- Email: [email protected]
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Novel approaches to the biosynthesis of vanillin Nicholas J Walton*, Arjan Narbad*, Craig B Faulds† and Gary Williamson† Microorganisms able to produce vanillin in excess of 6 g/l from ferulic acid have now been isolated. In Pseudomonas strains, the metabolic pathway from eugenol via ferulic acid to vanillin has been characterised at the enzymic and molecular genetic levels. Attempts to introduce vanillin production into other organisms by genetic engineering have begun. Addresses *Food Safety Science Division and † Diet, Health and Consumer Science Division, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK Correspondence: Nicholas J Walton; e-mail: [email protected] Current Opinion in Biotechnology 2000, 11:490–496 0958-1669/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations 4-CL 4-hydroxycinnamate:CoA ligase HCHL 4-hydroxycinnamoyl-CoA hydratase/lyase
Introduction Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the major component of vanilla flavour extracted from the fermented pods of Vanilla orchids. It is the world’s principal flavouring compound, used extensively in the food industry in ice cream, chocolate and confectionery products and also as a fragrance. This review assesses recent advances in vanillin biosynthesis and in realising the potential of biotechnology in this field. The emphasis here will be on the biochemical and molecular genetic literature from 1998 onwards. For earlier literature the reader is directed to several reviews addressing the microbial biosynthesis of flavour compounds [1–4]. A wide-ranging review that, within its coverage, summarises the terminology of vanilla and vanillin preparations has recently appeared [5•].
Why biotechnology? Vanilla pods are produced largely in Madagascar and Indonesia and contain 2–3% by weight of vanillin in the cured pod [6,7•]. The major markets are the US, France and Germany. Less than 0.5% of the market (~20–50 tons per year out of a total of 12,000 tons per year) is met by extraction of Vanilla pods [4,7•]; the overwhelming usage in the flavour industry is of synthetic vanillin, for example, produced from kraft lignin, with a market price below $15 per kilo [7•,8•]. The value of the vanillin extracted from Vanilla pods is in contrast variously calculated as being between $1200 per kilo and $4000 per kilo [7•,8•]. An opportunity for biotechnology therefore lies in producing a replacement for synthetic vanillin that is produced ‘naturally’ (i.e. non-chemically) from sources other than the ‘named material’ (Vanilla) [6] and at an economic price [4]. Ideally, the replacement will include some of the minor components of natural
vanilla, notably vanillic acid, vanillyl alcohol and 4-hydroxybenzaldehyde, thereby differentiating it further from the synthetic compound. Vanillin is essentially absent from the green Vanilla pod and is only released from its β-D-glucoside during curing of the pods after harvest. The pathway of formation of vanillin-β-D-glucoside is still unresolved. A probable precursor is the phenylpropanoid ferulic acid (4-hydroxy3-methoxycinnamic acid), which possesses the same ring substituents. Although some studies of phenylpropanoid biochemistry and molecular biology have been reported in Vanilla [9,10], the key chain-shortening reactions from the cinnamate to the benzaldehyde are not characterised, in common with the formation of benzaldehydes and benzoates generally in plants (e.g. see [11,12]). Vanilla is not ideal for biotechnology because, in particular, it is slow-growing in tissue culture and the vanillin pathway is not very actively expressed. There have been continual attempts to produce vanillin from Vanilla and also Capsicum (chilli pepper) in culture (reviewed in [5•]). Microorganisms, on account of their rapid growth rates and amenability to molecular genetics, are much better targets and can be selected for their ability to grow on a putative precursor of vanillin as a sole source of carbon and energy. This precursor should be available at low cost, for example, as an agricultural processing by-product.
Feedstocks and biosynthetic pathways Several potential feedstocks have been suggested, including curcumin, siam benzoin resin, phenolic stilbenes, eugenol and ferulic acid [3]. The latter two have received most attention. Eugenol is a principal aromatic constituent of clove oil. Its market price is ~$5 per kilo, making it an economically realistic feedstock [8•]. The pathway from eugenol to ferulic acid (Figure 1) has been characterised in a strain of Pseudomonas (HR199) [13,P1]. The reactions are catalysed, successively, by eugenol hydroxylase (ehyA and ehyB genes), coniferyl alcohol dehydrogenase (calA) and coniferyl aldehyde dehydrogenase (calB) [14••]. Eugenol hydroxylase is a heterodimeric flavocytochrome c enzyme; ehyA encodes the cytochrome c subunit. A corresponding enzyme, designated eugenol dehydrogenase, has been purified and characterised from Pseudomonas fluorescens E118 [15•]). The substrate specificity is wide and includes vanillyl alcohol, which is converted to vanillin. Conversely, a vanillyl alcohol oxidase from Penicillium simplicissimum [16–18] is a flavoprotein homooctamer; however, it too has broad substrate specificity and can oxidise eugenol, producing coniferyl alcohol. The crystal structure has been determined [19]. The cDNA has been isolated and the gene heterologously expressed in Escherichia coli [20].
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Figure 1
CH=CH–CH2OH
CH2–CH=CH2 Eugenol hydroxylase
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Conversion of eugenol to ferulic acid in Pseudomonas sp. strain HR199.
and an α-L-arabinofuranosidase, released a high proportion of the arabinose-linked ferulic acid from sugarbeet pulp [25]. Pseudomonas strains also produce cinnamoyl esterases [26], which can release both monomeric and dimeric ferulic acid from wheat bran and barley spent grain in the presence of a xylanase [27]. The recovery of ferulic acid has received considerable attention. One procedure yielded 1 g of ferulic acid from 1 kg of dry sugar beet pulp [28]. The sugar beet pulp contained ~6 g/kg dry weight of ferulic acid, so 1 g of ferulic acid represents ~17% yield. Ferulic acid also occurs in several dimeric forms in plant cell walls. In maize, the diferulate content is 25 g/kg [29]. In sugar beet pulp, the diferulate level is 1.7 g/kg, and in Chinese water chestnuts 4.5 g/kg [29,30]. However, enzymes for degrading diferulates into their constituent monomers have not yet been isolated or characterised.
