Shifting the biotransformation pathways of L-phenylalanine into benzaldehyde by Trametes suaveolens CBS using HP20 resin

Transcription

1 Letters in Applied Microbiology 2001, 32, 262±267 Shifting the biotransformation pathways of L-phenylalanine into benzaldehyde by Trametes suaveolens CBS using HP20 resin A. Lomascolo, M. Asther, D. Navarro, C. Antona, M. Delattre and L. Lesage-Meessen Unite de Biotechnologie des Champignons lamenteux de l'inra, IFR de Biotechnologie Agro-industriel de Marseille, UniversiteÂs de Provence et de la MeÂditerraneÂe, Marseille, France 2000/51: received 11 January 2001 and accepted 17 January 2001 A.LOMASCOLO,M.ASTHER,D.NAVARRO,C.ANTONA,M.DELATTREAND L. L E S A G E - M E E S S E N Aims: The biotransformation of L-phenylalanine into benzaldehyde (bitter almond aroma) was studied in the strain Trametes suaveolens CBS Methods and Results: Cultures of this fungus were carried out in the absence or in the presence of HP20 resin, a highly selective adsorbent for aromatic compounds. For the identi cation of the main catabolic pathways of L-phenylalanine, a control medium (without L-phenylalanine) was supplemented with each of the aromatic compounds, previously detected in the culture broth, as precursors. Trametes suaveolens CBS was shown to biosynthesize benzyl and p-hydroxybenzyl derivatives, particularly benzaldehyde, and large amounts of 3-phenyl-1-propanol, benzyl and p-hydroxybenzyl alcohols as the products of both cinnamate and phenylpyruvate pathways. Conclusions: The addition of HP20 resin, made it possible to direct the catabolism of L- phenylalanine to benzaldehyde, the desired target compound, and to trap it before its transformation into benzyl alcohol. In these conditions, benzaldehyde production was increased 21-fold, from 33 to 710 mg l )1 corresponding to a molar yield of 31%. Signi cance and Impact of the Study: These results showed the good potential of Trametes suaveolens as a biotechnological agent to synthesize natural benzaldehyde which is one of the most important aromatic aldehydes used in the avour industry. INTRODUCTION Flavour and fragrance chemicals used in the food and cosmetic industries represent a market of great interest today. Among them, vanillin, benzaldehyde (bitter almond aroma) and cinnamaldehyde (cinnamon aroma) are the most important aromatic aldehydes used in the avour industry (Welsh et al. 1989). The world consumption of benzalde- is approximately 7000 tons per year (Clark 1995). 2hyde For Correspondence to: Dr A. Lomascolo, Unite de Biotechnologie des Champignons lamenteux de l'inra, IFR de Biotechnologie Agro-industriel de Marseille, ESIL, UniversiteÂs de Provence et de la MeÂditerraneÂe, CP 925, 163, avenue de Luminy, Marseille cedex 09, France ( lomascolo@esil.univ-mrs.fr). several years, consumer preference for natural food additives has led to an increasing demand for natural aroma compounds. For a long time, essential plant oils were the sole sources of natural avours. Natural benzaldehyde was usually liberated by enzymatic hydrolysis from amygdalin, a cyanogenic glycoside present in the nut meat of apricots, peaches, kernel plums and bitter almonds. However, this process was capable of generating toxic by-products, such as hydrocyanic acid (Clark 1995). Biotechnological processes represent an alternative means of producing natural aromas, independent of the availability of plant material (weather, diseases, trade restrictions). For several decades, lamentous fungi have been known for their high ability to synthesize aromatic avours (Janssens et al. 1992; Feron et al. 1996). Some species of white-rot basidiomycetes were shown to produce traces of ã 2001 The Society for Applied Microbiology

