Clone of plipastatin biosynthetic gene cluster by transformation-associated recombination technique and high efficient expression in model organism Bacillus subtilis

Plipastatin, a cyclic lipopeptide, exhibits inhibitory activity against filamentous fungi and plays an important role in the prevention of plant diseases, and post-harvest preservation of fruits and vegetables. However, the application of plipastatin has been hampered by low yields in natural strains, while chemical synthesis is not feasible because of its complex chemical structure. In this study, a scarless genetic modification method was applied to construct a heterologous expression host (Bacillus subtilis 1A751 Δpps) by knocking out the natural plipastatin genes from B. subtilis strain 1A751. The core genes for plipastatin biosynthesis from B. amyloliquefaciens HYM12 were captured and assembled with sfp and degQ using transformation-associated recombination (TAR) cloning. The resultant gene cluster was introduced into B. subtilis 1A751 Δpps to generate strain B. subtilis 1A751 Δpps amyE::ppsA-E + sfp + degQ. Its fermentation products were analyzed and identified by high-performance liquid chromatography-electrospray ionization-mass spectrometry. The results showed that the gene cluster for plipastatin synthesis was expressed successfully. This is the first time three gene fragments with significantly different DNA sizes (38.4, 0.3 and 0.8 kb) have been assembled by the TAR technique, thus we simultaneously established a method to express recombined large fragments in B. subtilis.