Natural Product Reports

Ergot alkaloid biosynthesis

Journal: Natural Product Reports

Manuscript ID: NP-HIG-05-2014-000062.R1

Article Type: Highlight

Date Submitted by the Author: 03-Aug-2014

Complete List of Authors: Jakubczyk, Dorota; John Innes,

Cheng, John; John Innes,
O'Connor, Sarah; John Innes Centre, Norwich Research Park
Page 1 of 14 Natural Product Reports

Biosynthesis of the Ergot Alkaloids

Dorota Jakubczyk, Johnathan Z. Cheng, Sarah E. O’Connor
The John Innes Centre, Department of Biological Chemistry, Norwich NR4 7UH
[email protected]

1. History of Ergot Alkaloids

2. Ergot Alkaloid Classes
3. Ergot Alkaloid Producers
4. Ergot Alkaloid Biosynthesis
4.1 Proposed Ergot Alkaloid Biosynthetic Pathway
4.2 Ergot Alkaloid Biosynthetic Gene Clusters
4.3 Functional Characterization of Early Ergot Alkaloid Biosynthetic Enzymes
4.4 Functional Characterization of Late Ergot Alkaloid Biosynthetic Enzymes
5. Production of Ergot Alkaloids
6. Conclusions

The ergots are a structurally diverse group of alkaloids derived from tryptophan 7 and
dimethylallyl pyrophosphate (DMAPP) 8. The potent bioactivity of ergot alkaloids have resulted
in their use in many applications throughout human history. In this highlight, we recap some of
the history of the ergot alkaloids, along with a brief description of the classifications of the
different ergot structures and producing organisms. Finally we describe what the advancements
that have been made in understanding the biosynthetic pathways, both at the genomic and the
biochemical levels. We note that several excellent review on the ergot alkaloids, including one
by Wallwey and Li in Nat. Prod. Rep., have been published recently.(1-3) We provide a brief
overview of the ergot alkaloids, and highlight the advances in biosynthetic pathway elucidation
that have been made since 2011 in section 4.

1. History of Ergot Alkaloids

Ergot alkaloids were first identified in dark dense sclerotia produced upon the infection
of grass and grains by parasitic fungi of the genus Claviceps. However, ergot alkaloids are also
produced in a variety of other filamentous fungi, including species in the genus Aspergillus,
Neotyphodium, Arthroderma, Penicillium, Epichloe, Balansia and the recently described
Periglandula.(4,5) Ergot alkaloids have long been a part of human history. Ergot grain disease
and the bioactive properties of the ergots have been noted in parts of Egyptian, Assyrian,
Chinese and Greek history.(6) Ergot alkaloids impacted society during the Middle Ages in Central
Europe, as these alkaloids caused mass poisonings in both humans and animals that fed on
grains contaminated by ergot producing fungi.(7) Mass outbreaks of gangrene, convulsions, and
hallucinations as a result of ergot poisoning were collectively named “St. Anthony’s Fire”
otherwise now known as “Ergotism”. Ergot alkaloids were also associated with historical events
of mass hysteria during the Great Fear of French Revolution and were believed to play a role in
the Salem Witch Trials.(6,8) Ergotism was finally correlated to the consumption of infected rye
during the latter part of the 17th century, enabling steps to be taken to reduce the horrific
poisonings caused by these compounds.

1
 Natural Product Reports Page 2 of 14

The notorious history and abuse of ergot compounds have often overshadowed the
beneficial medicinal properties of these molecules. Clinical use of ergot compounds as medicine
for postpartum hemorrhage began to emerge in the early 19th century. Further research and
screening of ergot derivatives for oxytocic activity in 1938 resulted in the synthesis of lysergic
acid diethylamide (LSD) 3 hallucinogen that has become infamous for its use as an illicit
recreational drug.(6) Currently, ergot alkaloids are the inspiration behind numerous semi-
synthetic derivatives that have been applied for a wide range of medicinal purposes including
the treatment of migraines, parkinsonism, and tumor growth. The diverse bioactivity exhibited
by ergot alkaloids is related to its ability to act as an agonist or antagonist toward
neuroreceptors for dopamine, serotonin, and adrenaline.(9,10) In 2010, the total production of
these alkaloids was approximately 20,000 kg, of which field cultivation contributed about
50%.(11)Semi-synthetic derivatives of ergot alkaloids aim to tailor their activity toward specific
receptors while reducing their adverse side effects (Figure 1).

2. Ergot Alkaloid Classes

All ergot alkaloid structures contain the tetracyclic ergoline ring (Figure 2A). Ergot alkaloids
can be divided into classes based on the substituents attached on the ergoline scaffold; the
major classes include the clavines, simple lysergic acid derivatives and ergopeptides. The
clavines include such structures as agroclavine 1 or festuclavine 2 (Figure 2B). Simple lysergic
acid derivatives consist of the basic D-lysergic acid structure with attachment of an amide in the
form of an alkyl amide (Figure 2C). Ergopeptides consists of a D-lysergic acid and a cyclic
tripeptide moiety (Figure 2D).

3. Ergot Alkaloid Producers

Ergot alkaloid producing fungi occupy distinct ecological niches. Clavicipitaceous species
such as Claviceps purpurea and Neotyphodium lolii from the order Eurotiales are plant parasites
and biotrophic symbionts, while Aspergillus fumigatus from the order Eurotiales is an
opportunistic pathogen of mammals.(8,12-14) These distantly related fungi lineages, not
surprisingly, produce unique ergot alkaloid profiles. Ergot alkaloids that are derivatives of
lysergic acid and ergopeptides (Figure 2 C, D) are associated with Clavicipitaceous fungi
Claviceps purpurea and Neotyphodium lolii, and are believed to aid in protecting the fungi from
predation by mammals and insects. In contrast, clavine type ergot alkaloids (Figure 2B) are only
produced by A. fumigatus during conidiation but its biological role to aid in survival of conidia
during invasive aspergillosis is not completely understood.(15) Recently, fungi of the family
Arthrodermataceae have been studied for ergot production.(16) Arthroderma benhamiae has
been demonstrated to be a producer of chanoclavine-I aldehyde 14.(16) Notably, isolation of
the ergot alkaloid peptide, ergosinine, from the sea slug Pleurobranchus forskalii has been
reported, indicating that ergots may also be produced in aquatic organisms.(17)
Ergot alkaloids were also found in plant taxa Convolvulaceae (Solanales), which are
associated with Clavicipitaceous fungi.(18,19) (20) It has been shown that the morning glory
family (Convolvulaceae) are colonized by an ergot alkaloid-producing clavicipitaceous fungus
and are seed-transmitted.(18,21) Treatment of the colonized host leaves with fungicides led to
elimination of leaf-associated fungus and simultaneous loss of alkaloids from the plant.(22)
These endophytic fungi form mutualistic symbiosis with plants and cause no symptoms of