Isoeugenol is also a potential feedstock. Recently, the isolation of a Bacillus subtilis strain was reported that could produce 0.6 g/l of vanillin with a molar yield of 12.4% [21]. Ferulic acid is abundant in many cereals as a component of the cell-wall material [22•]. Wheat bran contains 4–7 g/kg dry weight of cell-wall material and maize bran ~30 g/kg. Rice endosperm contains 12 g/kg. Sugarbeet (~5–10 g/kg) is also a good source. Barley spent grain, a brewing by-product, is a poorer source (~0.1 g/kg). Ferulic acid in these materials is present esterified to carbohydrates and can be released by treatment with strong alkali, but production of a ‘natural’ product demands a microbiological or enzymic approach (Figure 2). Processes using cinnamoyl esterases [23] together with plant cell-wall glycosyl hydrolases have been developed. A cinnamoyl esterase from Aspergillus niger (FAEA), inducible upon growth on cerealderived material, released almost 100% of the ferulic acid from wheat bran when used in conjunction with a xylanase from Trichoderma viride [24]. Another cinnamoyl esterase from A. niger (CinnAE), in conjunction with an arabinanase
A wide range of soil-dwelling microbes can degrade ferulic acid. The ease with which ferulic acid can be catabolised to vanillic acid (though vanillin may be an intermediate) has led to interest in organisms and enzymes that might be used to
Figure 2 Ara Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Gal
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Xyl Xyl Xyl Xyl Xyl Xyl Xyl Ara Current Opinion in Biotechnology
A theoretical feruloylated cereal arabinoxylan. Arrows indicate points of cleavage by a cinnamoyl esterase, releasing monomeric and dimeric ferulic acid for further metabolism. Ara, arabinose; Gal, galactose; GlcA, glucuronic acid; Xyl, xylose.
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Figure 3
(a) COOH
CH=CH–COOH
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HO Aspergillus niger
OCH3
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Pycnoporus cinnabarinus
OCH3
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CHO
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(b) CH=CH–COOH
CH=CH–COSCoA fcs
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ech
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HO 4-Hydroxycinnamate: CoA ligase
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4-Hydroxycinnamoyl-CoA hydratase/lyase
OCH3 Feruloyl-CoA
HO OCH3 4-Hydroxy-3-methoxyphenyl-βhydroxypropionyl-CoA
ech
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(c) OH OH O HO
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Figure 3 legend
Microbial routes to vanillin. (a) A two-step process from ferulic acid using the filamentous fungi Aspergillus niger and Pycnoporus cinnabarinus. (b) Metabolism of ferulic acid via vanillin in Pseudomonas fluorescens AN103. The gene nomenclature from [39•]
is adopted. (c) Biocatalytic synthesis of vanillin from glucose. Recombinant E.coli KL7 strain converts glucose to vanillic acid, which is then reduced to vanillin in vitro using an aryl aldehyde dehydrogenase partially purified from Neurospora crassa.
reduce vanillic acid to vanillin. Li and Rosazza [31] have characterised the products of vanillic acid supplied to growing cultures of Nocardia sp. strain NRRL 5646. The main products were guaiacol (69% yield) and vanillyl alcohol (11% yield). An ATP- and NADPH-requiring carboxylic acid reductase that reduced vanillic acid to vanillin was purified.
substrates, 4-coumaroyl-CoA and caffeoyl-CoA, are also accepted by HCHL and give rise to 4-hydroxybenzaldehyde and protocatechuic aldehyde, respectively [36•]. The third enzyme of the pathway, catalysing the removal of vanillin, is vanillin:NAD+ oxidoreductase. The genes encoding these three enzymes are closely associated in the genome and appear co-induced in response to ferulic acid ([35]; A Narbad, unpublished data).
Using filamentous fungi, a two-stage process for vanillin formation was developed in which a strain of A. niger was first used to convert ferulic acid to vanillic acid, which was then reduced to vanillin by a laccase-deficient strain of Pycnoporus cinnabarinus [32] (Figure 3a). Laccase activity was found to be associated with the formation of ferulic polymers and the loss of phenolic monomers from the culture medium and so a laccase deficient strain was used to avoid these problems [7•]. The reduction of vanillic acid to vanillin (and also further to vanillyl alcohol) by P. cinnabarinus was promoted by cellobiose, which diminishes the concurrent decarboxylation of vanillic acid to a by-product, methoxyhydroquinone. The basis of this effect is not fully understood [33]. More recently, high-density cultures of P. cinnabarinus alone have been studied [6]. Using a glucose-phospholipid medium, 760 mg/l of vanillin was produced from ferulic acid over 15 days. Substantially higher levels of production of vanillin from ferulic acid have been reported with two strains of Amycolatopsis [P2], which were reported to accumulate as much as 11.5 g/l, and with Streptomyces setonii [8•]. This latter organism accumulated vanillic acid to around 200 mg/l and then proceeded to accumulate vanillin as a metabolic ‘overflow’ product, reaching levels of 6.4 g/l in shake-flask experiments, with a molar yield of 68%. Guaiacol, which imparts a smoky flavour, was also produced [P3]. The biochemical basis for these very high accumulations is not well understood. As with the metabolism of eugenol to ferulic acid, the mechanism of chain-shortening of ferulic acid to vanillin has so far been elucidated only in Pseudomonas (Figure 3b). Narbad and Gasson [34] isolated P. fluorescens AN103 on the basis of growth on ferulic acid as the sole carbon source. The initial enzyme in the pathway of this strain is a 4-hydroxycinnamate:CoA ligase (4-CL; AMP-forming) active with ferulate, 4-coumarate and caffeate. An enzyme denoted 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) then catalyses the hydration and subsequent retro-aldol cleavage of feruloyl-CoA to form vanillin and acetyl-CoA [35,P4]. This chain-shortening reaction is distinct from the β-oxidation that occurs in fatty acid degradation. Two closely related 4-hydroxycinnamoyl-CoA
Corresponding genes have been isolated independently from other Pseudomonas strains, including the eugenoldegrading strain HR199 [37,38,39•,P1]. An intriguing observation is that a gene (aat) encoding a putative β-ketoacyl-CoA thiolase is present in strain HR199 [39•], adjacent to the gene fcs encoding 4-CL. It is conceivable that this is a relic of a β-oxidative pathway to vanillic acid, the β-oxidative activity (4-hydroxyphenyl-β-hydroxypropionyl-CoA dehydrogenase) having been lost during evolution. Two other potential biotechnological routes to vanillin have received little recent attention. One is based on the cleavage of isorhapontin, a monoglucosylated stilbene constituent of spruce bark, by a dioxygenase isolated from Pseudomonas strain TMY1009 [40]. The second is based on the action of soybean lipoxygenase on esters of coniferyl alcohol (which can be formed from eugenol) [P5].
Product recovery Product recovery is a major factor in the technical and economic feasibility of a biotechnology process. Pervaporation, involving selective adsorption of a volatile solute onto a hydrophobic membrane and desorption into a vapour phase, has been described for vanillin [41,42]. The selective adsorption of a required product may aid production by limiting unwanted further reactions; for example, polystyrenic resins limited the further reduction of vanillin to vanillyl alcohol in cultures of Phanerochaete chrysosporium [43]. For the bioproduction of organic mol-ecules generally, aqueous–organic solvent two-phase systems offer considerable potential [44,45]. Decarboxylation of ferulic acid to 4-vinylguaiacol by Bacillus subtilis in such systems has been described [46]. It is possible that microbes selected for their ability to degrade eugenol or isoeugenol already display some degree of solvent resistance. This may be true also of Amycolatopsis [P2] and S. setonii [8•,P3] strains that can accumulate exceptionally high levels of vanillin from ferulic acid. As the genetic and biochemical basis of organic-solvent resistance in microorganisms is elucidated [47], the rational design of solvent-resistant microbes for biotechnology may become possible.
