REVIEWS

FUNGAL SECONDARY
METABOLISM  FROM
BIOCHEMISTRY TO GENOMICS
Nancy P. Keller*, Geoffrey Turner‡ and Joan W. Bennett§
Abstract | Much of natural product chemistry concerns a group of compounds known as
secondary metabolites. These low-molecular-weight metabolites often have potent
physiological activities. Digitalis, morphine and quinine are plant secondary metabolites,
whereas penicillin, cephalosporin, ergotrate and the statins are equally well known fungal
secondary metabolites. Although chemically diverse, all secondary metabolites are produced
by a few common biosynthetic pathways, often in conjunction with morphological
development. Recent advances in molecular biology, bioinformatics and comparative
genomics have revealed that the genes encoding specific fungal secondary metabolites are
clustered and often located near telomeres. In this review, we address some important
questions, including which evolutionary pressures led to gene clustering, why closely related
species produce different profiles of secondary metabolites, and whether fungal genomics will
accelerate the discovery of new pharmacologically active natural products.

The fungal kingdom includes many species with chemists. Secondary metabolites are often bioactive,
unique and unusual biochemical pathways. The usually of low molecular weight, and are produced as
products of these pathways include important pharma- families of related compounds at restricted parts of
ceuticals such as penicillin, cyclosporin and statins; the life cycle, with production often correlated with
*University of Wisconsin– potent poisons, including aflatoxins and tricho- a specific stage of morphological differentiation.
Madison, Department of thecenes; and some Janus-faced metabolites that Secondary metabolites share the enigmatic properties
Plant Pathology, are both toxic and pharmaceutically useful, such of cellular dispensability — producer organisms can
882 Russell Labs, 1630 as the ERGOT ALKALOIDS. All of these natural products, grow without synthesizing these metabolites — and
Linden Drive, Madison,
Wisconsin 53706, USA. along with many other low-molecular-weight fun- restricted taxonomic distribution (only a small group
‡
Department of Molecular gal metabolites, are classified together as secondary of organisms produces each metabolite)5,6.
Biology and Biotechnology, metabolites and have been reviewed elsewhere1–3. The systematic study of fungal secondary meta-
Firth Court, University of What are secondary metabolites? Julian Davies once bolites began in 1922 under the leadership of Harold
Sheffield, S10 2TN, UK.
§ joked that “the simplest definition is that they are not Raistrick, who eventually characterized more than
Department of Cell and
Molecular Biology, Tulane generally included in the standard metabolic charts”4. 200 mould metabolites7. However, it was not until
University, New Orleans, Secondary metabolites are customarily distinguished the discovery and development of penicillin BOX 1
Louisiana 70118, USA. from primary metabolites, which are the almost that widespread attention was focused on fungal
e-mails: universally distributed compounds of INTERMEDIARY metabolites. Pharmaceutical companies instigated
npk@plantpath.wisc.edu;
METABOLISM. Primary metabolism has been the domain extensive screening programmes, and by 1950 a
G.Turner@sheffield.ac.uk;
profmycogirl@yahoo.com of biochemists, whereas until recently, secondary treasure trove of microbial products with pharma-
doi:10.1038/nrmicro1286 metabolism has largely been the domain of organic ceutical applications had been discovered. This

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Box 1 | Penicillin
Penicillin, the first broad-spectrum antibiotic, is the most famous fungal secondary metabolite. Penicillin
transformed the practice of medicine, changed the trajectory of pharmaceutical research, influenced the course
of World War II and saved countless lives. The penicillin story has been told many times95–98. In 1929, Alexander
Fleming discovered the antibacterial action of a ‘mould juice’ from Penicillium notatum and named the
biological activity ‘penicillin’. A decade later, Howard Florey, working at Oxford University with Ernst Chain,
Norman Heatley and others, purified enough penicillin to demonstrate its clinical efficacy, completing their
first experiments during the evacuation of Dunkirk during World War II. Florey and Heatley, fearing a German
invasion, travelled clandestinely to the USA with cultures of the mould. Penicillin yields were low and the
surface-culture method used for growing the mould was cumbersome, so government scientists in Peoria,
Illinois screened fungi from all over the world in search of a higher-yielding strain that could grow in
submerged culture. An ardent technician named Mary Hunt, soon dubbed ‘Mouldy Mary’, scoured Peoria
markets for mouldy produce, and found the rotting cantaloupe that eventually yielded a strain of Penicillium
chrysogenum that was selected for large-scale production99. As scale-up methods were perfected in preparation
for D-Day, chemists worked hard to elucidate the chemical structure of the antibiotic. It became apparent that
there was more than one penicillin. The main metabolite obtained by surface culture of the Fleming strain of
P. notatum was different from the main metabolite obtained by submerged fermentation of the Peoria strain of
P. chrysogenum. These metabolites are now known, respectively, as 2-pentenylpenicillin (‘penicillin I’) and
benzylpenicillin (‘penicillin II’)100.
It had been expected that a complete chemical synthesis for penicillin would replace fungal biosynthesis in
commercial production. Instead, efficient industrial-scale fermentation methods were developed. These
fermentation technologies were not only good preparation for industrial production of the streptomycin family of
antibiotics by actinomycetes, but have also provided a platform for the biotechnological production of mammalian
hormones and other gene-encoded products using genetically engineered microorganisms.

