2.08 Natural Product Diversity from Marine Fungi Rainer Ebel, University of Aberdeen, Aberdeen, UK
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2.08 Natural Product Diversity from Marine Fungi Rainer Ebel, University of Aberdeen, Aberdeen, UK ª 2010 Elsevier Ltd. All rights reserved. 2.08.1 Introduction 223 2.08.2 Biology of Marine-Derived Fungi 223 2.08.3 Natural Product Chemistry of Marine-Derived Fungi 226 2.08.3.1 General Aspects of Secondary Metabolites in Marine-Derived Fungi 226 2.08.3.2 New Natural Products from Marine-Derived Fungi 227 2.08.3.2.1 Polyketides 227 2.08.3.2.2 Prenylated polyketides/meroterpenoids 240 2.08.3.2.3 Nitrogen-containing polyketides 240 2.08.3.2.4 Alkaloids 244 2.08.3.2.5 Diketopiperazines 245 2.08.3.2.6 Peptides 248 2.08.3.2.7 Terpenoids 251 2.08.3.2.8 Lipids 252 2.08.3.2.9 Shikimate-derived metabolites 253 2.08.3.2.10 Miscellaneous 253 2.08.3.2.11 Individual fungal strains producing different classes of natural products 254 2.08.4 Conclusions 257 References 258 2.08.1 Introduction In recent years, secondary metabolites obtained from marine-derived fungi have drawn considerable attention as many of them are structurally unique and possess interesting biological and pharmacological properties.1,2 Historically, the first secondary metabolite isolated from a marine-derived fungal strain was cephalosporin C, produced by a culture of a Cephalosporium sp., isolated in 1949 close to a sewage outlet off the Sardinian coast.3 However, this was a more or less incidental discovery, and it took another 30 years until marine-derived fungi were analyzed more systematically. It was only in the late 1980s that sizable quantities of new secondary metabolites were discovered from this long neglected source. The aim of this chapter is to give an overview of secondary metabolites from marine-derived fungi, focusing on the years 2007 and 2008. For earlier years, the reader is referred to a series of general reviews that focus on natural product chemistry of marine-derived fungi.4–7 In addition, more specialized overviews give insights into individual topics, including biotechnological aspects,8,9 screening strategies,10 individual therapeutical areas,11 and fungi from certain geographical areas.12 2.08.2 Biology of Marine-Derived Fungi According to a classical definition, marine fungi cannot be defined on a strictly physiological basis, but rather an ecological one: obligate marine fungi are those that grow and sporulate exclusively in a marine or estuarine habitat, while facultative marine fungi are those from freshwater or terrestrial milieus able to grow (and possibly also to sporulate) in the marine environment.13 However, on a practical basis, it is challenging to separate indigenous marine species (obligate and facultative) from ‘contaminants’ (sometimes also designated ‘transients’), which are terrestrial or freshwater species that are dormant in marine habitats, for example, in the form of spores or hyphal fractions. In principle, the best way of separating indigenous 223 224 Natural Product Diversity from Marine Fungi from nonindigenous species would be to test their germination ability.13 On the other hand, in many cases, it is difficult to obtain sexually reproducing forms, and there is a growing tendency to use molecular biology- based methods, for example sequencing of rDNA, instead of traditional approaches based on morphological characteristics. As the next sections reveal, isolation attempts by natural product chemists and also by trained marine mycologists tend to yield many strains belonging to genera or species well known from terrestrial habitats, while truly obligate marine fungi are obtained on a much rarer scale. Thus, after some vigorous discussions in the earlier days about the true origin of fungal strains isolated from marine habitats, it has now become a common practice to designate these strains ‘marine-derived fungi’. This neutral term does not imply any ecological role such as associates or true symbionts, and comprises any fungal strain obtained from marine environments using cultivation techniques with ‘marine’ media, but does not differentiate between facultative marine strains and contaminants from terrestrial habitats. Chemically characterized marine-derived fungal strains have been obtained from virtually every possible marine habitat, including inorganic matter (soil, sediments, sandy habitats, artificial substrates, and the water column), marine microbial communities, marine plants (algae, sea grasses, driftwood, and other higher plants, especially mangrove plants), marine invertebrates (most notably sponges, but also corals, ascidians, holothur- ians, bivalves, and crustaceans), and vertebrates (mainly fishes). However, it is worth mentioning that it is expected that the fraction of culturable isolates is very low, that is, in