CONSTRUCTION OF NEW INSECTICIDAL BACILLUS-THURINGIENSIS RECOMBINANT STRAINS BY USING THE SPORULATION NONDEPENDENT EXPRESSION SYSTEM OF CRYIIIA AND A SITE-SPECIFIC RECOMBINATION VECTOR

Bacillus thuringiensis (Bt) delta-endotoxins are safe biological insec ticidal proteins whose usefulness has long been recognized. The first commercialized Bt insecticidal formulations were composed of spore-cry stal preparations derived from wild-type strains. These products gener ally have a limited insecticidal host range and several genetically mo dified strains have, therefore, been constructed using transformation procedures. However, addition of a new delta-endotoxin gene to strains already harboring other delta-endotoxin genes often resulted in broad er-spectrum but less potent products because they produced significant ly less of each of the crystal proteins. We report expression of the c oding sequence of the sporulation specific cryIC gene from the non-spo rulation-dependent cryIIIA promoter. Large amounts of CryIC accumulate d in various Bt strains with different genetic backgrounds. Sporulatio n deficient SpoOA mutants, acrystalliferous derivatives and wild-type Bt strains expressing the engineered cryIII-cryIC gene were obtained. Introduction of the cryIII-cryIC gene whose product is highly active a gainst Spodoptera littoralis into the Kto strain harboring the cryIA(c ) gene active against Ostrinia nubilalis resulted in the construction of a new strain with increased potency and broader activity spectrum t han the parent strain. Large amounts of each toxin were produced and t he expression of the two genes seemed to be summed, presumably because the expression systems of the two genes are different. The plasmid sh uttle vector used to introduce the cryIII-cryIC gene into the differen t Bt hosts utilizes the specific resolution site df transposon Tn4430 to enable construction of recombinant Bt strains that are free of fore ign non-Bt DNA. This should facilitate the approval and acceptance for environmental release of the insecticidal recombinant products.