A carbonate-forming Baeyer-Villiger monooxygenase

Article abstract of DOI:10.1038/nchembio.1527

Despite the remarkable versatility displayed by flavin-dependent monooxygenases (FMOs) in natural product biosynthesis, one notably missing activity is the oxidative generation of carbonate functional groups. We describe a multifunctional Baeyer-Villiger monooxygenase, CcsB, which catalyzes the formation of an in-line carbonate in the macrocyclic portion of cytochalasin E. This study expands the repertoire of activities of FMOs and provides a possible synthetic strategy for transformation of ketones into carbonates.

Full text of DOI:10.1038/nchembio.1527

brief communication
puBLIsHed OnLIne: 18 MaY 2014 | dOI: 10.1038/nCHeMBIO.1527
a carbonate-forming Baeyer-Villiger monooxygenase
Youcai Hu1, david dietrich2, Wei Xu1, ashay patel3, Justin a J thuss2, Jingjing Wang1, Wen-Bing Yin1,4,
Kangjian Qiao1, K n Houk3, John C Vederas2* & Yi tang1,3*
Despite the remarkable versatility displayed by flavin-
The ccs biosynthetic gene cluster for 1 and 2 from A. clavatus is
dependent monooxygenases (FMOs) in natural product centered on a PKS-nonribosomal peptide synthetase CcsA13. CcsB
biosynthesis, one notably missing activity is the oxidative (ACLA_078650) is the only predicted FMO in the gene cluster, with
generation of carbonate functional groups. We describe a moderate sequence identity to well-characterized type I BVMOs7
multifunctional Baeyer-Villiger monooxygenase, CcsB, which (Supplementary Figs. 6 and 7). CcsB contains the conserved finger-
catalyzes the formation of an in-line carbonate in the macrocyclic print motif FXGXXXHXXXW14 and the strictly conserved active site
portion of cytochalasin E. This study expands the repertoire of arginine (Arg421) that stabilizes the Fl-4a-OO− anion through elec-
activities of FMOs and provides a possible synthetic strategy trostatic interactions15. The two remaining oxygenases encoded in the
for transformation of ketones into carbonates.
gene cluster, CcsG and CcsD, are both P450 monooxygenases and are
FMOs catalyze an enormous variety of substrate oxidations and possibly involved in the oxidation of other sites in 1, as reported for
reductions in both primary and secondary metabolism1. During the related chaetoglobosin16. We therefore propose CcsB is involved
natural product biosynthesis, following the assembly of the struc- in generation of the carbonate group in 1 starting from a ketone pre-
tural framework by upstream enzymes, FMOs have essential roles in cursor, via a mechanism previously not observed among BVMOs.
introducing structural complexity and biological activity2. FMOs are
To investigate the role of CcsB, we sought to inactivate the gene
versatile enzymes that can catalyze the formation of different types of ccsB in A. clavatus. First, the positive regulatory gene ccsR was over-
C-O bonds3–5; absent from the FMO product portfolio is the carbon- expressed in a Δlig4 modified A. clavatus strain to improve the titer of
ate moiety. To date, no enzymatic oxidation of a ketone or an ester to 1 and 2 (ref. 13). We also detected and structurally verified the pres-
the corresponding carbonate has been described, although there are ence of 5 in the culture extract (Fig. 2a and Supplementary Fig. 8),
abundant examples of oxidation of ketones to esters catalyzed by BV which is consistent with other co-isolation reports of 1 and 5 in
monooxygenases (BVMOs)6,7. Nonenzymatic transformation of an fungal strains that can produce 1 (ref. 17). Using the overproducing
ester to a carbonate is a similarly challenging synthetic transforma- strain (OE<ccsR, Δlig4), the ccsB gene was deleted, and one of the
tion. The difficulties in generation of the carbonate by synthetic and desired mutants (ΔccsB-37) was confirmed by PCR (Supplementary
enzymatic BV mechanisms are similar and include increased electron Fig. 9). Deletion of the ccsB gene abolished the production of 1, 2
density of the ester carbonyl that deters a second peroxide attack and and 5 and led to the production of 1,5 diketone–containing com-
the unlikelihood that the resulting Criegee complex will collapse via pound 7 (m/z 454[M + Na]+; Fig. 2a). The structure of 7 (named
C-C bond migration to form an additional C-O bond.