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Metabolic engineering The isolation of genes of vanillin-biosynthetic pathways presents several possibilities. An obvious target in Pseudomonas strains is the deletion of the vanillin:NAD+ oxidoreductase activity, in order to promote the accumulation of vanillin by preventing its oxidation to vanillic acid [48•,49]. In the case of strain HR199, this was only partially successful because vanillin was found also to be a substrate of coniferyl aldehyde dehydrogenase, an enzyme of the eugenol degradative pathway. This lead to difficulties in selecting for mutants unable to grow on vanillin and ‘leakiness’ of the derivative strains in which the gene had been specifically disrupted [48•]. In any event, these manipulated Pseudomonas strains do not match the levels of vanillin reportedly produced from ferulic acid by Amycolatopsis [P2] and by S. setonii [8•,P3]. A different approach is to express the genes in a host organism. 4-CL and HCHL have been expressed together in E. coli and vanillin formation at millimolar levels has been observed in resting cells supplied with ferulic acid [39•]. Vanillyl alcohol was also produced. Another possibility is introduction of the genes into food-fermentative organisms, for example Lactococcus or Saccharomyces, in order to produce flavour compounds directly in a food product. This would obviate the need for flavour extraction. The expression of HCHL in plants is of particular interest because the 4-hydroxycinnamoyl-CoA thioesters that are substrates for HCHL are also intermediates of the plant central phenylpropanoid and lignin biosynthetic pathways. Although HCHL has been expressed in plant systems (A Mitra, MJ Mayer, personal communication), so far no vanillin or vanillin-β-D-glucoside has been detected. This might reflect a low availability of endogenous feruloyl-CoA. Vanilla is unusual, if not unique, in accumulating vanillin-β-D-glucoside and more detailed study of its biochemistry might reveal the particular requirements for vanillin-β-D-glucoside formation. Li and Frost [50] recently attempted to generate vanillin from glucose via the shikimate pathway using genetically engineered E. coli in a fed-batch fermentation (Figure 3c). In essence, the basis of the study was a strain of E. coli KL7 carrying a mutated aroE (shikimate dehydrogenase) locus and an aroBaroZ (3-dehydroquinate synthase and 3-dehydroshikimate dehydratase) cassette, together with plasmid-localised COMT (catechol-O-methyltransferase) and aroFFBR (a 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase insensitive to feedback inhibition). The result was an organism with an increased capacity to generate 3-dehydroshikimate, but blocked in the further conversion to shikimate; instead, 3-dehydroshikimate dehydratase produced protocatechuate that was then methylated by catechol-O-methyltransferase to produce vanillate. The final conversion of vanillate to vanillin was catalysed extracellularly, following ethyl acetate extraction of the acidified broth, by an aryl dehydrogenase partially purified from Neurospora crassa (Figure 3c). Vanillate
formation was limited by protocatechuate methylation; increasing the activity of catechol-O-methyltransferase (by use of a plasmid with two copies of COMT) had little effect, but supplementation with L-methionine was effective, suggesting a limiting supply of S-adenosylmethionine. Under these conditions, 5 g/l of vanillate was formed after 48 h. The enzymic reduction of vanillate to vanillin proceeded in almost complete yield in 7 h, but required ATP and glucose-6-phosphate dehydrogenase to recycle the NADP cofactor. The obvious limitations of this scheme lie in the methylation of protocatechuate and in a separate vanillate reduction step using an expensive cofactor recycling system. The potential advantage is synthesis from a cheap and ubiquitous feedstock, glucose.
Conclusions Substantial progress has been made towards achieving a ‘natural’ vanilla flavour using biological ‘clean technology’. Might this erode the market for vanilla from Vanilla pods? This is a serious question; however, vanilla is governed by precise labelling regulations, notably in the US and France, and the quality and flavour complexity of vanilla are unlikely to be equalled by a biotechnological substitute. Furthermore, isotope-ratio mass spectrometry [51,52] and nuclear magnetic resonance [53] might be used to distinguish the biosynthetic origin of vanillin from Vanilla and from microbial sources. If, in addition, gene technology is used in the creation of potential novel foods, further issues are raised. Detailed evidence is required by regulatory authorities that harmful unintended effects of genetic manipulation are not present or unlikely to arise. Such evidence may be obtained by metabolic profiling and analysis of protein expression patterns. An armoury of analytical technologies is developing to address these questions and to provide a level of analysis and interpretation that, paradoxically, looks likely to exceed that either available or demanded for traditional foodstuffs. A vanilla-type flavour by genetic engineering is at present closer to technological reality than to commercial realisation. The study of vanillin biosynthesis has led to the discovery of several interesting new enzymes that raise more general evolutionary questions and offer novel scientific opportunities. More will undoubtedly follow if the characterisation recently achieved in Pseudomonas is extended to other organisms.
Acknowledgements The authors acknowledge support from the Biotechnology and Biological Sciences Research Council, through the Competitive Strategic Grant to the Institute of Food Research, and from the European Commission. We thank Delphine Couteau for helpful discussions.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
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3.
Benz I, Muheim A: Biotechnological production of vanillin. In Flavour science — Recent Developments. Edited by Taylor AJ, Mottram DS. Cambridge, UK: The Royal Society of Chemistry; 1996:111-117.
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Krings U, Berger RG: Biotechnological production of flavours and fragrances. Appl Microbiol Biotechnol 1998, 49:1-8.
5. •
Rao SR, Ravishankar GA: Vanilla flavour: production by conventional and biotechnological routes. J Sci Food Agric 2000, 80:289-304. This comprehensive review includes a particularly useful summary of the cultivation, chemistry and nomenclature of vanilla. 6.
Oddou J, Stentelaire C, Lesage-Meessen L, Asther M, Colonna Ceccaldi B: Improvement of ferulic acid bioconversion into vanillin by use of high-density cultures of Pycnoporus cinnabarinus. Appl Microbiol Biotechnol 1999, 53:1-6.
7. •
Lomascolo A, Stentelaire C, Asther M, Lesage-Meessen L: Basidiomycetes as new biotechnological tools to generate natural aromatic flavours for the food industry. Trends Biotechnol 1999, 17:282-289. This includes an account of vanillin biosynthesis using Aspergillus, Pycnoporus and Phanerochaete. 8. Muheim A, Lerch K: Towards a high-yield conversion of ferulic acid • to vanillin. Appl Microbiol Biotechnol 1999, 51:456-461. The authors give an account of vanillin formation by Streptomyces setonii undertaken by Givaudan-Roure; comparison with Pseudomonas. 9.
