microalgal metabolites: a new perspective

August 2, 1996 21:20 Annual Reviews SHIMTEXT.TRA AR15-14

Annu. Rev. Microbiol. 1996. 50:431–65Copyright c© 1996 by Annual Reviews Inc. All rights reserved

MICROALGAL METABOLITES: A NewPerspective

Yuzuru ShimizuDepartment of Pharmacognosy and Environmental Health Sciences, College ofPharmacy, University of Rhode Island, Kingston, RI 02881

KEY WORDS: secondary metabolites, phylogenic relationship, biosynthesis, genetic, symbio-sis, food chain

ABSTRACT

Occurrence of secondary metabolites in microalgae (protoctista) is discussed withrespect to the phylogenic or taxonomic relationships of organisms. Biosyntheticmechanisms of certain metabolites such as paralytic shellfish poisoning toxinsand polyether toxins are also discussed, and genetic aspects of the secondarymetabolite production as well.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432

METABOLITES FOUND IN MICROALGAE AND ALGAL TAXONOMY . . . . . . . . . . . . . 433Sterols and Related Compounds in Membranes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433Saxitoxin and Gonyautoxin Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435Polyether Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436Macrolides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439Open-Chain Polyketides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440Cyclic Oxylipins or Eicosanoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441Peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443Miscellaneous Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443

BIOSYNTHESIS OF MICROALGAL METABOLITES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446Biosynthesis of Nitrogenous Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447Biosynthesis of Polyketides in Microalgae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449Biosynthesis of Miscellaneous Algal Metabolites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454

GENETICS OF SECONDARY METABOLITES PRODUCTION AND SYMBIONTS’ROLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454

SIGNIFICANCE OF MICROALGAL METABOLITES IN THE FOOD CHAIN. . . . . . . . . . 458

CONCLUSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

4310066-4227/96/1001-0431$08.00

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432 SHIMIZU

INTRODUCTION

In recent years, there has been tremendous interest in microalgal metabolitesamong researchers such as natural products chemists, pharmacologists, andbiochemists. There are two major reasons for this surge of interest. First, it hasbeen recognized that microalgae can be a source of new types of metabolites orpotential drugs. In the past, drug searches have been focused mostly on organ-isms such as actinomycetes, fungi, and higher plants. Here, people are increas-ingly isolating known compounds or close analogues of known compounds,and the task is becoming more and more repetitive and wasteful. Meanwhile,microalgae have yielded new types of structures not found in higher plants orother traditional drugs sources. The second reason that microalgae have beenattracting so much attention is the realization that they may be a primary sourceof some exciting molecules found in marine invertebrates. In the past twodecades, a great number of new structures with unique biological activity havebeen found in marine invertebrates. Many of them are potential therapeuticdrugs, but their supplies are very limited. As described below, there is directevidence that some of the compounds are derived from microalgae such as di-noflagellates. Thus, the simple thinking that these compounds can be producedabundantly by culturing their source organisms has driven many researchers insearch of new algae and their metabolites.

Despite all this enthusiasm, the research of microalgal metabolites has a veryshort history. Only limited knowledge is available about their chemotaxonom-ical positions and biosynthetic mechanisms, which are very beneficial in thedrug search for and the cultural production of the desired metabolites. Thischapter is to put the available information in a new perspective in order to ben-efit current and future researchers as much as possible. It is not intended toprovide a glossary of the compounds which have been isolated from microal-gae. Such compilations have already been made by several authors (25, 46, 61,102, 130).

The word “microalgae” as used in this chapter needs an explanation. Theword usually refers to small photosynthetic organisms mostly found in aquaticenvironments. Recently, more and more people have limited their use of theword for eukaryotic organisms. Thus, blue-green algae, which are prokaryotes,are excluded from the category. Cyanobacteria is the word of choice for thosewho believe in the strict distinction between prokaryotes and eukaryotes. Onthe other hand, a great number of people are still using the term blue-green algaeor blue-greens, and papers and chapters are written under the title of blue-greenalgal metabolites. Actually, their macromorphology, life style, and environ-mental roles are not so different from eukaryotic microalgae. Moreover, someof their metabolites are also found in the primitive eukaryotes (mesokaryotes),

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MICROALGAL METABOLITES 433

and it is difficult to exclude blue-greens from this discussion. Therefore I referto some pertinent cyanobactrial metabolites as is necessary.

This taxonomical confusion also extends to eukaryotic microalgae. Exceptfor traditional green, red, and brown macro algae, the entire microalgal floraare in the overlapped area between the animal and plant kingdoms. Here, thephotosynthetic ability, which is commonly used to distinguish plants and ani-mals, is totally irrelevant because one can find autotrophic, heterotrophic, andmesotrophic forms in the same species. The new kingdom Protista convenientlycovers all unicellular microbes, and sometimes includes both prokaryotes andeukaryotes. On the other hand, the term Protoctista excludes prokaryotes butincludes macroforms of algae. As defined, “the protoctista comprises the entiremotley and unruly group of nonplant, nonanimal, [and] nonfungal organismsrepresentative of lineages of the earliest descendants of eukaryotes” (17). Thischapter covers mostly this category of organisms.

