Introduction

Soil bacteria belonging to the species Pseudomonas putida have been applied in technical biodegradation processes (Nusslein et al. 1992; Ramos et al. 1994; Ronchel et al. 1995), taking advantage of their extremely metabolic versatility. The strain P. putida KT2440 used in this study is a plasmid-cured, restriction-deficient derivative of the toluene degrader P. putida mt-2 (ATCC 33015) (Franklin et al. 1981; Ramos et al. 1994, 1997; Stanier et al. 1966). It is regarded as a biosafety strain for biotechnological applications, and has been authorized as such by the USA National Institute of Health in 1981. P. putida KT2440 colonizes the rhizosphere of many plants and has been extensively characterized both physiologically and genetically (Mermod et al. 1984; Ramos et al. 1995, Molina et al. 1998). To exploit the biotechnological potential of this strain, its genome sequence has recently been determined (www.tigr.org) in order to serve as the basis for functional genomic studies.

Since P. putida KT2440 is able to use a wide range of aromatic molecules as sole carbon sources, we decided to study its potential use in the biotechnological production of the flavor compound vanillin from ferulic acid. Ferulic acid, which is a known precursor for vanillin production using other microorganisms (Falconnier et al. 1994; Lesage-Meessen et al. 1996, 2002; Gasson et al. 1998; Achterholt et al. 2000), is an abundant aromatic residue found in the cell walls of woods, grasses, and corn husks (Escott-Watson and Marais 1992; Ishii 1997; Oosterveld et al. 2000).

In this study, we investigated the catabolism of ferulic acid in P. putida KT2440 and identified and functionally characterized those genes essential for ferulic acid degradation in this strain. The potential application of P. putida KT2440 and derived mutants in biotechnological vanillin production process is discussed.

Materials and methods

Bacterial strains and plasmids

The strains of Pseudomonas putida KT2440 and Escherichia coli and the plasmids used in this study are listed in Table 1.

Table. 1. Bacterial strains and plasmids used in this study
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Growth of bacteria

E. coli was cultivated at 37 °C in Luria-Bertani (LB) or in M9 mineral salts medium (Sambrook et al. 1989). P. putida KT2440 strains were cultivated at 30 °C either in a nutrient broth (NB) medium (0.8%, w/v; Bacto Difco) or in a mineral salts medium (MM) (Schlegel at al. 1961) supplemented with carbon sources as indicated. Ferulic acid, vanillin, and vanillic acid were dissolved in dimethyl sulfoxide and added to the medium at a final concentration of 0.1% (w/v). Tetracycline and kanamycin were used at final concentrations of 25 μg/ml and 100 μg/ml, respectively, for selection of recombinant P. putida KT2440 strains. Growth of bacteria was monitored with a Klett-Summerson photometer.

Analytical methods

Culture supernatants were analyzed for excreted intermediates chromatographically, without prior extraction, using an HPLC apparatus (Fa. Knauer, Berlin, Germany) as described previously (Overhage et al. 1999b).

Preparation of the soluble fractions of crude protein extracts

Cells were disrupted either by a two-fold French press passage at 96 MPa, or by sonication (1 min/ml of cell suspension with an amplitude of 40 μm) with a Bandelin Sonopuls GM200 ultrasonic disintegrator. Soluble fractions of crude protein extracts were obtained by centrifugation at 100,000 g at 4 °C for 1 h.

Enzyme assays and specific staining for vanillin dehydrogenase activity in polyacrylamide gels

Feruloyl coenzyme A (feruloyl-CoA) synthetase (Fcs) and vanillin dehydrogenase (Vdh) were assayed as described previously (Overhage et al. 1999a). The amount of soluble protein was determined as described by Bradford (1976).

Proteins were separated under non-denaturing conditions in 7.4% (w/v) polyacrylamide gels as described by Stegemann et al. (1973). To stain gels for Vdh activity, the gels were incubated at 30  °C in the dark in 100 mM potassium phosphate buffer (pH 7.0) containing 0.08% (w/v) NAD+, 0.04% (w/v) p-nitroblue tetrazolium chloride, 0.003% (w/v) phenazine methosulfate, and different concentrations of vanillin.

