Peniphenone and Penilactone Formation in Penicillium crustosum via 1,4-Michael Additions of ortho-Quinone Methide from Hydroxyclavatol to γ-Butyrolactones from Crustosic Acid

Article abstract of DOI:10.1021/jacs.9b00110

Penilactones A and B consist of a γ-butyrolactone and two clavatol moieties. We identified two separate gene clusters for the biosynthesis of these key building blocks in Penicillium crustosum. Gene deletion, feeding experiments, and biochemical investiga

Full text of DOI:10.1021/jacs.9b00110

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Communication
Peniphenone and penilactone formation in Penicillium
crustosum via 1,4-Michael additions of ortho-quinone methide
from hydroxyclavatol to #-butyrolactones from crustosic acid
Jie Fan, Ge Liao, Florian Kindinger, Lena Ludwig-Radtke, Wen-Bing Yin, and Shu-Ming Li
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b00110 • Publication Date (Web): 27 Feb 2019
Downloaded from http://pubs.acs.org on February 27, 2019
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Peniphenone and penilactone formation in Penicillium crustosum via 1,4-
Michael additions of ortho-quinone methide from hydroxyclavatol to γ-
butyrolactones from crustosic acid
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Jie Fan,1,† Ge Liao,1,† Florian Kindinger,1 Lena Ludwig-Radtke,1 Wen-Bing Yin,2 and Shu-Ming Li1,*
1 Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Robert-Koch-Strasse 4,
Marburg 35037, Germany
2 State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
Supporting Information Placeholder
For secondary metabolite (SM) production in PRB-2, several
culture conditions were tested and the extracts were analyzed
on HPLC (Figure S1 in the Supplement Information (SI)). Three
dominant peaks were detected in a 7 days-old PD culture
(Figure S1), which were identified as 9, 10,14 and terrestric acid
(11)15 after isolation and structure elucidation (See SI for
details, NMR data and spectra are given in Tables S6−S10 and
Figures S28−S45). The stereochemistry of 11 was confirmed by
determination of its optical rotation and comparison with the
published data.15 9 has not been described before and
therefore was confirmed by X-ray analysis (Table S11). Two
ABSTRACT: Penilactones A and B consist of a -butyrolactone
and two clavatol moieties. We identified two separate gene
clusters for the biosynthesis of these key building blocks in
Penicillium crustosum. Gene deletion, feeding experiments, and
biochemical investigations proved that a non-reducing PKS
ClaF is responsible for the formation of clavatol and the PKS-
NRPS hybrid TraA is involved in the formation of crustosic acid,
which undergoes decarboxylation and isomerization to the
predominant terrestric acid. Both acids are proposed to be
converted to -butyrolactones with involvement of
a
cytochrome P450 ClaJ. Oxidation of clavatol to hydroxyclavatol
by a non-heme FeII/2-oxoglutarate-dependent oxygenase ClaD
and its spontaneous dehydration to an ortho-quinone methide
initiate the two non-enzymatic 1,4-Michael addition steps.
Spontaneous addition of the methide to the -butyrolactones
led to peniphenone D and penilactone D, which undergo again
stereospecific attacking by methide to give penilactones A/B.
