APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2002, p. 4315–4321 0099-2240/02/$04.00⫹0 DOI: 10.1128/AEM.68.9.4315–4321.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 68, No. 9
Biotransformation of Eugenol to Ferulic Acid by a Recombinant Strain of Ralstonia eutropha H16 Jo ¨rg Overhage, Alexander Steinbu ¨chel, and Horst Priefert* Institut fu ¨r Mikrobiologie der Westfa ¨lischen Wilhelms-Universita ¨t Mu ¨nster, D-48149 Mu ¨nster, Germany Received 18 March 2002/Accepted 11 June 2002
The gene loci ehyAB, calA, and calB, encoding eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase, respectively, which are involved in the first steps of eugenol catabolism in Pseudomonas sp. strain HR199, were amplified by PCR and combined to construct a catabolic gene cassette. This gene cassette was cloned in the newly designed broad-host-range vector pBBR1-JO2 (pBBR1JO2ehyABcalAcalB) and transferred to Ralstonia eutropha H16. A recombinant strain of R. eutropha H16 harboring this plasmid expressed functionally active eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase. Cells of R. eutropha H16(pBBR1-JO2ehyABcalAcalB) from the lateexponential growth phase were used as biocatalysts for the biotransformation of eugenol to ferulic acid. A maximum conversion rate of 2.9 mmol of eugenol per h per liter of culture was achieved with a yield of 93.8 mol% of ferulic acid from eugenol within 20 h, without further optimization. feryl aldehyde dehydrogenase, which also catalyzes the oxidation of vanillin as a side activity (23). Thus, Pseudomonas sp. strain HR199 and derivatives seemed not to be the most suitable candidates for vanillin production. Other approaches were based on the biotransformation of ferulic acid to vanillin by the white-rot fungus Pycnoporus cinnabarinus (21) or the gram-positive bacterium Amycolatopsis sp. strain HR167 (2). Amycolatopsis sp. strain HR167 is used for the biotechnological production of vanillin (J. Rabenhorst and R. Hopp, 1997, patent application EP0761817). To use eugenol as a cheap resource to provide ferulic acid for this biotransformation, the industrially approved bacterium Ralstonia eutropha H16 was genetically modified to convert eugenol to ferulic acid, quantitatively.
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the characteristic aroma component of the vanilla pod and is used in a broad range of flavors for foods, confectionery, and beverages; as a fragrance ingredient in perfumes and cosmetics; and for pharmaceuticals. The main production of vanillin is done via chemical synthesis from guaiacol and lignin (9). The increasing customer-led demand for natural flavors has induced growing interest in producing vanillin from natural raw materials by biotransformation, which can then be regarded as a natural aroma chemical. This common trend in the production of flavors and fragrances has recently been reviewed (6, 15, 17, 27, 29, 30). Since vanillin occurs as an intermediate in the catabolism of phenolic stilbenes, eugenol (4-allyl-2-methoxyphenol), ferulate (4-hydroxy-3-methoxycinnamate), and lignin (7, 33, 35, 36), these compounds are potential substrates for biotransformation processes. A method for biotransformation of eugenol to vanillin was developed by Rabenhorst and Hopp (1991, patent application EP0405197), based on a new Pseudomonas sp. strain, HR199, which degrades eugenol via coniferyl alcohol (4-hydroxy-3-methoxycinnamyl alcohol), coniferyl aldehyde (4-hydroxy-3-methoxycinnamyl aldehyde), ferulic acid, vanillin, vanillic acid (4-hydroxy-3-methoxybenzoate), and protocatechuic acid (3,4-dihydroxybenzoate) (28). The genes involved in this conversion have been cloned (1, 22, 24, 25, 26; A. Steinbu ¨chel, H. Priefert, and J. Rabenhorst, 1998, patent application EP0845532). However, by using the wild type of strain HR199, no production of vanillin was observed. This was due to further conversion of vanillin to vanillic acid. Even the insertional inactivation of the vdh gene, encoding vanillin dehydrogenase in Pseudomonas sp. strain HR199 with an omega element, led to a strain that only transiently accumulated vanillin during transformation of eugenol. The lack of vanillin dehydrogenase was superimposed by the calB-encoded coni-
MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. All bacteria and plasmids investigated in this study are listed in Table 1. Cells of Escherichia coli were grown at 37°C in Luria-Bertani (LB) or M9 medium (31). Cells of Ralstonia eutropha H16 and Pseudomonas sp. strain HR199 were grown at 30°C either in a nutrient broth (NB) medium (0.8% [wt/vol] Bacto; Difco) or in mineral salt medium (MM) (32) or HR-MM (28)—MM supplemented with carbon sources as indicated in the text. Eugenol was added to the medium at a final concentration of 0.05% (vol/vol). Kanamycin was used at final concentrations of 300 and 50 g/ml for recombinant strains of R. eutropha H16 and E. coli, respectively. Cells of R. eutropha H16 and Pseudomonas sp. strain HR199 were grown in 250-ml Erlenmeyer flasks with baffles on a rotary shaker (RC-6-W; A. Ku ¨hner AG, Basel, Switzerland) at 150 rpm. Samples were taken from the cultures, and cells were removed by centrifugation. The culture supernatants obtained were analyzed by high-performance liquid chromatography (HPLC) with respect to the appearance or disappearance of catabolic intermediates as described below. Analytical methods. By using a Knauer HPLC apparatus, culture supernatants were analyzed for excreted intermediates of the eugenol catabolism by liquid chromatography without prior extraction. Intermediates were separated by reverse-phase chromatography on a Nucleosil-100 C18 column (particle size, 5 m; column, 250 by 4.0 mm) with a gradient of 0.1% (vol/vol) formic acid (eluant A) and acetonitrile (eluant B) in a range of 27 to 100% (vol/vol) eluant B and at a flow rate of 1 ml/min. For quantification, all intermediates were calibrated with external standards. The compounds were identified by their retention times, as well as the corresponding spectra, which were identified with a diode array detector (WellChrom Diodenarray-Detektor K-2150; Knauer, Berlin, Germany).
* Corresponding author. Mailing address: Institut fu ¨r Mikrobiologie, Westfa¨lische Wilhelms-Universita¨t Mu ¨nster, Corrensstrasse 3, D-48149 Mu ¨nster, Germany. Phone: 49-251-8339830. Fax: 49-2518338388. E-mail:
[email protected]. 4315
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APPL. ENVIRON. MICROBIOL. TABLE 1. Bacterial strains and plasmids used in this study
Strain or plasmid
Relevant characteristics
Source or reference
Strains Pseudomonas sp. strain HR199 (wild type)
Wild type, eugenol positive, ferulic acid positive
DSM 7063
R. eutropha H16 (wild type)
Wild type, eugenol negative, ferulic acid negative
DSM 428
recA1 endA1 gyrA96 thi-1 hsdR17 (rK⫺, mK⫹) supE44 relA1 ⫺ lac [F⬘ proAB lacI qZ⌬M15 Tn10(Tcr)] recA; harboring tra genes of plasmid RP4 in chromosome, pro thi-1
5
Apr, lacPOZ⬘, T7 and T3 promoter
Stratagene, San Diego, Calif. 16 This study 26 This study This study This study
E. coli XL1-Blue S17-1 Plasmids pBluescript SK⫺ pBBR1-MCS2 pBBR1-JO2 pSKehyAB pSKcalA pSKcalB pSKehyABcalAcalB pBBR1-JO2ehyABcalAcalB
34
Kmr, lacPOZ⬘, mob, broad host range Kmr, lacPOZ⬘, mob, broad host range pBluescript SK⫺ harboring PCR product comprising ehyAB as SacI-NotI fragment pBluescript SK⫺ harboring PCR product comprising calA as NotI-SpeI fragment pBluescript SK⫺ harboring PCR product comprising calB as SpeI-BamHI fragment pBluescript SK⫺ harboring PCR products comprising ehyAB, calA, and calB as a SacI-BamHI fragment pBBR1-JO2 harboring PCR products comprising ehyAB, calA, and calB as a SacI-BamHI fragment
