In 1982, the Faulkner group at the Scripps Institute of Oceanography reported the isolation of renieramycin A (Figure 1; 1) from the Eastern pacific sponge of the genus Reniera sp. (Frincke and Faulkner, 1982). This material had antibiotic properties and its structure was very similar to the known antitumor agents of the saframycin class that had been reported 5 years earlier by Takahashi and Kubo (1977), from the terrestrial microbe, S. lavendulae. Two later papers (Arai et al., 1979, 1980) gave the structures of saframycins B (Figure 1; 2) and C (Figure 1; 3), and then of saframycin A, (Figure 1; 4), respectively. These reports were then followed in 1988 by the isolation of saframycin Mx 1 (Figure 1; 5) from the myxobacterium Myxococcus xanthus strain Mx48 (Irschik et al., 1988). Thus in just over 10 years, closely related antibacterial and antitumor compounds had been isolated from a streptomycete, a myxobacterium (both terrestrial) and from a marine sponge. However, these were only the tip of the iceberg, as the base molecule, naphthyridinomycin, (Figure 1; 6) was initially reported from Canada in 1974 (Sygusch et al., 1974) and 1975 (Kluepfel et al., 1975), being isolated from the terrestrial streptomycete Streptomyces lusitanus AY B-1026.

Why is this early work so important from a marine perspective? In the middle 1980s to early 1990s, the Rinehart group at the University of Illinois at Urbana, the second group after the Scheuer group in Hawaii to systematically study marine-derived compounds, and then later, in conjunction with the Wright group at Harbor Branch Oceanographic Institution in Florida, published two back to back papers in the Journal of Organic Chemistry showing the structures of the cytotoxic agent ET743 (Figure 1; 7) and its congeners, isolated from the Caribbean tunicate, Ecteinascidia turbinata (Rinehart et al., 1990; Wright et al., 1990). These reports were an extension of the work reported by Holt (1986) in his Ph.D. thesis completed while in the Rinehart group. That this organism “produced” a cytotoxic compound or compounds had been reported in 1969 by Sigel et al. (1970). These compounds were obviously built on the same basic structure as reported for naphthyridinomycin, the saframycins, and renieramycin. Thus one now had multiple bioactive compounds that must have been produced by a similar set of biosynthetic clusters, though it was unknown at the time what the organism or organisms might be, but microbes were prime candidates.

ET743 became an approved antitumor drug under the aegis of the Spanish company PharmaMar and the methods used in its production included: massive large-scale collections; aquaculture of the tunicate in sea and in lakes; then partial synthesis using a marine bacterial product, cyanosafracin B (Figure 1; 8) to produce cGMP ET743. The story leading to the production of ET743 has been presented by a number of people, but the best publications are those from the PharmaMar team (Cuevas et al., 2000, 2012; Menchaca et al., 2003; Cuevas and Francesch, 2009).

In addition to the publications from the PharmaMar group on the semisynthetic processes, two other highly relevant reviews are the one in 2002 by Scott and Williams covering the chemistry and biology of the tetrahydroquinoline antibiotics (Scott and Williams, 2002), which was followed in 2015 by a very thorough review on the ecteinascidins themselves in 2015, again from the Williams group (Le et al., 2015).

From a microbial aspect there were suggestions that an as yet uncultured bacterium, Candidatus Endoecteinacidia frumentenis (AY054370), was involved in the production of these molecules. This organism was found in ET743-producing E. turbinata collected in both the Caribbean and the Mediterranean seas (Moss et al., 2003; Perez-Matos et al., 2007). By use of the suggestions made by Piel (2006) as to how to utilize symbionts from invertebrates, and then using the knowledge of the organization of the BGCs of the saframycins (Li et al., 2008) and safracin B (Figure 1; 9) (Velasco et al., 2005) as markers, the Sherman group at the University of Michigan, were able to identify the “contig” that encoded the NRPS biosynthetic enzymes involved in the ET743 complex, as well as the probable producing bacterium, the as yet uncultured microbe Candidatus Endoecteinascidia frumentensis present in both the Caribbean and Mediterranean E. turbinata organisms (Rath et al., 2011). Four years later, the same group directly confirmed the initial report (Schofield et al., 2015). In the process, they demonstrated that the producing bacterium, E. frumentensis, may well represent a member of a new family of Gammaproteobacteria and has an extensively streamlined genome as found in other symbiotic microbes (McCutcheon and Moran, 2012), with most of the genetic machinery being devoted to this complex of compounds (Kehr and Dittmann, 2015).