2 BENZALDEHYDE BIOSYNTHESIS BY TRAMETES SUAVEOLENS 263 benzaldehyde de novo (Gross and Asther 1989; Gallois et al. 1990; Abraham and Berger 1994) or by bioconversion of L-phenylalanine, which can be a natural precursor, in the range of 25 to 587 mg l ±1 (Berger et al. 1987; Kawabe and Morita 1993; Fabre et al. 1996; Krings et al. 1996; Lapadatescu et al. 1997). However, benzaldehyde is toxic towards fungal metabolism and its accumulation in the culture medium may highly inhibit mycelial growth (Lamer et al. 1996; Lomascolo et al. 1999a). The genus Trametes, and particularly the species T. suaveolens, is used for the commercial production of laccase (Decarvalho et al. 1999; Fakoussa and Frost 1999). Its ability to produce numerous aromatic avours de novo (Lomascolo et al. 1999b) prompted the testing of T. suaveolens for the production of benzaldehyde from L-phenylalanine. The present work reports the study of the biotransformation of L-phenylalanine into benzaldehyde by T. suaveolens CBS , and the improvement of the synthesis of this bitter almond aroma by the use of a selective resin. MATERIALS AND METHODS Fungal strain The strain used in this study, Trametes suaveolens CBS , was obtained from the Centraalbureau voor Schimmelcultures (Delft, The Netherlands) and was kept on malt agar slants at 4 C. Chemicals L-phenylalanine and the aromatic compounds used as external standards in HPLC were provided by Sigma. The HP20 resin, selective for aromatic compounds, was a styrene and divinylbenzene copolymer and was purchased from Mitsubishi (Mitsubishi Chemical Corporation, Milan, Italy). Medium and culture conditions Fungal cultures were grown in a basal medium (Lomascolo et al. 1999a) containing 4á5 gl ±1 L-phenylalanine. Inoculum was prepared as described by Falconnier et al. (1994). Incubations were carried out at 25 C in 250 ml baf ed asks containing 100 ml culture medium, with an agitation rate of 120±130 rev min ±1. The ph was not regulated. Each experiment was carried out in triplicate and repeated at least twice. The standard deviation of analyses was less than 10% of the mean value. In the case of the cultures with HP20 resin, the adsorbent (100 g l ±1 ) was added directly as free particles on day 5 of cultivation. Then, every two days of cultivation, culture medium and resin were removed from two asks and separated by ltration. The compounds adsorbed on the resin were extracted twice with pure ethanol and analysed by HPLC, as were the compounds remaining in the culture medium. When bioconversion of different aromatic metabolites was investigated, the control medium (without L-phenylalanine) Fig. 1 (a) Aromatic compound production from L-phenylalanine by Trametes suaveolens CBS without resin (h) with resin (j). Data are presented for the day of maximal concentration in the culture medium (number in brackets). (b) Time course of mycelial biomass production (r) in relation to residual glucose (e), ammonium ( ) and L-phenylalanine (+) concentrations

