A three-plasmid-containing CRISPR-Cas9 platform to engineer 916 as an efficient biocontrol agent.

(Bv) is a widely used biocontrol agent against plant diseases, mainly because its genome contains numerous non-ribosomal peptide synthetases (NRPS) gene clusters for the synthesis of various cyclic lipopeptides (CLPs). The domesticated strain Bv916, capable of co-producing four CLPs, has been successfully applied for green control of rice sheath blight and angular leaf spot. To enhance Bv916's biological control efficacy while maintaining environmental safety, it is essential to establish a food-grade gene editing platform in Bv916. Here, a three-plasmid CRISPR-Cas9 platform for Bv916 was constructed using the thermosensitive origin pET194ts, constitutive P43 promoters for Cas9, the specific promoter Psrf for single guide RNAs (sgRNAs), and three resistance gene expression cassettes. By replacing the native promoters of ComX and RecA in Bv916 with the strong promoters P43 and PrepU, respectively, this platform achieved a single-gene editing efficiency of 96%, while the simultaneous dual-gene editing efficiency reached 61%, with each round completed within five business days. Furthermore, this gene editing platform is used to replace promoters of four NRPS gene clusters (, , , and ) in Bv916 with strong constitutive promoters (PB, PA, P43, and PrepU), generating the derivative BvLSBF. Compared to Bv916, BvLSBF showed 6.8-fold, 5.9-fold, 10.9-fold, and 6.2-fold increases in locillomycin, surfactin, bacillomycin L, and fengycin, respectively. Its antagonistic activity against plant pathogens was also significantly enhanced. This system enables further development of Bv916 as a cell factory and integration of multiple biocontrol factors, offering significant potential for sustainable agriculture.IMPORTANCEIn this study, a food-grade three-plasmid CRISPR-Cas9 platform for Bv916 was established by incorporating the optimized BvCas9 under the constitutive promoter P43, single guide RNAs (sgRNAs), and homologous recombination fragments into three thermosensitive shuttle vectors. This gene editing system was used to achieve gene insertion, deletion, and replacement in Bv916, particularly by editing four non-ribosomal peptide synthetase (NRPS) gene clusters. This resulted in increased production of four cyclic lipopeptides and significantly enhanced antibacterial and antifungal activity.