search for bioactive secondary metabolites has
continued unabated, and thousands of compounds
that inhibit the growth of bacteria, fungi, protozoa,
parasites, insects, viruses and even human tumour
cells have been discovered. Many other molecules
with cytotoxic, mutagenic, carcinogenic, teratogenic,
immunosuppressive, enzyme inhibitory, ALLELOPATHIC
and other biological effects also have been found. A
recent literature survey of fungal metabolites, which
examined 1,500 compounds that were isolated and
characterized between 1993 and 2001, showed that
more than half of these molecules had antibacterial,
antifungal or antitumour activity8. Given the empha-
sis on physiological activity, one common classifica-
tion method for secondary metabolites is through
defining their impact on human interests, for exam-
ple, plant and animal toxins, growth hormones and
pharmaceuticals. Another, more chemically rational
system of classification reflects the fact that, despite
their enormous chemical complexity and diversity, all
secondary metabolites arise from a limited number
of precursors from primary metabolism. This review

ERGOT ALKALOID
Any of a group of about 30
indole alkaloids obtained from
the sclerotial phase of the
fungus Claviceps purpurea.
INTERMEDIARY METABOLISM
Enzyme-catalysed processes
within cells that metabolize
macronutrients, carbohydrate,
fat and protein.
ALLELOPATHIC
Describes secondary
metabolites released by plants,
bacteria, fungi or viruses that
have a direct or indirect,
harmful or even beneficial
effect on another organism.
Box 2 | Aflatoxins
Mycotoxins, or mould poisons, are less well known than mushroom poisons but cause a higher incidence of disease.
Eating toxic mushrooms is easier to avoid than the inadvertent consumption of mould-contaminated foods.
Synthetic contaminants, food additives and pesticide residues get more press attention, but mycotoxins are almost
certainly the main non-infectious dietary risk factor in the human food supply101. The most famous mycotoxins are
the aflatoxins. These molecules were discovered in the early 1960s when thousands of turkey poults mysteriously
died in hatcheries in and around London. All of the dead turkeys had been fed the same Brazilian peanut meal. The
meal was heavily contaminated with a common species of mould, so suspicion focused on the fungus, and soon a
family of toxic metabolites was isolated. These toxins were named aflatoxins after the producer species, Aspergillus
flavus. The four major aflatoxins — aflatoxin B1, B2, G1 and G2 — were identified based on their blue or green
fluorescence under ultraviolet light and their relative chromatographic mobility during thin-layer chromatography
with silica gel101. Further studies showed that aflatoxin B1 was one of the most toxic and carcinogenic compounds
ever discovered102,103. The ease with which A. flavus grew on most major crop plants and the prevalence of aflatoxin
contamination of foods and feeds led to major international research efforts that included elucidation of most of the
genes in the biosynthetic pathway13,104,105.
There is considerable evidence that the Iraqi government stockpiled aflatoxins as part of their chemical-warfare
programme during the 1980s106. Perhaps Saddam Hussein, or one of his henchmen, was influenced by Graham
Greene’s 1979 spy novel, The Human Factor107. The plot revolves around the toxicologically improbable murder of a
man whose whisky had been laced with aflatoxin. When the victim, a heavy drinker, succumbs to liver cancer, no
one suspects foul play. Nonetheless, despite their rightly deserved notoriety, aflatoxins are a poor choice of poison
for both novelists and bioterrorists. They do most of their damage in developing countries, where their prevalence
in the food supply is an all too common phenomenon108.

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a Peptides Classes of fungal secondary metabolites

CH3 O
O O There are several classes of secondary metabolites,
N CH3
NH discussed below.
HN N
S HO
O N
H3C O
O Polyketides. Polyketides are the most abundant
N
H3C N fungal secondary metabolites. The genetically
O
Penicillin G O OH best-characterized polyketides include the yellow
O A. nidulans spore-pigment intermediate naphtho-
N CH3 N CH3 pyrone (WA)9, the carcinogen aflatoxin BOX 2 and
O
O
O
the commercially important cholesterol-lowering
CH3 compound lovastatin10 (FIG. 1). Fungal polyketides are
S N HN NH synthesized by type I polyketide synthases (PKSs),
N S OH CH3 O
O
which are multidomain proteins that are related to
N N
O O H eukaryotic fatty-acid synthases and contain similar
OH
DOMAIN structures (FIG. 2). For both enzymes, short-
Gliotoxin Cyclosporin chain carboxylic acids — usually acetyl coenzyme A
b Alkaloids (acetyl CoA) and malonyl CoA — are condensed to
2
H R O OH form carbon chains of varying lengths. The main dif-
O
O N
O
ference between polyketides and fatty acids is the full
N
N H3C N reduction of the β-carbon in fatty acids, which is an
O
O N
N optional event in polyketide synthesis. In the fungal
N R3 H PKSs, the ketoacyl CoA synthase (KS), acyltransferase
CH3 O
(AT) and acyl carrier (ACP) domains are essential for
polyketide synthesis, whereas the ketoreductase (KR),
HN
Ergopeptides Fumitremorgen C dehydratase (DH) and enoyl reductase (ER) domains
R1
that are required for ketone reduction in fatty acids are
c Terpenes
OH
not present in all fungal PKS enzymes (FIG. 2). Bacteria
O
O also have multidomain, type I PKS enzymes11, for
O O OH
example, the multimodular enzyme DEBS (6-deoxy-
O
CO erythronolide B synthase) is required for biosynthesis
O O
HO of the antibiotic erythromycin A. It consists of three
O
O OH different subunits, each of which is composed of two
O
MODULES. The six modules function successively in a
Aristolochene Trichothecene T2 toxin Gibberellin GA3
six-step reaction to incorporate methylmalonyl CoA
d Polyketides O into a growing polyketide chain, which is then released
HO O
O
and cyclized through addition of a thioesterase domain
O
O at the end of module six12.
HO CH3
Unlike bacterial PKS type I enzymes, which have
HN O
O O O separate modules for each methylmalonyl CoA addi-
Fusarin C tion, fungal PKSs are limited to one module, with
O O which they can carry out repeated biosynthetic reac-
O
tions, and are therefore called ‘iterative PKSs’. For
OH OH O Lovastatin example, the WA PKS, which has the minimal domain
O set of KS–AT–ACP, uses repeated cycles of condensa-
OH
O
CH3 tion without reduction to generate a heptaketide. The
O HO O
C-terminal region of the enzyme, which has a thio-
Aflatoxin B1 WA esterase domain motif, is responsible for a CLAISENTYPE
Figure 1 | The main groups of fungal secondary metabolites. Ergopeptides such as CYCLIZATION that results in the formation of the aromatic
ergotamine are tryptophan-derived alkaloids to which peptides are added by a non-ribosomal polyketide naphthopyrone9. How the number of cycles
peptide synthetase (NRPS). T2 toxin is a trichothecene of Fusarium sporotrichioides. WA of condensation is controlled to stop at the hexaketide
(a naphthopyrone), is a yellow pigment produced by the polyketide synthase WA, encoded by is not understood for this or for any other fungal type I
the wA gene of Aspergillus nidulans. It is converted to the green spore pigment by a laccase.
PKS enzyme.
Fusarin C synthesis is dependent on a hybrid PKS (polyketide synthase)–NRPS.
The diversity of fungal polyketide structures results
from the number of iteration reactions, the number of
will start by classifying secondary metabolites reduction reactions, which extender unit is used and,
DOMAIN according to the enzyme classes involved in their bio- in the case of aromatic polyketides, cyclizations of the
In a polyketide synthase or synthesis, and ends with a description of the wealth nascent polyketide chain. Further variety is achieved
non-ribosomal peptide of putative metabolites that have been revealed by by the introduction of many different post-polyketide-
synthetase, a stretch of
conserved amino acids that
sequencing the genomes of three Aspergillus spe- synthesis steps. For example, in addition to the PKS
defines a specific biochemical cies: Aspergillus nidulans, Aspergillus fumigatus and and a fatty-acid synthase that is required for a hexanoyl
function or active site region. Aspergillus oryzae. CoA starter unit, in A. nidulans the genes in the cluster

