LAUSR

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, 1-methoxyphenazine, 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, 1-hydroxyphenazine 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.