2
Page 3 of 14 Natural Product Reports

infection. The defensive mutualism consists of production of bioactive ergot alkaloids by fungi
to protect the host plant from herbivores, while the fungi benefit from protected niche and
nutrition from the plant. This indicates that the ecological role of ergot alkaloids supports
environmental tolerance of plants, their fitness, resistance from drought and feeding deterrence
from mammals and insects.(20,23-30) The fungal synbionts are vertically transmitted through
seed of the host plant,(31) though the mechanism of how the fungi spread in the respective
host plant remains unclear. There are no signs of penetration of the plant epidermis by an
epibiotic fungus. Hypothetically, fungal hyphae, which are in close contact with the oil secretory
glands of the plant cuticle, may play a major role in the metabolic interaction fungus-host
plants.(32)

4. Ergot Alkaloid Biosynthesis

4.1 Proposed Ergot Alkaloid Biosynthetic Pathway
Biosynthesis of ergot alkaloids was initially investigated through extensive feeding
studies of isotopically labelled substrates to cultures of C. purpurea.(20) These studies led to a
proposed biosynthetic pathway for ergot compounds (Figure 3). The first committed step of
ergot alkaloid biosynthesis is the prenylation of L-tryptophan 7 by dimethylallyl pyrophosphate
(DMAPP) 8, to yield 4-(γ, γ-dimethylallyl)tryptophan (DMAT) 10.(33,34) The next step involves
the N-methylation of DMAT to yield 4-dimethyl-L-abrine (N-Me-DMAT) 11.(35) Subsequently, a
proposed series of successive oxidation steps catalyze the intramolecular cyclization of the
prenyl and indole moieties to form ring C in tricyclic chanoclavine-I 13.(36-39) Chanoclavine-I 13
in turn is oxidized to form chanoclavine-I-aldehyde 14, which is the last common precursor of all
classes of ergot alkaloid. At this first branch point, chanoclavine-I-aldehyde 14 can undergo
intramolecular cyclization to form either ring D of tetracyclic agroclavine 1 (C. purpurea, N.lolii)
or festuclavine 2 (A. fumigatus). Subsequent branch points derivatize agroclavine 1 and
festuclavine 2 into lysergic acid amides/peptides and fumigaclavines, respectively, as described
in section 4.4 (Figure 3).

4.2 Ergot Alkaloid Biosynthetic Gene Clusters

Fungal genes that code for the biosynthesis of secondary metabolites typically cluster on
a single genetic locus, in contrast with genes for primary metabolism, which are not localized in
clusters.(12) For fungi the clustering of genes for secondary metabolite production is believed to
give a selective advantage due to improved efficiency of gene regulation. Other hypotheses
propose that this clustering may be a remnant from horizontal gene transfer from prokaryotes
or mechanism to facilitate horizontal gene transfer.(12,40,41) Ergot alkaloid biosynthetic genes
have been shown to be clustered in A. fumigatus(14) (Figure 4A) and Clavicipitaceous fungi C.
purpurea(42,43) (Figure 4B), C. fusiformis(44) (Figure 4C), N. lolii(45) (Figure 4D) and
Arthroderma benhamiae(16) (Figure 4E). Homologues common among these species are
believed to participate in early steps of ergot biosynthesis, while species-unique genes are most
likely responsible for further downstream modifications to give the specific ergot alkaloid
classes distinct to each species, as discussed further in section 4.4 (Figure 4). Given the
similarities of the early biosynthetic genes, the distantly related A. fumigatus and
Clavicipitaceous fungi likely share a common origin for their ability to produce ergot alkaloids
(Figure 4).(46)

