Targeted Inactivation of an α/β Hydrolase Gene Enables Discovery of Heterodimeric Nenestatins

Nenestatins (NENs) belong to benzo[<i>b</i>]fluorene-containing atypical angucyclines, a structurally diverse class of microbial natural products. Bioinformatic analysis of the NEN biosynthetic gene cluster (<i>nes</i> BGC) from the deep-sea sediment-derived <i>Micromonospora echinospora</i> SCSIO 04089 implicated Nes5 as an <i>α/β</i> hydrolase. The targeted inactivation of the <i>nes5</i> gene led to the accumulation of five new analogs, NENs E-I (<b>1</b>-<b>5</b>), together with the known monomer homo-dehydrorabelomycin E (<b>6</b>). Their structures were elucidated by comprehensive spectroscopic analysis and electronic circular dichroism calculations. Notably, both NEN A and NEN B were absent in the Δ<i>nes5</i> mutant, indicating that Nes5 is essential for their biosynthesis; however, the exact function of Nes5 requires further exploration.