# 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
Abbreviations of metabolites: Ac-CoA—Acetyl-Coenzyme A; Ace—Acetate; AcP—acetyl phosphate; DAHP—3-deoxy-D-azabino-heptulosonate-7-phosphate; DHGA—3,4-dihydroxyproxyphenylglyoxylate; DHMA—3,4-dihydroxyromaletale; DHQ—3-dehydroquinic acid; DHS—3-dehydrodiskimic acid; 1,3 DPG—3-phospho-D-glycerolphosphate; E4P—erythrose-4-phosphate; F6P—fructose 6-phosphate; GAP—D-glyceroldehyde 3-phosphate; G6P—glucose 6-phosphate; HCys—homocysteine; HMA—hydroxymandelate; HPP—hydroxyphenylpyruvate; Met—Methionine; PEP—phosphoenolpyruvate; Pyr—pyruvate;  
SAH--S-adenosylyl-L-homocysteine; SAM--S-adenosylylmethionine; SRH--S-ribosyl-L-homocysteine; Trp--Tryptophan. Abbreviations of proteins: ACAR--Aromatic carboxylic acid reductase; AroB--DHQ synthase; AroD--DHQ dehydratase; AroE--Shikimate dehydrogenase; AroF, AroG, AroH--DAHP synthase feedback-regulated isoenzymes; AroZ, 3 DSD--3-dehydrodkshiamate dehydratase; BFD--Benzoylormate decarboxylase; CPL--Christamate-pyruvate lyase; GapC--GAP dehydrogenase; GDH1/GDH2--NADP\({}^{+}\)/NAD\({}^{+}\)-dependent glutamate dehydrogenases; GPPP1--glycerol-1-phosphate; HBH--Hydroxybenzoate hydroxylase; HmaS--Hydroxymandelate synthase; HMO--Hydroxymandelate oxidase; HpaBC--Two-component flavin-dependent monooxygenase; LuxS--S-ribosylhomocysteine lyase; MetE, MetH--Methionine synthases; MetJ--transcription regulator of methionine biosynthetic gene cluster; MetK--SAM synthetase; Mtm--5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase; OMT--O-methyltransferase; PDC1--pyruvate decarboxylase; Pos5c--cytosol-relocalised NADH kinase; PpsA--phosphoenolpyruvate synthase; PPTase--Phosphopantethe transferase; Pta--phosphotransferacetylase; Tkta--Transketolase; UGT--UDP-glycostransferase; Xfpk--phosphoketolase. [PERSON] et al. [109] implemented a similar metabolic pathway in fission and baker's yeasts. This pathway consists of three enzymes: a 3 DSD from _Podospora paucista_; an ACAR from the _Nocardiia_ genus; and an OMT from _Homo sapiens_ (Figure 12A Blue). Synthetic genes encoding these enzymes were introduced in alcohol dehydrogenase ADH6 knockout strains to prevent vanillin reduction to vanillyl alcohol. In _Schizosaccharomyces pombe_, the genes were cloned under the control of P\({}_{\mathit{a}\mathit{a}\mathit{H}\mathit{+}}\) and integrated at the _leu1\({}^{+}\)_ locus. In contrast, in _Saccharomyces cerevisiae_, the genes were cloned under the control of the P\({}_{\mathit{T}\mathit{T}\mathit{T}\mathit{T}}\) and directly integrated into the _TPII_ promoter region. In addition, a PPTase gene from _Corynebacterium glutamicum_ required for the post-translational activation of ACAR was cloned and expressed under the control of the TPI1 promoter in a low-copy-number-replicating plasmid (Figure 13(A3)). These recombinant strains produce, respectively, 65 and 45 mg/L of vanillin from 20 g/L glucose [109]. _S. pombe_ was further improved by introducing a UDP-glycostransferase (UGT) from _Arabidopsis thaliana_, which converts vanillin into vanillin \(\beta\)-D-glucose and consequently circumvents the toxicity of vanillin [109]. Similar pathways to the one described in this work and improved constructs were the subject of several patent applications from industrial companies, such as International Flavors and Fragrances Inc. and/or Evolva [108; 109; 110]. Instead of diverting 3-dehydrodkshizinic acid (DHS) from the shikimate pathway, [PERSON] et al. [111] engineered a _Corynebacterium glutamicum_ strain to produce vanillin from glucose by the diversion of chorismate (Figure 12A Orange).