Predominately Uncultured Microbes As Sources of Bioactive Agents

fmicb-07-01832 November 16, 2016 Time: 14:6 # 1 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Frontiers - Publisher Connector REVIEW published: 18 November 2016 doi: 10.3389/fmicb.2016.01832 Predominately Uncultured Microbes as Sources of Bioactive Agents David J. Newman* Newman Consulting LLC, Wayne, PA, USA In this short review, I am discussing the relatively recent awareness of the role of symbionts in plant, marine-invertebrates and fungal areas. It is now quite obvious that in marine-invertebrates, a majority of compounds found are from either as yet unculturable or poorly culturable microbes, and techniques involving “state of the art” genomic analyses and subsequent computerized analyses are required to investigate these interactions. In the plant kingdom evidence is amassing that endophytes (mainly fungal in nature) are heavily involved in secondary metabolite production and that mimicking the microbial interactions of fermentable microbes leads to involvement of previously unrecognized gene clusters (cryptic clusters is one name used), that when activated, produce previously unknown bioactive molecules. Keywords: natural product sources, uncultured microbes, poor producing microbes, endophytes, microbe– microbe interactions Edited by: Michael Thomas-Poulsen, INTRODUCTION University of Copenhagen, Denmark Reviewed by: Over the past twenty-plus years, data related to microbes from all sources being involved in the Leo Van Overbeek, production of bioactive agents, has grown from being a “suggestion or slight possibility” to now Wageningen University and Research being recognized, at least in the marine area, as probably being the source(s) of the majority of Centre, Netherlands bioactive agents that have been reported from marine invertebrates. However, in the vast majority Yasuo Yoshikuni, Joint Genome Institute, USA of cases, the microbe(s) cannot be cultivated under the usual conditions that had been used for many years. The advances in genomic analysis techniques, particularly in the last five or so years, *Correspondence: David J. Newman has however, permitted the amplification and then the subsequent analysis from a biosynthetic [email protected] perspective, of the genes within a single “as yet uncultured” microbe, and thus the identification of the “source” of the bioactive materials reported from the “host.” Specialty section: In the case of plant-derived bioactive compounds, what has now occurred with regularity, This article was submitted to are reports of mainly endophytic microbes, usually fungi, but frequently actinobacteria, that will Antimicrobials, Resistance produce low levels of the “plant metabolite” on fermentation but on subsequent sub-culturing, and Chemotherapy, the microbe apparently loses its ability to produce the “plant-metabolite.” However, in the last a section of the journal few years, investigators have begun to “relearn” techniques that used to be very common in the Frontiers in Microbiology pharmaceutical industry, but to the ultimate chagrin of academic researchers, were never published Received: 12 July 2016 in regular journals, that supplementation of fermentation broths with extracts of parts of the Accepted: 01 November 2016 “nominal producing source,” could “induce or maintain” production of the metabolite of interest. Published: 18 November 2016 The other component of “production” that has now come to the fore, is the belated recognition Citation: that in any microbial system, there is constant chemical messaging between microbes in the Newman DJ (2016) Predominately Uncultured Microbes as Sources microbiome of the host. Whereas microbiologists and co-workers want a single microbe to produce of Bioactive Agents. a given compound, Mother Nature prefers a consortium and thus the recognition that mixed Front. Microbiol. 7:1832. cultures might well aid in “production” has now become quite apparent, usually via the “switching doi: 10.3389/fmicb.2016.01832 on or off” of biosynthetic gene clusters (BGCs) in one or more of the consortium. Frontiers in Microbiology | www.frontiersin.org 1 November 2016 | Volume 7 | Article 1832 fmicb-07-01832 November 16, 2016 Time: 14:6 # 2 Newman Uncultured Microbes as Compound Sources This short review will cover various aspects of the above, not a marine bacterial product, cyanosafracin B (Figure 1; 8) to in depth but relevant up to date references will be provided so produce cGMP ET743. The story leading to the production that the reader can further investigate the areas discussed. 