Enzyme assay. Cells of R. eutropha H16 (wild type) and R. eutropha H16(pBBR1-JO2ehyABcalAcalB) were grown to the late exponential phase at 30°C in MM containing 1.0% (wt/vol) gluconate. Recombinant strains of E. coli were grown for 12 h at 37°C in LB in the presence of 1 mM isopropyl--Dthiogalactopyranoside (IPTG). Cells were disrupted either by a twofold French press passage at 96 MPa or by sonification (1 min/ml of cell suspension with an amplitude of 40 m) with a Bandelin Sonoplus GM200 ultrasonic disintegrator. The soluble fractions of crude extracts were obtained by centrifugation at 100,000 ⫻ g at 4°C for 1 h. Eugenol hydroxylase and coniferyl aldehyde dehydrogenase activities were measured as described previously (1, 25, 26). The coniferyl alcohol dehydrogenase activity was assayed photometrically at 30°C in 200 mM Tris-HCl buffer (pH 9.0) in the presence of 2 mM NAD and 4 mM coniferyl alcohol. The reaction was started by addition of NAD and was followed by measuring the initial change in A400 due to the oxidation of coniferyl alcohol (ε ⫽ 22.4 cm2/mol). A stock solution of coniferyl alcohol was prepared in dimethyl sulfoxide. One unit of enzyme activity was defined as the amount required to catalyze the conversion of 1 mol of substrate per min. The activities determined with the spectrophotometric tests were confirmed by measuring the eugenol, coniferyl alcohol, coniferyl aldehyde, and ferulic acid concentrations of corresponding stopped reaction tests by HPLC analysis. The amount of soluble protein present was determined as described by Lowry et al. (19). Isolation, manipulation, analysis, and transfer of DNA. All genetic techniques were performed as described by Sambrook et al. (31). Conjugations of E. coli S17-1 (donor) harboring a hybrid plasmid and of R. eutropha H16 (recipient) were performed on solidified NB medium (13). The identity and accuracy of the constructed hybrid plasmids were confirmed by DNA sequencing by the chain termination method with the automatic sequencer LI-COR model 4000L (MWG-Biotech, Ebersberg, Germany). Amplification and cloning of the structural gene (calA) of the coniferyl alcohol dehydrogenase from Pseudomonas sp. strain HR199. The gene calA encoding coniferyl alcohol dehydrogenase was amplified in a PCR with the primers PCRcalAup (5⬘-AAAAGCGGCCGCGGACCGTAAAAAGGAAAGAGCAT GCAACTG-3⬘) and PCRcalAdown (5⬘-AAAAACTAGTCGTCCACGAACTT ACACGTAGGTCGATG-3⬘) together with PstI-digested genomic DNA from Pseudomonas sp. strain HR199 as template DNA. The PCR product was digested with NotI (site present in the upstream primer) and SpeI (site present in the downstream primer) and cloned in pBluescript SK⫺. In the resulting hybrid plasmid, pSKcalA, the calA gene was arranged colinear to and downstream of the lacZ promoter. Amplification and cloning of the structural gene (calB) of the coniferyl aldehyde dehydrogenase from Pseudomonas sp. strain HR199. The gene calB, encoding coniferyl aldehyde dehydrogenase, was amplified in a PCR with PstIdigested genomic DNA from Pseudomonas sp. strain HR199 and the primers
This study
PCRcalBup (5⬘-AAAAACTAGTAATAACAATTGACTCCTCAGGAGGTCA GC-3⬘) and PCRcalBdown (5⬘-AAAAGGATCCCCACTACCAACGGTTCTA ACACTCCGTT-3⬘). Since the upstream primer exhibited an SpeI site and the downstream primer exhibited a BamHI site, the correspondingly digested PCR product was cloned in pBluescript SK⫺ with the gene calB colinear to and downstream of the lacZ promoter. Construction of broad-host-range vector pBBR1-JO2. A region comprising the lac promoter/operator region, the multiple cloning site (MCS), and the LacZ alpha peptide coding region was amplified as a 633-bp SspI fragment (MCSJO2) from pBluescript SK⫺ by PCR with the primers