ketocytochalasin; Fig. 2b) was characterized by UV, MS and NMR
The carbonate functionality is therefore very rarely found techniques (Supplementary Note 1, Supplementary Table 1 and
in natural products8. Remarkably, several members of the cyto- Supplementary Figs. 10–13) and was confirmed by X-ray diffrac-
chalasin family of fungal natural products contain an in-line car- tion (Supplementary Fig. 12 and Supplementary Data Sets 2 and 4;
bonate moiety in the macrocycle portion of the molecules (Fig. 1 Cambridge Crystallographic Data Centre (CCDC) 970431). The
and Supplementary Results, Supplementary Fig. 1). Both abolishment of 1, 2 and 5 upon ccsB inactivation and the recovery
cytochalasin E (1) and cytochalasin K (2) are polyketides pro- of 7 strongly indicate that CcsB is the enzyme involved in the oxida-
duced by Aspergillus clavatus. Related compounds such as tion reactions at C21. The structure of 7 also points to a relatively
phenochalasin B (3)9 and scoparasin A (4)10 also contain a vinyl early action of CcsB in the tailoring of the cytochalasin scaffold en
carbonate. Other members of the large cytochalasin family are less route to 1 and 2. Additional modifications, such as C18 hydroxyla-
oxidized at the corresponding carbonate carbon than 1–4, including tion and C6-C7 epoxidation, should take place subsequent to the
esters such as rosellichalasin (5) and ketones, as seen in cytochalasin oxidative ring expansion steps (Supplementary Fig. 14).
G (6; Fig. 1 and Supplementary Figs. 2 and 3)11.
To examine whether CcsB can generate the ester or carbonate
To study the enzymatic basis of the carbonate group in 1, we first product starting from 7, recombinant CcsB was cloned, expressed
investigated the biosynthetic origin of the oxygen atoms in 1 by and purified to homogeneity from Escherichia coli (Supplementary
growing A. clavatus either in medium supplemented with sodium Fig. 15). CcsB was purified with a light yellow hue, and its UV spec-
[1-13C, 1-18O2]acetate or in a closed system in which consumed oxy- trum showed the characteristic absorption of FAD (Supplementary
gen was replaced by 18O2. A slight upfield shift (Δδc ~0.05 p.p.m.) for Fig. 16). LC/MS analysis of the supernatant of denatured protein
13C connected to 18O was used as an indicator of the source of oxy- confirmed the presence of FAD at ~10% CcsB concentration, which
gen atoms in 1 (ref. 12). Results showed that the carbonyl oxygen could be reconstituted with addition of FAD (Supplementary Figs. 16
at C21 of 1 is derived from acetate during polyketide assembly. In and 17). We then performed an in vitro reaction with CcsB and 7
contrast, both carbonate oxygen atoms attached to C21 are derived in the presence of NADPH, FAD and SsuE (which functions as an
from molecular oxygen, thereby pointing to an insertion pathway FMN reductase; Online Methods). Whereas the control reaction
catalyzed by an oxygenase (Supplementary Figs. 4 and 5).
without CcsB did not lead to new products, CcsB catalyzed the
1department of chemical and biomolecular engineering, university of california–los Angeles, los Angeles, california, uSA. 2department of
chemistry, university of Alberta, edmonton, Alberta, canada. 3department of chemistry and biochemistry, university of california–los Angeles,
los Angeles, california, uSA. 4present address: State Key laboratory of Mycology, institute of Microbiology, chinese Academy of Sciences, beijing,
china. *e-mail: john.vederas@ualberta.ca or yitang@ucla.edu
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1

NaTurE ChEMiCal BiOlOgy dOI: 10.1038/nCHeMBIO.1527
brief communication
12
O
weight of 8 by 1 Da (Supplementary Fig. 26). Under aqueous
conditions, conjugation of the β-diketo system is expected to be
favored for the ketone (8) over the ester (11). Indeed, density
OH
11
7
5
1
22
H
H
8
10
13
9
15
3
17
O
2
O
functional theory calculations (DFT; Supplementary Note 2)
indicate that 8 is roughly 6 kcal mol−1 (ΔG) more stable than 11
(Supplementary Fig. 27). With shorter reaction times and lower
NADPH concentrations, we did detect an unstable compound with
the same molecular weight and UV absorbance as 8 (Supplementary
Fig. 28). However, attempts to purify this compound resulted in
rapid conversion to 8, thereby indicating the possible identity
as 11 (Supplementary Fig. 29).
O
21
O
HN
O
HN
O
19
O
OH
O
OH
23
R
R
O
O
Cytochalasin E (1): R = H
Phenochalasin B (3): R = OMe
Cytochalasin K (2): R = H
Scoparasin A (4): R = OMe
O
O
H
H
O
O
HN
HN
Examination of known cytochalasin compounds revealed
that the vinylogous 1,5-diketo system is absolutely required for
carbonate formation (Supplementary Fig. 1). Only esters or
ketone compounds are found when such configuration is altered
(Supplementary Figs. 2 and 3). Our experiments here also sup-
port that both an α,β-unsaturated ester and an acidic proton in
the γ position (α to a distal carbonyl) are required for this unique
reactivity. Testing CcsB with numerous cytochalasin analogs lack-
ing one or more of these features, including cytochalasin B and
dehydrozygosporin19 (Supplementary Note 3, Supplementary
O
O
Cytochalasin G (6)
O
O
Rosellichalasin (5)
O
Figure 1 | Cytochalasins with different oxidation outcomes in the
macrocyclic portion. compounds 1–4 contain a vinyl carbonate group
of interest at c21 within the 13-membered macrocycle that is fused to an
isoindolone bicyclic scaffold. other members of the large cytochalasin family
are less oxidized at the corresponding carbonate carbon than 1–4, including
esters such as rosellichalasin (5) and ketones as in cytochalasin G (6).
