Supporting information
Induced production of BE-31405 by co-culturing of Talaromyces siamensis FKA-61 with a variety of fungal strains
Kenichi Nonaka1,2a*, Masato Iwatsuki1,2a, Syunsuke Horiuchi1, Kazuro Shiomi1,2, Satoshi Ōmura1* and Rokuro Masuma1,2
1 Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan
2 Graduate School of Infection Control Sciences, Kitasato University, Tokyo, Japan
* Correspondence: Dr. K. Nonaka or Professor S. Ōmura, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
a The first two authors contributed equally to the work.
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Contents
S1. Taxonomy of the strain FKA-612
S2. Taxonomy of the strain FKA-62 and other fungal strains4
S3. Fermentation5
S4. Isolation and characterizaion of BE-31405 and other metabolites from a6
co-culture broth of FKA-61 and FKA-62
S5. Isolation and characterizaion of metabolites from a pure broth of FKA-626
S6. Analysis of metabolites by HPLC7
S1. Taxonomy of strain FKA-61
The strain FKA-61 was isolated from soilcollected on the main island of Okinawa, Japan.
For determination of the morphological characteristics of FKA-61 using the methodology of Pitt,1 the isolate was inoculated as 3-point cultures on Czapek yeast extract agar (CYA), malt extract agar (MEA) and 25% glycerol nitrate agar (G25N), and grown at 25°C (also at 5°C and 37°C on CYA)for 7 days in the dark. Colony colors were designated according to the Methuen Handbook of Colour.2
For determination of the micro-morphological characteristics of FKA-61, microscopic slides were prepared using colonies from MEA. The slides were examined with a Vanox-S AH-2 microscope (Olympus, Tokyo, Japan), and digital photomicrographs were taken with a DP25 digital camera (Olympus). For scanning electron microscopy (SEM) of the conidia and conidiophores of FKA-61, agar blocks (5 mm2) were cut from a 7-day old culture of the strains growing on MEA. The agar blocks were fixed with osmium tetroxide (TAAB, Berks, UK), air-dried and sputter coated with gold using a JFC-1200 Fine Coater (JEOL, Akishima, Tokyo, Japan). The samples were observed under a JSM-5600 scanning electron microscope (JEOL).
DNA extraction, polymerase chain reaction (PCR) amplification of the ITS region and sequencing of the strain FKA-61 were conducted under the conditions followed by Nonaka et al.3 Sequencing products were purified using BigDye XTerminator Purification Kit (Applied Biosystems, Foster City, CA, USA), and samples were analyzed on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Contigs were assembled using the forward and reverse sequences with the SeqMan Pro program from the Lasergene 10 package (DNASTAR Inc., Madison, WI, USA).
Colonies on CYA were 39–41 mm in diameter after 7 days at 25 °C (Figure S1a), floccose, plane, with light yellow (4A4) aerial mycelium, covered with greyish turquoise (24E4) conidia, with entire margin; orange to red exudate and pale yellow soluble pigment were produced; reverse were violet brown (11F5). Colonies on MEA were 36–37 mm in diameter after 7 days at 25 °C (Figure S1b), floccose, colliculose, white (1A1), with entire margin; clear exudate was produced, soluble pigment was not produced; reverse were greenish grey (1B2). Colonies on G25N were 9–10 mm in diameter after 7 days at 25 °C, floccose, plane, white (1A1), with entire margin; clear exudate was produced and soluble pigment was not produced; reverse were yellowish brown (5E7). Colonies on CYA were 25–28 mm in diameter after 7 days at 37 °C, veltinous, with white (1A1) aerial mycelium, covered with dark green (27F4) conidia, with entire margin; exudate and soluble pigment were not produced; reverse were blond (4C4). Colonies on CYA at 5 °C showed no growth.
