# 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
[96] recently reported the implementation a similar pathway in _S. cerevisiae_ (Figure 9). Only the constructs giving the highest vanillin yields are described here, even if the authors tested three candidate genes encoding enzymes catalysing the conversion of 4-cumarnic acid to caffe acid (CYP199A2, _lpnBC_, and _sam5_). The chosen genes (_sam8_ and _sam5_ from _Saccharotnik expansensis_, _comt_ from _Arabidopsis thaliana_, and _fc_s and _ecch_ from _Streptomyces_ sp. V1) were codon-optimised, chemically synthesised, and assembled each with a promoter and a terminator. The selected pathway was introduced simultaneously in two yeast strains, a haploid and a diploid, by the integration of modules into a neutral  
Figure 8: **Vanillin production from ferulic acid in _E. coli._ (A) CoA-dependent non-\(\beta\)-oxidative deacetylation pathway and schematic representation of the construction used for its implementation. (B) Table summarising the function of genes/enzymes used. The origin of genes is indicated under the schematic representation of plasmids. **  
locus [96]. The amount of vanillin produced by the haploid strain (50.2 ug/L) was higher than the diploid strain, suggesting that the haploid strain is more suitable for vanillin production (Figure 9). Moreover, tyrosine availability in the cell was increased by integrating ARO4 and ARO7 feedback-resistant mutants (encoding a DAHP synthase and a chorismite mutase, respectively) at the delta 22 locus of the haploid strain, leading to the production of 700 ug/L of vanillin [96]. Another metabolic pathway for the bioconversion of L-phenylalanine to vanillin was proposed by Eviagenics in a patent (Figure 10) [97]. It mimics the benzoate pathway occurring in plants (as shown in Figure 1) [7,10], which had previously been the object of David Michael & Co., Inc. patents concerning the modification of cell plants [23,98]. This pathway comprises six main steps: (i) phenylalanine deamination; (ii) trans-cinnamic acid hydroxylation at position 4; (iii) coumaric acid side-chain reduction; (iv) 4-hydroxybenzoic acid hydroxylation at position 3; (v) PCA O-methylation; and vi) vanillic acid reduction. In its patent, Eviagenics suggests the use of five plant enzymes, namely PAL, C4H, CPR, 4 CL, and OMT, to be implemented into a specific yeast cell [97]. The conversion of cinnamic acid into coumaric acid was improved by the co-expression of genes coding for C4H and CPR [97]. To convert coumaoyl-CoA into 4-hydroxybenzaldehyde, a bacterial crotonase is used. Suggested enzymes are, for example, ECH from _P. fluorescens_ or from _Azotobacter vinelandii_. To convert the 4-hydroxybenzaldehyde into protocatechualdehyde, the authors described the use of an HBH enzyme instead of the plant 3-monooxygenase. HBH enzymes, coded by _pobA_ genes, have been identified in various microorganisms, where they are known to be involved in the catabolism of 4-coumaric acid into PCA [99,99,100,101]. However, like in those natural pathways, it is assumed here that endogenous dehydrogenases would be necessary to first convert the 4-hydroxybenzaldehyde into the corresponding acid. Thus, this pathway is more likely to lead to vanillic acid rather than vanillin (Figure 10). Figure 9: **Vanillin production from L-tyrosine by a female biosynthetic pathway. ** (**A**) Schematic representation of the biosynthetic pathway and of the constructions used to implement it in _E. coli_ or _S. cerevisiae_. Different transcriptional terminators are represented by different colors. (**B**) Table summarising the function and origin of the genes/enzymes used. Genes encoding the required enzymes were assembled as monocistronic units (i.e., each gene under the control of an inducible promoter and followed by a terminator) in _S. cerevisiae_, yielding the Y00 VAN-engineered strain.