SUPPLEMENTAL MATERIAL

General Procedure

All reagents were obtained from Sigma-Aldrich (USA) and were used without further

purification. Preparative RP-HPLC separation was performed on a Shimadzu LC-20AT system,

detected by a UV detector at 203 nm and equipped with a YMC semipreparative C18 column

(10 µm, 10 × 250 mm) running with a flow rate of 3 mL/min. Melting point was measured with

a Fisher-Johns micromelting point apparatus (USA). 1H, 13C, and 2D NMR spectra were

recorded on a Bruker AM-400 NMR spectrometer and a Advance DRX-500 NMR spectrometer

with TMS as internal standard. FAB-MS data were obtained on a VG AutoSpec 3000

spectrometers (Micromass, UK). UV spectra were obtained on a Shimadzu double-beam 210A

spectrophotometer. The IR (KBr) spectra were recorded on a Bio-Rad FTS-135 spectrometer.

Chemical shifts were reported as parts per million (δ), using the residual C5D5N as an internal

standard, and coupling constants (J) in hertz. 1H and 13C NMR assignments were supported by

1H-1H COSY, HMQC and HMBC experiments. The negative FAB-MS and HR-FAB-MS

spectra were collected with a VG AutoSpec 3000 spectrometers in m/z (rel. %). Optical rotation

was measured on a Horiba SEAP-300 sensitive polarimeter. The Strain 4234 sample was dried

in an Hitachi ZS-2030 critical point dryer and gold-coated using a Hitachi Z-1010 Sputtering

System, and observed by examining the gold-coated dehydrated specimens with a Hitachi S-

3000N scanning electron microscope.

Microorganism and Media

All strains used in this work (Aspergillus flavus, Aspergillus niger, Absidia orchidis,

Aspergillus oryzae, Bacillus cereus, Bacillus megeterium, Bacillus mycoides, Bacillus subtilis,

Bacillus thuringiensis, Candida albicans, Candida utilis, Escherichia coli, Geotrichum

candidum, Gibberella zeae, Helminthosporium sigmoideum Cav, Magnaporthe grisea, Mucor

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ssp, Mucor sufu, Penicillium citrinum, Penicillium notatum, Plantain head blight, Pseudomonas

slolanacearum, Rhizoctonia solani, Rhizopus nigricans, Saccharomyces cerevisiae, Shigella

flexneri, Staphylococcus aureus, Streptomyces sp. JAU4234, Trichoderma viride,

Ustilaginoidea virens, Xanthomonas citri, Xanthomonas oryzae) were deposited at College of

Biological Science and Engineering, Jiangxi Agriculture University, China.

All cultures of bacteria were grown on LB agar (5 g of yeast extract, 10 g of tryptone, 10 g of

NaCl, and 20 g of agar in 1 liter of water). Strain JAU4234 and Fungi were used PDA agar (200

g of potato infusion, 20 g of glucose, and 20 g of agar in 1 liter of water). Yeast were

maintained on YPD agar (10 g of yeast extract, 20 g of peptone, 20 g of glucose, and 20 g of

agar in 1 liter of water). Seed medium of strain JAU4234 consisted of 10 g cornstarch, 30 g

maize meal, 10 g soybean meal, 0.5 g (NH4)2SO4, 6 g CaCO3 and 2 g bean oil in 1 liter of water,

adjusted to pH 8.0. Fermentation medium of strain JAU4234 was 20 g cornstarch, 20 g maize

meal, 10 g soybean meal, 20 glucose, 2.5 g (NH4)2SO4, 2.5 g KNO3, 0.3 g KH2PO4, 3 g NaCl, 6

g CaCO3 and 5 g bean oil in 1 liter of water, adjusted to pH 6.5 (5). A colony of strain JAU4234

grown on plate was picked up and transferred into a 500 ml flask containing 100 ml of seed

culture medium, and then incubated at 30±0.5°C and 220 rpm for ~24 h. The 500 liter

fermentor (model GUJS500C; EASTBIO, Zhenjiang, China) containing 200 liter of

fermentation medium was sterilized at 121°C for 20 min and inoculated with 5% (v/v) seed

cultures of strain JAU4234. Fermentation temperature was maintained at 30±0.5 °C at an

agitation speed of 600 rpm and an air flow rate of 1±0.2 vvm. Dissolved oxygen tension was

measured using an autoclavable O2 sensor (Mettler Toledo, Greifensee, Switzerland).

