Establishment of a Method for Biosynthesizing Rare Vitamin Ds by Overexpressing the Neverland Protein in Silkworm (<i>Bombyx mori</i>) using Baculovirus

Vitamin D is a fat-soluble vitamin with physiological effects that vary depending on differences in its side chains. However, rare forms of vitamin D (D₄-D₇) are poorly distributed in nature, making them difficult to obtain. This study aimed to evaluate the potential of recombinant baculovirus-injected silkworms to synthesize rare forms of vitamin D. Using the silkworm baculovirus expression vector system (BEVS) to overexpress the <i>Bombyx mori</i> neverland (BmNvd) protein, we detected provitamins D₃, D₅, and D₇ in these silkworms. We assessed the synthesis of vitamin D under two different ultraviolet (UV) exposure conditions and identified the synthesized vitamins D₃, D₅, and D₇ using gas chromatography-mass spectrometry (GC-MS) analysis. Silkworms achieved a maximum of 6910.81 ± 400.98 μg/100 g of vitamin D₃, with vitamin D₅ and D₇ levels of 1653.57 ± 102.98 and 243.22 ± 19.78 μg/100 g, respectively. The results demonstrated the potential of silkworms to synthesize vitamin D₃ and other rare forms of vitamin D, offering a new perspective on insects as viable sources of rare vitamin D.