AEM Accepts, published online ahead of print on 13 June 2008

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Appl. Environ. Microbiol. doi:10.1128/AEM.00598-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Production of an insecticidal crystal protein from Bacillus thuringiensis by the methylotroph Methylobacterium extorquens

Young J. Choi, J. Lawrence Gringorten, Louise Bélanger, Lyne Morel, Denis Bourque, Luke Masson, Denis Groleau, and Carlos B. Míguez^*

Microbial and Enzymatic Technology Group, Bioprocess Sector, and Environmental Sector, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada; and Canadian Forest Service, Great Lakes Forestry Centre, 1219 Queen St. E., Sault Ste. Marie, Ontario P6A 2E5, Canada

^* To whom correspondence should be addressed. Email: carlos.miguez{at}nrc-cnrc.gc.ca.

The Cry1Aa protein from Bacillus thuringiensis is an insecticidal protein that is highly active against several species of Lepidoptera. We have 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 -endotoxin crystals similar to the crystalline inclusions formed by B. thuringiensis. Both the protoxin protein and activated toxin were visualized by SDS-PAGE 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. 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.