Metabolic Engineering of Pseudomonas chlororaphis for De Novo Production of Iodinin from Glycerol

Iodinin, a natural phenazine N-oxide, is generally derived from phenazine-1,6-dicarboxylic acid (PDC) and has been widely used in the pharmacological and therapeutic fields because of its diverse biological activities. Despite the unique structural properties of N-oxides, which renders them attractive starting materials for the manufacture of chemotherapeutic agents, few studies have investigated their production using rationally engineered microorganisms from a renewable resource. Herein, Pseudomonas chlororaphis was metabolically engineered to produce iodinin from glycerol via an artificial pathway. First, the de novo biosynthesis pathway of iodinin was constructed by introducing four heterologous enzymes including EsmA1 and EsmA2 from Streptomyces antibioticus , monooxygenase PhzS(Pa) from Pseudomonas aeruginosa , and N-monooxygenase PhzNO1(Na) from Nocardiopsis sp. 13-12-13. The metabolic flux toward PDC was then improved via PhzG isozyme screening and synthesis module optimization. Through gene overexpression and culture optimization, the engineered strain produced 27.7 mg/L iodinin and 37.9 mg/L 1,6-dihydroxyphenazine from glycerol, respectively. To verify the availability of this PDC-producing kernel, the highest production of 1,6-dimethoxyphenazine to date was also achieved at 71.8 mg/L by introducing O-methyltransferase PhzM(La) from Lysobacter antibioticus. This work creates a promising alternative route that can offer selectivity and sustainability for producing aromatic N-oxides from renewable resources for the first time.