Molecular genetic manipulation of truncated Cry1C protein synthesis in Bacillus thuringiensis to improve stability and yield

Cry1 protoxins of Bacillus thuringiensis are insecticidal 135-kDa proteins synthesized and assembled into parasporal crystals during sporulation. Afte r ingestion, these crystals dissolve in the midgut and active toxins with m olecular masses of about 65-kDa are released from the N-terminal half of th e molecule by midgut proteases. Direct synthesis of the toxin-containing N- terminal half of Cry1 molecules using recombinant DNA techniques results in a low level of unstable truncated proteins that do not crystallize. In the present study, inclusions of truncated Cry1C (Cry1C-t) were obtained by co mbining genetic elements from other endotoxin genes and operons that enhanc e Cry protein synthesis and crystallization. Increased levels of Cry1C-t sy nthesis were achieved by using cyt1A promoters to drive expression of the 5 ' half of cry1C that included in the construct the 5' cry3A STAB-SD mRNA st abilizing sequence and the 3' stem-loop transcription terminator. RNA dot b lot analysis showed that the STAB-SD and 3' transcriptional termination seq uences were important for stabilization of truncated cry1C (cry1C-t) mRNA. A low level of cry1C-t mRNA was present when only the cyt1A promoters were used to express cry1C-t, but no accumulation of Cry1C-t was detected in Wes tern blots. The orientation of the transcription terminator was important t o enhancing Cry1C-t synthesis. Inclusion of the 20- and 29-kDa helper prote in genes in cry1C-t constructs further enhanced synthesis. The Cry1C-t prot ein was toxic to Spodoptera exigua larvae, though the toxicity (50% lethal concentration [LC50] = 13.2 mu g/ml) was lower than that of full-length Cry 1C (LC50 = 1.8 mu g/ml). However, transformation of the HD1 isolate of B. t huringiensis subsp. kurstaki with the cry1C-t construct enhanced its toxici ty to S. exigua as much as fourfold.