Understanding the phylogenic relationships of those organisms is importantto a chemist in order to anticipate their chemical constituents. In the literature,we see quite diverse schemes of phylogenic positions for various microalgae.Assumptions are made on a variety of factors, such as chlorophyll and otherpigment patterns, organelle compositions, and, more recently, RNA sequences(21, 60, 115, 120). Figure 1 shows an example of the phylogenic tree composedfrom a combination of these data.

METABOLITES FOUND IN MICROALGAE AND ALGALTAXONOMY

Sterols and Related Compounds in MembranesIf biological membranes are a very basic element of life, their structures andcomponents can also be intrinsic to the phylogenic positions of organisms.There have been many attempts to relate the membrane sterols to taxonomy.Generally speaking, prokaryotic membranes lack sterols. Animals use C27

sterols represented by cholesterol; Mycota (fungi), C28 sterols; and Chlorophyta(green algae) and higher plants, C29 sterols. Phaeophyta (brown algae) use C28

sterols such as fucosterol. Significantly, Rhodophyta (red algae), which arepresumed to have branched out at an early stage of phylogeny, use cholesteroland other C27 zoosterols.

In Protoctista, the membrane sterol patterns show a pattern of variation thatsuggests different phylogenic origins of the organisms or the membranes. TheeuglenoidTetrahymenaspp. use pentacyclic triterpene tetrahymenol and cili-ates use the closely related hopanol (1, 57). The biosynthesis of both tetrahy-menol and hopanol is done by the proton initiated cyclization of squalene itself

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434 SHIMIZU

Figure 1 Speculative phylogenic positions of proctista organisms discussed in this chapter.

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and does not go through 2,3-oxidosqualene, whose formation requires squalenemonooxygenase (1, 56). Hopanol derivatives are triterpenoids in ferns, but thehopane derivatives (in the form of C-glycoside) are also membrane componentsof eubacteria such asAcetobacter, Bacillus,andMethylobacterium(1). Here,we may be seeing a possible line of phylogenic connection among these organ-isms or the origins of the organelles that are involved in the biosynthesis of thesterols.

Dinoflagellate membranes contain cholesterol and dinosterol derivatives(103). Dinosterol was first found inAlexandrium(also known asGonyaulax)spp. (103) and later in many other dinoflagellates (127, 128). The structureof dinosterol has a 4α-methyl group and an unusual 23-methyl group sidechain. Dinosterol is highly resistant to chemical transformation, and the intactmolecule is well-preserved in old sediments as a chemical fossil of dinoflagel-lates. The 4α-methyl sterols are also found in Chlorophyta, Euglenophyta, andChrysophyta (33), but 23-methyl substitution seems to be characteristic onlyof dinoflagellate sterols. Certain genera of dinoflagellates do not have sterolswith a dinostane skeleton. For example, allAmphidiniumspp. lack sterolswith dinostane skeleton but have amphisterol instead (127). The 4α-methylC28 sterol skeleton of amphisterol is essentially identical with the Eugleno-phyta and Chrysophyta sterols, suggesting thatAmphidiniumis a link to thesedifferent phyla. Dinoflagellates in some genera such asGymnodiniumare alsodevoid of peridinin, the pigment considered to be characteristic to dinoflagel-lates. Those observations raise a question about the taxonomical homogeneityof the Dinophyceae family, which is very large and embraces many differenttypes of organisms. Figure 2 shows some representative skeletons of protoctistamembrane sterols and triterpenes.

Saxitoxin and Gonyautoxin DerivativesProbably the most prominent metabolites produced by dinoflagellates are saxi-toxin and gonyautoxin derivatives. Saxitoxin was first recognized as the cause ofparalytic shellfish poisoning (PSP) inAlexandrium catenella(92, 117). Neosax-itoxin and gonyautoxins were first found inA. tamarense(80, 95, 97, 101, 110).The compounds are introduced into the food chain and accumulate in shellfishand other marine organisms. They are important pharmacological tools as theyare highly selective sodium channel blockers (36). Saxitoxin analogues arealso found in other genera of dinoflagellates. However, the toxigenicity of theorganisms is not uniform, and even in the same species there is a wide variationof virulence (2, 58). The unique structure of PSP toxins is a tricyclic perhy-dropurine skeleton. Figure 3 shows some representative PSP toxins. Theirstructural variation comes from the presence or absence of an O-sulfate groupand/or an N-sulfate carbamoyl group. These structurally unique compounds

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436 SHIMIZU

Figure 2 Structural patterns of membrane sterols and triterpenes of protoctista.

are also found in cyanobacteria. Thus, saxitoxin and neosaxitoxin are foundin Aphanizomenon flos-aquae, a common freshwater cyanobacterium (3, 41,45). A group of gonyautoxins are found in another species,Anabaena circiralis(39, 76, 79). There is some evidence that the occurrence of PSP toxins amongblue-greens is more widely spread. It has been recently reported that suchcommon blue-green algae asLyngbya wollei(14) andOscillatoria morigeotiiproduce gonyautoxins and decarbamoylsaxitoxin (79). The occurrence of thesevery peculiar compounds both in prokaryotes and eukaryotes leads to severalinteresting speculations that are discussed later.

