Increased virulence using engineered protease-chitin binding domain hybrid expressed in the entomopathogenic fungus Beauveria bassiana
Increased virulence using engineered protease-chitin binding domain hybrid expressed in the entomopathogenic fungus Beauveria bassiana
复制标题
使用在昆虫病原真菌白僵菌中表达的工程蛋白酶-几丁质结合域杂交体提高毒力
DOI：
10.1016/j.micpath.2010.06.013
复制
发表时间：
2010-12-01
影响因子：
3.8
通讯作者：
Pei, Yan
中科院分区：
文献类型：
--
作者：
Fan, Yanhua;Pei, Xiaoqiong;Pei, Yan
昆虫角质层主要由蛋白质和高度不溶性多糖、几丁质的互连网络组成。昆虫病原真菌，如白僵菌，通过多种水解酶（包括蛋白酶和几丁质酶）与机械压力的作用，直接渗透宿主角质层，从而侵入昆虫。为了更好地靶向角质层蛋白-几丁质结构并加快渗透速度，通过将家蚕几丁质酶的几丁质结合域BmChBD与来自白僵菌的枯草杆菌蛋白酶样蛋白酶CDEP-1的C端融合，构建了杂合蛋白酶（CDEP-BmChBD）。与野生型相比，杂合蛋白酶能够结合几丁质并从昆虫角质层释放更多的肽/蛋白质。添加毕赤酵母产生的蛋白酶CDEP-1或CDEP:BmChBD作为添加剂可增强白僵菌的杀虫活性，但CDEP:BmChBD的增强效果显着高于CDEP-1。与野生型和过度表达天然蛋白酶的菌株相比，白僵菌中杂合蛋白酶的表达也显着增加了真菌毒力。这些结果表明，合理设计毒力因子是通过基因工程改良菌株的潜在策略。 (C) 2010 年，爱思唯尔有限公司出版。
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. (C) 2010 Published by Elsevier Ltd.