In vitro chemoenzymatic and in vivo biocatalytic syntheses of new beauvericin analogues

Article abstract of 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 ei

Full text of DOI:10.1039/c2cc31669b

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COMMUNICATION
In vitro chemoenzymatic and in vivo biocatalytic syntheses of new
beauvericin analoguesw
a
a
a
a
a
Diana Matthes, Lennart Richter, Jane Muller, Alexander Denisiuk, Sven C. Feifel,
¨
b
b
a
b
Yuquan Xu, Patricia Espinosa-Artiles, Roderich D. Sussmuth* and Istvan Molnar*
¨
´ ´
Received 6th March 2012, Accepted 17th April 2012
DOI: 10.1039/c2cc31669b
À1
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.
(351 kDa, B0.4 mg mL ) from E. 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
14–17
biosynthesis (MBS)
using a KIVR knockout strain of
À 12
18
B. bassiana (B. bassiana kivr ) or an E. coli strain expressing
+
BbBEAS (E. coli bbBeas ). Previous studies of enzymatic
1
The entomopathogenic fungus B. bassiana produces various
synthesis of CODs with related fungal NRPSs indicated a relaxed
2,3
secondary metabolites,
including the cyclooligomer depsi-
substrate specificity for the hydroxy acid-activating A
whereas amino acid activation was apparently more restricted.
Application of these observations to in vivo biosynthesis with
1
domain,
1
9,20
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
21
fungal cultures led to the isolation of new CODs of the enniatin,
22,23
the PF1022 and the beauvericin series.
4
bassianolide (Beauveria bassiana) and PF1022A (Rosellinia sp.).
Beauvericin synthetase BbBEAS was isolated from E. coli BL21
+
bbBeas (for cultivation conditions see ESIw, General techniques)
Fungal CODs are interesting pharmacophores that exhibit a
5
broad range of biological activities including antitumor, anti-
1
bacterial, antibiotic, antifungal, insecticidal, anthelmintic, anti-
6
7
malarial, anti-inflammatory and immunosuppressant activities.
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
4,8
(NRPS) enzymes in an iterative and recursive process.
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
D-Hiv]; A L-phenylalanine [L-Phe]) by aminoadenylation,
followed by covalent loading of these substrates onto BbBEAS
1
: D-2-hydroxyisovalerate
[
2
:
4,9–11
for subsequent assembly of beauvericin.
The beauvericin
precursor D-Hiv is synthesized by ketoisovalerate reductase
4,12,13
KIVR) from 2-ketoisovalerate from primary metabolism.
(
In this contribution we demonstrate, for the first time, the
isolation of recombinant beauvericin synthetase BbBEAS
a
Technische Universita¨t Berlin, Institut fu¨r Chemie,
Straße des 17. Juni 124, 10623 Berlin, Germany.
E-mail: suessmuth@chem.tu-berlin.de
SW Center for Natural Products Research and Commercialization,
b
School of Natural Resources and the Environment, The University of
Arizona, 250 E. Valencia Rd., Tucson, AZ 85706, USA.
E-mail: imolnar@email.arizona.edu
w Electronic supplementary information (ESI) available: Experimental
details, HPLC-ESI-MS, -MS/MS and -MRM data. See DOI: 10.1039/
c2cc31669b
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.
5
674 Chem. Commun., 2012, 48, 5674–5676
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by precipitation with 60% ammonium sulphate as described
Table 1 Beauvericin analogues produced by in vitro chemoenzymatic
synthesis with BbBEAS
2
4
recently for related COD synthetases. In vitro reconstitution
of the enzyme and detection of synthesised COD analogues
were carried out according to Zocher et al. by incorporation of
3
3
25
a [ H] label from [ H-methyl]-SAM. Accordingly, 10 mL of a
reaction mixture, containing 0.1 M ATP (pH 7), 1 M MgCl2
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
3
started by adding 0.55 mCi [ H-methyl]-SAM and 200 mL
BbBEAS (0.2 nmol). After 30 min of incubation at 25 1C the
2
reaction was stopped by adding 1 mL H O. The radioactively
1
2
RF value R =R = R
3
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
No. Precursor
Product
a
b
3
D-Hiv
Beauvericin
Beau-2
Beau-12
Beau-13
Beau-14
0.4
0.7
0.6
0.5
0.5
iPr
Et
Ethynyl
FMe
C1Me
2
12
D-Hbu
DL-Hpyn
1
1
3
4
D-Fluorolactate
D-Chlorolactate
F254 plates at room temperature (eluent EtOAc : MeOH : H O =
2
1
00 : 5 : 1) after equilibration of the chamber with eluent
a
b
Beauvericin produced by chemoenzymatic synthesis. Average relative
) calculated from three independent experiments.
