title: 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 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 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.
authors.affiliation: Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México.
authors.forename: Blanca I
authors.initials: BI
authors.lastname: García-Gómez
authors.forename: Jorge
authors.initials: J
authors.lastname: Sánchez
authors.forename: Diana L
authors.initials: DL
authors.lastname: Martínez de Castro
authors.forename: Jorge E
authors.initials: JE
authors.lastname: Ibarra
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
authors.forename: Mario
authors.initials: M
authors.lastname: Soberón
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics / Q000633:toxicity; D003545:Cysteine / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004731:Endotoxins / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007814:Larva; D007915:Lepidoptera; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010572:Pest Control, Biological; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics / Q000633:toxicity
chemical_list: D000083722:Bacillus thuringiensis Toxins; C029103:Bacillus thuringiensis protoxin; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004731:Endotoxins; D006460:Hemolysin Proteins; D011498:Protein Precursors; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D003545:Cysteine