The FR901464-producing strain Pseudomonas sp. No. 26632 was purchased from the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan. It was routinely activated from a glycerol stock on Luria-Bertani (LB) agar at 30 °C for 2 days as a starting plate. Several colonies from the plate were inoculated into a flask containing 250 ml of LB medium and incubated at 30 °C for 24 h on a rotary shaker (150 r.p.m.) to prepare a seed culture. For fermentation, the seed culture was inoculated at 2% (v/v) to each of the two fermentors (BioFlo IV, 20 l total volume, New Brunswick Scientific, Enfield, CT, USA); each contains 12 l of production medium consisting of soluble starch 1%, glycerin 1%, defatted soybean meal 1%, glucose 0.5%, corn steep liquor 0.5%, (NH4)2SO4 0.2%, CaCO3 0.2%, MgSO4·7H2O 0.006% and antifoam-204 0.01% (pH 7.0), slightly modified from the literature.2 The pH of bacterial culture was automatically maintained by the fermentor with 1N HCl or 1N NaOH, and the fermentation was proceeded at 25 °C for 48 h with an agitation of 200 r.p.m. and an air flow-rate of 4 l min−1. The repeated fermentation with two fermentors led to an accumulation of 72 l of fermentation broth.

Fermentation broth was extracted three times with ethyl acetate (3:2, v/v), and the extracts were pooled and concentrated to dryness with a rotary evaporator at 35 °C. The resulting crude extract (25.2 g) was subjected to two steps of silica gel chromatography and one step of octadecyl-silica C18 chromatography on an YFLC AI-580 flash chromatography system (Yamazen, San Bruno, CA, USA), with elution monitored at UV 235 nm (see Supplementary Information: Scheme S1). In the first step, each 5 g of crude extract was suspended in 25 ml of ethyl acetate and mixed with 30 g of silica gel and packed into an injection column (3.0 × 12.5 cm, Catalog No. W830 silica gel, Yamazen), which was mounted atop a silica gel Universal Column (4.8 × 18.5 cm, 200 g silica gel, 40 μm, 60 Å). The column system was sequentially eluted by 1.1 l of each of the following solvents: hexane, hexane:ethyl acetate (3:1, v/v); hexane:ethyl acetate (1:1, v/v); ethyl acetate, ethyl acetate:acetone (1:1, v/v); and acetone, all at a flow rate of 50 ml min–1. The ethyl acetate fraction containing 1 was concentrated to dryness at 35 °C. In the second step, the above resulting residue was suspended in 15 ml of acetone, mixed with 15 g of silica gel and packed into an injection column (2.0 × 6.5 cm, 14 g silica gel), which was mounted atop a silica gel Universal Column (2.6 × 12.0 cm, 40 g silica gel, 40 μm, 60 Å). The column system was sequentially eluted with chloroform, and 1, 2, 4 and 10% of acetone in chloroform, and finally acetone at 18 ml min–1. In the third step, the 1% acetone fraction containing 1 was again concentrated and further fractionated on an octadecyl-silica C18 column system equipped with an injection column (2.0 × 6.5 cm, 14 g gel) and a Universal Column (2.0 × 8.0 cm, 14 g gel, 50 μm, 120 Å). After loading the sample, the column system was first eluted with 5% acetonitrile for 3 min, then by a linear gradient of acetonitrile from 5 to 100% in 10 min, and finally by 100% acetonitrile for 14 min, with a flow rate of 20 ml min–1. The fraction containing 1 was eluted at 15.7 min. In the final purification step, acetonitrile solution of 1 was purified with a Varian ProStar HPLC system (210 binary pump and 330 photodiode array detector, Varian, Palo Alto, CA, USA) equipped with an Agilent Prep-C18 column (21.2 × 250 mm, 10 μm, Agilent, Santa Clara, CA, USA) to give 28 mg of pure 1. As shown in Supplementary Figure S1, 1 was present in significant amount in the culture grown in fermentor but barely in that grown in shaking flask, suggesting that production of 1 is heavily culture condition-dependent.

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