PCRMCSup (5⬘-AAAAAATA TTACCGAGCGCAGCGAGTCAGTGAGCG-3⬘) and PCRMCSdown (5⬘-AA AAAATATTTTACGCGTCCCATTCGCCATTCAGGCTACGC-3⬘). After digestion of the kanamycin resistance-conferring broad-host-range vector pBBR1MCS2 with SspI, a 704-bp fragment containing the MCS and lacZ gene of this vector was substituted by MCSJO2, resulting in the new broad-host-range vector pBBR1-JO2. Thus, the two vectors differed only in the orientation of the MCS. In pBBR1-MCS2, the orientation of the MCS corresponded to pBluescript KS⫺, and in pBBR1-JO2, it corresponded to pBluescript SK⫺. Materials. Restriction endonucleases, T4 DNA ligase, lambda DNA, and substrates used in the enzyme assays were obtained from either C. F. Boehringer & Soehne (Mannheim, Germany) or GIBCO/BRL-Bethesda Research Laboratories GmbH (Eggenstein, Germany). Agarose type NA was purchased from Pharmacia-LKB (Uppsala, Sweden). Synthetic oligonucleotides were purchased from MWG-Biotech (Ebersberg, Germany). All other chemicals were from Haarmann & Reimer (Holzminden, Germany), E. Merck AG (Darmstadt, Germany), Fluka Chemie (Buchs, Switzerland), Serva Feinbiochemica (Heidelberg, Germany), or Sigma Chemie (Deisenhofen, Germany). Nucleotide sequence accession numbers. The nucleotide sequence accession numbers of the ehyAB, calA (named “CADH” in the patent; Steinbu ¨chel et al., patent application EP0845532), and calB genes are AJ243941, A92130, and AJ006231, respectively.
RESULTS Tolerance of R. eutropha H16 to different eugenol concentrations. R. eutropha H16 neither is able to grow on eugenol or ferulic acid as the sole carbon source nor is able to convert eugenol or ferulic acid to coniferyl alcohol or vanillin, respectively. Consequently, it probably lacks the enzymes for the degradation of ferulic acid and seemed to be a suitable candidate to produce and accumulate ferulic acid from eugenol after
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TABLE 2. Tolerance of R eutropha H16 to different eugenol concentrationsa OD600 after incubation forb:
Eugenol concn (% [vol/vol])
0h
24 h
48 h
0 0.010 0.025 0.050 0.075 0.100
0.11 0.11 0.11 0.12 0.11 0.12
8.5 5.9 0.13 0.13 0.17 0.25
6 2.6 0.30 0.19 0.20 0.28
a Cells of R. eutropha H16 were precultured overnight in MM containing 0.5% (wt/vol) sodium gluconate as the carbon source. The cells were used for inoculation of a series of 50-ml cultures in 250-ml baffled Erlenmeyer flasks containing MM with 0.5% (wt/vol) sodium gluconate as the carbon source and different concentrations of eugenol. b Data represent the mean of three independent determinations.
heterologous expression of genes for eugenol conversion to ferulic acid introduced by genetic engineering. Since it is known that eugenol has antibacterial properties, the tolerance of the wild-type strain R. eutropha H16 to eugenol was studied. To investigate the eugenol tolerance of R. eutropha H16, cells were precultured overnight in MM containing 0.5% (wt/ vol) sodium gluconate as the carbon source. The cells were used for inoculation of several 50-ml cultures in 250-ml baffled Erlenmeyer flasks containing MM with 0.5% (wt/vol) sodium gluconate as the carbon source and increasing concentrations of eugenol ranging from 0.01% (vol/vol) to 0.1% (vol/vol), respectively (Table 2). The cultures were incubated for 48 h at 30°C, and growth was monitored by determination of the optical density at 600 nm (OD600). As shown in Table 2, R. eutropha H16 was highly sensitive to eugenol. Even at concentrations of about 0.01% (vol/vol), growth was significantly reduced. At concentrations higher than 0.05% (vol/vol), growth was completely inhibited. Based on these data, it was anticipated that the eugenol addition had to be strictly controlled in a biotransformation process based on a recombinant strain of R. eutropha H16.
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Cloning and functional expression of ehyAB, calA, and calB in E. coli. The genes that should be used to enable a recombinant R. eutropha H16 strain to convert eugenol to ferulic acid were amplified from the genomic DNA of Pseudomonas sp. strain HR199. To confirm the physiological function of these genes, they were expressed in E. coli XL1-Blue. The genes ehyA and ehyB were recently cloned together with open reading frame 2 in pBluescript SK⫺ (26). The resulting hybrid plasmid, pSKehyAB, conferred eugenol hydroxylase activity to the corresponding recombinant strain of E. coli XL1Blue after growth in the presence of the inducer IPTG (Table 3). The genes calA and calB were amplified and cloned in pBluescript SK⫺ as described in Materials and Methods. A recombinant strain of E. coli XL1-Blue harboring pSKcalA exhibited coniferyl alcohol dehydrogenase activity after growth in the presence of IPTG (Table 3). The hybrid plasmid pSKcalB conferred coniferyl aldehyde dehydrogenase activity to the corresponding recombinant strain of E. coli XL1-Blue after growth of this strain in the presence of IPTG (Table 3). Construction of hybrid plasmid pBBR1-JO2ehyABcalAcalB. To equip the industrially relevant strain R. eutropha H16 with the genetic information necessary for the biotransformation of eugenol to ferulic acid, ehyAB, calA, and calB from Pseudomonas sp. strain HR199 had to be transferred to this strain and were therefore combined in a vector with a broad host range, which could be replicated in R. eutropha H16. Since no broadhost-range vector exhibiting a kanamycin resistance gene combined with a suitable MCS was available, the vector pBBR1JO2 was constructed. The genes calA and calB were isolated from NotI-SpeIdigested pSKcalA and SpeI-BamHI-digested pSKcalB, respectively, and subsequently cloned in the NotI-BamHI-digested hybrid plasmid pSKehyAB (Fig. 1). The resulting hybrid plasmid, pSKehyABcalAcalB, conferred eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase activities to the corresponding recombinant E. coli XL1-Blue strain (Table 3). From the hybrid plasmid pSKehy-
TABLE 3. Eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase activities in R. eutropha H16 (wild type) and H16(pBBR1-JO2ehyABcalAcalB) and in recombinant strains of E. coli XL1-Bluea Sp act (U/mg of protein)b Strain
R. eutropha H16 H16(pBBR1-JO2ehyABcalAcalB) E. coli XL1-Blue(pSKehyAB) XL1-Blue(pSKcalA) XL1-Blue(pSKcalB) XL1-Blue(pSKehyABcalAcalB) XL1-Blue(pBluescript SK⫺) XL1-Blue(pBBR1JO2ehyABcalAcalB) XL1-Blue(pBBR1-JO2) Pseudomonas sp. strain HR199 a b c
Eugenol hydroxylase
Coniferyl alcohol dehydrogenase
Coniferyl aldehyde dehydrogenase
⬍0.01 0.21 ⫾ 0.01
⬍0.01 0.22 ⫾ 0.02
⬍0.01 0.04 ⫾ 0.005
0.16 ⫾ 0.01 ND ND 0.14 ⫾ 0.01 ⬍0.01 0.14 ⫾ 0.01
NDc 1.16 ⫾ 0.05 ND 0.29 ⫾ 0.02 ⬍0.01 0.13 ⫾ 0.01
ND ND 0.06 ⫾ 0.01 0.01 ⫾ 0.01 ⬍0.01 0.01 ⫾ 0.01
⬍0.01
⬍0.01
⬍0.01
0.016 (26)
0.24
0.46 (1)
The experiments and enzyme assays were done in triplicate, and the standard errors were calculated by standard statistics (P ⬍ 0.05). Cells were grown and enzyme activities were determined as described in Materials and Methods. References are shown in parentheses. ND, not determined.
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APPL. ENVIRON. MICROBIOL.
FIG. 1. Construction of hybrid plasmid pBBR1-JO2ehyABcalAcalB.
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culture supernatant was proven by HPLC analyses. In this experiment, a maximum conversion rate of 2.9 mmol per h per liter of culture was readily obtained without further optimization. The course of ferulic acid production is summarized in Fig. 2. Transconjugants that harbored only the vector pBBR1JO2 and that were treated in the same way as a negative control showed no conversion of eugenol. DISCUSSION
FIG. 2. Production of ferulic acid from eugenol by R. eutropha H16(pBBR1-JO2ehyABcalAcalB). Cells were grown in 50 ml of liquid MM with 1.0% (wt/vol) sodium gluconate in the presence of kanamycin overnight at 30°C to the late-exponential growth phase. Cells were further incubated at 30°C, and 25-l portions of eugenol (final concentration, 0.05% [vol/vol]) were added at the time points indicated (arrows). Samples were taken, and ferulic acid concentrations were determined by HPLC. Data represent the mean of three independent experiments. Variance is indicated by error bars.
ABcalAcalB, a SacI-BamHI fragment was isolated that comprised the genes ehyAB, calA, and calB. This fragment was cloned in the corresponding sites of the broad-host-range vector pBBR1-JO2, resulting in the hybrid plasmid pBBR1JO2ehyABcalAcalB (Fig. 1). Recombinant cells of E. coli XL1-Blue harboring the plasmid pBBR1-JO2ehyABcalAcalB exhibited eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase activity after growth in the presence of IPTG (Table 3). Transfer of plasmid pBBR1-JO2ehyABcalAcalB to R. eutropha H16 and physiological characterization of the recombinant strain. The mobilizing strain E. coli S17-1 was transformed with the hybrid plasmid pBBR1-JO2ehyABcalAcalB, which was isolated from the recombinant strain of E. coli XL1-Blue. E. coli S17-1(pBBR1-JO2ehyABcalAcalB) was used as a donor strain in a conjugation experiment, and the plasmid was transferred to R. eutropha H16. In the soluble fraction of the crude extract of gluconate-grown cells of the resulting recombinant strain R. eutropha H16(pBBR1-JO2ehyABcalAcalB), eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase activities were detected (Table 3). The results revealed that ehyAB, calA, and calB were functionally expressed in R. eutropha H16 with high efficiency. In contrast, the control strain R. eutropha H16 harboring only the vector pBBR1-JO2 exhibited none of the activities mentioned (Table 3). Production of ferulic acid from eugenol. To investigate the conversion of eugenol to ferulic acid by the transconjugant R. eutropha H16(pBBR1-JO2ehyABcalAcalB), cells were grown in 50 ml of liquid MM with 1.0% (wt/vol) sodium gluconate in the presence of kanamycin overnight at 30°C. After the lateexponential growth phase was reached, 25-l portions of eugenol (final concentration, 0.05% [vol/vol]) were added to this culture at different time intervals (Fig. 2), and the occurrence of coniferyl alcohol, coniferyl aldehyde, and ferulic acid in the
Vanillin is one of the most important aromatic compounds used in the production of flavors and fragrances. Due to an increasing demand for healthy and natural foods, several strategies were applied to produce vanillin from natural sources by biotransformation. The most promising substrates for this purpose were eugenol and ferulic acid. The biotransformation of eugenol was studied in detail in Pseudomonas sp. strain HR199 (24, 26, 28). This bacterium is able to grow with eugenol as the sole carbon source, despite its antibacterial property. Eugenol is degraded via coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillic acid, and protocatechuic acid. One promising approach to obtain a vanillin-producing strain from Pseudomonas sp. strain HR199 was based on the inactivation of the vanillin dehydrogenase gene (vdh) by site-directed mutagenesis. The resulting vanillin dehydrogenase-deficient vdh⍀Km Pseudomonas sp. strain actually accumulated vanillin up to 2.9 mM from 6.5 mM eugenol (23). However, vanillin was only transiently accumulated, due to further oxidation to vanillic acid catalyzed by a side activity of the coniferyl aldehyde dehydrogenase (23). Based on ferulic acid as a substrate, economically interesting vanillin production processes were established with Amycolatopsis sp. strain HR167 (2; J. Rabenhorst and R. Hopp, 1997, patent application EP0761817) or Streptomyces setonii (20) as a biocatalyst. With Amycolatopsis sp. strain HR167, 19.9 g of ferulic acid liter⫺1 was converted to 11.5 g of vanillin liter⫺1 within 32 h at a 10-liter scale, which corresponds to a molar yield of 77.8% (J. Rabenhorst and R. Hopp, 1997, patent application EP0761817). With S. setonii strain ATCC 39116, 13.9 g of vanillin liter⫺1 was obtained from 22.5 g of ferulic acid liter⫺1 after 17 h of incubation at a 10-liter scale, corresponding to a molar yield of 75% (B. Mu ¨ller, T. Mu ¨nch, A. Muheim, and M. Wetli, 1998, patent application EP0885968). Although ferulic acid is an extremely abundant cinnamic acid derivative found in the cell walls of woods, grasses, and corn hulls, it is not easily accessible from these natural sources, since it is covalently linked to the various carbohydrates as a glycosidic conjugate, or it occurs as an ester or amide. Thus, it can be released from these natural products only by alkaline hydrolysis (3). However, ferulic acid gained from this chemical process cannot be considered as “natural.” Attempts to release ferulic acid enzymatically from lignin employing ferulic acid esterases or cinnamoyl ester hydrolases (10–12) are still far from being an implementable process due to unacceptable yields. Thus, the enzymatical conversion of eugenol to ferulic acid is a promising alternative to gain “natural” ferulic acid as the substrate for the above-mentioned biotransformations with Amycolatopsis sp. strain HR167 or S. setonii, respectively. The catabolism of eugenol, which proceeds via ferulic acid, was investigated in detail in Pseudomonas sp. strain HR199 and
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Pseudomonas sp. strain OPS1, and the genes involved have recently been cloned and characterized (4, 24, 26). Pseudomonas sp. strain HR199 has even been used for ferulic acid production from eugenol (28). However, the molar yield after a 75-h biotransformation was only 52%, since ferulic acid was further degraded by this strain. Thus, the genes ehyAB, calA, and calB, encoding the enzymes eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase, respectively, which catalyze the conversion of eugenol to ferulic acid in Pseudomonas sp. strain HR199, were expressed in a suitable host unable to degrade ferulic acid. This prerequisite was met by R. eutropha H16, which is a well-characterized gram-negative bacterium. It is already used in the industry for the production of polyhydroxyalkanoates such as poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (8), which might also be used for the conversion of residual carbon sources by the chemical industry (14, 18). Moreover, it is easily accessible for genetic and metabolic engineering. Thus, this safe and easyto-use biocatalyst was an auspicious candidate for the establishment of the biotransformation process to produce ferulic acid. Although the R. eutropha wild-type strain H16, like other bacteria, appeared to be sensitive to high concentrations of eugenol (higher than 0.01% [vol/vol]), in biotransformation experiments with the corresponding recombinant R. eutropha strain H16(pBBR1-JO2ehyABcalAcalB) expressing the genes ehyAB, calA, and calB, fivefold-higher eugenol concentrations could be applied. However, the addition of eugenol had to be controlled in order not to exceed a final concentration of 3.25 mM (corresponding to 0.05% [vol/vol] eugenol) to avoid toxic effects. The eugenol addition mode was the only crucial parameter, and a conversion rate of 2.9 mmol per h per liter of culture was easily obtained without further optimization. This corresponded to a consumption rate of 0.48 g of eugenol per liter and per h, which is sufficiently high to further optimize a process based on a recombinant strain of R. eutropha H16. After a biotransformation time of 20 h, a total of 915 mol of ferulic acid was obtained from 975 mol of eugenol, corresponding to a molar yield of 93.8%. Thus, an effective biotransformation process leading from eugenol to ferulic acid was successfully established.
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6. 7. 8. 9. 10. 11.
12.
13. 14.
15. 16.
17. 18. 19. 20. 21.
22.
23.
ACKNOWLEDGMENTS
24.
H. Priefert and A. Steinbu ¨chel are indebted to Haarmann & Reimer GmbH for providing a collaborative research grant. We thank Daniela Rehder for proofreading the manuscript.
25.
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