# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 4 Genetic Engineering Approaches
### Genetic Engineering in Model Microorganisms
#### 4.3.3 Engineering of Chassis  
The major barrier to use _Escherichia coli_ and _Saccharomyces cerevisiae_ for vanillin biosynthesis is the rapid conversion of vanillin into undesired vanillyl alcohol by several endogenous enzymes. The degradation of desired aldehydes into undesired alcohols can be minimised by the deletion of alcohol dehydrogenases (ADH), aldosterone reductases (AKR), and/or aldehyde reductases (ALDR). However, it may be difficult to completely prevent vanillin reduction due to the catalytic promiscuity and presence of multiple candidate enzymes. [PERSON] et al. [108] generated a _E. coli_ RARE strain, with a reduced aromatic aldehyde reduction ability, by the rational target deletion of six genes, encoding three AKR and three ADH, based on the reported activities of these enzymes on benzaldehyde. This _E. coli_ RARE strain carrying a de novo vanillin biosynthetic pathway (Figure 12A Blue) and a feedback-resistant form of _E. coli_ AroG (_aroG_*, DAHP synthase) produced about 120 mg/L of vanillin from glucose (Figure 13(A2)), while the wild-type strain only produced trace amounts of vanillin [108]. Likewise, [PERSON] et al. [88] improved vanillin production from eugenol (Figure 7(B2)) by about 8-fold by the inactivation of two ALDRs (_yjB_ and _yqhD_) and a choline dehydrogenase (_betA_) in _E. coli_ BW25113. In _S. cerevisiae_, [PERSON] et al. [103] performed a genome-wide search of enzymes with vanillin reductase activity (ADH, AKR, and/or ALDR) and selected 29 candidates to study their ability to reduce vanillin. The analysis of the deletion mutants showed that ADH6 was the most active vanillin reductase in _S. cerevisiae_. Thus, the authors constructed an _adh6_ mutant strain for vanillin biosynthesis and suggested that the inactivation of additional reductases would result in a significant increase in vanillin production [103]. Later on, the same and/or additional vanillin reductase candidates were investigated by simple and/or multiple deletions [102; 113; 114]. Overall, these studies explored 36 candidates and  
Figure 14: **Implementation of a de novo biosynthetic pathway to produce vanillin from a PET-derived monomer (terephthalate acid). ** (**A**) Schematic representation of the biosynthetic pathway and the used constructions to produce vanillin in _E. coli_. (**B**) Table summarising the function and origin of the genes/enzymes used. showed that multiple incidences of the inactivation of enzymes with vanillin reductase activity is necessary to efficiently reduce the degradation of vanillin into vanillyl alcohol in _S. cerevisiae_[102; 109; 113; 114]. [PERSON] and [PERSON] [112] recently designed and constructed _S. cerevisiae_ MARE strains, with a minimal aromatic aldehyde reduction ability, in which more than 10 oxidoreductase genes were inactivated. The JS-MARE3 strain was obtained by the deletion of 12 genes, encoding five ADH, four AKR, and three ALDR. To confirm the potential of this yeast platform for the de novo biosynthesis of vanillin, the de novo biosynthetic pathway via DHS was tested (Figure 12A Blue). Here, this pathway was constituted by 3 DSD from _Podospora anserina_ (_aroZ_ gene), OMT from _Homo sapiens_, ACAR from _Segniliparus rotundus_, and PPTase from _Nocardia_. JS-VA1a (Figure 13A(6)) was obtained by the integration of genes into the JS-MARE3 chromosome, and produced 104 mg/L of vanillin with no detection of vanillyl alcohol, contrarily to the control strain, in which vanillyl alcohol was accumulated and only a trace amount of vanillin was found [112]. Optimisation of Metabolic Fluxes to Improve Cofactor and Precursor Supplies  
CoA-dependent vanillin biosynthetic pathways require the constant availability of this cofactor to achieve high-yield vanillin production. [PERSON] et al. [121] designed a CoA regeneration system in _E. coli_ BW25113 by the overexpression of _gltA_ and the deletion of _icdA_ genes.