conversion of 7 into two new products, 8 and 9, as evident in the Table 4 and Supplementary Fig. 30), did not lead to formation
time-course analysis (Fig. 3a). The masses of 8 (m/z 448 [M+H]+) of carbonate products (Supplementary Fig. 25). These obser-
and 9 (m/z 464 [M+H]+) are consistent with that of 7 containing one vations, combined with the lack of precedent for a second BV
and two additional oxygen atoms, respectively. Removal of NADPH oxidation of an ester moiety in both synthetic and biosynthetic
from the reaction abolished the conversion of 7. The ratio of 9 to literature, suggest alternative mechanisms to generate the car-
8 formed in the reaction strongly correlated to the molar ratio of bonate 9 (Supplementary Figs. 31–33). The first oxygen inser-
NADPH to 7 in the reaction (Supplementary Fig. 18).
tion step that oxidizes 7 to 11 follows that of the classic BVMO
Complete NMR and X-ray characterization (Supplementary mechanism and is in line with the rules of migratory aptitude.
Note 1, Supplementary Table 2, Supplementary Figs. 19 and 20 We propose that insertion of the second oxygen into 11 to form
and Supplementary Data Sets 1 and 3; CCDC 970432) of 9 the carbonate 9 could be initiated through a Michael addition
(Fig. 3b) verified the compound to indeed be the carbonate- from the peroxy-flavin anion (Fl-4a-OO−) on the β-carbon of
containing cytochalasin Z16 (ref. 18). The identity of 9 confirmed the vinyl ester to yield an α-β epoxide. (Supplementary Fig. 31).
that CcsB is the only enzyme required to convert the ketone 7 into Epoxide opening facilitated by abstraction of the C18 α-proton
carbonate 9, an unprecedented example in FMO chemistry. leads to an α-alcohol intermediate, which can form the epoxy
Mutation of the Fl-4a-OO−–stabilizing Arg421 to alanine alkoxide20 and readily rearrange across the 1,5-diketone system
(Supplementary Fig. 21) completely abolished the conversion of 7 to afford 9. This last step is analogous to the reported Lewis acid–
to 8 and 9 (Fig. 3a and Supplementary Fig. 15b), thereby confirm- promoted conversion of an α-hydroxy β-dicarbonyl compound
ing the catalytic role of CcsB in the reaction. Feeding of pure 9 to the to a carbonate21,22. Alternatively, direct addition of Fl-4a-OO− at
ΔccsB-37 strain led to the restored production of 1 and 2 (but not 5; C21 of 11 may also take place (Supplementary Figs. 32 and 33).
Fig. 3c and Supplementary Fig. 22), which verified that 9 is indeed Direct testing of these proposals, including trapping of the con-
an on-pathway intermediate in the ccs biosynthetic pathway.
jugated flavin intermediate or testing of molecules containing
Initially, we expected that 8 would be an ester corresponding to 11. similarly tuned functional groups, will be needed to conclusively
However, structural elucidation revealed a double bond shift to iso- define the mechanism.
precytochalasin (Fig. 3b, Supplementary Table 3, Supplementary
Sequence analysis of CcsB revealed that it is unremarkably
Figs. 23 and 24 and Supplementary Note 1). No conversion to 9 was grouped with well-characterized BVMOs (Supplementary Fig. 7).
observed when pure 8 was incubated with CcsB (Supplementary An intriguing question is therefore how intrinsically different
Fig. 25), suggesting that the β-γunsaturated ester
1
8 may be a shunt product formed from the spon-
taneous isomerization of the true intermediate
11 (Fig. 3b). Feeding of 8 to A. clavatus ΔccsB-37
strain led to the restored production of 5, along
with a new product cytochalasin Z17 (10)18
(Fig. 3c). Structurally, 5 and 10 represent the varia-
tions observed at C6-C7 in 1 and 2, respectively.
The simultaneous production of both 8 and 9
from the CcsB assay therefore rationalizes the
co-isolation of 5 and 1 in all fungal producers
(Supplementary Fig. 14).
a
b
UV: λ = 210 nm
2
i
5
m/z: 518.0 [M + Na]+
m/z: 486.0 [M + Na]+
m/z: 454.0 [M + Na]+
1
12
2
5
ii
11
7
5
1
22
H
10
15
19
9
1'
7
3
13
21
17
23
O
3'
iii
UV: λ = 210 nm
HN
O
5'
m/z: 518.0 [M + Na]+
m/z: 486.0 [M + Na]+
m/z: 454.0 [M + Na]+
O
7
Ketocytochalasin (7)
iv
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Time (min)
The proposed isomerization of 11 to yield
8 unveils a reactive vinylogous β-diketo–type
system facilitated through deprotonation of the
distal α-carbon C18, followed by C20 proton
abstraction from solution. As expected, when
the CcsB reaction was performed in D2O buf-
fers, we observed the increase in the molecular
Figure 2 | genetic confirmation of CcsB activity. (a) traces i and iii show Hplc analysis
(λ = 210 nm) of metabolites extracted from the oe<ccsR Δlig4 strain and from ΔccsB-37
mutant, respectively. traces ii and iv show selected ion monitoring of m/z 518 (1 and 2),
486 (5) and 454 (7) of traces i and iii, respectively. All of the m/z values are [M + na]+.
the metabolites are extracted from the day 4 cultures. the experiments were performed
three times, and representative results are shown here. (b) chemical structure of 7.
