Handbook of Vanilla Science and Technology

It is also probable that parallel pathways exist in some microorganisms and their expression is substrate and condition dependent. Microbial cells are typically in their stationary growth phase with an ample source of alternative carbon source. Although conditions for maintaining cell viability must be maintained throughout the bioconversion step, it is not necessary that active cell growth occurs. In some instances, additional co-factors have been suggested, such as, sulfhydro-compounds, phospholipids, or cellobiose.

GRAS status of microorganisms and of fermentation ingredients is preferred. Here the focus is on the microorganisms, which have been studied more extensively. The accumulated knowledge provides an insight as to which metabolic pathways are critical and how to improve them.

19.3.1 Bacteria

19.3.1.1 Pseudomonas

The ability of Pseudomonas strains to degrade a variety of aromatic compounds made them among the first to be studied for vanillin production.

It was P. fluorescence in which the genes coding the enzymes involved in the formation of vanillin from ferulic acid were identified as 4-CL and HCHL enzymes for the first time.

A close relationship of hydroxycinnamate catabolism to the β-oxidation of fatty acids was also demonstrated (Narbad and Gasson 1998; Gasson et al. 1998). In P. fluorescence, the HCHL belongs to the superfamily of fatty acid β-oxidation associated hydratases/ isomerases. However, a NAD⁺ binding domain typical of enoyl-SCoA hydratase/iso-merases was absent in the P. fluorescence genes.

Previous reports suggested either a side-chain reductive mechanism (Andreoni and Bestetti 1988) or a CoA-independent deacetylation (Jurova and Wurst 1993); however, the proposed mechanism was not fully elucidated and was only suggested based on intermediates.

The eugenol to ferulic acid pathway was characterized in Pseudomonas strain H199 and the genes encoding the enzymes were identified as eugenol hydroxylase (ehyA and ehyB genes), coniferyl alcohol dehydrogenase (calA), and coniferyl aldehyde dehydrogenase (Rabenhorst 1996; Priefert et al. 1999).

Under the optimized reaction conditions, P. putida IE27 cells produced 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20°C after a 24-hour incubation in the presence of 10% (v/v) dimethyl sulfoxide (Yamada et al. 2007).

Vanillin was shown to be further oxidized through vanillic acid and protocatechuic acid in Pseudomonas sp., which expresses a complete set of genes (vdh, vanA/vanB, pcaG/pcaH) needed for this metabolism.

19.3.1.2 Streptomyces

Streptomyces setonii is a strain reported to give the highest bioconversion yields ofvanillin. It was identified by Sutherland et al. (1983) and later showed an impressive tolerance towards high concentrations of this aromatic aldehyde (Muheim and Lerch 1999). Typical byproducts were vanillic alcohol, vanillic acid, guaiacol, 4-vinylguaiacol, and 2-methoxy-4-ethyl-phenol. These intermediates suggest that S. setonii employs a non-oxidative decarboxylation pathway that involves initial enzymatic isomerization to the quinoid intermediate, which subsequently decarboxylates to 4-vinylguaiacol (Figure 19.4).

Bioconversion outcomes depend heavily on the pH of the medium. A pH of 8.2 was shown to be ideal for accumulation of vanillin by S. setonii, reaching 90% conversion, whereas lower pH favored reduction of vanillin to vanillyl alcohol and higher pH favored oxidation to vanillic acid (Gunnarsson and Palmqvist 2006). The same study also showed the influence of the carbohydrate source on vanillin accumulation. The best yield of vanillin (9.2 g/l) was achieved when ferulic acid (13.1 g/l) and glucose were used at pH 8.2. On the other hand, with arabinose as a carbon source and pH 8.2, vanillin reached only 4.7 g/l, although ferulic acid bioconversion was reported to be several-fold higher than on glucose. There was no vanillyl alcohol formed when arabinose was used as a carbon source.

Another species, S. sannanensis, produced mostly vanillic acid (0.4 g/l) when grown only on ferulic acid (0.94 g/l) with no additional carbon source. Accumulation of vanillic acid slowed down after the inhibition of hydroxycinnamate-CoA-ligase, which suggested involvement of the CoA-dependent degradation pathway in S. sannanensis (Ghosh et al. 2007).

A soil isolate of Streptomyces sp. displayed activity to modify phenylpropanoids derived from lignin. Cloning of the genes vanA and vanB encoding vanillate demethylase into S. lividans demonstrated that veratric acid can be demethylated by vanillate demethylase into vanillic acid (Nishimura et al. 2006). Vanillic acid can be subsequently reduced to vanillin.