Angewandte
Chemie
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.
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3
These are not the final page numbers!
Liu, Chengwei
