Metabolic engineering of hairy root cultures in Beta vulgaris for enhanced production of vanillin, 4-hydroxybenzoic acid, and vanillyl alcohol

The flavor of vanilla is a complex blend of compounds, with vanillin as the most prominent, along with vanillyl alcohol and 4-hydroxybenzoic acid. Natural vanillin extracted from vanilla beans is expensive, so researchers use heterologous synthesis to produce nature-identical vanillin in plant hosts. Consequently, alternative traditional farming and gathering methods are required to bridge the significant disparity between supply and demand. The current research successfully developed a method to induce hairy root formation from leaves. It integrated the Vanillin synthase (VpVAN) gene into transgenic hairy root lines of <i>Beta vulgaris</i>, synthesizing vanillin-related compounds. The presence of the VpVAN gene in transgenic roots was confirmed using PCR analysis. Additionally, RT-qPCR analysis demonstrated the expression of the VpVAN gene in the transgenic root lines. The transgenic hairy root clones H1, H2, and H5 showed enhanced vanillin production, vanillyl alcohol, and 4-hydroxybenzoic acid. Elicitation with methyl jasmonate (MJ) and salicylic acid (SA) further improved the production of these compounds in <i>B. vulgaris</i> hairy roots. The maximum hairy root biomass was observed after 60 days, with the maximum synthesis of vanillin and 4-hydroxybenzoic acid obtained from hairy root clones H5 and HR2, respectively. Vanillyl alcohol HR2 was obtained on the 45th day of cultivation. Elicitation with wound-associated hormone methyl jasmonate and salicylic acid enhanced the yield of vanillin, vanillyl alcohol, and 4-hydroxybenzoic acid, with a 215-fold increase in vanillin, a 13-fold increase in vanillyl alcohol, and a 21 fold increase in 4-hydroxybenzoic acid. The study results indicate that establishing transgenic hairy root cultures with the VpVAN gene is a promising alternative method for enhancing the production of vanilla flavor compounds such as vanillin, vanillyl alcohol, and 4-hydroxybenzoic acid. A cost-effective protocol has been developed to mass-produce phenolic compounds using a hairy root culture of <i>B. vulgaris</i>. This approach addresses the increasing demand for these substances while reducing the cost of natural vanillin production, making it suitable for industrial-scale applications.