11. Schnitzler J-P, Madlung J, Rose A, Seitz HU: Biosynthesis of p-hydroxybenzoic acid in elicitor-treated carrot cell cultures. Planta 1992, 188:594-600. 12. Löscher R, Heide L: Biosynthesis of p-hydroxybenzoate from p-coumarate and p-coumaroyl-Coenzyme A in cell-free extracts of Lithospermum erythrorhizon cell cultures. Plant Physiol 1994, 106:271-279. 13. Rabenhorst J: Production of methoxyphenol-type natural aroma chemicals by biotransformation of eugenol with a new Pseudomonas sp. Arch Microbiol Biotechnol 1996, 46:470-474. 14. Priefert H, Overhage J, Steinbüchel A: Identification and molecular •• characterization of the eugenol hydroxylase genes (ehyA/ehyB) of Pseudomonas sp. strain HR199. Arch Microbiol 1999, 172:354-363. An elegant analysis of the genes and enzymes responsible for the conversion of eugenol to ferulic acid in a Pseudomonas strain. 15. Furukawa H, Wieser M, Morita H, Sugio T, Nagasawa T: Purification • and characterization of eugenol dehydrogenase from Pseudomonas fluorescens E118. Arch Microbiol 1998, 171:37-43. A detailed analysis of this enzyme, its substrate specificity and its relationship to other flavoenzymes. 16. De Jong E, Van Berkel WJH, Van der Zwan RP, De Bont JAM: Purification and characterization of vanillyl-alcohol oxidase from Penicillium simplicissimum. A novel alcohol oxidase containing covalently bound FAD. Eur J Biochem 1992, 208:651-657. 17.
20. Benen JAE, Sánchez-Torres P, Wagemaker MJM, Fraaije MW, Van Berkel WJH, Visser J: Molecular cloning, sequencing, and heterologous expression of the vao gene from Penicillium simplicissimum 170.90 encoding vanillyl-alcohol oxidase. J Biol Chem 1998, 273:7865-7872. 21. Shimoni E, Ravid U, Shoham Y: Isolation of a Bacillus sp. capable of transforming isoeugenol to vanillin. J Biotechnol 2000, 78:1-9. 22. Clifford MN: Chlorogenic acid and other cinnamates — nature, • occurrence and dietary burden. J Sci Food Agric 1999, 79:362-372. A critical and comprehensive review of the natural occurrence of plant phenolic compounds, including those that can be transformed to vanillin. It lists levels of these compounds and their derivatives found in a wide range of plants. 23. Williamson G, Kroon PA, Faulds CB: Hairy plant polysaccharides: a close shave with microbial esterases. Microbiology 1998, 144:2011-2023. 24. Faulds CB, Williamson G: Release of ferulic acid from wheat bran by a ferulic acid esterase (FAE-III) from Aspergillus niger. Appl Microbiol Biotechnol 1995, 43:1082-1087. 25. Kroon PA, Williamson G: Release of ferulic acid from sugar-beet pulp by using arabinanase, arabinofuranosidase and an esterase from Aspergillus niger. Biotechnol Appl Biochem 1996, 23:263-267. 26. Ferreira LMA, Wood TM, Williamson G, Faulds C, Hazlewood GP, Black GW, Gilbert HJ: A modular esterase from Pseudomonas fluorescens subsp. cellulosa contains a non-catalytic cellulosebinding domain. Biochem J 1993, 294:349-355. 27.
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28. Couteau D, Mathaly P: Purification of ferulic acid by adsorption after enzymic release from a sugar-beet pulp extract. Ind Crops Products 1997, 6:237-252. 29. Saulnier L, Thibault JF: Ferulic acid and diferulic acids as components of sugar-beet pectins and maize bran heteroxylans. J Sci Food Agric 1999, 79:396-402. 30. Parr AJ, Waldron KW, Ng A, Parker MJ: The wall-bound phenolics of Chinese water chestnut (Eleocharis dulcis). J Sci Food Agric 1996, 71:501-507. 31. Li T, Rosazza JPN: Biocatalytic synthesis of vanillin. Appl Environ Microbiol 2000, 66:684-687. 32. Lesage-Meessen L, Delattre M, Haon M, Thibault JF, Colonna Ceccaldi B, Brunerie P, Asther M: Two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus. J Biotechnol 1996, 50:107-113. 33. Bonnin E, Lesage-Meessen L, Asther M, Thibault JF: Enhanced bioconversion of vanillic acid into vanillin by the use of ‘natural’ cellobiose. J Sci Food Agric 1999, 79:484-486. 34. Narbad A, Gasson MJ: Metabolism of ferulic acid to vanillin using a novel CoA-dependent pathway in a newly-isolated strain of Pseudomonas fluorescens. Microbiology 1998, 144:1397-1405. 35. Gasson MJ, Kitamura Y, McLauchlan WR, Narbad A, Parr AJ, Parsons ELH, Payne J, Rhodes MJC, Walton NJ: Metabolism of ferulic acid to vanillin. A bacterial gene of the enoyl-SCoA hydratase/isomerase superfamily encodes an enzyme for the hydration and cleavage of hydroxycinnamic acid SCoA thioester. J Biol Chem 1998, 273:4163-4170. 36. Mitra A, Kitamura Y, Gasson MJ, Narbad A, Parr AJ, Payne J, • Rhodes MJC, Sewter C, Walton NJ: 4-Hydroxycinnamoyl-CoA hydratase/lyase (HCHL) – an enzyme of phenylpropanoid chain-cleavage from Pseudomonas. Arch Biochem Biophys 1999, 365:10-16. This paper describes the purification and kinetic properties of the vanillin-forming enzyme isolated from Pseudomonas fluorescens strain AN103. 37.
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38. Venturi V, Zennaro F, Degrassi G, Okeke BC, Bruschi CV: Genetics of ferulic acid bioconversion to protocatechuic acid in plant-growth-promoting Pseudomonas putida WCS358. Microbiology 1998, 144:965-973. 39. Overhage J, Priefert H, Steinbüchel A: Biochemical and genetic • analyses of ferulic acid catabolism in Pseudomonas sp. strain HR199. Appl Environ Microbiol 1999, 65:4837-4847. Detailed analysis of the genes of ferulic acid catabolism in this eugenoldegrading strain.
48. Overhage J, Priefert H, Rabenhorst J, Steinbüchel A: Biotransformation • of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene. Appl Microbiol Biotechnol 1999, 52:820-828. A detailed analysis of attempts to increase vanillin accumulation by specific gene disruption and of the pitfalls encountered. 49. Okeke BC, Venturi V: Construction of recombinants Pseudomonas putida BO14 and Escherichia coli QEFCA8 for ferulic acid biotransformation to vanillin. J Biosci Bioeng 1999, 88:103-106. 50. Li K, Frost JW: Synthesis of vanillin from glucose. J Am Chem Soc 1998, 120:10545-10546.
