[Objective] In Pseudomonas sp. M18, there are nine structural genes encoding pyoteorin (Plt) biosynthase, including pltLABCDEFG and pltM. In order to identify the gene incoding rate-limiting enzyme, 9 structural genes were overexpressed. [Methods] The entire ORFs of these nine genes were PCR amplified from the M18 strain chromosomal DNA template and respectively cloned into the Pseudomonas-E. coli plasmid pME6032, generating nine overexpression plasmids of plt genes. The M18 strains, carrying these overexpression plasmids, were assayed for Plt production in KMB media. [Results] It was showed that the Plt production of three strains respectively harboring pltC, pltD, pltF overexpression plasmids was raised by 96%, 78%, 75% as compared to the control. The optimal inducing concentration of IPTG was 1.0 mmol/L, and the optimal inducing time was at 6 hours after inoculation. [Conclusion] The genes pltC, pltD, pltF divergently encode polyketidesynthase, halogenase and acyl-CoA synthetase rate-limiting enzyme in Plt biosynthesis. At the optimal IPTG concentration and inducing time, pyoluteorin production of strains containing pltD, pltF overexpression plasmids was respectively increased by 77.5% and 159.1%.