additional minor peaks were proven to be
5 and
hydroxyclavatol ethyl ether (12) (Figures 1A and S1).14
A
O
O
HO
OH
O
OH
HO
OH
O
OH
O
O
OH
O
OH
O
O
O
O
O
O
O
O
O
OH
OH
OH
O
OH
OH
OH
OH
OH
O
4
1
2
3
)
penilactone D (
)
penilactone A (
OH
)
penilactone B (
OH
)
peniphenone D (
OH
O
O
OH
O
O
O
OH
OH
Penilactones A (1) and B (2) (Figure 1A) are rare fungal
metabolites and were firstly isolated from Penicillium
crustosum PRB-2.1 Together with their putative precursors
peniphenone D (3) and penilactone D (4) (Figure 1A), they
were also identified in other Penicillium species.2-4 Feeding
experiments suggested that 1 and 2 are derived from acetyl-
CoA and L-malic acid (Figure 1B).1 It was proposed that 1 and
2 are formed by 1,4-Michael additions of two clavatol (5)
molecules in its active form ortho-quinone methide (6) with a
γ-butyrolactone (tetronic acid), i.e. (R)-5-methyl (7) or (S)-5-
carboxylmethyltetronic acid (8).1,2 This hypothesis was
O
OH
OH
hydroxyclavatol
10
6
9
)
5
o-quinone methide (
OH
)
hydroxyclavatol (
clavatol (
O
)
methyl ether (
)
O
O
O
O
O
HO
O
O
OH
O
O
O
hydroxyclavatol
11
terrestric acid (
)
13
crustosic acid (
)
12
ethyl ether (
O
CH3CO-SCoA
)
B
O
O
O
O
HO
OH
OH
O
O
HO
pyruvic acid
O
7
(R)-5-methyltetronic acid (
)
O
O
CO2
O
OH
O
OH
OH
1
penilactone A (
)
O
biomimetic synthesis.5-7 Acetate of
O
OH
O
confirmed by
a
2
OH
O
HO
hydroxyclavatol (9) instead of 5 was used at 110 °C for the
synthesis. Hydroxyclavatol methyl ether (10) was also isolated
from P. crustosum.3
O
oxaloacetic acid
clavatol (5)
6
o-quinone methide ( )
HO
OH
OH
O
O
O
O
OH OH
O
O
O
O
HO
O
OH
5 can be considered as a polyketide synthase (PKS) product.
However, the responsible enzyme is unknown before. Neither
the direct precursor nor the biosynthesis of 7 and 8 has been
reported. Michael addition as a thermodynamically controlled
1,4-addition of active methylenes to activated olefins such as
α,β-unsaturated carbonyl derivatives8 are widely used in the
chemical synthesis9-12 and also involved in the biosynthesis of
natural products.13 However, the substrates, enzymes and
conditions for Michael addition involved in the formation of 1–
4 in nature have not been reported yet.
HO
O
OH
CH3CO-SCoA
O
(S)-5-caboxylmethyl
tetronic acid (
L-malic acid
8
)
2
penilactone B (
)
Figure 1. Metabolites from PRB-2 (A) and proposed biosynthetic
routes to 1 and 2 (B). 1
However, peniphenones and penilactones could only be
detected in extracted ion chromatograms (EICs, data not
shown). To increase their productivity, PRB-2 was cultivated in
PD surface culture for 14 days. LC-MS analysis revealed clear
accumulation of 1–4 (Figures 2, S1, and S2). A 30 days-old rice
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culture also accumulated 1–4 and was therefore used for
For understanding the role of 11 and 13 for 1–4, they were
isolated from ∆claF mutant and fed into ∆traA mutant. Feeding
11 only restored 1 and 3 production, while 1–4 were detected
after feeding with 13 (Figures 2C, S18 and S19). More
interestingly, 11 was also restored after feeding with 13, but
not vice versa (Figure 2D). This proved that 13 is the precursor
of both 8 and 11. 11 serves then as a precursor of 7 (Scheme
1).
1
2
3
4
5
6
7
8
isolation and structure elucidation by MS, NMR (Tables S6–S7
and Figures S28–S31), optical rotation, and CD spectra (Figures
S46−S49).1,3 The CD spectra of 1 and 2 (Figures S46 and S47)
correspond very well to those reported previously.1 The
stereochemistry of 3 and 4 was determined by chemical
synthesis from 7 and 8 with known configuration at C-5 (Figure
S21, see below for the formation of 3 from 7 and 4 from 8).
