# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 5 Discussion
\begin{table}
\begin{tabular}{c c c c c c} \hline
**Substrate** & **Strain** & **Figure/Section** & **Titre \({}^{*}\)** & **Yield \({}^{*}\)** & **Reference** \\ \hline Isoeugenol & _E. coli_ BL21 + [_item_] & Figure 7 & 28.37 g/L & 81\% & [85] \\ \hline Eugenol & _E. coli_ XL1-blue + [_vaoA,calA,calB,chf,cs_] & Sections Vanillin from & 0.3 g/L & 8\% & [87] \\ \cline{2-5}  & _E. coli_ BW25113 WT + [_vaoA,calA,cr,ch_] & _[PERSON]_ BW25113 Van-3 + [_vaoA,calA,cr,ch_] & Degradation & 10.68 mg/L \({}^{*}\) & 3.5\% & [88] \\ \cline{2-5}  & _[PERSON]_ JM109 construction A2 & & 1.1 g/L & 70\% & [90] \\ \cline{2-5}  & _[PERSON]_ JM109 construction A3 & Figure 8 & 0.36 g/L & 78\% & [91] \\ \cline{2-5}  & _[PERSON]_ JM109 construction A4 in & & & & \\  & fed-batch & & & & \\ \cline{2-5}  & _[PERSON]_ BW25113 WT + [_cchf,cs_] & Section Optimisation of & 0.79 g/L & 34\% & [121] \\ \cline{2-5}  & _[PERSON] coli_ BW25113 \(\Delta\)_icdA_ \(\uparrow\)_gltA_ + [_cchf,cs_] & Improve Cofactor and & 2.21 g/L & 92\% & \\ \cline{2-5}  & _[PERSON]. coli_ BL21 (DE3) + [_fdc_] & Figure 11 & & & \\  & _[PERSON]. [PERSON]_ BL21 (DE3) + [_cso_] & Section De Novo & 7.93 g/L & 69\% & [104] \\ \cline{2-5}  & _[PERSON]. coli_ BL21 (DE3) + [_ado,pad_] & from Ferulic Acid & 4.22 g/L & 92\% & [105] \\ \cline{2-5}  & _[PERSON]_ BL21 (DE3) + [_sam8,sam5,comt,fcs,ctl_] & Figure 9 & 97.2 mg/L & 5.8\% & [94] \\ \cline{2-5}  & _S. cerevisiae_ + [_sam8,sam5,comt,fcs,ctl_] & Section Vanillin from & 50.2 \(\mu\)g/L & NC & [96] \\ \cline{2-5}  & _s. cerevisiae_ + [_sam8,sam5,comt,fcs,ctl_] & & & & \\  & _aro_*4/no_\({}^{*}\)+ [_sam8,sam5,comt,fcs,ctl_] & & & & \\ \cline{2-5}  & _Phenylalanine_ & _S. cerevisiae_ YOOVAN & Figure 10 & - & NC & [97] \\ \cline{2-5}  & _S. cerevisiae_ YOOVAN & Section Vanillin from & & & \\  & YOCPVAN & Aromatic Amino Acids & 153 \(\mu\)g/L \({}^{*}\) & NC & [102] \\ \cline{2-5}  & _E. coli_ KL7 construction Figure 13(A1) + & & & & \\  & in vitro aldehyde carboxylic acid & & & & \\  & reductase & & & & \\ \cline{2-5}  & _E. coli_ RARE construction Figure 13(A2) & & 0.120 g/L & 1.2\% & [122] \\ \cline{2-5}  & _E. coli_ RARE \(\Delta\)_Metl_ construction & & & & \\  & Figure 13(A2) & & 0.256 g/L & 2.5\% & [122] \\ \cline{2-5}  & _S. pombe_\(\Delta\)_DH6_ construction & Figures 12 and 13 & & & \\ \cline{2-5} Glucose & Figure 13(A3) & Section De Novo & 65 mg/L & 0.4\% & [109] \\ \cline{2-5}  & _S. cerevisiae_\(\Delta\)_DH6_ construction & from Glucose & & & \\  & Figure 13(A3) & & 0.380 g/L & 1.1\% & [125] \\ \cline{2-5}  & _Coronpeacterium_ construction & & & & \\  & Figure 13(A4) & & & & \\ \cline{2-5}  & _S. cerevisiae_ JS-MARE3 VA1a & & 0.107 g/L & 0.6\% & \\ \cline{2-5}  & VA6 & & 0.217 g/L & 1.3\% & [112] \\ \cline{2-5}  & VA8 & & 0.262 g/L & 1.5\% & \\ \cline{2-5}  & VA9 & & 0.352 g/L & 3.5\% & \\ \hline \end{tabular}
\end{table} 
Table 1: _Cont._Even if not much is known about native regulation in these bacteria, approaches modifying the gene expression regulation might significantly affect vanillin production. Secondly, the heterologous expression of genes involved in vanillin biosynthetic pathways from phenylpropanoids has been achieved in _E. coli_[85; 86; 87; 89] and lactic acid bacteria [145]. Eugenol was converted to ferulic acid by genetically engineered _E. coli_ with a molar yield >90%; however, further transformation to vanillin was inefficient (Table 1) [87]. Several attempts to improve ferulic acid bioconversion in _E. coli_ have been tried and a final vanillin concentration of 4.3 g/L was obtained by the integration of the required genes (_fcs_ and _ech_) into the chromosome [92]. An even better yield (5.14 g/L) was obtained in _E. coli_ after the implementation of a CoA regeneration system, allowing a constant supply of this cofactor [121]. These investigations also allowed us to study the enzymes involved in ferulic acid bioconversion and to identify bottlenecks, including the substrate and product toxicity, the promiscuity of enzymes, and the genetic instability of recombinant strains, and remedy it.