Three new ophiobolin sesterterpenoids, (6R)-16,17,21,21-O-tetrahydroophiobolin G (1), (6R)-16,17-dihydroophiobolin H (2), and (5S,6S)-16,17-dihydroophiobolin H (3), and three new farnesylated phthalide derivatives farnesylemefuranones D–F (9–11), along with five known ophiobolin analogues (4–8), were isolated and identified from the culture extract of Aspergillus insuetus SD-512, a deep-sea-derived fungus obtained from cold seep sediments collected at a depth of 1331 m. Among them, compounds 9–11 are rare examples of phthalide derivatives linked with farnesyl moieties via ether bonds. Their structures were established on the basis of detailed interpretation of the NMR spectroscopic and mass spectrometric data. X-ray crystallographic analysis, ECD calculations, and DP4+ probability analysis were performed to confirm the structures and establish the relative and absolute configurations of compounds 1–4. Compounds 3 and 9–11 showed broad-spectrum antibacterial activities, and differences in potencies could be assigned to structural modifications. This is the first report of secondary metabolites obtained from a deep sea cold-seep-derived fungus.

1-Hydroxyphenazine derivatives are phenazine family chemicals with broad-spectrum antibacterial and potential biological activities. However, the lack of variety and low titer hinder their applications. In this research, three enzymes PhzS (monooxygenase), NaphzNO1 (N-monooxygenase), and LaphzM (methyltransferase) were heterologously expressed in a phenazine-1-carboxylic acid generating strain Pseudomonas chlororaphis H18. Four phenazines, 1-hydroxyphenazine, 1methoxyphenazine, 1-hydroxyphenazine N′ 10-oxide, and a novel phenazine derivative 1-methoxyphenazine N′ 10-oxide, were isolated, characterized in the genetically modified strains, and exhibited excellent antimicrobial activities. Next, we verified the hydroxyl methylation activity of LaphzM and elucidated the biosynthetic pathway of 1-methoxyphenazine N′ 10-oxide in vitro. Moreover, the titer of 1-hydroxyphenazine derivatives was engineered. The three compounds 1-methoxyphenazine, 1hydroxyphenazine N′ 10-oxide, and 1-methoxyphenazine N′ 10-oxide all reach the highest titer reported to date. This work provides a promising platform for phenazine derivatives' combinatorial biosynthesis and engineering.