Under these conditions, the production of 9 and 10 was
strongly reduced. In comparison, 11 was detected as the
predominant peak. Furthermore, a new peak was identified as
carboxylated derivative of 11, termed crustosic acid (13)
hereafter (Figures 1A, S44, and S45). 13 has an [α]20 D value
of -164.1, while that of 11 at +37.1. The configuration of 13 was
assigned by comparison with the optical rotation data of 5-
methyl- and 5-carboxylmethyltetronic acids.16
9
10
11
12
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For biosynthetic studies on 1 and 2, the genome of PRB-2 was
sequenced and the draft genome sequence was used for
prediction of putative gene clusters by using AntiSMASH.17 For
gene inactivation, we established a gene replacement protocol
using the split marker strategy and hygromycin B as selection
marker, which significantly enhances the homologous
recombination events at the target gene (Figure S3).18
Based on its aromatic character, 5 is expected to be assembled
by a non-reducing PKS (NR-PKS).19 One of the six NR-PKS genes
pcr3094 within a 36.2 kbp large cluster (Figure 2A and Table
S4) has
a SAT-KS-AT-PT-ACP-MeT-TE domain structure
(Abbreviations for PKS and NRPS domains as given before19,20).
It shares a sequence identity of 57.7 % with CitS from Monascus
ruber21 and 64.4 % with EAW12049.1 from Aspergillus clavatus
(Table S4). Deletion of pcr3094, termed claF (from the clavatol
cluster) hereafter, completely abolished the production of 1−5,
9, and 10 (see SI for manipulation). The two tetronic acids 11
and 13 accumulated with much higher yields in the ∆claF
mutant than in PRB-2 (Figures 2B, S4, and S6). Feeding 5 to the
mutant restored the production of 1–4 and 9 (Figure S15).
To provide more evidence for the function of ClaF as a clavatol
synthase, pcr3094 was cloned into pYH-wA-pyrG and
expressed in A. nidulans.22-24 The formation of 5 in the
transformant JF11 was confirmed by LC-MS (Figure S20) and
1H NMR analyses after isolation. These results proved that ClaF
is responsible for 5 formation in the biosynthesis of 1–4
(Scheme 1).
To identify the genetic potential for 7 and 8, we focused on PKS-
NRPS hybrid enzymes, because tetronic acids like carlosic acid
are usually assembled by such enzymes.25 Analysis of the draft
sequence revealed the presence of a candidate gene pcr11009,
termed traA (from the terrestric acid cluster), within a 33.6 kbp
large cluster. TraA with a domain structure KS-AT-DH-MeT-
KR-ACP-C-A-PCP (Figure 2A) shares a sequence identity of 69.6
% with CaaA in the carlosic acid biosynthesis (Table S5).25
Deletion of traA completely abolished the production of 1–4,
indicating its involvement in the biosynthesis. As expected, 5,
9, and 10 were accumulated in the ∆traA mutant (Figures 2C
and S8). Surprisingly, the production of 11 and 13 were also
totally blocked. To restore the production of 1–4, we
chemically synthesized 7 and 8 (Figure S21)5,7,26,27 and fed
them to the ∆traA mutant. LC-MS analysis revealed that feeding
7 restored the production of 1 and 3, but not 2 and 4. In
contrast, 2 and 4, but not 1 and 3 were detected in the culture
of ∆traA mutant fed with 8 (Figures 2C, S16 and S17). This
proved that TraA is involved in the formation of 7 and 8, which
cannot be converted to each other (Scheme 1).
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A
It can be concluded that 13 is the product of TraA with or
1
2
3
4
without other enzymes and mainly converted to the
predominant product 11 in PRB-2. Only small amounts of 11
and 13 undergo degradation to 7 and 8 for the formation of
1−4 (Figure 2D and Scheme 1).
5
6
7
8
Having the both backbone genes/enzymes identified, we
intended to investigate the conversion of 13 to 8 and 11, 11 to
7, and the metabolism of 5. Inactivation of the oxygenase gene
traH abolished the production of 1, 3, and 11, confirming its
involvement in the decarboxylation and isomerization of 13 to
11 (Figures 2C and S13). In the deletion mutants of the
cytochrome P450 traB and the dehydrogenase traD, no
accumulation of 11, 13, or 1–4 was detected (Figures S9 and
S10), proving their roles in the 13 formation (Scheme 1).
Deletion of traE and traF did not result in significant changes in
SM production (Figures S11 and S12).
