Handbook of Vanilla Science and Technology

Isoeugenol is metabolized through an epoxide-diol pathway (Figure 19.8). It undergoes side-chain epoxidation, epoxide hydrolysis to a diol, and diol cleavage to vanillin. Intermediates were identified as isoeugenol-epoxide and isoeugenol-diol, leading to vanillin or vanillyl alcohol. Vanillin undergoes downstream oxidation into vanillic acid. The biotransformation with resting cells of B. pumillus showed that vanillin was oxidized to vanillic acid and then to protocatechuic acid before the aromatic ring was broken. These findings suggest that isoeugenol is degraded through an epoxide-diol pathway (Hua et al. 2007). The same pathway was found in resting cells of Nocardia iowensis (Seshadri et al. 2008). The cell-free extract exhibiting activity of two enzymes, isoeugenol mono-oxygenase and vanillin oxidase, converted isoeugenol to vanillin and vanillic acid without cofactors.

Fig. 19.8 Suggested degradation pathway for isoeugenol.

The same enzyme, isoeugenol mono-oxygenase, from Pseudomonas putida was used to transform E. coli and thus enabled the recombinant cells to convert isoeugenol to vanillin without generating undesirable by-products (Yamada et al. 2008).

19.2.3 Lignin

Lignin is a cross-linked phenylpropanoid polymer and is a major plant component. It is well known that vanillin is formed in small quantities from aromatic compounds known to be precursors in the biosynthesis and degradation of lignin. Enzymes needed for degradation of lignin are associated with white-rot fungi such as Phanerochaete chrysosporium, which delignify and decompose lignin efficiently. It is known that lignin peroxidase and laccase play a role in the depolymerization of lignin. Guaiacol intermediates released from lignin are further converted into a number of phenolic compounds, such as coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillic acid, p-hydroxycinnamic aldehyde, dehydrodivanillin, etc. This microbial process is slow and the vanillin levels are too low to be competitive.

19.2.4 Sugars

Sugars as a carbon source play three important roles during vanillin formation:

I as an energy source for the producing organism;

II as a precursor of vanillin; and III as a co-factor.

Glucose is typically employed as a carbon source fed together with ferulic acid, eugenol, or another precursor. In some instances the type of carbohydrate influences vanillin formation, as demonstrated with cellobiose, glucose, fructose, and maltose during P. cinnabarinus bioconversion (Lesage-Meessen et al. 2002; Oddou et al. 1999). On the other hand, A. niger bioconversion of ferulic acid to vanillin was impacted very little by the type of carbohydrate used. Only slightly higher levels of vanillin accumulated with lactose and cellobiose than with glucose, galactose, or sucrose (Labuda et al. 1992a).

Using glucose as a vanillin precursor is economically a very attractive proposition. A number of studies constructing a recombinant E. coli strain capable of forming vanillin from glucose were successful and are described below. In some recombinant E. coli strains engineered with an arabinose inducible promoter, arabinose was used as an inducer. A recent paper by Hansen et al. (2009) describes successful generation of recombinant transgenic Schizosaccharomyces pombe and Saccharomyces cerevisiae capable of synthesizing vanillin from glucose through new de novo pathways, although the yields of vanillin were quite low, 65 and 45 mg/liter, respectively.

As arabinose is found in plant hydrolyzates, it is important to establish its impact on vanillin generation. S. setonii formed roughly half of the vanillin in the presence of arabinose (3.1 g/l) compared to that produced with glucose (6.1 g/l) (Gunnarsson and Palmqvist, 2006). In some instances, a carbohydrate such as cellobiose acts as an elicitor of the vanillin pathway (Lesage-Meessen et al. 2002).

19.3 MICROORGANISMS

Microorganisms selected for vanillin formation have been isolated based on their ability to metabolize phenolic precursors to vanillin as well as to tolerate higher concentrations of phenolic compounds. The substrates and reaction conditions have been identified to maximize the yield of vanillin. The literature suggests that genetically unrelated microorganisms share similar pathways to degrade phenolic precursors.