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tethered amino acids. The resulting peptide is then

KS AT (DH) (MT) (ER) (KR) ACP (CYC) (TE) released by a thioesterase-like domain that is usually
located at the C-terminal end of the final module.
Figure 2 | Fungal polyketide synthase (PKS) domain structure. The minimal structure is The domains that carry out these activities are named
KS–AT–ACP. Optional domains are in brackets. Polyketide synthesis is initiated when acetyl A (adenylation), P (pantothenylation/peptidyl carrier),
and malonyl coenzyme A (CoA) are loaded as thioesters on to the 4′-phosphopantotheine of
an acyl carrier (ACP) domain by means of the acyltransferase (AT) domain. Condensation then
C (condensation/peptide-bond formation) and TE
occurs with another thioester intermediate bound to the ketoacyl CoA synthase (KS) domain, (thioesterase) (FIG. 3). The first fungal NRPS identified,
and decarboxylation of the ACP-bound intermediate occurs. The resulting β-ketothioester δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase
can then be reduced by the action of the ketoreductase (KR) domain, followed by dehydration (ACVS), catalyses the first committed step in the bio-
by the dehydratase (DH) domain. If an enoyl reductase (ER) domain is present, an unsaturated synthesis of the β-lactam antibiotics penicillin and
intermediate is formed. Some PKSs contain a methyltransferase (MT) domain that methylates cephalosporin17 BOX 1. ACVS condenses l-α-amino-
the α-carbon of the thioester. CYC, cyclase; TE, thioesterase.
adipic acid, l-cysteine and l-valine, and epimerizes
l-valine to d-valine18 to form a linear peptide (ACV)
that is subsequently cyclized to isopenicillin N by the
MODULE that is required for sterigmatocystin synthesis encode action of isopenicillin N synthase.
In a polyketide synthase or five monoxygenases, four dehydrogenases, an esterase, Diversity among non-ribosomal peptides arises
non-ribosomal peptide an O-methyltransferase, a reductase, an oxidase and a in the length of peptides produced, whether the
synthetase, the complete set of DNA-binding protein13. Similar clusters of genes that peptide is cyclized, and variations in the functions
domains that is required for one
round of chain elongation and
code for PKS enzymes and 15 or more genes encoding of the domains. Another example of an NRPS that
modification. post-PKS-step enzymes are present in the clusters that produces a linear peptide is the peptaibol synthetase
are required for aflatoxin synthesis in Aspergillus flavus of Trichoderma virens19. This enzyme produces an
CLAISENTYPE CYCLIZATION and Aspergillus parasiticus14,15. 18-residue linear peptide that includes the rare amino
Claisen condensations are a
acid aminoisobutyric acid. The N terminus is acylated
common mechanism in
biological systems for synthesis Non-ribosomal peptides. Non-ribosomal peptides by an enzyme domain that resembles a fatty-acid
of carbon–carbon bonds. The are derived from both PROTEINOGENIC AMINO ACIDS and synthase, and the C-terminal amino acid is hydroxy-
product is a β-ketoester. A non-proteinogenic amino acids by multidomain, lated. In Tolypocladium niveum, an NRPS synthesizes
similar reaction is also used to multimodular enzymes named non-ribosomal pep- the cyclic undecapeptide cyclosporin20, which is an
cyclize the heptaketide product
of the wA gene to form an
tide synthetases16 (NRPSs) (FIGS 1,3). Each module immunosuppressive drug that is used to treat patients
aromatic ring. in an NRPS contains several domains that allow after organ-transplant surgery. This NRPS lacks a TE
recognition, activation and then covalent bind- domain. The peptide is released by cyclization, and
ing of a module-specific amino acid as a thioester some of the modules contain an additional methyl-
to the 4′-phosphopantetheine cofactor, which is ation domain between the A and P domains, which
attached to each module through a conserved serine. catalyses the methylation of amino acids. The enzyme
Subsequently, peptide bonds are formed between the modules and amino-acid sequences are collinear with
a repeating pattern (APC)n of domain structure. Other
NRPSs in bacteria and fungi have a more diverse
A P C A P C A P C TE domain arrangement. For example, in A. nidulans
the NRPS gene that is involved in the biosynthesis of
O S O S O S the siderophore ferricrocin, a cyclic hexapeptide with
the structure Gly–Ser–Gly–(N5-acetyl–N5-hydroxy-
H2N H2N
ornithine)3, has the domain structure (APC)3 (PC)2
REF. 21. It is not clear how the modules and domains
NH2 SH
L-cysteine L-valine
are used, although some iterative use of modules is
HO O
hypothesized.
L-α-aminoadipate
SH Terpenes. The best-known terpenes are odoriferous
H
H2N N plant metabolites such as camphor and turpentine,
O
but fungi also synthesize several important terpenes,
HO O O N
H
O including the aristolochenes, carotenoids, gibberellins,
HO
indole-diterpenes and trichothecenes. All terpenes are
δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine composed of several isoprene units, can be linear or
cyclic, saturated or unsaturated, and can be modified
in various ways (FIGS 1,4).
β-lactam antibiotics Common classes of terpenes include the mono-
Figure 3 | ACV synthetase, a trimodular non-ribosomal peptide synthetase. terpenes, which are generated from geranyl pyro-
δ-(L-α-aminoadipyl) L-cysteinyl-D-valine (ACV) synthetase catalyses the first committed step in phosphate (also known as geranyl diphosphate);
penicillin and cephalosporin biosynthesis. Each amino acid is recognized and activated by the sesquiterpenes, which are generated from farnesyl
cognate adenylation domain (A), and attached as a thioester to 4′-phosphopantetheine at the
pyrophosphate (also known as farnesyl diphosphate);
peptidyl carrier domain (P). Peptide bonds are formed with the involvement of the condensation
domain (C). The final tripeptide, attached to the peptidyl carrier domain of the C-terminal and diterpenes and carotenoids, which are generated
module, is released by the integrated thioesterase domain (TE), with the L-valine isomerized to from geranylgeranyl pyrophosphate (also known as
D-valine. The tripeptide is subsequently cyclized to isopenicillin N. geranylgeranyl diphosphate). The defining enzyme in

