Yi et al.
Concise Report
this kind of acyl transferase to determine their catalytic
mechanism (Figure S3), and only Cys/Ser-His was conserved in the
sequence alignment because their protein sequences were
obviously different with that of CerJ or FabD. Utilizing the
available proteomic information from the UniProt database, we
constructed the SSN and GNN of the KAS III-like acyl transferase
ChlB3 with the Enzyme Function Initiative-Enzyme Similarity Tool
(EFI-EST) and Genome Neighborhood Tool (Figure S4)[22]. More
than 1,500 biosynthetic gene clusters including possible KAS
III-like acyltransferases were found, and most of the secondary
and then coenzyme A (6.35 μmol, 1 eq) was dissolved in 0.5 mL of
H2O (O2 was removed by sonication) and added dropwise to the
reaction. After being stirred at room temperature for 2 hr, the
reaction mixture was directly purified by RP-HPLC (Venusil
XBP-C18 10.0 × 250 mm, 5 μm, 100 Å, 3ml/ml, 0-25 min 20%-60%
B, 25-29 min 60% B, 29-30 min 60%-20% B ; buffer A: 10 mM
NH4OAc in H2O; buffer B: CH3OH). These fractions containing
product were collected, and methanol was removed by
evaporation firstly, followed by the removal of water and NH4OAc
by lyophilization. More than 95% CoA was converted into its
metabolites from these gene clusters have not been excavated yet, derivative, and the mass of the product was confirmed by ESI-MS
form which possible molecular skeleton spanned polyketides,
non-ribosomal peptides and terpenes. Therefore, our exploration
will provide a reference for predicting the function of these
homologous proteins and discovering compounds with novel
structures.
[M-H]-or [M+H] +. For O-methylsalicyl-S-CoA, ESI-MS [M+H] +:cald.
902.2, found. 902.3; for 6-methyl-O-methylsalicyl-S-CoA, ESI-MS
[M-H]- cald. 914.2, found. 914.4.
Synthesis of CoA derivatives of salicyclic acid. Salicyclic acid
(100 μmol, 1.0 eq) in freshly distilled DMF (1 mL) was stirred with
PyBOP (1.5 eq), iPr2EtN (1.5 eq), and thiophenol (3.0 eq) under
argon. After being stirred at room temperature for 30 min, the
reaction mixture was directly purified by RP- HPLC column
(Venusil XBP-C18, 10.0 × 250 mm, 5 μm, 100 Å, 3ml/ml, 0-5 min
5%-70% B, 5-28 min 70%-100% B, 28-30 min 100%-5% B ; buffer A:
0.1% TFA in H2O; buffer B: 0.1% TFA in CH3CN). The solvent was
removed under reduced pressure to give the product of
salicyl-S-phenyl thioester. Then the salicyl-S-phenyl thioester
(60.8 μmol, 4.8 eq) and coenzyme A (12.7 μmol, 1.0 eq) were
stirred in phosphate buffer (8 mL, 50 mM, pH=8.5) under argon.
After being stirred at room temperature for 2 hr, the reaction
mixture was directly purified by reverse-phase HPLC (Venusil
XBP-C18, 10.0 × 250 mm, 5 μm, 100 Å, 3ml/ml, 0-25 min 20%-60%
B, 25-29 min 60% B, 29-30 min 60%-20% B ; buffer A: 10 mM
NH4OAc in H2O; buffer B: CH3OH). The fractions containing
product were pooled, and methanol was removed by evaporation
firstly, followed by the removal of water and NH4OAc by
lyophilization. More than 90% CoA was converted into its
derivative, and the mass of the product was confirmed by ESI-MS
[M-H]-: cald.888.1, found.888.2.
Conclusions
According to these data mentioned above, the biosynthesis
pathway of 1 should happen in the order showed in Scheme 1d.
Firstly ChlB1 synthesizes 6-methylsalicyl loaded on its ACP from
one acetyl CoA and three malonyl CoA. Secondly, ChlB3 transfers
6-methylsalicyl from 6-methylsalicyl-ChlB1 to holo-ChlB2. Thirdly,
ChlB6 immediately grafts 6-methylsalicyl from holo-ChlB2 to
DM-CHL, producing M-CHL. Finally ChlB4 and ChlB5 perform
chlorination and O-methylation on M-CHL to obtain DCM-CHL or
CHL. Manipulation of chain initiation has proven very useful for
generating novel products in vivo. However, manipulation of the
modified units is more powerful in increasing the structural
diversity of natural products at the biochemical level in vitro. And
our findings will provide a unique opportunity to understand
other similar functional modified enzymes and contribute to
generating new CHL analogs via combinatorial biosynthesis and
chemo-enzymatic synthesis.