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, MARINE-SOURCED BIOACTIVE AGENTS 2009). In addition to the publications from the PharmaMar group on AND MICROBES the semisynthetic processes, two other highly relevant reviews are the one in 2002 by Scott and Williams covering the chemistry The Naphthyridinomycin/ and biology of the tetrahydroquinoline antibiotics (Scott and Tetrahydroisoquinoline Derivatives Williams, 2002), which was followed in 2015 by a very thorough In 1982, the Faulkner group at the Scripps Institute of review on the ecteinascidins themselves in 2015, again from the Oceanography reported the isolation of renieramycin A Williams group (Le et al., 2015). (Figure 1; 1) from the Eastern pacific sponge of the genus From a microbial aspect there were suggestions that an as yet Reniera sp. (Frincke and Faulkner, 1982). This material had uncultured bacterium, Candidatus Endoecteinacidia frumentenis antibiotic properties and its structure was very similar to the (AY054370), was involved in the production of these molecules. known antitumor agents of the saframycin class that had This organism was found in ET743-producing E. turbinata been reported 5 years earlier by Takahashi and Kubo(1977), collected in both the Caribbean and the Mediterranean seas from the terrestrial microbe, S. lavendulae. Two later papers (Moss et al., 2003; Perez-Matos et al., 2007). By use of the (Arai et al., 1979, 1980) gave the structures of saframycins B suggestions made by Piel(2006) as to how to utilize symbionts (Figure 1; 2) and C (Figure 1; 3), and then of saframycin A, from invertebrates, and then using the knowledge of the (Figure 1; 4), respectively. These reports were then followed organization of the BGCs of the saframycins (Li et al., 2008) in 1988 by the isolation of saframycin Mx 1 (Figure 1; 5) and safracin B (Figure 1; 9) (Velasco et al., 2005) as markers, from the myxobacterium Myxococcus xanthus strain Mx48 the Sherman group at the University of Michigan, were able (Irschik et al., 1988). Thus in just over 10 years, closely related to identify the “contig” that encoded the NRPS biosynthetic antibacterial and antitumor compounds had been isolated from enzymes involved in the ET743 complex, as well as the probable a streptomycete, a myxobacterium (both terrestrial) and from a producing bacterium, the as yet uncultured microbe Candidatus marine sponge. However, these were only the tip of the iceberg, Endoecteinascidia frumentensis present in both the Caribbean as the base molecule, naphthyridinomycin, (Figure 1; 6) was and Mediterranean E. turbinata organisms (Rath et al., 2011). initially reported from Canada in 1974 (Sygusch et al., 1974) and Four years later, the same group directly confirmed the initial 1975 (Kluepfel et al., 1975), being isolated from the terrestrial report (Schofield et al., 2015). In the process, they demonstrated streptomycete Streptomyces lusitanus AY B-1026. that the producing bacterium, E. frumentensis, may well represent Why is this early work so important from a marine a member of a new family of Gammaproteobacteria and has perspective? In the middle 1980s to early 1990s, the Rinehart an extensively streamlined genome as found in other symbiotic group at the University of Illinois at Urbana, the second group microbes (McCutcheon and Moran, 2012), with most of the after the Scheuer group in Hawaii to systematically study marine- genetic machinery being devoted to this complex of compounds derived compounds, and then later, in conjunction with the (Kehr and Dittmann, 2015). 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 MARINE METABOLITES BASED UPON A agent ET743 (Figure 1; 7) and its congeners, isolated from the Caribbean tunicate, Ecteinascidia turbinata (Rinehart et al., 1990; TERRESTRIAL BEETLE TOXIN Wright et al., 1990). These reports were an extension of the work reported by Holt(1986) in his Ph.D. thesis completed Mycalamides, Onnamides, and Similar while in the Rinehart group. That this organism “produced” a Molecules cytotoxic compound or compounds had been reported in 1969 The structure of the toxin pederin (Figure 2; 10) “used” by rove by Sigel et al.(1970). These compounds were obviously built on beetles of the genus Paederus as a protective agent, was first the same basic structure as reported for naphthyridinomycin, identified chemically by Italian scientists studying this genus in a the saframycins, and renieramycin. Thus one now had multiple publication in 1952 (Pavan and Bo, 1952). The dermatitis caused bioactive compounds that must have been produced by a similar by the toxin has been well described in the literature, with a recent set of biosynthetic clusters, though it was unknown at the time publication by Cressey

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