Enhanced virulence through genetic engineering of Beauveria bassiana blastospores by overexpression of a cuticle-degrading endochitinase

Genetic engineering of entomopathogenic fungi combined with fast-acting infective blastospores grown by liquid fermentation offers an innovative approach to develop more efficient mycoinsecticides targeting a broad spectrum of arthropod pests, thus minimizing the over-reliance on synthetic chemical insecticides. In this research, Beauveria bassiana (Ascomycota: Cordycipitaceae) was genetically engineered by integrating multiple copies of an endogenously derived Chit1 gene, which encodes a constitutively expressed chitinase putatively involved in insect cuticle degradation, into its genome, with the aim at enhancing blastospore virulence. Genomic analysis of two highly virulent mutants overexpressing Chit1 identified 4 and 2 copies of this gene in their genomes, designated as ^OEBbChit1-3 and ^OEBbChit1-5, which were significantly upregulated by approximately ∼ 3- to 5-fold higher compared to the wild-type (WT) strain, thus confirming elevated gene expression and chitinase activity consistent with overexpression of the target gene. Compared with the WT, the median time to lethality (LT₅₀) of the ^OEBbChit1 mutants was shortened to 75.7 %, and the median lethal concentration (LC₅₀) decreased approximately 4.6-log₁₀ fold when blastospores were applied to Galleria mellonella larvae via the natural cuticle infection route. Among the phenotypic traits examined, oxidative stress was more pronounced in the highly virulent mutants, which exhibited slightly reduced blastospore production in vitro than the WT strain. Moreover, infections by the highly virulent mutants increased the density of total hemocytes relative to the WT and mock control, thus triggering a rapid insect immune response. Collectively, our findings provide compelling evidence of an innovative strategy based on the genetic improvement of B. bassiana by overexpression of the native chitinase in fast-acting blastospores, thus leading to enhanced virulence with minimal detrimental phenotypical effects in fungal development.