Vanillin accumulation by Vanilla planifolia: what do we think we know and what do we really know?

Natural vanilla is one of the most popular and valuable spice widely employed in food, beverages and cosmetics. This fragrance is produced in fruits of the orchid Vanilla planifolia and, from a chemical point of view, it is composed by a large number of molecules (more than 200), where vanillin, vanillic acid, vanillyl alcohol, p-hydroxybenzaldehyde and p-hydroxybenzoic acid are the most representative. These substances possess important antioxidant capacities and beneficial nutraceutical properties, as demonstrated by several works (Shyamala et al., 2007; Ueno et al., 2019). To overcome the high market demand of vanilla flavor, most of the vanilla commercial products are made of vanillin, obtained by chemical conversion of several substrates, most of which derive from by-products of lignin decomposition processes or from fossil fuels (Martău et al., 2021). This chemically synthetized vanillin is considered as a non-natural flavor, the resulting aroma covers only partially the wide and complex fragrance typical of the natural spice, and its low commercial value is not comparable to that of natural vanilla. In V. planifolia, vanillin biosynthetic pathway is described to start from ferulic acid and ferulic acid glucoside that would be respectively converted to vanillin and vanillin glucoside by the activity of VANILLIN SYNTHASE (VpVAN). Gallage and coworkers stated the activity of this enzyme through heterologous expression in yeasts and tobacco and suggested that VpVAN, according to its function, is highly expressed in the inner part of the vanilla pods and in particular in the phenyloplasts, compartments deriving from the conversion of chloroplasts during fruit ripening (Gallage et al., 2014; Gallage et al., 2018). However, the results obtained from Gallage and colleagues were questioned in another recent publication, in which the authors failed to produce vanillin by exploiting VpVAN, thus suggesting a re-evaluation of VpVAN role and the existence of more complex and extended pathway(s) for vanillin production in vanilla pods (Yang et al., 2017). In our project, bioinformatics researches and in-vitro experiments suggested that VpVAN is highly expressed in roots, stems, leaves and in-vitro protocorms of V. planifolia, even though these organs and the in vitro culture do not accumulate vanillin. On the other side, feeding the Vp in vitro culture with the putative vanillin precursor ferulic acid did not result in vanillin or vanillin derivatives accumulation, and the same results were obtained with Nicotiana benthamiana and Beta vulgaris transiently expressing the same gene. These evidences support the doubts of Yang and coworkers on the real VpVAN gene function. Thus, we started different approaches as feeding with various putative molecular precursors, transient expression of candidate genes and metabolite detection with high resolution LC-MS.