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　HT66　Delta　phzB　Delta　Nat.　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.