# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 2 Vanillin Biosynthesis in _Vanilla_
planifolia_ and reported converting ferulic acid into vanillin, but also coumaric acid into 4-hydroxybenzaldehyde and caffe acid into protocatechualdehyde in the presence of Fe\({}^{2+}\) and thiol reagents [21]. Notably, in the presence of glutathione, ferulic acid and 4-coumaric acid were converted at similar rates into vanillin and 4-hydroxybenzaldehyde, respectively [21]. In 2017, the [PERSON] group [22] re-evaluated the activity of the 4 HBS enzyme (which they previously described) since they had discovered that the gene encoding this enzyme was in fact the same as the _VpVan_ gene studied by [PERSON] et al. [20; 23]. Despite extensive efforts, the [PERSON] group failed to demonstrate any enzymatic activity for VpVAN in various in vitro and in vivo assays, although this could be attributed to a lack of codon optimisation of the _VpVan_ gene [22]. Yet, several independent studies using heterologous systems support the ability of VpVAN to convert ferulic acid into vanillin [15; 24; 25]. Enzymes catalysing the introduction of the C3-hydroxyl group have yet to be fully characterised. Studies on lignin biosynthesis in plants other than _Vanilla_ indicate that the 3-hydroxylation could be carried out by a polyphenol oxidase, a dioxygenase, or a cytochrome P450 reductase (CPR). For example, the CPR CYP98A3 from _Arabidopsis_, a 3-monooxygenase, can convert 4-coumaric acid into caffe acid, albeit very slowly [26]. Recombinant CYP98A3 expressed in yeast metabolised the 5-O-shikimate and 5-O-D-quinate esters of coumarate into the corresponding caffeine conjugates more efficiently than free 4-coumaric acid [27; 28]. These findings suggest that 3-hydroxylation may result from multiple reactions requiring several enzymes, including 4-coumaric acid-CoA ligases and hydroxycinnamoyl transferases [27]. Recently, [PERSON] et al. [29] identified and characterised an enzyme from maize that directly catalyses the 3-hydroxylation of 4-coumaric acid to caffe acid. This enzyme, possessing 4-coumaric acid 3-hydroxylase (C3H) activity, was annotated as ascorbate oxidase (APX1) with, putative orthologs identified in many plants, including in _V. planifolia_ (e.g., KAG0466359.1 with 81% identity). The analysis of APX1 deletion mutants in _Brachprodium_ and _Arabidopsis_ supports its involvement in the early steps of lignin biosynthesis [29]. The ability of CYP98A3 or APX1 to convert 4-hydroxybenzaldehyde to protocatechualdehyde (also known as 3,4-dihydroxybenzaldehyde) was not assessed. The methylation reaction is catalysed by an O-methyltransferasease (OMT) which transfers the methyl group of S-adenosyl-I-methionine (SAM) to a hydroxyl group. This kind of reaction is widespread in plants; for instance, 15 OMTs were identified by sequence analysis in _V. planifolia_[20]. [PERSON] et al. [30] isolated and characterised a multifunctional OMT from _V. planifolia_, showing a preference for caffeoyl aldehyde and 5-OH-coniferaldehyde as substrates over protocatechualdehyde or caffe acid [30]. The biochemical characterisation of four other O-methyltransferaseases from _V. planifolia_ revealed no link to vanillin synthesis [31; 32]. In vanilla pools, vanillin is stored as a nontoxic glucoside, which is more soluble and less toxic than vanillin. UDP-glycostransferases (UGTs), which transfer uridine-diphosphate-activated sugars (UDP-sugar) to low-molecular-weight substrates, are responsible for this reaction [10; 33]. The timing of this reaction is unknown and glycosylation may occur before the hydroxylation and/or methylation steps. During the curing of vanilla beans, vanillin is released by \(\beta\)-glucosidase, which hydrolyse the glycosidic bond between the sugar and the aglycone [10; 34]. How vanillin is biosynthesis in _Vanilla_ appears to be still disputed and unclear, possibly because vanillin biosynthesis may follow various routes under varying conditions [17]. Further research is needed to identify the missing enzymes and to clarify the biosynthetic pathway(s).