# 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
[65] reduced the formation of vanillyl alcohol over the entire course of the fermentation by deleting the _vtfG_ gene, encoding a putative oxidoreductase. In the same study, authors could also reduce the vanillic acid concentration using a mutant strain deleted for the _vdh_ gene and the putative ferulic acid \(\beta\)-oxidation gene cluster (_\(\Delta\)0689-0695_), including a _phdB_ gene encoding a 4-hydroxy-3-methoxyphenyl-\(\beta\)-hydroxypropionyl-CoA (HMPHP-CoA) dehydrogenase [65]. Similar results were recently obtained by [PERSON] et al. [74], who constructed and compared  
Figure 4: **Optimisation of vanillin biosynthesis from ferulic acid. ****(A)** Ferulic acid catabolic pathway and schematic representation of the constructions used to improve vanillin production; HMPHP-CoA: 4-hydroxy-3-methoxyphenyl-\(\beta\)-hydroxypropionyl CoA. (**B**) List of genes which have been inactivated in different microorganisms to increase vanillin production [60; 62; 65; 68; 69; 70; 73; 74]. vanillin production in the deletion mutants of _Amycolatopsis_ sp. CCTCC NO: M2011265. Compared to the single-mutant \(\Delta vdh\), the double mutant \(\Delta vdh\Delta pdh\) further increased vanillin and decreased vanillic acid production [74]. The valorisation of lignin-enriched residues for the production of high-value molecules, including vanillin, is very attractive for environmental, social, and economic reasons [76; 77]. Actinomycetes belonging to two genera, _Rhodococcus_ and _Arthrobacter_, have been studied for their potential to produce vanillin using lignin derivatives as sole sources of carbon and energy. A sequence analysis of _Rhodococcus_ I24 and PD630 led to the identification of _fcs_, _ech_, and _vdh_ genes. The inactivation of _fcs_ and _vdh_ genes in _Rhodococcus_ PD630 confirmed their involvement in the fertile acid catabolism and suggested vanillin as an intermediate of this catabolic pathway [36]. The whole genome sequencing of _Rhodococcus jostii_ RHA1 [78] allowed the identification and characterisation of genes involved in vanillin catabolism (_vdh_ and _vanAB_) by gene disruption and an enzyme activity assay [79]. Therefore, target pathway engineering approaches have been developed to improve vanillin production from lignin. For instance, [PERSON] et al. [80] noticed that a _vdh_ deletion mutant strain of _Rhodococcus jostii_ RHA1 (Figure 5A) accumulated 96 mg/L of vanillin when grown on a minimal medium containing wheat straw lignocellulosic. [PERSON] et al. [81] reported the development of recombinant strains of the lignin-degrading bacterium _Arthrobacter_ sp. C2 for the production of vanillin from corn stalks alkaline lignin. According to GC-MS analyses, alkaline lignin is first degraded into three types of monomers (H-, S-, and G-lignin units) through endogenous pathways of the _Arthrobacter_ sp. C2 strain (Figure 5B). G-units can be converted to vanill  
Figure 5: **Microbial vanillin biosynthesis from lignin. ** (**A**) Lignin degradation pathway with vanillin as an intermediate in _Rhodococcus jostii_ RHA1 and schematic representation of the method used for deletion of _vdh_, in order to increase vanillin production. (**B**) Lignin degradation pathway with vanillin as an intermediate in _Arthrobacter_ sp. C2 and schematic representation of the recombinant strain constructed to improve vanillin production. (**C**) Table summarising the function of the genes/enzymes used. transformed into vanillin by a vanillyl alcohol oxidase (_pchlF_, VAO). Vanillin is further degraded to vanillic acid, which is used as a carbon source after conversion into PCA by a benzaldehyde dehydrogenase (_xylC_, VDH) [81]. The authors have disrupted the _xylC_ gene and overexpressed the _pchlF_ gene under the control of the promoter P\({}_{\text{lac}}\) to optimise vanillin biosynthesis from alkaline lignin (Figure 5B). This recombinant strain produced about 57 mg/L vanillin, while the reference _Arthroucter_ sp. C2 strain produced only trace amounts (<4 mg/L) [81].