Reconstitution of biosynthetic machinery for the synthesis of the highly elaborated indole diterpene penitrem

Article abstract of DOI:10.1002/anie.201501072

Penitrem A is one of the most elaborated members of the fungal indole diterpenes. Two separate penitrem gene clusters were identified using genomic and RNA sequencing data, and 13 out of 17 transformations in the penitrem biosynthesis were elucidated by heterologous reconstitution of the relevant genes. These reactions involve 1) a prenylation-initiated cationic cyclization to install the bicyclo[3.2.0]heptane skeleton (PtmE), 2) a two-step P450-catalyzed oxidative processes forming the unique tricyclic penitrem skeleton (PtmK and PtmU), and 3) five sequential oxidative transformations (PtmKULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products. The gene cluster that is responsible for the biosynthesis of the indole diterpene penitrem A has been identified. Thirteen out of the seventeen involved transformations were elucidated by heterologous reconstitution of the relevant genes and found to feature a prenylation-initiated cationic cyclization (PtmE) and two successive P450-catalyzed oxidative reactions to install the bicyclo[4.2.0]octane skeleton.

Full text of DOI:10.1002/anie.201501072

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201501072
German Edition: DOI: 10.1002/ange.201501072
Biosynthesis
Hot Paper
Reconstitution of Biosynthetic Machinery for the Synthesis of the
Highly Elaborated Indole Diterpene Penitrem**
Chengwei Liu, Koichi Tagami, Atsushi Minami, Tomoyuki Matsumoto, Jens Christian Frisvad,
Hideyuki Suzuki, Jun Ishikawa, Katsuya Gomi, and Hideaki Oikawa*
Abstract: Penitrem A is one of the most elaborated members
of the fungal indole diterpenes. Two separate penitrem gene
clusters were identified using genomic and RNA sequencing
data, and 13 out of 17 transformations in the penitrem
biosynthesis were elucidated by heterologous reconstitution
of the relevant genes. These reactions involve 1) a prenylation-
initiated cationic cyclization to install the bicyclo[3.2.0]heptane
skeleton (PtmE), 2) a two-step P450-catalyzed oxidative
processes forming the unique tricyclic penitrem skeleton
(PtmK and PtmU), and 3) five sequential oxidative trans-
formations (PtmKULNJ). Importantly, without conventional
gene disruption, reconstitution of the biosynthetic machinery
provided sufficient data to determine the pathway. It was thus
demonstrated that the Aspergillus oryzae reconstitution system
is a powerful method for studying the biosynthesis of complex
natural products.
lin and paxilline (1) using five vectors with auxotrophic
markers in Aspergillus oryzae NSAR1.[2,3] To increase the
number of genes introduced into a single host, we developed
new vectors with two cloning sites and tandem transforma-
tions, which allow us to introduce four genes in a single
transformation. The use of this method enabled us to rapidly
achieve full reconstitution of seven aflatrem biosynthetic
genes in two rounds of transformations.[4]
To apply this reconstitution method to the elucidation of
the biosynthetic pathway of a complex natural product, we
chose tremogenic indole diterpene penitrem (2). Among the
indole diterpenes, such as 1, lolitrem, janthitrem, and
nodulisporic acid, 2 is one of the most highly elaborated
congeners and has a unique tricyclic system adjacent to the
paxilline core structure (Figure 1). Herein, we describe the
T
he increasing number of cryptic gene clusters prompted us
to explore a general method that applies the genome mining
approach to the functional analysis of individual biosynthetic
steps and the production of unidentified metabolites.[1]
Recently, we successfully employed the heterologous expres-
sion of entire biosynthetic genes for the diterpenes aphidico-
[*] Dr. C. Liu, K. Tagami, Dr. A. Minami, T. Matsumoto,
Prof. Dr. H. Oikawa
Division of Chemistry, Graduate School of Science
Hokkaido University
Sapporo 060-0810 (Japan)
E-mail: hoik@sci.hokudai.ac.jp
H. Suzuki
Kazusa DNA Research Institute
Kisarazu, Chiba 292-0818 (Japan)
Figure 1. Representative indole diterpenes from various fungi.
