Springer-Verlag 1997
Appl Microbiol Biotechnol (1997) 47: 393–397
ORIGINAL PAPER
L. Lesage-Meessen · M. Haon · M. Delattre J. -F. Thibault · B. Colonna Ceccaldi · M. Asther
An attempt to channel the transformation of vanillic acid into vanillin by controlling methoxyhydroquinone formation in Pycnoporus cinnabarinus with cellobiose
Received: 24 July 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996
Abstract The effects of adding cellobiose on the transformation of vanillic acid to vanillin by two strains of Pycnoporus cinnabarinus MUCL39532 and MUCL38467 were studied. When maltose was used as the carbon source in the culture medium, very high levels of methoxyhydroquinone were formed from vanillic acid. When cellobiose was used as the carbon source and/or added to the culture medium of P. cinnabarinus strains on day 3 just before vanillic acid was added, it channelled the vanillic acid metabolism via the reductive route leading to vanillin. Adding 3.5 g l)1 cellobiose to 3-dayold maltose cultures of P. cinnabarinus MUCL39532 and 2.5 g l)1 cellobiose to 3-day-old cellobiose cultures of P. cinnabarinus MUCL38467, yielded 510 mg l)1 and 560 mg l)1 vanillin with a molar yield of 50.2 % and 51.7 % respectively. Cellobiose may either have acted as an easily metabolizable carbon source, required for the reductive pathway to occur, or as an inducer of cellobiose:quinone oxidoreductase, which is known to inhibit vanillic acid decarboxylation.
L. Lesage-Meessen (&) · M. Haon · M. Delattre · M. Asther Laboratoire de Biotechnologie des Champignons Filamenteux, INRA, Centre d′Enseignement Supe´rieur en Biotechnologie, ESIL, Faculte´ des Sciences de Luminy, 163 Avenue de Luminy, Case Postale 925, 13288 Marseille cedex 09, France Fax: + (33) 04 91 82 86 01 e-mail:
[email protected] J. F. Thibault Laboratoire de Biochimie et Technologie des Glucides, INRA, rue de la Ge´raudie`re, BP1627, 44316 Nantes cedex 03, France B. Colonna Ceccaldi Pernod-Ricard, Centre de Recherche, 120 Avenue du Mare´chal Foch, 94015 Cre´teil, France
Introduction Vanillin is the most widely recognized aromatic chemical (Clark 1990). Vanillic acid is a well-known product of the degradation of lignin and lignin-related substances by the white-rot fungi (Kirk 1971) and other microorganisms (Sutherland et al. 1983). It was also found to be formed during the dissimilation of ferulic acid by the white-rot fungi (Nishida and Fukuzumi 1978; Gupta et al. 1981) and the fungi imperfecti (Rahouti et al. 1989). The metabolism of vanillic acid has been thoroughly studied in Sporotrichum pulverulentum (Ander et al. 1980; Buswell et al. 1982; Ander et al. 1983). It has been established that vanillic acid is either oxidatively decarboxylated into methoxyhydroquinone or reduced into vanillin and vanillyl alcohol. Oxidative decarboxylation into methoxyhydroquinone was originally described by Kirk and Lorenz (1973). The enzyme catalysing this reaction, vanillate hydroxylase, was purified from mycelial extracts of S. pulverulentum (Buswell et al. 1981). Decarboxylation of vanillic acid can also be obtained with laccase and peroxidase (Krisnangkura and Gold 1979). The metabolism of vanillic acid has been studied in wild-type S. pulverulentum and in three different mutants, and was found to depend on the culture conditions (Ander et al. 1980). The reducing pathway was not expressed unless the fungus had access to an easily metabolized carbon source such as glucose or cellobiose, while decarboxylation took place in the cultures when only vanillic acid was present. In a study on ferulic acid metabolism by Pycnoporus cinnabarinus (Falconnier et al. 1994), it was established that vanillic acid underwent oxidative decarboxylation into methoxyhydroquinone, as well as reductive conversions into vanillin and vanillyl alcohol (Fig. 1). On the basis of this study, a process was developed for producing vanillin with P. cinnabarinus (Gross et al. 1991); at the optimum, 64 mg l)1 vanillin was obtained from ferulic acid, which was too low for realistic
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Fig. 1 Different pathways of vanillic acid metabolism by Pycnoporus cinnabarinus (from Falconnier et al. 1994)
industrial applications to be feasible. In order to improve the vanillin yield from ferulic acid, a new strategy, in which the complementary bioconversion abilities of two filamentous fungi were combined, was developed (Lesage-Meessen et al. 1996). In the first step, the micromycete Aspergillus niger transforms ferulic acid into vanillic acid, while in the second step, vanillic acid is metabolized into vanillin by the white-rot fungus, P. cinnabarinus. In this way, more than 200 mg l)1 vanillin was obtained with maltose as carbon source, but high levels of methoxyhydroquinone considerably limited the vanillin production . In order to by-pass partly the pathway leading to methoxyhydroquinone and thus to favour the reductive pathway leading to vanillin, it was proposed in the present study to investigate the metabolism of vanillic acid by P. cinnabarinus strains in the presence of cellobiose.
