Mining and engineering of active PB transposons in Hymenoptera.

PiggyBac (PB) transposons, such as the canonical transposon from Trichoplusia ni and the more recently discovered Mage from Lepidoptera, are "cut-and-paste" mobile genetic elements widely harnessed as non-viral vector systems for gene therapy and transgenesis. Despite the ecological and economic significance of the insect order Hymenoptera, the evolutionary history and functional diversity of PB transposons within this group remain largely unexplored. To address this gap, we conducted a systematic investigation of PB transposons across 287 hymenopteran species. Our analysis revealed a widespread distribution, with PB elements detected in nearly half (137/287) of the genomes examined. Phylogenetic reconstruction identified four distinct clades, indicating a significant and previously unappreciated evolutionary diversity among hymenopteran PB transposases, and suggesting they have been highly active and dynamic. From this bioinformatic screen, we selected three PB transposons from Holcopasites calliopsidis (hcPB), Macropis europaea (mePB), and Andrena dorsata as strong candidates for recent activity. Functional validation using a binary transposition assay demonstrated that hcPB and mePB possess remarkable activity, comparable to the engineered hyperactive hyPB transposase. In contrast, adPB exhibited minimal transposition activity under the same conditions. We further engineered these systems based on successful strategies used for hyPB, which involves deleting an inhibitory N-terminal phosphorylation region and appending a second C-terminal domain. While this modification enhanced hyPB activity, it had variable effects on the new transposases: hcPB activity was maintained, but mePB activity decreased by approximately 20%. Intriguingly, merely deleting the N-terminal region severely impaired or abolished the activity of both hcPB and mePB, indicating distinct regulatory mechanisms. In summary, we have uncovered a rich diversity of active PB transposons in Hymenoptera and characterized two powerful new transposases (hcPB and mePB). These findings significantly advance the genome engineering toolkit, providing highly efficient tools with great promise for basic research and therapeutic applications.