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Engineering of multiple trypsin/chymotrypsin sites in Cry3A to enhance its activity against Monochamus alternatus Hope larvae
Author(s) -
Guo Yajie,
Wang Yafang,
O'Donoghue Anthony J,
Jiang Zhenze,
CarballarLejarazú Rebeca,
Liang Guanghong,
Hu Xia,
Wang Rong,
Xu Lei,
Guan Xiong,
Zhang Feiping,
Wu Songqing
Publication year - 2020
Publication title -
pest management science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.296
H-Index - 125
eISSN - 1526-4998
pISSN - 1526-498X
DOI - 10.1002/ps.5866
Subject(s) - trypsin , toxin , midgut , biology , bacillus thuringiensis , toxicity , biochemistry , proteolytic enzymes , proteases , microbiology and biotechnology , chymotrypsin , spodoptera , enzyme , larva , chemistry , botany , recombinant dna , bacteria , genetics , organic chemistry , gene
BACKGROUND Bacillus thuringiensis Cry3 toxins exhibit specific toxicity against several coleopteran larvae. However, owing to its low toxicity to Monochamus alternatus , Cry3A toxin is not useful for managing M. alternatus larvae. Here we assessed the proteolytic activation of Cry3Aa toxin in M. alternatus larval midgut and increased its toxicity by molecular modification. RESULTS Our results indicated that insufficient processing of Cry3Aa protoxin and non‐specific enzymatic digestion of Cry3Aa toxin in the midgut of M. alternatus larvae led to low toxicity. The results of transcriptome analysis, enzymatic assay with fluorogenic substrates, and multiplex substrate profiling by mass spectrometry showed that the main digestive enzymes in M. alternatus larval midgut were trypsin‐like proteases that preferentially cleaved peptides with arginine and lysine residues. Consequently, trypsin recognition sites were introduced into the Domain I of Cry3Aa protoxin in the loop regions between α‐helix 3 and α‐helix 4 to facilitate proteolytic activation. Multiple potential trypsin cleavage sites away from the helix sheet and functional regions in Cry3Aa proteins were also mutated to alanine to prevent non‐specific enzymatic digestion. Bioassays indicated that a modified Cry3Aa‐T toxin (K65A, K70A, K231A, K468A, and K596A) showed a 9.5‐fold (LC 50 = 12.3 μg/mL) increase in toxicity to M. alternatus larvae when compared to native Cry3Aa toxin. CONCLUSION This study highlights an effective way to increase the toxicity of Cry3Aa toxin to M. alternatus , which may be suitable for managing the resistance of transgenic plants to other pests, including some of the most important pests in agriculture. © 2020 Society of Chemical Industry