Isolation, manipulation and transfer of DNA

Plasmid DNA and DNA restriction fragments were isolated and analyzed by standard methods (Sambrook et al. 1989). Competent cells of E. coli were prepared and transformed using the CaCl2 procedure (Hanahan 1983). Conjugations of E. coli S17-1 (donor) harboring hybrid plasmids and of P. putida KT2440 (recipient) were carried out on solidified NB medium as described by Friedrich et al. (1981).

Computer-assisted localization of fcs, ech, and vdh in the genome sequence of P. putida KT2440

To identify genes involved in ferulic acid catabolism in P. putida KT2440, translated BLAST Search (blastx) from the National Center forBiotechnology Information (NCBI) was applied with sequences of corresponding genes from Pseudomonas sp. HR199, previously described (Overhage et al. 1999a), using a database with the genome sequence of strain KT2440 (generously provided by the Institute for Genomic Research, TIGR).

Amplification and cloning of fcs, ech, and vdh from genomic DNA of P. putida KT2440

The genes fcs, ech, and vdh were amplified in separate PCRs with genomic DNA of P. putida KT2440 as template DNA. To amplify fcs, oligonucleotides PCR2440fcsuB (5′-AAAAGGATCCCAAGAACAAGGAGCAACCTGCGTG-3′) and PCR2440fcsdE (5′-AAAAGAATTCCAGGTCGATGGGCGAGATCGCGGC-3′) were used as primers in the PCR (introduced restriction enzyme sites are underlined). To amplify ech, oligonucleotides PCR2440echuE (5′-AAAAGAATTCCCCGTTAGCGTGGCCCCATCA CC-3′) and PCR2440echdK (5′-AAAAGGTACCGTCGTTATAGAGAAATACCCGCGG GC-3′) were used as primers, and to amplify vdh, oligonucleotides PCR2440vdhuB (5′-AAA AGGATCCCGACAACAAGAGGAATGAGCATGTTG-3′) and PCR2440vdhdE (5′-AAA AGAATTCCTAGATGGGATAGTGACGCGGGCCG-3′) were used. The PCR products were isolated and digested with EcoRI and BamHI (in the case of fcs and vdh) or EcoRI and KpnI (in the case of ech) and ligated to correspondingly digested pBluescript SK- DNA. E. coli XL1-Blue was transformed with the ligation mixtures, and transformants harboring the hybrid plasmid pSKvdhBE, pSKfcsBE, or pSKechEK were obtained.

Expression of ech, fcs, and vdh from P. putida KT2440 in E. coli

The cloned ech and fcs PCR products were recovered from pSKfcsBE and pSKechEK after BamHI/EcoRI (fcs), and EcoRI/KpnI (ech) digestion, respectively, and ligated to BamHI/KpnI digested pBluescript SK−. E. coli XL1-Blue was transformed with the ligation mixture, and transformants harboring the hybrid plasmid pSKfcs/echBEK with the genes ech and fcs colinear to and downstream of the lacZ promoter were obtained. E. coli XL1-Blue harboring plasmid pSKfcs/echBEK was grown overnight in 50 ml of LB medium containing 12.5 μg of tetracycline per ml, 100 μg of ampicillin per ml, and 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cells were harvested by centrifugation, washed twice in 100 mM potassium phosphate buffer (pH 7.0), and resuspended in 50 ml of MM containing 5.2 mM ferulic acid. Bioconversion of ferulic acid with these resting cells was monitored by HPLC analysis of cultural supernatants. In addition, feruloyl-CoA synthetase activity was determined in soluble fractions of crude protein extracts of the corresponding cells. E. coli XL1-Blue harboring the hybrid plasmid pSKvdhBE was grown overnight in 50 ml of LB medium containing 12.5 μg of tetracycline per ml, 100 μg of ampicillin per ml, and 1 mM IPTG. Soluble fractions of crude protein extracts of harvested and washed cells exhibited vanillin dehydrogenase, which was determined by the corresponding enzyme assay and by specific activity staining.

Inactivation of fcs and ech by replacement with ΩKm

For inactivation of the fcs gene by replacement with ΩKm, a region upstream and downstream of fcs was amplified from genomic DNA of P. putida KT2440. To obtain the upstream region of fcs, oligonucleotides PCRfcsu1EB (5′-AAAAGGATCCGAATTCCCAA GGGCGCGCGCCTGGTCAGCG-3′) and PCRfcsu2EV (5′-AAAAGATATCGCGCAGCAC GTCGTCGACGTGACTG-3′) were used as primers in the PCR. To obtain the downstream region of fcs, oligonucleotides PCRfcsd1EV (5′-AAAAGATATCGGCCGGGCTGTGCACC GTCTTCGAG-3′) and PCRfcsd2E (5′-AAAAGAATTCCCTCGTCGAACCAGCCCTGCAC CTG-3′) were used as primers in the PCR. The isolated upstream and downstream PCR products were digested with BamHI/EcoRV and EcoRI/EcoRV, respectively, and ligated with BamHI/EcoRI-digested pBluescript SK− (Stratagene, San Diego, Calif.) and ΩKm, recovered from SmaI-digested pSKsymΩKm (Overhage et al. 1999a). E. coli XL1-Blue was transformed with the ligation mixture, and transformants harboring the hybrid plasmid pSKfcsΩKm were obtained. Fragment fcsΩKm was isolated from EcoRI-digested pSKfcsΩKm and ligated to EcoRI-digested vector pSUP202, which harbors a tetracycline resistance gene. Resulting pSUPfcsΩKm was transferred from tetracycline- and kanamycin-resistant recombinant E. coli S17-1 strains to P. putida KT2440 by conjugation. Due to homologous recombination, the complete functional fcs gene was replaced with fcsΩKm by a double cross-over event, resulting in a kanamycin-resistant but tetracycline-sensitive phenotype of the mutant P. putida KT2440 fcsΩKm.

Ech was inactivated in a similar manner, using oligonucleotides PCRechu1EB (5′-AAAAGGATCCGAATTCCCTTTCGTCATCCGAGAGGTTGGATG-3′) and PCRechu2EV (5′-AAAAGATATCGGTTCTGCACTCTTGTTGTTCGAGGTG-3′) to amplify the upstream region, and oligonucleotides PCRechd1EV (5′-AAAAGATATCGCTG AGTCGCACCGCGGGCGGCGG-3′) and PCRechd2E (5′-AAAAGAATTCGCCGATGGA CCGCCGGGCTCAGCTC-3′) to amplify the downstream region. The isolated upstream and downstream PCR products were combined with pBluescript SK− and ΩKm to obtain the hybrid plasmid pSKechΩKm. EchΩKm was isolated and cloned in pSUP202. The resulting pSUPechΩKm was transferred to P. putida KT2440 as aforementioned to replace the complete functional ech by echΩKm and to obtain mutant P. putida KT2440echΩKm.

The genotypes of mutant strains KT2440fcsΩKm and KT2440echΩKm were confirmed by amplification of the corresponding genes from genomic DNA of these mutants by PCR and sequencing of the obtained single PCR products.

Inactivation of vdh by insertion of ΩKm

For inactivation of vdh by insertion of ΩKm, the gene was first amplified from genomic DNA of P. putida KT2440. To obtain a gene flanked by EcoRI sites, the aforementioned oligonucleotide PCR2440vdhdE and the oligonucleotide PCR2440vdhuE (5′-AAAAGAATT CCGACAACAAGAGGAATGAGCATGTTG-3′) were used as primers in the PCR. The isolated PCR product was digested with EcoRI and ligated to EcoRI-digested pBluescript SK− to obtain hybrid plasmid pSKvdhEE. pSKvdhEE was digested with StuI and ligated with ΩKm, resulting in plasmid pSKvdhΩKm. Subsequently, vdhΩKm was recovered from EcoRI-digested pSKvdhΩKm, and the functional vdh gene was exchanged with the inactivated gene vdhΩKm in P. putida KT2440 by homologous recombination as described for the exchange of fcs with fcsΩKm. The success of the gene replacement in mutant P. putida KT2440vdhΩKm was confirmed as described before.

