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Supplementary Information
Cloning of the biosynthetic gene cluster for naphthoxanthene antibiotic FD-594 from
Streptomyces sp. TA-0256*
Fumitaka Kudo, Takanori Yonezawa, Akiko Komatsubara, Kazutoshi Mizoue, Tadashi Eguchi
List of SI
Detail experimental procedure for identification of the pnx gene cluster
Enzymatic characterization of PnxGT2
Enzymatic characterization of PnxMT2
Preparation of DNA probe to identify the FD-594 biosynthetic gene cluster: Genomic DNA of
Streptomyces sp. TA-0256 was used as a template for the PCR amplification of
TDP-glucose-4,6-dehydratase (4,6-DH) gene and NDP-hexose-2,3-dehydratase (2,3-DH) gene. As a
result,
PCR
with
2,3-DH3:
5'-GAYGTSCTCCAGTCCGAGCA-3'
and
2,3-DH4C:
5'-GAASCGSCCGCCCTCCTCSGA-3' (S: a mixture of C and G; Y: a mixture of C and T) gave a
desired size (ca. 700 bp) of DNA amplification. PCR conditions were 25 cycles of 94°C for 30 s,
55°C for 30 s and 72°C for 30 s in all 10 l of reaction mixtures [1 l of Takara Ex Taq polymerase
buffer, 1.6 l of dNTP (2.5 mM each), 0.2 l of 2,3-DH3 (0.1 mM), 0.2 l of 2,3-DH4C (0.1 mM), 0.5
l of DMSO, 6.3 l of distilled water, 0.1 l of Takara Ex-Taq DNA polymerase (Takara Bio Inc.,
Ohtsu, Japan), and 0.1 l of template DNA (2 ng l-1 of chromosomal DNA)]. Amplified PCR
products were sub-cloned with T vector of pT7-blue (Takara Bio Inc.) to obtain plasmids containing a
partial sequence of 2,3-DH genes. DNA sequences of the cloned plasmids were analyzed using a
DNA sequencer (Long Reader 4200; Li-Cor Inc., NE, USA) and SequiTherm Excel™ II DNA
Sequencing Kit-LC (for 66-cm gels; Epicentre Biotechnologies, WI, USA) with M13 Fwd
(-29)/IRDye™ 800 and M13 Reverse (-29)/IRDye™ 800 (Li-Cor Inc.) according to the
manufacturer’s protocol.
Identification of the FD-594 biosynthetic gene (pnx) cluster: The chromosomal DNA of
Streptomyces sp. TA-0256 was partially digested with Sau3AI and the restriction enzyme was
denatured with the phenol-chloroform extraction. After ethanol precipitation, the digested DNA
fragments of the chromosomal DNA were treated with calf intestine alkaline phosphatase (CIAP) at
50°C; the CIAP was denatured with phenol–chloroform extraction. A cosmid vector pOJ446 was
digested separately with HpaI, and after the treatment of CIAP, followed by digestion with BamHI.
After the phenol–chloroform extraction and ethanol precipitation, the resulting vector DNA was
dissolved with the TE buffer. The digested pOJ446 and the partially digested chromosomal DNA
were ligated using a DNA ligation kit ver. 2 (Takara Bio Inc.) at 4°C overnight. After ethanol
precipitation, the ligated DNA was dissolved with the TE buffer. The resulting ligation mixture was
packaged into  phage, with subsequent phage transfection to E. coli XL1 Blue MRF’ using Gigapack
III XL Gold Packaging Extract (Stratagene, USA) according to the manufacturer’s protocol. The host
strain E. coli XL1 Blue MRF’ was cultured in 3 ml of LB containing 10 mM MgSO4 and 0.2% maltose
using OD600 0.5 – 1. The culture was centrifuged; the resulting wet cells were suspended in a 10 mM
MgSO4 up to OD600 1 for transfection. A cosmid library of 880 clones in E. coli grown on 50 g ml-1
of geneticin and 10 g ml-1 of tetracycline containing LB medium was screened using standard
colony hybridization with a Dig-labeled DNA probe for the 2,3-DH gene described above, which was
produced using a DIG DNA labeling kit (Roche, Mannheim, Germany). Hybridized spots were
detected with the DIG Nucleic Acid Detection Kit and NBT/BCIP solution (Roche) according to the
manufacturer’s protocol.
