Handbook of Vanilla Science and Technology

Wine related LABs were reported to produce small levels of vanillin when wine was made in wood barrels (de Revel et al. 2005). This implies that lignin in the wood would be the trigger of vanillin formation. LABs, such as Oenococcus oeni or Lactobacillus sp. used in wine-making, convert ferulic acid to vanillin at low levels. However, these two LABs are not able to form vanillin from eugenol, isoeugenol, or vanillic acid. Other LABs, such as Lactobacillus sp. and Pediococcus sp. strains, were able to produce significant quantities of 4-vinylguaiacol from ferulic acid. Also, LABs reduce vanillin non-enzymatically to the corresponding vanillyl alcohol. Lactobacillus lactis has been engineered to convert ferulic acid to vanillin (Bloem et al. 2007).

Heterologous expression of 4-hydroxy cinnamate:CoA ligase (4CL) from the plant Arabidopsis thaliana in LAB was successfully achieved (Martinez-Cuesta et al. 2005); however, the yield of the 4CL was very low in comparison with the formation of this enzyme in Pseudomonas; when grown on ferulic acid.

19.3.1.8 Clostridium

Clostridium aerotolerans and C. xylanolyticum, closely related species, were reported to transform cinnamic acid to 3-phenylpropionic acid anaerobically. Both species also reduced a wide range of cinnamic acid derivatives, including o-, m- and p-coumaric, o-, m- and p-methoxycinnamic, p-methylcinnamic, caffeic, ferulic, isoferulic; and 3,4,5-trimethoxy-cin-namic acids to their corresponding 3-phenylpropionic acid derivatives (Chamkha et al. 2001).

19.3.2 Fungi and yeasts

Selected fungi and yeasts are capable of converting ferulic acid, isoeugenol, or eugenol to vanillin. Not as many pathways have been fully elucidated to confirm the existence of enzymes and their specific intermediates as has been done for bacteria. Decarboxylation, side-chain reduction, CoA-independent, and β-oxidative decarboxylation were all reported as mechanisms of ferulic acid bioconversion by fungi and yeasts.

The major asset of fungi useful for vanillin production is their capacity to express enzymes, such as feruloyl esterase and cinnamic acid hydrolases, peroxidases, and decarboxylases. Esterases and hydrolases are instrumental in releasing ferulic acid from plant cell walls. Peroxidases, especially lignin peroxidases from basidiomycetes Phanerochaete chrysos-porium, are involved in the depolymerization of lignin type of materials during which some traces of vanillin are produced (ten Have et al. 1998).

Decarboxylases catalyze elimination of the carboxylic group of ferulic acid to form 4-vinylguaiacol (4-hydroxy-3-methoxystyrene). This has been reported in yeasts such as Brettanomyces anomalus (Edlin et al. 1998), S. cerevisiae (Clausen et al. 1994), and Rhodotorula glutinis (Labuda et al. 1992b). The corresponding genes from S. cerevisiae have been cloned and expressed in E. coli (Clausen et al. 1994; Ago and Kikuchi 1998).

The yeasts R. glutinis transformed 1 g/l ferulic acid to 4-vinylguaiacol with the conversion rate of 90% within 24 hours. Extended time led to accumulation of vanillic acid with about 14% after 95 hours of incubation. A vanillin conversion rate of about 8.5% was observed only in the presence of sulfhydro-reagents. Besides vanillin, vanillic acid, and 4-vinylguaiacol, LC/MS chromatography identified dihydroferulic acid and 3,4-dihydroxy benzoic acid in the broth (Labuda, unpublished data). Based on the intermediates, it appears that R. glutinis utilizes two parallel pathways, decarboxylation and side-chain reductive pathways.

p-Oxidation of ferulic acid employing hydroxycinnamate decarboxylase of Rhodotorula rubra led to accumulation of vanillic acid (Huang et al. 1993). This pathway, which is analogous to the β-oxidation of fatty acid, is thought to include a thiolytic cleavage of

4-hydroxy-3-methoxyphenyl-β-ketopropionyl.

As with R. glutinis, A. niger seems to utilize parallel metabolic pathways, decarboxylation, and side-chain reductive pathways. LC/MS identified vanillin, vanillic acid, 4-vinylguaiacol, protocatechuic aldehyde, protocatechuic acid, and 3,4-dimethoxy benzaldehyde (veratri-caldehyde) in Aspergillus niger medium after 150 hours incubation with ferulic acid. The vanillin conversion rate was 5.6% (Labuda, unpublished data).