9
10
11
12
13
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11
B
4
3
5
and
2
10
13
WT
WT
1
280 nm
9
EICs
11
Regarding Michael addition, we presumed a more active
intermediate than 5 for the formation of 6. Detailed inspection
of the cla cluster (Figure 2A, Table S4) revealed the presence of
genes coding for an oxygenase (claD) and a cytochrome P450
(claJ). ClaD comprises 338 amino acids and shares a sequence
identity of 53.8 % with CitB in the citrinin biosynthesis.21 It also
contains the typical conserved 2-His-1-Asp ion-binding triad
(His184, His202 and Asp187) of non-heme FeII/2-oxoglutarate-
dependent oxygenases (Figure S22). Deletion of claD abolished
the production of 1–4 and 9, whereas 5 was clearly
accumulated (Figures 2B and S5). Feeding 9 in the ∆claD
mutant restored the production of 1–4 (Figure S14), proving
its role in the conversion of 5 to 9.
13
13

claD
claD
5
280 nm
EICs

11
11
claF
claF
280 nm
EICs
13
9
5 10
claJ
280 nm
EICs
claJ
10
15
13
20
25
30
35
For biochemical characterization, claD was amplified and
cloned into pET28a (+). The purified ClaD (Figure S23) was
used for incubation with 5 in the presence of ascorbate (AA),
Fe[(NH4)2(SO4)2], and 2-oxoglutarate (2OG).28,29 HPLC analysis
confirmed the oxidation of 5 to 9 with a conversion yield of
22.5 % after incubation with 2 µg protein at 37°C for 30 min
(Figure 3A). Nearly no consumption of 5 was detected in the
assays without ascorbate or 2-oxoglutarate. Replacing
ascorbate by dithiothreitol (DTT) or without additional FeII
reduced the activity significantly. These results proved that
t/min
C
10
4
traH
traH
2
9
9
280 nm
5
EICs
10
traA
traA
5
280 nm
EICs
3
5
and
a
non-heme FeII/2-oxoglutarate-dependent
D
ClaD acts as
10
1
oxygenase and oxidizes 5 to yield 9. Determination of kinetic
parameters gave a KM of 0.30 mM toward 5 and a turnover
number (kcat) of 0.26 s−1 (Figure 3B).
9
9
280 nm
traA + 7
+ 7
traA
EICs
5
2
To prove the conversion between 9 and 6, 9 was incubated in
H2O and H218O at 25°C for 16 h. MS data in positive and negative
modes confirmed the incorporation of 18O into 9 and therefore
the equilibration (Figures 3C and S24).
10
+ 8
+ 8
traA
traA
4
280 nm
EICs
11
11
3
5
and
9
9
10
traA
traA
+ 11
+ 11
1
280 nm
EICs
13
2
5
3
and
1
10
4
280 nm
traA + 13
traA + 13
EICs

10
15
20
25
30
35
t/min
Figure 2. Schematic representation of clavatol and terrestric acid
clusters in PRB-2 (A) and LC-MS results of deletion mutants (B and C)
as well as of ∆traA mutant fed with putative precursors (D). EICs refer
[M+H]+ ions of 1–6 and 11, 13 or [M+Na]+ of 9 and 10 with tolerance
ranges of ± 0.005.
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3
3
280 nm
A
A
C
5
22.5 %
9
7
1
2
3
4
5
6
7
+AA, FeII, 2OG
9
9
i
O
OH
7+9 in H2O
1
1
O
OH
CH2OH
OH
O2
CO2
+FeII, 2OG
+AA, 2OG
ii
ClaD
Fe2+
4.3 %
6.5 %
OH
iii
CH3
2-oxoglutarate succinate
3+9 in H2O
8+9 in H2O
+AA, FeII
hydroxyclavatol (9)
iv
v
clavatol (5)
4
4
[M+Na]+: 219.0628
[M-H]-: 195.0663
+DTT, FeII, 2OG
denat. enzyme
+AA, FeII, 2OG
9
9
8
vi
vii
H2
O
standard 5
8
9
2
2
4+9 in H2O
viii
standard 9
O
OH
O
OH
10
15
20
25
4
standard 1
standard 2
standard 3
standard 4
1
CH218OH H218O
OH
3
t/min
50
40
30
20
10
0
B
10
11
12
13
14
15
16
17
18
19
20
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O
CH3
Km=0.30 mM
kcat=0.26 s-1
o-quinone methide (6)
[
18O]-hydroxyclavatol (9)
[M+Na]+: 221.0670
[M-H]-: 197.0705
9
8
7
standard 7
standard 8
standard 9
[M+H]+: 179.0703
[M-H]-: 177.0557
0.0
0.5
1.0
1.5
2.0
concentration of 5 [mM]
0
5
10
15
20
25
30
35
t/min
Figure 3. Functional proof of ClaD as a non-heme FeII/2-oxoglutarate-
dependent clavatol oxidase (A and B) and determination of the
equilibration between 9 and 6 (C).