The structure of the toxin pederin (Figure 2; 10) “used” by rove beetles of the genus Paederus as a protective agent, was first identified chemically by Italian scientists studying this genus in a publication in 1952 (Pavan and Bo, 1952). The dermatitis caused by the toxin has been well described in the literature, with a recent publication by Cressey et al. (2013) demonstrating the problem with this toxin.

Following on from the original 1952 publication, in 1965 Carani et al. (Cardani et al., 1965) published an initial structure which was then revised 3 years later by Matsumoto et al. (1968) giving the structure shown (Figure 2; 10). This is where the story might have languished with the compound simply becoming an interesting molecule to synthesize by demonstrating novel chemical methods. Such syntheses have been reported in some relatively recent publications in the synthetic chemistry literature (Jewett and Rawal, 2007; Wu et al., 2011). However, in the late 1980s, the Blunt and Munro group at the University of Canterbury in New Zealand, published their finding that an extract of a relatively deep water sponge of the genus Mycale, collected in cold water, produced bioactive molecules that had antiviral and cytotoxic biological activities, and encompassed the pederin nucleus within the overall structure. The molecules were also powerful vesicants, as is pederin. The two molecules were named mycalamide A (Figure 2; 11) and B (Figure 2; 12) differing only by a methyl group, but with a 10-fold difference in biological activity (Perry et al., 1988, 1990).

What is the relationship of such findings to pederin and Paederus beetles? The following reports in the insect physiology literature will help uncover the reasons why this was a very important finding, that led, many years later, to the identification of the actual “source” of the pederin-related molecules found in the Mycale sp., and in other sponges, in particular, the Theonella swinhoei “Yellow variant.” In 1999, a German entomologist, Rupert Kellner, published a very interesting paper that asked; “what was the basis of pederin polymorphism in the rove beetle Paederus riparius?” His suggestion, with data, was that an endosymbiont was the actual producer of the toxin (Kellner, 1999). Then 2 years later, he reported that pederin biosynthesis was suppressed in the closely related species, Paederus sabaeus, if antibiotics were used to remove endosymbionts (Kellner, 2001). Thus there was significant evidence implying a bacterial component in the production of pederin, and since this occurred in two different species of the beetle, it might be common to all.

However, to bring the story to its climax, one now has to return to the marine environment. From 1988 to early 2000, there were reports that a significant number of sponge extracts contained pederin-related molecules such as the onnamides, with onnamide F (Figure 2; 13) being a good example (Vuong et al., 2001), together with the theopederins, with compounds such as theopederins K (Figure 2; 14) and L (Figure 2; 15) being examples of the structural similarities (Paul et al., 2002). Kellner (2002b) reported that he had identified the endosymbiont as a very close relative to Pseudomonas aeruginosa and showed that interspecific transmission of the endosymbionts was related to the different genetic makeup of individual isolates (Kellner, 2002a).

