Journal of Biotechnology 48 (1996) 81-96 Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector Vincent Sanchisa,b,*, Her& Agaisse”, Josette Chaufauxb, Didier Lereclusa,b aUnitP de Biochimie Mierobienne, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France ‘Station de Recherches de Lutte Biologique, INRA, L.u Mini&e, 78285 Guyancourt Cedex, France Received 11 October 1995; revised 31 January 1996; accepted 2 February 1996 zyxwvutsrqponmlkjihgfedcba Abstract zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Bacillus thuringiensis (Bt) b-endotoxins are safe biological insecticidal proteins whose usefulness has long been recognized. The first commercialized Bt insecticidal formulations were composed of spore-crystal preparations derived from wild-type strains. These products generally have a limited insecticidal host range and several genetically modified strains have, therefore, been constructed using transformation procedures. However, addition of a new b-endotoxin gene to strains already harboring other 8-endotoxin genes often resulted in broader-spectrum but less potent products because they produced significantly less of each of the crystal proteins. We report expression of the coding sequence of the sporulation specific cryZC gene from the non-sporulation-dependent cryZZZA promoter. Large amounts of CryIC accumulated in various Bt strains with different genetic backgrounds. Sporulation deficient SpoOA mutants, acrystalliferous derivatives and wild-type Bt strains expressing the engineered cryZZZ-cryZC gene were obtained. Introduction of the cryZZZ-cryZC gene whose product is highly active against Spodoptera littoralis into the Kto strain harboring the cryZA(c) gene active against Ostrinia nubilalis resulted in the construction of a new strain with increased potency and broader activity spectrum than the parent strain. Large amounts of each toxin were produced and the expression of the two genes seemed to be summed, presumably because the expression systems of the two genes are different. The plasmid shuttle vector used to introduce the cryZZZ-cryZC gene into the different Bt hosts utilizes the specific resolution site of transposon Tn4430 to enable construction of recombinant Bt strains that are free of foreign non-Bt DNA. This should facilitate the approval and acceptance for environmental release of the insecticidal recombinant products. Keywords: Bacillus thuringiensis; Biopesticides; Sporulation; Site-specific recombination; Gene-expression Abbreviations: BHI, brain heart infusion; Bt Bacillus thuringiensis; CFU, colony forming units; IR, inverted repeat; IRS, internal resolution site; LB, Luria Broth; PCR, polymerase chain reaction; RSP, reverse sequencing primer; SOE, splicing by overlap extension. * Corresponding author. Tel.: + 33 145 688812; fax: + 33 145 688938. 0168-1656/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SO168-1656(96)01404-6