Improving Mycoinsecticide Of Beauveria Bassiana By Molecular Evolution Of Cuticle Degrading Chitinase And Protease Genes

Entomopathogenic fungi are key natural regulators in insect pest populations, providing the only practical microbial control of insects that feed by sucking plant or animal juices, and for many acridid and coleopteran pests which have no known bacterial or viral diseases. However, mycoinsecticides constitute a very small percentage of the total insecticide market for the reason of requiring a longer time to kill an insect pest after application, during which time the infected insect would cause serious damage to the crop. Improvements in virulence of entomopathogenic fungi can be achieved by understanding mechanisms of pathogenesis and thereafter by genetically modifying targeted virulence factors. Research indicated that penetration of host's cuticle is one of the key steps in infection for entomopathogenic fungi. Entry of fungi to insect cuticle involved both mechanical pressure and enzymatic degradation. During this step, insect pathogenic fungi secreted several hydrolases, including chitinases, proteases and lipases, which degrade host's cuticle and help fungi to penetrate cuticle. Among these hydrolases, chitinase and protease play important roles in infection, supported by the facts that overexpressing chitinase and protease gene can enhance the virulence of entomopathogenic fungi.In this paper, two molecular evolution approaches, i.e., DNA shuffling and domain combination were employed to improve the virulence of Beauveria bassiana, a widely distributed entomopathogenic fungi. Mutants of Bbchitl with higher chitinase activity were obtained by DNA shuffling. Heterologous chitin binding domains were fused to a chitinase gene Bbchitl and a protease gene CDEP-1 (both of them come from B.bassiana), respectively. The enzymatic characteristics of hybrid enzymes were analyzed. Finally, the hybrid genes were delivered into B.bassina and the virulence of the genetically engineered fungi was assayed.The main results were as fallowings:1. Enhanced the hydrolytic activity of chitinase (Bbchitl) from B. bassiana by DNA shufflingIn this experiment, Escherichia coli was used as display system. Mutant library resulted from DNA shuffling was screened by the form of clear zone on chitosan7B plate, which depends on the gene expression in host bacterium, E. coli, and the secretion of Bbchitl protein into medium. Conditions, including five signal peptides and different culture temperatures, which maybe affect the secretion of Bbchitl in E. coli, were investigated. The results indicated different signal peptides have various efficiencies to direct the secretion of Bbchitl, and the secretion efficiency of pelB signal peptide was highest among them. Furthermore, the secretory efficiency of Bbchitl at low...