# 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
coli_, a hydroxymandelate oxidase (HMO) from _Streptomyces coelicolor_, and a benzoylormate decarboxylase (BFD) from _Pseudomonas putida_[112]. The plasmid-based expression of this pathway confirmed its functionality in _S. cerevisiae_ MARE, yielding 20 mg/L of vanillin (Figure 13(A5)). [PERSON] and [PERSON] [112] also proposed the implementation of dual precursor biosynthetic pathways. First, a vanillin biosynthetic pathway diverting DHS was implemented and optimised, yielding the JS-VA6 strain (Figure 13(A6)), which produced 217 mg/L of vanillin from 20 g/L of glucose (see Section Optimisation of Metabolic Fluxes to Improve Cofactor and Precursor Supplies). Then, one of the two other de novo biosynthetic pathways were introduced into the JS-VA6 strain. It appeared that the hydroxymandelate degradation pathway for the overproduction of protocatechualdehyde was not compatible since the ACAR activity seemed to be repressed by hydroxymandelate analogues [112]. Alternatively, the compatibility of the chorismate deviation for the overproduction of PCA was tested by the integration of _ubiC_ from _E. coli_ and _pobA_ (encoding hydroxybenzoate hydroxylase) from _P. putida_ at the _gdh1_ (encoding a glutamate dehydrogenase) locus to further reduce NADPH consumption. The resulting strain JS-VA8 increased vanillin production (262 mg/L) compared to the parental strain JS-VA6 (Figure 13(A6)). From PET Monomers  
A circular economy aims at minimising adverse environmental impacts of manufacturing. In this context, the use of plastic waste to obtain value-added products has attracted significant attention in recent years [117; 118]. Polyvinyl alcohol and polyethylene terephthalate (PET) are polymers with widespread applications (e.g., textile industry, pigment binder, and plastic bottle production). However, these polymers are important pollutants and new methods to degrade and valorise them are required for an efficient circular economy [117; 118]. [PERSON] et al. [119] have identified a possible enzyme implicated in the bioconversion of polyvinyl alcohol to vanillin by _Pharerachaete chrysosporium_. Recently, [PERSON] and [PERSON] [120] reported the development of an engineered _E. coli_ for the direct bioconversion of terephthalatic acid (TA), a PET-derived monomer, into vanillin. To this end, they implemented in _E. coli_ RARE a de novo pathway comprising four steps (Figure 14). First, the _thpA1_, _tphA2_, _tphB2_, and _dcddh_ genes, encoding the enzymes involved in the conversion of TA into PCA, were assembled in a single operon under the control of the T7 promoter in pET21. The _comt_ and _car_ genes, required for the transformation of PCA to vanillin, were each cloned under the control of the T7 promoter in pRSF-Duet. In addition,a third plasmid comprising the _sfp_ gene, encoding a PPTase, was constructed [120]. Cells co-transformed with all three vectors (Figure 14) produced 0.76 mg/L of vanillin from 5 mM of TA. After extensively optimising the bioconversion conditions (such as the culture media, pH, temperature, and the addition of the cofactor's precursors), a maximum vanillin titre of 119 mg/L was obtained [120].