Conidiophores on MEA were borne on a basal felt or directly from the agar, stipes were simple, 100–250 × 3.0–4.0(–4.5) µm, with a heavy wall. Penicilli were typically biverticillate (Figure S1c). Metulae in whorls of 2–6, which were usually rather appressed, sometimes slightly divergent when forced apart by larger whorls, about 9.0–12.5(–16.0) × 2.2–3.5 µm across the top, individually more or less cylindrical. Phialideswereacerose, 10.0–14.0 × 2.2–3.2 µm and collula were 0.5–1.5 m wide. Conidia borne in chains, were ellipsoidal to fusiform slightly roughened, 3.2–4.2 × 2.0–2.8 µm.
From the above morphological characteristics, strain FKA-61 was classified into the genus Penicillium subgenus Biverticillium section Simplicium seriesMiniolutea using Pitt’s classification.1 Recently, species of Penicillium subgenus Biverticillium were transferred to the genus Talaromyces.4
To determine the most closely-related Talaromyces species, the DNA sequence of the ITS region of FKA-61 was compared to sequences in the GenBank database by BLASTN 2.2.29 analysis,5 the strain FKA-61 having a 99.6% similarity with Talaromyces siamensis 2,6 (GenBank accession number JN899385).
From the results of morphological characteristics and BLAST search, the strain FKA-61 was identified as Talaromyces siamensis. This species is the first report in Japan.
Figure S1 Talaromyces siamensis FKA-61. (a) Photograph of colonies grown on Czapek yeast extract agar (CYA) after 7 days. (b) Photograph of colonies grown on malt extract agar (MEA) after 7 days.(c) Scanning electron micrograph of conidiophores grown on MEA. Scale bar, 10 m.
S2. Taxonomy of the strain FKA-62 and other fungal strains
The strain FKA-62 and other fungal strains were isolated from soils collected in various island locations in Japan (e.g., Hachijo Island, Chichi-jima and Haha-jima).
Taxonomic studies of five of nine co-cultured fungi, Penicillium pinophilum (current name; Talayomyces pinophilus) FKI-5653, Trichoderma harzianum FKI-5655, Staphylotrichum boninense FKI-4751, Gymnascella aurantiaca FKI-6588 and Mariannaea elegans var. punicea FKI-6644, have been reported previously.7-10
To determine the most closely related species, the DNA sequences of the ITS region of remaining co-cultured fungal strains were compared to sequences in the GenBank database by BLASTN 2.2.29 analysis,5 Strain FKA-62 had a 91.9% similarity withPhomopsis asparagi pa1100 (GenBank accession number JQ070363). As a result, it was classified into the genus Phomopsis. In addition, FKI-6685 had a 99.3% similarity withPhomopsis grabrae SCHM 3622 (GenBank accession number AY601918), FKI-6651 had a 100% similarity withPestalotiopsis foedans 3.9144 (GenBank accession number JN943631) and FKI-4665 had a 99.3% similarity withPaecilomyces lilacinus (current name; Purprocillium lilacinum) ATCC 10114 (GenBank accession number AY213665), confirming their identity.
S3. Fermentation
The strains FKA-61 and FKA-62 were each grown and maintained on an agar slant consisting of 0.1% glycerol, 0.08% KH2PO4, 0.02% K2HPO4, 0.02% MgSO4·7H2O, 0.02% KCl, 0.2% NaNO3, 0.02% yeast extract and 1.5% agar (adjusted to pH 6.0 before sterilization). A loopful of spores of each strain was inoculated into 100 ml of the seed medium consisting of 2.0% glucose, 0.5% Polypepton (Nihon Pharmaceutical Co., Tokyo, Japan), 0.2% yeast extract, 0.2% KH2PO4, 0.05% MgSO4·7H2O and 0.1% agar (adjusted to pH 6.0 before sterilization) in a 500-ml Erlenmeyer flask. The flask was incubated on a rotary shaker (210 rpm) at 27°C for 3 days. For the co-culture, a 1-ml portion of each seed culture of FKA-61 and FKA-62 was transferred to thirty 500-ml Erlenmeyer flasks each containing 100 ml of production medium of 2.4% potato dextrose broth (PDB, Difco Laboratories, Detroit, MI, USA) (adjusted to pH 6.0 before sterilization), and the fermentation on a rotary shaker (210 rpm) was carried out at 27°Cfor 6 days. For the pure culture, a 1-ml portion of each seed culture of FKA-61 or FKA-62 was transferred to three 500-ml Erlenmeyer flasks containing the production medium, and fermentation on a rotary shaker (210 rpm) was carried out at 27°Cfor 6 days.
