Increased virulence using engineered protease-chitin binding domain hybrid expressed in the entomopathogenic fungus Beauveria bassiana.
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.
Fan
Yanhua
Y
Biotechnology Research Center, Southwest University, Beibei, 400715, Chongqing PR China.
Pei
Xiaoqiong
X
Guo
Shujuan
S
Zhang
Yongjun
Y
Luo
Zhibing
Z
Liao
Xinggang
X
Pei
Yan
Y
D000818:Animals; D052982:Beauveria / Q000201:enzymology* / Q000472:pathogenicity*; D001665:Binding Sites / Q000235:genetics; D012831:Bombyx / Q000201:enzymology / Q000235:genetics; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000235:genetics; D019476:Insect Proteins / Q000378:metabolism; D007313:Insecta / Q000382:microbiology; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism*; D010572:Pest Control, Biological; D010843:Pichia / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016019:Survival Analysis; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics* / Q000378:metabolism
D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; C047663:cuticle proteins, insects; D002686:Chitin; D002688:Chitinases; D010447:Peptide Hydrolases