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Mevalonate pathway Carotenoid biosynthesis has been studied in

Neurospora crassa25. Only one geranylgeranyl diphos-
phate synthase has been found in the genome of
P P O N. crassa, in contrast to the two distinct genes encoding
P P
O geranylgeranyl diphosphate synthase that are found in
Dimethylallyl diphosphate Isopentenyl diphosphate gibberellin- and indole-diterpene-producing fungi.
Secondary metabolites Primary metabolites
Of the genes encoding these cyclases, one (named
ggs2 in G. fujikuroi and paxG in Penicillium paxilli) is
Indole alkaloids P P dedicated to secondary metabolic pathways for gib-
O
berellin and paxilline, respectively; this might reflect
Dimethylallyl diphosphate (DMAPP) compartmentalization of primary and secondary
O
P P
terpene metabolism26.

Indole alkaloids. Indole alkaloids are usually derived

Monoterpenes P P
O from tryptophan and dimethylallyl pyrophosphate,
Geranyl diphosphate (GPP)
O
P P
Sesquiterpenes P P Steroids
O synthetase (DMATS). Following methylation of
Farnesyl diphosphate (FPP)
O proceed through agroclavine to lysergic acid. Lysergic
P P
Diterpenes P P Carotenoids
O
Geranylgeranyl diphosphate (GGPP)
C50 Coenzyme Q
Figure 4 | Terpene biosynthetic pathway. Isopentenyl diphosphate and its isomer dimethylallyl
diphosphate (DMAPP), products of the mevalonate pathway, are the building blocks (5C isoprene
units) for the linear polyprenyl diphosphates, which are precursors of steroids, carotenoids and
coenzyme Q in many species. A family of isoprenyl diphosphate synthases is responsible for
chain elongation. DMAPP and the isoprenoid intermediates are also the starting points for a wide
range of secondary metabolites, including indole alkaloids, monoterpenes, sesquiterpenes and
diterpenes. The terpenes are produced by cyclization of the isoprenoids. For example, farnesyl
diphosphate is cyclized to produce a large variety of sesquiterpenes, depending on the cyclase
structure and function. DMAPP can be added as a side chain to various aromatic compounds.
For example, ergotamine is derived from dimethylallyl tryptophan.
terpene synthesis is terpene cyclase, which is essential
for the production of different terpenes from different
diphosphates. Although terpene cyclases have struc-
tural homology, they have little primary sequence
similarity and seem to have diverged relatively rapidly
from a common ancestor.
Several fungal terpene cyclases have been char-
acterized, including a bifunctional terpene cyclase
from Gibberella fujikuroi22, a trichodiene synthase
from Fusarium sporotrichioides23 and an aristolo-
chene cyclase from Aspergillus terreus and Penicillium
PROTEINOGENIC AMINO ACIDS roquefortii24. The crystal structures of the trichodiene
Those amino acids that are
synthase and aristolochene cyclase have been solved.
found in proteins and that are
coded for in the standard A conserved DDXXD/E motif (where X is any amino
genetic code. Proteinogenic acid) is involved in binding Mg2+ ions, which are
means ‘protein-building’. required for the enzyme to bind to prenyl phosphates.
Trichothecene and aristolochene are the substrates
PRENYLATION
The enzymatic addition of
for further modifications, including acetylations,
prenyl moieties to secondary esterifications and oxygenations that generate various
metabolic intermediates. sesquiterpenoid mycotoxins.
although sometimes amino acids other than tryptophan
are used as precursors BOX 3. The best-understood
pathway is ergotamine synthesis in Claviceps purpurea
and related species27. The first committed step is the
PRENYLATION of tryptophan by dimethylallyl tryptophan
dimethylallyl tryptophan, a series of oxidation steps
acid is then activated by a single-module NRPS, con-
densed with a tripeptide that is produced by a second
NRPS, and released as ergotamine.
Other tryptophan-derived alkaloids such as the
fumigaclavines and fumitremorgens of A. fumigatus
undergo one or more prenylation steps. The details of
these pathways are yet to be elucidated, but it is likely
that the fumigaclavine biosynthetic pathway proceeds
through agroclavine and might therefore have some
early steps in common with the ergotamine pathway.
Fumitremorgens are derived from proline, tryptophan
and dimethylallyl pyrophosphate28. Tryptophan and
proline are condensed to form a diketopiperazine called
brevianamide F, possibly by means of an unidentified
NRPS. It is hypothesized that brevianamides E and A
from Penicillium brevicompactum and fumitremorgens
A, B and C, and tryprostatins from A. fumigatus are
derived from brevianamide F by diverse pathways
that require various oxidases, methylases and prenyl
transferases.
Genetics
Gene clusters. Until recently, research into fungal
secondary metabolism was dominated by drug com-
panies, which have screening programmes for new
and commercially viable bioactive products that are
coupled with the structural elucidation of bioactive
secondary metabolites. The biochemical pathways
that lead to the production of a few specific second-
ary metabolites, including aflatoxin, penicillins and
ergot alkaloids, have however been analysed using
blocked mutants and isotopic tracers29–31. In general,
biochemical and genetic studies were rare owing to
the formidable technical challenges: the enzymes that
synthesize secondary metabolites are only present in
minute quantities, and many of the producer fungal
species lacked the sexual systems that were required
for genetic analysis. The advent of recombinant DNA

