Efficient and accurate BmNPV bacmid editing system by two-step golden gate assembly.

The silkworm-baculovirus expression vector system (silkworm-BEVS), using Bombyx mori nucleopolyhedrovirus (BmNPV) and silkworm larvae or pupae, has been used as a cost-effective expression system for the production of various recombinant proteins. Recently, several gene knockouts in baculoviruses have been shown to improve the productivity of recombinant proteins. However, the gene editing of the baculovirus genome (approximately 130 kb) remains challenging and time-consuming. In this study, we sought to further enhance the productivity of the silkworm-BEVS by synthesizing and gene editing the BmNPV bacmid from plasmids containing fragments of BmNPV genomic DNA using a two-step Golden Gate Assembly (GGA). The BmNPV genome, divided into 19 fragments, was amplified by PCR and cloned into the plasmids. From these initial plasmids, four intermediate plasmids containing the BmNPV genomic DNA were constructed by GGA with the type IIS restriction enzyme BsaI. Subsequently, the full-length bacmid was successfully synthesized from the four intermediate plasmids by GGA with another type IIS restriction enzyme PaqCI with a high efficiency of 97.2 %. Furthermore, this methodology enabled the rapid and straightforward generation of the BmNPV bacmid lacking six genes, resulting in the suppression of degradation of recombinant proteins expressed in silkworm pupae. These results indicate that the BmNPV bacmid can be quickly and efficiently edited using only simple cloning techniques and enzymatic reactions, marking a significant advancement in the improvement of the silkworm-BEVS.