Journal of Natural Products
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m/z 790.1, 772.1, 754.0, 736.1, 576.0; (+)-HR-MS m/z 828.4326 [M
+ K]+ (calcd for C43H67KNO12, 828.4300).
In summary, we have discovered the parallel pathways
responsible for the postmodification step in the biosynthesis of
FK506. Our results clearly demonstrate that there are two
independent biosynthetic routes to FK506, and it has been
shown that 9-deoxoFK506 derivatives, 9-deoxo-31-O-deme-
thylFK506 (3) and 9-deoxoFK506 (4), can be used as
substrates for FkbD, whereas FkbM can utilize 31-O-demethyl
derivatives, 31-O-demethylFK506 (2) and 9-deoxo-31-O-
demethylFK506 (3), as substrates. These substrate-flexible
post-PKS modification enzymes, FkbD and FkbM, can provide
a potential tool for the combinatorial biosynthesis of novel
macrolide derivatives.
9-Deoxo-31-O-demethylFK506 (3): amorphous white powder;
1H and 13C NMR (see Table 1); (+)-ESIMS m/z 793.1 [M + NH4]+;
(+)-MS/MS m/z 776.1, 758.0, 740.0, 722.0, 561.9; (+)-HR-MS m/z
814.4510 [M + K]+ (calcd for C43H69KNO11, 814.4508).
1
9-DeoxoFK506 (4): amorphous white powder; H and 13C NMR
(see Table 1); (+)-ESIMS m/z 807.1 [M + NH4]+; (+)-MS/MS m/z
772.0, 754.0, 736.1, 561.9; (+)-HR-MS m/z 828.4677 [M + K]+ (calcd
for C44H71KNO11, 828.4664).
9-HydroxyFK506 (5): (+)-ESIMS m/z 823.3 [M + NH4]+;
(+)-MS/MS m/z 788.2, 770.2, 752.0, 578.0; (+)-HR-MS m/z
844.4616 [M + K]+ (calcd for C44H71KNO12, 844.4613).
9-Hydroxy-31-O-demethylFK506 (6): (+)-ESIMS m/z 809.3 [M
+ NH4]+; (+)-MS/MS m/z 792.1, 774.3, 756.2, 738.2, 578.0; (+)-HR-
MS m/z 830.4467 [M + K]+ (calcd for C43H69KNO12, 830.4457).
In Vitro Characterization of FkbD and FkbM. The details
regarding the preparation and purification of recombinant FkbD and
FkbM are described in the Supporting Information. Recombinant
FkbD (1 mM) was incubated with 9-deoxo-31-O-demethylFK506 (3)
or 9-deoxoFK506 (4) in reaction buffer (50 mM sodium phosphate,
pH 7.4, 1 mM NADPH, 50 μg/mL ferredoxin, 0.1 U of ferredoxin
reductase) at 30 °C for 1 h or 3 h. FkbM (1 mM) was incubated with
31-O-demethylFK506 (2) or 9-deoxo-31-O-demethylFK506 (3) in
reaction buffer (50 mM potassium phosphate buffer, pH 7.4, 5 mM
SAM, 5 mM MgSO4) at 30 °C for 3 h. Reactions were monitored
using HPLC−ESI-MS/MS analysis, as previously described.15
EXPERIMENTAL SECTION
■
General Experimental Procedures. NMR spectra were acquired
using a Varian INOVA 500 spectrometer operating at 500 MHz for 1H
and 125 MHz for 13C nuclei. Chemical shifts are given in ppm using
tetramethylsilane (TMS) as an internal reference. All NMR data
processing was done using Mnova software (Mestrelab Research S.L.).
Samples for NMR analysis were prepared by dissolving each
compound in 250 μL of CDCl3 (Sigma) and placing the solutions
in 5 mm Shigemi advanced NMR microtubes (Sigma) matched to the
solvent. HPLC−ESI-MS/MS spectra using an ACQUITY UPLC BEH
C18 column (50 × 2.1 mm, 1.7 μm; Waters) column were recorded on
a Waters/Micromass Quattro micro MS interface consisting of a
Waters 2695 separation module connected directly to a Micromass
Quattro micro MS. The HR-MS data was obtained using a Waters
SYNAPT G2-S mass spectrometer coupled with UPLC. HPLC
purification was performed using preparative Spherisorb S5 ODS2
(Waters, 250 × 20 mm, 5 μm) and semipreparative Watchers 120
ODS-BP (250 × 10 mm, 5 μm) columns on an Acme 9000 HPLC
system (YL Instrument Co. Ltd., Korea) consisting of a SP930D
gradient pump coupled with a UV730D UV detector set to 205 nm
and a CTS30 column oven set to 50 °C.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details; tables for bacterial strains, plasmids, and
primers used in this study; SDS−PAGE analysis of purified
proteins; and ESI-MS/MS, 1D NMR, and 2D NMR spectra of
1−6. This material is available free of charge via the Internet at
Construction of Mutants and Culture Conditions. The fkbD
and fkbM genes were inactivated in the FK506-producing strain
Streptomyces sp. KCTC11604BP by in-frame deletion or gene
replacement with an apramycin-resistance gene via double crossover
homologous recombination. Details regarding DNA manipulation and
construction of plasmids for gene deletion and heterologous
expression as well as the resulting mutant strains are described in
the Supporting Information (see also Table S1). Spores of Streptomyces
sp. KCTC 11604BP and its gene deletion mutants were generated on
ISP4 agar plates, and a seed culture was prepared in R2YE24 broth. 50
mg of vegetative cells grown in the seed culture were inoculated into
250 mL baffled flasks containing 50 mL of R2YE medium and
cultivated on an orbital shaker (set at 180 rpm) for 6 days at 28 °C.
Escherichia coli BL21(DE3) and E. coli BL21(DE3)pLysS (Novagen)
were used as heterologous hosts for the expression of recombinant
FkbD and FkbM. The E. coli strains were grown in LB liquid medium.
Ampicillin (100 μg/mL), apramycin (50 μg/mL), and kanamycin (50
μg/mL) were selectively added to the growth media as required.
Extraction and Isolation. Wild-type strain Streptomyces sp.
KCTC11604BP and mutant strains were cultured in production
media and processed separately. The individual culture broths of each
strain were centrifuged, and the supernatant was subjected to solvent−
solvent partition with ethyl acetate. The ethyl acetate extracts were
evaporated, and the resultant brown residues were either separated by
open column chromatography or directly injected to preparative
reversed-phase HPLC to obtain fractions containing target com-
pounds. These partially separated fractions were subjected to
semipreparative reversed-phase HPLC to afford individual com-
pounds, which were again purified using semipreparative reversed-
phase HPLC to yield pure compounds. The details regarding the
isolation and purification of products obtained are described in the
Supporting Information.
AUTHOR INFORMATION
Corresponding Author
+82-2-3277-3419.
■
Author Contributions
‡Y.H.B. and P.B.S. contributed equally to this paper.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a grant from the National
Research Foundation (NRF), funded by the Korean govern-
ment (MEST) (20120006243 and 20120000650); the
Intelligent Synthetic Biology Center of the Global Frontier
Project, funded by the MEST (2012054879); and the Seoul
R&BD Program (ST110024).
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31-O-DemethylFK506 (2): amorphous white powder; 1H and 13
NMR (see Table 1); (+)-ESIMS m/z 807.1 [M + NH4]+; (+)-MS/MS
C
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