Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance

Nature. 2016 May 5;533(7601):58-63. doi: 10.1038/nature17938. Epub 2016 Apr 27.


The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacillus thuringiensis / genetics*
  • Bacillus thuringiensis Toxins
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism*
  • Bacteriophages / genetics
  • Biotechnology
  • Cadherins / metabolism
  • Cell Death
  • Consensus Sequence
  • Crops, Agricultural / genetics
  • Crops, Agricultural / metabolism
  • Directed Molecular Evolution / methods*
  • Endotoxins / genetics*
  • Endotoxins / metabolism*
  • Genetic Variation / genetics*
  • Hemolysin Proteins / genetics*
  • Hemolysin Proteins / metabolism*
  • Insecticide Resistance*
  • Insecticides / metabolism
  • Molecular Sequence Data
  • Moths / cytology
  • Moths / physiology*
  • Mutagenesis / genetics
  • Pest Control, Biological / methods*
  • Plants, Genetically Modified
  • Protein Binding / genetics
  • Protein Stability
  • Selection, Genetic


  • Bacillus thuringiensis Toxins
  • Bacterial Proteins
  • Cadherins
  • Endotoxins
  • Hemolysin Proteins
  • Insecticides
  • insecticidal crystal protein, Bacillus Thuringiensis