PhD defense by Nethaji J Gallage – Københavns Universitet

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PhD defense by Nethaji J Gallage

Title: Elucidation of the vanillin biosynthetic pathway in the vanilla orchid

Academic supervisors: 
Professor Birger Lindberg Møller, PLEN 
Research Scientist, Esben Halkjær Hansen, Evolva Biotech A/S
Postdoc Rubini Maya Kannangara

Michael Broberg Palmgren, PLEN 
Daniele Werck, Universite de Strasbourg 
Thomas Vogt, Leibniz Institute of Plant Biochemistry

Reception in room R161 after the defense


Vanilla is one of the most widely used flavouring agents in the world. Natural vanilla is extracted from the pods of vanilla orchid, Vanilla planifolia. Vanilla planifolia is a vine with a large, green, fleshy and succulent stem. The flowers are yellow and bisexual. When the flowers are pollinated, each flower develops into a long slender fruit, commonly called a pod. The pod reaches its full size 10-15 weeks after pollination. Vanilla pods are harvested 8-9 months after pollination. The immature green vanilla pods are almost odourless as flavour components are stored as glycosides. Freshly harvested pods are processed by curing to stop the natural vegetative process and to initiate the enzymes that are responsible for the formation of the well-known aromatic flavour constituents.

Vanillin (3-methoxy-4-hydroxybenzaldehyde) is the main flavour component of vanilla extract from cured vanilla pods. As vanillin is toxic to living organisms in high concentrations, vanilla plants store vanillin almost entirely as the glucose conjugated form, vanillin-β-D-glucoside. The highest concentration of vanillin glucoside is localized in the inner part of the pod including mesocarp and placenta 6 months after the pollination. Subcellular localization of vanillin and its glucoside was speculated to be in the vacuole. Despite the popularity of the flavour, the vanillin biosynthetic pathway has remained elusive, presumably due to lack of genetic and genomic resources during past few decades.

The research presented in this PhD elucidates the pathway for synthesis of vanillin in the pods of Vanilla planifolia. A single hydratase/lyase type enzyme, designated vanillin synthase (VpVAN), is able to catalyze the direct conversion of ferulic acid glucoside and ferulic acid into vanillin glucoside and vanillin, respectively. This reaction is envisioned to proceed by two sequential partial reactions composed of an initial hydration addition reaction followed by a retro-aldol elimination reaction. (Chapter 2; Gallage et al., 2014). The enzyme is processed by a second maturation step where 137 amino acids are cleaved off. Both pre-mature and mature forms of VpVAN are expressed in the vanilla pods 6 months after pollination and in the leaves of Nicotiana benthamiana, in which VpVAN was transiently expressed. Moreover, VpVAN were found to localize in the cytosol both before and after pro peptide cleavage, while pro peptide is transported into the vacuole for potential degradation (Chapter 3 – Manuscript in preparation).

This PhD thesis also includes a review (Chapter 4), which represents the current state of biotechnology-derived vanillin synthesis based on ferulic acid, eugenol and glucose using microorganisms. Vanillin is a compound of major interest in the flavour and fragrance industry. In 2010, the annual world sales of vanillin reached more than 15,000 tons, Nowadays, less than 1 % of the global production of vanillin is derived from vanilla pods, since the production and isolation of vanillin from vanilla pods is a laborious and expensive process and has a stagnant supply of vanilla pods of around 2000 tons. The worldwide increasing demand for natural vanillin flavour can therefore no longer be met by the pods of vanilla orchids alone. Consequently, research in bioengineering of microorganisms for flavour production is expanding. Several major issues have challenged the bioconversion of various substrates to vanillin by microorganisms. Examples are: cytotoxicity of the flavour products and of their precursors, inefficient metabolic flow, formation of undesired by-products, the complexity of product discovery and downstream processing methods due to physicochemical properties of the substrate, the product and the nature of microorganisms used. Hence, bioengineering tools have been employed to circumvent these drawbacks. The review emphasizes the major issues encountered and the solutions obtained

Finally, the uses of VpVAN for future industrial applications are claimed in the Chapter 5, which is a patent application.