3 264 A. LOMASCOLO ET AL. was supplemented with each of the following molecules: p-hydroxybenzoic acid, p-hydroxybenzaldehyde, p-hydroxybenzyl alcohol, benzaldehyde, benzyl alcohol, benzoic acid, cinnamic acid, cinnamaldehyde and cinnamyl alcohol, which were added to the culture medium on day 3 of cultivation at 50 mg l ±1. For phenylpyruvic, phenyllactic, phenylglyoxylic and phenylacetic acids, and 3-phenyl-1-propanol, daily additions of 100 mg l ±1 were made from day 4 to day 9 of cultivation. All the acid precursors were added as sodium salts. Growth measurements, glucose and ammonium determinations Glucose concentrations in the culture medium were measured quantitatively using the GOD±PAP reagent kit (Boehringer Mannheim, Meylan, France). Growth measurements and ammonium concentrations were determined as described by Lesage-Meessen et al. (1996). HPLC quantitative analyses of aromatic metabolites Each day, 1 ml of culture medium was analysed by HPLC according to the method described by Lomascolo et al. (1999a). For phenylglyoxylic and phenyllactic acids, separation was achieved on a Merck (Darmstadt, Germany) C18 reversed-phase column (Licrospher, mm). The column temperature was maintained at 30 C, and the detector wavelengths were set at 220, 280 and 326 nm. The mobile phase, at a ow rate of 0á4 ml min ±1, comprised a mixture of two solvents: A, water with 0á01% (v/v) acetic acid and B, methanol. The following elution pro le was used: a 80:20 mixture of solvents A and B for the rst 2 min, changed linearly to 100% B over 26 min, and 100% B held for 4 min. The column was equilibrated for 5 min in the 80:20 mixture of solvents A and B before each run. Isocratic HPLC analysis on an ion-exchange column maintained at 65 C (Biorad, Richmond, CA, USA; Aminex fast acids, 100 7á8 mm) was carried out for phenylpyruvic acid. The eluant was water with Aromatic precursor added in the culture medium Amounts of metabolites produced* (%) Table 1 Metabolism of aromatic intermediates or derivatives of L-phenylalanine by Trametes suaveolens CBS cinnamic acid cinnamyl alcohol 6; 3-phenyl-1-propanol 32; p-hydroxybenzoic acid 12; methyl cinnamate and cinnamaldehyde (traces) cinnamaldehyde cinnamyl alcohol 4; 3-phenyl-1-propanol 50; p-hydroxybenzoic acid 14 cinnamyl alcohol p-hydroxybenzaldehyde 3; p-hydroxybenzoic acid 21; 3-phenyl-1-propanol 54; cinnamaldehyde (traces) 3-phenyl-1-propanol not metabolized phenylpyruvic acid p-hydroxybenzyl alcohol 5; p-hydroxybenzaldehyde 3; benzyl alcohol 13; 3-phenyl-1-propanol 13; phenylacetaldehyde 7; 2-phenylethanol 7 phenyllactic acid mandelic acid 6; benzyl alcohol 7; 3-phenyl-1-propanol 7; p-hydroxybenzaldehyde 2; p-hydroxybenzyl alcohol 3 phenylglyoxylic acid mandelic acid 9; benzaldehyde 21; benzyl alcohol 53; p-hydroxybenzaldehyde (traces) mandelic acid phenylglyoxylic acid 7; benzyl alcohol 7; phenylacetic acid mandelic acid 5; phenylglyoxylic acid 12; 2-phenylethanol 58 2-phenylethanol not metabolized benzaldehyde benzyl alcohol 34; p-hydroxybenzaldehyde 2; p-hydroxybenzoic acid (traces) benzyl alcohol p-hydroxybenzaldehyde 1; p-hydroxybenzoic acid 10; benzaldehyde (traces) benzoic acid benzyl alcohol 19; p-hydroxybenzoic acid (traces); benzaldehyde (traces) p-hydroxybenzoic acid p-hydroxybenzaldehyde 3; p-hydroxybenzyl alcohol 18 p-hydroxybenzaldehyde p-hydroxybenzyl alcohol 52; p-hydroxybenzoic acid 38 p-hydroxybenzyl alcohol p-hydroxybenzoic acid 8; p-hydroxybenzaldehyde (traces) * The aromatic metabolite amounts were calculated, on day 6 of cultivation, as percentages of the total amount of precursor added.