3
 Natural Product Reports Page 4 of 14

Using a reverse genetics approach, Tsai et al. successfully identified and cloned the gene
coding for L-tryptophan dimethylallyl prenyl transferase (DmaW) from C. purpurea.(47) This
initial discovery allowed the identification of the ergotamine biosynthesis cluster (68.5kb) from
C. purpurea – the first ergot gene cluster – via chromosome walking (Figure 4B).(42) Gene open
reading frames were assigned putative functions based on sequence similarity to previously
characterized enzymes.(42) Importantly, this gene cluster included open reading frames
encoding non-ribosomal peptide synthetase (NRPS) modules (Lps1 and Lps2) that would be
expected to be involved with the later biosynthetic pathway formation of ergopeptides.(48-50)
Additionally it was also observed that comparison of cluster sequences within C. purpurea strain
P1 (ergotamine producer) with strain C. purpurea ECC93 (ergocristine producer) displayed
conservation of most genes associated with the early pathway formation of the ergoline ring,
yet displayed high variation in genes associated with the NRPS production of the peptide ergot
moiety. An excellent study by Schardl et al. compares ergot alkaloid profiles, their gene contents
and arrangements of those genes among 15 Clavicipitaceae.(2,5) The dramatic differences in
ergot alkaloid profiles are now believed to be caused by specific mid-pathway or late-pathway
genes and differences in substrate or product specifity due to gene sequence variations. Notably,
there seems to be a strong tendency for alkaloid loci to have conserved cores that specify the
skeleton structure, whereas the peripheral genes determine the chemical derivatizations of
these core skeletons that impact the biological specificity of these molecules. For example, the
authors have correlated chemotypes of Claviceps species with presence or absence of the genes
lpsA, lpsB, lpsC, easH, easO and easP and with the position of these genes in the clusters. In
general, location at the periphery of the cluster means that the gene is near transposon-derived,
AT-rich repeat blocks, which facilitates gene losses, duplications, and neofunctionalizations. The
organization of the ergot biosynthetic genes strongly suggest that these fungi are under
selection for alkaloid diversification, which is likely related to the variable life cycles and
environments of these fungi.
Clustered genes for ergot biosynthesis were subsequently found in Neotyphodium sp.
Lp1 (a natural hybrid Neotyphodium lolii x Epichloe typhina), initially studied by Panaccione et
al.,(51) where disruption of the NRPS Lps1 homologue (LpsA) involved in ergopeptide
biosynthesis resulted in the loss of downstream alkaloid ergovaline 6. Wang et al. further
demonstrated that disruption of a dmaW homologue led to loss of ergot alkaloid production for
6 in this species.(52) Complementation of the gene with the dmaW homologue from C.
fusiformis restored ergot alkaloid production.(52,53) Later, Fleetwood et al. identified part of
the ergot alkaloid cluster for ergovaline biosynthesis (~19kb) in N. lolli using both chromosome
walking and southern blot (Figure 4D).(45) Notably, it was demonstrated that the LpsB gene in N.
lolli, a homologue of the C. purpurea Lps2, was associated with ergovaline 6 production.(45)
The A. fumigatus ergot biosynthetic gene cluster (22kb), the discovery of which was
facilitated by the published genome seqeunce of A. fumigatus, is associated to the production
of fumigaclavines A, B, C, (21, 20, 22 respectively) and festuclavine 2.(14) The gene cluster that
is responsible for the production of these ergot alkaloids had been previously identified via gene
disruption of dmaW in A. fumigatus and heterologous expression and characterization of the of
the dimethylallyltryptophan synthase dmaW gene (annotated as fgaPT2) in Saccharomyces
cerevisiae.(14,54) Further analysis of gene function in this cluster led to the characterization of
easF and easD gene products that are attributed to the early step ergot pathway.(55,56)

4
Page 5 of 14 Natural Product Reports

Notably, no homologues for the later pathway lysergyl peptide synthase genes were
observed,(42,45) which correlates with the lack of lysergic acid 18 derived ergopeptides in A.
fumigatus. A recent survey of various isolates of the A. fumigatus family were shown to have
variable production of ergot alkaloids, which could be linked to changes in the ergot gene
cluster.(57)
Genome sequence analysis of fungi of the family Arthrodermataceae revealed the
presence of a gene cluster consisting of five genes in several species with high sequence
similarity to those involved in the early common steps of ergot alkaloid biosynthesis in
Aspergillus fumigatus and Claviceps purpurea.(16)

4.3 Biochemical Characterization of Early Ergot Alkaloid Biosynthetic Enzymes

A number of genes in the ergot alkaloid biosynthetic clusters have been biochemically
characterized. The first step into the ergot biosynthetic pathway is catalyzed by the
dimethylallyl prenyltransferase (DmaW) enzyme(58) purified to homogeneity from cultures of
ergot alkaloid producing C. fusiformis.(33,34) DmaW prenylates L-tryptophan via an electrophilic
aromatic substitution reaction.(34,59) Recent work suggests that the mechanism may entail a
Cope rearrangement (Figure 3),(60) and two lysine aamino acids have been implicated in the
mechanism.(61) DmaW homologues from A. fumigatus and other Clavicipitaceous fungi such as
C. purpurea and N. lolli have also been characterized.(47,52,54) The structure of this enzyme
has been solved recently, which will has improved our understanding of this enzyme’s specificity
for the substrate and regioselectivity.(62) Recent work has indicated that alternate substrates,
4-methyltryptophan, 4-methoxytryptophan and 4-aminotryptophan, can be prenylated by
DmaW.(63) Intriguingly, several tryptophan prenyl transferases have also been shown to display
aminopeptidase activity.(64)
The next early pathway enzyme EasF is responsible for the N-methylation of DMAT 10
and was first purified by Otsuka et al. from cell free cultures of C. purpurea.(35) EasF methylates
the amine nitrogen of dimethylallyl tryptophan using the S-adenosyl methionine (SAM) co-
factor. Later, after the identification of the ergot biosynthetic gene cluster in A. fumigatus, the
easF gene was successfully cloned and heterologously expressed and also methylated DMAT to
yield N-Me-DMAT 11 (dimethylallyl L-abrine).(55) Following the N-methyltransferase EasF, two
successive oxidations are proposed to transform N-Me-DMAT 11 to chanoclavine-I 13, thus
forming ergoline ring C. These two oxidation steps of the pathway are predicted based on
feeding studies conducted by Kozikowski et al.(38) Two notable observations from these studies
were (1) observation that a proposed diene intermediate 12 was incorporated into downstream
ergot alkaloids of C. purpurea(38) and (2) molecular oxygen was incorporated into chanoclavine-
I 13.(37) Enzyme candidates of the ergot clusters that were capable of carrying out oxidation
reactions were proposed to be EasC and EasE, which display protein sequence similarity to
catalases and FAD oxygenases, respectively. The involvement of the easE and easC gene
products in the oxidations of N-Me-DMAT 11 to chanoclavine-I 13 in C. purpurea has also been
demonstrated by gene disruption experiments.(65) The disruption of EasE and EasC genes in A.
fumigatus and the subsequent interpretation of the resulting ergot alkaloid profiles indicate
that EasC and EasE are both required for ring C formation.(66) Heterologous expression of EasC
yielded a protein with catalase activity.(66)