So how did this work then relate to the “marine-derived pederin-like compounds”? In a series of papers in the time frame from 2002 to 2005, Piel demonstrated that he could find the biosynthetic clusters for pederin in the putative pseudomonad identified and isolated by Kellner (Piel, 2002), and that these had an unprecedented diversity of catalytic domains in the first four clusters in the process (Piel et al., 2004c). Since Piel had the relevant genetic probes, he collaborated with the Japanese group led by Fusetani and Matsunaga at the University of Tokyo, to investigate the production of the closely related onnamides, isolated from the Japanese sponge Theonella swinhoei (yellow variant) in warm, shallow waters off of Okinawa, in contrast to the mycalamide-producer. The pseudomonal probes “lit up” parts of the sponge metagenome, and they were able to locate the nexus of the biosynthesis to an as yet uncultivated symbiont (Piel et al., 2004b). Concomitantly Piel also demonstrated evidence for what is now known as a “symbiosis island” that permitted horizontal acquisition of the pederin biosynthetic capabilities in Paederus fuscipes (Piel et al., 2004a). The details as of that time were published in a short review in the Journal of Natural Products in 2005 (Piel et al., 2005), and then in 2011, Kador et al. (2011) published separate oligonucleotide probes that could be used to detect pederin producers in Paederus beetles.

Following on from these seminal studies, Wilson and Piel (2013) demonstrated potential approaches to the study of uncultivated, or not yet cultivatable microbes, as resources for novel biosynthetic enzymology. This paper demonstrated the potential for performing genomic work on very small numbers of uncultivated bacteria isolated from invertebrate hosts, in this case, sponges, and tunicates. This was exactly the type of investigation that Bewley and Faulkner (1998) wished to perform in the late 1990s when they identified the production of the cyclic peptides swinholide A (Figure 2; 16) and theopalauamide (Figure 2; 17) by Theonella swinhoei, and their suggestion, using the techniques then available (microscopy in general), that a microbial consortium might be responsible for their “production.”

In 2014, 1 year after their earlier paper, the Piel group demonstrated in a seminal publication in Nature, that the producing organism in T. swinhoei was an as yet uncultured microbe, and that there were two subtly distinct variations isolated, TSY-1 and TSY-2 via single cell separation of the sponge and contents. Both contained a plasmid that contained the onnamide and polytheonamide BGCs, but further investigation showed that they differed significantly when BGC clusters that were chromosomally encoded were studied. Thus the TSY-1 variant in addition to the plasmid-encoded BGCs, also contained the genes for a further 28 BGCs including cyclotheonamides, proteusins, and ceramides, plus others. The other strain, TSY-2 only had seven other BGCs with very little overlap with TSY-1 metabolites. As a result of these and other studies on different sponge taxa, the suggestion was made that these were representative of a new phylum “Tectomicrobia.” Thus these two microbes contained the necessary genetic machinery to produce 31 of the then 32 known cytotoxins to have been isolated from this particular sponge at that time (Wilson et al., 2014).

Recently, the Piel group have published the enzymology involved in the formation of the long peptides such as the polytheonamides which have repeating D and L amino-acids, but are ribosomally produced peptides (Morinaka et al., 2014), with a recent publication by Hayata et al. (2016) demonstrating syntheses around this structure to produce new ion-channel cytotoxins. Morionka et al. (2014), was then followed in 2016 by a review demonstrating the metabolic potential of the as yet uncultivated “Entotheonella” where more information is given as to the multiplicity of structures that result from this microbe (Freeman et al., 2016).

Looking at an area where many marine-derived bioactive compounds have been reported, but none have yet made into clinical trials, Ueoka et al. (2015) published an extensive paper on the source of misokinolide A, a compound that differs from the well-known swinholide A by removal of one double bond in the ring structure (Figure 2; 18), demonstrating that it too, came from a non-cultivated Entotheonella but not from T. swinhoei but from a Discodermia species. Interestingly the protein phosphatase inhibitor calyculin A (Figure 2;19) produced by Discodermia calyx, was shown by Wakimoto et al. (2014) to be a product of the same microbial genus, then in a more thorough paper published in 2016, they demonstrated that it is actually produced as a pro-drug (Wakimoto et al., 2016). Such a pro-drug approach may well be a protection method to avoid killing the host invertebrate or even the producing microbe.