DNA sequence determination

DNA sequences were determined by the dideoxy chain-termination method (Sanger et al. 1977) with a 4000L DNA sequencer (LI-COR, Biotechnology Division, Lincoln, Neb., USA). A thermo sequenase fluorescence-labeled primer cycle-sequencing kit with 7-deaza-dGTP (Amersham Life Science, Little Chalfont, UK) was used as specified by the manufacturer, together with synthetic fluorescently labeled oligonucleotides as primers.

Results

Growth of Pseudomonas putida KT2440 with ferulic acid as carbon source

P. putida KT2440 is able to utilize ferulic acid as sole carbon source for growth. A complete degradation of 4.5 mM ferulic acid by P. putida KT2440 cells was observed during the 7-h lag growth phase (Fig. 1). After ferulic acid was depleted from the medium, cells entered the exponential growth phase. Vanillin and vanillic acid were identified as intermediates that were excreted into the culture medium transiently in trace amounts.

Fig. 1.
figure 1

Growth of Pseudomonas putida KT2440 in 50 ml MM with 0.1% (v/v) ferulic acid, and intermediates detected in the culture supernatant. Growth was monitored with a Klett-Summerson photometer. Cultures were incubated at 30 °C, samples were taken from the cultures, and supernatants were analyzed by HPLC as described in Materials and methods. × Absorbance, ■ ferulic acid, ◯ vanillic acid, ▲ vanillin

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Identification of the genes ech, fcs, and vdh

The nucleotide sequences of fcs, ech, and vdh from Pseudomonas sp. HR199 (fcs HR199, ech HR199 , vdh HR199) (Overhage et al. 1999b) were compared with the genome sequence of P. putida KT2440, which revealed the arrangement of three ORFs on fragment 10767 (length=128,524 bp) of strain KT2440 exhibiting highest homologies to fcs HR199, ech HR199 and vdh HR199 (Fig. 2). The amino acid sequence deduced from ech HR199 exhibited 88% identity to the corresponding gene product from P. putida KT2440 (ech KT2440) in a 276-amino-acid overlap. The translational start codon ATG of vdh KT2440 was located at a distance of 76 bp downstream of the translational stop codon TGA of ech KT2440. The gene product of vdh KT2440 exhibited 80% identity with the Vdh of strain HR199 in a 482-amino-acid overlap. The translational start codon GTG of fcs KT2440 was located at a distance of 92 bp downstream of the translational stop codon TAG of vdh KT2440. The gene product of fcs KT2440 exhibited 75% identity with the Fcs of strain HR199 in a 584-amino-acid overlap. Typical ribosomal binding sites (AAGGAG, AAGAGG, and AAGAG) preceded the translational start codons of fcs, vdh, and ech at distances of 8, 7, and 9 nucleotides, respectively. Ech and fcs, which are located in close proximity in a variety of other bacteria, were separated by the vdh gene in P. putida KT2440 (Fig. 2). Thus, the organization of these genes in strain KT2440 resembles that in Pseudomonas sp. HR199.

Fig. 2.
figure 2

Organization of the structural genes of the enoyl-CoA hydratase/aldolase (ech), feruloyl-CoA synthetase (fcs), and vanillin dehydrogenase (vdh) in different bacteria

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Heterologous expression of ech KT2440, fcs KT2440, and vdh KT2440 in E. coli and biotransformation of ferulic acid to vanillin by recombinant strains

To assign enzymatic functions to the products of ech, fcs, and vdh of P. putida KT2440, these genes were heterologously expressed in E. coli. Ech, fcs, and vdh were amplified and cloned colinear to and downstream of the lacZ promoter of pBluescript SK− as described in Materials and methods. Resulting recombinant strains of E. coli XL1-Blue harboring pSKfcs/echBEK exhibited feruloyl-CoA synthetase activity (Table 2). Recombinant strains harboring pSKvdhBE exhibited vanillin dehydrogenase activity (Table 3). The occurrence of feruloyl-CoA and vanillic acid as the products of the Fcs- and Vdh-catalyzed reactions, respectively, was proven by HPLC-analyses of the incubated enzyme assay reaction mixtures.

