Construction of a Conditionally Asporogenous Bacillus thuringiensis Recombinant Strain Overproducing Cry Protein by Deletion of the leuB Gene

Abstract

One of the common shortcomings with Bacillus thuringiensis (Bt) biopesticides in field application is their instability under UV irradiation. In Bt, the leuB gene encodes the 3-isopropylmalate dehydrogenase. In addition to its role in leucine biosynthesis, LeuB would be likely recruited to catalyze the dehydrogenation of malate in the final step of tricarboxylic acid cycle during sporulation. In this study, we constructed a Bt recombinant strain in which the gene leuB was deleted by using the markerless gene deletion system. The leuB mutant strain showed a conditionally asporogenous phenotype while overproducing insecticidal crystal proteins and retaining its insecticidal activity well in both fermentation and LB media. Furthermore, the metabolic regulation mechanisms of LeuB was elucidated by iTRAQ-based quantitative proteomics approach. Evidences from proteomics data suggested that the inhibited supply of pyruvate (carbon source) was an important factor related to the conditionally asporogenous feature of the mutant. Consistently, the mutant regained its ability to sporulate in LB medium by adding 1% glucose or 1% sodium pyruvate. Taken together, our study demonstrated that deletion of the leuB gene resulted in delayed or completely blocked mother cell lysis, allowing the crystals encapsulated within cells, which makes this recombinant strain a good candidate for developing Bt preparations with better UV-stability.

Development of a Safe and Effective Bacillus thuringiensis-Based Nanobiopesticide for Controlling Tea Pests

“…Bt produces Cry proteins with high insecticidal activity against lepidopteran, coleopteran, and dipteran insects and nematodes during the sporulation phase . Cry proteins can be hydrolyzed into active toxins by midgut proteases when ingested by insects and then bind to membrane receptors in the midgut cells to induce membrane pore formation, leading to the death of insects. − However, Cry proteins have poor stability, and the application of Bt in agriculture has been greatly limited as a result of its intolerance to harsh environmental conditions, , leading to reduced efficacy and inability to meet biological pest control. Thus, improving the utilization rate of effective ingredients and controlling their loss are urgent considerations in Bt preparation.…”