5
 Natural Product Reports Page 6 of 14

EasD, is an NAD+ binding oxidase capable of oxidizing the hydroxyl of chanoclavine-I 13

to yield chanoclavine-I-aldehyde 14. EasD was successfully cloned and characterized from A.
fumigatus by Wallwey et al.(67) A homologous gene in the cluster from Arthroderma
benhamiae was heterologously expressed and also shown to oxidize chanoclavine-I 13 in the
presence of NAD+ to form chanoclavine-I aldehyde 14.(16) The next enzymes of the pathway,
EasA and EasG, are required in the cyclization of chanoclavine-I-aldehyde 14 to form ergoline
ring D, representing the branching point of ergot alkaloid biosynthesis into either festuclavine 2
(A. fumigatus) or agroclavine 1 derived alkaloids (C. purpurea / N. lolii). Homologues of EasA in
the ergot cluster show protein sequence similarity to enzymes of the Old Yellow Enzyme (OYE)
family. OYE enzymes display activity toward the reduction of alpha beta unsaturated ketones
and aldehydes,(68) making this a likely candidate capable of reducing the alpha beta
unsaturated carbonyl of chanoclavine-I-aldehyde 14 to give the cyclized iminium intermediates
15, 19 in ring D formation (Figure 3).(56,69,70) A notable difference between the ergot alkaloid
classes is the fully saturated D ring of (Figure 2A) the clavine type alkaloids compared to the
unsaturated ergoline D ring of the ergotamine type alkaloids.(71) An EasA homolog from N. lolli
and its role in the cyclization of ring D to produce agroclavine 1 as opposed to the EasA homolog
from A. fumigatus which forms festuclavine 2 has been documented.(72) In addition, mutational
analysis suggests the mechanistic rationale behind this critical branch point in ergot alkaloid
biosynthesis and created an EasA homolog capable of producing both festuclavine 2 and
agroclavine 1 products.(72) The EasG protein encoded by the cluster displays similarity to
Rossman fold NADPH reductases and its function is to reduce the proposed cyclized iminium
products 15, 19 of EasA to form festuclavine 2 (A. fumigatus) or agroclavine 1 (C. purpurea / N.
lolii).(72-74)

4.4 Biochemical Characterization of Late Ergot Alkaloid Biosynthetic Enzymes

Early pathway steps define ergoline ring biosynthesis up to either festuclavine 2 or
agroclavine 1 intermediates. The enzymes involved with the transformations in later step ergot
alkaloid pathway biosynthesis are attributed to the pathway divergence of ergot alkaloid
profiles among different fungi species.
The Clavicipitaceous fungi C. purpurea and N. lolli carry late step pathway genes
encoding non-ribosomal peptide synthases (NRPS) domains for the conversion of agroclavine 1
into ergopeptides. Several of these genes have been studied by gene disruption or in vitro
characterization (Figure 3). These studies have shown evidence that ergopeptide formation
occurs via an enzyme complex composed of NRPS subunits D-lysergyl peptidyl synthetase (Lps2)
that activates lysergic acid and (Lps1) which forms the tripeptide moiety.(48-51,53,75-78) The
enzyme CloA was also demonstrated to be critical for the oxidation of elymoclavine 16 to yield
paspalic acid 17, which either spontaneously or via an isomerase enzyme rearranges to form
lysergic acid 18 (Figure 3).(79) Recently, Havemann et al. have expressed EasH (C. purpurea)
annotated as nonheme-iron dioxygenase, which cyclizes dihydrolysergyl-ala-phe-pro-lactams to
dihydroergotamine by catalyzing a hydroxylation and subsequent lactol formation.(80)
In contrast, the A. fumigatus fumigaclavine C 22 biosynthetic gene cluster carries late
ergot pathway genes that have been demonstrated to show acetylation and reverse prenyl
transferase activities for the conversion of festuclavine 2 into later pathway fumigaclavines A 21,
B 20, and C 22.(81,82) A. fumigatus does not appear to carry any genes that encode for NRPS

6
Page 7 of 14 Natural Product Reports

domains that are observed in ergot biosynthetic clusters of N. lolii and C. purpurea (Figure 3).
Recently however, the nonribosomal peptide synthetases PesL and Pes1, previously thought to
be involved in biosynthesis of fungal quinazoline containing natural products, have been shown
to be essential for fumigaclavine C 22 biosynthesis in A. fumigatus by gene deletion
experiments.(83) Notably, these synthetases are not found in the core ergot cluster. A.
fumigatus also produces Fumitremorgin B, which requires an N-prenylation step, the enzyme
for which has also been identified.(84)

5. Production of Ergot Alkaloids

Production of ergot alkaloids in A. fumigatus is restricted to conidiating cultures.(85) Cultures
typically accumulate several pathway intermediates at once, with most of the alkaloid content
associated with the fungal colonies rather than being exported to the media. A two-stage
culture process that combines shake culture with static culture was shown to enhance the
production of fumigaclavine C 22 to 60 mg/L.(86,87) A recent review highlights the challenges
and progress associated with the use of Claviceps as a source for biotechnological production of
ergot alkaloids.(11) Very recently, heterologous reconstitution of these pathways presents
another option for expression of the ergot alkaloids. The early steps of this pathway– DmaW,
EasF, EasE, EasC– have been reconstituted in Aspergillus nidulans (a non-producer of ergots)(88)
and Saccharomyces cerevisea (in press). Finally, a recent review highlighting the methods
required for isolation of ergot alkaloids has been recently published.(89)

6. Conclusions
The ergot alkaloids are a group of structurally diverse and biologically active natural products.
As additional genomes of fungal species are reported, undoubtedly more gene clusters,
biosynthetic enzymes and subsequently new compounds and their biosynthetical mechanisms
will be discovered. Many of these fungi, particularly those that are plant associated, are difficult
to culture. Therefore production of these ergot alkaloids by heterologous expression of the
genes clusters (a synthetic biology approach) is a powerful tool to access new ergot alkaloids
from species that are hard to culture. Moreover, biotrophic relations of fungi and plants from
diverse caldes, organization of ecological communities, evolution and diversification of
mutualisms will continue to provide new insights into the biological activity and evolution of the
ergot alkaloids.

Figure Legends

Figure 1. Natural and semi-synthetic ergot alkaloids displaying diverse bioactivity by interactions
with vary neurotransmitter receptors.

Figure 2. A. 6,8-Dimethylergoline tetracyclic ring structure with conventional numbering and

lettering. B. Examples of clavines. C. Simple lysergic acid derivatives. D. Ergopeptides consists of
D-lysergic acid with a cyclic tripeptide moiety.

