# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 4 Genetic Engineering Approaches
### Genetic Engineering in Model Microorganisms  
#### 4.3.1 Implementation of Nature-Inspired Bioconversion Pathways Vanillin from Phenylpropanoids  
The second approach to the microbial production of vanillin involves the introduction of the vanillin biosynthetic pathway in model microorganisms. When considering the implementation of phenylpropanoid bioconversion pathways, _Escherichia coli_ has served as the primary chassis microorganism. During the past two decades, a number of engineered _E. coli_ strains have been developed to produce vanillin from eugenol, isoeugenol, or ferulic acid. The conversion of isoeugenol via the epoxide-diol pathway requires only an IEM (Figure 7A). [PERSON] et al. [85] chose to clone the _item_ gene of _Pseudomonas putida_ IE27 under the control of the T7 promoter (P\({}_{\overline{17}}\)) in _E. coli_ BL21(DE3). Resting cells of this recombinant strain produced 186 mM (\(\approx\)28 g/L) of vanillin from 230 mM of isoeugenol in 6 h [85]. Although the yield obtained in this experiment is very interesting (\(>\)80%), the use of resting cells at an industrial level is difficult due to the need for large-volume washing and centrifugation cycles. Recently, [PERSON] et al. [86] employed semi-rational protein engineering using a protein IEM720 (encoded by a homolog of _item_ isolated from metagenomic DNA) to create a F281Q variant with better enzymatic properties (higher \(k_{\text{cat}}\) and specific activity) than the wild-type enzyme [86]. Thus, protein engineering offers new opportunities for the industrially relevant optimisation of vanillin biosynthetic pathways. [PERSON] et al. [87] reported the development of a two-stage process for the biotransformation of eugenol to vanillin (Figure 7B). First, a recombinant _E. coli_ XL1-blue co-expressing _twoA_ from _Penicillium simplicissumum_ CBS 170.90 and _calA_ and _calB_ genes from _Pseudomonas_ sp. HR199 converted eugenol to ferulic acid with a molar yield \(>\)90%. Then, the conversion of ferulic acid to vanillin was tested by co-cultivating this strain with a second one co-expressing _fc_s and _ech_ from _Pseudomonas_ sp. HR199 (Figure 7(B1)) at a higher temperature to inactivate CALDH (which also possesses vanillin dehydrogenase activity). However, the conversion yield was only 8% and vanillin was further reduced to vanill alcohol [87]. Recently, [PERSON] et al. [88] showed that coniferly aldehyde inhibits the conversion of ferulic acid into vanillin by FCS and ECH and confirmed that CALDH oxidases vanillin to vanillic acid. To circumvent these drawbacks and simplify the pathway, the authors proposed the use of a cinnamyl-CoA reductase (CCR) from _Leucaemia leucoccephala_, which can directly convert coniferyl aldehyde to feruoly-CoA. The complete pathway, composed by VAO, CADH, CCR, and ECH enzymes, was implemented into _E. coli_ BW25113 (Figure 7(B2)), yielding 0.07 mM of vanillin and 1.92 mM of vanillyl alcohol from 2 mM of eugenol [88]. The reduction of vanillin to vanillic alcohol was catalysed by endogenous reductases, whose inactivation improved vanillin yields (see Section Reduction of Vanillin Degradation). Ferulic acid bioconversion by non-\(\beta\)-oxidative deacetylation (CoA dependent) pathway involves the expression of the _ech_ and _fc_s genes (Figure 8). [PERSON] et al. [89] cloned _ech_ and _fc_s genes from _Amycolatopsis_ sp. HR104 or _Delftia acidovorans_ under the control of the arabinose-inducible promoter (P\({}_{\text{BAD}}\)) into the pBAD24 vector. The highest vanillin production (0.58 g/L of vanillin from 1 g/L of ferulic acid) was obtained with the _E. coli_ strain carrying the _Amycolatopsis_ sp. HR104 genes (Figure 8(A1)) in LB medium [89]. In another study, this group cloned, the _Amycolatopsis_ sp. HR104 genes under the control of the _trc_ promoter (P\({}_{trc}\)) into the pTrc99A vector (Figure 8(A2)). The _E. coli_ strain carrying this construct produced 1.1 g/L of vanillin after 48 h of cultivation in 2 YT medium [90]. [PERSON] et al. [91] reported the use of resting cells of _E.