# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 4 Genetic Engineering Approaches
### Genetic Engineering in Vanillin-Producing Microorganisms  
#### 4.2.1 Overexpression or Inactivation of Endogenous Genes  
Bacteria known to naturally convert phenylpropanoids into vanillin are primarily soil-dwelling bacteria that contribute to the decomposition of organic matters. They belong to the actinomycetes (_Amycolatopsis_ spp., _Streptomyces_ spp., _Rhodococcus_ spp., and _Arthobacter_ spp.) and gammaproteobacteria (_Pseudomonas_ spp.) classes. Efforts to optimise the conversion of ferulic acid to vanillin and/or limit vanillin catabolism have essentially focused on the bacteria of these genera. In _P. putida_ KT2440, both strategies (the optimisation of ferulic acid conversion and the limitation of vanillin degradation) have been carried out (Figure 4). To prevent vanillic acid formation, [PERSON] et al. [68] inactivated _vdh_ (encoding a vanillin dehydrogenase) and _mobABC_ (coding for a molybdate transporter). The inactivation of this transporter reduces the availability of molybdate, a cofactor required by oxidorectudases [68]. After 5 h conversion, the double mutant GN276 converted 6 mM ferulic acid into 4.8 mM vanillin and 0.3 mM of vanillic acid, while the wild-type strain only produced vanillic acid (3.9 mM). Vanillin bioconversion was further optimised by overexpressing _fcs_ and _ech_ under the control of the strong _tac_ promoter (A1 in Figure 4). The resulting strain, _P. putida_ GN299, converted nearly 10 mM of ferulic acid into 8.3 mM of vanillin, 0.2 mM of vanillic acid, and 1.1 mM of vanillyl alcohol after 5 h of conversion. To further reduce the vanillic acid formation, the authors deleted three other dehydrogenases, yielding the GN442 strain [68]. Recently, the expression of vanillin reductase genes (identified in the genome of _Pseudomonas_ sp. 9.1 and _P. putida_ KT2440 by sequence analysis and annotated as aromatic aldehyde reductase, _areA_) in _E. coli_ evidenced their capacity to reduce vanillin. Moreover, the deletion of the _areA_ gene in the _P. putida_ GN442 strain resulted in a decrease in vanillyl alcohol and an increase in vanillin production [69]. In _Amycolatopsis_ strains, the optimisation of vanillin production has also been achieved by the overexpression of the genes involved in bioconversion (Figure 4). For instance, the introduction of additional copies of _ech_ and _fcs_ under the control of the _ermE\({}^{*}\)_ promoter into the genome of _Amycolatopsis_ sp. ATCC 39116 [70] or _Amycolatopsis_ sp. _ZYL926_[71] resulted in an increase in vanillin production (A2 in Figure 4). Moreover, a recent patent application described an increase in vanillin production from ferulic acid following the inactivation of a gene encoding a ROK-family transcriptional regulator (_ROK-TR1_) in _Amycolatopsis_ sp. HM-141 [72]. This regulator might repress, directly or indirectly, the transcription of the genes involved in vanillin biosynthesis, as its overexpression reduces vanillin production compared to the wild-type strain. Several studies also aimed at limiting vanillin degradation (Figure 4). The genome sequencing of _Amcyclatoposis_ sp. ATCC 39116 [75] led to the identification of a vanillin dehydrogenase candidate gene _vdh_ whose deletion reduced by more than 90% vanillin degradation to vanillic acid [62; 70]. Additionally, the deletion of vanillic acid decarboxylase (_vdcBCD_) and vanillate demethylylase (_vanAB_) gene clusters showed that in _Amcyclatoposis_ sp. ATCC 39116, vanillic acid is mainly converted into guaiaocl by decarboxylation [60]. However, _vdh_ mutants can still produce vanillyl alcohol under fermentation conditions. Researchers from Rhodia Operations used five genes susceptible to encode enzymes reducing vanillin into vanillyl alcohol [73]. These aryl-alcohol dehydrogenases have been named \"vanillin reductases\" after the validation of their catalytic activity. According to the Rhodia Operations patent, the inactivation of the five _vr_ genes yielded an _Amcyclatoposis_ sp. ATCC 39116 strain that did not produce vanillyl alcohol [73]. In 2018, [PERSON] et al.