Sequencing and phylogenetic analysis of 16S rDNA

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Genomic DNA of strain JAU4234 was prepared using AxyPrep bacterial genomic DNA

Miniprep kit (Axygen biotechnology Ltd., Hangzhou, China). The 16S rDNA was amplified by

PCR using forward primer 9–27f (5’-GAGTTTGATCCTGGCTCAG-3’) and reverse primer

1541–1525r (5’-AAGGAGGTGATCCAGCC-3’) (1). The PCR product (~1.5 kb) was directly

sequenced by BGI-Shanghai (Shanghai, China) after purification by AxyPrep gel extraction kit

(Axygen biotechnology Ltd.). CLUSTAL X software (version 2.0, Conway Institute, USA) was

used to generate alignment of strain JAU4234 and other members of the genus Streptomyces

(2). Phylogenetic analysis was carried out by the neighbor-joining method using MEGA

software (version 4.0, Center for Evolutionary Medicine and Informatics, Biodesign Institute,

USA) (4).

Minimum Inhibitory Concentration (MIC)

Actinomycin X2 (1), fungichromin (2) and antifungalmycin 702 (3) purified from culture

filtrates of strain JAU4234 were bioassayed in Petri dishes to determine the MICs (over 50%

inhibition and over 90% inhibition) against all tested microorganism described in Table 1 and 2.

The specific method was performed following the procedures described in Shih et al., 2003 (3)

Reference

1. Kurosawa, K., V. P. Bui, J. L. VanEssendelft, L. B. Willis, P. A. Lessard, I.

Ghiviriga, T. G. Sambandan, C. K. Rha, and A. J. Sinskey. 2006. Characterization

of Streptomyces MITKK-103, a newly isolated actinomycin X2-producer. Appl

Microbiol Biotechnol 72:145-154.

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2. Larkin, M. A., G. Blackshields, N. P. Brown, R. Chenna, P. A. McGettigan, H.

McWilliam, F. Valentin, I. M. Wallace, A. Wilm, R. Lopez, J. D. Thompson, T. J.

Gibson, and D. G. Higgins. 2007. Clustal W and Clustal X version 2.0. Bioinformatics

23:2947-8.

3. Shih, H. D., Y. C. Liu, F. L. Hsu, V. Mulabagal, R. Dodda, and J. W. Huang. 2003.

Fungichromin: a substance from Streptomyces padanus with inhibitory effects on

Rhizoctonia solani. J Agric Food Chem 51:95-99.

4. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular

Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596-

9.

5. Xiong, Z. Q., X. R. Tu, and G. Q. Tu. 2008. Optimization of medium composition for

actinomycin X2 production by Streptomyces spp JAU4234 using response surface

methodology. J Ind Microbiol Biotechnol 35:729-34.

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FIG. S1. Morphology of strain JAU4234 grown on PDA agar at 28 °C for 7 days. (A) aerial

mycelium by Optical Microscope (1600×); (B) scanning electron micrograph of strain JAU4234.

Bar, 20 μm; (C) scanning electron micrograph of spores. Bar, 5 μm.

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FIG. S2. UV/visible spectrum of actinomycin X2 (1), fungichromin (2) and antifungalmycin 702

(3).

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FIG. S3. The key HMBC correlations of compound 3.

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TABLE S1. The 1H and 13C NMR spectral data for 3 in pyridine-d5 (500 MHz and 125 MHz, J in

Hz, in ppm)

No. δH δC No. δH δC

1 172.9 19 6.33 (dd, 14.5, 11.0) 135.2

2 3.24 (t, 8.0) 59.6 20 6.52 (dd, 14.6, 11.0) 131.8

3 5.00 (m) 72.2 21 6.40 (m) 134.1

4 2.25 (m) 42.1 22 6.72 (dd, 14.8, 10.8) 131.5

5 4.58 (m) 72.7 23 7.03 (dd, 14.8, 10.8) 131.8

6 1.90 (m) 45.3 24 6.47 (m) 134.5

7 4.67 (m) 72.4 25 5.13 (brs) 84.3

8 1.87 (m) 45.6 26 4.35 (m) 71.6

9 4.50 (m) 72.8 27 5.60 (t, 7.1) 76.3

10 1.84 (m) 43.4 28 1.76 (d, 6.2) 18.4

11 4.13 (m) 71.8 29 1.65 (s) 25.7

12 2.43 (m) 42.5 1′ 4.56 (m) 72.3

13 4.80 (m) 69.8 2′ 2.01 (m) 35.9

14 4.68 (m) 77.3 3′ 1.88 (m) 25.8

15 4.61 (d, 5.8) 84.6 4′ 1.27 (m) 32.2

16 83.3 s 5′ 1.25 (m) 23.0

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17 6.02 (d, 15.0) 139.6 6′ 0.79 (t, 6.8) 14.3

18 6.75 (dd, 15.0, 11.0) 124.1

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