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Box 3 | Ergot alkaloids

Ergot alkaloids are found in the sclerotia of Claviceps, a genus of plant pathogens that parasitizes rye, wheat and
other grasses. When sclerotia are ground and baked into bread, inadvertent human consumption can lead to
convulsions, vasoconstriction and possible hallucinations, a disease syndrome variously called St Anthony’s Fire or
ergotism. Claviceps sclerotia contain a cocktail of ergot alkaloids from which modern scientists have identified three
main groups: the clavine type, the lysergic-acid type and the peptide alkaloids. Members of each group have
different physiological properties, some of which have been exploited for human use. For example,
ethnomycologists have hypothesized that the Eleusian mysteries, holy rituals carried out in ancient Greece, used an
elixir containing water-soluble, hallucinogenic ergot alkaloids. Medieval midwives adopted Claviceps sclerotia to
hasten labour and to induce abortion. In the twentieth and twenty-first centuries, ergot alkaloids have been used in
obstetrics as well as in the treatment of migraine headaches109.
The famous hallucinogen lysergic acid diethyl amide (LSD) was discovered by Hofmann at the Sandoz
Laboratories in 1938, in a research project that involved making novel derivatives of methergine R, a compound
then widely used to assuage haemorrhage during childbirth. Hofmann accidentally swallowed a minute quantity of
his semi-synthetic derivative and discovered its hallucinogenic properties. For a while, LSD was marketed to
psychiatrists, used unsuccessfully to treat schizophrenia, and adopted by the American CIA as a truth serum for
investigating suspected communists110. Most famously, during the 1960s, LSD was the favourite recreational drug of
the counterculture movement. Popular songs such as the Beatles’ ‘Lucy in the Sky with Diamonds’ and Jefferson
Airplane’s ‘White Rabbit’ glamourized its psychedelic effects. By the mid-1970s, however, government efforts to
criminalize its use, coupled with increased public awareness about the possibility of ‘bad trips’, succeeded in making
LSD less socially acceptable.
One provocative hypothesis developed by historians of the era was that ergotism might have contributed to the
notorious seventeenth-century witchcraft trials at Salem, Massachusetts111,112. More recently, Robin Cook, a popular
novelist, used the Salem–ergot hypothesis as the plot line for Acceptable Risk113. In this thriller, biotechnologists
isolate Claviceps spores from a damp New England cellar, culture the fungus, and extract a new alkaloid that
enhances intellect, stamina, libido and ego. Too late, they discover that their ‘billion-dollar drug’ has side effects,
turning those who consume it into flesh-eating zombies. In a crude way, this story highlights the paradox of most
powerful drugs: often the distinction between a toxin and a remedy is a fraction of a decimal point, the addition of a
methyl group or the removal of a double bond.