7
 Natural Product Reports Page 8 of 14

Figure 3. Early and late biosynthetic pathways of ergot alkaloids. Clavicipitaceous fungi C.
purpurea and N. lolii are associated with production of ergot alkaloids with an unsaturated
ergoline D ring whereas A. fumigatus is associated with production of saturated D ring.
Ergotamine 5 derives from agroclavine 1 and fumigaclavine C 22 derives from festuclavine 2.

Figure 4. Representative ergot alkaloid gene clusters. A. A. fumigatus. B. C. purpurea. C. C.

fusiformis. D. N. lolli. E. A. benhamiae.

References

1. Panaccione, D. G., Ryan, K. L., Schardl, C. L., and Florea, S. (2012) Analysis and
modification of ergot alkaloid profiles in fungi. Meth. Enzymol. 515, 267-290
2. Schardl, C. L., Florea, S., Pan, J., Nagabhyru, P., Bec, S., and Calie, P. J. (2013) The
epichloae: alkaloid diversity and roles in symbiosis with grasses. Curr. Opin. Plant Biol. 16,
480-488
3. Wallwey, C., and Li, S. M. (2011) Ergot alkaloids: structure diversity, biosynthetic gene
clusters and functional proof of biosynthetic genes. Nat. Prod. Rep. 28, 496-510
4. Steiner, U., Leibner, S., Schardl, C. L., Leuchtmann, A., and Leistner, E. (2011)
Periglandula, a new fungal genus within the Clavicipitaceae and its association with
Convolvulaceae. Mycologia 103, 1133-1145
5. Schardl, C. L., Young, C. A., Hesse, U., Amyotte, S. G., Andreeva, K., Calie, P. J., Fleetwood,
D. J., Haws, D. C., Moore, N., Oeser, B., Panaccione, D. G., Schweri, K. K., Voisey, C. R.,
Farman, M. L., Jaromczyk, J. W., Roe, B. A., O'Sullivan, D. M., Scott, B., Tudzynski, P., An,
Z., Arnaoudova, E. G., Bullock, C. T., Charlton, N. D., Chen, L., Cox, M., Dinkins, R. D.,
Florea, S., Glenn, A. E., Gordon, A., Guldener, U., Harris, D. R., Hollin, W., Jaromczyk, J.,
Johnson, R. D., Khan, A. K., Leistner, E., Leuchtmann, A., Li, C., Liu, J., Liu, J., Liu, M., Mace,
W., Machado, C., Nagabhyru, P., Pan, J., Schmid, J., Sugawara, K., Steiner, U., Takach, J. E.,
Tanaka, E., Webb, J. S., Wilson, E. V., Wiseman, J. L., Yoshida, R., and Zeng, Z. (2013)
Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae
reveals dynamics of alkaloid loci. PLoS Genetics 9, e1003323
6. Schiff, P. L. (2006) Ergot and its alkaloids. Am. J. Pharmaceut. Ed. 70, 98
7. Dewick, P. M. Medicinal Natural Products: A Biosynthetic Approach, 3rd Edition. John
Wiley
8. Schardl, C. L., Panaccione, D. G., and Tudzynski, P. (2006) Ergot alkaloids — biology and
molecular biology. The Alkaloids: Chemistry and Biology 63, 45-86
9. Buzzi, M. G., and Moskowitz, M. A. (1991) Evidence for 5-HT1B/1D receptors mediating
the antimigraine effect of sumatriptan and dihydroergotamine. Cephalalgia 11, 165-168
10. Stone, T. W. (1974) Further evidence for a dopamine receptor stimulating action of an
ergot alkaloid. Brain Research 72, 177-180

8
Page 9 of 14 Natural Product Reports

11. Hulvova, H., Galuszka, P., Frebortova, J., and Frebort, I. (2013) Parasitic fungus Claviceps
as a source for biotechnological production of ergot alkaloids. Biotechnology Adv. 31, 79-
89
12. Keller, N. P., Turner, G., and Bennett, J. W. (2005) Fungal secondary metabolism - from
biochemistry to genomics. Nat. Rev. Microbiol. 3, 937-947
13. Brookman, J. L., and Denning, D. W. (2000) Molecular genetics in Aspergillus fumigatus.
Curr. Opin. Microbiol. 3, 468-474
14. Coyle, C. M., and Panaccione, D. G. (2005) An ergot alkaloid biosynthesis gene and
clustered hypothetical genes from Aspergillus fumigatus. Appl. Environ. Microbiol. 71,
3112-3118
15. Coyle, C. M., Kenaley, S. C., Rittenour, W. R., and Panaccione, D. G. (2007) Association of
ergot alkaloids with conidiation in Aspergillus fumigatus. Mycologia 99, 804-811
16. Wallwey, C., Heddergott, C., Xie, X., Brakhage, A. A., and Li, S. M. (2012) Genome mining
reveals the presence of a conserved gene cluster for the biosynthesis of ergot alkaloid
precursors in the fungal family Arthrodermataceae. Microbiology 158, 1634-1644
17. Wakimoto, T., Tan, K. C., and Abe, I. (2013) Ergot alkaloid from the sea slug
Pleurobranchus forskalii. Toxicon 72, 1-4
18. Ahimsa-Muller, M. A., Markert, A., Hellwig, S., Knoop, V., Steiner, U., Drewke, C., and
Leistner, E. (2007) Clavicipitaceous fungi associated with ergoline alkaloid-containing
convolvulaceae. J. Nat. Prod. 70, 1955-1960
19. Hofmann, A. (1961) Solanaceae and Convolvulaceae: Secondary Metabolites:
Biosynthesis, Chemotaxonomy, Biological and Economic Significance. Planta Med. 9,
354–367
20. Groger, D., and Floss, H. G. (1998) Biochemistry of ergot alkaloids - achievements and
challenges. Alkaloids (Academic Press) 50, 171-218
21. Steiner, U., Ahimsa-Muller, M. A., Markert, A., Kucht, S., Gross, J., Kauf, N., Kuzma, M.,
Zych, M., Lamshoft, M., Furmanowa, M., Knoop, V., Drewke, C., and Leistner, E. (2006)
Molecular characterization of a seed transmitted clavicipitaceous fungus occurring on
dicotyledoneous plants (Convolvulaceae). Planta 224, 533-544
22. Kucht, S., Gross, J., Hussein, Y., Grothe, T., Keller, U., Basar, S., Konig, W. A., Steiner, U.,
and Leistner, E. (2004) Elimination of ergoline alkaloids following treatment of Ipomoea
asarifolia (Convolvulaceae) with fungicides. Planta 219, 619-625
23. Schultes RE, H. A. (1992) Plants of the Gods e Their Sacred, Healing and Hallucinogenic
Powers. Healing Arts Press, Rochester.
24. White Jr JF, B. C., Hywel-Jones NL, Spatafora JW. (2003) Clavicipitalean Fungi,
Evolutionary Biology, Chemistry, Biocontrol, and Cultural Impacts. Marcel Dekker, New
York
25. Schulz B, B. C., Sieber T. (2006) Microbial Root Endophytes. Springer, Berlin, Heidelberg,
New York
26. Varma A, A. L., Werner D, Hampp R. (2008) Plant Surface Microbiology. Springer, Berlin,
Heidelberg
27. Tudzynski, P., and Scheffer, J. (2004) Claviceps purpurea: molecular aspects of a unique
pathogenic lifestyle. Mol. Plant Pathol. 5, 377-388