Polyether CompoundsAnother very characteristic group of metabolites produced by Protoctista arepolycyclic ethers (Figure 4). The spectacular linear arrangement of all-transcyclic ethers represented by brevetoxin B (55) and brevetoxin A (104) was first

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found in the dinoflagellateGymnodinium breve(4, 55, 87, 96, 104) and wasfound later in several other dinoflagellate species (70, 78, 130). Ciguatoxin andits congeners, which are responsible for ciguatera fish poisoning, are producedby Gambierdiscus toxicus(54, 67, 132). The same organism also produces astrong Ca2+ channel activator, maitotoxin, that has a structure of three cyclicsystems linked together (68). These polycyclic ethers are not reported in ter-restrial organisms and are thought to be specific to dinoflagellates, but recentlyPrymnesium parvumin Prymnesiophyceae was also found to produce similarcompounds (40) (Figure 5). The taxonomic position of Prymnesiophyceae maybe debatable, but it is usually placed in the class Chrysophyta or Haptophytaand is different from Dinophyceae, which is in Pyrrhophyta. The chemicalconstituents of Prymnesiophyceae are not well investigated, but further inves-tigation may reveal the closeness of these families.

Okadaic acid and related toxins are strong protein phosphatase 1 and proteinphosphatase 2A inhibitors used as tools in cell biology research (26). The com-pounds were first found in marine invertebrates such as sponges and shellfish(93, 119, 131). Dinoflagellates such asDinophysisspp.,Prorocentrum lima,andP. concavumproduce these compounds (19, 38, 66, 134). The structuresof okadaic acid and their derivatives hold some resemblance to those of thepolyether antibiotics such as monensin and nonactin (Figure 6).

Figure 3 Representative structures of PSP toxins produced by both dinoflagellates and cyanobac-teria. They are divided into two series: saxitoxin and neosaxitoxin series. Further structurevariation comes from the presence or absence of O- and N-sulfate moieties and stereochemicaldifferences.

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438 SHIMIZU

Figure 4 Examples of linear polycyclic ethers produced by dinoflagellates. Brevetoxins areproduced by the Florida red tide organismGymnodinium breveand ciguatoxin congeners byGam-bierdiscus toxicus, a tropical dinoflagellate.

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MacrolidesMacrolides are one of the structural types often seen inStreptomycesmetabo-lites, and several major antibiotics belong to this class. Dinoflagellates producesimilar macrolides.Amphidiniumspp. isolated from theAmphiscolopsflat-worm produce a series of macrolides represented by amphidinolide B (Figure7). They have varied lactone ring sizes, and most of them, such as amphidino-lide B and caribenolide I, are reported to be highly cytotoxic and antitumor (5, 7,42, 46). It is interesting that not all strains in anAmphidiniumspecies can pro-duce macrolide compounds. At this moment, no taxonomic or morphologicalcharacteristics can be correlated with macrolide productivity of the organisms.The symbiotic nature of the organisms was considered to be related to the

Figure 5 The structures of maitotoxin from the dinoflagellate,Gambierdiscus toxicus(Dino-phyceae), and prymnesin fromPrymnesium parvum(Haptophyceae). The two compounds pro-duced by organisms in the two different families show remarkable resemblance.

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440 SHIMIZU

Figure 6 The structures of okadaic acid and its derivatives, potent protein phosphatase 1 and 2Ainhibitors produced by the dinoflagellatesProrocentrumspp. andDinophysisspp.

biosynthetic ability of the organisms, but a nonsymbiotic free-swimming straincan also produce the macrolides (5). Goniodomin A is an antifungal com-pound found in the dinoflagellateGoniodoma(also known asAlexandrium)sp. (65). Prorocentrolide is an unusual nitrogen-containing macrolide fromProrocentrum lima(121). More recently, another nitrogen-containing lactone,gymnodimine, was isolated fromGymnodiniumsp. as a foodborne toxin (94)(Figure 8).

Macrolides are also produced by blue-greens, a group of which has been iso-lated: scytophycins (Figure 9) fromScytonemaspp. (48), and tolytoxin fromTolypothrix conglutinatavar. colorata(11). Scytophycins are highly cytotoxic21-membered macrolides, and their structures are closely related to cytotoxicor antitumor macrolides found in marine invertebrates (10). Tolytoxin is recog-nized as a potent microfilament-depolymerizing agent. Debromoaplysiatoxinproduced by several strains ofLyngbya majuscula(62, 69) is a potent proteinkinase C activator (26, 27).

The presence of macrolides in cyanobacteria and dinoflagellates raises someintriguing questions. First, are the dinoflagellate macrolides and the cyanobac-teria macrolides related? Second, is there any genetic relationship between theactinomycetes and the microalgae with respect to the capability of macrolidebiosynthesis? Do the various macrolides discovered in marine invertebrateshave their origins in microalgae or cyanobacteria? These questions are ad-dressed further below.