methodologies in the 1980s enabled dramatic progress
in the genetics and biochemistry of fungal secondary
metabolism. This rapid progress was facilitated by what
is now considered a hallmark characteristic of second-
ary metabolic biosynthetic pathways — the grouping
of pathway genes in a contiguous cluster.
Metabolic gene clustering was not predicted by the
fungal research community. In fact, in the era between
the discovery of the bacterial operon and the advent
of large-scale eukaryotic gene cloning, it had become
dogma that eukaryotic genes that are involved in
functionally related pathways are not linked. By 1990
however, this tenet had been abandoned owing to the
almost routine discovery of gene clusters in fungi for
phenotypes as varied as nutrient use, mating type,
pathogenicity and secondary metabolism32,33. In less
than a decade, it was shown that the genes for the pro-
duction of a broad range of secondary metabolites were
located adjacent to one another; in addition, a pathway-
specific regulatory gene was often embedded in these
gene clusters. Fungal secondary metabolite clusters
characterized by gene cloning include aflatoxins14,15,
cephalosporin 34 , compactin 35,36 , ergot alkaloids27 ,
fumonisin37, gibberellins38,39, HC toxin40, lovastatin10,
melanin41,42, paxillin26, penicillin43,44, sterigmatocystin13,
sirodesmin45 and trichothecenes46,47.
Transcription factors. The co-regulation of the clusters
of genes that code for the synthesis of natural products
can, in part, be explained by coordinated transcrip-
tional control of biosynthetic genes by ‘narrow’- or
‘broad’-domain transcription factors. The narrow
pathway-specific regulators are usually found in the
cluster and positively regulate gene expression. These
proteins are often Zn(II)2Cys6 zinc binuclear cluster
proteins48–50, which are a class of proteins so far only
found in fungi. The archetypal protein in this group
is AflR (aflatoxin regulator), the Zn(II)2Cys6 protein
that is required for aflatoxin and sterigmatocystin bio-
synthetic gene activation49–52. Typical for this group of
DNA-binding proteins, AflR recognizes and binds to
a palindromic sequence found in the promoters of
the biosynthetic genes50. Other transcription factors
that are encoded in biosynthetic gene clusters include
Cys2His2 zinc-finger proteins (Tri6 and MRTRI6 for
trichothecene production48) and an ankyrin repeat
protein (ToxE for HC-toxin production53). Cluster
regulators not found in the cluster itself include
a two-peptide forkhead complex (AcFKH1 and
CPCR1) for cephalosporin production 54 and an
HAP-like transcriptional complex (PENR1) for peni-
cillin55. Additionally, PENR1 has also been shown to
be important in taka-amylase, xylanase and cellobio-
hydrolase production56.
Secondary metabolite biosynthesis has long been
known to be responsive to environmental cues,
including the carbon and nitrogen source, ambient
temperature, light and pH57,58. Broad-domain factors
are transcription factors that are important in inte-
grating cellular responses to these parameters. Several
studies59,60 indicate that responses to environmental
signals are transmitted through Cys2His2 zinc-finger

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© 2005 Nature Publishing Group

