Costa Rica - Part 4 - Vanilla

During our recent visit to Costa Rica, a highlight was our visit to Villa Vanilla, a small biodynamic, organic farm specializing in growing various spices (including black pepper, cinnamon, cacao and vanilla) using sustainable practices. Vanilla, Vanilla planifolia, is my favorite spice. Given a choice among hundreds of ice cream flavors, I will choose natural vanilla every time! Therefore it was of great interest to me to see how this spice is grown and processed. Because the major aroma and flavor compounds in vanilla are derived from phenylalanine, and my own research has entailed studies on phenylalanine biosynthesis and metabolism in plants, I was doubly excited!

Vanilla planifolia is grown as a vine, supported by young trees at Villa Vanilla.

Vanilla is an orchid, producing clusters of greenish yellow flowers that must be pollinated manually in order to set fruit. The flowers last for only one day. The vanilla orchid vines were not in bloom at the time of our visit (January 2019) and so the following image of vanilla flowers is taken from the Wikipedia article on Vanilla planifolia:

Vanilla Flower Image Source:

The pollinated ovaries develop into pods (also called beans) which take several months to mature

The ripe pods are harvested, wrapped in burlap and placed in a box to cure and ferment

After fermentation the pods (beans) are dried

The major chemical constituents of the dried pods contributing to their flavor and aroma include vanillin (4-hydroxy-3-methoxybenzaldehyde), vanillic acid (4-hydroxy-3-methoxybenzoic acid), 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid, with vanillin being the key component:

Structures of vanillin, vanillic acid, 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid

Vanillin can be conjugated with glucose to form vanillin glucoside, glucovanillin (not shown).

There has been substantial interest in elucidating the biosynthetic pathway of vanillin in Vanilla planifolia. In 2014, Gallage et al. reported the identification of a single enzyme (a hydratase/lyase type enzyme designated vanillin synthase (VpVAN)) isolated from Vanilla planifolia that catalyzes the conversion of ferulic acid to vanillin.

Ferulic acid structure

This enzyme also is reported to act on ferulic acid glucoside producing vanillin glucoside (glucovanillin).

Gallage, N.J., Hansen, E.H., Kannangara, R., Olsen, C.E., Motawia, M.S., Jørgensen, K., Holme, I., Hebelstrup, K., Grisoni, M., Møller, B.L. Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme. Nat. Commun. 5: 4037 (2014)

Radiotracer experiments conducted by Gallage et al. (2014) and by Negishi et al. (2009) suggest that the pathway of vanillin biosynthesis involves the following steps in mature vanilla pods, with methionine (via S-adenosylmethionine) providing the methyl group (CH₃) of the 3-methoxy moiety of ferulic acid and vanillin:

phenylalanine -> cinnamic acid -> 4-coumaric acid -> -> caffeic acid -> ferulic acid -> vanillin -> glucovanillin

Negishi, O., Sugiura, K., Negishi, Y. Biosynthesis of vanillin via ferulic acid in Vanilla planifolia. J. Agric. Food Chem. 57: 9956-9961 (2009)

The entire pathway appears to be localized in the chloroplast:

Gallage, N.J., Jørgensen, K., Janfelt, C., Nielsen, A.J.Z., Naake, T., Dunski, E., Dalsten, L., Grisoni, M., Møller, B.L. The intracellular localization of the vanillin biosynthetic machinery in pods of Vanilla planifolia. Plant Cell Physiol. 59: 304-318 (2018)

However, Yang et al. (2017) have raised concerns about the proposed "vanillin synthase" identified by Gallage et al. (2014):

• The proposed vanillin synthase exhibits high identity to cysteine proteases, and is identical at the protein sequence level to a protein identified in 2003 as being associated with the conversion of 4-coumaric acid to 4-hydroxybenzaldehyde.

• The recombinant cysteine protease-like protein, whether expressed in an in vitro transcription-translation system, E. coli, yeast, or plants, is unable to convert ferulic acid to vanillin.

• The protein is a component of an enzyme complex that preferentially converts 4-coumaric acid to 4-hydroxybenzaldehyde, as demonstrated by the purification of this complex and peptide sequencing.

• RNA sequencing provides evidence that this protein is expressed in many tissues of V. planifolia irrespective of whether or not they produce vanillin.

Yang et al. (2017) conclude:

V. planifolia does not appear to contain a cysteine protease-like "vanillin synthase" that can, by itself, directly convert ferulic acid to vanillin. The pathway to vanillin in V. planifolia is yet to be conclusively determined.

Yang, H., Barros-Rios, J., Kourteva, G., Rao, X., Chen, F., Shen, H., Liu, C., Podstolski, A., Belanger, F., Havkin-Frenkel, D., Dixon, R.A. A re-evaluation of the final step of vanillin biosynthesis in the orchid Vanilla planifolia. Phytochemistry 139: 33-46 (2017)

Clearly, further research is needed to clarify this biosynthetic pathway leading to one of the most popular natural spices, and second in value only to Saffron.

Previous articles in this series:

Costa Rica - Part 1 - The Sloth Institute Costa Rica

Costa Rica - Part 2 - Scintillating Sunsets

Costa Rica - Part 3 - Superb Scenery