# 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 implementation of this CoA regeneration system enhanced vanillin production from ferulic acid by 2.8 times, with the molar conversion yield increasing from 34% to 94% compared to the parental strain (Figure 8A2). The overexpression of _gltA_, encoding a citrate synthase, increases the conversion of acetyl-CoA (generated during vanillin biosynthesis from ferulic acid) and oxaloacetate into CoA and citrate, respectively. The deletion of _icdA_, encoding an isocitrate dehydrogenase, results in isocitrate flowing through the glyoxylate bypass, a shorter route than the tricarboxylic acid cycle to obtain oxaloacetate [121]. Similarly, [PERSON] et al. [88] improved the CCR activity and, consequently, vanillin production from eugenol (Figure 7B2) by the overexpression of _gltA_ and the deletion of _ptsH_, encoding a phosphocharrier protein whose inactivation leads to phosphoenolpyruvate accumulation, stimulating the conversion of acetyl-CoA into CoA and ATP. To achieve a better vanillin yield, the identification of the limiting steps in de novo vanillin biosynthetic pathways and their optimisation by diverse strategies are essential. In 1998, [PERSON] and [PERSON] [107] reported that the improvement of the methylation step was essential for the do novo biosynthesis of vanillin and suggested that S-adenosyl-L-methionine (SAM, a cofactor required for methylation) availability may be limiting in vivo. Later, aiming to improve vanillin yields, [PERSON] et al. [122] increased the availability of DHS precursors and SAM. For that, first, the authors overexpressed the _ppsA_ and _tktA_ genes (encoding a PEP synthase and transketolase, respectively) to increase the formation of DAHP in _E. coli_ RARE. Then, they designed and implemented a SAM regeneration system (Figure 12B) based on the deletion of _metl_ (a transcription regulator of the methionine biosynthetic cluster) and the overexpression of the native _mtn_ and _luxS_ genes, encoding a 5\({}^{\prime}\)-methylthioadenosine/S-adenosylhomocysteine nucleoside and a S-ribosylhomocysteine lyase, respectively. The resulting strain (Figure 13A2)) synthetised 256 mg/L of vanillin, and its supplementation with methionine (SAM precursor) resulted in 419 mg/L of vanillin [122]. To avoid vanillin toxicity and improve vanillin yields in _S. cerevisiae_, [PERSON] et al. [123] expressed the _UGT_ gene and applied an in silico metabolic modelling analysis to identify bottlenecks in the metabolic flow for vanillin \(\beta\)-D-glucoside biosynthesis. Based on this analysis, two genes, _PDCI_ (pyruvate decarboxylase) and _GDH1_ (glutamate dehydrogenase), were identified as target genes whose inactivation might improve vanillin \(\beta\)-D-glucose production. The deletion of these genes was predicted to increase the availability of ATP and NADPH [124], two cofactors required for vanillin biosynthesis. In addition, researchers overexpressed GDH2 to ensure sufficient nitrogen uptake (Figure 12C). In batch cultivation, a 1.6-fold higher synthesis of vanillin \(\beta\)-D-glucose was obtained using this engineered strain (Figure 13A3)) in comparison to the parental strain (expressing the _UGT_ gene) [123]. Despite this yield improvement, the accumulation of PCA and protocatechualdehyde intermediates suggested a limiting step of downstream PCA formation [123]. [PERSON] and [PERSON] [125] reduced the accumulation of protocatechualdehyde and increased the production of vanillin \(\beta\)-D-glucose by up to 380 mg/L by overexpressing the OMT gene in the previously engineered strain (Figure 13(A3)). [125]. Later on, the implementation of the de novo biosynthetic pathway via DHS into the _S. cerevisiae_ JS-MARE3 chromosome by [PERSON] and [PERSON] [112] resulted in 104 mg/L of vanillin and a high accumulation of PCA (375 mg/L), indicating that ACAR and/or OMT reactions are limiting steps.