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A new shunt product of actinorhodin biosynthesis
Supplementary Information
Structure and biosynthetic implication of 5R-(N-acetyl-Lcysteinyl)-14S-hydroxy-dihydrokalafungin from a mutant of the
actVA-ORF4 gene for actinorhodin biosynthesis in Streptomyces
coelicolor A3(2)
Takaaki Taguchi1, Tomoki Maruyama1, Ryuichi Sawa2, Masayuki Igarashi2, Susumu
Okamoto3, * and Koji Ichinose1, *
1Research
Institute of Pharmaceutical Sciences, Musashino University, Shinmachi,
Nishitokyo-shi, Tokyo 202-8585, Japan,
2Institute
of Microbial Chemistry (BIKAKEN), Tokyo, Kamiosaki, Shinagawa-ku,
Tokyo 141-0021, Japan,
3National
Food Research Institute, Kannondai, Tsukuba 305-8642, Japan
Correspondence: Dr. S. Okamoto, National Food Research Institute, Kannondai, Tsukuba
305-8642, Japan and Prof. K. Ichinose, Research Institute of Pharmaceutical Sciences,
Musashino University, Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan.
E-mail addresses: [email protected] (S. Okamoto) [email protected] (K.
Ichinose)
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A new shunt product of actinorhodin biosynthesis
Construction of the ΔactVA-4 mutant
S. coelicolor A3(2) strain M510 (M145 DredD)1 was used as the wild-type strain. An
unmarked, in-frame deletion of actVA-ORF4 in M510 was constructed as follows. A
1.2-kb region 5’ of actVA-ORF4 was amplified by PCR with primers ΔactVA4_UP-F
(5’-TCGAGCTCGGTACCCATGGAACG-TACGCG-GTATCGAAGTG-3’)
and
ΔactVA4_UP-R (5’-GATATCCTTGTTCTCATCGG-GCATCG-3’). A second 1-kb
region 3’ of actVA-ORF4 was amplified with primers ΔactVA4_DOWN-F (5’GAGAACAAGGATATCCTCAACGGCATCGGCTATC-AC-3’) and ΔactVA4_DOWNR
(5’-CTCTAGAGGATCCCCAGCGTGTGCTCG-TACACCA-3’).
The
PCR
fragments were assembled and cloned into pUC18 (linearized with SmaI) using the InFusion cloning system (Takara Bio). Following sequence confirmation, the actVAORF4 in-frame deletion construct was shuttled into pGM160::oriT (NcoI-XbaI) to yield
pGMDactVA4. E. coli ET12567/pUZ8002 was transformed with the recombinant
plasmid and mated into S. coelicolor M510. Transformants were selected with
thiostrepton and purified by streaking onto thiostrepton-containing plates. To obtain
single crossover recombinants, purified transformants were cultured on thiostreptoncontaining plates at 37°C (pGM160::oriT carries a temperature-sensitive replicon
derived from pSG5 and cannot replicate at 37°C). Thiostrepton-resistant singlecrossover recombinants were subcultured by two rounds of streaking in the absence of
thiostrepton at 37°C to obtain double-crossover recombinants in which the delivery
plasmid was lost from the cells. Serial dilutions of the resulting spores were plated, and
the resulting colonies were tested for thiostrepton sensitivity. Colonies with a
thiostrepton-sensitive phenotype were selected, and the deletion of actVA-ORF4 was
confirmed by PCR with primers primer-F (5’-TGCGA-AGGCTGTCGGTGCTCA-3’)
and primer-R (5’-TACGAAGTGGGCGGCGAAAC-3’). The precise gene deletion was
further verified by DNA sequencing.
Reference:
1) Floriano, B. & Bibb, M. J. afsR is a pleiotropic but conditionally required for
regulatory gene for antibiotic production in Streptomyces coelicolor A3(2). Mol.
Microbiol., 21, 385-396 (1996).
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A new shunt product of actinorhodin biosynthesis
Figure S1. Construction of the deletion mutant of actVA-ORF4 (DactVA-4). (A) The
organizations of genes in the wild-type and ∆actVA-4 strains are shown. Arrows
indicate the position of the primers used for genotyping. (B) Agarose gel analysis of
PCR products generated from genomic DNA of selected S. coelicolor strains. The wildtype strain yields a 1.1-kbp PCR product when the prime-F and primer-R are used. The
DactVA-4 strains show a 0.37-kbp PCR product demonstrating the deletion of the
actVA-ORF4 gene.
A new shunt product of actinorhodin biosynthesis
A
B
Comp. Z
THK-OH
E
D
C
DHK-OH
OH O
O
O
O
O
O
5R,14S-epoxykalafungin
Figure S2. UV-VIS spectra of Comp. Z, 5R-AcCys-14S-OH-DHK (A), THK-OH (B),
DHK-OH (C) and 5R, 14S-epoxykalafungin. (D). (E) The structure of 5R,14S-epoxykalafungin.
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A new shunt product of actinorhodin biosynthesis
A
16
11-OH
15
8 10
9
*
14-OH
2’-NH
3
10
2’
3’ 3’
2 2
PPM
5
16
4b
4a
15
3
5
14-OH
10
PPM
B
Figure S3. (A) The Reuslts of 2D-ROESY
H-16
analysis. ROEs between 14-OH (δH 7.10)
and H-4α (δH 2.15) and between 14-OH and
H-3’
H-3
H-15
H-15 (δH 4.15) are shown by red lines. (B)
The 3D structure of 5R-AcCys-14S-OH-
H-4b
DHK constructed by employing Molecular
14-OH
Operating Environment (MOE) 2012.1001
H-4a
(Chemical Computing Group Inc., Canada).
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