To convert the lipopeptide non-producer strain Bacillus subtilis pB2 into a plipastatin and surfactin coproducer, a gene expression cassette composed of a constitutive promoter (P43), functional gene sfp, and pleiotropic regulatory gene degQ was integrated into the chromosomal amyE locus of strain B. subtilis pB2 by homologous recombination, which generated a plipastatin and surfactin co-producer. Thirteen plipastatins and fifteen surfactins were identified in lipopeptide extracts using analytical techniques, and their effects on microorganisms were described by microscopic, cytoskeleton analysis and flow-cytometry, respectively. Plipastatins isolated from the engineered strain pB2-L exhibited strong antifungal activity (MIC 16 μg ml) by disrupting the cell walls, membranes and cytoskeleton of Fusarium oxysporum f. sp. cucumerinum hyphae. Surfactins affected the cell membrane of Staphylococcus aureus (MIC 20 μg ml), resulting in nucleic acid leakage and ultimately, cell death. Based on the convenience of genetic manipulation in the engineering strain, this work could be useful for the rational design of lipopeptide synthetases via the recombination of gene fragments to generate arrays of peptide derivatives and thus expand the diversity of microbial-produced lipopeptides.