S4. Isolation and characterizaion of BE-31405 and other metabolites from a co-culture broth of FKA-61 and FKA-62
Purification was guided by antifungal activity against Phomopsis sp. FKA-62. The 6-day old co-culture broth (2.9 liters) was added to ethanol (2.9 liters) and centrifuged (3,000 rpm). After evaporation to remove EtOH in vacuo, the aqueous solution was extracted with ethyl acetate twice, followed by concentration in vacuo. The ethyl acetate extract (2.3 g) was applied to a silca-gel column (33 × 120 mm, Merck KGaA., Darmstadt, Germany). After washing with CHCl3 (300 ml), the active fraction was eluted with CHCl3-MeOH (100:1, 300 ml) and concentrated in vacuo. This material (283 mg) was purified by HPLC on an ODS column (Capcell Pak C18, 20 × 250 mm, Shiseido. Co. Ltd., Tokyo, Japan) with 40% CH3CN/0.1% TFA aq at 7.0 ml min–1 detected at UV 210 nm. The peaks at retention times of 18, 32, 33, 37, 45, 59 and 62 minutes were collected and concentrated in vacuo to dryness to afford sclerotinin A (1.0 mg),11 griseofulvin (3.0 mg),12 vermistatin (1.9 mg),13 chrodrimanin F (2.4 mg),14 chrodrimanin E (0.8 mg),14 BE-31405 (55 mg),15 and thailandolide B (0.8 mg), 16 which were identified by the comparison with MS and NMR data with those in the literature, respectively. Among them, BE-31405 showed potent antifungal activity against Phomopsis sp. FKA-62.
S5. Isolation and characterizaion of metabolites from a pure broth of FKA-62
The 6-day old pure culture broth of FKA-62 (400 ml) was added to 400 ml of EtOH, centrifuged (3000 rpm) and EtOH was removed in vacuo. The aqueous solution (300 ml) was extracted with ethyl acetate twice, followed by concentration in vacuo. The ethyl acetate extract (32.5 mg) was diluted with a small amout of 30% MeOH aq and applied to an ODS cartridge column (Sep-Pak Plus C18, WAT020515, Waters Co., Milford, MA, USA) and eluated with 30%, 60% and 100% MeOH aq (each 180 ml). The 30% and 60% MeOH aq fractions were combined and concentrated in vacuo. The obtained material (18.6 mg) was purified by HPLC on an ODS column (Inertsil C8-4, 5 µm, 14 × 250 mm, GL Sciences Inc., Tokyo, Japan) with 30% CH3CN/0.05% TFA aq at 5.0 ml min–1 detected at UV 210 nm. The peaks at retention times of 11, 17 and 36 minutes were collected and concentrated in vacuo to dryness to afford dihydrogladiolic acid (5.8 mg),17 diaparthinol (0.7 mg),18 and mycoepoxydiene (0.8 mg),19 which were identified by the comparison of MS and NMR data with those of each in the literature.
S6. Analysis of metabolites by HPLC
The production of metabolites was measured by analytical HPLC under the following conditions; column: Symmetry C18 (2.1× 150 mm, Waters, Milford, MA, USA), UV detection: 210 nm, flow rate: 0.2 ml/minute, mobile phase: acetonitrile-water with 0.05% phosphoric acid, 5–100% (20 minutes) in pure culture broth of FKA-61 for detection of sclerotinin A, griseofulvin, vermistatin, chrodrimanin F, chrodrimanin E and thailandolide B with retention times of 11.2, 12.7, 12.8, 13.0, 14.3 and 14.6 minutes, respectively, in pure culture broth of FKA-62 for detection of dihydrogladiolic acid, diaparthinol and mycoepoxydiene with retention times of 7.9, 9.5 and 10.8 minutes, respectively, or in co-culture broth for detection of BE-31405 with retention time of 14.1 minute.
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