2
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NaTurE ChEMiCal BiOlOgy dOI: 10.1038/nCHeMBIO.1527
brief communication
a
c
7
2
1
10
5
Figure 3 | reactions catalyzed by CcsB and
7
8
9
i
chemical complementation of DccsB-37.
no CcsB
i
(a) Hplc analysis of products of the ccsb reaction
(λ = 210 nm). In vitro reaction conditions are
50 mM potassium phosphate buffer (pH 7.0),
4 mM nAdpH, 20 μM FAd, 6 μM Ssue,
ii
CcsB + 7 (3 h)
CcsB + 7 (7 h)
ii
iii
iv
v
iii
CcsB + 7 (11 h)
iv
10 μM ccsb and 0.4 mM 7. trace i shows control
reaction without ccsb, traces ii–v show time
course analysis of the conversion of ketone 7
to ester 8 and carbonate 9, trace vi shows the
reaction with the ccsb mutant R421A, and trace
vii shows the hexane extract of E. coli used for
biotransformation of 7 to 8 and 9. (b) chemical
structures of compounds 8–11. compound 11
was not isolated in this study because of its
isomerization to 8. (c) Hplc analysis (λ = 210 nm)
of extract from the chemical complementation of
8 and 9 to A. clavatus ΔccsB-37. traces i–iv show
standards of 1, 5, 7 and 10; trace v shows
∆ccsB-37
CcsB + 7 (24 h)
v
∆ccsB-37 + 9
vi
vii
CcsB R421A + 7 (24 h)
vi
vii
BL21 (DE3)/pYC01 + 7
∆ccsB-37 + 8
32.0
33.0
34.0
Time (min)
35.0
36.0
37.0
22.5
25.0
27.5
30.0
Time (min)
32.5
35.0
b
6
OH
7
H
H
H
H
14
16
17
13
21
O
O
O
O
O
O
O
O
HN
O
HN
HN
HN
19
18
O
O
20
O
O
O
O
O
O
Cytochalasin Z16 (9)
Cytochalasin Z17 (10)
Iso-precytochalasin (8)
Precytochalasin (11, not isolated)
A. clavatus ΔccsB-37; and trace vi and vii show A. clavatus ΔccsB-37 complemented with 9 or 8 in growth medium, respectively. 9 is a precursor to 1 and 2,
whereas 8 is a precursor to 5 and 10. All of the experiments were performed three times, and representative results are shown here.
CcsB is from the canonical BVMO in catalytic function. Close 11. Scherlach, K., Boettger, D., Remme, N. & Hertweck, C. Nat. Prod. Rep. 27,
869–886 (2010).
homologs of CcsB are widely found in sequenced fungal genome
12. Vederas, J.C. J. Am. Chem. Soc. 102, 374–376 (1980).
databases, with several highly homologous hits embedded in gene
13. Qiao, K., Chooi, Y.H. & Tang, Y. Metab. Eng. 13, 723–732 (2011).
clusters that resemble the ccs pathway. It is also expected that for
14. Mirza, I.A. et al. J. Am. Chem. Soc. 131, 8848–8854 (2009).
all of the ester-containing cytochalasins, such as cytochalasin B,
a CcsB-like enzyme must be present to perform the first BV oxida-
tion. Such activities were demonstrated in the classical feeding studies
of deoxaphomin to Phoma sp. and the recovery of cytochalasin B23.
We therefore propose that formation of the carbonate product by
CcsB is the fortuitous result of the perfectly arranged functional
groups near the ketone site in 7 and 11. The only comparable BVMO
examples are those that can convert an acyl-carrier protein–bound
thioester into a thiocarbonate for subsequent product release, such
15. Malito, E., Alfieri, A., Fraaije, M.W. & Mattevi, A. Proc. Natl. Acad. Sci. USA
101, 13157–13162 (2004).