Table. 2. Feruloyl-CoA synthetase activities in mutant strains of Pseudomonas putida KT2440 and in recombinant strains of Escherichia coli XL1-Blue. Cells of P. putida KT2440 (wild-type) and mutant strains of P. putida KT2440 were grown to the late-exponential phase at 30 °C in H16-MM containing 0.5% (w/v) sodium gluconate and 0.1% (v/v) ferulic acid. Recombinant strains of E. coli were grown for 12 h at 37 °C in LB in the presence of 1 mM IPTG. Feruloyl-CoA synthetase was assayed as described in Materials and methods. Specific activities are given as units per milligram of protein. Data represent the mean of three determinations
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Table 3. Vanillin dehydrogenase activities in P. putida KT2440, P. putida KT2440 vdhΩKm and in recombinant strains of E. coli XL1-Blue. Cells of P. putida KT2440 (wild-type) and P. putida KT2440vdhΩKm were grown to the late-exponential phase at 30 °C in H16-MM containing 0.5% (w/v) sodium gluconate and 0.1% (v/v) ferulic acid or 0.1% (v/v) vanillin, respectively (as indicated).Recombinant strains of E. coli were grown for 12 h at 37 °C in LB in the presence of 1 mM IPTG. Vanillin dehydrogenase was assayed as described in Materials and methods. Specific activities are given as units per milligram of protein. Data represent the mean of three determinations
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With resting cells of E. coli XL1-Blue harboring plasmid pSKfcs/echBEK, a conversion of 4.3 mM ferulic acid to vanillin was obtained with a maximum rate of about 1.2 mmol per ml per h (data not shown). Beside vanillin, vanillyl alcohol was detected in the medium as a result of vanillin reduction by the E. coli cells. This reduction was also observed in a control experiment in which cells of E. coli XL1-Blue harboring only the vector pBluescript SK- were incubated in MM in the presence of 2 mM vanillin (data not shown).

Extracts of E. coli XL1-Blue harboring pSKvdhBE or only the vector pBluescript SK− were analyzed in polyacrylamide gels together with an extract of ferulic acid-grown P. putida KT2440 cells by specific activity staining for vanillin dehydrogenase. Protein bands of similar migration were detected in extracts of E. coli XL1-Blue (pSKvdhBE) and P. putida KT2440, whereas no band occurred in the extract of E. coli XL1-Blue (pBluescript SK−) (Fig. 3).

Fig. 3.
figure 3

Detection of vanillin dehydrogenase in gluconate-, ferulic acid-, and vanillin-grown cells of different strains of P. putida KT2440, and in recombinant E. coli strains, respectively. Cytoplasmic fractions were separated under non-denaturating conditions in a 7.4% (w/v) polyacrylamide gel as described in Materials and methods, and were stained for vanillin dehydrogenase. Lanes: 1 E. coli XL1-Blue (pBluescript SK−), 2 E. coli XL1-Blue pSKvdhΩKm, 3 P. putida KT2440 (gluconate/vanillin), 4 E. coli XL1-Blue pSKvdhBE, 5 P. putida KT2440 (gluconate/ferulic acid), 6 P. putida KT2440vdhΩKm (gluconate/vanillin), 7 P. putida KT2440vdhΩKm (gluconate/ferulic acid)

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Construction and characterization of ech-, fcs-, and vdh-deficient mutants of P. putida KT2440

To confirm the essential involvement of ech, fcs, and vdh in the catabolism of ferulic acid of P. putida KT2440, each gene was deleted separately and exchanged with an ΩKm-element. The homogenote genotypes of the resulting kanamycin-resistant and tetracycline-sensitive mutant strains KT2440echΩKm, KT2440fcsΩKm, and KT2440vdhΩKm were confirmed by PCR analysis and DNA sequencing. The phenotypes of the aforementioned mutants were investigated on solidified MM with ferulic acid, vanillin, vanillic acid, or sodium gluconate as sole carbon sources. All mutants were unable to grow on ferulic acid, but retained the ability to grow on vanillin or vanillic acid.

