Genome-Wide Simplification of the AcMNPV Genome Using Synthetic Biology

Large-scale genome simplification represents a fundamental goal in synthetic biology. Baculoviruses, with their biphasic life cycle and inherent genomic plasticity, have emerged as ideal models for synthetic genome engineering. Although modified baculovirus genomes are widely used as expression vectors for robust recombinant protein production, many genomic regions are dispensable for in vitro budded virus (BV) production. In this study, guided by the synthetic biology "design-build-test-learn" framework, we systematically reduced the genome of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and obtained synthetic viruses capable of producing BVs. Building upon our previous work on whole-genome synthesis and partial genome reduction, we developed a strategy to rescue viruses by cotransfecting linearized genome fragments into host cells, thereby accelerating the iterative evaluation of genomic deletions. A total of 35 reduced genomes of varying sizes were synthesized, and the titers of the corresponding rescued viruses were measured. The most reduced functional genome, AcMNPV-Syn-mini, corresponds to the deletion of approximately 28 kb encompassing 39 nonessential genes. We analyze and discuss the gene organization and characteristics of this minimized genome. Our findings provide a foundation for the development of high-capacity baculoviral vectors and contribute to a deeper understanding of baculovirus functional genomics.