Development of a simplified and innovative CRISPR/Cas9 methodology for Bacillus amyloliquefaciens FZB42 genome editing: an alternative for the biocontrol of agricultural pathogens

Abstract

Agricultural production is constantly affected by the occurrence of crop pests, causing economic losses and instability in food production. An alternative to minimize these problems is the development of research that may enhance the biocontrol capacity of Bacillus amyloliquefaciens strains, such as genomic editing using the popular CRISPR/Cas9 technique. Therefore, the main objective of this proposal is to develop an efficient, innovative and simplified methodology of genomic editing with the CRISPR/Cas9 system in vitro and to apply it in the optimization of the production of bioactive metabolites in B. amyloliquefaciens FZB42. The proposed methodology has as its main differential being independent of plasmids and using ribonucleoprotein complexes (RNPs) produced in vitro from purified Cas9 protein and gRNA molecules. B. amyloliquefaciens FZB42 will have the genome edited to optimize the srf and bac operons, promoting increased expression of surfactin and bacilysin, two metabolites with important antibacterial and antifungal activity for biocontrol. The Psrf promoter of the srf operon will be replaced by the constitutive promoter P43, which is known to have high expression level in the exponential and stationary phase of growth for B. subtilis. The alteration of the bac operon will be done by optimizing the Ribosomal Binding Site (RBS) sequence and eliminating the first binding site of the negative ScoC regulator in the Pbac promoter. The resulting strain will be analyzed for the production of surfactin and bacilysin, as well as used in experiments to verify the antimicrobial capacity against Rhizoctonia solani and Agrobacterium tumefaciens. The execution of this work will also create the possibility of using the CRISPR/Cas9 methodology developed here in other bacterial species, even those that do not have well established genetic systems.