Pyoluteorin (Plt) is a PKS-NRPS hybrid antibiotic that is produced by Pseudomonas spp. and shows strong antifungal and antibacterial activities. Pseudomonas protegens H78, which was isolated from the rape rhizosphere in Shanghai, can produce a large array of secondary metabolites, including antibiotics and siderophores. Plt is produced at low levels in the H78 wild-type strain. This study aimed to improve Plt production through combinatory genetic engineered strategies. Plt production was significantly enhanced (by14.3-fold) in the strain engineered by the following steps: (1) deletion of the translational repressor gene rsmE in the Gac/Rsm-RsmE pathway; (2) deletion of the ATP-dependent protease gene lon that encodes a potential enzyme that degrades positive regulators; (3) deletion of the negative regulatory gene pltZ of the Plt ABC-type transporter operon pltIJKNOP; (4) deletion of an inhibitory sequence within the operator of the transcriptional activator gene pltR; and (5) overexpression of the pltIJKNOP transport operon. The Plt production of the final engineered strain was increased to 214 from 15gml(-1) in the H78 wild-type strain. In addition, the pltA gene in the pltLABCDEFG biosynthetic operon was characterized as the gene encoding the rate-limiting enzyme in the Plt biosynthetic pathway of H78. However, overexpression of the rate-limiting enzyme gene pltA or the transcriptional activator gene pltR did not further improve Plt biosynthesis in the above multiple-gene knockout strains.