Angewandte
Chemie
DOI: 10.1002/anie.201410002
Biosynthesis
Discovery and Reconstitution of the Cycloclavine Biosynthetic
Pathway—Enzymatic Formation of a Cyclopropyl Group**
Dorota Jakubczyk, Lorenzo Caputi, Anaꢀlle Hatsch, Curt A. F. Nielsen, Melanie Diefenbacher,
Jens Klein, Andrea Molt, Hartwig Schrçder, Johnathan Z. Cheng, Michael Naesby,* and
Sarah E. OꢁConnor*
Abstract: The ergot alkaloids, a class of fungal-derived natural
products with important biological activities, are derived from
a common intermediate, chanoclavine-I, which is elaborated
into a set of diverse structures. Herein we report the discovery
of the biosynthetic pathway of cycloclavine, a complex ergot
alkaloid containing a cyclopropyl moiety. We used a yeast-
based expression platform along with in vitro biochemical
experiments to identify the enzyme that catalyzes a rearrange-
ment of the chanoclavine-I intermediate to form a cyclopropyl
moiety. The resulting compound, cycloclavine, was produced
in yeast at titers of > 500 mgLÀ1, thus demonstrating the
feasibility of the heterologous expression of these complex
alkaloids.
Cycloclavine (6) has been observed in only one species of
filamentous fungus, Aspergillus japonicus.[3a] Inspection of the
A. japonicus genome revealed a 16.8 kbp biosynthetic cluster
containing eight genes (for the organization of the cluster, see
Figure S1 in the Supporting Information),[4] seven of which
(dmaW, easF, easE, easC, easD, easA, easG) are homologous
to genes previously implicated in the biosynthesis of festu-
clavine (4) or agroclavine (5) in other filamentous fungi
(Figure 1a).[2] We set out to validate whether this cluster was
responsible for cycloclavine biosynthesis by reconstitution of
the eight genes in S. cerevisiae. Synthetic genes were used for
the construction of all strains, and a combination of GPD1
promoter/CYC1 terminator, PGK1 promoter/ADH2 termi-
nator, PDC1 promoter/FBA1 terminator, TEF1 promoter/
ENO2 terminator, and TEF2 promoter/PGI1 terminator was
used for the expression cassettes (see the Supporting Infor-
mation).[5,6] A previously reported S. cerevisiae strain produ-
ces the early ergot-alkaloid intermediate chanoclavine-I (2)
from the biosynthetic genes dmaW (A. japonicus), easF
(Aspergillus fumigatus), easE (A. japonicus), and easC
(A. japonicus) in titers of 0.75 mgLÀ1.[5] This relatively low
level was associated with the failure of N-methyl-4-dimethyl-
allyl-l-tryptophan (N-Me-DMAT, 1) to be converted effi-
ciently into chanoclavine-I (2; Figure 1a). Qualitative
increases in the levels of chanoclavine-I (2) were observed
in response to growth at decreasing temperatures and may
correspond to improved folding of the proteins responsible
for the conversion of N-Me-DMAT (1) into chanoclavine-I
(2; see Figure S2). The increase in chanoclavine-I production
provided a basis for extending the ergot-alkaloid pathway in
yeast.
T
he ergot alkaloids, produced by filamentous fungi, are an
important class of indole alkaloids with a range of pharmaco-
logical and agrochemical activities.[1,2] All ergot alkaloids are
derived from the common biosynthetic intermediate chano-
clavine-I (2), and the structural diversity within the ergot
alkaloids results from the elaborate chemical derivatization of
this intermediate.[2] However, the mechanisms of most of
these downstream elaborations are unknown. Notably, the
biosynthetic pathway of cycloclavine (6), which contains an
unusual cyclopropyl moiety (Figure 1a), remains cryptic.[3]
Herein we report the discovery of the biosynthetic pathway of
cycloclavine (6) and the reconstitution of this eight-enzyme
pathway in Saccharomyces cerevisiae at excellent production
levels (> 500 mgLÀ1). We further propose possibilities for the
mechanistic basis of cyclopropyl formation in cycloclavine
biosynthesis by the analysis of three enzymes in vitro.
We transformed this chanoclavine-I-producing strain with
combinations of expression vectors carrying the remaining
genes of the A. japonicus cluster (easD, easA, easG, and easH;
Figure 1a). When easD, easA, and easG were added, festu-
clavine (4) was observed (see Figure S3), which was not
unexpected, since festuclavine (4) is produced by homologues
of these seven genes found in other filamentous fungi, such as
A. fumigatus.[7–10] Gratifyingly, when easH, for which no role
was previously known, was added along with easD, easA, and
easG, the predominant product was cycloclavine (6), thus
clearly demonstrating that easH is necessary for cycloclavine
biosynthesis (Figure 2; see also Figure S4). Notably, concom-
itant production of festuclavine (4) was also observed. Since
both cycloclavine (6) and festuclavine (4) have been isolated
from A. japonicus,[3a] we hypothesize that this gene cluster
produces a mixture of these two compounds in the native
host, though how this ratio is impacted by environmental
[*] Dr. D. Jakubczyk, Dr. L. Caputi, Dr. J. Z. Cheng, Prof. S. E. O’Connor
Department of Biological Chemistry, John Innes Centre
Colney Lane, Norwich (UK)
E-mail: sarah.oconnor@jic.ac.uk
A. Hatsch, C. A. F. Nielsen, M. Diefenbacher, J. Klein, Dr. M. Naesby
Evolva SA
Duggingerstrasse 23, Reinach (Switzerland)
E-mail: michaeln@evolva.com
A. Molt, H. Schrçder
BASF SE
GVF/D—A030, Ludwigshafen (Germany)
[**] Funds to support this research were made available by the BBSRC
(BB/J018171/1) to S.E.O. We thank Dr. Stephen Bornemann for
helpful discussions.
Supporting information for this article, including all experimental
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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