Optimized cloning and expression workflow for Cry proteins from Bacillus wiedmannii biovar thuringiensis

Efficient heterologous expression of insecticidal Cry proteins, such as those produced by the bacterium Bacillus thuringiensis (Bt), remains essential for their structural and functional characterization. This article describes an optimized workflow for the amplification, cloning, and expression of cry genes identified in Bacillus wiedmannii biovar thuringiensis (Bwt). The Bwt genome harbors multiple cry genes that display high sequence conservation, especially in their flanking regions, and feature a high adenine/thymine content organized in short domains surrounding the start and stop codons. These regions often share homology with other cry genes, pseudogenes, or chromosomal fragments, making targeted amplification and cloning challenging. To overcome these difficulties, a nested PCR strategy was implemented to ensure high sequence specificity and fidelity. As a proof of concept, the cry4Ea1 and cry4Fa1 genes were cloned into the pSTAB vector, which combines the sporulation-dependent cyt1Aa promoter with the STAB-SD stabilizing sequence, and expressed in the Bt-derived acrystalliferous strain 4Q7. For genes containing internal restriction sites, the ligation-independent AQUA method provided an efficient alternative to conventional cloning. Optimization of the culture conditions demonstrated that inoculum physiology and nutrient availability significantly influenced biomass accumulation and Cry protein production, using the recombinant Bacillus thuringiensis subsp. israelensis 4Q7 strain expressing Cry4Fa1 as a representative case. This article analyzes these findings and compares them with other Bt expression systems, highlighting similarities, limitations, and complementary advances reported in other studies. Altogether, these approaches expand the molecular toolbox for Cry protein expression and characterization, offering promising biotechnological applications.