Scientists amplify Vip3A in Bacillus thuringiensis

Researchers have developed a strategy to increase the effectiveness of bioinsecticides based on the bacteria Bacillus thuringiensis (Bt). The study revealed that manipulating enzymes involved in the degradation of the bacterial cell wall can greatly increase the secretion of the Vip3A protein.

Vip3A belongs to the group of insecticidal vegetative proteins produced by Bt during its growth phase. Unlike the well-known Cry toxins, synthesized in the sporulating phase, Vip3A acts by another mechanism. It does not present cross-resistance with Cry and has a broad spectrum against lepidopterans.

The challenge faced by the scientific community was the retention of Vip3A inside the bacterial cell, even after the use of strong genetic promoters. The solution now found was to intervene in the protein secretion system, focusing on enzymes called cell wall hydrolases (CWHs).

Among these enzymes, the murein transglycosylase MltE stood out. Its overexpression weakened the bacterial cell wall and increased the release of membrane vesicles, structures that transport proteins such as Vip3A to the external environment. The result was a significant increase in the concentration of the toxin in the fermentation broth within 12 hours.

Tests with caterpillars of Spodoptera exigua showed the impact of this innovation. The culture broth of the strain modified with MltE, diluted four times, was able to cause up to 70% functional mortality in the larvae after only half a day of fermentation. In comparison, a strain previously modified by another method needed 36 hours to achieve a similar effect.

In addition to efficiency, the new method proposes a reduction in fermentation time. This represents a considerable gain for the industrial-scale production of bioinsecticides, potentially reducing costs and increasing competitiveness compared to synthetic products.

The research also reinforces a line of studies on membrane vesicles in Gram-positive bacteria. Until recently, it was considered that this group did not produce such structures due to the thickness of the cell wall. However, the work with Bt suggests that changes in the enzymatic balance of the wall may favor the formation of vesicles, with implications beyond agricultural toxicology.

The team also observed that proteins other than Vip3A – such as phospholipase C and hemolysin HBL – increased their presence in the vesicles of the modified strain, suggesting that the technique could be useful for the production of several recombinant proteins.

The study did not identify involvement of phage genes or other extrinsic cellular stress mechanisms, as occurs in other bacteria. This indicates that modulation of CWHs alone can induce vesicle formation in Bt, opening new biotechnological possibilities.

The team suggests that different types of CWHs exert distinct effects on the cell wall, depending on where they act. Hydrolases that break glycosidic or amide bonds, such as MltE, BioL and YgiM, favored the secretion of Vip3A. Enzymes such as EnvC and YkfC, which act on peptide bonds, showed the opposite effect.

The authors propose that the process of vesicle and protein release may be linked to the dynamics of cell division and the restructuring of the bacterial wall. Although further studies are needed, the hypothesis is that certain enzymes "open up space" physically in the cell wall, facilitating the formation and release of vesicles.

More information at doi.org/10.1016/j.pestbp.2025.106515