OPTIMIZATION OF CRY3A YIELDS IN BACILLUS-THURINGIENSIS BY USE OF SPORULATION-DEPENDENT PROMOTERS IN COMBINATION WITH THE STAB-SD MESSENGER-RNA SEQUENCE

The insecticidal activity of Bacillus thuringiensis strains toxic to c oleopterous insects is due to Cry3 proteins assembled into small recta ngular crystals. Toxin synthesis in these strains is dependent primari ly upon a promoter that is active in the stationary phase and a STAB-S D sequence that stabilizes the cry3 transcript-ribosome complex. Here we show that significantly higher yields of Cry3A can be obtained by u sing dual sporulation-dependent cyt1Aa promoters to drive the expressi on of cry3Aa when the STAB-SD sequence is included in the construct. T he Cry3A yield per unit of culture medium obtained with this expressio n system was 12.7-fold greater than that produced by DSM 2803, the wil d-type strain of B. thuringiensis from which Cry3Aa was originally des cribed, and 1.4-fold greater than that produced by NB176, a mutant of the same strain containing two or three copies of cry3Aa, which is the active ingredient of the commercial product Novodor, used for control of beetle pests. The toxicities of Cry3A produced with this construct or the wild-type strain were similar when assayed against larvae of t he cottonwood leaf beetle, Chrysomela scripts. The volume of Cry3A cry stals produced with cyt1Aa promoters and the STAB-SD sequence was 1.3- fold that of typical bipyramidal Cry1 crystals toxic to lepidopterous insects. The dual-promoter/STAB-SD system offers an additional method for potentially improving the efficacy of insecticides based on B. thu ringiensis.