Structural characterization and synergistic mechanism of Bacillus thuringiensis G033A exopolysaccharides enhancing...

Bacillus thuringiensis (Bt), a widely studied entomopathogenic bacterium, synthesizes diverse insecticidal proteins and extracellular polysaccharides (EPSs). The G033A strain, registered as the first genetically engineered Bt product registered in China for controlling Spodoptera frugiperda, produces EPSs during fermentation. However, the structure and function of the EPS remain uncharacterized. This study aimed to analyze the characteristics of EPS-G033A, and explore their synergistic effects with Bt insecticidal proteins. EPS-G033A had an average molecular weight of 71.5 kDa and was primarily composed of mannose, galactose, glucose, glucuronic acid, galactosamine, and glucosamine. Notably, EPS-G033A significantly enhanced the insecticidal activity against S. frugiperda, showing an approximately 1.7- to 2.1-fold improvement for Vip3Aa, a about 2.1-fold increase for Cry1Ac, and a 1.4- to 1.7-fold for the G033A wettable powder formulations. Binding affinity analysis demonstrated specific interactions between EPS-G033A and Vip3Aa protein, with a dissociation constant (Kd) of 10.68 ± 1.06 nmol/L. Western blotting indicated that EPS-G033A promoted the binding of Vip3Aa protein to brush border membrane vesicles (BBMVs) from the S. frugiperda midgut. These findings collectively demonstrate that EPS-G033A potentiates Bt bioinsecticides by directly interacting with the Vip3Aa protein, which in turn enhances the binding affinity of the toxin to BBMVs of target insects' midgut epithelium. Utilizing residual EPS from Bt fermentation byproducts presents a cost-effective strategy to enhance product performance while reducing biopesticide manufacturing costs.