B
O
O
OH
O
O
O
O
H
3
ClaJ shares clear sequence homology with fungal cytochrome
P450 enzymes, e.g. 42.0 % identity with BAJ04372.1 from
Aspergillus oryzae.30 Deletion of claJ resulted in the
abolishment of 1−4 (Figures 2B and S7), but still retained the
production of 5, 9, 11, and 13. This indicates its role in the C-C
double bond cleavage of 11 and 13 (Scheme 1). However, ClaJ
could also catalyzes the connection of the two building blocks
via Michael addition.
O
O
4
H
HO
OH
H
OH
O
7
O
H
3
O
6
OH
H2
8´
O
O
OH
O
OH
H
O
O
H
H2O
O
OH
O
H
O
O
OH
O
O
O
O
O
O
O
OH
O
O
O
OH
O
O
O
H
H
H
6
O
OH
O
O
O
H
O
OH
HO
H
H
HO
OH
OH
O
O
O
O
O
OH
O
O
O
O
O
O
OH
O
OH
O
OH
1
O
O
O
OH
O
3
O
H
O
O
4
COOH
O
H
O
HO
O
OH
OH
COOH
8
O
H
COOH
4
O
OH
H2
O
6
OH
O
H
OH
H
O
H
O
H2O
O
OH
O
O
O
H
H
O
OH
8´
O
O
O
O
O
O
O
COOH
O
OH
O
COOH
OH
O
O
HOOC
COOH
O
H
H
6
O
OH
O
O
O
OH
HO
O
H
H
H
HO
OH
OH
O
OH
O
O
O
O
OH
O
O
O
O
O
O
OH
O
OH
O
OH
COOH
2
COOH
COOH
Figure 4. Non-enzymatic formation of penilactones and peniphenone.
(A) LC-MS analysis of 48 h-incubation mixtures. Absorptions at 254 nm
(1−4, 9) or EICs (7 and 8) are illustrated. (B) Proposed mechanism of
non-enzymatic formation of 1−4 via Michael addition.
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ClaF
ClaF
1
2
3
4
5
6
7
non-enzymatic conversions
O
OH
O
OH
O
OH
O
OH
MeT
O
ACP
TE
MeT
SAT
KS
AT PT
TE
AT PT ACP
S
SAT
S
KS
OH
ClaD
OH
OCH3
OH
S
OH
hydroxyclavatol
10
OH
OH
O
methyl ether (
)
+H2O
-CH3OH
O
O
9
hydroxyclavatol (
+H2O -H2O
)
O
5
clavatol (
)
9
hydroxyclavatol (
-H2O
)
O
+CH3OH
O
OH
O
O
O
OH
O
OC2H5
-C2H5OH
+C2H5OH
spontaneous
3 x malonyl-
SCoA
3 x CO2, 3 x HSCoA
2 x S-adenosyl-
homoserine
OH
O
O
OH
2 x SAM
hydroxyclavatol
12
6
o-quinone methide (
)
ethyl ether (
)
O
8
9
6
o-quinone methide (
)
O
HO
OH
OH
O
TraB,
TraD
O
OH
6
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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40
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43
44
45
46
47
48
49
50
51
52
53
54
55
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60
O
O
ClaJ
TraA
O
O
HO
O
O
O
AT
KS
OH
MeT
A
OH
non-enzymatic
Michael addition
OH
ACP
S
non-enzymatic
Michael addition
PCP
C
HOOC
COOH
O
DH
KR
O
HOOC
COOH
S
13
penilactone D (
4
)
2
penilactone B ( )
crustosic acid (
)
(S)-5-carboxylmethyl-
8
tetronic acid ( )
S
O
O
O
L-malic acid
HO
O
O
O
TraH
3 x malonyl-
SCoA
OH
O
HO
OH
6
OH
6
O
O
OH
O
O
O
ClaJ
O
3 x CO2
3 x HSCoA
O
O
O
HO
O
non-enzymatic
Michael addition
non-enzymatic
Michael addition
O
O
OH
OH
OH
3
1
)
11
terrestric acid (
(R)-5-methyl-
peniphenone D (
)
penilactone A (
)
7
tetronic acid (
)
Scheme 1. Proposed biosynthetic pathways of penilactones and peniphenones in P. crustosum.