the range of 1% or less, with regard to the overall estimated biodiversity, similar to the situation with bacteria. While there is increasing evidence that marine-derived fungi are frequently encountered as associates of other organisms including animals and plants (algae and mangroves), comparatively little is known about free- living representatives found in the open seas, or their role in abiotic substrata such as marine sediments. A recent investigation indicates that the role of marine fungi in sediments probably has so far been under- estimated considerably, simply because they tend to escape detection by microscopical techniques due to the formation of aggregates.14 Virtually any isolation attempt yields new and sometimes novel strains, and the total biodiversity can only be estimated. Similar to the situation encountered with marine bacteria, molecular biological approaches very often give evidence for new taxonomic groups having no known closely related cultivated isolates, for example, when analyzing methane hydrate-bearing deep-sea marine sediments.15 On the other hand, classical isolation techniques typically yield multiple fungal strains for one given biological source, exemplified by a recent report on fungal endophytes of the mangrove plant Kandelia candel, which resulted in more than 50 taxonomically distinct isolates for this one host species, sampled at a single nature reserve in Hong Kong.16 Fungal communities living in marine invertebrates are so far less characterized, although from a chemical point of view, they are among the most prolific producers of secondary metabolites. In the course of a study of marine ascomycetes, only one strain, Abyssomyces hydrozoicus, has been found to be associated with hydrozoans, while the remaining obligate marine fungi were mostly detected in marine algae.17 Although a variety of fungal isolates can usually be obtained from most marine sponges using classical cultivation techniques, the true origin of these sponge-derived fungal strains remains a matter of debate. Since most genera encountered upon cultivation studies are well known from terrestrial habitats, and sponges are known to filter considerable amounts of seawater per day, it is difficult to decide whether a given isolate is truly a sponge associate or even symbiotic, or rather derives from spores washed into the sea and merely trapped inside the sponge during filter feeding. One major caveat is the fact that so far no unequivocal evidence of a fungal associate actively living inside a marine sponge, that is, as fungal hyphae, has been presented. It is interesting to note that this assumption with regard to the origin of sponge- derived fungi is in sharp contrast to the situation of sponge-associated bacteria. Even though sponges feed on bacteria accumulated through filtering of seawater, they have been shown to harbor large quantities of bacteria, some of which seem to be sponge-specific. The finding that some of these bacterial symbionts occur in different sponges collected independently from various geographical locations and are more closely related to each other than to any other known bacterial taxa18,19 has led to the introduction of a new candidate bacterial phylum – Poribacteria.20 However, there is growing evidence that a true symbiotic association between sponges and fungi might indeed exist. On a molecular level, horizontal gene transfer of a mitochondrial intron from a putative fungal Natural Product Diversity from Marine Fungi 225 donor to the sponge Tetilla sp. (Spirophorida) has recently been demonstrated.21 In addition, it had been shown before, again by molecular biological methods, that sponges possess a cell surface receptor that recognizes (1 ! 3)- -D-glucans and thus are able to detect fungi in their environment via the D-glucan carbohydrates on their surface.22 By transmission electron microscopy, an endosymbiotic yeast was discovered in demosponges of the genus Chondrilla from various locations including the Mediterranean, the Caribbean, and the Australian Pacific.23 This symbiont is a chitinous-walled fission yeast, and was interpreted as a yolk body in previous ultrastructural studies. Using immunocytochemical techniques, it could be demonstrated that symbiotic yeast cells were transmitted from the soma through the oocytes to the fertilized eggs, and thus are directly propagated by vertical transmission. However, this example represents a symbiotic relationship that appears to be rather specific for the sponge genus Chondrilla, since similar phenomena were not detected in other demosponge genera.23 The association of marine algae and their endophytic fungi has been studied for a number of years, also employing molecular techniques such as denaturing gradient gel electrophoresis (DGGE). One particularly well-characterized example is the brown alga