40. Kamoda S, Habu N, Samejima M, Yoshimoto T: Purification and α,β β-dioxygenase responsible for some properties of lignostilbene-α the Cα-Cβ cleavage of a diarylpropane type lignin model compound from Pseudomonas sp. TMY1009. Agricultural Biol Chem 1989, 53:2757-2761.
51. Kaunzinger A, Juchelka D, Mosandl A: Progress in the authenticity assessment of vanilla. 1. Initiation of authenticity profiles. J Agricultural Food Chem 1997, 45:1752-1757.
41. Bengston G, Bödekker KW: Extraction of bioproducts with homogeneous membranes. In Bioflavour 95. Edited by Étiévant P, Schreier P. Paris: INRA Editions; 1995:393-403.
52. Dennis MJ, Wilson P, Kelly S, Parker I: The use of pyrolytic techniques to estimate site-specific isotope data of vanillin. J Anal Appl Pyrol 1998, 47:95-103.
42. Böddeker KW, Gatfield IL, Jahnig J, Scorm C: Pervaporation at the vapor pressure limit: vanillin. J Membrane Sci 1997, 137:155-158.
53. Martin GJ: Recent advances in site-specific natural isotope fractionation studied by nuclear magnetic resonance. Isotopes Environ Health Studies 1998, 34:233-243.
43. Stentelaire C, Lesage-Meessen L, Delattre M, Haon M, Sgoillot JC, Colonna Cecaldi B, Asther M: By-passing of unwanted vanillyl alcohol formation using selective adsorbents to improve vanillin production with Phanerochaete chrysosporium. World J Microbiol Biotechnol 1998, 14:285-287. 44. Salter GJ, Kell DB: Solvent selection for whole cell biotransformations in organic media. Crit Rev Biotechnol 1995, 15:139-177. 45. Schmid A, Kollmer A, Mathys RG, Witholt B: Developments toward large-scale bacterial processes in the presence of bulk amounts of organic solvents. Extremophiles 1998, 2:249-256. 46. Lee I-Y, Volm TG, Rosazza JPN: Decarboxylation of ferulic acid to 4-vinylguaiacol by Bacillus pumilus in aqueous-organic solvent two-phase systems. Enzyme Microb Technol 1998, 23:261-266. 47.
Aono R: Improvement of organic solvent tolerance level of Escherichia coli by overexpression of stress-responsive genes. Extremophiles 1998, 2:239-248.
Patents P1. Steinbüchel A, Priefert H, Rabenhorst J: Syntheesenzyme für die Herstellung von Coniferylalkohol, Coniferylaldehyd, Ferulasäure, Vanillin und Vanillinsäure und deren Verwendung. European Patent Application 1998, EP 0 845 532 A2. P2. Rabenhorst J, Hopp R: Verfahren zur Herstellung von Vanillin und dafür geeignete Mikroorganismen. European Patent Application 1997, EP 0 761 817 A2. P3. Muheim A, Müller B, Münch T, Wetli M: Process for the production of vanillin. European Patent Application 1998, EP 0 885 968 A1. P4. Narbad A, Rhodes MJC, Gasson MJ, Walton NJ: Production of vanillin. PCT Patent Application 1997, WO 97/35999. P5. Markus PH, Peters ALJ, Roos R: Process for the preparation of phenylaldehydes. European Patent Application 1992, EP 0 542 348 A2.
Novel approaches to the biosynthesis of vanillin Nicholas J Walton*, Arjan Narbad*, Craig B Faulds† and Gary Williamson† Microorganisms able to produce vanillin in excess of 6 g/l from ferulic acid have now been isolated. In Pseudomonas strains, the metabolic pathway from eugenol via ferulic acid to vanillin has been characterised at the enzymic and molecular genetic levels. Attempts to introduce vanillin production into other organisms by genetic engineering have begun. Addresses *Food Safety Science Division and † Diet, Health and Consumer Science Division, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK Correspondence: Nicholas J Walton; e-mail: [email protected] Current Opinion in Biotechnology 2000, 11:490–496 0958-1669/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations 4-CL 4-hydroxycinnamate:CoA ligase HCHL 4-hydroxycinnamoyl-CoA hydratase/lyase
Introduction Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the major component of vanilla flavour extracted from the fermented pods of Vanilla orchids. It is the world’s principal flavouring compound, used extensively in the food industry in ice cream, chocolate and confectionery products and also as a fragrance. This review assesses recent advances in vanillin biosynthesis and in realising the potential of biotechnology in this field. The emphasis here will be on the biochemical and molecular genetic literature from 1998 onwards. For earlier literature the reader is directed to several reviews addressing the microbial biosynthesis of flavour compounds [1–4]. A wide-ranging review that, within its coverage, summarises the terminology of vanilla and vanillin preparations has recently appeared [5•].
Why biotechnology? Vanilla pods are produced largely in Madagascar and Indonesia and contain 2–3% by weight of vanillin in the cured pod [6,7•]. The major markets are the US, France and Germany. Less than 0.5% of the market (~20–50 tons per year out of a total of 12,000 tons per year) is met by extraction of Vanilla pods [4,7•]; the overwhelming usage in the flavour industry is of synthetic vanillin, for example, produced from kraft lignin, with a market price below $15 per kilo [7•,8•]. The value of the vanillin extracted from Vanilla pods is in contrast variously calculated as being between $1200 per kilo and $4000 per kilo [7•,8•]. An opportunity for biotechnology therefore lies in producing a replacement for synthetic vanillin that is produced ‘naturally’ (i.e. non-chemically) from sources other than the ‘named material’ (Vanilla) [6] and at an economic price [4]. Ideally, the replacement will include some of the minor components of natural
vanilla, notably vanillic acid, vanillyl alcohol and 4-hydroxybenzaldehyde, thereby differentiating it further from the synthetic compound. Vanillin is essentially absent from the green Vanilla pod and is only released from its β-D-glucoside during curing of the pods after harvest. The pathway of formation of vanillin-β-D-glucoside is still unresolved. A probable precursor is the phenylpropanoid ferulic acid (4-hydroxy3-methoxycinnamic acid), which possesses the same ring substituents. Although some studies of phenylpropanoid biochemistry and molecular biology have been reported in Vanilla [9,10], the key chain-shortening reactions from the cinnamate to the benzaldehyde are not characterised, in common with the formation of benzaldehydes and benzoates generally in plants (e.g. see [11,12]). Vanilla is not ideal for biotechnology because, in particular, it is slow-growing in tissue culture and the vanillin pathway is not very actively expressed. There have been continual attempts to produce vanillin from Vanilla and also Capsicum (chilli pepper) in culture (reviewed in [5•]). Microorganisms, on account of their rapid growth rates and amenability to molecular genetics, are much better targets and can be selected for their ability to grow on a putative precursor of vanillin as a sole source of carbon and energy. This precursor should be available at low cost, for example, as an agricultural processing by-product.