Materials and methods Two strains of Pycnoporus cinnabarinus, MUCL38467 and MUCL39532, obtained from the Mycothe`que de l′Universite´ Catholique de Louvain (Louvain-La-Neuve, Belgique) were used. The fungi were grown in a basal medium containing maltose (20 g l)1) or cellobiose (5 g l)1) as the carbon source, diammonium tartrate (1.842 g l)1) as the nitrogen source, yeast extract (0.5 g l)1), KH2PO4 (0.2 g l)1), CaCl2 (0.0132 g l)1) and MgSO4 (0.5 g l)1). Cultures were inoculated with mycelium fragments of P. cinnabarinus as described by Falconnier et al. (1994). After growth on 2.5 g l)1 cellobiose medium for 10 days, mycelium was collected, mixed with sterile water and disintegrated into small fragments using an Ultra-Turrax T25 blender (Janke & Kunkel, Gmbh. & Co. KG, Staufen, Germany). A 5-ml sample of this suspension was inoculated into basal medium. Incubation was carried out at 30 °C in 250-ml baffled flasks, containing 100 ml medium and shaken at 120 rpm. After 3 days of incubation, 2.5 g l)1 or 3.5 g l)1 cellobiose was added to the culture medium, followed by 0.3 g l)1 vanillic acid (used as filtered salt solution). From day 4 to day 6, the culture medium was supplemented daily
with 0.3 g l)1 vanillic acid. Various concentrations of cellobiose (1–10 g l)1) were used as the carbon sources and/or added on day 3 to the culture medium; only the best results obtained in terms of the amount of vanillin produced were reported. Flasks without any cellobiose supplementation served as controls. Each experiment was run in triplicate and repeated at least twice. The standard deviation of the analyses was less than 5 %. Growth was measured in terms of the dry weight of mycelium after filtration on glass-fiber filters (GF/D, Whatman, Maidstone, England) and overnight drying at 105 °C. Isocratic HPLC analysis was carried out directly on culture filtrates for the maltose, cellobiose and glucose determinations. An ion-exchange column maintained at 80 °C (BioRad, Richmond, Calif., USA; Aminex ion-exclusion HPX-87P, 300 × 7.8 mm) and a model 1050 HPLC (Hewlett-Packard, Rockville, Md., USA), equipped with a refractive-index detector were used. Water served as eluant at a flow rate of 0.4 ml min)1. Quantification was performed using external standards. Ammonium was measured quantitatively using the Spectroquant 14752 ammonium method (Merck, Darmstadt, Allemagne) with alkaline chloride as the standard. Phenolic metabolites were quantified by HPLC as described by Falconnier et al. (1994).
