# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 4 Genetic Engineering Approaches
### Genetic Engineering in Model Microorganisms
#### 4.3.1 Implementation of Nature-Inspired Bioconversion Pathways Vanillin from Phenylpropanoids
coli_ JM109 harbouring a low-copy number vector (Figure 8(A3)) based on the RK2 replicon with _ech_ and _fc_s genes from _Pseudomonas fluorescens_ BF13 under the control of their native promoter (P\({}_{\text{frc}}\)) to achieve a yield of 0.36 g/L of vanillin [91]. Recently, the same group [92] used an integrative vector to introduce the _exl/fcs_ gene cassette into the _E. coli_ JM109 chromosome (Figure 8(A4)). The amount of vanillin obtained increased by 1.52-fold compared to the strain harbouring the low copy RK2 derivative vector. An even higher vanillin amount (4.26 g/L, molar yield \(\approx\) 88%) was obtained when the experiment was carried in fed-batch conditions [92]. In the above-mentioned studies, the conversion yield of ferulic acid into vanillin varies between 70 and 88%, with important differences in the final amount of vanillin obtained. The reasons explaining these differences cannot be deduced from these studies, since several parameters (origin of the genes, promoters, expression vectors, strains, culture conditions, etc.) differ from one study to the other. In the future, systematic studies will be needed to establish the contribution of each factor to the final yield. The effective activity of CoA-dependent enzymes requires the constant availability of this cofactor. Therefore, to improve the vanillin yield, some strains carrying vanillin biosynthetic pathways have been engineered to overproduce coenzyme-A (see Section Optimisation of metabolic fluxes to improve cofactor and precursor supplies). Figure 7: Vanillin production from eugenol or isoeugenol in _E. coli._ (**A**) Pathway from isoeugenol and schematic representation of the genetic construction used for its implementation. (**B**) Pathways from eugenol and schematic representation of the genetic constructions used for their implementation. (**C**) Table summarising the function and origin of the genes/enzymes used. Recently, based on sequence and structure analysis of ECH from _Streptomyces_ sp. V1, [PERSON] et al. [93] performed rational protein engineering and generated nine mutants, of which three (F74W, R147Q, and \(\Delta\)N1-11) exhibited a better vanillin yield than the wild-type enzyme. An in silico analysis could partially explain the negative results obtained by the six other mutants, but suggested that a balance between catalytic activity and enzyme stability must be considered to optimise the vanillin yield [93]. Vanillin from Aromatic Amino Acids  
Inspired by the proposed vanillin biosynthetic pathways occurring in _Vanilla_ and in vanillin-producing microorganisms, some researchers reported the use of synthetic biology approaches to produce, in model microorganisms, vanillin from aromatic amino acids, which have a lower price and higher availability than ferulic acid. For instance, [PERSON] et al. [94] produced vanillin from accessible carbon sources in _E. coli_ by mimicking the ferulate pathway (Figure 9). They assembled a vanillin biosynthetic pathway, previously proposed in an iGEM project [95], comprising five enzymes: TAL, C3H, OMT, FCS, and ECH. In this work, the authors tested two candidate genes encoding enzymes catalysing the conversion of tyrosine to 4-cumarnic acid (_RsTAL_ and _sam8_) and 4-cumarnic acid to caffe acid (_lpnBC_ and _sam5_). Here, only the constructions giving the best vanillin yields are described. The _sam8_ and _sam5_ genes (from _Saccharotnik expansensis_) were cloned in pACYC-Duettogether with a _comt_ synthetic gene (codon-optimised for _E. coli_) encoding the caffe acid O-methyltransferasearse (COMT) from _Arabidopsis thaliana_. The _ech_ and _fc_ genes were amplified from the genomic DNA of _Streptomyces_ sp. V-1 and cloned into pET-Duet with an extra copy of _sam5_. The introduction of both vectors in _E. coli_ BL21 (DE3) allowed the production of 97 mg/L of vanillin from tyrosine (Figure 9A) [94]. Moreover, the introduction of the same genes in a tyrosine overproducing strain yielded 19 mg/L of vanillin from glucose, 13 mg/L from xylose, and 25 mg/L from glycerol [94]. [PERSON] et al.