Benzofluorene-containing angucyclines, such as lomaiviticins, kinamycins, and fluostatins, have attracted increasing attentions on chemical synthesis and biosynthesis due to their unique structures and potent biological activities. Our continuous searching for novel natural products from deep-sea derived actinomycetes led to the discovery of nenestatin A (1), a benzo[b]fluorene compound possessing a carbon scaffold similar to the monomeric lomaiviticins, from the deep sea-derived Micromonospora echinospora SCSIO 04089. In addition, the biosynthetic gene cluster of nenestatin A (nes) was identified by a genome sequencing approach and comparative bioinformatics analysis with the lomaiviticin biosynthetic gene clusters. The nes-gene cluster was further verified by in vivo gene disruption experiments to afford a biosynthetic intermediate homo-dehydrorabelomycin E (2). This study adds a new member to the growing family of benzofluorene angucyclines and paves the way for further biosynthetic investigations.