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
retentions (R
F
Table 2 Beauvericin analogues obtained by in vivo whole cell bio-
catalytic synthesis.
2
6
thioester, similar to that observed earlier by Zocher et al.
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).
B. bassiana E. coli
+
bbBeas
À
kivr
a
a
1
R =R = R R
2
3
4
No. Precursor Product Yield
Yield
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
12,22
been performed as described by Xu et al.
3
2
4
5
8
9
D-Hiv
Beauvericin 1.11
3.33
0.005
2.18
0.32
1.20
2.76
0.04
iPr
Et
nPr
nBu
allo-sBu
sBu
tBu
Ethynyl
msBu
Me
Me
Me
Me
Me
Me
Me ,H
Me
Me
D-Hbu
D-Hval
D-Hcap
D-allo-Hmv Beau-8
D-Hmv Beau-9
Beau-2
Beau-4
Beau-5
0.23
0.48
0.34
7.82
5.19
À
b
c
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
10 DL-Htbu Beau-10 0.10
d
1
1
2
6
DL-Hpyn Beau-12 0.17
D-Hmsbu Beau-16 0.04
ND
NT
e
a
À1
Average yields of the product in mg L are calculated from two
independent experiments. Final concentrations of the precursors during
+
E. coli BL21 bbBeas was based on the constructs and conditions
18,22
of Xu et al.
fermentation: B. bassiana, 40 mM; E. coli, 30 mM (D-Hiv: 15 mM).
b
The feeding regimen included simultaneous
À
c
+
d
e
B. bassiana kivr . E. coli bbBeas . ND, not detected. NT, not tested.
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.
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
1
8
described by Xu et al. 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
Out of the 37 a-hydroxy acids tested, eight were shown to
support mutational biosynthesis with the B. bassiana kivr
À
strain (Table 2). In spite of the radically different cell wall
structures of the fungus and the Gram-negative bacterium,
À
analogue, indicating that both the B. bassiana kivr strain
This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 5674–5676 5675

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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-
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.
À
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,
Notes and references
with the corresponding MS/MS spectrum providing a finger-
print where each peak can be assigned to one fragment of the
1 R. L. Hamill, C. E. Higgens, H. E. Boaz and M. Gorman,
Tetrahedron Lett., 1969, 10, 4255–4258.
2
3
A. A. Yakasai, J. Davison, Z. Wasil, L. M. Halo, C. P. Butts,
C. M. Lazarus, A. M. Bailey, T. J. Simpson and R. J. Cox, J. Am.
Chem. Soc., 2011, 133, 10990–10998.
L. M. Halo, J. W. Marshall, A. A. Yakasai, Z. Song, C. P. Butts,
M. P. Crump, M. Heneghan, A. M. Bailey, T. J. Simpson,
C. M. Lazarus and R. J. Cox, ChemBioChem, 2008, 9, 585–594.
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
4
5
6
¨ ¨
¨
R. Sussmuth, J. Muller, H. von Dohren and I. Molnar, Nat. Prod.
´
Rep., 2011, 28, 99–124.
À
J. Zhan, A. M. Burns, M. X. Liu, S. H. Faeth and A. A. L.
Gunatilaka, J. Nat. Prod., 2007, 70, 227–232.
G. A. Conder, S. S. Johnson, D. S. Nowakowski, T. E. Blake,
F. E. Dutton, S. J. Nelson and E. M. Thomas, J. Antibiot., 1995,
48, 820–823.
F. Sarabia, S. Chammaa, A. S. Ruiz, L. M. Ortiz and F. J. Herrera,
Curr. Med. Chem., 2004, 11, 1309.
M. A. Fischbach and C. T. Walsh, Chem. Rev., 2006, 106,
3468–3496.
A. Koglin and C. T. Walsh, Nat. Prod. Rep., 2009, 26, 987–1000.
estimated by HPLC-ESI-MS (for quantification see ESIw,
General techniques) to range between 0.04 and 7.82 mg L
À1
(
Table 2, and Fig. S3a–i, and Table S2, ESIw). Upon compar-
À1
ison, we find that the same strain produces 1.1 mg L
7
8
9
beauvericin upon feeding the natural hydroxy acid 3 (D-Hiv)
under identical fermentation conditions. Product yields in
À1
À1
E. coli ranged from B0.005 mg L to B2.8 mg L for
Beau-2, -4, -5, -8, -9 and -10, as compared to that of beauvericin
À1
10 R. Finking and M. A. Marahiel, Annu. Rev. Microbiol., 2004, 58,
53–488.
