# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 4 Genetic Engineering Approaches
### Genetic Engineering in Model Microorganisms
#### 4.3.3 Engineering of Chassis
To improve the production of vanillin, the authors further conducted several successive genetic modifications aiming to: (1) improve the phosphopantetheinylation of ACAR by the additional expression of two PPTase from _Bacillus subtilis_ and _E. coli_ (_Sfp_ and _EntD_, respectively); (2) increase the amount of NADPH and ATP cofactors by the introduction of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapC) from _Clostridium acetobutylcium_ and a cytosol-relocalised NADH kinase (PosSc) from _S. cerevisiae_ (Figure 12C); and (3) improve the SAM supply and methylation step by the integration of a mutant _metK_* from _E. coli_, encoding a SAM synthetase variant (I303V, with higher activity and reduced product inhibition) together with an additional copy of OMT (Figure 12D). The resulting strain JS-VA6 (Figure 13(A6)) produced 217 mg/L of vanillin and significantly reduced PCA accumulation (\(\approx\)200 mg/L) compared to the parental strain JS-VA1a [112]. Additionally, a dual precursor biosynthetic pathway was implemented and the supply of D-erythrose 4-phosphate, a precursor of DHS, was improved by the introduction of two heterologous genes, _xfpk_ from _Bifidobacterium breve_ and _pta_ from _Clostridium kuyveri_ (encoding a phosphoketolase and a phosphotransferacetylase, respectively), at the _gpp1_ (encoding a glycerol-1-phosphate) locus to minimise acetate formation (Figure 12D). The final strain JS-V9 (Figure 13(A6)) produced 352 mg/L of vanillin, which represents the highest vanillin yield from glucose in yeast [112]. Improvement of Vanillin Tolerance  
A major problem when using model microorganisms as cell factories for vanillin production is the toxicity of this compound. Vanillin, bearing an aldehyde functional group, is highly reactive, causing an accumulation of reactive oxygen species (ROS). ROS mediates the oxidative degradation of unsaturated fatty acids on the membrane, which leads to cell death due to the disruption of the membrane integrity and the production of highly toxic aliphatic aldehydes derived from endogenous lipids [126]. Moreover, in _S. cerevisiae_, it was reported that vanillin represses the translation due to incorrect ribosome assembly, which causes the accumulation of processing bodies and stress granules composed of non-translating mRNAs and various proteins [127]. As described before, common vanillin resistance mechanisms for microorganisms are vanillin efflux and/or its degradation into less toxic compounds such as vanillyl alcohol and vanillin acid. Vanillin-tolerant yeast strains were obtained by over-expressing genes encoding reductases or dehydrogenases involved in vanillin detoxification [128]. However, as it decreases vanillin yields, such approaches are not suitable for vanillin production. Recently, transcriptomic, proteomic, and metabolomic studies were employed to understand the vanillin metabolic network and elucidate vanillin resistance mechanisms in microbes [57; 58; 65; 129; 66]. For instance, in a proteomic study supported by multiple physiological experiments and mutant analyses, [PERSON] et al. [130] have identified that an RND efflux gene (_acrD_) and two aromatic acid efflux genes (_aaeA_ and _aaeB_) are involved in vanillin resistance in _E. coli_. Similarly, vanillin tolerance was increased in _S. cerevisiae_ by the deletion of the YRR1 gene, which upregulated genes encoding ABC transporters, dehydrogenases, and proteins involved in ribosome biogenesis [131]. Therefore, increasing vanillin efflux by overexpressing vanillin-dedicated membrane transport proteins might be an effective strategy for engineering vanillin-tolerant hosts for vanillin synthesis. However, the identification of vanillin-specific transporters is difficult due to the presence of multiple transport proteins and their wide substrate specificity. For instance, dozens of transporter genes have been identified in vanillin-producing organisms (e.g., _P. putida_ KT440, _Amyclatoppsis_ sp. ATCC 39116) [65; 132].