9
 Natural Product Reports Page 10 of 14

28. Bacon CW, L. P. (2005) Ecological fitness factors for fungi within the Balansieae and
Clavicipitaceae In: Dighton J, White JF, Oudemans P (eds), The Fungal Community, its
Organization and Role in the Ecosystem. CRC Taylor & Francis, Boca Raton, 519-532
29. Schardl C, L. A. (2005) The Epichloe endophytes of grasses and the symbiotic continuum.
In: Dighton J, White JF, Oudemans P (eds), The Fungal Community, its Organization and
Role in the Ecosystem. RC Taylor and Francis, Boca Raton, 475-503
30. Schardl, C. L., Panaccione, D. G., and Tudzynski, P. (2006) Ergot alkaloids--biology and
molecular biology. Alkaloids Chem. Biol. 63, 45-86
31. Panaccione, D. G., Beaulieu, W. T., and Cook, D. (2014) Bioactive alkaloids in vertically
transmitted fungal endophytes. Functional Ecology 28, 299-314
32. Steiner, U., and Leistner, E. (2012) Ergoline alkaloids in convolvulaceous host plants
originate from epibiotic clavicipitaceous fungi of the genus Periglandula. Fungal Ecology
5, 316-321
33. Gebler, J. C., and Poulter, C. D. (1992) Purification and characterization of dimethylallyl
tryptophan synthase from Claviceps purpurea. Arch. Biochem. Biophys. 296, 308-313
34. Gebler, J. C., Woodside, A. B., and Poulter, C. D. (1992) Dimethylallyltryptophan synthase.
An enzyme-catalyzed electrophilic aromatic substitution. J. Am. Chem. Soc. 114, 7354-
7360
35. Otsuka, H., Quigley, F. R., Groeger, D., Anderson, J. A., and Floss, H. G. (1980) In vivo and
in vitro evidence for N-methylation as the second pathway-specific step in ergoline
biosynthesis. Planta Med. 40, 109-119
36. Floss, H. G., Tcheng-Lin, M., Chang, C.-J., Naidoo, B., Blair, G. E., Abou-Chaar, C. I., and
Cassady, J. M. (1974) Biosynthesis of ergot alkaloids. Mechanism of the conversion of
chanoclavine-I into tetracyclic ergolines. J. Am. Chem. Soc. 96, 1898-1909
37. Kobayashi, M., and Floss, H. G. (1987) Biosynthesis of ergot alkaloids: origin of the
oxygen atoms in chanoclavine-I and elymoclavine. J. Org. Chem. 52, 4350-4352
38. Kozikowski, A. P., Chen, C., Wu, J. P., Shibuya, M., Kim, C. G., and Floss, H. G. (1993)
Probing ergot alkaloid biosynthesis: intermediates in the formation of ring C. J. Am.
Chem. Soc. 115, 2482-2488
39. Kozikowski, A. P., Wu, J. P., Shibuya, M., and Floss, H. G. (1988) Probing ergot alkaloid
biosynthesis: identification of advanced intermediates along the biosynthetic pathway. J.
Am. Chem. Soc. 110, 1970-1971
40. Cramer, R. A., Jr., Shwab, E. K., and Keller, N. P. (2009) Genetic regulation of Aspergillus
secondary metabolites and their role in fungal pathogenesis. in Aspergillus fumigatus
and aspergillosis, ASM Press, pp. 185-199
41. Walton, J. D. (2000) Horizontal gene transfer and the evolution of secondary metabolite
gene clusters in fungi: an hypothesis. Fungal Genet. Bio.l 30, 167-171
42. Haarmann, T., Machado, C., Lubbe, Y., Correia, T., Schardl, C. L., Panaccione, D. G., and
Tudzynski, P. (2005) The ergot alkaloid gene cluster in Claviceps purpurea: extension of
the cluster sequence and intra species evolution. Phytochemistry 66, 1312-1320
43. Tudzynski, P., Holter, K., Correia, T., Arntz, C., Grammel, N., and Keller, U. (1999)
Evidence for an ergot alkaloid gene cluster in Claviceps purpurea. Mol. Gen. Genet. 261,
133-141