For preparing feeding experiments in ∆claJ, we carried out
control incubations of 7 with 9 and 8 with 9 in water at 25°C,
which delivered surprising results, i.e. the non-enzymatic
Michael addition under these mild conditions (Figure 4A). In
AUTHOR INFORMATION
Corresponding Author
*E-mail: shuming.li@staff.uni-marburg.de.
the first combination, 3 was detected as the major and 1 as a
minor product, while 4 as the major and 2 as a minor product
in the case of 8 with 9. When 3 and 4 were incubated with 9, 1
and 2 were detected (Figure 4A). Formation of 3 and 4 is time-
and pH-dependent (Figures S25 and S26). 3 and 4 are formed
under neutral or acidic conditions. When pH values were
higher than 5.0, diclavatol3 was also detected (Figure S26). It is
obvious that the active intermediate 6 can be easily formed
from 9 in aqueous system and initiates the Michael additions
(Figure 4B), which was confirmed by incubation of 9, 10, and
12 in different solvents. They are stable in acetonitrile.
Alcohols determined the end products of 6 (Figure S27, Scheme
1). All these results indicate that ClaJ is likely not involved in
the Michael addition, probably in the conversion of 11 to 7 and
13 to 8 (Scheme 1).
ORCID
Shu-Ming Li: 0000-0003-4583-2655
Wen-Bing Yin: 0000-0002-9184-3198
Author Contributions
†These authors contributed equally to this work.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We thank Tianjiao Zhu (Ocean University of China, Qingdao) for
providing strain PRB-2, Rixa Kraut, Stefan Newel, and Andreas
Heine (University of Marburg) for taking MS, NMR spectra and
X-ray crystal analysis, respectively. This project was financially
funded in part by the Deutsche Forschungsgemeinschaft (DFG,
Taken together, 1–4 are formed by enzymes from independent
pathways of two separate gene clusters (Scheme 1). The tra
cluster assembles 13, which is converted to 11. Both acids
deliver the two γ-butyrolactones 7 and 8. The cla cluster
provides the highly active 6 by a spontaneous dehydration of 9.
This initiates the two step non-enzymatic Michael additions by
the intermolecular nucleophile attacking of 6 to 7 or 8 and
subsequent reaction with 3 and 4. Thus, this study provides an
excellent example for SM with complex structures that are
formed by enzymes from different pathways and by
combination of enzymatic and non-enzymatic reactions.
German Research Foundation)
− Li844/11-1 and INST
160/620-1 as well as the National Natural Science Foundation
of China − 31861133004. Jie Fan (201507565006) and Ge Liao
(201607565014) are scholarship recipients from the China
Scholarship Council.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
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Table of Contents
1
2
ACP
A
MeT
AT
3
4
5
6
O
DH
KR
C
KS
PCP
OH
HO
OH
O
O
O
O
O
O
O
HOOC
R=CH3 or
CH2COOH
O
HO
O
O
O
O
O
R
O
OH
-butyrolactones
crustosic acid
terrestric acid
penilactone A
7
O
OH
8
9
O
O
OH
O
OH
HO
OH
OH
O
OH
O
2
spontaneous
hydroxyclavatol
ortho-quinone methide
OH
OH
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
clavatol
AT PT
O
OH
TE
ACPMeT
SAT
KS
COOH
penilactone B
7
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