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

Area of Science:

• Biotechnology
• Molecular Biology
• Insect Molecular Virology

Background:

• The silkworm-baculovirus expression vector system (silkworm-BEVS) is a cost-effective platform for recombinant protein production.
• Gene knockouts in baculoviruses can enhance recombinant protein yield, but baculovirus genome editing is challenging.
• Efficient gene editing of the Bombyx mori nucleopolyhedrovirus (BmNPV) genome is needed to optimize silkworm-BEVS.

Purpose of the Study:

• To develop a streamlined and efficient method for synthesizing and gene editing the BmNPV bacmid.
• To enhance the productivity of the silkworm-BEVS through improved bacmid engineering.
• To demonstrate the rapid generation of gene-edited BmNPV bacmids for protein production.

Main Methods:

• A two-step Golden Gate Assembly (GGA) strategy was employed to synthesize the BmNPV bacmid from 19 genomic fragments.
• Intermediate plasmids containing BmNPV genomic DNA were constructed using the type IIS restriction enzyme BsaI.
• The full-length BmNPV bacmid was assembled with high efficiency (97.2%) using the type IIS restriction enzyme PaqCI.

Main Results:

• A highly efficient (97.2%) method for synthesizing the full-length BmNPV bacmid was established using a two-step GGA.
• This methodology allowed for the rapid and straightforward generation of BmNPV bacmids with targeted gene deletions (six genes).
• The gene-edited BmNPV bacmid suppressed the degradation of recombinant proteins expressed in silkworm pupae, enhancing yield.

Conclusions:

• The developed GGA approach significantly simplifies and accelerates BmNPV bacmid engineering.
• This advancement offers a powerful tool for improving the silkworm-BEVS for efficient recombinant protein production.
• The rapid gene editing capability is a major step forward for baculovirus expression systems.