Journal of the American Chemical Society
ARTICLE
molecular modeling, it exhibited improved in vitro neurite out-
growth activity in comparison to 1. We thus are exploring the
potential of this novel neuroregenerative agent as a new molec-
ular probe to investigate the detailed mechanism underlying neu-
roregeneration by neuroimmunophilin ligands. Significantly, our
procedure exemplifies a new combinatorial biosynthetic method
to modify structurally complex molecules such as 1 as an
alternative method to chemical synthesis and to create improved
biological agents.
from both 1- and 2-producing strains were analyzed by HPLC-ESI-MS/
MS as described (see the Supporting Information).33 Details regarding
three-dimensional modeling, docking, and molecular dynamics simula-
tion are described in the Supporting Information. The evaluation of
immunosuppressive and neurite outgrowth activities of 1 and its
analogues is also described in the Supporting Information.
Accession Codes. The 1 gene clusters of Streptomyces sp. ATCC
55098, Streptomyces sp. KCTC 11604BP, and S. kanamyceticus KCTC
9225 were deposited in GenBank under accession numbers HM116537,
HM116536, and HM116538, respectively.
’ EXPERIMENTAL SECTION
’ ASSOCIATED CONTENT
General. Bacterial strains, identification and sequencing of the
FK506 (1) biosynthetic gene clusters, culture conditions, and materials
are described in the Supporting Information.
Chemicals. The chemical synthesis and structure of allylmalonyl-
CoA (9), trans-2-pentenyl-CoA (11), 3-oxopentanoyl-SNAC (13), trans-
2-pentenyl-SNAC (14), pentanoyl-SNAC (15), allylmalonyl-SNAC
(16), propylmalonyl-SNAC (17), and 4-fluorocrotonic acid (22) are de-
scribed in the Supporting Information.
Construction of Plasmids and Mutants. tcs genes were in-
activated in the 1-producing strain Streptomyces sp. KCTC 11604BP by
in-frame deletion via double crossover homologous recombination.
Details regarding DNA isolation and 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 Tables S7 and S8).
S
Supporting Information. Isolation and sequencing of gene
b
clusters; deduced functions of ORFs; gene deletion experiments;
phylogenetic analyses; construction of plasmids and mutants;
synthesis of SNAC esters and their H and 13C NMR spectra;
1
1
synthesis of trans-2-pentenyl-CoA and its H NMR spectrum;
13C enrichment experiment; expression, purification, and analysis of
enzymes; analysis of intracellular acyl-CoAs; isolation of novel
analogues and their ESI-MS/MS and NMR (1H, 13C, COSY,
HMQC, HMBC, and 19F) spectra; in silico docking experiments;
and immunosuppressive and neurite outgrowth assay. This material
’ AUTHOR INFORMATION
Analysis of FK506 Congeners and Their Analogues. The 1-
related biosynthetic intermediates and their analogues, which were
generated by 1-producing Streptomyces sp. KCTC 11604BP, its deletion
mutants, and deletion mutants supplemented with the SNAC thioesters
13, 14, 15, 16, and 17, and a series of carboxylic acids, as well as 2-
producing S. hygroscopicus var. ascomyceticus ATCC 14891, were ex-
tracted with EtOAc from the fermentation broth (see the Supporting
Information), and then analyzed by HPLC-ESI-MS/MS as described.13
Samples were separated on an ACQUITY UPLC BEH C18 column (50ꢀ
2.1 mm, 1.7 μm; Waters) interfaced with a Waters/Micromass Quattro
micro/MS instrument tracing by MS/MS using a gradient of MeCN at a
flow rate of 0.2 mL/min over 50 min starting with 40% (v/v) aqueous
MeCN containing 10 mM ammonium acetate and 0.1% acetic acid.
Tracing was done by MS/MS operated in multiple reactions monitoring
mode choosing mass pairs specific for the selected analytes to detect the
transition from parent ion as an ammonium adduct to product ion: 821 >
576 for 1; 809 > 564 for 2; 795 > 550 for 12; 823 > 578 for 18; 837 > 592
for 23; 835 > 590 for 24; and 827 > 582 for 25. Three separate cultivations
and independent extractions were performed.
Corresponding Author
slim@genotech.co.kr; joonyoon@ewha.ac.kr
Author Contributions
OThese authors contributed equally.
’ ACKNOWLEDGMENT
This work was supported by the National Research Labora-
tory (NRL) program (R0A-2008-000-20030-0) through the
National Research Foundation of Korea (NRF), NRF grants
(2010-0001487, 2010-0017984, and 2009-0083522), the Advanced
Biomass R&D Center (ABC) (ABC-2010-0029800), the 21C
Frontier Microbial Genomics and Applications Center Program
(11-2008-16-001-00), Brain Korea 21 project funded by the
Ministry of Education, Science & Technology (MEST), and the
Marine and Extreme Genome Research Center Program of
the Ministry of Land, Transportation and Maritime Affairs, Republic
of Korea. B.S.M. gratefully acknowledges a grant from the National
Institutes of Health (CA127622). We thank the Korea Basic Science
Institute (KBSI) for recording 900 MHz NMR spectra.
Isolation and Structural Identification of FK506 Analo-
gues. Details regarding the isolation and characterization of new 1
analogues obtained by supplementing the tcsB deletion mutant with
three different carboxylic acids (20, 21, and 22) are described in the
Supporting Information.
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