Feedstocks and biosynthetic pathways Several potential feedstocks have been suggested, including curcumin, siam benzoin resin, phenolic stilbenes, eugenol and ferulic acid [3]. The latter two have received most attention. Eugenol is a principal aromatic constituent of clove oil. Its market price is ~$5 per kilo, making it an economically realistic feedstock [8•]. The pathway from eugenol to ferulic acid (Figure 1) has been characterised in a strain of Pseudomonas (HR199) [13,P1]. The reactions are catalysed, successively, by eugenol hydroxylase (ehyA and ehyB genes), coniferyl alcohol dehydrogenase (calA) and coniferyl aldehyde dehydrogenase (calB) [14••]. Eugenol hydroxylase is a heterodimeric flavocytochrome c enzyme; ehyA encodes the cytochrome c subunit. A corresponding enzyme, designated eugenol dehydrogenase, has been purified and characterised from Pseudomonas fluorescens E118 [15•]). The substrate specificity is wide and includes vanillyl alcohol, which is converted to vanillin. Conversely, a vanillyl alcohol oxidase from Penicillium simplicissimum [16–18] is a flavoprotein homooctamer; however, it too has broad substrate specificity and can oxidise eugenol, producing coniferyl alcohol. The crystal structure has been determined [19]. The cDNA has been isolated and the gene heterologously expressed in Escherichia coli [20].
Novel approaches to the biosynthesis of vanillin Walton et al.
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Figure 1
CH=CH–CH2OH
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Conversion of eugenol to ferulic acid in Pseudomonas sp. strain HR199.
and an α-L-arabinofuranosidase, released a high proportion of the arabinose-linked ferulic acid from sugarbeet pulp [25]. Pseudomonas strains also produce cinnamoyl esterases [26], which can release both monomeric and dimeric ferulic acid from wheat bran and barley spent grain in the presence of a xylanase [27]. The recovery of ferulic acid has received considerable attention. One procedure yielded 1 g of ferulic acid from 1 kg of dry sugar beet pulp [28]. The sugar beet pulp contained ~6 g/kg dry weight of ferulic acid, so 1 g of ferulic acid represents ~17% yield. Ferulic acid also occurs in several dimeric forms in plant cell walls. In maize, the diferulate content is 25 g/kg [29]. In sugar beet pulp, the diferulate level is 1.7 g/kg, and in Chinese water chestnuts 4.5 g/kg [29,30]. However, enzymes for degrading diferulates into their constituent monomers have not yet been isolated or characterised.
Isoeugenol is also a potential feedstock. Recently, the isolation of a Bacillus subtilis strain was reported that could produce 0.6 g/l of vanillin with a molar yield of 12.4% [21]. Ferulic acid is abundant in many cereals as a component of the cell-wall material [22•]. Wheat bran contains 4–7 g/kg dry weight of cell-wall material and maize bran ~30 g/kg. Rice endosperm contains 12 g/kg. Sugarbeet (~5–10 g/kg) is also a good source. Barley spent grain, a brewing by-product, is a poorer source (~0.1 g/kg). Ferulic acid in these materials is present esterified to carbohydrates and can be released by treatment with strong alkali, but production of a ‘natural’ product demands a microbiological or enzymic approach (Figure 2). Processes using cinnamoyl esterases [23] together with plant cell-wall glycosyl hydrolases have been developed. A cinnamoyl esterase from Aspergillus niger (FAEA), inducible upon growth on cerealderived material, released almost 100% of the ferulic acid from wheat bran when used in conjunction with a xylanase from Trichoderma viride [24]. Another cinnamoyl esterase from A. niger (CinnAE), in conjunction with an arabinanase
A wide range of soil-dwelling microbes can degrade ferulic acid. The ease with which ferulic acid can be catabolised to vanillic acid (though vanillin may be an intermediate) has led to interest in organisms and enzymes that might be used to
Figure 2 Ara Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl Gal
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A theoretical feruloylated cereal arabinoxylan. Arrows indicate points of cleavage by a cinnamoyl esterase, releasing monomeric and dimeric ferulic acid for further metabolism. Ara, arabinose; Gal, galactose; GlcA, glucuronic acid; Xyl, xylose.
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Figure 3
(a) COOH
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Pycnoporus cinnabarinus
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Figure 3 legend
Microbial routes to vanillin. (a) A two-step process from ferulic acid using the filamentous fungi Aspergillus niger and Pycnoporus cinnabarinus. (b) Metabolism of ferulic acid via vanillin in Pseudomonas fluorescens AN103. The gene nomenclature from [39•]
is adopted. (c) Biocatalytic synthesis of vanillin from glucose. Recombinant E.coli KL7 strain converts glucose to vanillic acid, which is then reduced to vanillin in vitro using an aryl aldehyde dehydrogenase partially purified from Neurospora crassa.
reduce vanillic acid to vanillin. Li and Rosazza [31] have characterised the products of vanillic acid supplied to growing cultures of Nocardia sp. strain NRRL 5646. The main products were guaiacol (69% yield) and vanillyl alcohol (11% yield). An ATP- and NADPH-requiring carboxylic acid reductase that reduced vanillic acid to vanillin was purified.
substrates, 4-coumaroyl-CoA and caffeoyl-CoA, are also accepted by HCHL and give rise to 4-hydroxybenzaldehyde and protocatechuic aldehyde, respectively [36•]. The third enzyme of the pathway, catalysing the removal of vanillin, is vanillin:NAD+ oxidoreductase. The genes encoding these three enzymes are closely associated in the genome and appear co-induced in response to ferulic acid ([35]; A Narbad, unpublished data).
