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

Citation
Hw. Park et al., Molecular genetic manipulation of truncated Cry1C protein synthesis in Bacillus thuringiensis to improve stability and yield, APPL ENVIR, 66(10), 2000, pp. 4449-4455
Citations number
26
Categorie Soggetti
Biology,Microbiology
Journal title
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
ISSN journal
00992240 → ACNP
Volume
66
Issue
10
Year of publication
2000
Pages
4449 - 4455
Database
ISI
SICI code
0099-2240(200010)66:10<4449:MGMOTC>2.0.ZU;2-9
Abstract
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.