Consequently, three positive clones (c594A, c594B, and c594C) were obtained and the
corresponding cosmids were extracted using a standard protocol. Southern hybridization with the
same probe, and a partial sequence analysis indicated that these cosmids contained overlapped DNA
sequences including the 2,3-DH gene. Because cosmid c594B seemed to include a majority of a gene
cluster, c594B was sequenced randomly using a shotgun sequence method on double-stranded DNA
templates with more than ten-fold coverage and minimum three times each portion of the DNA
sequence (Shimadzu Biotech, Kyoto, Japan). To connect all contigs from the shotgun sequence
analysis, three parts of DNA fragments from c594B were cloned separately and sequenced on
double-stranded DNA templates using a Long Reader 4200. Furthermore, several DNA fragments
from c594A were sub-cloned into the plasmid vector LITMUS28 and sequenced. Results revealed a
43,026-bp of DNA sequence containing a type II PKS gene cluster. The ORFs were determined using
FramePlot analysis (http://www.nih.go.jp/~jun/cgi-bin/frameplot.pl) and a BLAST homology search
using the NCBI BLAST server. The determined DNA sequence data of the pnx gene cluster in
Streptomyces sp. TA-0256 has been deposited to the DDBJ databases under accession number
AB469194.
Enzymatic
characterization
of
PnxGT2:
5'-GGAAGGCATATGAGAATCCTCATGACG-3'
A
set
of
and
primers,
GT2F:
GT2R:
5'-GCTGCTCGGATCCGCGCCGGTGCGTGCC-3', was used to amplify pnxGT2. PCR conditions
were 30 cycles of 98°C for 10 s, 55°C for 5 s, 72°C for 90 s in 2 l of 5 × PrimeSTAR buffer, 0.8 l of
dNTP (2.5 mM each), 0.5 l of DMSO, 0.2 l of primer 1 (0.1 mM), 0.2 l of primer 2 (0.1 mM), 0.1
l of PrimeSTAR polymerase (Takara Bio Inc.), 1 l of template DNA (cosmid c594B, 0.1 ng l-1),
and 5.2 l of water. Amplified PCR products were sub-cloned into the EcoRV site of LITMUS28.
After confirmation of the DNA sequences, the desired plasmids were digested with appropriate
restriction enzymes and the resulting DNA fragments were inserted into the corresponding site of an
expression vector pColdI (Takara Bio Inc.) resulting pnxGT2/pColdI. The expression plasmid
pnxGT2/pColdI was introduced into E. coli BL21(DE3) by a standard chemical transformation. The
PnxGT2 needed to be co-expressed with molecular chaperones GroES and GroEL (pREP4-groESL)
in E. coli to obtain its soluble form.1 Then, the E. coli harboring pnxGT2/pColdI/pREP4-groESL was
cultured in LB medium with 50 g ml-1 of ampicillin and 30 g ml-1 of kanamycin at 37°C by OD600
0.7 and a final 0.2 mM isopropyl -D-thiogalactoside (IPTG) was then added for induction of
overexpression. The culture was continued at 24°C overnight; the cells were harvested by
centrifugation (8,000 rpm × 10 min).
The wet cells were suspended in 50 mM Tris buffer (pH 7.5) containing 10% glycerol, 1 m M
MgCl2, and 1 mM MnCl2 (buffer A), and were disrupted by sonication. After removal of the cell
debris by centrifugation at 15,000 rpm for 30 min at 4°C, the supernatant was applied directly to
His-bind resin (Novagen Inc., Darmstadt, Germany). The resin was washed with 20 mM of imidazole
and eluted using 200 mM of imidazole in buffer A. To remove imidazole, the purified fraction was
passed through a 5-ml desalting column (HiTrap; GE Healthcare, Buckinghamshire, UK) with buffer
A. Then the PnxGT2 containing solution was concentrated into ca. 1 ml by centrifugation (7,000 rpm
× 2 hr) with an ultrafiltration membrane (10,000 MW, Vivaspin 20; Sartorius AG, Gottingen,
Germany). The PnxGT2 concentration was estimated using Lowry methods with BSA as a standard.
Usually, 1 ml of pure PnxGT2 (8.2 mg ml-1, 1.2 mM) was obtained from 6.6 g of wet cells.
The FD-594 aglycon was prepared using acid hydrolysis of FD-594, according to procedures
described in a previous report.2 TDP-D-olivose was chemically synthesized according to the previous
method.3 The PnxGT2 enzymatic solution consisted of 0.5 mM of FD-594 aglycon, 2.0 mM of
TDP-olivose, 2.5% DMSO, and 70 M of pure PnxGT2 (total 100 l). PnxGT2 enzymatic reaction
was performed at 28°C for 24 hr; then the reaction products were extracted three times with 100 l of
ethyl acetate after acidification of the reaction mixture to pH 5.0. After removal of the solvent using a
centrifugal evaporator, the residue was dissolved with 100 l of methanol for HPLC (UV or PDA)
and LC-ESI-MS analysis.
Then HPLC was performed using a pump (L-7100 LC; Hitachi Ltd.) equipped with a UV
detector (L-7405; Hitachi Ltd.) and a chromato-integrator (D-2500; Hitachi Ltd.). Alternatively, we
used an intelligent pump instrument (L-6250; Hitachi Ltd.) equipped with a photodiode array (996;
Waters Corp.) that was controlled by a software (Empower 2; Waters Corp.). An aliquot of the
PnxGT2 reaction products (5 l) was injected into the HPLC systems equipped with a column (4.6 ×
250 mm, Pegasil ODS; Senshu, Tokyo Japan). The elution was made by 40% aqueous acetonitrile
containing 0.1% TFA for 10 min, followed by a linear gradient of 40% to 60% aqueous acetonitrile
containing 0.1% TFA for 10 min, and 100% acetonitrile containing 0.1% TFA for 10 min, with a flow
rate 1 ml min-1. The elution was monitored at 360 nm.
Subsequently, LC–ESIMS analysis was performed using a mass spectrometer (LCQ; Finnigan
MAT GmbH) coupled with Nanospace HPLC (Shiseido Co. Ltd.) equipped with a Nanospace SE-1
UV detector (Shiseido Co. Ltd.). An aliquot of the solution (2 l) was injected into the LC system
equipped with a column (RP-18 GP; Kanto Chemical Co. Inc.). The elution was made using 10%
aqueous acetonitrile for 10 min, followed by a linear gradient of 40% to 100% aqueous acetonitrile
for 30 min, and 100% acetonitrile for 10 min at a flow rate of 50 l min-1. The elution was monitored
at 360 nm. An ESI mass spectrometer was operated in negative-ion mode.
Enzymatic characterization of PnxMT2: pnxMT2 gene was amplified using a set of primers,
MT2-F:
5'-GGAGAGATCATATGACCGGACC-3'
and
MT2-R:
5'-CAGGGGGGTACCGACGCTC-3', according to the same PCR conditions as for the pnxGT2 gene.
Similarly, the desired DNA fragment of the pnxMT2 gene was inserted into pColdI. The resulting
expression plasmid pnxMT2/pColdI was introduced into E. coli BL21(DE3) for expression. The E.
coli harboring pnxMT2/pColdI was cultured in LB medium with 50 g ml-1 of ampicillin at 37°C by
OD600 0.7 and a final 0.2 mM IPTG was then added for induction of overexpression. The culture was
continued at 15°C overnight and the cells were harvested by centrifugation (8,000 rpm × 10 min). The
wet cells were suspended in 50 mM Tris buffer (pH 7.5) containing 10% glycerol and disrupted by
sonication. The PnxMT2 was purified using the same method as for PnxGT2. Usually, 1 ml of pure
PnxMT2 (8.8 mg ml-1, 0.31 mM) was obtained from 1.0 g of wet cells.
The PnxMT2 enzymatic reaction coupled with the PnxGT2 enzymatic reaction was conducted
as follows. The enzymatic solution consisted of 0.2 mM of FD-594 aglycon, 10 mM of TDP-olivose,
5 mM of SAM, 2% DMSO, 25 M of PnxGT2, and 90 M of PnxMT2 (total 100 l). The enzymatic
reaction was performed at 28°C for 14 hr. The analytical conditions for the enzymatic reaction were
identical to the method described above for the PnxGT2 enzymatic reaction. For the stepwise
enzymatic reaction analysis, the first reacted enzyme was heat-inactivated at 80°C for 5 min. The
PnxGT2 reaction was first conducted in 0.2 mM of FD-594 aglycon, 10 mM of TDP-olivose, 2%
DMSO, and 25 M of PnxGT2 (total 100 l) at 28°C for 18 hr. Then, the mixture was heat-treated at
80°C for 5 min; a final 3.3 mM of SAM and 75M of PnxMT2 were added to the mixture (total 150
l), and the reaction was continued for 24 hr.
1
Cole, P. A. Chaperone-assisted protein expression. Structure 4, 239-242 (1996).
2
Kondo, K., Eguchi, T., Kakinuma, K., Mizoue, K. & Qiao, Y. F. Structure and biosynthesis of
FD-594; a new antitumor antibiotic. J. Antibiot. 51, 288-295 (1998).
3
Minami, A. & Eguchi, T. Substrate flexibility of vicenisaminyltransferase VinC involved in
the biosynthesis of vicenistatin. J. Am. Chem. Soc. 129, 5102-5107, (2007).