Discovery and Biosynthesis of Cinnamoyl-Containing Pepticinnamins Q–V Produced by the Marine-Derived <i>Streptomyces</i> sp. SCSIO 68065

Guided by comprehensive bioinformatic analysis and global molecular networking, four previously undescribed peptidic natural products, pepticinnamins Q-T (<b>1</b>-<b>4</b>), along with two known analogues (<b>5</b>, <b>6</b>), were isolated from cultures of the marine-derived <i>Streptomyces</i> sp. SCSIO 68065. Heterologous expression of the <i>pcn</i> biosynthetic gene cluster in the engineered chassis strain <i>Streptomyces atratus</i> ZH16NSEPK enabled the production of pepticinnamin analogues and led to the targeted isolation of two undescribed biosynthetic intermediates, pepticinnamins U and V (<b>7</b>, <b>8</b>), as well as the known compound pepticinnamin M (<b>9</b>). The structures of these compounds were elucidated by spectroscopic analyses (including 1D and 2D NMR), HRESIMS, time-dependent density functional theory electronic circular dichroism (TDDFT-ECD) calculations, single-crystal X-ray diffraction studies, and advanced Marfey's method. Pepticinnamins Q-S (<b>1</b>-<b>3</b>) and U (<b>7</b>) are characterized by an unusual epoxidized cinnamoyl moiety. Comparative genomic analysis with homologous gene clusters allowed the proposal of their plausible biosynthetic pathways. Moreover, the cytochrome P450 monooxygenase Pcn29 was experimentally confirmed to catalyze the key epoxidation of the cinnamoyl moiety through a combination of targeted gene deletion and <i>in vitro</i> enzymatic reconstitution studies.