Handbook of Vanilla Science and Technology

A. niger deacetylates ferulic acid to vanillic acid very efficiently. In a two-step bioconversion process, vanillic acid is subsequently reduced to vanillin by Pycnoporus cinnabar-inus (Lesage-Meessen et al. 2002). Cellobiose is suggested to enhance channeling of the bioconversion of vanillic acid into vanillin by controlling methoxyhydroquinone formation in P. cinnabarinus. This process could be further improved by introducing a chimeric bifunctional enzyme that increases the synergistic effect on the degradation of complex substrates. For example, a chimeric construct composed of the sequences encoding the feruloyl esterase A (FAEA) is fused to the endoxylanase B (XYNB) of A. niger, which allows for efficient release of ferulic acid from plant materials (Levasseur et al. 2005). Another example is the improved secretion efficiency of a fused dockerin-feruloyl esterase protein, which consists of feruloyl esterase of A. niger and dockerin of Clostridium thermocellum, by introducing 514 amino acid sequence of glucoamylase and a dibasic proteolytic processing site upstream of the chimeric protein (Levasseur et al. 2004).

Mutation and genetic manipulation of A. niger, A. flavus, and Penicillium chrysogenum resulted in constructed strains, which exhibited both ferulic acid esterase and alkene cleavage activities. These strains converted ferulic acid glycoside directly into vanillic acid (Cheetham et al. 2005).

Eugenol and isoeugenol are tolerated and metabolized by few fungi. The fungus Bysso-chlamys fulva V107 converted eugenol to 21.9 g/l of coniferyl alcohol within 36 hours, representing 94.6% molar yield (Furukawa et al. 1999).

Induction of vanillyl alcohol oxidase was reported for Penicillium simplicissimum when grown on veratryl alcohol, anisyl alcohol, or 4-(methoxymethyl)-phenol (Fraaije et al. 1997). In addition to vanillyl-alcohol oxidase, an intracellular catalase peroxidase was induced. Induction of vanillyl-alcohol oxidase in P. simplicissimum was prevented by the addition of isoeugenol to veratryl alcohol-containing media, but growth was unaffected. The vanillyl alcohol oxidase gene (vaoA) from P. simplicissimum CBS 170.90 cloned into Amycolapsis enabled this bacterium to metabolize eugenol.

The fungus Phanerochaete chrysosporium gained interest mostly as a micro-organism able to decompose lignin into vanillin and other phenolic metabolites (Kirk and Farell 1987). However, the levels of vanillin were very low. Vanillin triggered metabolic shifts in P. chrysosporium identified by proteomic differential display technique during which a set of reductases and dehydrogenases, as well as extracellular peroxidases were upregulated. A metabolic shift from the glyoxylate cycle to the tricarboxylic cycle was detected (Shimizu et al. 2005). Recently, P. chrysosporium was used to produce vanillin from green coconut husks. Vanillin was detected in the range between 44 to 52 mg/g of solid support after solvent extraction (Barbosa et al. 2008).

A recent publication (Hansen et al. 2009) describes a different approach, by creating new transgenic de novo pathways to form vanillin from glucose in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The new pathway combines bacterial, mold, plant, and human genes. The engineered pathway included 3-dehydroshikimate dehydratase from Podospora pauciseta, an aromatic carboxylic acid reductase (ACAR) from Nocardia sp., and a human O-methyl transferase. In S. cerevisiae co-expression of a Corynebacterium glutamicum phosphopantetheinyl, transferase was additionally required. Elimination of the yeast alcohol dehydrogenase gene (adg6) prevented the reduction of vanillin to vanillyl alcohol. In addition, to eliminate vanillin toxicity and improve yield, a gene coding for UDP-glycosyltransferase from Arabidopsis thaliana was used to create vanillyl-b-D-glucoside. This work demonstrates the capability of creating engineered pathways in organisms that normally do not possess the capacity to produce vanillin and other flavor compounds.

In general, filamentous fungi and yeasts are grown more slowly than bacteria, and the process conditions are more challenging.

19.4 PROCESSES

Metabolic engineering offers several possibilities to improve vanillin yield:

• reduce the downstream degradation of vanillin;

• improve the expression of existing genes;

• gene transfer into other production microorganisms or plants, etc.