# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 4 Genetic Engineering Approaches
### Genetic Engineering in Model Microorganisms
#### 4.3.2 Implementation of De Novo Bioconversion Pathways  
De Novo Bioconversion Pathway from Ferulic Acid  
Implementing phenylpropanoloid bioconversion pathways in _E. coli_ allowed us to achieve high yields of vanillin (>70%) from ferulic acid. Moreover, the production of trace amounts of vanillin from aromatic amino acids in _E. coli_ and S. cerevisiae_ was achieved by mimicking ferulate and benzoate pathways. However, the industrial implementation of these processes is complicated and energetically costly for the producing microorganism since the applied biosynthetic pathways require ATP and CoA as cofactors, which must be continuously supplied in large amounts to achieve high-yield production. The design of de novo coenzyme A-independent biosynthetic pathways can circumvent this limitation. In this context, [PERSON] et al. [103] reported the introduction of a de novo biosynthetic pathway in _E. coli_ to produce vanillin from ferulic acid via a CoA-independent route (Figure 11). Here, ferulic acid is first converted to 4-vinylguaiacol by a CoA-independent ferulic acid decarboxylase (FDC), which is then transformed into vanillin by a carotenoid oxygenase (CSO). In this study, 6.8 mM of vanillin was synthetised from 10 mM of ferulic acid using an _E. coli_ strain expressing the genes _fdc_ and _cso2_. Moreover, researchers demonstrated that the optimal conditions for both reactions are largely different [103]. Later on, the same group obtained a yield of 52 mM of vanillin (from 75 mM of ferulic acid) in a two-stage process using two recombinant _E. coli_ BL21 (DE3) (Figure 11(A1)) [104]. The first recombinant strain, harbouring the _fdc_ gene from _Bacillus pumilus_ under the control of the T7 promoter, completely converted the ferulic acid into 4-vinylguaiacol. The second one, expressing the _cso2_ gene (from _Caulobacter segnis_) and overexpressing chaperonins, converted a maximum of 69% of 4-vinylguaiacol into vanillin when the reaction was carried out in a biphasic system to avoid substrate and product toxicity [104]. The co-overexpression of the _groES_ and _groEL_ genes (encoding the chaperonin GroEL and the cochaperonin GroES) was required to efficiently produce CSO as a soluble protein [103]. Figure 11: Vanillin production from ferulic acid by a de novo CoA-independent decarboxylation pathway. (**A**) Schematic representation of the pathway and of the constructions used. (**B**) Table summarising the function and origin of genes/enzymes used. ADO* represents a modified ADO table to convert ferulic acid directly into vanillin. In both studies by [PERSON] et al. [103; 104], the limiting step was the one catalysed by Cso2, even when optimal conditions (a low temperature and alkaline pH) were used. Thus, the identification of efficient and robust oxygenases is vital to improve vanillin yields using this pathway. [PERSON] et al. [105] identified a thermoresistant aromatic dioxygenase (ADO) from _Thermothelemoyces thermophile_, able to convert 4-vinylguaiacol into vanillin. The conversion of ferulic acid to vanillin was tested using _E. coli_ BL23 (DE3) co-expressing _ado_ and _pad_ (PAD, phenolic acid decarboxylase from _Bacillus coagul_s) genes (Figure 11(A2)). This strain could produce 7.1 mM of vanillin from 30 mM of ferulic acid. However, the reaction catalysed by ADO was still the limiting step and endogenous ADHs were very active in these conditions since ~13 mM of 4-vinylguaiocl was accumulated and 9.7 mM of vanillin acid was formed. The optimisation of cultures conditions resulted in the production of 27.7 mM of vanillin and limited the accumulation of vanillic acid and 4-vinylguaiocl to 0.2 mM and ~2 mM, respectively [105]. The molar vanillin yield obtained in this study (92%) is higher than the ones obtained in studies describing the use of the natural CoA-dependent pathway, so the industrial implementation of CoA-independent pathways may have significant advantages. [PERSON] et al.