Handbook of Vanilla Science and Technology

Based on the intermediates, it seems logical that parallel pathways exist in several microorganisms. For example, the intermediates vinylguaiacol and dihydroferulic acid found in C. glutamicum suggest that decarboxylation and reductive conversion take place at the same time. An experiment studying the Rhodococcus genome and its expression supports this phenomenon. A genome sequence data of Rhodococcus sp. I24 suggested a CoA-dependent, non-β-oxidative pathway for ferulic acid bioconversion. However, the expression and functional characterization of corresponding structural genes from I24 also suggested that degradation of ferulic acid in this strain proceeds via a β-oxidative pathway (Plaggenborg et al. 2006).

Besides being the most suitable precursor, ferulic acid exhibits many physiological functions, including antioxidant, antimicrobial, anti-inflammatory, anti-thrombosis, and anti-cancer activities. It also protects against coronary disease, lowers cholesterol, and increases sperm viability. It is well regarded as a free radical scavenger that greatly reduces oxidative damage effect (Kanski et al. 2002).

It is ferulic acid that is utilized as a precursor in commercial processes to produce vanillin, for example, by companies such as Firmenich, Symrise, Rhodia, and Comax.

19.2.2 Eugenol and isoeugenol

Clove oil is an inexpensive source of eugenol (2-methoxy-4-(2-propenyl)-phenol), a main component of the essential oil of the clove tree Syzygium aromaticum. Both eugenol and isoeugenol are produced by plants as a part of their biodefense mechanism. Eugenol’s antiseptic properties are widely used in dentistry. Although structurally similar to vanillin and economically viable, eugenol is the most challenging substrate for conversion due to its toxicity and low water solubility. There are two strategies that could decrease toxicity and improve solubility. The first strategy is to change the genetic make-up of the producing micro-organisms to increase the tolerance to higher concentrations of eugenol. The second strategy is to use bi-phasic or immobilized microorganisms where the substrate and the product are continuously removed. Some microorganisms, such as Pseudomonas or E. coli, are more tolerant to eugenol than other bacteria and fungi.

Isoeugenol (2-methoxy-4-(1-propenyl)-phenol) is more readily metabolized. Although present in essential oils, it is usually prepared from eugenol via a chemical route. Isomerization of eugenol to isoeugenol is catalyzed by metal ions at high temperature in potassium hydroxide (KOH). Generally the end product is not considered natural. A newer method using microwave technology requires less harsh conditions to perform this alkaline catalysis (Sinha et al. 2002). Pathways, intermediates, and enzymes involved in eugenol and isoeugenol metabolism have been identified (Hua et al. 2007; Xu et al. 2007; Zhang et al. 2007; Yamada et al. 2007).

The eugenol degradation pathway through a quinone intermediate and coniferyl alcohol was confirmed (Figure 19.7). The genes encoding the enzymes needed for the oxidative catabolism of eugenol were isolated: eugenol hydroxylase/vanillyl alcohol oxidase (ehyA, ehyB, and vaoA), coniferyl alcohol dehydrogenase (calA), and coniferyl aldehyde dehydrogenase (calB) (Priefert et al. 2001; Overhage et al. 2003, Xu et al. 2007). Eugenol hydroxylase/vanillyl alcohol oxidase catalyzes the first and second steps during which a transient epoxide, eugenol oxide, is formed and then transformed into coniferyl alcohol. The major intermediates identified are coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillyl alcohol, and vanillic acid. The intermediate ferulic acid is then transformed into vanillin following non-oxidative deacetylation, employing 4-hydroxycinnamate CoA ligase (4-CL) (fcs) and 4-hydroxycinnamate CoA-hydratase/lyase (HCHL) (ech) (Gasson et al. 1998; Overhage et al. 2003). Another epoxide-diol pathway suggested as intermediates eugenol epoxide, eugenol diol, and their further metabolism into coniferyl alcohol (Priefert et al. 2001).

Fig. 19.7 Suggested degradation pathway for eugenol.