1 H. von Do
12 Y. Xu, E. M. K. Wijeratne, P. Espinosa-Artiles, A. A. L.
Gunatilaka and I. Molnar, ChemBioChem, 2009, 10, 345–354.
3 C. Lee, H. Gorisch, H. Kleinkauf and R. Zocher, J. Biol. Chem.,
992, 267, 11741–11744.
14 S. Weist and R. D. Sussmuth, Appl. Microbiol. Biotechnol., 2005,
8, 141–150.
5 S. Weist, B. Bister, O. Puk, D. Bischoff, S. Pelzer, G. J. Nicholson,
W. Wohlleben, G. Jung and R. D. Sussmuth, Angew. Chem., Int.
Ed., 2002, 41, 3383–3385.
16 S. Weist, C. Kittel, D. Bischoff, B. Bister, V. Pfeifer,
G. J. Nicholson, W. Wohlleben and R. D. Sussmuth, J. Am. Chem.
Soc., 2004, 126, 5942–5943.
at B3 mg L with the native substrate 3 in this strain (Table 2,
4
and Fig. S4a–g, and Table S3, ESIw). Surprisingly, precursor
analogues 8 and 9 provide for beauvericin analogues yields in
1
¨
hren, Adv. Biochem. Eng./Biotechnol., 2004, 88, 217–264.
À1
´
B. bassiana of up to 7.8 and 5.2 mg L , respectively, exceeding
1
¨
that of the native product beauvericin. Although the same
+
precursor analogues also performed well in E. coli bbBeas ,
1
¨
6
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
1
¨
¨
1
7 S. Eichner, T. Knobloch, H. G. Floss, J. Fohrer, K. Harmrolfs,
J. Hermane, A. Schulz, F. Sasse, P. Spiteller, F. Taft and
A. Kirschning, Angew. Chem., Int. Ed., 2012, 51, 752–757.
1
1
2
2
8 Y. Xu, R. Orozco, E. M. K. Wijeratne, A. A. L. Gunatilaka,
S. P. Stock and I. Molna
9 S. C. Feifel, T. Schmiederer, T. Hornbogen, H. Berg, R. D.
Sussmuth and R. Zocher, ChemBioChem, 2007, 8, 1767–1770.
0 J. Muller, S. C. Feifel, T. Schmiederer, R. Zocher and
R. D. Sussmuth, ChemBioChem, 2009, 10, 323–328.
1 M. Krause, A. Lindemann, M. Glinski, T. Hornbogen, G. Bonse,
P. Jeschke, G. Thielking, W. Gau, H. Kleinkauf and R. Zocher,
J. Antibiot., 2001, 54, 797–804.
´
r, Chem. Biol., 2008, 15, 898–907.
¨
¨
¨
2
7
22 Y. Xu, J. Zhan, E. M. Wijeratne, A. M. Burns, A. A.
L. Gunatilaka and I. Molnar, J. Nat. Prod., 2007, 70, 1467–1471.
whole cell biocatalytic approaches 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
´
3 C. Nilanonta, M. Isaka, P. Kittakoop, S. Trakulnaleamsai,
2
M. Tanticharoen and Y. Thebtaranonth, Tetrahedron, 2002, 58,
3
355–3360.
2
2
2
4 H. Peeters, R. Zocher, N. Madry, P. B. Oelrichs, H. Kleinkauf and
G. Kraepelin, J. Antibiot., 1983, 36, 1762–1766.
5 R. Zocher, U. Keller and H. Kleinkauf, Biochemistry, 1982, 21,
43–48.
6 R. Zocher, U. Keller, H. Kleinkauf, in Peptide Antibiotics,
Biosynthesis and Functions, ed. H. Kleinkauf, H. v. Dohren, Walter
¨
de Gruyter & Co, Berlin, New York, 1982, pp. 269–274.
27 A. Kirschning and F. Hahn, Angew. Chem., 2012, 124, 2–13.
28 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed.,
28,29
ribosomally synthesized peptide antibiotics.
2001, 40, 2004–2021.
9 F. Oldach, R. Al-Toma, A. Kuthning, T. Caetano, S. Mendo,
N. Budisa and R. D. Sussmuth, Angew. Chem., Int. Ed., 2012, 51,
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.
2
¨
415–418.
5
676 Chem. Commun., 2012, 48, 5674–5676
This journal is c The Royal Society of Chemistry 2012