J. C. Frisvad
reconstitution of 13 out of the 17 penitrem biosynthetic genes
in the heterologous host A. oryzae to produce 2. This process
revealed a long biosynthetic pathway and a four-step reaction
sequence for the construction of the characteristic tricyclic
core scaffold, which features a cyclobutane and an eight-
membered cyclic ether.
The tremogenic indole diterpene family consists of
various members.[5,6] These metabolites have a common
hexacyclic core structure, which is frequently modified by
prenylation, aside from further modifications that give carbo-
cycles and cyclic ethers (Figure 1).[5,6] Penitrem A (2) has
been isolated from several Penicillium species, such as
P. crustosum,[7] P. janczewskii,[8] and P. simplicissimum.[9]
Among these strains, P. crustosum produces the penitrem
homologues A–F[7] and paxilline-related metabolites, such as
paspaline (3),[10] PC-M5 (4), and PC-M4[11] (5; Scheme 1).
Center for Eukaryotic Biotechnology
Departments of Systems Biology
Technical University of Denmark
Biocentrum-DTU, Building 221, 2800 Kgs. Lyngby (Denmark)
J. Ishikawa
Department of Bioactive Molecules
National Institute of Infectious Diseases
Shinjuku 162-8640 (Japan)
K. Gomi
Graduate School of Agricultural Science, Tohoku University
Sendai 981-8555 (Japan)
[**] This work was supported by a MEXT research grant on innovative
areas (22108002) to H.O. We are grateful to Prof. Hideo Hayashi
(Osaka Prefecture University) for providing Penicillium simplicissi-
mum AK-40 (ATCC 90288).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201501072.
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Scheme 1. Elucidated biosynthetic pathway of the penitrems.
Biosynthetic studies with isotopically labeled precursors,
including paxilline,[8,12] suggested that the characteristic
penitrem core scaffold is derived from a diprenylated paxil-
line derivative. Isolation of the structurally related metabo-
lites 4 and 5 allowed us to propose that the characteristic
bicyclo[4.2.0]octane system is constructed by cyclization of
diprenylated paxilline to bicyclo[3.2.0]heptane, subsequent
oxidative ring expansion, and formation of the cyclic ether.
Considering these findings, we divided the penitrem biosyn-
thetic pathway into three stages: conversion of indole-3-
glycerol phosphate and farnesyl diphosphate (FPP) into
1 (stage I), transformation of 1 into 5 (stage II), and
conversion of 5 into 2, which entails epoxidation, hydroxyl-
ation, and chlorination reactions after construction of the
penitrem skeleton (stage III; Scheme 1).
To determine the biosynthetic gene cluster of 2, a genomic
analysis of P. simplicissimum was employed. Using gene
cluster searching methods (a local Blast search and the
2ndFind program),[13] we identified the contiguous sequence
that contained genes homologous to those of the paxilline
gene cluster. Using our proposed biosynthetic pathway, we
speculated that this cluster (44 kb) consisting of the 15
putative genes lacked several genes for the oxidative modi-
fications of the indole ring (Figure 2; see also the Supporting
Information, Table S1). Therefore, another penitrem pro-
ducer, namely P. crustosum, was also subjected to an RNA
sequencing analysis, which identified 15 orthologous genes
Figure 2. Biosynthetic gene clusters of penitrem in Penicillium simplicis-
simum. The genes ptmGAQMBCP are highly homologous to those of
the paxilline gene cluster.
and 5 further genes that showed close correlation to the
production of penitrems in the expression profile (Table S2).
Using these sequences, we identified a second 11 kb cluster
consisting of five additional genes in the P. simplicissimum
genome. The penitrem gene cluster designated as ptm
consisted of seven orthologous paxilline biosynthetic genes,
ptmGCMBPQA, the transcriptional regulator ptmS and the
transporter ptmT, two prenyltransferase genes ptmDE, and
six genes ptmJKLNOU that encode the oxidative trans-
formation enzymes (four cytochrome P450 monooxygenases
and two flavin adenine dinucleotide (FAD) dependent
monooxygenases). The cluster also included three additional
genes, namely ptmH (oxidoreductase), ptmV (acetyltransfer-
ase), and ptmI, which showed very low homology (< 20%
identity) to the aromatase/cyclase genes in the biosynthesis of
bacterial aromatic polyketides.
