Plipastatin, an antimicrobial peptide produced by , exhibits remarkable antimicrobial activity against a diverse range of pathogenic bacteria and fungi. However, the practical application of plipastatin has been significantly hampered by its low yield in wild species. Here, the native promoters of both the plipastatin operon and the gene in the mono-producing strain M-24 were replaced by the constitutive promoter P, resulting in plipastatin titers being increased by 27% (607 mg/mL) and 50% (717 mg/mL), respectively. Overexpression of long chain fatty acid coenzyme A ligase (LCFA) increased the yield of plipastatin by 105% (980 mg/mL). A new efflux transporter, YoeA, was identified as a MATE (multidrug and toxic compound extrusion) family member, overexpression of enhanced plipastatin production to 1233 mg/mL, an increase of 157%, and knockout of decreased plipastatin production by 70%; in contrast, overexpression or knockout of in mono-producing surfactin and iturin engineered strains only slightly affected their production, demonstrating that YoeA acts as the major exporter for plipastatin. Co-overexpression of and improved plipastatin production to 1890 mg/mL, which was further elevated to 2060 mg/mL after gene deletion. Lastly, the use of optimized culture medium achieved 2514 mg/mL plipastatin production, which was 5.26-fold higher than that of the initial strain. These results suggest that multiple strain engineering is an effective strategy for increasing lipopeptide production, and identification of the novel transport efflux protein YoeA provides new insights into the regulation and industrial application of plipastatin.