Characterization of ChlB3 and ChlB6 activities on holo-ChlB2
and small molecule acyl-S-ChlB1-ACP. To generate the small
molecule acyl-S-ChlB1-ACP, the reaction was carried out in 75 mM
MOPS (pH 7.5), 10 mM MgCl2, 1 mM TCEP (pH 8.0), 100 μM
ChlB1-ACP, 150 μM indicated CoA derivatives, and 2 μM sfp for 1
hr at 30°C. To produce the holo-ChlB2, a similar reaction was
performed in 75 mM MOPS (pH 7.5),10 mM MgCl2, 1 mM TCEP
(pH 8.0), 300 μM ChlB2, 450 μM CoA, and 5 μM sfp for 1 hr at
30°C. For the transferring of small molecule acyl group, the
reaction harboring 75 mM MOPS (pH 7.5), 10 mM MgCl2, 1 mM
TCEP (pH 8.0), 30 μM indicated small molecule acyl-S-ChlB1-ACP
and 30 μM holo-ChlB2, was initiated by addition of 0.2 μM ChlB3
or ChlB6, and quenched with 0.25 volume 10% formic acid at
indicated time. The quenched reaction was directly analyzed by
reverse-phase HPLC (Vydac 218TP54 C18 , 250 × 4.6 mm, 5 μm,
300 Å, 1ml/min, 0-3 min 20% B, 3-5 min 20%-35% B, 5-25min
35%-55% B, 25-26 min 55%-99% B, 26-29 min 99% B, 29-30 min,
99%-20% B; buffer A: 0.1% TFA in H2O; buffer B: 0.1% TFA in
CH3CN).
Characterization of ChlB3 and ChlB6 activities on
desmethylsalicyl CHL (DM-CHL) and small molecule acyl-S-ChlB2.
To generate the small molecule acyl-S-ChlB2, the reaction was
carried out in 75 mM MOPS (pH 7.5), 10 mM MgCl2, 1 mM TCEP
(pH 8.0), 300 μM apo-ChlB2, 450 μM indicated CoA derivatives,
and 5 μM sfp for 1 hr at 30°C. The reaction containing 75 mM
MOPS (pH 7.5), 10mM MgCl2, 1 mM TCEP (pH 8.0), 1 mg/mL BSA,
5% DMSO,150 μM small molecule acyl-S-pantetheinyl ChlB2 and
150 μM DM-CHL, was initiated with 20.0 μM ChlB3 or ChlB6, and
quenched with 0.25 volume 10 % formic acid or 2 volume
methanol at indicated time. The reaction quenched with formic
acid was directly analyzed by reverse-phase HPLC (Vydac 218TP54
Experimental
Bacterial Strains, Plasmids, Biochemicals, and chemicals. E.
coli DH5α was used for general gene clone, and E. coli BL21 (DE3)
was used for protein expression (Novagen). The Chlorothricin
producer, S. antibioticus DSM40725 was purchased from DSMZ.
Cloning vectors pSP72 (Promega) and expression vectors pET28a,
pET37b (Novagen) and pMal-c2X (NEB) were originally from
commercial sources. HCoA was from Sigma, and all the other
common biochemicals and reagents came from standard
commercial sources.
DNA Isolation and Manipulation. DNA isolation and
manipulation in E. coli and Streptomyces were carried out
according to standard methods. PCR amplifications were carried
out on an Authorized Thermal Cycler (Eppendorf AG) using either
Taq DNA polymerase or PfuUltra High-Fidelity DNA polymerase.
Primer synthesis and DNA sequencing were performed at the
Shanghai Invitrogen Biotech Co., Ltd., and Chinese National
Human Genome Center.
Expression and purification of sfp, ChlB1-ACP, ChlB2, ChlB3,
and ChlB6. Sfp, ChlB1-ACP, ChlB2, ChlB3 and ChlB6 were
expressed and purified as we have reported previously.[15]
Synthesis of CoA derivatives of 5-chloro-6-methyl-O-
methylsalicyclic acid and 6-methyl-O-methylsalicyclic acid. CoA
derivatives of 5-chloro-6-methyl-O-methylsalicyclic acid and
6-methyl-O-methylsalicyclic acid were synthesized and purified as
we reported previously. [15]
Synthesis of CoA derivatives of O-methylsalicyclic acid.
6-methyl-O-methylsalicyclic acid or O-methylsalicyclic acid (10
μmol, 1.6 eq), PyBOP (10 μmol, 1.6 eq) and K2CO3 (25.4 μmol, 4.0
eq) were dissolved in 0.5 mL of freshly distilled THF under argon,
4
© 2019 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2019, 37, XXX-XXX
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