FlbA FadA PkaA Colony
growth
Post- Transcriptional
transcriptional
BrlA AflR LaeA
Conidiation Sterigmatocystin Penicillin
Figure 5 | Integrating signal-transduction controls in
spore production (conidiation) and secondary
metabolism in Aspergillus nidulans. The model shown
uses solid lines to indicate known pathways and dashed lines
to indicate hypothesized pathways. FadA, α subunit
heterotrimeric G protein; FlbA, a regulatory protein that has
an RGS (regulator of G protein signalling) motif; PkaA,
catalytic subunit of protein kinase A; BrlA, a conidiation-
specific transcription factor; AflR (aflatoxin regulator), a
sterigmatocystin/aflatoxin-specific transcription factor.
global transcription factors that mediate carbon61
(CreA), nitrogen62 and pH63,64 (PacC) signalling. Broad
transcription factors can positively (PacC regulation of
penicillin) or negatively (CreA regulation of penicillin)
regulate metabolite production and are conserved in all
fungi and other eukaryotes. Regulation by both narrow-
and broad-domain transcription factors ensures
that secondary metabolite pathways can respond to
the demands of general cellular metabolism and the
presence of specific pathway inducers.
Secondary metabolism and fungal development. An
association between natural product formation and
morphological development has been observed for
decades57,65. For example, one unusual class of mutants
in A. parasiticus named ‘sec’ are deficient in both
sporulation and aflatoxin production66. Although the
genes that encode the enzymes for aflatoxin synthesis
are present67, expression of the pathway-specific regu-
lator, aflR, is 5–10-fold reduced in the toxigenic strains
compared with parental strains68. A similar example
involves a mutation in the A. parasiticus fluP gene
that results in a fluffy hyphal morphology phenotype,
a reduction in the number of asexual spores produced
and a reduction in the level of aflatoxin production69.
The correlation of secondary metabolism with
fungal development has recently been reviewed70, and
the authors made special note of the joint regulation of
these processes through a G-protein–protein kinase A
signal-transduction pathway. FadA, a G protein in
A. nidulans, and PkaA, a protein kinase that is down-
stream in the FadA regulatory pathway, are particularly
well characterized. Both proteins negatively regulate
aflatoxin and sterigmatocystin synthesis71,72 (FIG. 5).
PkaA regulates production of sterigmatocystin tran-
scriptionally and post-transcriptionally through aflR
and its gene product72, and it is postulated that a simi-
lar regulatory cascade controls aflatoxin regulation in
NATURE REVIEWS | MICROBIOLOGY
© 2005 Nature Publishing Group
REVIEWS
A. parasiticus73. AflR is inactivated by PkaA-mediated
phosphorylation, and the transcription of aflR is repressed
by PkaA activity74; the repression of aflR transcription is
mediated by PkaA through a novel methyltransferase,
LaeA75 (discussed later).
Increasing research into the roles of G-protein
signalling in fungal development indicates that signal-
transduction pathways often positively or negatively
regulate secondary metabolism. For example, FadA
negatively regulates aflatoxin and sterigmatocystin
synthesis, but positively regulates penicillin produc-
tion in A. nidulans, and a FadA homologue also
positively regulates trichothecene production in
F. sporotrichioides76. G proteins also regulate secondary
metabolism in Botrytis cinerea and Trichoderma atro-
viride, among others77,78. In addition to its role in the
regulation of secondary metabolism and sporulation,
G-protein signalling is crucial for pathogenicity. For
instance, deletion of cpg1, which encodes the α-subunit
of a heterotrimeric G protein in the chestnut blight
fungus Cryphonectria parasitica, results in a marked
reduction in the fungal growth rate, reduced levels of
spore production, decrease in pigmentation and a loss
of virulence79.
Epigenetic controls? The role of narrow pathway-
specific regulators and broad global transcription
factors such as PacC in penicillin biosynthesis63 and
in regulation of secondary metabolite gene clusters
— coupled with the G-protein/cAMP/protein-kinase-
mediated regulation of sterigmatocystin and aflatoxin
biosynthesis — provided the research community with
insights into the mechanisms of secondary-metabolite
regulation in fungi. None of these findings, however,
could explain why metabolite-specific synthesis and
regulatory genes were clustered.
The evolution and maintenance of gene clusters has
received a great deal of attention in bacteria80–82 and
fungi32,33,83,84. Evidence for horizontal transfer of the
penicillin cluster from bacteria to fungi was met with
great excitement, as it suggested a reason for both the
origin and maintenance of metabolic clustering83,84,85.
However, other functionally conserved metabolites,
such as gibberellin production in the fungus G. fujikuroi
and in plants, are unlikely to have arisen from hori-
zontal transfer between kingdoms22, and it is likely
that several evolutionary mechanisms account for the
presence of secondary-metabolite clusters. Although a
unifying model to explain the clustering of secondary-
metabolite genes in fungi is lacking, the preservation
of clusters, now known to be extensive through scru-
tiny of fungal genomes, might indicate an underlying
advantage to maintaining clustering.
An exciting advance in support of a global require-
ment for fungal secondary-metabolite gene clustering
has arisen from identification of an Aspergillus methyl-
transferase, LaeA75. LaeA was originally identified by
complementation of a sterigmatocystin biosynthetic
mutant. Deletion and overexpression of laeA in
A. nidulans, A. fumigatus and A. terreus strains either
silences or increases, respectively, the production
VOLUME 3 | DECEMBER 2005 | 943
REVIEWS
Heterochromatin nucleosome
Euchromatin Heterochromatin Euchromatin
LaeA
Euchromatin Euchromatin Euchromatin
Figure 6 | Model of LaeA function. Secondary metabolite gene clusters such as the
sterigmatocystin gene cluster are maintained in silenced heterochromatin but are surrounded
by expressed euchromatin. The LaeA protein functions to initiate a process that converts
heterochromatin to euchromatin, perhaps by interfering with methylases or deacetylases that
are associated with heterochromatin. Sterigmatocystin genes are designated with thick
arrows, thin arrows indicate genes that are located on either side of the sterigmatocystin
cluster and that are not regulated by LaeA.
of several secondary metabolites and the expression
of their respective genes 75. Sequence analysis of laeA
and analysis of the encoded protein indicated that it
is a protein methyltransferase75. The highest sequence
similarity is to histone and arginine methyltransferases,
which have important roles in the regulation of gene
expression, such as defining the boundaries of EUCHRO
MATIC and HETEROCHROMATIC chromosomal domains
in the mating locus of yeast and the β-globin locus in
mice86–88. Although LaeA functions are not yet fully
characterized, we speculate that this protein is involved
in chromatin modification, perhaps reminiscent of
mating-type locus expression in yeast. Methylation
is involved in both gene repression and activation of
heterochromatic and euchromatic regions of eukaryo-
tic chromosomes89, so one model of LaeA-mediated
regulation of clusters invokes a role in heterochromatin
repression (FIG. 6).
Bioinformatics and gene predictions
Automated and manual annotations of the N. crassa,
A. fumigatus, A. nidulans and A. oryzae genomes
predict gene functions based on homology to char-
acterized genes and their products. It is easy to find
genes encoding putative NRPSs and PKSs based on
protein domain structures. Terpene and alkaloid
Table 1 | Summary of secondary-metabolite gene classes in Aspergillus
EUCHROMATIC
Describes chromosome regions Protein Aspergillus oryzae
with actively transcribed genes. PKS 30
Generally these regions stain
poorly or not at all. NRPS 18
FAS 5 1
HETEROCHROMATIC
Describes chromosomal regions Sesquiterpene cyclase 1 Not detected
that are generally genetically
inert. The chromatin is tightly DMATS 2 7
coiled throughout the cell cycle PKS, polyketide synthase; NRPS, non-ribosomal peptide synthetase; FAS, fatty-acid synthase; DMATS, dimethylallyl tryptophan
and stains well. synthetase.
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© 2005 Nature Publishing Group
biosynthetic genes are detected by sequence similar-
ity to fungal terpene cyclases and with the DMATSs
of Claviceps species. As terpene cyclase primary
sequences have diverged, it is more difficult to detect
the genes that encode these proteins.
Oxidoreductases, methylases, acetylases, esterases
and transporters are not exclusive to secondary metab-
olism, so homology searches are more problematic for
these genes. Nevertheless, if good candidate genes
encoding these enzymes are found adjacent to signa-
ture secondary metabolic genes such as NRPS, PKS
and DMATS homologues, there is an improved chance
that they are involved in production of a secondary
metabolite.
Using these criteria, several important genes have
been identified from fungal genomic-DNA-sequence
data; a summary is given in TABLE 1. Most of these are
present in putative gene clusters. In some cases, we
know the function of the cluster from pre-genomic
studies, but the function of most of these putative
clusters will remain speculative until functional stud-
ies have been carried out. More putative secondary
metabolite clusters are present than are needed to
account for the known products, and some of these
clusters might not be expressed under laboratory con-
ditions. For example, the aflatoxin cluster of A. oryzae
is not expressed under conditions that are favourable
to aflatoxin expression in A. flavus and A. parasiticus
(reviewed in REF. 33).
Comparative studies allow a confident predic-
tion of the likely gliotoxin cluster of A. fumigatus,
based on its resemblance to the sirodesmin cluster
of Leptosphaeria maculans. Both compounds are
piperazines. Sirodesmin is derived from tyrosine and
serine, and gliotoxin is derived from phenylalanine
and serine 45 . Each cluster includes a bimodular
NRPS, which is presumably required for condensa-
tion of the precursor amino acids. The A. fumigatus
genome also has seven significant hits in the public
databases (ranging from 22%, expect 5e-15, to 52%,
expect e-115 in a BLAST search) with the DMATS of
C. purpurea, and the best hit (TIGR assigned number
Afu2g18040 accessible at the Central Aspergillus Data
Repository (see Online links box)) is present in a clus-
ter that contains other genes resembling those found
in C. purpurea. No NRPS is present in the putative
A. fumigatus cluster, as expected from the absence
of reports of ergopeptine synthesis from this species,
Aspergillus fumigatus Aspergillus nidulans
14 27
14 14
6
1
2
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 REVIEWS