Open-Chain PolyketidesDinoflagellates in the genusAmphidiniumproduce long chain–oxygenatedalkyl compounds (Figures 10 and 11). These amphidinols (82, 90) are highly

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antifungal against certain organisms.Amphidiniumspp. also produce the inter-esting free 1,4-polyketides, amphiketide I and II (6). The symbiotic dinoflagel-lateSymbiodiniumspp. produce zooxanthellatoxins, which are reported to beCa++ channel activators (71, 72). Palytoxins, long-chain toxins found in thezoanthusPalythoaspp., may also have their origins in dinoflagellates. Recently,its congeners were reported in the free-swimming dinoflagellateOstreopsis sia-mensis(123).

Cyanobacteria also produce open-chain polyketides having various biologi-cal activities. For example, a Hawaiian strain ofLyngbya majusculaproducesnitrogen-containing polyketides called majusculamides (61). A strain ofL.majusculafrom Curaoa produces curacins, which are potent inhibitors of mi-crotubulin assembly (32). The presence of thiazolidine rings in the moleculesis reminiscent ofStreptomycesmetabolites.

Cyclic Oxylipins or EicosanoidsOxylipins are highly bioactive compounds derived from polyunsaturated fattyacids like arachidonic acid. They play a role in important biological functions,

Figure 7 The structure of macrolides produced by the dinoflagellatesAmphinidiumspp. Themolecular and lactone sizes vary. Most macrolides are potent cytotoxins, and some are antitumorcompounds.

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442 SHIMIZU

Figure 8 Examples of macrocycle lactones produced by different genera of dinoflagellates. Go-niodomin is produced byAlexandrium(formerly Goniodoma) spp., prorocentrolide byProrocen-trum lima, and gymnodinine by aGymnodiniumsp.

such as chemotaxis. A number of oxylipins have been identified in macroal-gae (see reference 30 for an excellent review); however, there are a limitednumber of reports on such microalgal compounds. This is rather surprisingbecause microalgae are known to be rich in polyunsaturated fatty acid deriva-tives, especially arachidonate and eicosapentenoate, and because microalgaeare considered to be the primary source of unsaturated acids in the food chain.In conjunction with prostaglandins found in marine invertebrates such as thesoft coralPlexaura homomalla(126), symbiotic algae have been examined asthe possible source of the oxylipin compounds, but there is no proof that thesymbionts are involved in the biosynthesis of the marine prostaglandins (16).

A new type of carbocyclic eicosanoids, bacillariolides I and II, were isolatedfrom the pennate diatomPseudonitzschia pungensf. multiseries(124, 125).They have a ring structure closed between C2 and C6 of eicosapentenoic acidinstead of between C8 and C12 as in prostaglandins (Figure 12). BacillariolideI has significant inhibitory activity against phospholipase A2 (unpublished data)but its function in the algae is yet to be clarified.

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PeptidesA large number of cyclic and open-chain peptides and depsipeptides (i.e. mixedpeptides and esters) have been isolated from cyanobacteria. Many of them areknown as toxins hazardous to public health (13, 64). One characteristic featureof these peptides is the inclusion of unusual amino acids such asβ-amino acids,D-amino acids, and dehydro amino acids in the molecules. Another charac-teristic feature is the occurrence of thiazol and oxazol or their hydrogenatedrings. These features are very similar to some of the cyclic peptides producedby Streptomycesspp. In a surprising contrast, there are few reports on thesepeptides in eukaryotic macro- and microalgae. The only similar metabolite iskahalalide F, which was isolated from the macro marine green algaBryopsissp. (37).

Miscellaneous CompoundsThe diatomNitzchia pungensf. multiseriesand some other diatoms produce aprenylated amino acid called domoic acid (28, 51, 59, 107) (Figure 13). Thecompound, which was first isolated from the red macroalgaChondria armataofRhodomelaceae (18), is a potent excitatory toxin and is responsible for the so-

Figure 9 Examples of cyanobacterial macrolides: scytophycins fromScytonemaand other genera,and debromoaplysiatoxin fromLyngbya majuscula.

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444 SHIMIZU

Figure 10 Long-chain polyoxyalkyl compounds produced by dinoflagellates: zooxanthellatoxinfrom Symbiodiniumsp. and amphidinol fromAmphinidiumspp. Other similar metabolites includepalytoxin derivatives fromOstreopsis siamensis.

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Figure 11 Structures of unusual alkyl chain compounds produced by the dinoflagellateAmphini-diumsp. and curacin A and majusculamide A from the cyanobacteriumLyngbya majuscula.

called amnesic shellfish poisoning (ASP) (129). The monoprenylated analogue,called kainic acid, is found in the red macroalgaeDigenea simplex, Palmeriapalmata,andCentrocetos clavulatum.

Prenylated amino acid derivatives are also seen in a cyanobacterium.Lyngbyamajusculafrom Hawaii produces protein kinase C activators: lyngbyatoxinsA, B, and C (9) (Figure 13). The resemblance of some blue-green metabolitesto Streptomycesmetabolites is probably best exemplified by these compounds,which are essentially identical to teleocidins produced byStreptomyces medio-cidicus.

