# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 4 Genetic Engineering Approaches  
### Overview  
Bio-vanillin can be produced by vanillin-producing or model microorganisms. The advantages and drawbacks of each host chassis for vanillin biosynthesis are summarised in Figure 3. On the one hand, vanillin-producing microorganisms can efficiently produce vanillin and are highly tolerant to vanillin, to the precursors commonly used, and the biosynthetic intermediates. These strains contain the bioconversion pathways for vanillin synthesis from eugenol, isoeigenenol, and/or ferulic acid. Vanillin-producing strains used for the industrial bioconversion of ferulic acid into vanillin are not laboratory strains. Most of the time, efficient genetic tools are lacking or limited, hampering their genetic manipulation [60; 64]. Moreover, little is known about the native metabolic pathways in which the vanillin biosynthetic genes are involved or the regulation of their expression in these microorganisms [65; 66].  
On the other hand, model microorganisms, such as _Escherichia coli_, _Saccharomyces cerevisiae_, _Corynebacterium glutamicum_, and _Bacillus subtilis_, commonly used in industrial bioconversion processes, benefit from optimised fermentation conditions. A wealth of genetic and synthetic biology tools are available for pathway identification, prediction, reconstruction, and/or implementation in these species [67]. However, vanillin toxicity represents a major problem, as model microorganisms are highly sensitive to this compound and its aldehyde biosynthetic intermediates. Moreover, the intensive optimisation of strains/pathways is often required to reach high vanillin yields.  
Despite their different levels of resistance to vanillin, both vanillin-producing and model microorganisms possess resistance mechanisms to reduce the strong antimicrobial activity of vanillin. Common defence mechanisms are the transport of vanillin to an extracellular medium or/and the degradation of vanillin into less toxic compounds.  
Figure 3: Vanillin production using different chassis: Advantages; Drawbacks; and Genetic engineering approaches.  
Vanillin degradation in microorganisms can be performed by reductases and oxidases, which are able to convert it into vanilllyl alcohol and vanillic acid, respectively, reducing vanillin yields.  
To overcome the aforementioned limitations, genetic engineering strategies are implemented in both types of chassis. In vanillin-producing microorganisms, they aim at: (i) improving vanillin yields by overexpressing biosynthetic genes or by inactivating genes involved in vanillin catabolism to avoid its degradation; and (ii) reducing production costs by introducing genes directing the synthesis of biosynthetic precursors from simple or cheaper substrates. In model microorganisms, genetic engineering approaches aim at: (i) implementing nature-inspired vanillin biosynthetic pathways to produce vanillin from aromatic amino acids; (ii) implementing and optimising de novo vanillin biosynthetic pathways; and (iii) engineering the chassis to develop strains with lower vanillin degradation activity and higher vanillin productivity and tolerance. The following sections describe in detail each of these approaches.