Unit 2 – Natural Vanillin – Bioprocesses As We Have Seen

Unit 2 – Natural Vanillin – Bioprocesses

As we have seen before Vanillin is the flavour molecule with the by far largest production volume. Nearly 100 tons are produced by fermentation each year and we will have today a closer look at the different ways to obtain vanillin biotechnologically. But at first we will get to know something about the properties of vanillin.

Vanillin has a typical Vanilla like, sweet, creamy, chocolate like smell. The taste is descriped as Vanilla like, Vanillin, sweet, creamy and phenolic. Therefor the main applications in food are in Ice cream, chocolates, bakery and dairy products, sweets as well as beverages. But now we will come back to the production processes. There are mainly two chemical production processes. The largest process starts with guaiacol, a fossil oil based chemical, while the second one uses lignin, a waste product of paper pulping. In the past also Eugenol was used as a starting material. All these processes will result not in a natural flavour. As the demand for a natural vanillin is high and the vanilla pods are not a sufficient source, despite the exorbitant price, there are still a lot of efforts for biotechnological processes to obtain natural vanillin.

When we look at the molecule, we see that it is a relatively simple small molecule. It is a substituted phenolic aldehyde. In general there are two possibilities to obtain vanillin. The first one is the de novo synthesis from glucose as it is also done in the Vanilla plant. The second is a biotransformation of a natural occurring molecule with a similar structure. Both ways have been gone and we will have now a closer look at the different processes. Let’s start with the de novo synthesis from glucose. Already in 1998 Li and Frost from the Michigan State University described a two‐step process for the production of vanillin. They used a recombinant E. coli strain and obtained in a fed‐batch fermentation 5 g/L of Vanillic acid after 48 Hours. Vanillic acid has then be reduced by an aryl aldehyde dehydrogenase isolated from Neurospora crassa with a 92 % conversion rate within 7 hours to vanillin.

They received a patent for this process in 2002. The authors themselves realized that this process realized that this process was not sufficient for a commercial production and that further optimization, especially the integration of the aryl aldehyde dehydrogenase into an microbial host would be necessary, as this enzyme is NADP dependent.

A second process based on glucose was published in 2009 by Hansen et al., who used Saccharomyces cervisiae and the fission yeast Schizosaccharomyces pombe as a host for the recombinant process. They introduced the genes encoding 3‐dehydroshikimate dehydratase (3DSD) from the dung mould Podospora pauciseta to catalyze the formation of protocatechuic acid from 3‐dehydroshikimate. From protocatechuic acid, the pathway then proceeds either via vanillic acid formed by O‐methylation by the O‐methyltransferase from Homo sapiens (OMT) (EC 2.1.1.6) and then by reduction… …by the aromatic carboxylic acid reductase (ACAR) from Nocardia iowensis to obtain vanillin. Alternatively, the reaction

scheme of the two last steps can be exchanged. For good expression of the genes it was necessary to adopt the codon usage of the foreign genes to that of the yeasts.

To avoid the toxicity of vanillin it was glycosylated by an Arabidopsis thaliana UDP‐ glycosyltransferase, converting the form vanillin into vanillin β‐D‐glucoside. This process was developed by the Swiss company Evolva together with IFF. They announced in 2014 that their vanillin was brought to market, but up to now no significant amounts were seen on the market. The ways to vanillin by biotransformation are manifold. There are numerous natural aromatic molecules which can act as a precursor for the formation of vanillin and for all of these processes, methods have been described. In 1990 Dolfini et al. from the Mallinckrodt company filed a patent for the hydrolysis of curcumin to obtain vanillin. Curcumin is the main compound of turmeric oleoresin from the herb Curcuma longa and is used as a yellowish orange dye in foodstuffs. As you can see, when looking at the chemical structure, it is possible to obtain two molecules of vanillin from one molecule curcumin. The hydrolysis is done at 316 °C and 21,8 bar. This can only be considered as a US natural process, but it would not be accepted under the EU Regulation.

But there are also some biotechnological approaches to obtain vanillin from curcumin. In 2011 Bharti & Gupta described the conversion of curcumin with a strain of Rhodococcus rhodochrous. They obtained 3,56 mg vanillin from 29,02 mg of natural curcumin. This is by far too low for a commercial process.

