Handbook of Vanilla Science and Technology

Glucose was transformed into vanillin in two steps: in the first step vanillic acid was formed through shikimate pathways; secondly vanillic acid was enzymatically reduced to vanillin. Recombinant E. coli required five enzymes: shikimate dehydrogenase (mutated aroE), 3-dehydroquinate synthase, 3-dehydroshikimate (aroBaroZ), catechol-O-methyl-transferase (COMT), and 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase (aroFFBR) (Frost 2002; Li and Frost 1998). The recombinant microbe performed dehydration of 3-dehydroshikimic acid and regioselectively methylated the resulting protocatechuic acid into vanillic acid. This methylation was a rate limiting step, which was not overcome by doubling the plasmid with COMT. However, the addition of l-methionine improves the vanillate formation. The last step, the reduction of vanillic acid to vanillin, is done enzymatically by purified aryl-aldehyde dehydrogenase from Neurospora crassa. This enzymatic step requires two cofactors ATP and NADP, which make the cost quite prohibitive. Nevertheless, the yield of 5 g/l of vanillin from glucose is impressive especially when E.coli does not produce any vanillin without the above described genetic changes.

Ferulic acid biotransformation needed a different strategy to develop recombinant E. coli. It was done by inserting the fcs (feruloyl-CoA synthetase) and ech (enoyl-CoA hydratase/ aldolase) genes from Amycolatopsis sp. strain HR104 and Delftia acidovorans under the control of the arabinose-inducible promoter Pb_AD into the pBAD24 expression vector. Optimized cell growing conditions led to the highest vanillin production with the E. coli strain carrying the Amycolatopsis genes, 0.58 g/l of vanillin from 1 g/l ferulic acid (Yoon et al. 2005).

Eugenol is another substrate for which a recombinant E. coli strain was developed (Overhage et al. 2003). Two recombinant strains were constructed. The first step allowed eugenol to be transformed into ferulic acid by inserting the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp. and the Penicillium simplicissimum vanillyl alcohol oxidase. The second step converted ferulic acid to vanillin by inserting the genes encoding feruloyl-CoA synthetase (ech) and enoyl-CoA hydratase/aldolase (fcs) of Pseudomonas sp. strain HR199 into another E. coli (Overhage et al. 1999). These two recombinant strains of E. coli when used together successfully converted eugenol to vanillin. First, the resting cells of recombinant strain E. coli XL1-Blue (pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91%. The maximum concentration of ferulic acid was 14.7 g/l after 30 hours fermentation. This corresponded to 93.3% molar biotransformation yield with respect to concentration of eugenol. The maximum production rate from ferulic acid was achieved by feeding ferulic acid at a rate 2.8 g/hour/l. This process led to 0.3 g/l of vanillin besides 0.1 g/ l of vanillyl alcohol and 4.6 g/l of ferulic acid. This yield is rather low and needs to be improved.

The attempt to use genes encoding eugenol hydroxylase and its electron acceptor from Pseudomonas sp. strain HR199, in E. coli XL1-Blue, were shown to be unsuitable for performing the eugenol bioconversion.

The isoeugenol mono-oxygenase gene of P. putida IE27 was inserted into the recombinant plasmid in E. coli BL21. It enabled the transformed cells to produce vanillin with the yield of 28.3 g/l from 230 mM isoeugenol. The molar conversion rate was 81% after 6 hours (Yamada et al. 2008).

These studies indicate that recombinant strains could represent an interesting alternative to natural strains in the field of vanillin production. However, higher productivity is needed.

19.3.1.6 Amycolatopsis sp

Using Amycolatopsis sp. strain HR167,11.5 g of vanillin per liter was produced from 19.9 g/l of ferulic acid within 32 hours on a 10-liter scale, corresponding to a molar yield of 77.8% (Rabenhorst and Hopp 2000).

Recombinant vanillin tolerant bacteria Amycolatopsis sp. HR167 was made by cloning a vanillyl alcohol oxidase (vaoA) from Penicillium simplicissimum CBS 170.90 into a hybrid plasmid. This new recombinant strain gained the capability to grow on eugenol. Resting cells produced downstream intermediates from eugenol with the conversion rate of 0.36 g/l/h. Intermediates were identified as coniferyl alcohol (4.7 g/l), coniferyl aldehyde, ferulic acid, guaiacol, and vanillic acid. Vanillin was present only in minute amounts.

19.3.1.7 Lactic Acid Bacteria (LAB)

LABs are a main staple of human nutrition and therefore their application for vanillin production would be very logical.