Handbook of Vanilla Science and Technology

A β-oxidation deacetylation analogous pathway was suggested for P. putida (Zenk et al. 1980) and Rhodotorula rubra (Huang et al. 1993) (Figure 19.3). The first step is the formation of feruloyl-SCoA by 4-hydroxycinnamate-SCoA ligase (fcs). The second step involves a reduction catalyzed by 4-hydroxycinnamate-CoA-hydratase/lyase (ech) to give 4-hydroxy-3-methoxyphenyl-β-ketopropionyl-SCoA. The next step is a typical β-oxidation thiolytic cleavage yielding vanillyl-SCoA and acetyl-SCoA. Vanillyl-SCoA hydrates and splits off CoASH yielding vanillin. This step is catalyzed by β-ketoacyl-CoA-thiolase (aat). Limited experimental evidence is available to support this route to vanillin. Besides, a contrarian experiment proving that the deletion of p—ketothiolase gene aat adjunct to other genes (fcs, ech, vdh) does not influence ferulic acid catabolism into vanillin in Pseudomonas sp. HR199 has been reported (Overhage et al. 1999).

Fig. 19.3 β-oxidative deacetylation (CoA-dependent).

19.2.1.3 Non-oxidative decarboxylation

The first step involves decarboxylase, which isomerizes ferulic acid to a quinoid intermediate and subsequently decarboxylates the intermediate to 4-vinylguaiacol (Huang et al. 1993) (Figure 19.4). Further downstream intermediates between 4-vinylguaiacol and vanillin have not been identified. The specificity of decarboxylase varies, based on the producing microorganism. For example, Bacillus pumilus has two decarboxylases active towards ferulic and p-coumaric acids, but with no activity against caffeic acid. On the other hand, Lactobacillus plantarum has two inducible decarboxylases, one for ferulic acid and one for caffeic and p-coumaric acids (Barthelmebs et al. 2000). In some microorganisms, such as the yeast Brettanomyces anomalus, the enzyme is constitutively produced at low levels and induced upon addition of the substrate (Edlin et al. 1998). R. glutinis and R. rubra were reported to metabolize ferulic acid via decarboxylation (Huang et al. 1993; Labuda et al. 1992a). R. glutinis converts 90% of ferulic acid to 4-vinylguaiacol within 2 hours (Labuda et al. 1992b). This mechanism was also reported for Bacillus coagulans (Karmakar et al. 2000). Pestalotiapalmarum bioconversion broth contained also methoxyhydroquinone as a result of oxidative decarboxylation of vanillic acid in addition to vanillin, vanillic acid, vanillyl alcohol, and protocatechuic acid (Rahouti et al. 1989).

Fig. 19.4 Non-oxidative decarboxylation.

19.2.1.4 CoA-Independent Deacetylation

During this degradation pathway, the trans double bond of ferulic acid is hydrated to yield a transient intermediate 4-hydroxy-3-methoxy-p-hydroxypropionic acid (Figure 19.5). This step is followed by aldolase cleavage of the acetate portion, yielding directly vanillin. The transient intermediate can be also reduced to 3(4-hydroxy-3-methoxyphenyl)3-ketopropio-nic acid and then deacetylated into vanillin. An enzyme ferulic acid deacetylase (fca) has been identified in P putida strain WCS358 as an catalyst deacetylating ferulic acid into acetate and vanillin (Venturi et al. 1998). The existence of the transient intermediate and the precise mechanism of this pathway have not been fully confirmed. CoA-independent deacetylation was suggested for Delftia acidovorans, Pseudomonas sp., P mira, Bacillus subtilis, Corynebacterium glutamicum, E. coli, Streptomyces setonii, Aspergillus niger, Fomes fomentarius, Fusarium solani, Polysporus versicolor, and Rhodotorula rubra.

Fig. 19.5 CoA-independent deacetylation.

19.2.1.5 Side-Chain Reductive Pathway

As with decarboxylation, this pathway involves isomerization of ferulic acid to a transient quinoid intermediate, which is reduced to dihydroferulic acid (4-hydroxy-3-methoxy phenylpropionic acid) (Rosazza et al. 1995) (Figure 19.6). This pathway was reported for S. cerevisiae expressed under anaerobic conditions (Huang et al. 1993) or L. plantarum (Barthelmebs et al. 2000). However, this side-chain reduction was also reported under aerobic conditions in Phanerochaete chrysosporium (Gupta et al. 1981). Depending on the microorganism, dihydroferulic acid is further metabolized to homovanillic, or vanillic acid. Vanillic acid can be further reduced to vanillin. Ferulic acid can be also directly reduced to coniferyl alcohol or demethylated to caffeic acid.

Fig. 19.6 Side-chain reductive pathway.