The group of Berger published in 2015 a process with three isolated enzymes. First the phenolic hydroxyl groups were protected via acetylation, in a second step the molecule was hydrolysed by a laccase, and in a third step the acetyl group was removed with a feruloyl esterase. This process is also not commercially feasible due to low concentrations and yields. Isoeugenol was one of the first precursors, applied for production of natural vanillin. In 1991 when I was at Haarmann & Reimer, we found that species of the genera Serratia, Enterobacter and Klebsiella were able to convert isoeugenol to vanillin. With Serratia marcescens we obtained after 9 days a concentration of 3,8 g/L vanillin. This process was commercially not attractive, as most of the producing organisms belong to the security level 2. A second drawback was that natural isoeugenol is in contrast to eugenol only found as a minor component in a number of special essential oils like Ylang‐Ylang and nutmeg.

Another natural precursor is eugenol. It is the main constituent of the essential oil of the clove tree, Syzygium aromaticum. It is available in high volumes and at relatively low prices. A number of Pseudomonas species are able to metabolize eugenol, despite the fact that it is a relatively high antimicrobial active compound. In the 1990ies we isolated at Haarmann & Reimer Pseudomonas species HR 199, which was able to degrade Eugenol. The metabolites detected analytically indicate the following pathway. Eugenol is oxidized in the first step to coniferyl alcohol. And then via conferyl aldehyde to ferulic acid. Which is de-acidulated to vanillic acid. Together with a group of Prof. Steinbüchel from the University of Münster we analysed the genetics. As it can be seen in the chart, vanillin is an intermediate in the degradation pathway. But the vanillin dehydrogenase is so active that it was not possible to

detect even trace amounts of vanillin in the wild type strain. By constructing a deletion mutant of the vdh‐gene it was possible to produce vanillin. In the flask scale the concentration was very low due to the limited concentration of eugenol which can be added. In a carefully controlled fed‐batch fermentation process it was possible to increase the concentration significantly. But the problem remains that the coniferyl aldehyde dehydrogenase has also a vdh activity. The commercially successful process is based on the microbial biotransformation of ferulic acid. Ferulic acid is an abundant phenolic phytochemical mainly found in plant cell walls. There it is esterified with polysaccharides, flavonoids, hydroxycarboxylic acids, and long chain alcohols. Commercially available it is either from the bran of rice (Oryza sativa), wheat (Triticum aestivum ) or from corn (Zea mays). Several groups have studied a number of different organisms, were vanillin is an intermediate in the degradation of ferulic acid. At Haarmann & Reimer we developed in the mid 1990ies a process with Amycolatopsis sp. HR 167. This strain was able to produce a high concentration of vanillin from ferulic acid in a fed‐batch fermentation process. After optimisation, about 11,5 g/L vanillin with a molar yield of 77,8 % was obtained within 1,5 days. A few months later a patent from Muheim & Lerch from Givaudan patented a very similar process using Streptomyces setonii ATCC 39116. Later on, it became clear by genetic analysis, that both strains belong to the same species, which is now classified as Amycolatopsis setonii. This process is nowadays the only commercialized process. The H&R patent was sold in 2012 to Rhodia, which was later bought by Solvay. Solvay is now the world largest producer of natural vanillin from ferulic acid. The advantage of this process is that it is a natural, not genetically engineered strain, which is at least in Europe a significant advantage for products which are used in the food area.

In the meantime, the production strain has been optimized by genetic engineering by the group of Steinbüchel on behalf of Symrise. By deletion the vdh gene and the constitutive expression of the vanillin anabolism genes ech and fcs a final vanillin concentration of 19.3 g/liter, with a molar yield of 94.9%, was obtained. They achieved an even higher final vanillin concentration of 22.3 g/liter, at the expense of a lower molar yield, by using an improved feeding strategy. This is the highest vanillin concentration by biotransformation disclosed so far. Zheng et al published in 2005 a method using two fungal strains. In a first step a strain of Aspergillus niger produced vanillic acid, which was then in a second step reduced to vanillin by a strain of Pycnoporus cinnabarinus. Only 2,2 g/L of vanillic acid was obtained in the first step, in the second step 2,8 g/L vanillin were formed. This is too low for a commercial process.