Designing an Artificial Pathway for the Biosynthesis of a Novel Phenazine N‑Oxide in Pseudomonas chlororaphis HT66

Aromatic N-oxides are valuable due to their versatile chemical, pharmaceutical, and agricultural applications. Natural phenazine N-oxides possess potent biological activities and can be applied in many ways; however, few N-oxides have been identified. Herein, we developed a microbial system to synthesize phenazine N-oxides via an artificial pathway. First, the N-monooxygenase NaphzNO1 was predicted and screened in Nocardiopsis sp. 13–12–13 through a product comparison and gene sequencing. Subsequently, according to similarities in the chemical structures of substrates, an artificial pathway for the synthesis of a phenazine N-oxide in Pseudomonas chlororaphis HT66 was designed and established using three heterologous enzymes, a monooxygenase (PhzS) from P. aeruginosa PAO1, a monooxygenase (PhzO) from P. chlororaphis GP72, and the N-monooxygenase NaphzNO1. A novel phenazine derivative, 1-hydroxyphenazine N′10-oxide, was obtained in an engineered strain, P. chlororaphis HT66-SN. The phenazine N-monooxygenase NaphzNO1 was identified by metabolically engineering the phenazine-producing platform P. chlororaphis HT66. Moreover, the function of NaphzNO1, which can catalyze the conversion of 1-hydroxyphenazine but not that of 2-hydroxyphenazine, was confirmed in vitro. Additionally, 1-hydroxyphenazine N′10-oxide demonstrated substantial cytotoxic activity against two human cancer cell lines, MCF-7 and HT-29. Furthermore, the highest microbial production of 1-hydroxyphenazine N′10-oxide to date was achieved at 143.4 mg/L in the metabolically engineered strain P3-SN. These findings demonstrate that P. chlororaphis HT66 has the potential to be engineered as a platform for phenazine-modifying gene identification and derivative production. The present study also provides a promising alternative for the sustainable synthesis of aromatic N-oxides with unique chemical structures by N-monooxygenase.