Biotechnology Research Center, Southwest University, Beibei, 400715, Chongqing PR China.
Microb Pathog. 2010 Dec;49(6):376-80. doi: 10.1016/j.micpath.2010.06.013. Epub 2010 Jul 30.
Insect cuticles consist mainly of interlinked networks of proteins and the highly insoluble polysaccharide, chitin. Entomopathogenic fungi, such as Beauveria bassiana, invade insects by direct penetration of host cuticles via the action of diverse hydrolases including proteases and chitinases coupled to mechanical pressure. In order to better target cuticle protein-chitin structures and accelerate penetration speed, a hybrid protease (CDEP-BmChBD) was constructed by fusion of a chitin binding domain BmChBD from Bombyx mori chitinase to the C-terminal of CDEP-1, a subtilisin-like protease from B. bassiana. Compared to the wild-type, the hybrid protease was able to bind chitin and released greater amounts of peptides/proteins from insect cuticles. The insecticidal activity of B. bassiana was enhanced by including proteases, CDEP-1 or CDEP:BmChBD produced in Pichia pastoris, as an additive, however, the augment effect of CDEP:BmChBD was significantly higher than that of CDEP-1. Expression of the hybrid protease in B. bassiana also significantly increased fungal virulence compared to wild-type and strains overexpressing the native protease. These results demonstrate that rational design virulence factor is a potential strategy for strain improvement by genetic engineering.
昆虫外骨骼主要由相互连接的蛋白质网络和高度不溶的多糖几丁质组成。昆虫病原真菌,如球孢白僵菌,通过多种水解酶(包括蛋白酶和几丁质酶)的作用以及机械压力的直接穿透宿主外骨骼来入侵昆虫。为了更好地靶向外骨骼蛋白-几丁质结构并加速穿透速度,通过将来自家蚕几丁质酶的几丁质结合结构域 BmChBD 融合到来自球孢白僵菌的枯草杆菌蛋白酶样蛋白酶 CDEP-1 的 C 端,构建了一种杂合蛋白酶(CDEP-BmChBD)。与野生型相比,该杂合蛋白酶能够结合几丁质并从昆虫外骨骼中释放出更多的肽/蛋白质。将蛋白酶、CDEP-1 或在巴斯德毕赤酵母中产生的 CDEP:BmChBD 作为添加剂包含在内,增强了球孢白僵菌的杀虫活性,然而,CDEP:BmChBD 的增强效果明显高于 CDEP-1。与野生型和过表达天然蛋白酶的菌株相比,在球孢白僵菌中表达杂合蛋白酶也显著提高了真菌的毒力。这些结果表明,合理设计的毒力因子是通过遗传工程改良菌株的潜在策略。