Pseudomonas putida KT2442, a natural producer of polyhydroxyalkanoate, spends a lot of energy and carbon sources to form flagella and pili; therefore, deleting the genes involved in the biosynthesis and assembly of flagella and pili might improve PHA productivity. In this study, two novel deletion systems were constructed in order to efficiently remove the 76 genes involved in the biosynthesis and assembly of flagella and pili in P. putida KT2442. Both systems combine suicide-plasmid-based homologous recombination and mutant lox site-specific recombination and involve three plasmids. The first includes pK18mobsacB, pWJW101, and pWJW102; and the second includes pZJD29c, pDTW202, and pWJW103. These newly constructed systems were successfully used to remove different gene clusters in P. putida KT2442 and showed a high deletion efficiency (above 90%) whether for the second-round or the third-round recombination. Both systems could efficiently delete the gene PP4378 encoding flagellin in putida KT2442, resulting in the mutant strain WJPP01. The second system was used to remove the pili-forming gene cluster PP2357-PP2363 in putida KT2442, resulting in the mutant strain WJPP02, and also used to remove the flagella-forming gene cluster PP4329-PP4397 in WJPP02, resulting in the mutant strain WJPP03. Compared with the wild-type KT2442, the 1.2% genome reduction mutant WJPP03 grew faster, lacked flagella and motility, showed sharply decreased biofilm and 3',5'-cyclic diguanylic acid (c-di-GMP), but accumulated more polyhydroxyalkanoate. The biomass, polyhydroxyalkanoate yield, and content of WJPP03 increased 19.1, 73.4, and 45.6%, respectively, with sodium hexanoate supplementation, and also increased 11.4, 53.6, and 37.9%, respectively, with lauric acid supplementation.