian/microbial consortium, the natural source of ET-743/Yondelis (78) was determined to be the bacteria, Candidatus Endoecteinascidia frumentensis, which is associated with Ecteinascidia turbinate [16].
The known diterpene glycoside sordarin (150) was produced by Talaromyces sp. CMB TU011 isolated from an unidentified ascidian, and it presented antifungal activity [85].   3.6. Summary of Natural Products 150 natural products have been isolated from microorganisms associated with ascidians up to 2017. Natural products originating from ascidian-associated microorganisms is a hot research topic, as evidenced by the surge of publications in this area beginning in the 2000 (Figure 3). Among them, polyketides and alkaloid compounds represent 43.3% of the total number (Figure 4). Most of these compounds have potent bioactivities, and induce in vitro cytotoxicity, or have antimicrobial, anti-inflammatory, antioxidant, and antifouling properties, to name only a few properties ( Figure 5). Some compounds have in vivo antitumor activity, and several promising drugs have been used in preclinical evaluation and clinical trials. How far have we progressed in the understanding of the molecular mechanisms of action of these compounds?

Summary of Natural Products
Microorganisms are a promising source of bioactive compounds, and the discovery of new strains is vital for new or more active compounds [116]. As discussed in this review, most compounds have been isolated from bacteria, cyanobacteria, actinobacteria, and fungi associated with ascidians ( Figure 6). Some compounds were initially considered to be from ascidians, but later confirmed to be produced by ascidian-associated bacteria, such as the well-known ET-743 and Didemnin B; others were isolated directly from the symbiotic microorganisms [32]. Because of the large number of compounds isolated from ascidians, approximately 1080 in 2012 [117], it is important to confirm the true source of more compounds. We think that only the tip of the iceberg has been explored in this regard.
150 natural products have been isolated from microorganisms associated with ascidians up to 2017. Natural products originating from ascidian-associated microorganisms is a hot research topic, as evidenced by the surge of publications in this area beginning in the 2000 (Figure 3). Among them, polyketides and alkaloid compounds represent 43.3% of the total number (Figure 4). Most of these compounds have potent bioactivities, and induce in vitro cytotoxicity, or have antimicrobial, antiinflammatory, antioxidant, and antifouling properties, to name only a few properties ( Figure 5). Some compounds have in vivo antitumor activity, and several promising drugs have been used in preclinical evaluation and clinical trials. How far have we progressed in the understanding of the molecular mechanisms of action of these compounds?
Microorganisms are a promising source of bioactive compounds, and the discovery of new strains is vital for new or more active compounds [116]. As discussed in this review, most compounds have been isolated from bacteria, cyanobacteria, actinobacteria, and fungi associated with ascidians ( Figure 6). Some compounds were initially considered to be from ascidians, but later confirmed to be produced by ascidian-associated bacteria, such as the well-known ET-743 and Didemnin B; others were isolated directly from the symbiotic microorganisms [32]. Because of the large number of compounds isolated from ascidians, approximately 1080 in 2012 [117], it is important to confirm the true source of more compounds. We think that only the tip of the iceberg has been explored in this regard.

Conclusions
The marine environment supplies many kinds of habitats that support marine life. It provides an extremely distinct environment for its living organisms. The diverse conditions enable high microbial diversity, and this in turn is associated with biological elaboration of more novel chemical structures [118]. This review has presented 150 natural products produced by ascidian-associated microorganisms. These secondary metabolites belong to polyketides, terpenoids, peptides, alkaloids and other types, and showed a good range of bioactivities. These results indicates the potential of the

Conclusions
The marine environment supplies many kinds of habitats that support marine life. It provides an extremely distinct environment for its living organisms. The diverse conditions enable high microbial diversity, and this in turn is associated with biological elaboration of more novel chemical structures [118]. This review has presented 150 natural products produced by ascidian-associated microorganisms. These secondary metabolites belong to polyketides, terpenoids, peptides, alkaloids and other types, and showed a good range of bioactivities. These results indicates the potential of the microorganisms associated with ascidians as sources of bioactive natural products.
In recent years, new approaches to the isolation of microorganisms have been greatly improved. High-throughput cultivation of microorganisms using microcapsules provides an approach to cultivate more biomass. Flow cytometry can then be used to select the microcapsules containing microcolonies. This method can obtain more than 10,000 bacterial and fungal isolates per environmental sample [119]. In 2009, microorganism samples from the coral mucus were encapsulated within agar spheres, encased in a polysulphonic polymeric membrane, and incubated on the mucus surface of coral Fungia granulosa. Massive microorganisms obtained shared only 50% similarity (85-96%) with previously identified microorganisms [120]. Alternatively, diffusion growth chambers (DGCs) provide another approach to isolate 'uncultivable' microorganisms, as they can be implanted in the tissue of the organism of choice. In 2014, DGCs were first utilized for the cultivation of marine sponge-associated bacteria. Two hundred and fifty-five 16S rRNA gene sequences were obtained, among which 15 sequences were from previously undescribed bacteria [121]. The successful application of new, effective, and efficient approaches in isolating microorganisms will surely contribute to the discovery of novel natural products. However, there are few reports on isolating approaches for ascidian-associated microorganisms. Ongoing studies in our laboratory have been designed to accelerate the isolation of new microorganisms and novel compounds from ascidians.
In closing, we note that with further biotechnological advances, new methods in chemical and biological synthesis will contribute to the discovery of novel and complex drug leads. During the process of finding new compounds, researchers are now sufficiently empowered by such advances that they can think creatively about the drug discovery process. Once the microorganism biosynthetic gene clusters and chemical synthetic routes have been characterized, they can be cloned, artificially modified, and expressed in order to efficiently produce larger amounts of specific compounds, or structurally novel chemical tools [122].