Natural Product Biotechnology Group
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Theme 4: Regulation of fragrant methoxybenzaldehyde biosynthesis in root organs of Hemidesmus indicus

Hemidesmus indicus (Asclepiadaceae) is an important medicinal plant known for multi-purpose uses since ancient times. The plant is a perennial twiner or creeper, rootstock woody, fragrant. A phenolic compound, 2-hydroxy-4-methoxy-benzaldehyde (MBALD) and its corresponding benzoic acid derivatives are responsible for the fragrance in root organs. The MBALD structure showed apparent similarity with the structure of vanillin.

We made DBT convinced on this apparently explored root fragrance and obtained a research grant to do further study. We developed a validated HPLC method for the first time to determine fragrant methoxybenzaldehyde and its acid-derivative from H. indicus roots, which was fast, sensitive and reproducible. Different elicitors (e.g., chitosan, methyl-jasmonate, yeast extract) were tested for their relative efficiency in uplifting MBALD accumulation in roots, amongst which, treatment with yeast extract showed maximum accumulation at a defined period of incubation, as shown by HPLC analysis.

Tt appeared that these unusual phenolic-derivatives are presumably derived from phenylpropanoid pathway. This formed a basis for predicting the biosynthetic route; a phenylpropanoid chain-cleavage is likely to be the basis for the formation of MBALD. We aim to explore this novel biosynthetic route(s) of MBALD formation, including the characterization of the principal catalytic enzyme(s) responsible for the production of this phenolic-fragrance at unusually high level. We demonstrated that a general phenylpropanoid pathway is involved in the formation of MBALD in H. indicus roots. Subsequently, in a DAE-BRNS-funded project, we have looked in-depth on the enzymatic route to this root-localized hydroxybenzoate metabolite formation. We found that regulation at shikimate pathway plays an important role in enhancing MBALD accumulation in H. indicus roots.

Chitosan/yeast-extract-elicited excised roots were used to stimulate this enzymatic route. Using cell-free root extract, we have shown that a phenylpropanoid side chain-shortening followed by a hydroxylation in the aromatic ring at 2-position, and subsequently a methylation at 4-position are involved in the formation of MBALD, the principal phenolic fragrance, of H. indicus roots. This biochemical information would provide the basis for characterizing these enzymes at cDNA level in near future.

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