16. Ishiuchi, K. et al. J. Am. Chem. Soc. 135, 7371–7377 (2013).
17. Kimura, Y., Nakajima, H. & Hamasaki, T. Agric. Biol. Chem. 53, 1699–1701 (1989).
18. Lin, Z.J. et al. Helv. Chim. Acta 92, 1538–1544 (2009).
19. Minato, H. & Matsumoto, M. J. Chem. Soc. Perkin 1 1, 38–45 (1970).
20. Rubin, M.B. & Inbar, S. J. Org. Chem. 53, 3355–3358 (1988).
21. Hong, X., Mejia-Oneto, J.M. & Padwa, A. Tetrahedr. Lett. 47, 8387–8390 (2006).
22. Mejía-Oneto, J.M. & Padwa, A. Helv. Chim. Acta 91, 285–302 (2008).
23. Robert, J.L. & Tamm, C. Helv. Chim. Acta 58, 2501–2504 (1975).
24. Frank, B. et al. J. Mol. Biol. 374, 24–38 (2007).
as that catalyzed by FR9H-Ox in the FR901464 pathway24,25
.
25. Tang, M.C., He, H.Y., Zhang, F. & Tang, G.L. ACS Catal. 3, 444–447 (2013).
In conclusion, we identified the enzymatic basis for form-
ing the in-line carbonate moiety in cytochalasins isolated from
fungi. The conversion of a ketone to a carbonate is unprecedented
in natural product biosynthesis and is a completely new type
of transformation discovered for the large, well-characterized
FMO family.
acknowledgments
This work was supported by the US National Institutes of Health (1R01GM085128 and
1DP1GM106413) to Y.T., the Natural Sciences and Engineering Research Council of
Canada and the Canada Research Chair in Bioorganic and Medicinal Chemistry to
J.C.V. and the US National Science Foundation (NSF) CHE-1059084 to K.N.H. A.P.
thanks the Chemistry Biology Interface program (T32GM008496) for support. NMR
instrumentation was supported by the NSF equipment grant CHE-1048804. We thank
Y.-H. Chooi and N.K. Garg for helpful discussions. We thank S.I. Khan at the University
of California–Los Angeles Department of Chemistry and Biochemistry crystallography
facility for solving the X-ray structures. We thank D. Li at Chinese Ocean University
for providing the standards for 5, 9 and 10. We acknowledge the Extreme Science and
Engineering Design Environment program (TG-CHE040013N) for providing high-
performance computing resources.
received 8 november 2013; accepted 12 april 2014;
published online 18 may 2014
Methods
Methods and any associated references are available in the online
version of the paper.
Accession codes. CCDC: Crystallographic data of 7 and 9 were
allocated under deposition numbers CCDC 970431 and CCDC
970432, respectively.
author contributions
Y.H., D.D., J.C.V. and Y.T. developed the hypothesis and designed the study. D.D.
performed the isotopic labeling studies in A. clavatus, and J.A.J.T. and D.D. prepared
selected substrate analogs. Y.H. performed the compound isolation and characterization.
Y.H. performed the in vitro analysis of CcsB functions. A.P. and K.N.H. performed and
interpreted the density functional theory calculations. All of the authors analyzed and
discussed the results. Y.H., J.C.V. and Y.T. prepared the manuscript.
references
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additional information
Supplementary information and chemical compound information is available in the
online version of the paper. Reprints and permissions information is available online at
http://www.nature.com/reprints/index.html. Correspondence and requests for materials
should be addressed to J.C.V. or Y.T.
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3

ONliNE METhODS
KH2PO4 1.52 g/l, MgSO4·7H2O 0.52 g/l, glucose 10 g/l, sorbitol 218.6 g/l and
agar 18 g/l, supplemented with a mixture comprising ZnSO4·7H2O 22.0 mg/l,
H3BO3 11.0 mg/l, MnCl2·4H2O 5 mg/l, FeSO4·7H2O 5 mg/l, CoCl2·5H2O
1.6 mg/l, CuSO4·7H2O 1.6 mg/l, (NH4)6Mo7O24·4H2O 1.1 mg/l and Na2EDTA
50.0 mg/l and adjusted to pH 6.5 with 1 N KOH) with a suitable selection
agent and overlaid with an SMMT soft agar medium. The transformants were
transferred to fresh SMMT agar plates and grown at 25 °C for another 7 d. For
selection of transformants, zeocin (200 μg/ml), hygromycin (100 μg/ml) or
glufosinate (8 mg/ml) was used as the selection marker for ccsR overexpression
integration, lig4 knockout or ccsB knockout, respectively. Correct insertion of
the bar marker in the ccsB gene among transformants was screened using PCR,
as shown in Supplementary Figure 9. ΔccsB-37 was found to be the correct
mutant (Supplementary Table 6).
Strains and culture condition. The A. clavatus strain was obtained from
Agriculture Research Service (NRRL 1) Culture Collection and was used as
the parental strain in our study. Both the wild-type and the mutant strains
were grown on malt extract peptone agar (MEPA) (30 g/l malt extract, 3 g/l
papaic digest of soybean meal and 15 g/l agar) at 25 °C. E. coli strain XL1-Blue
(Stratagene) and E. coli TOPO10 (Invitrogen) were used for cloning. E. coli
strain BL21(DE3) (Novagen) was used for protein expression.
