# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 5 Discussion
Thus, resting cells have been used to bypass isoeugenol toxicity and produce a high concentration of vanillin (28.3 g/L) [85]. The use of scaffolds to co-localise enzymes and avoid the loss of intermediates has been proposed [146]. Ferulic acid can be efficiently converted into vanillin by non-recombinant vanillin-producing strains and by recombinant strains. However, the availability of ferulic acid is low and variable, and the price of it can fluctuate significantly, increasing production costs. Thus, the biosynthesis of vanillin from simple carbon sources (such as sugars or amino acids) or alternative sources (e.g., agro-waste and plastic waste) has been investigated to avoid this problem and reduce costs. The implementation of the ferulate [94; 96] and benzoate [97] biosynthetic pathways for vanillin production using aromatic amino acids has been reported in _E. coli_ and yeast. Moreover, de novo biosynthetic pathways have been designed to produce vanillin from glucose in specific _E. coli_[108], _Corynebacterium glutamicum_[111], and yeast strains [109; 112]. In addition, a de novo pathway was established in _E. coli_ to produce vanillin from terephthalic acid (TA), a PET-derived monomer [120]. In all cases, even after extensive optimisation, yields were below 0.5 g/L (Table 1), suggesting that optimisations are still required to achieve industrially attractive yields. Explanations for these low yields include poor gene expression, low enzyme activity and specificity, low cofactor availability, the deviation of intermediates to undesired pathways, and vanillin toxicity. The enzymes involved in these pathways have low activity and selectivity. For instance, the enzymes FCS and ECH, which convert ferulic acid to vanillin, can also convert 4-coumaric acid and caffe acid into 4-hydroxybenzaldehyde and protocatechualdehyde, respectively [59; 147]. Moreover, enzymes catalysing a 3-hydroxylation reaction, such as cytochromes P450 or monooxygenases, have been reported to have a broad spectrum of substrates, acting on different phenolic compounds [148; 149]. [PERSON] et al. [150] performed site-directed mutagenesis in the gene encoding CYP199A2 from _Rhodospseudomonas palustris_ to obtain cytochrome P450 mutants with improved catalytic properties. A F185L mutant exhibits 5.5-fold higher activity on 4-coumaric acid than the wild-type enzyme. _E. coli_ cells expressing this mutated enzyme produced 2.5 g/L of 4-coumaric acid in 24h [150]. Therefore, identifying and characterising new enzymes [151; 152], or mutagenesis of the existing ones [153], to increase their activity and selectivity is a prerequisite to improving vanillin yields. Vanillin-biosensors, enabling the efficient and rapid detection of vanillin facilitates the screening of strains for vanillin production optimisation, allowing the development of high throughput methods. For example, vanillin-inducible promoters were used to construct a vanillin-sensing _E. coli_, which was used to screen new enzymes able to degrade lignin and convert its derivates to vanillin [88; 154]. Similarly, vanillin-sensing _E. coli_ was designed based on the VanR-VanO system and has been used to select an efficient OMT for the conversion of protocatechuate to vanillate [155]. Recently, the vanillin-inducible promoter _ADH7_ from _S. paradoxus_ was used to screen aromatic transporter candidates and to construct a vanillin-induced bidirectional transport system in _E. coli_[135]. Compared to vanillin-producing microorganisms, _E. coli_ and yeast are highly sensitive to vanillin. Common vanillin detoxification mechanisms are based on vanillin degradation, which is not desired during vanillin production processes. Thus, suitable recovery strategies have mainly prevented vanillin toxicity [133; 134]. The overexpression of vanillin-dedicated membrane transport proteins might be an effective genetic engineering strategy to develop vanillin-tolerant strains [130; 156].