Handbook of Vanilla Science and Technology

Fig. 18.1 Scheme of potential pathways to vanillin, in comparison to monolignol and ferulic acid formation. The pathway on the left-hand side of the figure shows the formation of ferulic acid from frans-cinnamic acid, according to recent studies on monolignol biosynthesis and the formation of ferulate inArabidopsis.Vanillin is shownarising fromtwo mechanisticallydifferent routes: directlyfrom coumaricacid by non-oxidative chain shortening, orvia anyone ofthree CoenzymeAesters by β-oxidation. The numbers in bold represent different enzyme types that should be recognizable in EST datasets. Those involved in the formation offerulate from cinnamate haveall been functionally identified; it is assumed that similartypes of enzymes (or even possibly the same enzymes) could be involved in the hydroxylation, O-methylation and reduction of benzoyl CoA or benzaldehyde intermediates.

18.2 MULTIPLE PATHWAYS TO VANILLIN?

Past work on the vanillin pathway, and pathways leading to related benzenoids, has been reviewed in more detail elsewhere (Dignum et al. 2001; Walton et al. 2003; Wildermuth 2006). It is generally agreed that vanillin is a product of the phenylpropanoid pathway from

Table 18.1 A timeline for the development of concepts related to vanillin biosynthesis

System and approach | Concept | Reference

Radiolabeling of V. planifola pods | Vanillin is formed directly from ferulic acid | Zenk1965

Radiolabeling of V. planifolia tissue cultures | Intermediacy of isoferulic acid (which is subsequently demethylated) | Funk and Brodelius 1990a,b

Enzyme assay in cell free extracts from Lithospermum erythrorhizon | Non-oxidative chain-shortening of coumaric acid to 4-hydroxybenzaldehyde | Yazaki et al. 1991

Measuring metabolite levels in V. planifolia pods | Intermediacy of tartrate esters | Kanisawa et al. 1994

Enzyme isolation and assay from cell cultures of Hypericum androaemum | Involvement of a cinnamoyl CoA hydratase/lyase in non-oxidative chain shortening | El-Mawla et al. 2002

Enzyme isolation from V. planifolia cell cultures | Thiol-dependent non-oxidative conversion of 4-coumarate to benzaldehyde | Podstolski et al. 2002

L-phenylalanine, and that the hydroxyl group at the 4-position of the aromatic ring (para to the side chain) therefore originates through the action of the cytochrome P450 enzyme cinnamate 4-hydroxylase (C4H, Figure 18.1). This model is supported by labeling studies (Zenk 1965). The conversion of coumarate (4-hydroxycinnamate) to vanillin then “simply” requires four steps:

I shortening of the side chain by two carbons, catalyzed by a “chain shortening” enzyme or enzyme complex (CSE);

II introduction of the aldehyde function to the side chain (in some models this may occur as an integral part of chain shortening;

III introduction of the 3-hydroxyl group; and

IV 3-O-methylation (Figure 18.1).

Clearly the O-methylation reaction has to occur after the 3-hydroxylation, but, apart from this, these reactions could theoretically occur in any order. However, the number of possible theoretical pathways to vanillin is increased beyond three factorial by the fact that there is more than one mechanism for chain shortening of hydroxcycinnamic acids, and these lead to products with different oxidation states of the terminal group of the side-chain. Furthermore, if the model assumes a shared pathway to that involved in monolignol biosynthesis in which the first reactions are the ring modifications, additional reactions associated with formation of different types of ester intermediates could likely also be involved (Figure 18.1).