Pyrrophyta (dinoflagellates), Euglenophyta, and Cryptophyta are closely re-lated classes considered to be primitive eukaryotes. Among them, the majorityof reports on secondary metabolites are concentrated on Pyrrophyta, and thereare very few reports on organisms in the other classes, mainly due to the lack

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of proper culture technique for metabolic studies. There are some hints thatthey are as resourceful as those in Pyrrophyta. Interesting styrylchromonederivatives, hormothamniones, were isolated from a Puerto Rican isolate of themarine cryptophyteChrysophaeum taylosi(29, 31). The highly cytotoxic com-pounds are partially of polyketide origin (Figure 14). In another close class,heterotrophic Ciliophora (ciliates),Euplotesspp. were found to produce thesesquiterpenoids called euplotins (20, 86). Euplotins have masked trialdehydes,a structural feature seen in some natural antifeedant compounds. Euplotins arestructurally very close to udoteatrial (Figure 14) and its analogs found in themacro green algaeUdotea flabellum(74) andHalimedaspp. (83). However,there is no evidence for the presence of a common vector in the biosynthesisof these sesquiterpenoids. Rather, it was conclusively reported that Euplotesbiosynthesize euplotins de novo, independent of the types of bacteria on whichthey feed (86).

BIOSYNTHESIS OF MICROALGAL METABOLITES

The most striking aspect of microalgal metabolites is the unforeseen ways inwhich some of the compounds are biosynthesized. A substantial amount ofknowledge is now available about the biosyntheis of terrestrial and microalgalmetabolites, and it is now customary to classify compounds according to their

Figure 12 New carbocyclic eicosanoids, bacillariolide I and II, from a diatom and their proposedbiosynthetic pathway.

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Figure 13 Metabolites formed from amino acids and prenyl groups: domoic acid from diatoms,and lyngbyatoxin A from a cyanobacterium. Teleocidins are closely relatedStreptomycesmetabo-lites.

biosynthetic origins. Therefore it is rather surprising to see the apparentlyaberrant biosynthetic patterns in these lower eukaryotes and close prokaryoticneighbors, i.e. cyanobacteria.

Biosynthesis of Nitrogenous CompoundsAs soon as the structure of saxitoxin had been elucidated by X-ray crystallog-raphy (91), speculations were made on the biosynthesis of its peculiar perhy-dropurine skeleton. The most conventional hypothesis was that the moleculewould be formed by C1 addition and Michael-type addition of C3 acrylic acidor its equivalent on an ordinary purine derivative (111). In reality, however, themolecule is built in a completely different manner. According to feeding exper-iments carried out with the dinoflagellateAlexandrium tamarense(85) and theblue-green algaAphanizomenon flos-aquae, the molecule is built from arginineand acetate in an extraordinary manner (98–100, 106, 108, 109) (Figure 15).

First, an acetate moiety condenses to theα-carbon of an arginine unit. ThisClaisen-type condensation on an amino acid is uncommon. A few examples areknown, including the condensation of succinate on glycine to form aminole-vulinic acid (ALA) in the first step of porphyrine biosynthesis. Thus, [2-13C-2-

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15N-] arginine (ornithine) was incorporated intact into the toxin molecule (112).The connectivity of13C-15N was preserved as proved by the spin-spin couplingin the NMR spectrum. The amino group is then transformed into a guanidogroup by the transfer of an amidine moiety from another molecule of arginine.The guanido carbon of arginine is also incorporated in the two guanidine groupsin the nucleus and the side-chain carbamate. The side-chain C13 comes fromthe methionine methyl group. Some more detailed mechanisms have been pos-tulated, as suggested by feeding experiments using double-labeled precursors(35). Over all, three arginine, one acetate, and one methionine molecules areneeded to build a saxitoxin molecule. Figure 16 illustrates the entire proposedpathway for PSP toxins.

Figure 14 Structures of hormothamniones from the chrysophyteChrysophaeum tayloriand eu-plotin from the ciliateEuplotes crassus. Only a few metabolites are known from Chrysophytaand Ciliophora. Euplotin is structurally related to udoteatrials fromUdoteaandHalimedaspp.(Chlorophyta).

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Figure 15 Biosynthetic origins of saxitoxin derivatives (PSP toxins) as proven by feeding studies.Three arginine molecules are required to build the molecule containing seven nitrogen atoms.

While the biosynthesis of saxitoxin derivatives exemplifies the uniquenessof the microalgal guanidium metabolism, two prominent cyclic guanidium-containing toxins from cyanobacteria are also biosynthesized in peculiar man-ners from arginine (64). The biosynthesis of anatoxin-A produced by thecyanobacteriumAnabaena flos-aquaeinvolves the loss of glycine from argi-nine. Significantly, this retroaldol-type cleavage is analogous to the Claisencondensation of acetate to arginine in the saxitoxin biosynthesis. The cyto-toxic toxin cylindrospermopsin, from the cyanobacteriumCylindrospermopsisraciborskii, has a tricyclic guanidium skeleton and a ureido ring system. Thecompound is also formed from arginine by a process involving a retro-Claisencondensation (Figure 17).

The uniqueness of nitrogen metabolism in cyanobacteria is further demon-strated in the biosynthesis of methylaspartic acid found in the cyclic peptidesmicrocystin and nodularin (63). These potent protein phosphatase inhibitorsfromMicrocystisandNodulariaspp. contain a 2R,3S-methylaspartic acid moi-ety. The known biosynthetic pathway to 2S,3R-methylaspartic acid involves amethyl rearrangement of glutamate by methylaspartate mutase. The blue-greencompound is formed by the condensation of pyruvate and acetate followed byisomerization and amination.

