# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 2 Vanillin Biosynthesis in _Vanilla_  
Early studies have established that vanillin is present in green _Vanilla_ beans as vanillin glucoside, which is hydrolys during curing to form the active flavour compound [10; 14; 15]. To elucidate the biosynthetic route to vanillin glucoside, green _Vanilla_ beans were fed with several [14C]-radiolelled molecules, including L-phenylalanine, L-tyrosine, shikimate intermediates, or lignin precursors [10; 16]. Based on these first experiments, vanillin biosynthesis in _Vanilla_ was assumed to be synthesised by the phenylpropanoid route [17]. It is generally accepted that vanillin is mainly produced from L-phenylalanine. A phenylalanine ammonia-lyase (PAL) converts L-phenylalanine to trans-cinnamic acid, which is then converted into 4-coumaric acid by a cinnamate-4-hydroxyylase (C4H) [18]. PAL enzymes may also exhibit tyrosine ammonia-lyase activity (TAL), forming 4-coumaric acid directly from tyrosine. Feeding experiments with [14C]-L-tyrosine or [14C]-L-phenylalanine confirmed the existence of both PAL and TAL activities in _Vanilla_ and suggested that PAL activity is ten times stronger than TAL activity [15]. The conversion of 4-coumaric acid to vanillin requires four steps: (a) the shortening of the side chain by two carbons; (b) the introduction of the aldehyde function; (c) the introduction of the hydroxyl group at position 3; and (d) the O-methylation of this hydroxyl group (Figure 1). While O-methylation must occur after the 3-hydroxylation, the actual order of these reactions is still unresolved [17]. Some models propose that the aldehyde function is introduced during the chain-shortening reaction [2; 11; 19]. Some of the enzymes catalysing these reactions are still unidentified or poorly characterised in _Vanilla_, and their role in vanillin biosynthesis has been inferred based on studies of orthologous enzymes in other plants [10]. Based on the [14C]-radiolbeck precursor incorporation and the analysis of in vitro enzyme activities, two main routes have been proposed: the \"benzoate pathway\" and the \"ferulate pathway\" (Figure 1). The benzoate pathway involves the initial reduction of 4-coumaric acid to 4-hydroxybenzaldehyde, followed by hydroxylation and methylation. In contrast, the ferulate pathway proposes the hydroxylation and methylation of 4-coumaric acid prior to the side-chain reduction of ferulic acid. The benzoate pathway was proposed by the [PERSON] group [16] who partially purified from _V. planifolia_ tissue cultures an enzyme, designated as 4-hydroxybenzaldehyde synthetase (4 HBS), catalysing in vitro the conversion of 4-coumaric acid into 4-hydroxybenzaldehyde. Conversion of 4-hydroxybenzaldehyde into vanillin was then proposed to proceed via hydroxylation at C3 followed by the methylation of the newly introduced hydroxyl group. On the other hand, in support of the ferulate pathway, [PERSON] et al. [20] identified in _V. planifolia_ an enzyme, which they named vanillin synthase, encoded by the _V.P.Van_ gene. Figure 1: **Proposed vanillin biosynthetic pathways through 4-hydroxybenzaldehyde and ferulic acid in _V. planifolia_**. Based on [PERSON] et al. [16], [PERSON] et al. [20], and [PERSON] and [PERSON] [21]. PAL: phenylalanine ammonia- lyase; C4H: cinnamate-4-hydroxyase; TAL: tyrosine ammonia- lyase; 4 HBS: 4-hydroxybenzaldehyde synthetase; PPDO: phenylpropanol 2,3-dioxygenase; CPR: cytochrome P450 reductase; OMT: O-methyltransferase; 4 CL: 4-hydroxycinnamoyl-CoA ligase; HCT: hydroxcinnamoyl transferase; C3H: 4-coumaric acid 3-hydroxyylase; VpVAN: vanillin synthase; UGT: UDP-glycosyltransferase; \(\beta\)-Gluc: \(\beta\)-glucosidase. When expressed in vitro, _Vp_VAN converted ferulic acid to vanillin, but showed no activity with 4-coumaric or caffe acids. Later on, another side chain shortening enzyme, named phenylpropanol 2,3-dioxygenase, was partially purified from _V.