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Initially, we examined the stage I transformation. Scott
and co-workers had shown that 1, aflatrem, and lolitrem are
biosynthesized via a common intermediate, namely paspaline
(3), based on a series of gene disruption experiments with four
orthologous genes paxGCMB.[14] On the other hand, oxida-
tive modifications of 3 with orthologous P450 gene sets
(paxPQ, atmPQ, and ltmPQ) gave slightly different results.[14]
As 2 shares a common hexacyclic core structure with these
metabolites, we speculated that 3 is a precursor of 2 as in the
case of aflatrem.[4] To confirm the putative functions of the
ptmPQ genes, we introduced these genes to the paspaline-
producing transformant AO-paxGCMB.[4] The incubation of
the resultant strain AO-paxGCMBptmPQ produced 1 (Fig-
ure S1), confirming its intermediacy (Scheme 1; stage I: titer
of 1: 8.8 mgkgꢀ1). This result was in good agreement with
previous biosynthetic studies[8,12] and excluded the possibility
that b-paxitriol (6) is directly formed by PtmP/PtmQ-depen-
dent oxidative modifications.[15]
genes, namely ptmE (prenyltransferase), ptmI (unknown),
and ptmV (acetyltransferase), into AO-ptmHD to yield
AO-ptmHDV, AO-ptmHDVI, AO-ptmHDIE, and AO-
ptmHDVIE. Whereas incubation of AO-ptmHDV and AO-
ptmHDIE with 1 did not give any predominant products,
incubation of AO-ptmHDVI gave new product 8 (Figure 3;
Figure S4). Based on HR-MS data (8: m/z 487.2811 [M+H]+),
the molecular formula of 8 was determined to be C32H41NO3,
which corresponds to the dehydration product of 7. The
structure was determined by spectroscopic analysis to be the
prenylated form of penijanthine (9),[17] which was isolated
from P. janthinellum (Scheme 1). Both PtmV and PtmI are
likely required for the elimination. Similar two-step elimi-
nation (acetylation and elimination) processes have been
reported for the biosynthesis of tetronate antibiotics,[18]
although different types of enzymes are involved in the
penitrem and tetronate biosyntheses. To determine the exact
reaction sequence of stage II, we employed an in vitro
functional analysis of PtmD. The enzymatic reactions of the
three substrates 1, 6, and 9, which were synthesized from
paxilline, with PtmD in the presence of dimethylallyl
diphosphate (DMAPP) showed a clear preference in the
order of 6, 9, and 1 (Figures S2 and S3). This result suggests
that the three-step process consists of C10 ketoreduction
(PtmH) followed by C20 prenylation (PtmD) and then
dehydration (PtmV, PtmI). For the AO-ptmHDVIE reaction,
the HPLC profile was essentially identical to that of AO-
ptmHDVI, indicating that the timing of the second prenyla-
tion is critical for the construction of the bicyclo[4.2.0]octane
skeleton. Thus, we turned our attention to the functional
analysis of the oxidation enzyme genes to reduce the number
of possible reaction pathways.
