Efficient Production of Bacillus thuringiensis Cry1AMod Toxins under Regulation of cry3Aa Promoter and Single Cysteine Mutations in the Protoxin Region

Abstract

Bacillus thuringiensis Cry1AbMod toxins are engineered versions of Cry1Ab that lack the amino-terminal end, including domain I helix ␣-1 and part of helix ␣-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 to Escherichia coli cells, since the cry1A promoter that drives its expression in B. thuringiensis has readthrough expression activity in E. 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 the cry3A promoter region to drive its expression in B. thuringiensis without expression in E. coli cells. However, p3A-Cry1Ab(c)Mod construction promotes the formation of Cry1AMod crystals in B. thuringiensis cells that were not soluble at pH 10.5 and showed no toxicity to Plutella xylostella larvae. 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 to P. xylostella. These results show that combining the cry3A promoter expression system with single cysteine mutations is a useful system for efficient expression of Cry1AMod toxins in B. thuringiensis.

Saturation mutagenesis and self‐inducible expression of trehalose synthase in Bacillus subtilis

“…cry3Aa promoter is one of the cry promoters. A previous study demonstrated that Cry1AMod toxins from B. thuringiensis were efficiently produced under the regulation of the cry3Aa promoter . It was reported that an extracellular enzyme cellulase was highly upregulated during the stationary phase when under control of the cry3Aa promoter .…”

“…A previous study demonstrated that Cry1AMod toxins from B. thuringiensis were efficiently produced under the regulation of the cry3Aa promoter. 52 It was reported that an extracellular enzyme cellulase was highly upregulated during the stationary phase when under control of the cry3Aa promoter. 46 The srfA promoter has been used to enable comK and pnpA gene expression in B. subtilis, 60,61 which is an operon needed for the development of genetic competence in B. subtilis.…”

“…Cry3Aa is a toxin produced by the bacterium Bacillus thuringiensis . A previous study demonstrated that Cry1AMod toxins from B. thuringiensis were efficiently produced under the regulation of the cry3Aa promoter . srfA is an operon needed for the development of genetic competence in B. subtilis , which is required for the nonribosomal biosynthesis of the cyclic lipopeptide, surfactin .…”

“…A previous study demonstrated that Cry1AMod toxins from B. thuringiensis were efficiently produced under the regulation of the cry3Aa promoter. 52 srfA is an operon needed for the development of genetic competence in B. subtilis, which is required for the nonribosomal biosynthesis of the cyclic lipopeptide, surfactin. 53 To date, there is no report of production of recombinant enzymes using a fusion promoter of Cry3Aa and srfA.…”

A Novel Regulator PepR Regulates the Expression of Dipeptidase Gene pepV in Bacillus thuringiensis

“…For example, the cry1Ac promoter can direct the expression of a variety of cry -like genes [ 4 ], such as cry1Ac , cry1Ab , cry1Ac5 , cry1Ac - av3 , cry2Ab27 , and cry8 . Similarly, the expression of Cry1AbMod/Cry1AcMod [ 5 ], Cry1Ac [ 6 ], Cry1c [ 7 ], Cry3A [ 8 ], Cry8Ga [ 9 ], and Cry69Aa1 [ 10 ] can be directed by the cry3A promoter. Recent reports have shown that some non- cry gene promoters with high-level activity were utilized for the expression of cry genes [ 11 , 12 ].…”