4 BENZALDEHYDE BIOSYNTHESIS BY TRAMETES SUAVEOLENS 265 0á03% (v/v) sulphuric acid at a ow rate of 1 ml min ±1. Detection was achieved with a refractive index detector. Identity of the aromatic metabolites was veri ed by HPLC±mass spectrometry using a Perkin-Elmer API150EX apparatus (Perkin-Elmer Applied Biosystems, Courtaboeuf, France) with an ion spray source and a 20 volt ori ce voltage. RESULTS L-phenylalanine catabolism in the absence or in the presence of HP20 resin In the presence of L-phenylalanine and glucose as the carbon source, the metabolism of L-phenylalanine by T. suaveolens CBS appeared to be directed towards the production of aryl alcohols, since this strain produced a maximum of 300 mg l ±1 benzyl alcohol, 50 mg l ±1 p-hydroxy-benzylalcohol and 75 mg l ±1 3-phenyl-1-propanol during the exponential growth phase (Fig. 1a). The maximal concentration of benzaldehyde synthesized was 33 mg l ±1 on day 12 of cultivation. At the end of cultivation (on day 21), 30 mg l ±1 3benzoic acid was synthesized. Traces of phenylpyruvic and cinnamic acids were also detected. These aromatic metabolites were only detected when L-phenylalanine was included in the culture medium. At maximal benzaldehyde concentration, glucose and ammonium were completely consumed while 2 g l ±1 L-phenylalanine was present in the culture medium. Mycelial biomass reached a maximum of 5á5 gl ±1 on day 10 of cultivation and decreased thereafter (Fig. 1b). In order to improve the concentration of benzaldehyde, HP20 resin, an adsorbent known to trap aromatic compounds (Lomascolo et al. 1999a), was added to the culture medium of T. suaveolens. Under these conditions, various aromatic metabolites from L-phenylalanine were determined and the concentration of several of them was increased (Fig. 1a). Thus, a high benzaldehyde concentration was obtained, with a maximum of 710 mg l ±1 on day 14 and a molar yield of 31%. In addition, cinnamic acid derivatives, such as cinnamyl alcohol (153 mg l ±1 ) and cinnamaldehyde (71 mg l ±1 ), were recovered with maximal concentrations on days 10 and 14, respectively; 2-phenylethanol, phenylacetic acid, and traces of phenyllactic, mandelic and phenylglyoxylic acids were also detected. Fig. 2 Proposed diagram for the metabolism of L-phenylalanine by Trametes suaveolens CBS The arrows in dotted lines represent supposed pathways according to the literature (Kawabe and Morita 1993; Jensen et al. 1994; Krings et al. 1996; Lapadatescu et al. 2000)

5 266 A. LOMASCOLO ET AL. Identi cation of catabolic pathways of L-phenylalanine The main catabolic pathways of L-phenylalanine were identi ed using the aromatic compounds previously detected in the culture medium of T. suaveolens as precursors. The results are shown in Table 1. Cinnamic acid, cinnamaldehyde and cinnamyl alcohol were converted mainly to 3-phenyl-1-propanol and, in minor quantities, to p-hydroxy derivatives, showing the break of the propenoic chain and the hydroxylation of the aromatic ring. 3-Phenyl-1-propanol and 2-phenylethanol were not biotransformed to another avouring compound. Phenyllactic, phenylglyoxylic and phenylacetic acids were converted into mandelic acid while phenylacetic acid led to the formation of both mandelic and phenylglyoxylic acids. Only phenylglyoxylic and benzoic acids were biotransformed directly into benzaldehyde. Benzyl alcohol, p-hydroxybenzyl alcohol and cinnamyl alcohol were very slightly biotransformed into the corresponding aldehyde. In contrast, all the aromatic aldehydes added to the culture medium were biotransformed in a preferential way to the corresponding alcohol: 34% benzyl alcohol, 50% 3-phenyl-1-propanol and 52% p-hydroxybenzyl alcohol for benzaldehyde, cinnamaldehyde and p-hydroxybenzaldehyde, respectively. Otherwise, benzaldehyde and benzyl alcohol were never converted to benzoic acid. DISCUSSION Trametes suaveolens was shown to biotransform