To obtain information on the biosynthetic reaction
sequence in stage II, and especially on the timing of the two
prenylation reactions, two genes ptmHD that encode
NAD(P+)-dependent oxidoreductase and prenyltransferase
(31%/55% identity/similarity to paxD)[14a,16] were introduced
into A. oryzae NSAR1 with pUSA2-ptmHD. The resultant
transformant AO-ptmHD converted 1 into the new metabo-
lites
6 and 7 (Figure 3). The molecular formulae (6:
C27H35NO4; 7: C32H43NO4) were determined by HR-MS, and
the structures of 6 and 7 were determined to be b-paxitriol[15]
and its monoprenylated analogue by extensive spectroscopic
analysis (Scheme 1). We hypothesized that 7 would be further
modified by a second prenylation (reverse mode) and
dehydration. Therefore, we introduced possible candidate
To address the critical issue of whether 4 and 5 are shunt
metabolites or actual intermediates, we prepared the trans-
formants AO-ptmKULNJ and AO-ptmKULNO. The micro-
bial transformations of 5 with AO-ptmKULNJ and AO-
ptmKULNO afforded 2 and penitrem F (14), respectively
[Stage III; conversions: 20% (5!2), 40% (5!13)]. These
results showed that 5 is an intermediate in penitrem
biosynthesis and also that five genes ptmKULNJ are essential
for the transformations in stage III, and that PtmJ catalyzes
the last benzylic hydroxylation (Scheme 1). To elucidate the
detailed biosynthetic pathway in stage III, we prepared the
transformants AO-ptmKUN, AO-ptmK, AO-ptmN, and AO-
ptmJ. The reaction of 5 with AO-ptmKUN yielded penitrem C
(12; Figure 3, Figure S4), indicating that these genes are
involved in the formation of the key bicyclic ring and that
PtmL catalyzes the epoxidation. Incubation of AO-ptmK with
5 gave new product 10 (Figure 3, Figure S4). Its molecular
formula C37H47NO4 was determined by HR-MS (m/z 569.3577
[M+H]+), which gave an m/z value that was 2 units lower than
1
that of 5, and the H NMR spectrum showed additional exo
olefin signals at 4.73 and 4.84 ppm. Together with other
spectral data, the structure of 10, designated as secopeni-
trem D, was determined to be the one shown in Scheme 1.
Each biosynthetic reaction was further confirmed by the
transformant carrying a single gene (Figure 3, Figure S4).
As the penitrem gene cluster encodes six oxidation
enzymes, the functional analysis of the five genes ptmKULNJ
Figure 3. HPLC profiles of extracts from biotransformations with
A. oryzae transformants and of in vitro reactions: i) AO-ptmHD with 1;
ii) AO-ptmHDVI with 1; iii) AO-ptmHDVIE with 1; iv) AO-ptmHDVIEO
with 1; v) PtmO reaction with 8; vi) PtmE reaction with 4; vii) AO-
ptmKULNJ with 5; viii) AO-ptmKULNO with 5; ix) AO-ptmKUN with 5;
x) AO-ptmK with 5; xi) AO-ptmN with 13; xii) AO-ptmJ with 14.
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left a putative flavin-dependent monooxygenase gene ptmO.
The in vitro transformation of 8 with recombinant PtmO gave
a single, acid-labile product, which was identical to 4[11a]
(Figure 3, Scheme 1). Based on this result, we introduced
two genes ptmOE into AO-ptmHDVI, and the resultant AO-
ptmHDVIOE was incubated with 1. HPLC analysis of the
extract showed a prominent product that was identical to 5
(Figure 3). To confirm this highly unusual reaction, we
employed in vitro analysis of PtmE. Incubation of 4 with
recombinant PtmE in the presence of Mg2+ and DMAPP gave
5 (Figure 3; for the proposed reaction mechanism, see
Figure 4). PtmE did not accept any other precursors, includ-
options will greatly facilitate the study of biosynthetic path-
ways of fungal natural products. This powerful method
enabled us to not only characterize each individual enzyme
reaction step, but also to synthesize intermediates and the
final natural product. We would like to emphasize that the
functional analysis of 13 out of the 17 genes involved in the
penitrem biosynthesis was achieved without using the con-
ventional gene disruption method. In turn, this indicates that
the A. oryzae heterologous expression system constitutes
a highly reliable method for studying fungal natural product
biosynthesis.[26]
Keywords: biosynthesis · heterologous expression ·
indole diterpenes · natural products · penitrem
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Received: February 4, 2015
Published online: && &&, &&&&
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Communications
Biosynthesis
C. Liu, K. Tagami, A. Minami,
T. Matsumoto, J. C. Frisvad, H. Suzuki,
J. Ishikawa, K. Gomi,
H. Oikawa*
&&&& — &&&&
Reconstitution of Biosynthetic Machinery
for the Synthesis of the Highly Elaborated
Indole Diterpene Penitrem
The gene cluster that is responsible for
the biosynthesis of the indole diterpene
penitrem A has been identified. Thirteen
out of the seventeen involved transfor-
mations were elucidated by heterologous
reconstitution of the relevant genes and
found to feature a prenylation-initiated
cationic cyclization (PtmE) and two suc-
cessive P450-catalyzed oxidative reac-
tions to install the bicyclo[4.2.0]heptane
skeleton.
6
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Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!