# 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
The strain used was designed to overproduce PCA from chorismate by modulating the primary metabolism through multiple gene deletion, including _trpE_ (encoding an anthranilate synthase, involved in tryptophan synthesis), _csm_ (encoding a chorismate mutase, responsible for the conversion of chorismite into prephenate), and _pcaHG_ (encoding a protocatechuate dioxygenase, responsible for the degradation of PCA into \(\beta\)-carboxy-muconic acid), and the heterologous expression of genes encoding feedback-resistant variants of AroG (_aroG\({}^{\text{FRB}}\)_, DAHP synthase) and CPL (_ubiC_, chorismate-pyruvate lyase) from _E. coli_. The conversion of PCA to vanillin involved two enzymes: an ACAR from the _Nocardiia_ genus and a mutated OMT from _Rattus norvegicus_. Synthetic genes encoding these enzymes were introduced in compatible self-replicative plasmids and transferred into the host strain (Figure 13(A4)). Vanillin could only be produced when the genes _vdh_, _vanAB_, and _NCgl0324_ (encoding a putative aromatic aldehyde reductase) were also deleted to prevent vanillin degradation [111]. Figure 13: **Implementation of de novo biosynthetic pathways to produce vanillin from glucose in different model chassis. ****(A1–A6)** Schematic representation of the genetic constructions used to implement these pathways. Different transcriptional terminators are represented by different colors. Coloured arrows represent genes encoding enzymes involved in different metabolic steps. Blue—conversion of dehydrohoshiximic acid into vanillin; Orange—conversion of chorismate into protocatechic acid; Yellow—conversion of HPP into protocatechaldehyde; Magenta—conversion of vanillin in vanillin glucoside; Grey—genes involved in primary metabolism which are overexpressed for chassis optimisation; Red rectangle within an arrow or \(\Delta\)—endogenous genes deleted for chassis optimisation; **(B)** Table summarising the origin of the heterologous genes. List of genes deleted in model microorganisms to improve vanillin production: _adh6_—alcohol dehydrogenase; _aroE_—shikimate dehydrogenase; _gdh1_—glutamate dehydrogenase I; _gpp1_—glycerol-1-phosphates; MARE—multiple-aldehyde reductases; _melt_—transcription regulator of methionine biosynthetic cluster; _NCgl0324_—aromatic aldehyde reductase; _pcaHGC_—protocatechuate dioxygenase; _pdc1_—pyruvate  
decarboxylasease; _serA_--phosphoglycerate dehydrogenase; RARE--multiple-aldehyde reductaseases; _trpE_--anthranulate synthase; _csm_--chorismate mutase; _vdh_--vanillin dehydrogenase; _vnuAB_--vanillate demethylase. List of endogenous genes overexpressed (in bold) for chassis optimisation: _aroB_--DHQ synthase; _aroC_--3-dehydroshikimate dehydratase; _gdh2_--glutamate dehydrogenase; II; _luxS_--S_-ribosylhomocysteine lyase; _mtn_ -5_'-methylthioadenosine/S-adenosyl/homocysteine nucleosidase; _ppsA_--phosphoenolpyruvate synthase; _serA_--phosphoglycerate dehydrogenase; _tktA_--transketolase. Recently, [PERSON] and [PERSON] [112] explored, in a _S. cerevisiae_ MARE strain, with a minimal aromatic aldehyde reduction ability (see Section Reduction of Vanillin Degradation), the implementation of these two de novo bioconversion pathways from the shimikate pathway: the one diverting DHS (Figure 12A Blue), previously applied in a _S. cerevisiae_ strain [109; 110; 113; 114], and the one based on the chorismate deviation for the overproduction of PCA, previously applied in _Corynebacterium_[111] (Figure 12A Orange); but also of a third one, diverting hydroxyphenylpyruvate (HPPP) from the prephenate pathway. This third biosynthetic pathway from glucose to vanillin is based on the hydroxymandelate degradation pathway for the overproduction of protocatechualdehyde (Figure 12A Yellow), and had previously been implemented by the same group in _E. coli_[115; 116]. Here, the hydroxymandelate degradation pathway consisted of four enzymes: a hydroxymandelate synthase (HmaS) from _Amyclatopopsis orientalis_, a two-component flavin-dependent monooxygenase (HpaBC) from _E.