PARALOGUES
Genes that are derived from a
common ancestor by
duplication. They can have
related functions.
ORTHOLOGUES
Genes that are derived from a
common ancestor by a
speciation event. They usually
have equivalent function in
their respective species.
which produces its own group of clavines90,91. Further
homologues of DMATS genes are found in this and
another cluster, and these might be prenyl transferases
that are required for prenylation steps required in
fumigaclavine and fumitremorgen biosynthesis.
There are still many PKS and NRPS genes (and
associated clusters) for which the putative products
cannot be predicted. In bacteria, prediction of NRPS
products has been partially successful through the use
of a derivation of a module-recognition code that is
based on correlation of NRPS adenylation domain
structure to a particular amino acid92. However, this
algorithm is not sufficiently robust to predict the
amino-acid specificity of fungal NRPS modules, partly
because of the limited experimental information
available. Similarly, it is not yet possible to predict the
structure of any polyketide of an iterative, type I fungal
PKS through analysis of domain and motif structures
alone. For example, automated annotation might label
some fungal PKSs as ‘lovastatin biosynthesis’ genes.
This classification simply reflects similarity to one
of the lovastatin PKS genes, and probably indicates a
related PARALOGUE rather than a true ORTHOLOGUE.
Several sequenced fungal genomes contain a hybrid
PKS–NRPS gene. This arrangement has been observed
previously in bacteria, and illustrates how genome data
add to our knowledge of the diversity of PKS and NRPS
structures found in nature. A hybrid gene in Fusarium
verticilliodes (formerly Fusarium moniliforme) produces
a precursor of the toxin fusarin C 93, and a PKS/NRPS is
a virulence factor in the rice blast fungus Magnaporthe
grisea94. Genes of this hybrid class can be detected in
several fungal genomes; however, sequence compari-
sons indicate that most are paralogues to the fusarin C
gene, and in most cases the products are unknown.
Bioinformatic analysis of the Aspergillus genomes
has revealed few terpene biosynthetic genes. A. nidulans
has a homologue of the aristolochene cyclase gene found
in P. roquefortii. A. terreus and A. oryzae have a homo-
logue of trichothecene cyclase, but no obvious terpene
cyclase is detectable in A. fumigatus.
Conclusions
Secondary metabolites are low-molecular-weight natural
products with restricted taxonomic distribution, often
synthesized after active growth has ceased, which do
not have an obvious function in producer species. The
β-lactam antibiotic penicillin, which is synthesized by
an NRPS, and the polyketides aflatoxin and sterigmato-
cystin, which are synthesized through a polyketide
pathway, are among the best studied fungal secondary
metabolites, and their pathways have become paradigms.
The discovery that genes involved in their production
are clustered, as are the genes that code for the produc-
tion of the vast majority of other secondary metabolites
that have been studied, has important implications for
gene regulation and evolution. Pragmatically, it means
that putative biosynthetic pathway genes for secondary
metabolism can easily be detected by in silico analysis
of genomic data. Although bioinformatics alone can-
not predict pathway products, or even determine if the
genes are expressed, these analyses can reveal molecular
evidence of many hitherto undiscovered pathways.
The recent characterization of LaeA, which is a
global regulator of secondary metabolism, provides
a tool for detecting gene clusters, and might reveal
novel chromatin-based mechanisms of transcriptional
control of these clusters. The laeA gene has been found
in all aspergilli examined to date; however, it remains
to be seen if LaeA functions in other fungi.
Note added in proof
Three recent papers describe the genomes of A. fumi-
gatus114, A. nidulans115 and A. oryzae116.

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Acknowledgements
Genomic data for Aspergillus fumigatus were provided by The
Institute for Genomic Research and The Wellcome Trust Sanger
Institute; genomic data for Aspergillus nidulans were provided
by The Broad Institute; and genomic data for Aspergillus oryzae
were provided by The National Institute of Advanced Industrial
Science and Technology. Coordination of the analyses of these
data was enabled by an international collaboration involving
more than 50 institutions from 10 countries and coordinated
from Manchester, UK.
Competing interests statement
The authors declare no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez: http://www.ncbi.nlm.nih.gov/Entrez
Aspergillus flavus | Aspergillus parasiticus | Aspergillus terreus |
Fusarium sporotrichioides | Magnaporthe grisea |
Neurospora crassa
FURTHER INFORMATION
Nancy P. Keller’s homepage:
http://www.plantpath.wisc.edu/NewFacPage/faculty.htm
Geoffrey Turner’s homepage:
http://www.shef.ac.uk/mbb/staff/turner
The Aspergillus website:
http://www.aspergillus.man.ac.uk
The Aspergillus nidulans Database:
http://www.broad.mit.edu/annotation/fungi/aspergillus
Central Aspergillus Data Repository:
http://www.cadre.man.ac.uk
Database of the Genomes Analysed at the National
Institute of Advanced Industrial Science and Technology:
http://www.bio.nite.go.jp/dogan/Top
The TIGR Aspergillus fumigatus Genome Project:
http://www.tigr.org/tdb/e2k1/afu1
The Wellcome Trust Sanger Institute Aspergillus fumigatus
Genome Project:
http://www.sanger.ac.uk/Projects/A_fumigatus
Access to this interactive links box is free online.

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