Multiple active-sites editing of adenylation domains of srfA operon for constructing diverse surfactin lipopeptide

The srfA operon, which encodes the surfactin synthase SrfA in Bacillus rhizobacteria, is essential for surfactin biosynthesis. As a typical non-ribosomal peptide synthetase (NRPS), the adenylation domain of SrfA determines the structure of the synthesized peptide. A major challenge in the field lies in the precise and efficient engineering of NRPS adenylation domains to generate diverse surfactin variants with enhanced bioactivities. Herein, we applied a non-specific guide RNA system (UgRNA/Cas9) for multi-site and diversified editing of the srfA operon in the high-secreting strain Bacillus pumilus LG3145. UgRNAs targeting four conserved motifs around the SrfA active pocket were designed to direct Cas9-mediated replacement of Stachelhaus codes with codons for 28 different amino acids. This strategy generated two engineered strains: WA1, which carries a Val→Phe mutation in module 2 and produces [Ser²]surfactin, and WA2, which harbors three mutations (Thr→Ser, Glu→Lys, Asn→Gly) across modules 3 and 6, yielding [Lys^3,6]surfactin. The two variants displayed distinct antifungal profiles: WA1 exhibited strong activity against Ustilaginoidea virens, whereas WA2 inhibited both Pyricularia oryzae and U. virens. In contrast, the parent strain LG3145 was only effective against Rhizoctonia solani. This work demonstrates the potential of UgRNA/Cas9-driven editing of NRPS domains to expand structural and functional diversity of surfactins.