Handbook of Vanilla Science and Technology

Marine actinomycetes Streptomyces maritimus are one of few microorganisms that express the gene encP encoding bacterial phenylalanine ammonia lyase (PAL), an enzyme that catalyzes the conversion of the amino acid l-phenylalanine to trans-cinnamic acid. PAL is a part of the phenylpropanoid pathway found in higher plants (Xiang and Moore 2005). This could provide an opportunity to combine the bacterial and plant phenylpropanoid pathways.

Streptomyces halstedii was selected among 535 actinomycetes from soil. It converted cinnamic acid into cinnamamide (95% molar conversion from 2 g/l of substrate) and ferulic acid into vanillic acid (80% molar conversion from 1 g/l of substrate) with low quantities of vanillin (0.10-0.15 g/l) (Brunati et al. 2004).

19.3.1.3 Bacillus

It was found that bacilli degrade a wide range of aromatic acids, such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(p-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids. Ferulic acid was metabolized with a non-β-oxidative mechanism with intermediates 4-hydroxy-3-methoxyphenyl-p-hydroxypropionic acid, vanillin, and vanillic acid (Peng et al. 2003). On the other hand, isoeugenol is metabolized through an epoxide-diol pathway (Hua et al. 2007).

Bacillus coagulans BK07 decarboxylates ferulic acid within 7 hours to 4-vinylguaiacol, which is immediately converted to vanillin and then oxidized to vanillic acid. Vanillic acid is further demethylated to protocatechuic acid (Karmakar et al. 2000). The very high conversion rate (95%) of ferulic acid to 4-vinylguaiacol is similar to that of Rhodotorula glutinis.

A soil isolate of Bacillus subtilis sp. is able to grow on isoeugenol as a sole carbon source and converts it into vanillin. The bioconversion has been achieved with cells as well as with the cell free extract. It was reported that growing cells of B. subtilis B2 produced 0.61 g/l of vanillin, whereas cell free extracts yielded 0.9 g/l vanillin from isoeugenol (Shimoni et al. 2000). Isoeugenol biotransformation by B. pumillus led to vanillin as a main product. The growing cells of B. pumillus S-1 converted 10 g/l isoeugenol to 3.75 g/l vanillin in 150 hours, with a molar yield of 40.5%, which is the highest up to now (Hua et al. 2007).

19.3.1.4 Corynebacterium

Corynebacterium glutamicum utilizes a number of aromatic compounds, such as ferulate, vanillate, and vanillin as the sole carbon source. This bacterium has outstanding ability to switch between metabolic pathways based on the exposure of the cells to a specific aromatic compound. Induction of four genes, vanA, vanB, and vanK genes, and the divergently transcribed vanR gene, is needed for catabolism of ferulic acid (Merkens et al. 2005). Expression of these genes is not catabolically suppressed when ferulic acid is co-metabolized with glucose. The metabolic channeling of ferulate, vanillin, and vanillate into the proto-catechuate branch of the β-ketoadipate pathway is controlled by a PadR-like transcriptional regulatory protein repressor (Brinkrolf et al. 2006).

Besides vanillin, 4-vinylguaiacol, vanillic acid, and dihydroferulic acid were identified by LC/MS chromatography (Labuda et al. 1993). These intermediates suggest that ferulic acid is metabolized in parallel via decarboxylation and side-chain decarboxylation. Other compounds, such as 3,4-dihydroxybenzoic acid, 3,4-dihydroxybenzaldehyde, 3,4-dihydroxy cinnamic acid, and 3,4-dimethoxy benzaldehyde, were formed by C. glutamicum from ferulic acid as well. It is assumed that these intermediates are the products of the downstream metabolism of vanillic acid.

19.3.1.5 Escherichia coli

Non-recombinant E. coli has not displayed the ability to form vanillin from glucose, ferulic acid, eugenol, or isoeugenol. At the same time, E. coli is easy to manipulate with highly developed genetic engineering tools and is relatively easy to scale up. The combination of these features makes it ideal to exploit E. coli for vanillin production.

Three substrates and metabolic pathways have been considered to achieve vanillin formation in E. coli, glucose, eugenol, and ferulic acid.