Transgenic Ocimum sanctum plants were engineered to produce vanillin by overexpressing the VpVAN gene using Agrobacterium-mediated transformation. Positive transformants developed shoots within 4-5 weeks and were transferred to a root induction medium and four independent transformants with no observed adverse effects were kept for anlysis. Quantitative RT-PCR indicated significantly higher VpVAN expression in transgenic lines AG_3 and AG_1, impacting the phenylpropanoid pathway and phenolic compound accumulation. Molecular docking studies indicated ferulic acid's higher binding affinity to vanillin synthase than eugenol. LC-MS/MS analysis revealed a marked increase in vanillin production in transgenic lines compared to wild type, with AG_3 exhibiting the highest vanillin content (1.98 ± 0.0047 mg/g extract) and AG_1 following (1.49 ± 0.0047 mg/g extract). AG_3 also showed elevated levels of benzoic acid, 4-hydroxy benzyl alcohol, and ferulic acid. This study highlights the potential of metabolic engineering in O. sanctum for enhanced vanillin production, suggesting pathways for large-scale production of natural vanillin and other valuable compounds in transgenic plants.
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