Genome Mining in <i>Streptomyces avermitilis</i>: A Biochemical Baeyer−Villiger Reaction and Discovery of a New Branch of the Pentalenolactone Family Tree JiangJiaoyang TetzlaffCharles N. TakamatsuSatoshi IwatsukiMasato KomatsuMamoru IkedaHaruo CaneDavid E. 2009 Incubation of 1-deoxy-11-oxopentalenic acid (<b>12</b>) with recombinant PtlE protein from <i>Streptomyces avermitilis</i> in the presence of NADPH and catalytic FAD gave the Baeyer−Villiger oxidation product, the previously unknown compound neopentalenolactone D (<b>13</b>), representing a new branch of the pentalenolactone biosynthetic pathway. The structure and stereochemistry of the derived neopentalenolactone D methyl ester (<b>13-Me</b>) were fully assigned by a combination of GC-MS and NMR analysis and confirmed by X-ray crystallography. Neopentalenolactone D (<b>13</b>) was also isolated from engineered cultures of <i>S. avermitilis</i> from which the <i>ptlD</i> gene within the 13.4-kb (<i>neo</i>)-<i>ptl</i> biosynthetic gene cluster had been deleted. The <i>ΔptlEΔptlD</i> double deletion mutant accumulated <b>12</b>, the substrate for the <i>ptlE</i> gene product, while the corresponding single <i>ΔptlE</i> mutant produced <b>12</b> as well as the related oxidation products <b>14</b> and <b>15</b>. Engineered strains of <i>S. avermitilis</i>, SUKA5 and pKU462::ermRp-<i>ptl</i> cluster, harboring the complete (<i>neo</i>)<i>ptl</i> cluster produced the oxidized lactone <b>18</b> and the closely related seco acid hydrolysis products <b>16</b> and <b>17</b>.