DNA manipulation and construction of plasmids. Genomic DNA of
A. clavatus NRRL1 was isolated from mycelium grown in stationary liquid
culture supplied with malt extract peptone for 48 h at 25 °C. The mycelia
collected were lyophilized overnight and were ground to a fine powder using
toothpicks. Then, 700 μl LETS buffer (10 mM Tris-HCl, pH 8.0, 20 mM EDTA,
0.5% SDS, 0.1 M HCl) was added, and the samples were mixed by using the
toothpick as well as by inverting the tubes several times before leaving the sam-
ple on the bench for 5 min. After that, 700 μl of phenol/chloroform/isoamyl
alcohol (25:24:1) was added and mixed by inverting 10–15 times before centri-
fuging at 13,200 r.p.m. for 10 min. The supernatant was then transferred into a
new 1.5-μl centrifuge tube and an equal volume of phenol/chloroform/isoamyl
alcohol (25:24:1) was added and mixed by inverting 10–15 times. The mixture
was centrifuged at 13,200 r.p.m. for 10 min, and the supernatant was then
transferred into a new 1.5-μl centrifuge tube and 1 ml 95% ice-cold EtOH was
added. DNA was pelleted by centrifugation at 4 °C (13,200 r.p.m., 10 min), the
supernatant was removed and the pellet was washed twice with ice-cold 70%
ethanol, centrifuging briefly and discarding supernatant after each wash. The
resulting pellet was allowed to air dry for 10 min before resuspending in 40 μl
of 10 mM Tris buffer (pH 8.0). 0.5 μl RNase (10 mg/ml stock) was added,
and the mixture was incubated at 50 °C for 30 min before using for molecular
cloning. Platinum Pfx DNA polymerase (Invitrogen) was used to perform PCR
reactions from genomic DNA. The sequences of PCR products were confirmed
by DNA sequencing (Retrogen, CA). The primers for cloning ccsB and for the
construction of knockout cassettes are shown in Supplementary Table 4. The
gene encoding ccsB was amplified and inserted into pET23 to yield pYC01.
The ccsB gene was fused to an N-terminal Flag peptide for protein purifica-
tion. The first 36 residues at the N terminus of CcsB were removed in this
construct as these were predicted to be a membrane signal peptide. To perform
site-directed mutagenesis of ccsB, PCR-directed mutagenesis was used. To
construct the knockout cassette for ccsB, the selection marker bar gene with
the trpC promoter was amplified from the plasmid pBARKS1. This marker
was then flanked by two fragments of ccsB using fusion PCR and inserted into
the pCR-Blunt (Invitrogen) vector. PCR was then used to amplify up to 10 μg
of the entire knockout cassette for fungal transformation.
Protein expression and purification. The expression plasmid for CcsB pYC01
(Supplementary Table 7) was transformed into E. coli strain BL21 (DE3) for
expression of CcsB. Cells in LB medium (1 l) supplemented with ampicillin
(100 mg/l) inoculated with BL21(DE3)/pYC-1 were grown to an OD600 of
0.6. Protein expression was then induced with 0.12 mM of isopropylthio-
β-D-galactoside (IPTG, Sigma-Aldrich), followed by further incubation
with shaking at 250 r.p.m. at 16 °C for 16 h. All of the enzyme purification
steps were conducted at 4 °C. E. coli cells were harvested by centrifugation
(3,750 r.p.m., 15 min, 4 °C), resuspended in 20 ml TBS buffer and lysed with
sonication on ice. Cellular debris was removed by centrifugation (14,000g,
0.5 h, 4 °C). Flag-tagged proteins were purified by using Anti-Flag M1 Agarose
Affinity Gel (Sigma-Aldrich), following the supplied protocols. The cleared
cell lysate was applied onto a gravity flow column with packed anti-Flag
Agarose Affinity Gel. After extensive washing steps, CcsB was eluted with the
Flag peptide elution buffer (100 μg/ml Flag peptide, 50 mM Tris-HCl, pH 7.4,
100 mM NaCl). Purified proteins were concentrated, buffer exchanged into
50 mM potassium phosphate buffer (pH 7.0) plus 20% glycerol, concentrated,
aliquoted and flash frozen. Protein concentrations were determined using the
Bradford dye-binding assay (Bio-Rad).
In vitro activity test for CcsB. CcsB activity was assayed by monitoring the
conversion of substrates into products as analyzed by LC/MS. A typical 100-μl
assay solution contained 50 mM potassium phosphate buffer (pH 7.0), 4 mM
NADPH (Sigma-Aldrich), 20 μM FAD (Sigma-Aldrich), 6 μM SsuE and 10 μM
CcsB. The in vitro reaction was initiated by adding 0.4 mM of substrate, such
as rosellichalasin (5), cytochalasin B (6; Sigma-Aldrich), ketocytochalasin (7),
iso-precytochalasin (8) or cytochalasin D derivative (12). The reactions were
performed at 25 °C for 3 h, 7 h, 11 h and 24 h. At each time point, the reac-
tion was terminated by the addition of an equal volume of MeOH. Protein
precipitate was removed by centrifugation. The substrates and products were
extracted with 200 μl hexane twice, dried and redissolved in 60 μl methanol.