L-phenylalanine, an amino acid produced by biotechnological means (Evans et al. 1987), into benzaldehyde. Moreover, the strain studied was shown to biosynthesize benzyl alcohol (accumulated in the medium) and p-hydroxy derivatives, which have also been detected in culture broths of Bjerkandera adusta (Lapadatescu et al. 2000) and Pycnoporus cinnabarinus (Lomascolo et al. 1999a), and 3-phenyl-1-propanol also detected in culture uids of Ischnoderma benzoinum (Krings et al. 1996) and Polyporus tuberaster (Kawabe and Morita 1993). Comprehensive knowledge about the metabolic pathways of L-phenylalanine will be of value to improve the yield of the bioconversion of this amino acid into benzaldehyde. In the light of the present work and reports in the literature, a diagram of the catabolism of L-phenylalanine of the white-rot fungus T. suaveolens CBS , is proposed (Fig. 2). Two microbial routes for L-phenylalanine degradation have been suggested (Wat and Towers 1977; Casey and Dobb 1992; Feron et al. 1996). One route is oxidative, leading to phenylpyruvic acid via a transaminase; the other is non-oxidative, leading to cinnamic acid via a phenylalanine ammonia lyase. Both pathways seemed to occur in the case of T. suaveolens. L-phenylalanine could possibly be transformed into cinnamic acid and further reduced to cinnamaldehyde, cinnamyl alcohol and 3-phenyl- 1-propanol (Fig. 2). The latter compound ( oral aroma) may be the accumulated avouring product of this pathway, as described for I. benzoinum CBS (Krings et al. 1996). The deamination of L-phenylalanine to phenylpyruvic acid might be the major degradation pathway. Two additional divergent routes could exist, the rst leading to 2-phenylethanol and the second, to benzyl derivatives via phenylacetaldehyde, phenylacetic, mandelic and phenylglyoxylic acids (Fig. 2). There was no evidence for b-oxidation of L-phenylalanine to benzoic acid, as in the case of B. adusta CBS 595á78 (Lapadatescu et al. 2000), and no direct link between cinnamic and benzoic acids (Jensen et al. 1994). It is worth noting that in numerous metabolic steps, biotransformation pathways were orientated towards the synthesis of alcohols (accumulated in the culture medium) rather than aldehydes. Under basal culture conditions, benzaldehyde concentration was very low and led to the development of a strategy using a selective adsorbent to trap benzaldehyde before its transformation to benzyl alcohol. HP20 resin, a styrene divinylbenzene copolymer highly selective for aromatic compounds, could have several roles in the culture medium of T. suaveolens CBS ; while increasing the total production of aromatic compounds, it limits the possible toxicity of benzaldehyde towards the fungus (Lamer et al. 1996; Lomascolo et al. 1999a). Trapping this aromatic aldehyde could also facilitate its recovery in industrial processes. The use of this adsorbent allowed a 21-fold increase in the concentration of benzaldehyde (33 mg l ±1 without resin and 710 mg l ±1 with resin) and a 17-fold increase in productivity (3 mg l ±1 day ±1 without resin and 51 mg l ±1 day ±1 with resin). Thus, HP20 resin could possibly direct the metabolism of L-phenylalanine to the phenylpyruvate pathway, leading to high concentrations of benzaldehyde, the desired target compound of the bioconversion. ACKNOWLEDGEMENTS This work was supported by the Conseil ReÂgional Provence- Alpes-CoÃte d'azur (France). REFERENCES Abraham, B.G. and Berger, R.G. (1994) Higher fungi for generating aroma components through novel biotechnologies. Journal of Food and Agricultural Chemistry 42, 2344±2348. Berger, R.G.. NeuhaÈuser, K. and Drawert, F. (1987) High productivity fermentation of volatile avours using a strain of Ischnoderma benzoinum. Biotechnology and Bioengineering 30, 987. Casey, J. and Dobb, R. (1992) Microbial routes to aromatic aldehydes. Enzyme Microbiology and Technology 14, 739±747.