For further physiological characterization, cells of the mutant strains KT2440echΩKm, KT2440fcsΩKm, and KT2440vdhΩKm were precultured overnight in MM containing 0.5% (w/v) gluconate as sole carbon source. Cells were harvested, washed twice with MM and used for inoculation of 50 ml MM containing 5.2 mM ferulic acid as sole carbon source. The cultures were incubated for 30 h at 30 °C, and 1-ml samples were taken and analyzed by HPLC with respect to the appearance or consumption of catabolic intermediates. Cells of the wild type were incubated under identical conditions as a control. In wild type cultures, ferulic acid was completely depleted from the medium within 7 h and the occurrence of traces of the intermediates vanillin and vanillic acid was observed. In cultures of the mutant strains KT2440echΩKm, KT2440fcsΩKm, and KT2440vdhΩKm, no decrease of the ferulic acid concentration and no appearance of intermediates were observed within 30 h (data not shown).

The effect of the inactivation of ech, fcs, and vdh by Ω-element insertion in P. putida KT2440 was also investigated by enzyme assays for feruloyl-CoA-synthetase (Fcs) and vanillin dehydrogenase (Vdh) activity. Cells of the mutants and the wild type were grown in MM containing 0.5% (w/v) sodium gluconate plus 0.1% (w/v) ferulic acid, harvested in the late exponential growth phase, and soluble fractions of crude extracts were analyzed for Fcs and Vdh activity. In contrast to the wild type, no Fcs activity was detectable in extracts of the mutants (Table 2). In mutant strain KT2440fcsΩKm, this indicated successful inactivation of the feruloyl-CoA synthetase gene. In mutant strains KT2440echΩKm and KT2440vdhΩKm the lack of Fcs activity might be attributed to a "downstream" effect of the Ω-element insertions in the ech or vdh gene, respectively, both of which are located upstream of the fcs gene (Fig. 2). With respect to the Vdh activities, it was remarkable that extracts of the mutant strain KT2440vdhΩKm still exhibited about 10% of the Vdh activity obtained with extracts of the wild type (Table 3). To confirm the complete inactivation of Vdh in P. putida KT2440vdhΩKm, extracts of this mutant were separated under non-denaturing conditions in a polyacrylamide gel together with an extract of the wild type, and the gel was specifically stained for vanillin dehydrogenase (Fig. 3). In extracts of the mutant, only a faint band was observed, which co-migrated with the Vdh band obtained with the extract of wild type cells grown in the presence of ferulic acid. However, this band was due to an unspecific reaction, since similar bands were observed in all P. putida KT2440-derived extracts in control experiments, omitting the substrate vanillin in the staining solution (data not shown in detail).

Remarkably, the mutant P. putida KT2440vdhΩKm retained the ability to grow on vanillin as sole carbon source despite the lack of a functional vdh gene. To investigate this phenotype, cells of the mutant and the wild type were grown in MM containing 0.5% (w/v) sodium gluconate plus 0.1% (w/v) vanillin, harvested in the late exponential growth phase, and soluble fractions of crude extracts were analyzed for vanillin dehydrogenase activity by enzyme assay and activity staining. The extract derived from the mutant as well as the extract of the wild type showed similar, unexpected low Vdh activity (Table 3) and gave rise to only faint bands of similar migration in the activity staining (Fig. 3). These data indicated that vdh KT2440 was only expressed during growth of P. putida KT2440 in the presence of ferulic acid, but a different enzyme was responsible for vanillin oxidation during growth in the presence of vanillin.

Discussion

Due to their high adaptation capacity and their catabolic potential to metabolize an enormous range of natural organic compounds, pseudomonads play a crucial role in degradation processes of organic matter in nature (Stanier et al. 1966) and are frequently applied to the biotechnological production of valuable chemicals. One of the pseudomonads with current applications in many areas of biotechnology is the nonpathogenic, Gram-negative, biosafety microorganism P. putida KT2440. Sequencing of its genome, and the body of knowledge accumulated in the past on its genetics, physiology and biochemistry render this bacterium as a model organism for functional genomic studies of metabolic versatility.

