# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 3 Identification of Genes and Pathways in Vanillin-Producing Microorganisms  
The world of microorganisms is vast, and their metabolism is highly versatile. Numerous microorganisms are used for the production of natural food components. Strains of interest are selected based on their growth properties, production potential, and tolerance to high concentrations of both substrates and products. Vanillin-producing microorganisms have been identified for their natural ability to produce vanillin as a catabolic intermediate of phenolic compound degradation pathways. For instance, some species of actinomycetes belonging to the genera _Amycolatopsis_, _Rhodococcus_, and _Streptomyces_ were reported to convert ferulic acid into vanillin [35; 36; 37]. Some species of Gram-negative and Gram-positive bacteria belonging to the _Pseudomonas_ and _Bacillus_ genera, respectively, were shown to produce vanillin from ferulic acid, eugenol, or isoeguenol [38; 39; 40; 41; 42; 43]. Finally, some species of fungi, including _Aspergillus niger_, _Phanterochate chrysosporium_, and _Pycnoporus cinnobarinus_, have been studied for their capacity to produce vanillin by the bioconversion of agricultural and industrial lignin-enriched residues (e.g., waste residues from rice bran [44], fruit peels [45; 46], coconut husk [47], and wheat bran [48]). The bioconversion ability to produce vanillin is strain-specific as other species of the same microbial genus are not capable of the same conversions. Genes and enzymes involved in vanillin biosynthesis from phenolic compounds have been characterised in some microorganisms (Figure 2A). Isoeugenol is readily transformed into vanillin through an epoxide-diol pathway by an isoeugenol monooxygenase (gene name: _iem_, enzyme name: IEM) that was found in some _Pseudomonas_ strains [49; 50; 51]. Genes encoding the enzymes involved in the eugenol bioconversion pathway were first identified in _Pseudomonas_ sp. HR199 [52]. The transformation of eugenol into ferulic acid comprises three steps: (1) the heterodimeric eugenol hydroxylase (_EthyA_, _EthyB_, EUGH) catalyses the oxidation of eugenol into coniferyl alcohol [52], and this reaction can also be catalysed by a vanillyl alcohol oxidase (_xuoA_, VAO) [53]; (2) and (3) coniferyl alcohol is then oxidised into coniferyl aldehyde and subsequently into ferulic acid by coniferyl alcohol and coniferyl aldehyde dehydrogenase (_calA_ and _calB_, CADH and CALDH) [54]. Ferulic acid can then be catabolismed by microorganisms, and vanillin is obtained as an intermediate of the catabolic route. Five different ferulic acid catabolic pathways have been proposed in various microorganisms [2; 8; 11]. The most efficient for vanillin production is the non-\(\beta\)-oxidative deacetylation (CoA-dependent) route (Figure 2A), which requires two enzymes, a feruloyl-CoA synthetase (_fcs_, FCS) and an enoyl-CoA hydratase/4-hydroxycinnamate CoA-hydratase/lyase (_ech_, ECH/HCHL). Genes and enzymes involved in this pathway have been identified and characterised in _Pseudomonas_[55] and _Amycolatopsis_ strains [35]. Finally, several microorganisms, including fungi and soil bacteria, have been studied for their ability to degrade lignin and produce vanillin [56]. However, genes and enzymes involved in this bioconversion have yet to be fully characterised [57]. While in _Vanilla_, the biosynthesis of vanillin appears to be connected to the biosynthesis of lignin monomers, in microorganisms, vanillin production seems to be related to phenyl-propanol degradation pathways, in which vanillin is an intermediate. Thus, vanillin might be further catabolised to vanillyl alcohol, vanillic acid, protocatechuric acid (PCA), or guaiaicol (Figure 2B). A set of genes implicated in this catabolic pathway was identified in some _Pseudomonas_[38; 58; 59], _Rhodococcus_[36], and _Amycolatopsis_[60; 61; 62] strains. The various precursors (isoeugenol, eugenol, ferulic acid, and lignin) are not equivalent for the biotechnological production of vanillin.