A 20-μl sample was then analyzed by LC/MS. LC/MS was conducted with a
Shimadzu 2010 EV liquid chromatography mass spectrometer by using both
positive and negative electrospray ionization and a Phenomenex Luna 5 μm,
2.0 mm × 100 mm C18 reverse-phase column. Samples were separated on a
linear gradient of 5% to 95% acetonitrile (CH3CN) (v/v) in H2O at a flow rate
of 0.1 ml/min. To examine the effect of the relative concentrations of NADPH
and 7 on the distribution of products 8 and 9, separate assays containing 7
(0.4 mM) and NADPH of varying concentrations (3.2 mM, 2.0 mM, 1.2 mM,
0.8 mM, 0.4 mM and 0.2 mM) were performed and analyzed. The products
were extracted and monitored by LC/MS after 1 h, 3 h, 7 h and 12 h. To per-
form the reaction in D2O, all of the buffers and stock solutions were prepared
with D2O. The remaining conditions were kept the same as above.
Fungal transformation and genetic manipulation in A. clavatus. Preparation
of A. clavatus protoplasts and polyethylene glycol–mediated transformation
of A. clavatus was performed as described previously26 with some modifica-
tion. In detail, A. clavatus spores were collected from culture grown for 2–3 d
and inoculated in 50 ml sterile liquid minimal medium and incubated at 28 °C
(280 r.p.m.) for 14 h. The culture was harvested by centrifuging at 4 °C
(3,750 r.p.m.) for 10 min. The supernatant was decanted, and the aggregated
germilings were transferred to a 250-ml flask containing 3 mg/ml lysing
enzymes (Sigma-Aldrich) and 2 mg/ml yatalase (Sigma-Aldrich) in 12 ml
of osmotic medium (1.2 M MgSO4, 10 mM sodium phosphate buffer) and
incubated at 25 °C (80 r.p.m.) for 12 h. Cells were poured directly in a sterile
30-ml glass Corex tube and overlaid very gently with 10 ml of trapping
buffer (0.6 M sorbitol, 0.1 M Tris-HCI, pH 7.0) before centrifuging at 4 °C
(5,000 r.p.m.) for 15 min. Protoplasts at the buffer interface were removed
with a pipette and diluted with 1 volume of STC buffer (1.2 M sorbitol/10 mM
Tris HCl, pH 7.5/10 mM CaCl2) before centrifuging at 4 °C (6,000 r.p.m.) for
5 min. The supernatant was then decanted, and the protoplasts were diluted
with 1 volume of STC buffer before using for transformation. Then 100 μl of
PEG solution at pH 8.0 (400 mg/ml polyethylene glycol 4000, 50 mM calcium
chloride and 10 mM Tris-HCl) was added to the protoplast suspension. The
mixture was subsequently combined with 10 μg of the DNA fragment with
which the cells were to be transformed. The mixture was incubated at 4 °C for
20 min to allow the transformation to proceed. After incubation on ice, 1 ml
of the PEG solution was added to the reaction mixture, and the mixture was
incubated at room temperature for an additional 5 min. The resulting cells
were plated on a SMMT agar medium ((NH4)2C4H4O6 1.84 g/l, KCl 0.52 g/l,
Chemical analysis and characterization of compounds from A. clavatus and
A. clavatus DccsB-37 mutant. For small-scale analysis, A. clavatus strains were
grown in stationary liquid surface culture as a surface mat on 100 × 15 mm2
Petri dishes with 15-ml malt extract peptone (MEP) medium for 4 d at 25 °C.
The cultures were extracted with the same volume of ethyl acetate (EtOAc)
and evaporated to dryness. The dried extract was dissolved in methanol and
analyzed by LC/MS (for conditions, see above). To purify intermediate 7,
A. clavatus ΔccsB-37 mutant strain was grown in stationary liquid surface cul-
ture condition in 4 l MEP liquid medium divided into 120 large 150 × 15 mm2
Petri dishes for 5 d at 25 °C. The resulting mycelia were extracted two
times with equal volumes of acetone, whereas the culture medium was treated
with XAD16 resin. Absorbed compounds on XAD16 were eluted by washing
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doi:10.1038/nchembio.1527

with acetone. The organic extracts were combined and evaporated by Rotovap.
