# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 4 Genetic Engineering Approaches
### Genetic Engineering in Model Microorganisms
#### 4.3.2 Implementation of De Novo Bioconversion Pathways
[105] demonstrated that ADO could also catalyse the oxidative cleavage of a C=C bond in other aromatic compounds (e.g., isoeugenol, anothele, and O-methyl isoeugenol). Recently, [PERSON] et al. [106] performed rational protein engineering of ADO, based on in silico analyses, to generate enzyme variants able to catalyse the direct conversion of ferulic acid to vanillin. Variants harboring the F82Y, V332R, and F334R mutations exhibited high catalytic activity on ferulic acid and produced 7.0 mM of vanillin from 10 mM of ferulic acid in 24 h [106]. De Novo Bioconversion Pathways from Glucose  
The design of de novo coenzyme A-independent biosynthetic pathways allowing the production of vanillin from glucose by the diversion of intermediates from the shikimate pathway (Figure 12) and their introduction in model organisms has been reported in a few studies. For instance, [PERSON] and [PERSON] [107] engineered a recombinant _E. coli_ strain able to convert glucose into vanillic acid by diverting 3-dehydrochikimic acid (DHS) from the shikimate pathway (Figure 12A Blue). The engineered strain contained a mutated _aroE_ gene (encoding a shikimate dehydatase) and an _aroBaroZ_ cassette (encoding a DHQ synthase and a 3-dehydrochikimate dehydatase: 3 DSD) inserted into the _serA_ locus, allowing the accumulation of DHS and promoting its conversion into PCA. This strain also harboured a plasmid carrying an _aroF\({}^{\text{FBR}}\)_ gene (encoding a feedback-resistant DAHP synthase), two cassets of the _COMT_ gene under the control _lac_ promoter, and a _serA_ gene (a gene required for L-serine synthesis, which allows one to distinguish recombinant bacteria in minimal medium). The recombinant strain (Figure 13(A1)) produced 3 g/L of vanillic acid and 12.9 g/L of PCA from 20 g/L of glucose. The reduction of these aromatic acids was performed in vitro using a purified ACAR, aryl aldehyde carboxylic acid reductase from _Neurospora crassa_, to synthetise vanillin and protocatechualdehyde, which were then purified by chemical extraction. The same report suggests that the industrial relevance of this metabolic pathway depends on the ability to synthesise vanillin by in vivo reactions implemented in a single microorganism [107]. Later on, [PERSON] et al. [108] generated a _E. coli_ RARE strain with a reduced aromatic aldehyde reduction ability (see Section Reduction of Vanillin Degradation), in which the complete metabolic pathway (Figure 12A Blue), including the ACAR step, was introduced, allowing the production of 120 mg/L of vanillin from 12 g/L of glucose (Figure 13(A2)). The vanillin yield was further improved by chassis optimisation (see Section Optimisation of Metabolic Fluxes to Improve Cofactor and Precursor Supplies) [108]. Figure 12: **Vanillin production from glucose by de novo biosynthetic pathways. ****(A)** Vanillin biosynthetic pathways. The arrows representing the reactions are coloured as follows: grey—primary metabolism (e.g., shikimate pathway); blue—conversion of dehydrodiskimic acid into vanillin; orange—conversion of chorismate into protocatechuic acid; yellow—conversion of hydroxyyphenylpyruvate into protocatechuialdehyde; and magenta—conversion of vanillin in vanillin glucoside. Green arrows and enzyme names indicate that the enzymes catalysing the reactions are overexpressed in optimised chassis. Red dotted arrows marked with an X and red enzyme names indicates that the enzyme catalysing the reactions are absent in optimised chassis, following the inactivation of the endogenous genes encoding the corresponding enzymes. (B) Strategies to improve the SAM supply for methylation step. (C) Modified steps of glycolysis or nitrogen metabolism to improve the NADPH supply. (D) Strategies to improve the E4P precursor supply. For panels B, C, and D, the green and red arrows and protein names are used, as in panel A, to indicate overexpression or absence, respectively.