To conclude the microbial chemistry aspect of this section, but now moving back to the terrestrial sphere, there was a very intriguing report showing the presence of a pederin-like compound, nosperin (Figure 2; 20) found in a lichen, where the bacterium was a cyanobacterium, a Nostoc sp., so these biosynthetic genes are extremely widespread (Kampa et al., 2013).

As mentioned earlier, the structures of pederin and its derivatives certainly excited synthetic organic chemists and over the years, effectively all of the molecules that contain the pederin backbone have been synthesized, even when ring-opened as in irciniastatin A (Figure 2; 21). This compound was initially reported by the Pettit group in 2004 (Pettit et al., 2004) and subsequently reported as psymberin by the Crews group the same year (Cichewicz et al., 2004) from a different sponge genus. Careful inspection of the supporting information in the Crews paper showed that they knew of the same compound under a different name/genus from the Pettit group, published before their submission. Thus the Pettit group has priority in the discovery of this compound structure. Representative examples of these compounds have been synthesized by considerable numbers of organic chemists in the last 10 plus years. The following papers should be consulted by readers interested in the synthetic processes used (Jewett and Rawal, 2007; Jiang et al., 2007; Nishii et al., 2009; Wan et al., 2011; Wu et al., 2011, 2012; Mosey and Floreancig, 2012; Floreancig, 2014; Uesugi et al., 2015). Of these, the review by Mosey and Floreancig (2012) gives a relatively thorough overview of the isolation, biological activities, and medicinal chemistry of these agents.

Thus what began as a discussion of the toxin produced by the blister beetle that was known in Brazilian forests/jungle and in other parts of the World, led to the ability to identify and express genetic loci related to the biosynthesis of the agent, but then “migrated” into areas not even thought to be possible; that the beetle toxin was in fact, used by biodiversity (aka Mother Nature) to generate molecules in organisms as diverse as shallow and deep water marine sponges, in warm (close to 35°C) and cold (2°C) water environments and even in terrestrial lichens. None of these were even thought of in the wildest dreams of the original scientists working on beetle toxins.

In the encrusting ascidians such as Lissoclinum species, investigators over the years have found a series of bioactive agents mainly based on cyclic peptides. For many years, the actual source of the agents, in particular the patellamides, was not known. In a presentation at a Society of Industrial Microbiology Meeting very late in 2004, Long et al. (2005) presented evidence that the probable producer was an as yet uncultivated cyanophyte, a “Prochloron” species. Unfortunately, this paper was not included in the printed list of papers presented (only discovered after the presentation at the meeting), thus in the absence of a dated presentation, the absolute priority to this group could not be formally assigned. Long et al. (2005) demonstrating that the complete Prochloron sp. genome could be moved into an E. coli host using shotgun cloning techniques. Prochloron samples were extracted from the cloacal cavities of ten specimens of L. patella, each showing a matching patellamide composition according to HPLC analysis of the holobiont extracts. Using bacterial artificial chromosome techniques and then attempting to express the patellamide producing genes using primers failed. However, by use of standard fermentation techniques, clones were obtained that produced patellamide D Figure 3; 22) and ascidiacylamide (Figure 3; 23).

Whilst this paper was in press, the Schmidt group published similar results using a different L. patella source from that used by Long et al. (2005; Schmidt et al., 2005). The Schmidt group used material from the Republic of Palau instead of the Great Barrier Reef (Long et al., 2005) and identified the relevant gene clusters in the isolated Prochloron species. Since they had been working on a full genomic analysis of this symbiont, they had information available pointing to potential gene biosynthetic clusters, enabling a rapid assessment of the potential biosynthetic clusters by expressing them in E. coli. Thus two independent groups came up with the same source of the patellamides.