The dried extract was partitioned between methanol and hexane. The hexane
Small needles of 9 were obtained from MeOH/CHCl3 (10:1) by slow evapora-
tion with an initial concentration of 1 mg/ml. Iso-precytochalasin (8) was a
1
fraction was evaporated to dryness and purified by ISCO-CombiFlash Rf 200 white solid with [α]D +83° (c 0.2, CHCl3); H NMR (500 MHz, CDCl3) and
(Teledyne Isco, Inc.) with a gradient of hexane and acetone (linear gradient
13C NMR (125 MHz, CDCl3); (Supplementary Table 3 and attached spectra).
of 0% to 30% acetone over 30 min at 10 ml/min) on normal phase silica gel. ESI-MS m/z 448 [M + H]+ and 470 [M + Na]+. HRESIMS m/z 448.24674
The fractions containing 7 were combined and further purified by reverse- [M + H]+ (calcd for 448.24878, C28H34NO4,); 446.23307[M - H]- (calcd for
phase HPLC (Phenyl-Hexyl 5μm, 250 × 10 mm) on a linear gradient of 60%
446.23368, C28H32NO4,). Cytochalasin Z16 (9) was a colorless needle with
[α]D +37° (c 0.8, CHCl3); 1H NMR (500 MHz, CDCl3) and 13C NMR(125 MHz,
to 90% CH3CN (v/v, free of acid) over 30 min at a flow rate of 2.5 ml/min to
1
give a pure white solid (7, tR = 16.2 min, 10 mg/12 l). H, 13C and 2D NMR CDCl3) (Supplementary Table 2 and attached spectra). ESI-MS m/z 464
spectra were obtained using CDCl3 as solvent on Bruker AV500 spectrometer
with a 5-mm dual cryoprobe at the UCLA Molecular Instrumentation Center.
Pure 7 was crystallized from MeOH/CHCl3 (5:1) with an initial concentra-
tion of 2 mg/ml. Ketocytochalasin (7) is a colorless crystal with [α]D –148°
(c 1.2, CHCl3); 1H NMR (500 MHz, CDCl3) and 13C NMR(125 MHz, CDCl3)
(Supplementary Table 1 and attached spectra); ESI-MS m/z 432 [M + H]+ and
454 [M + Na]+. HRESIMS m/z 432.25183 [M + H]+ (for 432.25387, C28H34NO3);
430.23815[M - H]-(calcd. for 430.23877, C28H32NO3).
[M + H]+ and 486 [M + Na]+.
Isotopic labeling study: feeding with 1-13C, 1-18O2 acetate or 18O2 gas. To
test the origin of oxygen at C17 in cytochalasin E (1), doubly labeled acetate
(1-13C and 18O2) was fed to A. clavatus grown in potato-dextrose (PD) liquid
stationary cultures. Labeled 1 was isolated by extraction with EtOAc, purified
by SiO2 gel column chromatography (97-3, CH2Cl2-MeOH) and character-
ized by 1H and 13C-NMR. To confirm the molecular origin of oxygen at C17,
1 was isolated from A. clavatus grown in a closed system in which the oxygen
Biotransformation of 7 to yield 8 and 9. To obtain sufficient amount of 8 and 9 consumed was replaced by 18O2 (apparatus is shown in Supplementary
for structural characterization, in vivo biotransformation using E. coli that over- Fig. 4a). A. clavatus was inoculated in fresh PD medium (4 × 50 ml) and incu-
produces CcsB was performed. The E. coli BL21(DE3)/pYC01 strain was grown bated in a closed system at 30 °C. The closed system is connected via Tygon
in 0.5 l of LB medium supplemented with ampicillin (100 mg/l) to an OD600
=
tubing to a condensation trap, a stirring 3 M KOH trap (to remove excess CO2)
0.6 at 37 °C and 250 r.p.m. Expression was induced with 0.12 mM IPTG. and an aquarium pump (controlled by a Variac, 3–5 V). A pressure-equalizing
After induction, the culture was shaken for an additional 16 h at 16 °C and burette (containing 0.5 g/l CuSO4) was filled with either 16O2 or 18O2 to create
then concentrated 20-fold to 25 ml. To initiate the biotransformation, 2 mg/l a slightly over-pressurized system. As the oxygen was consumed, more was
of 7 was added to the concentrated culture, and the culture was shaken at added. In the first 24 h of growth, 50 ml residual 16O2 was consumed. In the
250 r.p.m. for an additional 24 h at 16 °C. To monitor the conversion of 7, 1-ml following 49 h, 1,130 ml 18O2 was consumed, and, finally, the system is purged
aliquots were removed at 3 h, 7 h, 13 h and 24 h and extracted with EtOAc for with 170 ml 16O2 in the final 14 h. Labeled 1 was isolated and characterized
LC/MS analysis. After 24 h, the culture was extracted with 25 ml of hexane by 1H and 13C-NMR.
twice, dried and redissolved in 1 ml of MeOH. The crude extract was then
purified by reverse-phase HPLC (phenyl-hexyl 5 μm, 250 × 10 mm) on a linear
gradient of 60% to 90% CH3CN (v/v, free of acid) over 30 min at a flow rate
of 2.5 ml/min, to give 8 (tR = 16.7 min, 0.3 mg) and 9 (tR = 17.7 min, 1.5 mg).
26. Yelton, M.M., Hamer, J.E. & Timberlake, W.E. Proc. Natl. Acad. Sci. USA 81,
1470–1474 (1984).
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doi:10.1038/nchembio.1527