Fluostatins Produced by the Heterologous Expression of a TAR Reassembled Environmental DNA Derived Type II PKS Gene Cluster

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Cultures were grown in ISP-4 containing 5% HP-20 at 30 °C for 2 weeks. A crude extract was eluted (100% MeOH) from resin that had been washed with 50% MeOH. The eluant was partitioned by silica gel flash chromatography (CHCl3/MeOH step gradient). Individual compounds were purified by reversed-phase HPLC (XBridge 150 mm × 10 mm, 5 μm) using a linear gradient of 10:90 (H2O/MeOH, 0.1% formic acid) to 100% (MeOH, 0.1% formic acid) over 30 min (7 mL/min). Fluostatins G and H were separated in a second reversed-phase HPLC step using a linear gradient of 45:55 (H2O/ACN, 0.1% formic acid) to 50:50 (H2O/ACN, 0.1% formic acid) over 30 min.

Fluostatin F (4) HRMS-ESI (m/z): [M−H]−: calcd for C19H13O6337.0712; found 337.0696.1H NMR (600 MHz, CD3OD) δ 7.31 (H5, s, 1H), 7.14 (H9, dd, 7.1, 8.0 Hz, 1H), 7.06 (H10, d, 7.1 Hz, 1H), 6.89 (H8, d, 8.0 Hz, 1H), 5.82 (H1, d, 2.5 Hz, 1H), 3.89 (H2, d, 2.5 Hz, 1H), 3.49 (H13, s, 3H), 1.55 (H12, s, 3H).13C NMR (150 MHz, CD3OD) δ 195.8 (C11), 195.7 (C4), 156.4 (C6), 155.3 (C7), 137.8 (C6a), 136.7 (C10a), 133.7 (C11a), 133.5 (C4a), 132.1 (C9), 128.9 (C6b), 127.8 (C11b), 125.8 (C8), 123.3 (C5), 116.1 (C10), 69.8 (C1), 61.3 (C2), 59.3 (C3), 57.7 (C13), 15.2 (C12). Fluostatin G (5) HRMS-ESI (m/z): [M−H]−: calcd for C24H21O7421.1287; found 421.1257.1H NMR (600 MHz, CD3CN) δ 7.47 (H5, s, 1H), 7.23 (H9, dd, 7.2, 8.1 Hz, 1H), 7.14 (H10, dd, 7.2, 0.8 Hz, 1H), 7.04 (H8, dd, 8.1, 0.8 Hz, 1H), 6.92 (H1, d, 2.2 Hz, 1H), 3.88 (H2, d, 2.2 Hz, 1H), 2.40 (H14, sext, 7.0 Hz, 1H), 1.54 (H12, s, 3H), 1.32 (H15, m, 2H), 1.26 (H16, m, 2H), 1.07 (H18, d, 7.0 Hz, 3H), 0.78 (H17, t, 7.1 Hz, 3H).13C NMR (150 MHz, CD3CN) δ 193.7 (C4), 193.3 (C11), 176.5 (C13), 153.9 (C6), 153.1 (C7), 136.8 (C6a), 136.5 (C10a), 133.4 (C14a), 133.3 (C11a), 132.6 (C9), 127.7 (C6b), 126.3 (C11b), 125.2 (C8), 122.1 (C5), 116.9 (C10), 63.9 (C1), 60.7 (C2), 59.4 (C3), 40.0 (C14), 36.5 (C15), 20.8 (C16), 17.2 (C18), 15.0 (C12), 14.1 (C17). Fluostatin H (6) HRMS-ESI (m/z): [M−H]−: calcd for C24H21O7421.1287; found 421.1248.1H NMR (600 MHz, CD3CN) δ 7.47 (H5, s, 1H), 7.23 (H9, dd, 7.3, 8.0 Hz, 1H), 7.14 (H10, d, 7.3 Hz, 1H), 7.04 (H8, d, 8.0 Hz, 1H), 6.91 (H1, d, 2.2 Hz, 1H), 3.89 (H2, d, 2.2 Hz, 1H), 2.39 (H14, sext, 7.0 Hz, 1H), 1.54 (H12, s, 3H), 1.36 (H15, m, 2H), 1.28 (H16, m, 2H), 1.06 (H18, d, 7.0 Hz, 3H), 0.82 (H17, t, 7.1 Hz, 3H).13C NMR (150 MHz, CD3CN) δ 193.7 (C4), 193.4 (C11), 176.7 (C13), 154.0 (C6), 153.2 (C7), 136.9 (C6a), 136.6(C10a), 133.5 (C4a), 133.4 (C11a), 132.6 (C9), 127.7 (C6b), 126.3 (C11b), 125.2 (C8), 122.1 (C5), 117.0 (C10), 64.0 (C1), 60.8 (C2), 59.4 (C3), 39.9 (C14), 36.7 (C15), 20.9 (C16), 17.4 (C18), 15.0 (C12), 14.2 (C17).

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Equal portions (10) of [1-13C] acetate were added daily from day 3 through day 12 to attain a final acetate concentration of 10 mM.

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