A Cry1Ac toxin variant generated by directed evolution has enhanced toxicity against Lepidopteran insects.
Cry1Ac insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) have become an important natural biological agent for the control of lepidopteran insects. In this study, a cry1Ac toxin gene from Bacillus thuringiensis 4.0718 was modified by using error-prone PCR, staggered extension process (StEP) shuffling combined with Red/ET homologous recombination to investigate the insecticidal activity of delta-endotoxin Cry1Ac. A Cry1Ac toxin variant (designated as T524N) screened by insect bioassay showed increased insecticidal activity against Spodoptera exigua larvae while its original insecticidal activity against Helicoverpa armigera larvae was still retained. The mutant toxin T524N had one amino acid substitution at position 524 relative to the original Cry1Ac toxin, and it can accumulate within the acrystalliferous strain Cry-B and form more but a little smaller bipyramidal crystals than the original Cry1Ac toxin. Analysis of theoretical molecular models of mutant and original Cry1Ac proteins indicated that the mutation T524N located in the loop linking β16-β17 of domain III in Cry1Ac toxin happens in the fourth conserved block which is an arginine-rich region to form a highly hydrophobic surface involving interaction with receptor molecules. This study showed for the first time that single mutation T524N played an essential role in the insecticidal activity. This finding provides the biological evidence of the structural function of domain III in insecticidal activity of the Cry1Ac toxin, which probably leads to a deep understanding between the interaction of toxic proteins and receptor macromolecules.
Shan
Shiping
S
Key Laboratory of Microbial Molecular Biology of Hunan Province, College of Life Science, Hunan Normal University, Changsha, 410081, People's Republic of China.
Zhang
Youming
Y
Ding
Xuezhi
X
Hu
Shengbiao
S
Sun
Yunjun
Y
Yu
Ziquan
Z
Liu
Shiquan
S
Zhu
Zhou
Z
Xia
Liqiu
L
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001413:Bacillus thuringiensis / Q000201:enzymology* / Q000472:pathogenicity*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000633:toxicity*; D004269:DNA, Bacterial / Q000235:genetics; D019020:Directed Molecular Evolution; D004731:Endotoxins / Q000235:genetics* / Q000633:toxicity*; D006460:Hemolysin Proteins / Q000235:genetics* / Q000633:toxicity*; D007306:Insecticides / Q000633:toxicity*; D007814:Larva / Q000187:drug effects; D007915:Lepidoptera / Q000187:drug effects*; D008958:Models, Molecular; D016296:Mutagenesis; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction / Q000379:methods; D016019:Survival Analysis
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis