Biocatalytic Synthesis of Vanillin - Applied and Environmental ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2000, p. 684–687 0099-2240/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 66, No. 2

Biocatalytic Synthesis of Vanillin TAO LI

AND

JOHN P. N. ROSAZZA*

Division of Medicinal and Natural Products Chemistry, and Center for Biocatalysis and Bioprocessing, College of Pharmacy, University of Iowa, Iowa City, Iowa 52242 Received 1 July 1999/Accepted 17 November 1999

The conversions of vanillic acid and O-benzylvanillic acid to vanillin were examined by using whole cells and enzyme preparations of Nocardia sp. strain NRRL 5646. With growing cultures, vanillic acid was decarboxylated (69% yield) to guaiacol and reduced (11% yield) to vanillyl alcohol. In resting Nocardia cells in buffer, 4-Obenzylvanillic acid was converted to the corresponding alcohol product without decarboxylation. Purified Nocardia carboxylic acid reductase, an ATP and NADPH-dependent enzyme, quantitatively reduced vanillic acid to vanillin. Structures of metabolites were established by 1H nuclear magnetic resonance and mass spectral analyses. In this report, we describe biotransformations of vanillic acid by growing cells of Nocardia sp. strain NRRL 5646 and a pure aryl aldehyde oxidoreductase (carboxylic acid reductase) from this organism. We also show that a complicating vanillic acid decarboxylation reaction can be overcome by use of a vanillic acid derivative as a substrate in whole-cell bioconversions.

Vanilla is one of the most widely used flavors in food industry. Natural vanilla extracted from the cured pods of the flowers of Vanilla planifolia has an estimated net value of more than $ 1 billion annually (18). Vanillin (3-methoxy-4-hydroxybenzaldehyde) is the most important organoleptic component in vanilla. More than 12,000 tons of synthetic vanillin are produced each year from petrochemical and wood pulping industries (7). Strong market demand for natural and environmentally friendly products has spawned efforts to produce vanillin by microbial transformation from natural substrates, including phenolic stibenes (27), eugenol (19, 26), and ferulic acid (14, 17). Ferulic acid [3-(4-hydroxy-3-methoxyphenyl)-propenoic acid] is an extremely abundant plant product available from corn kernel hulls obtained from wet milling (21). Vanillic acid is the major product obtained by ␤-oxidation of ferulic acid by species of Bacillus (6), Pseudomonas (11, 23), Polyporus (9), Rhodotorula (8), and Streptomyces (17, 22). Thus, vanillic acid is an abundant, readily available precursor for the biocatalytic synthesis of vanillin. Microbial reductions of carboxylic acids are widely observed in bacteria and fungi (1, 2, 4, 5, 10, 12, 13, 24, 25). One common mechanism of enzymatic carboxylic acid reduction involves activation of carboxyl groups with ATP to highly reactive carbonyl-AMP intermediates which are readily reduced by NADPH to aldehydes (5, 13, 16) (see Fig. 1). Both the adenylation and carbonyl-AMP reduction steps are catalyzed by a single enzyme (5, 13, 16). A separate alcohol oxidoreductase reduces the resulting aldehydes to their corresponding alcohol products (16). Although carboxylic acid reductases have been purified from two species of Nocardia (13, 16), properties of the reported enzymes are significantly different. It is possible to envision an enzymatic synthesis of vanillin beginning either with ferulic acid (8, 17, 21) or with vanillic acid as the starting materials. Such approaches are attractive because the natural carboxylic acid starting materials are abundant and inexpensive and are soluble in aqueous media. Furthermore, reductions of carboxylic acids to aldehydes are difficult to achieve by chemical means, and the reduction of vanillic acid to vanillin has not been widely reported.

MATERIALS AND METHODS Biocatalysts. Nocardia species strain NRRL 5646 is maintained in the University of Iowa College of Pharmacy culture collection and is grown and maintained on slants of Sabouraud-dextrose agar or sporulating agar (ATCC no. 5 medium). The carboxylic acid reductase used in enzymatic reductions of vanillic acid was isolated from Nocardia sp. strain NRRL 5646 by our procedure (16). The enzyme used in this work was pure, based on sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis (15). Chemicals. Vanillic acid (4-hydroxy-3-methoxybenzoic acid) (compound 1a [Fig. 1]), vanillin (4-hydroxy-3-methoxybenzaldehyde) (compound 3a), vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) (compound 4a), guaiacol (2-methoxyphenol) (compound 2), and benzyl bromide were purchased from Aldrich Chemical Co. (Milwaukee, Wis.). Other chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.) and Aldrich unless otherwise indicated. 4-O-Benzylation of vanillic acid was carried out by adding 11 ml of 1 N NaOH to 900 mg of vanillic acid (5.4 mmol) in 16 ml of ethanol, followed by dropwise addition of 1 g (5.8 mmol) of benzyl bromide in 2.5 ml of ethanol over 60 min. The reaction mixture was refluxed for 2 h before being poured into 150 ml of water and acidified to pH 2 with 6 N HCl. The precipitate was collected by filtration and crystallized in xylene to afford 802 mg of compound 1b (3.1 mmol, 56% yield). The structure of compound 1b was confirmed by low-resolution electron impact mass spectrometry (EIMS) with the following parameters: m/z 258 (8%, M⫹) and 91 (100%, benzonium ion); 1H nuclear magnetic resonance (NMR) at 360 MHz (d6-acetone), ␦H 3.83 (3H, s, OCH3), 5.21 (2H, s, C-7⬘), 7.12, 7.14 (1H, d, J ⫽ 8.3 Hz, H-5), 7.33 to 7.52 (5H, m, H-2⬘, -3⬘, -4⬘, -5⬘, and -6⬘), 7.58, 7.57 (1H, d, J ⫽ 2.0 Hz, H-2), and 7.63 to 7.65 (1H, dd, J ⫽ 8.3, 2.0 Hz, H-6). 4-O-Benzylvanillin (compound 3b) was prepared by refluxing 32 g of vanillin (210 mmol) with 26 ml (213 mmol) of benzyl bromide, and 30 g of potassium bicarbonate in 200 ml of ethanol for 5 h. After cooling, the filtrate was crystallized at 4°C overnight. Recrystallization in ethanol gave 45.3 g (187 mmol, 85% yield) of compound 3b confirmed by EIMS: m/z 242 (4%, M⫹), 91 (100%, benzonium ion); 1H NMR at 360 MHz (CDCl3), ␦H 3.95 (3H, s, OCH3), 5.25 (2H, s, C-7⬘), 7.00, 6.98 (1H, d, J ⫽ 8.3 Hz, H-5), 7.34 to 7.43 (7H, m, H-2, -6, -2⬘, -3⬘, -4⬘, -5⬘, and -6⬘), and 9.84 (1H, s, H-7). 4-O-Benzylvanillyl alcohol (compound 4b) was prepared by refluxing a mixture of 15 g of potassium bicarbonate, 16 g (104 mmol) of vanillyl alcohol, and 18.3 g of benzyl bromide (107 mmol) in 115 ml of ethanol for 3 h. The reaction workup was like that for 4-O-benzylvanillic acid to give pure product. Recrystallization from cyclohexane gave 17 g of compound 4b (64% yield). The low-resolution, fast atom bombardment (FAB) mass spectrum gave m/z 244 (17%, M⫹), 227 (9%, M⫹ ⫺ OH), and 91 (100%, benzonium ion); 1H NMR at 360 MHz (CDCl3), ␦H 3.89 (3H, s, OCH3), 4.58 (2H, s, C-7), 5.16 (2H, s, H-7⬘), 6.76 to 6.79 (1H, dd, J ⫽ 8.3, 1.9 Hz, H-6), 6.82, 6.84 (1H, d, J ⫽ 8.2 Hz, H-5), 6.91, 6.92 (1H, d, J ⫽ 1.7 Hz, H-2), and 7.26 to 7.43 (5H, m, H-2⬘, -3⬘, -4⬘, -5⬘, and -6⬘). Chromatography. Thin-layer chromatography (TLC) was carried out on silica gel GF254 plates (E. Merck, Darmstadt, Germany). Developed chromatograms were directly visualized under 254-nm UV light to observe fluorescence quenching. Phenolic compounds were also visualized with Pauly’s reagent, which consisted of solution A (0.5% sulfanilic acid in 2 N HCl), solution B (0.5% sodium

* Corresponding author. Mailing address: Division of Medicinal and Natural Products and Center for Biocatalysis and Bioprocessing, College of Pharmacy, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-8842. Fax: (319) 335-8766. E-mail: john-rosazza@uiowa .edu. 684

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FIG. 1. Whole-cell and enzyme transformation pathways for vanillic acid and O-benzyl vanillic acid by Nocardia sp. strain NRRL 5646.

nitrite in water), and solution C (1 N potassium hydroxide in 50% ethanolwater). Developed plates were first sprayed with a freshly prepared equal-volume mixture of solutions A and B and then sprayed with solution C before being heated with a heat gun to develop colors from yellow to orange. Aldehydes such as vanillin were also detected with 2,4,-dinitrophenylhydrazine (0.4% in 2 N HCl [wt/vol]) spray. With dichloromethane-acetonitrile-formic acid (75:25:1 [vol/vol/ vol]), Rf values for vanillic acid, vanillin, vanillyl alcohol, and guaiacol were 0.5, 0.4, 0.8, and 0.9, respectively. With dichloromethane-acetic acid-benzene (100: 2:2 [vol/vol/vol]), Rf values for 4-O-benzylvanillic acid (compound 1b), 4-Obenzylvanillin (compound 3b), and 4-O-benzylvanillyl alcohol (compound 4b) were 0.7, 0.8, and 0.2, respectively. High-performance liquid chromatography (HPLC) was performed with a Shimadzu liquid chromatograph equipped either with a LC-10AD system controller, four FCV-10AL pumps, and a photodiode array UV-Vis detector (SPD-M6A) or with a SCL 6-B system controller, two LC-6A pumps, and a variable-wavelength UV detector. Eluted peaks were detected at 273 to 276 nm or at 290 nm and identified by comparison with authentic compounds. Separations carried out under isocratic conditions over a Versapack C18 column (250 by 4.6 mm, 10-␮m particle size; Alltech, Deerfield, Ill.) with acetonitrile-water-formic acid (20:80:1) at a flow rate of 0.9 ml/min gave retention volumes (Rv) of 5.0 ml for vanillyl alcohol, 8.1 ml for vanillic acid, 13.2 ml for vanillin, 16.1 ml for o-anisic acid, and 18.7 ml for guaiacol. Standard curves for vanillic acid (compound 1a), vanillin (compound 3a), vanillyl alcohol (compound 4a), and guaiacol (compound 2) were established over the range of 0.3 to 8 ␮g. For quantitation of 4-O-benzylvanillic acid biotransformations, separations were carried out under isocratic conditions over a 10 ␮m C18 Versapack column (300 by 4.1 mm; Alltech) with acetonitrile-water-formic acid (30:70:1) at a flow rate of 2 ml/min. Rv values were 8.1 ml for p-anisic acid, 21.9 ml for 4-O-benzylvanillyl alcohol (compound 4b), 33.4 ml for 4-O-benzylvanillic acid (compound 1b), and 61.1 ml for 4-O-benzylvanillin (compound 3b). Standard curves for these compounds were established over the range of 0.3 to 3 ␮g. Spectroscopy. Mass spectra were obtained by using a Trio-1 MS linked with a 5890 Hewlett-Packard gas chromatograph. EIMS was performed at an ionization voltage of 70 eV. For direct inlet probe analysis, the probe temperature was set at 30°C for 1 min, raised to 300°C at 150°C/min, and held at 300°C for 10 min for analysis. For gas chromatography-mass spectrometry, separations were carried out on an OV-1 capillary column (10 m by 0.25 mm; 1-␮m film thickness) with helium as carrier gas at a flow rate of 20 ml/min. The column oven temperature was held at 50°C for 1 min, raised to 250°C at a rate of 15°C/min, and held at that temperature for 10 min. Injector and detector temperatures were 220 and 270°C, respectively. FAB mass analyses were performed by using a ZAB-HF mass spectrometer (VG Analytical, Inc.). Ionizing matrices were either 3-nitrobenzyl alcohol or thioglycerol. NMR spectra were obtained with a Bruker WM 360-MHz high-field spectrometer equipped with an IBM Aspect-2000 processor. Tetramethylsilane was used as the internal standard. Chemical-shift values are reported in parts per million (ppm), and coupling constants (J values) are given in hertz. Abbreviations for NMR are as follows: s, singlet; d, doublet; t, triplet; dd, doublet of doublets; and m, multiplet. Biotransformation of vanillic acid by Nocardia isolates. A preparative scale incubation was grown by our standard two-stage incubation protocol (3) in 200-ml volumes of sterile soybean flour-glucose medium held in stainless-steelcapped, 1-liter, DeLong culture flasks. The medium contained 20 g of glucose, 5 g of yeast extract, 5 g of soybean flour, 5 g of NaCl, and 5 g of K2HPO4 per liter

in distilled water and was adjusted to pH 7.2 with 6 N HCl before being autoclaved at 121°C for 20 min. Cultures were incubated by shaking at 250 rpm at 28°C on G25 Gyrotory shakers (New Brunswick Scientific Co., Edison, N.J.). A 10% inoculum derived from a 72-h-old first-stage culture was used to initiate second-stage cultures, which were incubated as described above. After 24 h of incubation in the second stage, 160 mg of vanillic acid (in 2 ml of dimethyl sulfoxide) was added to each of three flasks. Control cultures included everything except vanillic acid. Samples were removed at various time intervals, adjusted to pH 2 with 6 N HCl, extracted with 1 ml of ethyl acetate, and centrifuged for 1 min at 2,500 rpm. Organic layers were removed, evaporated to dryness, and reconstituted in 0.5 ml of methanol. The samples were spotted onto TLC plates for analysis. Samples of 3 ml were also taken over the course of vanillic acid transformation for HPLC analysis. To each sample, 200 ␮l of o-anisic acid solution (30 mg/ml in acetonitrile) was added as an internal standard. Culture samples were then acidified to pH 2 with 6 N HCl, 1 ml was loaded onto solid-phase extraction cartridges (Chem Elut CE 1001, 1-ml aqueous solution capacity; Varian, Harbor City, Calif.). After 5 min, cartridges were extracted twice with 3 ml each of dichloromethane-acetonitrile (90:10). These organic extracts were combined and used for HPLC analysis. The microbial reaction was terminated 40 h after the addition of vanillic acid. The culture was acidified to pH 2 with 6 N HCl. After centrifugation to remove cells and other solids, the supernatant from 200 ml of culture was loaded onto a solid-phase extraction cartridge (Chem Elut 1200, 200-ml aqueous solution capacity). After 5 min of equilibration, the cartridge was washed three times with 200 ml of hexane and then three times with 200 ml of dichloromethane. The organic extracts were separately concentrated by rotary evaporation to give 45 mg of oil (from hexane) and 90 mg of residue (from dichloromethane). The dichloromethane extracts were streaked onto a 1-mm-thick preparative TLC plate (20 by 20 cm), which was developed in dichloromethane-acetonitrile-formic acid (75:25:1 [vol/vol]). The separated bands after development were scraped from the plate, and compounds were eluted from silica gel with a mixture of dichloromethane and acetonitrile (70:30 [vol/vol]). The band extracts were checked for purity by TLC, and concentrated for mass spectrometry and 1H NMR spectroscopy. Reduction of vanillic acid to vanillin by Nocardia carboxylic acid reductase. The reduction was carried out in a reaction mixture of 200 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 34 mg of vanillic acid, 59 mg of NADPH, 110 mg of ATP, and 100 ␮g of purified carboxylic acid reductase (0.6 U) (16). The reaction mixture was incubated at 30°C with gentle shaking (50 rpm) for 24 h after which the entire reaction mixture was loaded onto a solid-phase extraction cartridge (Chem Elut 1200, 200-ml aqueous solution capacity). After 5 min of equilibration, the cartridge was washed twice with 100 ml of hexane, followed by two washes 100 ml of dichloromethane. The dichloromethane solution was concentrated to less than 2 ml. The solution was transferred to a vial, and the solvent was removed with a stream of nitrogen to give pure vanillin as determined by TLC, EIMS, and 1H NMR analyses. Nocardia resting cell conversions of 4-O-benzylvanillic acid. Nocardia sp. was grown by using the standard two-stage incubation protocol. After 24 h in the second-stage culture, 5 mg of benzoic acid per ml was added as an inducer for carboxylic acid reductase (16). The cells were harvested by centrifugation at 8,000 ⫻ g for 20 min at 24 h after the benzoate addition. Cells were washed twice with 0.9% saline solution and pelleted before use. For preparative scale biotransformations, 2.8 g of cells (wet weight) were

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FIG. 2. Time course of growing cell biotransformations of vanillic acid by Nocardia sp. strain NRRL 5646. Triplicate sample averages for vanillic acid (F), vanillyl alcohol (■), vanillin (䊐), and guaiacol (E) had variances of not more than ⫾5%. suspended in 400 ml of 50 mM Tris HCl (pH 7.4) containing 1% glucose, 10 mM MgCl2, and 5 mM phosphate. 4-O-Benzylvanillic acid (0.7 g in 2 ml of dimethyl sulfoxide) was added to the cell suspension, which was incubated with shaking at 150 rpm at 30°C for 15 min. The reaction mixture was acidified to pH 2, centrifuged to remove cells and other unwanted solids, and loaded onto two solid-phase extraction cartridges (Chem Elut 1200, 200 ml on each). After 5 min of equilibration, each cartridge was washed three times with 200 ml of ethyl acetate. The ethyl acetate solution was concentrated by rotary evaporation to give 670 mg of crude product, which was reconstituted to 2.0 ml with dichloromethane and loaded onto a silica gel column (0.9 by 20 cm). 4-O-Benzylvanillin (170 mg, 24% yield) was eluted with dichloromethane, while 4-O-benzylvanillyl alcohol (210 mg, 30% yield) was eluted with dichloromethane-benzene-acetic acid (100:4:6 [vol/vol/vol]). These compounds were spectrally (EIMS and NMR) and chromatographically identical to synthetic standards described earlier. 4-O-Benzylvanillin obtained from the preparative-scale incubation was dissolved in 4 ml of ethanol, 5 ml of concentrated HCl was added, and the mixture was refluxed for 2 h. The solvent was removed by rotary evaporation, and the residue dissolved in 0.5 ml was purified by silica gel column chromatography (0.9 by 5 cm) eluted with dichloromethane to give 65 mg of pure vanillin as determined by TLC, EIMS, and 1H NMR. Analytical scale biotransformations were conducted in the same medium as for preparative biotransformations. Suspensions of cells (0.05 g [wet weight]/ml) containing 0.6 mg of 4-O-benzylvanillic acid per ml were incubated at 30°C. Samples (1 ml) taken during the course of incubation received 50 ␮l of p-anisic acid (4 mg/ml in methanol) as an internal standard. Samples were acidified to pH 2 with 6 N HCl and loaded onto solid-phase extraction cartridges (Chem Elut CE 1001), which were equilibrated for 5 min and then eluted twice with 3 ml of ethyl acetate. Pooled eluates were analyzed by HPLC.

APPL. ENVIRON. MICROBIOL.

M⫹ ⫺ CH3 ⫺ H2O), and 81 (100%, M⫹ ⫺ CH3 ⫺ CO), and an 1H NMR spectrum at 360 MHz (CDCl3), ␦H 3.76 (3H, s, OCH3), 6.80, 6.85 (3H, m, H-3, H-4, H-6), and 6.90 to 6.93 (1H, m, H-5). These properties were identical to those for an authentic sample of guaiacol. The dichloromethane extract gave two compounds (4 mg each) by preparative TLC. The compound at Rf 0.77 was spectrally identical to authentic vanillin (compound 3a): low-resolution EIMS, m/z 152 (88%, M⫹), 151 (100%, M⫹ ⫺ H), 137 (6%, M⫹ ⫺ CH3), 123 (13%, M⫹ ⫺ CHO); 1H NMR at 360 MHz (CDCl3), ␦H 3.96 (3H, s, OCH3), 6.22 (1H, s, OH), 7.03, 7.06 (1H, d, J ⫽ 8.5, H-5), 7.26 to 7.44 (2H, m, H-2, H-6), and 9.83 (1H, s, CHO). The compound at Rf ⫽ 0.35 was identical to vanillyl alcohol (compound 4a): low-resolution EIMS, m/z 154 (28%, M⫹), 136 (50%, M⫹ ⫺ H2O); 1H NMR at 360 MHz (CDCl3), ␦H 3.90 (3H, s, OCH3), 4.61 (2H, s, CH2OH), and 6.82 to 6.92 (3H, m, H-2, H-5, H-6). Reduction of vanillic acid by purified Nocardia carboxylic acid reductase. The enzymatic reaction gave 8 mg (53 ␮mol) of vanillin by TLC, EIMS, and 1H NMR analyses, all of which were identical to an authentic sample of vanillin. Based upon the estimated amount of NADPH consumed (51 ␮mol, [change in optical density at 340 nm]) during the course of the reaction, the yield of vanillin was essentially quantitative. The remainder of the vanillic acid substrate was unreacted. Transformation of 4-O-benzylvanillic acid with resting cells of Nocardia sp. By HPLC, benzoate-induced cells of Nocardia sp. rapidly transformed 4-O-benzylvanillic acid to metabolites within 40 min (Fig. 3). In the first 15 min, the aldehyde 3b accumulated in transient fashion to a maximum 30% yield (175 ␮g/ml) after which compound 3b was further quantitatively reduced to the corresponding alcohol 4b. In the preparative scale incubation reaction, TLC analysis indicated that benzoate-induced Nocardia cells produced two major metabolites corresponding to 4-O-benzylvanillin (compound 3b, 170 mg, 24% yield) and 4-O-benzylvanillyl alcohol (compound 4b, 210 mg, 30% yield). These products were spec-

RESULTS Biotransformation of vanillic acid with growing cells of Nocardia sp. HPLC analyses of vanillic acid biotransformation by Nocardia sp. (Fig. 2) showed that almost all vanillic acid was consumed within 48 h to give guaiacol (69% yield) by decarboxylation and vanillyl alcohol (11% yield) by carboxylic acid reduction. Only traces of vanillin were detected in this analytical experiment. To properly characterize vanillic acid metabolites, a preparative scale incubation gave 45 mg of a sample from the hexane extract of a 40-h biotransformation culture. The product from the hexane extract gave a single spot by TLC (Rf ⫽ 0.89), was orange with Pauly’s phenolic spray reagent, gave an EIMS spectrum data as follows: m/z (percent relative abundance) 124 (78%, M⫹), 109 (92%, M⫹ ⫺ CH3), 91 (44%,

FIG. 3. Time course of resting cell biotransformations of O-benzylvanillic acid by Nocardia sp. strain NRRL 5646. Triplicate sample averages for O-benzylvanillic acid (F), O-benzylvanillyl alcohol (■), and O-benzylvanillin (䊐) had variances of not more than ⫾5%.

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trally and chromatographically comparable to synthetic compounds 3b and 4b. Thus, 4-O-benzylvanillic acid was reduced first to the aldehyde 3b and subsequently to the alcohol 4b (Fig. 1). To confirm the identity of compound 3b, chemical removal of the benzyl group from the microbial metabolite gave 65 mg of vanillin identified by spectral and chromatographic comparisons with authentic vanillin. DISCUSSION Many microbial transformation approaches have been used to produce vanillin. In most cases, the yields of vanillin were low, and times for biotransformation reactions are long (7, 14, 17, 19, 26, 28). The synthesis of vanillin from ferulic acid has been reported with several microorganisms (6, 9, 17, 22, 23). In P. acidivorans (23), manometric studies suggested that vanillin was produced by a two-step reaction including water addition to the cinnamoyl side chain and subsequent retro-aldol condensation to give vanillin. In general, yields were low because vanillin was further oxidized to vanillic acid which was O-demethylated to protocatechuic acid or decarboxylated to guaiacol. The addition of sulfhydryl compounds to ferulic acid transformation medium increased vanillin accumulations by Pseudomonas putida ATCC 55180 (14). However, the mechanism by which sulfhydryl reagents improved vanillin yields was not clear. Mulheim and Lerch recently described a relatively high yield direct conversion of ferulic acid to vanillin by cultures of Streptomyces setonii (17). With this organism, yields of vanillin were directly proportional to ferulic acid concentrations in culture media. In our laboratory, the mechanism of ferulic acid transformation to vanillic acid by R. rubra IFO 889 occurred by ␤-oxidation (8). The ready availability of vanillic acid by this process, and the discovery of a vanillic acid reduction pathway in Nocardia sp. strain NRRL 5646 suggested the potentials for whole cells or the pure carboxylic acid reductase from this microorganism for vanillin synthesis. With pure Nocardia carboxylic acid reductase, the ATP- and NADPH-dependent reduction of vanillic acid was quantitative, yielding only vanillin and no complicating byproducts. In wholecell Nocardia biotransformation reactions, vanillic acid decarboxylation to guaiacol was the major complicating pathway. The identification of guaiacol and vanillyl alcohol as metabolites confirmed that Nocardia sp. strain NRRL 5646 possesses two different metabolic pathways for the biotransformation of vanillic acid. These pathways are (i) decarboxylation to guaiacol and (ii) reduction to vanillin and further reduction to vanillyl alcohol (Fig. 1). We also observed vanillic acid decarboxylation in Rhodotorula rubra (8) and in Bacillus pumilis (21). Deuterium isotope incorporation studies revealed that the decarboxylation reaction involved the enzymatic tautomerization of vanillic acid through the para-hydroxyl group to a quinoid intermediate before decarboxylation. Blocking the p-phenolic group was considered as a possible means of preventing the enzymatic tautomerization reaction and subsequent decarboxylation. To test this hypothesis, we employed the benzylic functional group, which is readily removed by simple acid treatment. However, other alternative blocking groups, such as esters, could function equally well. 4-O-Benzylvanillic acid was not decarboxylated and was quantitatively reduced to 4-O-benzylvanillin and 4-O-benzylvanillyl alcohol. We know that Nocardia sp. also contains another reductase that converts aldehydes to alcohols

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(unpublished data). We are now exploring the possibility of cloning the carboxylic acid reductase in a suitable biocatalytic host. ACKNOWLEDGMENTS Tao Li acknowledges support through a Center for Biocatalysis and Bioprocessing Fellowship, and we are grateful for financial support from USDA through the Byproducts for Biotechnology Consortium. REFERENCES 1. Arfman, H. A., and W. R. Abraham. 1993. Microbial reduction of aromatic carboxylic acids. Z. Naturforsch. 48c:52–57. 2. Bachman, D. M., B. Dragoon, and S. John. 1960. Reduction of salicylate to saligenin by Neurospora. Arch. Biochem. Biophys. 91:326. 3. Betts, R. E., D. E. Walters, and J. P. Rosazza. 1974. Microbial transformations of antitumor compounds. 1. Conversion of acronycine to 9-hydroxyacronycine by Cunninghamella echinulata. J. Med. Chem. 17:599–602. 4. Chen, Y., and J. P. N. Rosazza. 1994. Microbial transformation of ibuprofen by a Nocardia species. Appl. Environ. Microbiol. 60:1292–1296. 5. Gross, G. G. 1972. Formation and reduction of intermediate acyl-adenylate by aryl-aldehyde NADP oxidoreductase from Neurospora crassa. Eur. J. Biochem. 31:585–592. 6. Gurujeyalakshmi, G., and A. Mahadevan. 1987. Dissimilation of ferulic acid by Bacillus subtilis. Curr. Microbiol. 16:69–73. 7. Hagedorn, S., and B. Kaphammer. 1994. Microbial biocatalysis in the generation of flavor and fragrance chemicals. Annu. Rev. Microbiol. 48:773–800. 8. Huang, Z., L. Dostal, and J. P. N. Rosazza. 1993. Mechanisms of ferulic acid conversions to vanillic acid and guaiacol by Rhodotorula rubra. J. Biol. Chem. 268:23954–23958. 9. Ishikawa, H., W. J. Schubert, and F. F. Nord. 1963. The degradation by Polyporus versicolor and Formes fomentarius of aromatic compounds structurally related to softwood lignin. Archiv. Biochem, Biophys. 100:140–149. 10. Jezo, I., and J. Zemek. 1986. Enzymatische Reducktion einiger aromatischer Carboxysa¨uren. Chem. Papers 40:279–281. 11. Jurkova ´, M., and M. Wurst. 1993. Biodegradation of aromatic carboxylic acids by Pseudomonas mira. FEMS Microbiol. Lett. 111:245–250. 12. Kato, N., H. Konishi, K. Uda, M. Shimao, and C. Sakazawa. 1988. Microbial reduction of benzoate to benzyl alcohol. Agric. Biol. Chem. 52:1885–1886. 13. Kato, N., E. H. Joung, H. C. Yang, M. Masuda, M. Shimao, and H. Yanase. 1991. Purification and characterization of aromatic acid reductase from Nocardia asteroides JCM 3016. Agric. Biol. Chem. 55:757–762. 14. Labuda, I. M., S. K. Goers, and K. A. Keon. July 1992. Bioconversion process for the production of vanillin. U.S. patent 5,128,253. 15. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685. 16. Li, T., and J. P. N. Rosazza. 1997. The purification, characterization, and properties of an aldehyde oxidoreductase from Nocardia sp. NRRL 5646. J. Bacteriol. 179:3482–3487. 17. Mulheim, A., and K. Lerch. 1999. Towards a high-yield bioconversion of ferulic acid to vanillin. Appl. Microbiol. Biotechnol. 51:456–461. 18. Prince, R. C., and D. E. Gunson. 1994. Just plain vanilla? Trends Biol. Sci. 19:521. 19. Rabenhorst, J., and R. Hopp. May 1991. Process for the preparation of vanillin. U.S. patent 5,017,388. 20. Raman, T. S., and E. R. B. Shanmugasundaram. 1962. Metabolism of some aromatic acids by Aspergillus niger. J. Bacteriol. 84:1340–1341. 21. Rosazza, J. P. N., Z. Huang, L. Dostal, T. Volm, and B. Rousseau. 1995. Biocatalytic transformation of ferulic acid: an abundant aromatic natural product. J. Ind. Microbiol. 15:457–471. 22. Sutherland, J. B., D. L. Crawford, and A. L. Pometto III. 1983. Metabolism of cinnamic, p-coumaric, and ferulic acids by Streptomyces, setonii. Can. J. Microbiol. 29:1253–1257. 23. Toms, A., and J. M. Wood. 1970. The degradation of trans-ferulic acid by Pseudomonas acidovorans. Biochemistry 9:337–343. 24. Tsuda, Y., K. Kawai, and S. Nakajima. 1984. Asymmetric reduction of 2methyl-2-aryloxyacetic acids by Glomerella cingulata. Agric. Biol. Chem. 48: 1373–1374. 25. Tsuda, Y., K. Kawai, and S. Nakajima. 1985. Microbial reduction of 2-phenylpropionic acid, 2-benzyloxypropionic acid and 2-(2-furfuryl)propionic acid. Chem. Pharm. Bull. 33:4657–4661. 26. Washisu, Y., A. Tetsushi, N. Hashimoto, T. Kanisawa. 1993. Manufacture of vanillin and related compounds with Pseudomonas. Japanese Patent No. 5,227,980 27. Yoshimoto, T., M. Samejima, N. Hanyu, and T. Koma. August 1990. Dioxygenase for styrene cleavage manufactured by Pseudomonas. Japanese patent 2,195,871.