In the present study, we analyzed the growth behavior of P. putida KT2440 with ferulic acid as an aromatic precursor for biotechnological vanillin production. Ferulic acid is very abundant in nature, since it is the oxidation product of the major aromatic constituent of lignin coniferyl alcohol (Crawford and Crawford 1980). It was found to be an excellent precursor for the production of vanillin that can be used for flavoring purposes (Muheim and Lerch 1999; Achterholt et al. 2000; Priefert et al. 2001). Four major pathways of ferulic acid degradation can be distinguished with respect to the initial reaction: (1) non-oxidative decarboxylation, (2) side-chain reduction, (3) coenzyme-A-independent deacetylation, and (4) coenzyme-A-dependent deacetylation (reviewed in Priefert et al. 2001).

The genome sequence of P. putida KT2440 was compared with known genes of the ferulic acid degradation pathway of Pseudomonas sp. HR199 (Overhage et al. 1999b), and the presence of the genes fcs KT2440, ech KT2440, and vdh KT2440 in the genome of strain KT2440 was revealed. The enzymatic functions of the corresponding gene products from P. putida KT2440 as feruloyl-CoA synthetase, enoyl-CoA hydratase/aldolase, and vanillin dehydrogenase, respectively, were physiologically confirmed by expression of the corresponding genes in E. coli. Resting cells of the recombinant strain of E. coli expressing the genes fcs and ech simultaneously could be used for a biotransformation of ferulic acid to vanillin. However, the efficiency of this biotransformation was limited due to further disproportionation of vanillin, as observed before (Labuda et al. 1994; Overhage et al. 1999). Because of their rather high chemical reactivity, aldehydes like vanillin are often toxic and their accumulation is seldom observed (Lindahl et al. 1992). Actually, vanillin was found to inhibit cell metabolism and thus, most microorganisms either quickly oxidize or reduce the compound for detoxification.

To confirm the assumption that the ferulic acid catabolism in P. putida KT2440 proceeds via the same coenzyme A-dependent, non-β-oxidative pathway as in Pseudomonas sp. HR199, fcs, ech, and vdh were separately inactivated in P. putida KT2440 and the corresponding mutants were characterized physiologically, revealing the essential involvement of the corresponding enzymes in ferulic acid catabolism.

With respect to obtain a promising strain for a biotechnological process for the production of vanillin, investigation of the vdh knock-out mutant P. putida KT2440vdhΩKm was of special interest, since it was expected to accumulate vanillin in the medium during growth on ferulic acid. However, this mutant was unable to grow with ferulic acid as sole carbon source and thus vanillin did not accumulate. Results obtained with this phenotype were in notably surprising contrast to those obtained with Pseudomonas sp. HR199. Although the arrangement of the ferulic acid degradation genes is essentially the same in both pseudomonads (Fig. 2), inactivation of vdh in Pseudomonas sp. HR199 did not influence its ability to degrade ferulic acid (Overhage et al. 1999a, b). However, like the corresponding mutant of Pseudomonas sp. HR199, mutant strain KT2440vdhΩKm with inactivated vdh was still able to grow on vanillin as sole carbon source, just as the wild type strain. Oxidation of vanillin by Pseudomonas sp. HR199vdhΩKm is due to a side activity of the calB-encoded coniferyl aldehyde dehydrogenase (Overhage et al. 1999a), which catalyzes a reaction in the eugenol degradation pathway in Pseudomonas sp. HR199 leading from eugenol to ferulic acid (Priefert et al. 1999; Achterholt et al. 1998). However, this pathway is missing in P. putida KT2440, which is not able to grow with eugenol as sole carbon source (data not shown). Thus, degradation of vanillin by the mutant P. putida KT2440vdhΩKm must be attributed to a different enzyme exhibiting vanillin dehydrogenase activity in this strain. Surprisingly, even the wild type P. putida KT2440 exhibited only very low Vdh activity in extracts of cells grown in the presence of vanillin and did not show the Vdh band after activity staining, characteristic for cells grown in the presence of ferulic acid. Thus, an enzyme different from Vdh seems to be responsible for the oxidation of vanillin during growth in the presence of this substrate and vdh expression occurred only during growth in the presence of ferulic acid.

The present work will be the basis for the rational development of an efficient biocatalyst for the production of vanillin. This might be achieved by introducing a pathway leading from eugenol to ferulic acid in P. putida KT2440vdhΩKm in order to extend the substrate range of KT2440 for the exploitation of this cheaper and more abundant vanillin precursor.