Results Vanillic acid metabolism by P. cinnabarinus MUCL39532 in the presence of cellobiose The metabolism of vanillic acid by P. cinnabarinus MUCL39532, in terms of the amount of cellobiose added, the mycelial biomass, and the carbon and nitrogen consumption, is given in Fig. 2. At the metabolite production optimum (on day 7), only low amounts of vanillic acid were detectable in the medium. The major aromatic product formed in media containing maltose or cellobiose as the carbon source was methoxyhydroquinone. The pattern of vanillin and vanillyl alcohol accumulation differed very little. Under these conditions, the nitrogen was consumed faster in cellobiose medium than in maltose medium, but 10 mM nitrogen was present at the end of both experiments. As regards the carbon consumption, only 6 g l)1 maltose was dissimilated on day 7, which led to a continuous increase in the mycelial biomass, while the total amount of cellobiose was depleted by day 3, leading to a decrease in the biomass on that day. When cellobiose was added to 3-day-old cultures of strain MUCL39532 grown on maltose medium, vanillic acid was largely metabolized into vanillin, while only low amounts of methoxyhydroquinone and vanillyl alcohol were formed. The best result was obtained by adding 3.5 g l)1 cellobiose to 3-day-old maltose-containing cultures 2 h before vanillic acid addition, which led to a 1.6-fold increase in the vanillin production, corresponding to 510 mg l)1 and to a 2.2-fold decrease in methoxyhydroquinone formation as compared to that recorded in cellobiose-free medium. The molar yield of vanillin production from vanillic acid reached 50.2 %. The cellobiose added on day 3 was depleted very fast, but no change in the fungal biomass was observed as
395 b Fig. 2A–C Vanillic acid transformation into vanillin in 7-day-old cultures of Pycnoporus cinnabarinus MUCL39532 supplemented daily from day 3 to day 6 with 300 mg l)1 vanillic acid in relation to mycelial biomass, carbon and nitrogen consumption: A with maltose as carbon source (20 g l)1); B with cellobiose as carbon source (5 g l)1); C with maltose as carbon source (20 g l)1) and 3.5 g l)1 cellobiose supplemented on day 3. d Nitrogen, j maltose, r cellobiose, m biomass
compared to maltose medium. The maltose and nitrogen consumptions increased significantly, however, but large amounts of carbon and nitrogen were measured at the end of the experiment. Vanillic acid metabolism by P. cinnabarinus MUCL38467 in the presence of cellobiose The metabolism of vanillic acid by P. cinnabarinus MUCL38467, in terms of the amount of cellobiose added, the mycelial biomass, and the carbon and nitrogen consumption, at the metabolite production optimum (on day 7) is given in Fig. 3. Using maltose as the carbon source led to a very strong accumulation of methoxyhydroquinone, attaining 709 mg l)1, while only low levels of vanillin and vanillyl alcohol were produced. Maltose and nitrogen were slowly consumed, reaching 7 g l)1 and 12 mM respectively at the end of the experiment, which led to a continuous increase in the biomass. When 5 g l)1 cellobiose was substituted for 20 g l)1 maltose, a 1.4-fold decrease in the methoxyhydroquinone level was recorded, while the vanillin production increased 1.8-fold, up to 305 mg l)1. Adding cellobiose on day 3 to cellobiose-containing medium resulted in a very high level of vanillin production, along with a significant decrease in the methoxyhydroquinone. The best vanillin production (560 mg l)1, with a molar yield of 51.7 %) was obtained by adding 2.5 g l)1 cellobiose to 3-day-old cellobiose-containing cultures. Under both growth conditions where cellobiose was used as the carbon source, nitrogen was consumed in a similar way and, as previously described with MUCL39532, cellobiose was dissimilated very quickly leading to a decrease in the biomass on day 4. In the experiment in which cellobiose was added on day 3, the biomass obtained was significantly higher.