Biosynthesis of Polyketides in MicroalgaeThe biosynthesis of brevetoxins has been a subject of enormous interest. Themechanism conceived by most people is that the all-translinear ring system can

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Fig

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Figure 17 Two cyanobacterial toxins: anatoxin-A(s) and cylindrospermopsin biosynthesizedfrom arginine. The pathways involve unusual catabolism of arginine and have features similarto PSP toxin biosynthesis.

be formed by a cascade of opening of the properly arranged all-transepoxides,which can be derived from atrans-polyene chain (73, 79). The concomitantopenings of the epoxides can start from either side of the chain, but the discov-ery of hemibrevetoxin, which constitutes only the right half of the brevetoxinmolecule suggests the cyclization starts from the right end of the molecule(Figure 18). The trigger mechanism of the cascade is probably the opening ofacis-epoxide, since all brevetoxins have aβ-hydroxyl group on the last ring.

The carbon chain backbone seems to belong to a typical acetogenin or polyke-tide. The polyketide biosynthesis has been extensively studied with terres-trial plants, animals, and microbial metabolites. In fact, polyether antibioticssuch asStreptomyces cinnamonensis–produced monensin were shown to bebiosynthesized via epoxide intermediates from normal polyketide chains (8).However, feeding experiments with13C-labeled acetate resulted in inexplica-ble incorporation patterns (15, 53) (Figure 18). Pulse feeding experiments with[2-13C-]acetate caused speculation that the acetate was first incorporated intodicarboxylic acids before being utilized in the toxin biosynthesis (15). Also,some of the methyl side chains seem to be derived from acetate methyl groups

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452 SHIMIZU

(52). Accordingly, a new type of polyketide biosynthesis, which involves di-carboxylic acid building blocks, has been proposed for brevetoxin biosynthesis(15, 52).

Similar observations have been made with other dinoflagellate polyketides.A recent report on the biosynthesis of amphidinolide J suggests that its carbonbackbone is also formed from a mixture of acetate, succinate, andβ-keto-glutarate (47) (Figure 19). Okadaic acid and its derivatives are reported tobe formed mostly from acetate units using glycolic acid as the starter unit,but there are building blocks that cannot be explained by the normal polyke-tide biosyntheis pathway (75, 77, 135). Interestingly, in both okadaic acidand amphidinolide J, the methyl branches seem to be derived from acetate bynucleophilic substitution and decarboxylation, a methylation mechanism seen

Figure 18 The proposed mechanism of linear all-transpolycyclic ether formation in dinoflagel-lates and speculative building blocks of the carbon backbone of brevetoxin B.

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Figure 19 Examples of unusual acetate incorporation patterns into the dinoflagellate polyketides.Amphidinolide J is produced byAmphidiniumspp. and okadaic acid byProrocentrumspp. Thebiosynthesis may involve mixed building blocks such as dicarboxylic acids.

in certain bacterial metabolites. The amphiketides I and II, open-chain ke-tides from anAmphidiniumsp., have rare poly-1,4-ketone structure (6). Thecompounds can be formed by the condensation of C4 dicarboxylic acids orthe collapse of epoxides, which are similar to the intermediates postulated forbrevetoxin biosynthesis.

The biosynthesis of dinoflagellate polyketides is far from settled. Most prob-lems arise from difficulties in feeding experiments. The organisms are highlyselective in accepting exogenous organic compounds; in addition, considerablerandomization takes place in most cases (113, 114).

In comparison, the biosynthesis of blue-green algal polyketides is more inline with the known pathways in microorganisms. Tolytoxin is biosynthesized

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Figure 20 Incorporation patterns of acetates, glycine, and methionine methyl groups into thecyanobacterial macrolide tolytoxin.

from all acetate units using glycine as the starter unit (12). Feeding experimentsshow that the labels on acetate including the oxygens were incorporated intactinto the molecule. The methyl branches also come from methionine in theorthodox manner, and no acetate methyl groups are utilized (Figure 20).

Biosynthesis of Miscellaneous Algal MetabolitesThe diatom metabolite domoic acid and the red algal metabolite kainic acid wereassumed to be formed by the condensation of prenyl groups and glutamate. Infact, the absolute configuration at C2 in domoic acid retains the L-configurationof glutamate. Feeding13C-labeled acetate to the culture ofPseudonitzschiapungensf. multiseriesresulted in incorporation patterns consistent with glu-tamate biosynthesis, furnishing indirect evidence for the glutamate precursorhypothesis (24). There was no observed incorporation of acetate into the prenylmoiety in this experiment.

Bacillariolides produced by the same organismP. pungensf. multiseriesare a new type of cyclopentane eicosanoid. The biosynthesis is explained by asequence of reactions initiated by perhydroxylation of eicosapentenoic acid with5-lipoxygenase (124). However, it was revealed that the absolute configurationof the compounds’ stereochemistry is opposite to that predicted from the5Sconfiguration expected from the known lipoxygenase stereospecificity (125).