10
Page 11 of 14 Natural Product Reports

44. Lorenz, N., Wilson, E. V., Machado, C., Schardl, C. L., and Tudzynski, P. (2007)
Comparison of ergot alkaloid biosynthesis gene clusters in Claviceps species indicates
loss of late pathway steps in evolution of C. fusiformis. Appl. Environ. Microbiol. 73,
7185-7191
45. Fleetwood, D. J., Scott, B., Lane, G. A., Tanaka, A., and Johnson, R. D. (2007) A complex
ergovaline gene cluster in Epichloe endophytes of grasses. Appl. Environ. Microbiol. 73,
2571-2579
46. Liu, M., Panaccione, D. G., and Schardl, C. L. (2009) Phylogenetic analyses reveal
monophyletic origin of the ergot alkaloid gene dmaW in fungi. Evolution. Bioinformatics
5, 15-30
47. Tsai, H.-F., Wang, H., Gebler, J. C., Poulter, C. D., and Schardl, C. L. (1995) The Claviceps
purpurea gene encoding dimethylallyltryptophan synthase, the committed step for ergot
alkaloid biosynthesis. Biochem. Biophys. Res. Commun. 216, 119-125
48. Correia, T., Grammel, N., Ortel, I., Keller, U., and Tudzynski, P. (2003) Molecular Cloning
and Analysis of the Ergopeptine Assembly System in the Ergot Fungus Claviceps
purpurea. Chem. Biol. 10, 1281-1292
49. Riederer, B., Han, M., and Keller, U. (1996) D-Lysergyl peptide synthetase from the ergot
fungus Claviceps purpurea. J. Biol. Chem. 271, 27524-27530
50. Walzel, B., Riederer, B., and Keller, U. (1997) Mechanism of alkaloid cyclopeptide
synthesis in the ergot fungus Claviceps purpurea. Chem. Biol. 4, 223-230
51. Panaccione, D. G., Johnson, R. D., Wang, J., Young, C. A., Damrongkool, P., Scott, B., and
Schardl, C. L. (2001) Elimination of ergovaline from a grass-Neotyphodium endophyte
symbiosis by genetic modification of the endophyte. Proc. Natl. Acad. Sci. U S A 98,
12820-12825
52. Wang, J., Machado, C., Panaccione, D. G., Tsai, H.-F., and Schardl, C. L. (2004) The
determinant step in ergot alkaloid biosynthesis by an endophyte of perennial ryegrass.
Fungal Genet. Biol. 41, 189-198
53. Panaccione, D. G., Tapper, B. A., Lane, G. A., Davies, E., and Fraser, K. (2003) Biochemical
outcome of blocking the ergot alkaloid pathway of a grass endophyte. J. Agri. Food Chem.
51, 6429-6437
54. Unsold, I. A., and Li, S. M. (2005) Overproduction, purification and characterization of
FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus. Microbiology
151, 1499-1505
55. Rigbers, O., and Li, S. M. (2008) Ergot alkaloid biosynthesis in Aspergillus fumigatus.
Overproduction and biochemical characterization of a 4-dimethylallyltryptophan N-
methyltransferase. J. Biol. Chem. 283, 26859-26868
56. Wallwey, C., Matuschek, M., Xie, X. L., and Li, S. M. (2010) Ergot alkaloid biosynthesis in
Aspergillus fumigatus: Conversion of chanoclavine-I aldehyde to festuclavine by the
festuclavine synthase FgaFS in the presence of the old yellow enzyme FgaOx3. Org.
Biomolec. Chem. 8, 3500-3508
57. Robinson, S. L., and Panaccione, D. G. (2012) Chemotypic and genotypic diversity in the
ergot alkaloid pathway of Aspergillus fumigatus. Mycologia 104, 804-812

11
 Natural Product Reports Page 12 of 14

58. Lee, S.-L., Floss, H. G., and Heinstein, P. (1976) Purification and properties of
dimethylallylpyrophosphate:tryptophan dimethylallyl transferase, the first enzyme of
ergot alkaloid biosynthesis in Claviceps. sp. SD 58. Arch. Biochem. Biophys. 177, 84-94
59. Luk, L. Y. P., and Tanner, M. E. (2009) Mechanism of Dimethylallyltryptophan Synthase:
Evidence for a Dimethylallyl Cation Intermediate in an Aromatic Prenyltransferase
Reaction. J. Am. Chem. Soc. 131, 13932-13933
60. Luk, L. Y., Qian, Q., and Tanner, M. E. (2011) A cope rearrangement in the reaction
catalyzed by dimethylallyltryptophan synthase? J. Am. Chem. Soc. 133, 12342-12345
61. Stec, E., Steffan, N., Kremer, A., Zou, H., Zheng, X., and Li, S. M. (2008) Two lysine
residues are responsible for the enzymatic activities of indole prenyltransferases from
fungi. Chembiochem 9, 2055-2058
62. Metzger, U., Schall, C., Zocher, G., Unsold, I., Stec, E., Li, S.-M., Heide, L., and Stehle, T.
(2009) The structure of dimethylallyl tryptophan synthase reveals a common
architecture of aromatic prenyltransferases in fungi and bacteria. Proc. Natl. Acad. Sci. U
S A 106, 14309-14314
63. Rudolf, J. D., Wang, H., and Poulter, C. D. (2013) Multisite Prenylation of 4-Substituted
Tryptophans by Dimethylallyltryptophan Synthase. J. Am. Chem. Soc. 135, 1895-1902
64. Kremer, A., and Li, S. M. (2008) Tryptophan aminopeptidase activity of several indole
prenyltransferases from Aspergillus fumigatus. Chem. Biol. 15, 729-738
65. Lorenz, N., Olsovska, J., Sulc, M., and Tudzynski, P. (2010) Alkaloid cluster gene ccsA of
the ergot fungus Claviceps purpurea encodes chanoclavine I synthase, a flavin adenine
dinucleotide-containing oxidoreductase mediating the transformation of N-methyl-
dimethylallyltryptophan to chanoclavine I. Appl. Environ. Microbiol. 76, 1822-1830
66. Goetz, K. E., Coyle, C. M., Cheng, J. Z., O'Connor, S. E., and Panaccione, D. G. (2011) Ergot
cluster-encoded catalase is required for synthesis of chanoclavine-I in Aspergillus
fumigatus. Curr. Gen. 57, 201-211
67. Wallwey, C., Matuschek, M., and Li, S. M. (2010) Ergot alkaloid biosynthesis in
Aspergillus fumigatus: conversion of chanoclavine-I to chanoclavine-I aldehyde catalyzed
by a short-chain alcohol dehydrogenase FgaDH. Archives of microbiology 192, 127-134
68. Toogood, H. S., Gardiner, J. M., and Scrutton, N. S. (2010) Biocatalytic Reductions and
Chemical Versatility of the Old Yellow Enzyme Family of Flavoprotein Oxidoreductases.
ChemCatChem 2, 892-914
69. Cheng, J. Z., Coyle, C. M., Panaccione, D. G., and O'Connor, S. E. (2010) A role for Old
Yellow Enzyme in ergot alkaloid biosynthesis. Journal of the American Chemical Society
132, 1776-1777
70. Xie, X., Wallwey, C., Matuschek, M., Steinbach, K., and Li, S. M. (2011) Formyl migration
product of chanoclavine-I aldehyde in the presence of the old yellow enzyme FgaOx3
from Aspergillus fumigatus: a NMR structure elucidation. Magnetic resonance in
chemistry : MRC 49, 678-681
71. Coyle, C. M., Cheng, J. Z., O'Connor, S. E., and Panaccione, D. G. (2010) An old yellow
enzyme gene controls the branch point between Aspergillus fumigatus and Claviceps
purpurea ergot alkaloid pathways. Appl. Environ. Microbiol. 76, 3898-3903

