# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 5 Discussion  
Vanillin is the flavouring agent most used in the world. Besides extraction from vanilla pods, vanillin can be produced using chemical or biotechnological approaches (Figure 15). According to flavourists, vanillin alone does not constitute vanilla flavour, characterised by floral, smoky, sweet, butter, tobacco-like, and woody notes [136]. Therefore, bio-vanillin will not replace vanilla extract, but might replace synthetic vanillin with a \"natural flavour\" at an affordable price. Several biotechnological methods, summarised in Figure 15, have been established and optimised to produce bio-vanillin. Biotechnological methods have the advantage of using low quantities of safe chemicals and, therefore, are environmentally friendly. These methods have been developed based on the knowledge acquired on the reactions/enzymes involved in vanillin biosynthesis in _Vanilla_ and vanillin-producing microorganisms. They can be classified as plant-, enzyme-, or microorganism-based approaches according to the platform used for production [11]. Plant-based approaches involve the use of plant tissues to biosynthesis earvanillin. Some examples reported the heterologous gene expression in beetroot [137], chilli pepper [24], and rice [25] of genes involved in the conversion of ferulic acid to vanillin. However, the yield of vanillin production was insignificant due to the slow growth of plant cells and the weak expression of the biosynthetic enzymes. Enzyme-based approaches generate better vanillin yields and have been described for vanillin production from ferulic acid [104; 138], curcumin [139], vanillyl alcohol [140; 141], and isoeigenpol [142]. However, the high costs associated with enzyme production, purification, and formulation reduce the use of these approaches to produce vanillin industrially because of their low economic viability [11]. In this context, to bypass enzyme purification costs, some studies proposed the use of whole-cell catalysis for vanillin production. For instance, the conversion of 4-n-propylguaiacol (4 PG), one of the main components of lignin oil, into vanillin was reported using recombinant _E. coli_ resting cells [143]. First, 4 PG was transformed into isoeigenpol, using an _E. coli_ strain expressing a bacterially engineered eugenol oxygenase. Then, a second strain carrying a non-heme-dependent dioxygenase mutant was used to convert isoeigenpol into vanillin [143]. In another study, lignin-based vanilly alcohol was converted into vanillin using recombinant _E. coli_ resting cells expressing 5-hydroxymethylfurfural oxidase [144]. Microorganism-based approaches for bio-vanillin production are more attractive due to the metabolic versatility, the ease of the genetic manipulation of microorganisms, and the low production cost. Different genetic engineering approaches have been explored to improve endogenous vanillin biosynthetic pathways or implement exogenous ones in microorganisms. First, vanillin-producing bacteria have been genetically modified to improve the bioconversion of ferulic acid into vanillin. On the one hand, the overexpression of endogenous genes involved in vanillin biosynthesis (_fcs_ and _ech_) allowed an improvement of the final vanillin concentration and of the molar yield (Table 1) [71]. An even better molar yield (95%) was obtained after the deletion of _vdh_ and the overexpression of _ech_ and _fcs_ genes in _Amyclatoppsis_ sp. ATCC39116 [70]. These approaches were efficient since the molar yields were very satisfactory (close to 100%). On the other hand, an improvement in vanillin production could be achieved by deleting