Efficient Production of Bacillus thuringiensis Cry1AMod Toxins under Regulation ofcry3AaPromoter and Single Cysteine Mutations in the Protoxin Region

Abstract

ABSTRACTBacillus thuringiensisCry1AbMod toxins are engineered versions of Cry1Ab that lack the amino-terminal end, including domain I helix α-1 and part of helix α-2. This deletion improves oligomerization of these toxins in solution in the absence of cadherin receptor and counters resistance to Cry1A toxins in different lepidopteran insects, suggesting that oligomerization plays a major role in their toxicity. However, Cry1AbMod toxins are toxic toEscherichia colicells, since thecry1Apromoter that drives its expression inB. thuringiensishas readthrough expression activity inE. coli, making difficult the construction of these CryMod toxins. In this work, we show that Cry1AbMod and Cry1AcMod toxins can be cloned efficiently under regulation of thecry3Apromoter region to drive its expression inB. thuringiensiswithout expression inE. colicells. However, p3A-Cry1Ab(c)Mod construction promotes the formation of Cry1AMod crystals inB. thuringiensiscells that were not soluble at pH 10.5 and showed no toxicity toPlutella xylostellalarvae. Cysteine residues in the protoxin carboxyl-terminal end of Cry1A toxins have been shown to be involved in disulfide bond formation, which is important for crystallization. Six individual cysteine substitutions for serine residues were constructed in the carboxyl-terminal protoxin end of the p3A-Cry1AbMod construct and one in the carboxyl-terminal protoxin end of p3A-Cry1AcMod. Interestingly, p3A-Cry1AbMod C654S and C729S and p3A-Cry1AcMod C730S recover crystal solubility at pH 10.5 and toxicity toP. xylostella. These results show that combining thecry3Apromoter expression system with single cysteine mutations is a useful system for efficient expression of Cry1AMod toxins inB. thuringiensis.