Discussion As previously reported (Ander et al. 1980; Falconnier et al. 1994; Lesage-Meessen et al. 1996), P. cinnabarinus degrades vanillic acid in a similar way to S. pulverulentum, via either a reduction pathway leading to vanillin and vanillyl alcohol, or an oxidative decarboxylation pathway leading to methoxyhydroquinone. One of the main problems encountered, when P. cinnabarinus is used in a process of vanillin production from vanillic acid as a precursor, has been the predominance of the
396 b Fig. 3A–C Vanillic acid transformation to vanillin in 7-day-old cultures of Pycnoporus cinnabarinus MUCL38467 supplemented daily from day 3 to day 6 with 300 mg l)1 vanillic acid in relation to mycelial biomass, carbon and nitrogen consumption: A with maltose as carbon source (20 g l)1); B with cellobiose as carbon source (5 g l)1); C with cellobiose as carbon source (5 g l)1) and 2.5 g l)1 cellobiose supplemented on day 3. d Nitrogen, j maltose, r cellobiose, m biomass
vanillic acid oxidative system providing methoxyhydroquinone, which yielded relatively low levels of vanillin (Lesage-Meessen et al. 1996). Adding cellobiose has been found to channel the vanillic acid metabolism via the reductive pathway leading to vanillin. Strong decarboxylation and formation of methoxyhydroquinone occurred when maltose was used as the carbon source in P. cinnabarinus cultures, whereas cellobiose channelled the transformation of vanillic acid into vanillin, giving a yield of more than 500 mg l)1. However the behaviour of the strains studied has differed depending on how the cellobiose was applied. Using cellobiose or maltose as carbon sources in P. cinnabarinus MUCL39532 cultures led to similar patterns of vanillic acid metabolism; the vanillin production increased in maltose-containing medium only when cellobiose was applied to 3-day-old cultures before the addition of vanillic acid. With P. cinnabarinus MUCL38467, the vanillin level increased when cellobiose was used as the carbon source as well as when it was added on day 3, before vanillic acid was added. The amount of cellobiose present at the moment of vanillic acid supplementation seems to be the decisive factor. Under those culture conditions yielding high levels of vanillin, there may be sufficient remaining cellobiose, while in cultures with high levels of methoxyhydroquinone, the cellobiose may be completely consumed. The maltose level seems, on the contrary, to have no effect on the expression of the reducing pathway. Our results are in agreement with those obtained by Ander et al. (1980), who showed that S. pulverulentum cultures grown in the presence of vanillic acid did not produce vanillin and vanillyl alcohol without the presence of an energy source such as cellobiose. Similar energy requirements were also described for the reduction of veratric acid by Trametes versicolor (Zenk and Gross 1965). Ander et al. (1980) also established that vanillic acid decarboxylation occurs much earlier than the vanillic acid reduction. On the other hand, fungal growth on cellulose or cellobiose is known to result in cellobiose:quinone oxidoreductase production (Buswell et al. 1982). The synthesis of this extracellular enzyme has been reported in cellulolytic cultures of the white-rot fungi S. pulverulentum and T. versicolor (Westermark and Eriksson 1974) as well as in a variety of different cellulose-degrading fungi such as Phlebia radiata and P. cinnabarinus (Ander and Eriksson 1977). The cellobiose:quinone oxidoreductase induced by cellulose reduces the quinones to the corresponding phenols and requires the presence of cellobiose as the electron donor (Westermark and Eriksson 1974). Ander et al. (1990) have re-
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ported that active cellobiose:quinone oxidoreductase inhibited the vanillic acid decarboxylation catalysed by laccase or peroxidases. By inducing cellobiose:quinone oxidoreductase, cellobiose might indirectly limit the formation of methoxyhydroquinone by inhibiting vanillic acid decarboxylation, and consequently might favour vanillin production. The results of the present study show that adding cellobiose to the culture medium of P. cinnabarinus made it possible largely to by-pass the pathway leading to methoxyhydroquinone formation, thus favouring the production of vanillin. This finding confirms the industrial potential of a recently patented two-step process (Lesage-Meessen et al. 1994) in which Aspergillus niger, used to biotransform ferulic acid into vanillic acid, is combined with P. cinnabarinus to convert vanillic acid efficiently into vanillin in the presence of cellobiose. Considering the high cost of natural vanilla flavour (U.S.$ 4000 kg)1), vanillin is mainly of synthetic origin (for a price of U.S. $ 15 kg)1). With interest in ‘‘natural’’ products increasing, the biotechnological production of vanillin using a two-step bioconversion process offers a promising alternative, since the EEC directives incorporate under the term ‘‘natural products’’ those produced by filamentous fungi. Acknowledgements This study was supported by the Commission of the European Communities, Directorate-General XII for Research, Technological Development and Demonstration in the Field of Agriculture and Agro-Industry and the Conseil Re´gional Provence-Alpes-Coˆte d’Azur (France).
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