Production of an insecticidal crystal protein from Bacillus thuringiensis by the methylotroph Methylobacterium extorquens.
The Cry1Aa protein from Bacillus thuringiensis is an insecticidal protein that is highly active against several species of Lepidoptera. We cloned and expressed the cry1Aa gene in a plant-colonizing methylotroph, Methylobacterium extorquens, under the control of the strong M. extorquens AM1 methanol dehydrogenase promoter, P(mxaF). Transmission electron microscopy revealed characteristic bipyramidal intracellular delta-endotoxin crystals similar to the crystalline inclusions formed by B. thuringiensis. Both the protoxin protein and the activated toxin were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis. In single-dose assays of the recombinant against the silkworm, Bombyx mori, both whole cells and cell lysates caused rapid feeding inhibition followed by mortality. The biomass and growth rate of recombinant cells in shake flask culture were similar to those of the wild-type strain, indicating a lack of fitness cost to the recombinant under controlled culture conditions. Recombinant Cry1Aa was expressed at a level of 4.5% of total M. extorquens cell protein. The potential benefits of modifying M. extorquens to deliver insecticidal Cry proteins for crop and forest protection are discussed.
Choi
Young J
YJ
Microbial and Enzymatic Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada.
Gringorten
J Lawrence
JL
Bélanger
Louise
L
Morel
Lyne
L
Bourque
Denis
D
Masson
Luke
L
Groleau
Denis
D
Míguez
Carlos B
CB
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis*; D012831:Bombyx / Q000382:microbiology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000096:biosynthesis*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006460:Hemolysin Proteins / Q000096:biosynthesis*; D020601:Methylobacterium extorquens / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D010572:Pest Control, Biological; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis