Production of Antibacterial Questiomycin A in Metabolically Engineered Pseudomonas chlororaphis HT66

Pseudomonas chlororaphis has been demonstrated as a valuable source of antimicrobial metabolites for plant disease biocontrol and biopesticide development. Although phenazine-1-carboxylic acid (PCA) secreted by P. chlororaphis has been commercialized as an antifungal biopesticide, it shows poor antibacterial activity. Questiomycin A, with versatile antibacterial activities, is mainly discovered in some well-known phenazine-producing strains but not in Pseudomonas. Its low titer hinders practical applications. In this work, a metabolite was first identified as Questiomycin A in P. chlororaphis-derived strain HT66ΔphzBΔNat. Subsequently, Questiomycin A has been elucidated to share the same biosynthesis process with PCA by gene deletion and in vitro assays. Through rational metabolic engineering, heterologous phenoxazinone synthase introduction, and medium optimization, the titer reached 589.78 mg/L in P. chlororaphis, the highest production reported to date. This work contributes to a better understanding of Questiomycin A biosynthesis and demonstrates a promising approach to developing a new antibacterial biopesticide in Pseudomonas.