Using filamentous fungi, a two-stage process for vanillin formation was developed in which a strain of A. niger was first used to convert ferulic acid to vanillic acid, which was then reduced to vanillin by a laccase-deficient strain of Pycnoporus cinnabarinus [32] (Figure 3a). Laccase activity was found to be associated with the formation of ferulic polymers and the loss of phenolic monomers from the culture medium and so a laccase deficient strain was used to avoid these problems [7•]. The reduction of vanillic acid to vanillin (and also further to vanillyl alcohol) by P. cinnabarinus was promoted by cellobiose, which diminishes the concurrent decarboxylation of vanillic acid to a by-product, methoxyhydroquinone. The basis of this effect is not fully understood [33]. More recently, high-density cultures of P. cinnabarinus alone have been studied [6]. Using a glucose-phospholipid medium, 760 mg/l of vanillin was produced from ferulic acid over 15 days. Substantially higher levels of production of vanillin from ferulic acid have been reported with two strains of Amycolatopsis [P2], which were reported to accumulate as much as 11.5 g/l, and with Streptomyces setonii [8•]. This latter organism accumulated vanillic acid to around 200 mg/l and then proceeded to accumulate vanillin as a metabolic ‘overflow’ product, reaching levels of 6.4 g/l in shake-flask experiments, with a molar yield of 68%. Guaiacol, which imparts a smoky flavour, was also produced [P3]. The biochemical basis for these very high accumulations is not well understood. As with the metabolism of eugenol to ferulic acid, the mechanism of chain-shortening of ferulic acid to vanillin has so far been elucidated only in Pseudomonas (Figure 3b). Narbad and Gasson [34] isolated P. fluorescens AN103 on the basis of growth on ferulic acid as the sole carbon source. The initial enzyme in the pathway of this strain is a 4-hydroxycinnamate:CoA ligase (4-CL; AMP-forming) active with ferulate, 4-coumarate and caffeate. An enzyme denoted 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) then catalyses the hydration and subsequent retro-aldol cleavage of feruloyl-CoA to form vanillin and acetyl-CoA [35,P4]. This chain-shortening reaction is distinct from the β-oxidation that occurs in fatty acid degradation. Two closely related 4-hydroxycinnamoyl-CoA
Corresponding genes have been isolated independently from other Pseudomonas strains, including the eugenoldegrading strain HR199 [37,38,39•,P1]. An intriguing observation is that a gene (aat) encoding a putative β-ketoacyl-CoA thiolase is present in strain HR199 [39•], adjacent to the gene fcs encoding 4-CL. It is conceivable that this is a relic of a β-oxidative pathway to vanillic acid, the β-oxidative activity (4-hydroxyphenyl-β-hydroxypropionyl-CoA dehydrogenase) having been lost during evolution. Two other potential biotechnological routes to vanillin have received little recent attention. One is based on the cleavage of isorhapontin, a monoglucosylated stilbene constituent of spruce bark, by a dioxygenase isolated from Pseudomonas strain TMY1009 [40]. The second is based on the action of soybean lipoxygenase on esters of coniferyl alcohol (which can be formed from eugenol) [P5].
Product recovery Product recovery is a major factor in the technical and economic feasibility of a biotechnology process. Pervaporation, involving selective adsorption of a volatile solute onto a hydrophobic membrane and desorption into a vapour phase, has been described for vanillin [41,42]. The selective adsorption of a required product may aid production by limiting unwanted further reactions; for example, polystyrenic resins limited the further reduction of vanillin to vanillyl alcohol in cultures of Phanerochaete chrysosporium [43]. For the bioproduction of organic mol-ecules generally, aqueous–organic solvent two-phase systems offer considerable potential [44,45]. Decarboxylation of ferulic acid to 4-vinylguaiacol by Bacillus subtilis in such systems has been described [46]. It is possible that microbes selected for their ability to degrade eugenol or isoeugenol already display some degree of solvent resistance. This may be true also of Amycolatopsis [P2] and S. setonii [8•,P3] strains that can accumulate exceptionally high levels of vanillin from ferulic acid. As the genetic and biochemical basis of organic-solvent resistance in microorganisms is elucidated [47], the rational design of solvent-resistant microbes for biotechnology may become possible.
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Metabolic engineering The isolation of genes of vanillin-biosynthetic pathways presents several possibilities. An obvious target in Pseudomonas strains is the deletion of the vanillin:NAD+ oxidoreductase activity, in order to promote the accumulation of vanillin by preventing its oxidation to vanillic acid [48•,49]. In the case of strain HR199, this was only partially successful because vanillin was found also to be a substrate of coniferyl aldehyde dehydrogenase, an enzyme of the eugenol degradative pathway. This lead to difficulties in selecting for mutants unable to grow on vanillin and ‘leakiness’ of the derivative strains in which the gene had been specifically disrupted [48•]. In any event, these manipulated Pseudomonas strains do not match the levels of vanillin reportedly produced from ferulic acid by Amycolatopsis [P2] and by S. setonii [8•,P3]. A different approach is to express the genes in a host organism. 4-CL and HCHL have been expressed together in E. coli and vanillin formation at millimolar levels has been observed in resting cells supplied with ferulic acid [39•]. Vanillyl alcohol was also produced. Another possibility is introduction of the genes into food-fermentative organisms, for example Lactococcus or Saccharomyces, in order to produce flavour compounds directly in a food product. This would obviate the need for flavour extraction. The expression of HCHL in plants is of particular interest because the 4-hydroxycinnamoyl-CoA thioesters that are substrates for HCHL are also intermediates of the plant central phenylpropanoid and lignin biosynthetic pathways. Although HCHL has been expressed in plant systems (A Mitra, MJ Mayer, personal communication), so far no vanillin or vanillin-β-D-glucoside has been detected. This might reflect a low availability of endogenous feruloyl-CoA. Vanilla is unusual, if not unique, in accumulating vanillin-β-D-glucoside and more detailed study of its biochemistry might reveal the particular requirements for vanillin-β-D-glucoside formation. Li and Frost [50] recently attempted to generate vanillin from glucose via the shikimate pathway using genetically engineered E. coli in a fed-batch fermentation (Figure 3c). In essence, the basis of the study was a strain of E. coli KL7 carrying a mutated aroE (shikimate dehydrogenase) locus and an aroBaroZ (3-dehydroquinate synthase and 3-dehydroshikimate dehydratase) cassette, together with plasmid-localised COMT (catechol-O-methyltransferase) and aroFFBR (a 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase insensitive to feedback inhibition). The result was an organism with an increased capacity to generate 3-dehydroshikimate, but blocked in the further conversion to shikimate; instead, 3-dehydroshikimate dehydratase produced protocatechuate that was then methylated by catechol-O-methyltransferase to produce vanillate. The final conversion of vanillate to vanillin was catalysed extracellularly, following ethyl acetate extraction of the acidified broth, by an aryl dehydrogenase partially purified from Neurospora crassa (Figure 3c). Vanillate
formation was limited by protocatechuate methylation; increasing the activity of catechol-O-methyltransferase (by use of a plasmid with two copies of COMT) had little effect, but supplementation with L-methionine was effective, suggesting a limiting supply of S-adenosylmethionine. Under these conditions, 5 g/l of vanillate was formed after 48 h. The enzymic reduction of vanillate to vanillin proceeded in almost complete yield in 7 h, but required ATP and glucose-6-phosphate dehydrogenase to recycle the NADP cofactor. The obvious limitations of this scheme lie in the methylation of protocatechuate and in a separate vanillate reduction step using an expensive cofactor recycling system. The potential advantage is synthesis from a cheap and ubiquitous feedstock, glucose.