12
Page 13 of 14 Natural Product Reports

72. Cheng, J. Z., Coyle, C. M., Panaccione, D. G., and O'Connor, S. E. (2010) Controlling a
structural branch point in ergot alkaloid biosynthesis. J. Am. Chem. Soc. 132, 12835-
12837
73. Matuschek, M., Wallwey, C., Wollinsky, B., Xie, X., and Li, S.-M. (2012) In vitro conversion
of chanoclavine-I aldehyde to the stereoisomers festuclavine and pyroclavine controlled
by the second reduction step. RSC Advances 2, 3662
74. Matuschek, M., Wallwey, C., Xie, X., and Li, S. M. (2011) New insights into ergot alkaloid
biosynthesis in Claviceps purpurea: an agroclavine synthase EasG catalyses, via a non-
enzymatic adduct with reduced glutathione, the conversion of chanoclavine-I aldehyde
to agroclavine. Org. Biomol. Chem. 9, 4328-4335
75. Annis, S. L., and Panaccione, D. G. (1998) Presence of peptide synthetase gene
transcripts and accumulation of ergopeptines in Claviceps purpurea and Neotyphodium
coenophialum. Can. J. Microbiol. 44, 80-86
76. Damrongkool, P., Sedlock, A. B., Young, C. A., Johnson, R. D., Goetz, K. E., Scott, B.,
Schardl, C. L., and Panaccione, D. G. (2005) Structural analysis of a peptide synthetase
gene required for ergopeptine production in the endophytic fungus Neotyphodium lolii.
DNA sequence 16, 379-385
77. Haarmann, T., Lorenz, N., and Tudzynski, P. (2008) Use of a nonhomologous end joining
deficient strain (Deltaku70) of the ergot fungus Claviceps purpurea for identification of a
nonribosomal peptide synthetase gene involved in ergotamine biosynthesis. Fungal
Genet. Biol. 45, 35-44
78. Ortel, I., and Keller, U. (2009) Combinatorial assembly of simple and complex D-lysergic
acid alkaloid peptide classes in the ergot fungus Claviceps purpurea. J. Biol. Chem. 284,
6650-6660
79. Haarmann, T., Ortel, I., Tudzynski, P., and Keller, U. (2006) Identification of the
cytochrome P450 monooxygenase that bridges the clavine and ergoline alkaloid
pathways. Chembiochem 7, 645-652
80. Havemann, J., Vogel, D., Loll, B., and Keller, U. (2014) Cyclolization of D-Lysergic Acid
Alkaloid Peptides. Chem. Biol. 21, 146-155
81. Liu, X., Wang, L., Steffan, N., Yin, W.-B., and Li, S.-M. (2009) Ergot alkaloid biosynthesis in
Aspergillus fumigatus: FgaAT catalyzes the acetylation of fumigaclavine B. Chembiochem
10, 2325-2328
82. Unsoeld, I. A., and Li, S.-M. (2006) Reverse prenyltransferase in the biosynthesis of
fumigaclavine C in Aspergillus fumigatus: Gene expression, purification, and
characterization of fumigaclavine C synthase FGAPT1. Chembiochem 7, 158-164
83. O'Hanlon, K. A., Gallagher, L., Schrettl, M., Jochl, C., Kavanagh, K., Larsen, T. O., and
Doyle, S. (2012) Nonribosomal peptide synthetase genes pesL and pes1 are essential for
Fumigaclavine C production in Aspergillus fumigatus. Appl. Environ. Microbiol. 78, 3166-
3176
84. Grundmann, A., Kuznetsova, T., Afiyatullov, S., and Li, S. M. (2008) FtmPT2, an N-
prenyltransferase from Aspergillus fumigatus, catalyses the last step in the biosynthesis
of fumitremorgin B. Chembiochem 9, 2059-2063

13
 Natural Product Reports Page 14 of 14

85. Mulinti, P., Allen, N. A., Coyle, C. M., Gravelat, F. N., Sheppard, D. C., and Panaccione, D.
G. (2014) Accumulation of ergot alkaloids during conidiophore development in
Aspergillus fumigatus. Curr. Microbiol. 68, 1-5
86. Yao, L.-Y., Zhu, Y.-X., Jiao, R.-H., Lu, Y.-H., and Tan, R.-X. (2014) Enhanced production of
fumigaclavine C by ultrasound stimulation in a two-stage culture of Aspergillus fumigatus
CY018. Bioresour. Technol. 159, 112-117
87. Zhu, Y.-X., Yao, L.-Y., Jiao, R.-H., Lu, Y.-H., and Tan, R.-X. (2014) Enhanced production of
Fumigaclavine C in liquid culture of Aspergillus fumigatus under a two-stage process.
Bioresour. Technol. 152, 162-168
88. Ryan, K. L., Moore, C. T., and Panaccione, D. G. (2013) Partial reconstruction of the ergot
alkaloid pathway by heterologous gene expression in Aspergillus nidulans. Toxins 5, 445-
455
89. Wallwey, C., and Li, S. M. (2012) Production, detection, and purification of clavine-type
ergot alkaloids. Meth. Molecular Biol. 944, 121-131

14