GENETICS OF SECONDARY METABOLITESPRODUCTION AND SYMBIONTS’ ROLE

Simply stated, the biogenesis of secondary metabolites in microalgae is anunpredictable phenomenon. In many cases, the biosynthetic capability is notuniversal to a species; rather, it is exceptional and limited to certain strains.

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Take the example of saxitoxin and related compounds: The toxin-producingcapability ofAlexandriumspp. is not uniform, and there are many nontoxicstrains of these species in nature. Only two strains of the ubiquitous cyanobac-terium speciesAphanizomenon flos-aquaeare known to produce PSP toxins,and the vast majority of the species are nontoxic. The same is true for otherPSP-producing organisms such asPyrodinium bahamense. Furthermore, thetoxigenic organisms occur scattered throughout different phyla (Table 1). Ofvarious attempts to explain this seemingly whimsical nature of their toxigenic-ity, the so-called bacterial theory seems the most attractive but remains the mostcontroversial.

Some dinoflagellates have endosymbiotic bacteria, and attempts have beenmade to relate this to the toxigenic property since early days (116). A Japanesegroup isolated a gram-negative rod from a Japanese toxic strain ofAlexandriumtamarenseand demonstrated that the strain produces a small amount of PSPtoxins when cultured in a seawater medium (48, 49). Doucette & Trick reportedsimilar results (22). However, there are two serious drawbacks to the theorythat bacteria alone are responsible for the PSP production in algae. First,electron microscopy shows that the bacterial symbionts are not in every toxicdinoflagellate cell, while individual cells in a cloned culture seem to haveuniform toxin productivity. Thus, the cultures newly started by picking upa single cell from a cloned culture consistently show the same toxin profiles,suggesting that the toxin profile of the dinoflagellate is a phenotype. Second,experiments conducted by other Japanese groups present genetic evidence thatgoes against the bacterial production theory (44, 88, 89).

The dinoflagellates form conjugal cells (planozygotes) that split into newvegetative cells. The conjugation of gametes from twoAlexandriumcloneswith different toxin profiles resulted in new F1 clones, which inherited the toxinprofiles according to Mendelian rule (Figure 21). Thus, when two gametes with

Table 1 Microbes reported to produce PSP toxins

Organisms Phyla

Alexandriumspp. Pyrrhophyta Eukaryote, dinoflagellatesPyrodinium bahamensevar. Pyrrhophyta Eukaryote, dinoflagellate

compressaGymnodinium catenatum Pyrrhophyta Eukaryote, dinoflagellateAphanizomenon flos-aquae Cyanophyta Prokaryote, cyanobacteriumAnabaena circiralis Cyanophyta Prokaryote, cyanobacteriumLyngbya wollei Cyanophyta Prokaryote, cyanobacteriumOscillatoria morigeotii Cyanophyta Prokaryote, cyanobacteriumMoraxellaspp. Eubacteria Prokaryote, gram negative

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Figure 21 Mating of two different PSP toxin-producing strains ofAlexandriumspp. resulted inMendelian inheritance of the parental toxin profiles. The toxin analysis was done with F1 culturesstarted from four individual cells derived from the zygote. The parental profiles were expressedin the F1 population in a 1:1 ratio. F2 had the same toxin profiles as F1. No unilateral or randomexpression was observed (44).

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similar toxin profiles were mated, all F1 clones had the same parental profiles.On the other hand, when gametes with different profiles were conjugated, theF1 clone cultures inherited each parental profile in a 1:1 ratio. The F2 clonesshowed the same profiles as the F1. The inheritance of the toxin profiles is notrelated to the sexuality of the cells. Thus, the toxin producing genes are noton the mating type locus. A similar observation was made with another PSP-producing dinoflagellate,Gymnodinium catenatum(81). These results rule outthe bacterial theory and strongly suggest that the toxigenic genes are located onthe chromosomal DNA. Owing to these findings, we need a new explanationfor the observed bacterial production of PSP in culture.

Studies conducted over the years with a clone culture ofAlexandriumtamarensefrom the East Coast of the United States indicated that only 2 of40 bacterial clones of an identical type isolated from the organism showed theproduction of PSP toxins, only in a very minute quantity (105). Thus the PSPproduction by the bacteria associating with the dinoflagellate also seems to beexceptional or accidental. In earlier studies, a vigorous search for a plasmid asa common denominator failed to detect any in both toxin-producing dinoflagel-lates and cyanobacterium (105). All these results point to the horizontal transferof the genes required for toxin biosynthesis from the host organisms to the asso-ciated bacteria by means of transposons or phages. According to the elucidatedbiosynthetic pathway described earlier, the biosynthesis of PSP toxins requiresmore than a dozen enzymes. The observed transfer of the biosynthetic abilityto bacteria, therefore, suggests that the biosynthetic genes are clustered in asmall region. Such a transfer of genes for a complex secondary metabolitefrom a eukaryote to an associated microorganism was exemplified by the iso-lation of an epiphytic fungus from aTaxustree: the fungus can produce theantitumor compound taxol while the tree itself biosynthesizes the compound(118).