Conclusions Substantial progress has been made towards achieving a ‘natural’ vanilla flavour using biological ‘clean technology’. Might this erode the market for vanilla from Vanilla pods? This is a serious question; however, vanilla is governed by precise labelling regulations, notably in the US and France, and the quality and flavour complexity of vanilla are unlikely to be equalled by a biotechnological substitute. Furthermore, isotope-ratio mass spectrometry [51,52] and nuclear magnetic resonance [53] might be used to distinguish the biosynthetic origin of vanillin from Vanilla and from microbial sources. If, in addition, gene technology is used in the creation of potential novel foods, further issues are raised. Detailed evidence is required by regulatory authorities that harmful unintended effects of genetic manipulation are not present or unlikely to arise. Such evidence may be obtained by metabolic profiling and analysis of protein expression patterns. An armoury of analytical technologies is developing to address these questions and to provide a level of analysis and interpretation that, paradoxically, looks likely to exceed that either available or demanded for traditional foodstuffs. A vanilla-type flavour by genetic engineering is at present closer to technological reality than to commercial realisation. The study of vanillin biosynthesis has led to the discovery of several interesting new enzymes that raise more general evolutionary questions and offer novel scientific opportunities. More will undoubtedly follow if the characterisation recently achieved in Pseudomonas is extended to other organisms.
Acknowledgements The authors acknowledge support from the Biotechnology and Biological Sciences Research Council, through the Competitive Strategic Grant to the Institute of Food Research, and from the European Commission. We thank Delphine Couteau for helpful discussions.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Casey J, Dobb R: Microbial routes to aromatic aldehydes. Enzyme Microb Technol 1992, 14:739-747.
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Hagedorn S, Kaphammer B: Microbial biocatalysis in the generation of flavor and fragrance chemicals. Annu Rev Microbiol 1994, 48:773-800.
3.
Benz I, Muheim A: Biotechnological production of vanillin. In Flavour science — Recent Developments. Edited by Taylor AJ, Mottram DS. Cambridge, UK: The Royal Society of Chemistry; 1996:111-117.
4.
Krings U, Berger RG: Biotechnological production of flavours and fragrances. Appl Microbiol Biotechnol 1998, 49:1-8.
5. •
Rao SR, Ravishankar GA: Vanilla flavour: production by conventional and biotechnological routes. J Sci Food Agric 2000, 80:289-304. This comprehensive review includes a particularly useful summary of the cultivation, chemistry and nomenclature of vanilla. 6.
Oddou J, Stentelaire C, Lesage-Meessen L, Asther M, Colonna Ceccaldi B: Improvement of ferulic acid bioconversion into vanillin by use of high-density cultures of Pycnoporus cinnabarinus. Appl Microbiol Biotechnol 1999, 53:1-6.
7. •
Lomascolo A, Stentelaire C, Asther M, Lesage-Meessen L: Basidiomycetes as new biotechnological tools to generate natural aromatic flavours for the food industry. Trends Biotechnol 1999, 17:282-289. This includes an account of vanillin biosynthesis using Aspergillus, Pycnoporus and Phanerochaete. 8. Muheim A, Lerch K: Towards a high-yield conversion of ferulic acid • to vanillin. Appl Microbiol Biotechnol 1999, 51:456-461. The authors give an account of vanillin formation by Streptomyces setonii undertaken by Givaudan-Roure; comparison with Pseudomonas. 9.
11. Schnitzler J-P, Madlung J, Rose A, Seitz HU: Biosynthesis of p-hydroxybenzoic acid in elicitor-treated carrot cell cultures. Planta 1992, 188:594-600. 12. Löscher R, Heide L: Biosynthesis of p-hydroxybenzoate from p-coumarate and p-coumaroyl-Coenzyme A in cell-free extracts of Lithospermum erythrorhizon cell cultures. Plant Physiol 1994, 106:271-279. 13. Rabenhorst J: Production of methoxyphenol-type natural aroma chemicals by biotransformation of eugenol with a new Pseudomonas sp. Arch Microbiol Biotechnol 1996, 46:470-474. 14. Priefert H, Overhage J, Steinbüchel A: Identification and molecular •• characterization of the eugenol hydroxylase genes (ehyA/ehyB) of Pseudomonas sp. strain HR199. Arch Microbiol 1999, 172:354-363. An elegant analysis of the genes and enzymes responsible for the conversion of eugenol to ferulic acid in a Pseudomonas strain. 15. Furukawa H, Wieser M, Morita H, Sugio T, Nagasawa T: Purification • and characterization of eugenol dehydrogenase from Pseudomonas fluorescens E118. Arch Microbiol 1998, 171:37-43. A detailed analysis of this enzyme, its substrate specificity and its relationship to other flavoenzymes. 16. De Jong E, Van Berkel WJH, Van der Zwan RP, De Bont JAM: Purification and characterization of vanillyl-alcohol oxidase from Penicillium simplicissimum. A novel alcohol oxidase containing covalently bound FAD. Eur J Biochem 1992, 208:651-657. 17.
20. Benen JAE, Sánchez-Torres P, Wagemaker MJM, Fraaije MW, Van Berkel WJH, Visser J: Molecular cloning, sequencing, and heterologous expression of the vao gene from Penicillium simplicissimum 170.90 encoding vanillyl-alcohol oxidase. J Biol Chem 1998, 273:7865-7872. 21. Shimoni E, Ravid U, Shoham Y: Isolation of a Bacillus sp. capable of transforming isoeugenol to vanillin. J Biotechnol 2000, 78:1-9. 22. Clifford MN: Chlorogenic acid and other cinnamates — nature, • occurrence and dietary burden. J Sci Food Agric 1999, 79:362-372. A critical and comprehensive review of the natural occurrence of plant phenolic compounds, including those that can be transformed to vanillin. It lists levels of these compounds and their derivatives found in a wide range of plants. 23. Williamson G, Kroon PA, Faulds CB: Hairy plant polysaccharides: a close shave with microbial esterases. Microbiology 1998, 144:2011-2023. 24. Faulds CB, Williamson G: Release of ferulic acid from wheat bran by a ferulic acid esterase (FAE-III) from Aspergillus niger. Appl Microbiol Biotechnol 1995, 43:1082-1087. 25. Kroon PA, Williamson G: Release of ferulic acid from sugar-beet pulp by using arabinanase, arabinofuranosidase and an esterase from Aspergillus niger. Biotechnol Appl Biochem 1996, 23:263-267. 26. Ferreira LMA, Wood TM, Williamson G, Faulds C, Hazlewood GP, Black GW, Gilbert HJ: A modular esterase from Pseudomonas fluorescens subsp. cellulosa contains a non-catalytic cellulosebinding domain. Biochem J 1993, 294:349-355. 27.
Brodelius PE, Xue ZT: Isolation and characterisation of a cDNA from cell suspension cultures of Vanilla planifolia encoding 4-coumarate: coenzyme A ligase. Plant Physiol 1997, 35:497-506.
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