5674 Chem. Commun., 2012, 48, 5674–5676 This journal is cThe Royal Society of Chemistry 2012
Cite this:
Chem. Commun
., 2012, 48, 5674–5676
In vitro chemoenzymatic and in vivo biocatalytic syntheses of new
beauvericin analoguesw
Diana Matthes,
a
Lennart Richter,
a
Jane Mu
¨ller,
a
Alexander Denisiuk,
a
Sven C. Feifel,
a
Yuquan Xu,
b
Patricia Espinosa-Artiles,
b
Roderich D. Su
¨ssmuth*
a
and Istva
´n Molna
´r*
b
Received 6th March 2012, Accepted 17th April 2012
DOI: 10.1039/c2cc31669b
New beauvericins have been synthesized using the nonribosomal
peptide synthetase BbBEAS from the entomopathogenic fungus
Beauveria bassiana. Chemical diversity was generated by in vitro
chemoenzymatic and in vivo whole cell biocatalytic syntheses
using either a B. bassiana mutant or an E. coli strain expressing
the bbBeas gene.
The entomopathogenic fungus B. bassiana
1
produces various
secondary metabolites,
2,3
including the cyclooligomer depsi-
peptide (COD) beauvericin. This COD is a cyclic trimer of
D-Hiv-N-methyl-L-phenylalanine dipeptidol monomers. Beauver-
icin displays structural analogies to the cyclohexadepsipeptide
enniatin (Fusarium oxysporum) and to the cyclooctadepsipeptides
bassianolide (Beauveria bassiana) and PF1022A (Rosellinia sp.).
4
Fungal CODs are interesting pharmacophores that exhibit a
broad range of biological activities including antitumor,
5
anti-
bacterial, antibiotic,
1
antifungal, insecticidal, anthelmintic,
6
anti-
malarial, anti-inflammatory and immunosuppressant activities.
7
Therefore, analogues and derivatives of these small bioactive
natural products may acquire important roles in modern medi-
cine to treat a variety of diseases.
CODs are produced by nonribosomal peptide synthetase
(NRPS) enzymes in an iterative and recursive process.
4,8
Beauvericin synthetase (BbBEAS) contains two NRPS modules
harbouring domains that catalyze adenylation (A), thiolation (T),
methylation (M) and condensation (C) reactions. The A domains
activate their dedicated substrates (A
1
:D-2-hydroxyisovalerate
[D-Hiv]; A
2
:L-phenylalanine [L-Phe]) by aminoadenylation,
followed by covalent loading of these substrates onto BbBEAS
for subsequent assembly of beauvericin.
4,9–11
The beauvericin
precursor D-Hiv is synthesized by ketoisovalerate reductase
(KIVR) from 2-ketoisovalerate from primary metabolism.
4,12,13
In this contribution we demonstrate, for the first time, the
isolation of recombinant beauvericin synthetase BbBEAS
(351 kDa, B0.4mgmL
1
)fromE. coli and generate new
beauvericin analogues by a chemoenzymatic approach (Fig. 1A).
In a subsequent in vivo whole cell biocatalytic approach (Fig. 1B),
we also investigate the production of beauvericins by mutational
biosynthesis (MBS)
14–17
using a KIVR knockout strain of
B. bassiana (B. bassiana kivr
)
12
or an E. coli strain expressing
BbBEAS (E. coli bbBeas
+
).
18
Previous studies of enzymatic
synthesis of CODs with related fungal NRPSs indicated a relaxed
substrate specificity for the hydroxy acid-activating A
1
domain,
whereas amino acid activation was apparently more restricted.
19,20
Application of these observations to in vivo biosynthesis with
fungal cultures led to the isolation of new CODs of the enniatin,
21
the PF1022 and the beauvericin series.
22,23
Beauvericin synthetase BbBEAS was isolated from E. coli BL21
bbBeas
+
(for cultivation conditions see ESIw, General techniques)
Fig. 1 Synthesis of beauvericin analogues. (A) Concept of the in vitro
chemoenzymatic synthesis. An SDS-PAGE gel of recombinant
BbBEAS isolated from E. coli bbBeas
+
is also shown. (B) Concept
of the in vivo whole cell biocatalytic synthesis.
a
Technische Universita
¨t Berlin, Institut fu
¨r Chemie,
Straße des 17. Juni 124, 10623 Berlin, Germany.
E-mail: [email protected]e
b
SW Center for Natural Products Research and Commercialization,
School of Natural Resources and the Environment, The University of
Arizona, 250 E. Valencia Rd., Tucson, AZ 85706, USA.
E-mail: [email protected]
wElectronic supplementary information (ESI) available: Experimental
details, HPLC-ESI-MS, -MS/MS and -MRM data. See DOI: 10.1039/
c2cc31669b
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by precipitation with 60% ammonium sulphate as described
recently for related COD synthetases.
24
In vitro reconstitution
of the enzyme and detection of synthesised COD analogues
were carried out according to Zocher et al. by incorporation of
a[
3
H] label from [
3
H-methyl]-SAM.