Another interesting aspect of PSP production is that the toxin-producingstrains ofAphanizomenon flos-aquaealso produce totally unrelated other sec-ondary metabolites: a normorphinane alkaloid called aphanorphine (34) anda number of cyclic peptides (Y Shimizu, unpublished data). This is in clearcontrast to the nontoxic strains that do not produce any noticeable secondarymetabolites. This trend is true with other species of cyanobacteria, such asScytonemaandMicrocystis(RE Moore, private communication), and dinoflag-ellates in the genusAmphidinium. Examination of more than 40 strains ofthe dinoflagellatesAmphidinium carteraeandA. klebsiishowed that those thatproduce secondary metabolites also produce more than one kind of compound(5–7), and the other morphologically indistiguishable strains remain completelybarren with respect to secondary metabolite production. This suggests that those

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strains that can produce secondary metabolites may contain DNA sites (opengenomes) that easily accept various secondary metabolite-producing genes. Inthis respect, the chemical variation seen in the filamentous cyanobacteriumLyn-gbya majusculaalso warrants in-depth investigation. This seemingly taxonom-ically singular organism shows enormous chemical variation. The similarity ofsome of its metabolites andStreptomycesmetabolites such as teleocidin hintsat possible gene sharing between them.

Douglas et al (23) have further examined the involvement of bacteria in thebiosynthesis of domoic acid. The production of the conspicuous molecule bya red microalga and distantly classified diatoms hinted at the possibility ofbacteria as the common vector. In fact, in the near axenic culture ofNitzschiapungensf. multiseries, the production of domoic acid fell remarkably (23).The addition of the original bacteria to the culture restored the production.However, they found that the addition of bacteria from an unrelated sourcecould also restore the productivity. Therefore, in this case, the role of bacteriawas considered to be that of a nutrients supplier or an elicitor.

SIGNIFICANCE OF MICROALGAL METABOLITES INTHE FOOD CHAIN

Microalgal metabolites are probably most recognized as foodborne toxins orenvironmentally harmful compounds. Saxitoxin and related compounds thatcause paralytic shellfish poisoning were the first foodborne toxins whose ori-gins were traced to dinoflagellates. It is widely accepted that the toxins areaccumulated in bivalves and other invertebrates as a result of filter feeding ofthe toxin-producing algae. However, some of the accumulation mechanismsof the toxin are not so straightforward. Increasingly, we have been finding thetoxins in various non–filter feeders (50, 133).

Okadaic acid derivatives also cause “diarrhetic shellfish poisoning” (131).The compounds are produced in the dinoflagellateDinophysisspp. and areaccumulated in the shellfish (132, 134). Some other dinoflagellates, such asProrocentrum limaandProrocentrum concavum,are also known to produceokadaic acid (19, 38). Sponges in both temperate and tropical waters containthe compounds (93, 119), but the origin and the acquisition mechanism of thecompounds are not well understood.

Polycyclic ethers are also introduced into the food chain. Brevetoxins pro-duced byGymnodinium breveare the culprit of neurotoxic shellfish poisoning(NSP). Only very recently have the toxins and their metabolites been identifiedin shellfish (43). Ciguatoxin produced by the dinoflagellateGambierdiscustoxicusaccumulates in carnivorous fish probably through smaller herbivorous

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fish and causes ciguatera poisoning (54, 132). Again, however, it is not clearwhether all ciguatera poisons take this pathway (122).

Symboisis is also suspected as a mechanism of secondary metabolite transfer.The zoanthusPalythoaspp. contain potent palytoxins. Symbionts, bacteria, ordinoflagellates have been suspected to be the progenitors of the toxins. How-ever, the symbiont inPalythoais a symbiotic dinoflagellate,Symbiodiniumsp.,that to date has not been proven to produce palytoxins, although analogues ofthe highly oxygenated long-chain compounds are produced by a free-swimmingdinoflagellate,Ostreopsis siamensis(123).

Blue-green algal metabolites are also found in marine animals. The spongeTheonella swinhoeialso contains swinholides that are closely related to theblue-green metabolites scytophycins and tolytoxins (10). The exact source ofthe compounds is not clear, although some sponges are known to have algalsymbionts. Several bioactive peptides found in marine slags,Aplysiaor Dolla-bellaspp., have unmistakable structural features of blue-green metabolites (84,85), but again the exact sources of the compounds are not known.

CONCLUSION

We have only very limited knowledge about the origin and fate of secondarymetabolites found in marine or other aquatic environments. In the past, thefood chain and symbiosis were used to explain the occurrence of similar com-pounds in different organisms. In view of the evidence for the transfer of genesresponsible for the production of secondary metabolites, it is quite possiblethat two closely associated organisms produce identical metabolites indepen-dently. I believe that the biogenesis of the entire marine metabolites should beexamined with a new perspective.

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3. Alam M, Shimizu Y, Ikawa M, Sasner JJ.1978. Reinvestigation of the toxin fromAphanizomenon flos-aquaeby high per-

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5. Bauer I, Maranda L, Shimizu Y, PetersonRW, Cornell L, et al. 1994. The structuresof amphidinolide B isomers: strongly cy-totoxic macrolides produced by a free-swimming dinoflagellate,Amphidiniumsp.J. Am. Chem. Soc.116:2657–58

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microalgal metabolites: a new perspective

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