6 BENZALDEHYDE BIOSYNTHESIS BY TRAMETES SUAVEOLENS 267 Clark, G.S. (1995) Benzaldehyde. An aroma chemical pro le. Perfumer and Flavorist 20, 53±60. Decarvalho, M.E.A., Monteiro, M.C. and Santanna, G.L. (1999) Laccase from Trametes versicolor. Stability at temperature and alkaline conditions and its effect on biobleaching of hardwood kraft pulp. Applied Biochemistry and Biotechnology 77, 723±733. Evans, C.T., Gleeson, M., Aoki, H. et al. (1987) A novel, ef cient biotransformation, for the production of L-phenylalanine. Biotechnology 5, 818±823. Fabre, C., Blanc, P. and Goma, G. (1996) Production of benzaldehyde by several strains of Ischnoderma benzoinum. Sciences des Aliments 16, 61±68. Fakoussa, R.M. and Frost, P.J. (1999) In vivo-decolorization of coal derived humic acids by laccase-excreting fungus Trametes versicolor. Applied Microbiology and Biotechnology 52, 60±65. Falconnier, B., Lapierre, C., Lesage-Meessen, L. et al. (1994) Vanillin as a product of ferulic acid biotransformation by the white-rot fungus Pycnoporus cinnabarinus I-937: identi cation of methabolic pathways. Journal of Biotechnology 37, 123±132. Feron, G., Bonnarme, P. and Durand, A. (1996) Prospects for the microbial production of food avours. Trends in Biotechnology 7, 285±293. Gallois, A., Gross, B., Langlois, D., Spinnler, H.E. and Brunerie, P. (1990) In uence of culture conditions on production of avour compounds by 29 lignolytic Basidiomycetes. Mycological Research 4, 494±504. Gross, B. and Asther, M. (1989) AroÃmes de basidiomycetes: caracteâristiques, analyse et production. Sciences des Aliments 9, 427±454. Janssens, L., De Pooter, H.L., Schamp, N.M. and Vandamme, E.J. (1992) Production of avours by microorganisms. Process Biochemistry 27, 195±215. Jensen, K.A., Evans, K.M.C., Kirk, T.K. and Hammel, K.E. (1994) Biosynthetic pathway for veratryl alcohol in the lignolytic fungus Phanerochaete chrysosporium. Applied and Environmental Microbiology 60, 709±714. Kawabe, T. and Morita, H. (1993) Volatile components in culture uid of Polyporus tuberaster. Journal of Food and Agricultural Chemistry 41, 637±640. Krings, U., Hinz, M. and Berger, R.G. (1996) Degradation of [ 2 H] phenylalanine by the basidiomycete Ischnoderma benzoinum. Journal of Biotechnology 51, 123±129. Lamer, T., Spinnler, H.E., Souchon, I. and Voilley, A. (1996) Extraction of benzaldehyde from fermentation broth by pervaporation. Process Biochemistry 31, 533±542. Lapadatescu, C., Feron, G., Vergoignan, C., Dijian, A. and Bonnarme, P. (1997) In uence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens. Applied Microbiology and Biotechnology 47, 708±714. Lapadatescu, C., GinieÁs, C., Le QueÂreÂ, J.-L. and Bonnarme, P. (2000) Novel scheme for biosynthesis or aryl metabolites from L-phenylalanine in the fungus Bjerkandera adusta. Applied and Environmental Microbiology 66, 1517±1522. Lesage-Meessen, L., Delattre, M., Haon, M. et al. (1996) A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus. Journal of Biotechnology 50, 107±113. Lomascolo, A., Lesage-Meessen, L., Labat, M., Navarro, D., Delattre, M. and Asther, M. (1999a) Enhanced benzaldehyde formation by a monokaryotic strain of Pycnoporus cinnabarinus using a selective solid adsorbent in the culture medium. Canadian Journal of Microbiology 45, 653±657. Lomascolo, A., Stentelaire, C., Asther, M. and Lesage-Meessen, L. (1999b) Basidiomycetes as new biotechnological tools to generate natural aromatic avours for the food industry. Trends in Biotechnology 17, 282±289. Wat, C.-K. and Towers, G.H.N. (1977) Metabolism of the aromatic amino acids by fungi. Recent Advances in Phytochemistry 12, 371±432. Welsh, F.W., Murray, W.D. and Williams, R.E. (1989) Microbiological and enzymatic production of avor and fragrance chemicals. Critical Reviews in Biotechnology 9, 105±169.