Designing an Artificial Pathway for the Biosynthesis of a Novel Phenazine <i>N</i>-Oxide in <i>Pseudomonas chlororaphis</i> HT66

Abstract

Aromatic <i>N</i>-oxides are valuable due to their versatile chemical, pharmaceutical, and agricultural applications. Natural phenazine <i>N</i>-oxides possess potent biological activities and can be applied in many ways; however, few <i>N</i>-oxides have been identified. Herein, we developed a microbial system to synthesize phenazine <i>N</i>-oxides <i>via</i> an artificial pathway. First, the <i>N</i>-monooxygenase NaphzNO1 was predicted and screened in <i>Nocardiopsis</i> 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 <i>N</i>-oxide in <i>Pseudomonas chlororaphis</i> HT66 was designed and established using three heterologous enzymes, a monooxygenase (PhzS) from <i>P. aeruginosa</i> PAO1, a monooxygenase (PhzO) from <i>P. chlororaphis</i> GP72, and the <i>N</i>-monooxygenase NaphzNO1. A novel phenazine derivative, 1-hydroxyphenazine <i>N</i>'10-oxide, was obtained in an engineered strain, <i>P. chlororaphis</i> HT66-SN. The phenazine <i>N</i>-monooxygenase NaphzNO1 was identified by metabolically engineering the phenazine-producing platform <i>P. chlororaphis</i> HT66. Moreover, the function of NaphzNO1, which can catalyze the conversion of 1-hydroxyphenazine but not that of 2-hydroxyphenazine, was confirmed <i>in vitro</i>. Additionally, 1-hydroxyphenazine <i>N</i>'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 <i>N</i>'10-oxide to date was achieved at 143.4 mg/L in the metabolically engineered strain P3-SN. These findings demonstrate that <i>P. chlororaphis</i> 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 <i>N</i>-oxides with unique chemical structures by <i>N</i>-monooxygenase.