25
Accordingly, 10 mLofa
reaction mixture, containing 0.1 M ATP (pH 7), 1 M MgCl
2
and 0.1 M L-Phe in 1 M Tris–HCl (pH 8), was added to 3 mL
of 0.1 M D-2-hydroxyisovalerate (D-Hiv, 3). The reaction was
started by adding 0.55 mCi [
3
H-methyl]-SAM and 200 mL
BbBEAS (0.2 nmol). After 30 min of incubation at 25 1C the
reaction was stopped by adding 1 mL H
2
O. The radioactively
labelled depsipeptide was extracted with 2 mL EtOAc. 100 mL
of the organic phase was mixed with 4 mL LumaSafe Plus and
measured in a scintillator. The remaining organic extract was
analysed by thin layer chromatography (TLC) using silica 60
F
254
plates at room temperature (eluent EtOAc : MeOH : H
2
O=
100 : 5 : 1) after equilibration of the chamber with eluent
vapour. Detection and quantification were performed by using
a Radio-TLC Scanner (Raytest). Efficient chemoenzymatic
synthesis of beauvericin by recombinant BbBEAS was demon-
strated using this protocol, and the structure of the product
was confirmed by HPLC-ESI-MS and MS/MS (ESIw, Fig. S1
and S2, and General techniques). To our surprise, small
amounts of beauvericin were also produced by the BbBEAS
enzyme in vitro in control reactions where L-Phe was omitted
from the reaction mixture (Fig. S1B, ESIw). We propose that
L-Phe is present in the beauvericin synthetase preparation,
probably as an activated enzyme-bound adenylate or as a
thioester, similar to that observed earlier by Zocher et al.
26
Subsequently, we applied this chemoenzymatic synthesis strategy
to the production of beauvericin analogues by replacing D-Hiv
with one of 10 synthetic a-hydroxy acids (Table S1, ESIw).
Four a-hydroxy acids, 2-hydroxy-butyric acid (D-Hbu, 2), DL-2-
hydroxy-pent-4-ynoic acid (DL-Hpyn, 12), D-fluorolactate (13),
and D-chlorolactate (14), were successfully incorporated into
CODs (Table 1 and Fig. S1, ESIw).
In an attempt to expand the repertoire of beauvericin analogues,
we synthesized and tested 37 hydroxy acids in an in vivo whole cell
biocatalytic format (Table S1, ESIw). Precursor analogue feeding
experiments with B. bassiana kivr
(mutational biosynthesis) have
been performed as described by Xu et al.
12,22
As an alternative, we also evaluated the BbBEAS-expressing
E. coli strain as a whole cell biocatalyst because of its faster
growth rate and ease of genetic manipulation. Heterologous
production and extraction of beauvericin and its analogues from
E. coli BL21 bbBeas
+
was based on the constructs and conditions
of Xu et al.
18,22
The feeding regimen included simultaneous
supplementation with the natural amino acid L-Phe and one
of the synthetic hydroxycarboxylic acids. HPLC-ESI-MS was
carried out to identify the expected beauvericin analogues by their
characteristic molecular masses and retention times. Product
structures were confirmed by ESI-MS/MS experiments, providing
characteristic fragmentation pattern fingerprints. Further Multi-
ple Reaction Monitoring (MRM) experiments were conducted to
estimate the yields of the beauvericin analogues obtained.
Out of the 37 a-hydroxy acids tested, eight were shown to
support mutational biosynthesis with the B. bassiana kivr
strain (Table 2). In spite of the radically different cell wall
structures of the fungus and the Gram-negative bacterium,
six of these eight hydroxy acids were also accepted by E. coli
bbBeas
+
for biocatalytic conversion into beauvericin-like
products. Five novel beauvericin analogues (Beau-4, -5, -10,
-12, -16) were obtained by using these in vivo approaches,
while a further three (Beau-2, -8, -9) have previously been
described by Xu et al.
18
Beau-2 and Beau-12 were also detected
during in vitro chemoenzymatic synthesis (Table 1). All the
beauvericin analogues described here had all three of their
a-hydroxy acid positions occupied by the fed precursor
analogue, indicating that both the B. bassiana kivr
strain
Table 1 Beauvericin analogues produced by in vitro chemoenzymatic
synthesis with BbBEAS
No. Precursor Product R
F
value R
1
=R
2
=R
3
3D-Hiv Beauvericin
a
0.4
b
iPr
2D-Hbu Beau-2 0.7 Et
12 DL-Hpyn Beau-12 0.6 Ethynyl
13 D-Fluorolactate Beau-13 0.5 FMe
14 D-Chlorolactate Beau-14 0.5 C1Me
a
Beauvericin produced by chemoenzymatic synthesis.
b
Average relative
retentions (R
F
) calculated from three independent experiments.
Table 2 Beauvericin analogues obtained by in vivo whole cell bio-
catalytic synthesis.