The Schmidt group then proceeded over the next few years to further analyses of the producing symbiont, uncovering a fascinating series of complex interactions that permitted substitution of individual aminoacids in these ribosomally produced products, that had geographic relationships defined in terms of what mixture of patellamides and the closely related trunkamides were produced by individual ascidians. For the interested reader, a significant number of publications related to this symbiotic relationship have been published by the Schmidt group and their collaborators. These should be perused in detail to demonstrate the complexity of the interactions involved (Donia et al., 2011; Kwan et al., 2012, 2014; Schmidt et al., 2012; Tianero et al., 2012, 2015; Adnani et al., 2014; Schmidt, 2014, 2015). The most current paper is one published very recently in Applied and Environmental Microbiology demonstrating the intimate involvement of the microbe in this process (Lin et al., 2016).

The bryozoan B. neritina first came to the attention of natural product chemists when Pettit reported the isolation of bryostatin 3 (Figure 3; 24), and then over the next few years, elucidated the structures of 18 bryostatins, all isolated from massive wild collections of B. neritina either in the Gulf of Florida, or other coastal areas of the US, with a massive collection in Palos Verdes, California that led to the isolation of gram quantities of bryostatin 1 by workers at NCI-Frederick (Schaufelberger et al., 1991). The story up through late 2011 was given by in a review by Newman published as a chapter in the second edition of “Anticancer Agents from Natural Products” in 2012 (Newman, 2012). Over 80 clinical trials of bryostatin 1 (Figure 3; 25) with or without other cytotoxic agents have been reported over the years at Phase I or Phase II levels against various cancers, but none have demonstrated activities that have warranted continuation. Currently there is one trial shown at the Phase II level in Alzheimer’s disease (NCT02431468) under the aegis of Neurotrope Inc.

The actual producing agent in B. neritina was unknown until the report by Haygood and Davidson (1997), demonstrating that the larvae of bryostatin-producing bryozoans contained a previously unknown bacterium that could not be cultured, but was observable in the pallial sinus of the larvae. This microbe was named as “Candidatus Endobugula sertula” by Haygood, and over the next few years, this microbe was found in other examples of B. neritina but appeared to have a latitudinal restriction and strain variation with depth. These phenomena were well investigated by both the Haygood group on the Pacific Coast, and by the Lopanik group in the Atlantic Coast of the USA. The relevant publications are (Davidson and Haygood, 1999; Davidson et al., 2001; Lopanik et al., 2004, 2006; Sharp et al., 2007; Linneman et al., 2014).

The role of bryostatin within the larvae appears to be a protective measure. This was shown by investigation of the effect of removing the symbiont via antibiotic treatment, followed by predation studies on larvae containing the symbiont, versus those without it. The levels of bryostatins were also measured in these experiments and they demonstrated that without bryostatin production, the larvae were “food” for predators (Lopanik et al., 2004). What was of immense import, however, was the work performed by the Sherman group at the University of Michigan, in conjunction with the Haygood and Lopanik groups, where they identified the putative bryostatin gene cluster from “Candidatus Endobugula sertula” in 2007 (Sudek et al., 2007). This initial paper was followed up by the Sherman group in 2008 (Lopanik et al., 2008) and 2010 (Buchholz et al., 2010), leading to a very recent paper demonstrating the “holobiont fitness” via specific interactions with host organism PKC enzymes from the Lopanik group (Mathew et al., 2016). Thus even if the organism’s symbiont cannot yet be cultivated, its effect and product can be measured by modern techniques.

Finally, a very interesting paper was published recently demonstrating that molecules based on the bryostatin skeleton can have significant effects on viral replication. Paul Wender at Stanford University, who for many years has been involved in modifying the basic structure of the bryostatins and other potent marine-derived biologically active natural products, producing bioactive truncated structures. In the case of the bryostatin-based compounds they are known colloquially as “bryologs,” and Wender et al. published a paper in 2016 demonstrating the inhibition of Chikungunya virus-induced cell death, by a relatively simple bryostatin analog, that does not appear to use a PKC pathway, in contrast to the usual mechanism of action of bryostatins (Staveness et al., 2016). Thus close to 50 years since the first reports of bryostatins, they are still an active structural class for synthetic chemists to modify.