No. Precursor Product
B. bassiana
kivr
E. coli
bbBeas
+
R
1
=R
2
=R
3
R
4
Yield
a
Yield
a
3D-Hiv Beauvericin 1.11 3.33 iPr Me
2D-Hbu Beau-2 0.23 0.005 Et Me
4D-Hval Beau-4 0.48 2.18 nPr Me
5D-Hcap Beau-5 0.34 0.32 nBu Me
8D-allo-Hmv Beau-8 7.82 1.20 allo-sBu Me
9D-Hmv Beau-9 5.19 2.76 sBu Me
10 DL-Htbu Beau-10 0.10 0.04 tBu Me
b
,H
c
12 DL-Hpyn Beau-12 0.17 ND
d
Ethynyl Me
16 D-Hmsbu Beau-16 0.04 NT
e
msBu Me
a
Average yields of the product in mg L
1
are calculated from two
independent experiments. Final concentrations of the precursors during
fermentation: B. bassiana,40mM;E. coli,30mM(
D-Hiv: 15 mM).
b
B. bassiana kivr
.
c
E. coli bbBeas
+
.
d
ND, not detected.
e
NT, not tested.
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5676 Chem. Commun., 2012, 48, 5674–5676 This journal is cThe Royal Society of Chemistry 2012
and the BbBEAS-expressing E. coli are devoid of other accep-
table 2-hydroxy carboxylic acids. Both producer strains also
fully methylated all three amino acid positions of these beau-
vericin analogues, with the exception of Beau-10 from E. coli
where one of the amino acid positions remained unmethylated.
For example, feeding hydroxy acid 12 (DL-2-hydroxy-pent-4-
ynoic acid) to B. bassiana kivr
yields Beau-12 with a molecular
mass of [M + H]
+
= 772.6 and a retention time of 5.3 min,
with the corresponding MS/MS spectrum providing a finger-
print where each peak can be assigned to one fragment of the
molecule (Fig. S3h, ESIw). Similarly, feeding hydroxy acid 4
(D-2-hydroxy-pentanoic acid) to E. coli bbBeas
+
yields Beau-4
[M+H]
+
= 784.4 with a retention time of 5.7 min. Character-
istic fragments from MS/MS experiments were assigned accord-
ingly (Fig. S4c, ESIw). The yields of the beauvericin analogues
Beau-2, -4, -5, -8, -9, -10, -12 and -16 from B. bassina kivr
were
estimated by HPLC-ESI-MS (for quantification see ESIw,
General techniques) to range between 0.04 and 7.82 mg L
1
(Table 2, and Fig. S3a–i, and Table S2, ESIw). Upon compar-
ison, we find that the same strain produces 1.1 mg L
1
beauvericin upon feeding the natural hydroxy acid 3 (D-Hiv)
under identical fermentation conditions. Product yields in
E. coli ranged from B0.005 mg L
1
to B2.8mgL
1
for
Beau-2, -4, -5, -8, -9 and -10, as compared to that of beauvericin
at B3mgL
1
with the native substrate 3 in this strain (Table 2,
and Fig. S4a–g, and Table S3, ESIw). Surprisingly, precursor
analogues 8 and 9 provide for beauvericin analogues yields in
B. bassiana of up to 7.8 and 5.2 mg L
1
, respectively, exceeding
that of the native product beauvericin. Although the same
precursor analogues also performed well in E. coli bbBeas
+
,
the yields of Beau-8 and Beau-9 did not exceed that of
beauvericin in this host. In contrast to chemoenzymatic
synthesis, product analogue yields are determined not only by
the innate substrate preferences of BbBEAS during in vivo
biosynthesis. Rather, the variability of precursor uptake,
precursor and product toxicity, and catabolism of the precursor
or even the product can all reduce or boost beauvericin
analogue yields to different extents in different host strains.
Thus, Beau-13 and Beau-14 were only detectable during in vitro
chemoenzymatic synthesis, but not during in vivo biocatalysis.
Conversely, while precursor analogues 4, 5, 8, 9, and 10 were
apparently acceptable for BbBEAS in vivo, the corresponding
CODs remained below the detection limits of the chemoenzymatic
assay. Such unexpected and unpredictable results emphasize the
complementary nature of in vitro chemoenzymatic and in vivo
whole cell biocatalytic approaches
27
towards natural product
diversification and ‘‘unnatural product’’ biosynthesis. While the
full characterization of the substrate specificity of BbBEAS
requires further studies, our results show that the tolerance
spectrum of this enzyme includes hydroxy acids with various
aliphatic branched side chains, and extends even to halogenated
hydroxy acids. Particularly interesting is the biosynthesis, albeit at
a low yield, of Beau-12 with the ethynyl side chain amenable to
further derivatization by 1,3-dipolar cycloaddition reactions
known as ‘‘click chemistry’’ as has also been shown recently for
ribosomally synthesized peptide antibiotics.
28,29
In summary, we successfully generated seven novel beau-
vericins by in vitro chemoenzymatic and in vivo biocatalytic
syntheses using custom-synthetic hydroxy acid precursor analogues.
The production of these new beauvericins could now be
optimized and scaled up for characterization in various
biological assays.
This work was supported by the Cluster of Excellence ‘‘Unifying
concepts of catalysis’’ and coordinated by the TU Berlin.
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