CA2348962A1 - Construction of production strains for producing substituted phenols by specifically inactivating genes of the eugenol and ferulic acid catabolism - Google Patents

Abstract

The invention relates to a transformed and/or mutagenated unicellular or multicellular organism which is characterized in that enzymes of the eugenol and/or ferulic acid catabolism are deactivated in such a manner that the intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and/or vanillinic acid are accumulated.

Description

Constructing production strains for the preparation of substituted phenols by specifically inactivating genes of eu~enol and ferulic acid catabolism The present invention relates to the construction of production strains and to a process for preparing substituted methoxyphenols, in particular vanillin.
DE-A 4 227 076 (process for preparing substituted methoxyphenols, and microorganism which is suitable for this purpose) describes the preparation of substituted methoxyphenols using a novel Pseudomonas sp.. The starting material in this context is eugenol and the products are ferulic acid, vanillic acid, coniferyl alcohol and coniferyl aldehyde.
An extensive review of the biotransformations which were possible using ferulic acid, which was written by Rosazza et al. (Biocatalytic transformation of ferulic acid:
an abundant aromatic natural product; J. Ind. Microbiol. 15:457-471), also appeared in 1995.
The genes and enzymes for synthesizing coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillic and vanillin acid from Pseudomonas sp. were described in EP-A
0 845 532.
The enzymes for converting traps-ferulic acid into traps-feruloyl-SCoA ester and subsequently into vanillin, and also the gene for cleaving the ester, were described by the Institute of Food Research, Norwich, GB, in WO 97/35999. In 1998, the content of the patent also appeared in the form of scientific publications (Gasson et al. 1998.
Metabolism of ferulic acid to vanillin. J. Biol. Chem. 273:4163-4170; Narbad and Gasson 1998. Metabolism of ferulic acid via vanillin using a novel CoA-dependent pathway in a newly isolated strain of Pseudomonas fluorescens. Microbiology 144:1397 - 1405).
if a AJi DE-A 195 32 317 describes the use of Amycolatopsis sp. for obtaining vanillin from ferulic acid fermentatively in high yields.
The known processes suffer from the disadvantage that they either achieve only very low yields of vanillin or make use of relatively expensive starting compounds.
While the last-mentioned process (DE-A 195 32 317) does achieve high yields, the use of Pseudomonas sp. HR199 and Amycolatopsis sp. HR167 for biotransforming eugenol into vanillin requires a fermentation which is carried out in two steps, consequently leading to substantial expense and consumption of time.
to The object of the present invention is therefore to construct organisms which are able to convert the relatively inexpensive raw material eugenol into vanillin in a one-step process.
This object is achieved by means of constructing production strains of unicellular or multicellular organisms, which strains are characterized in that enzymes of eugenol and/or ferulic acid catabolism are inactivated such that the intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid accumulate.
.... 2o The production strain may be unicellular or multicellular.
Accordingly, the invention can relate to microorganisms, plants or animals. Furthermore, use can also be made of extracts which are obtained from the production strain. According to the invention, preference is given to using unicellular organisms. These latter organisms can be microorganisms or animal or plant cells. According to the invention, particular preference is given to using fungi and bacteria. The highest preference is given to bacterial species. Those bacteria which may in particular be used, after their eugenol and/or ferulic acid catabolism has/have been altered, are species of Rhodococcus, Pseudomonas and Escherichia.
In the simplest case, known, conventional microbiological methods can be used for isolating the organisms which may be employed in accordance with the invention.

Thus, the enzymic activity of the proteins involved in eugenol and/or ferulic acid catabolism can be altered by using enzyme inhibitors. Furthermore, the enzymic activity of the proteins involved in eugenol and/or ferulic acid catabolism can be altered by mutating the genes which encode these proteins. Such mutations can be generated in a random manner by means of classical methods, for example by using UV irradiation or mutation-inducing chemicals.
Recombinant DNA methods, such as deletions, insertions and/or nucleotide exchanges, are likewise suitable for isolating the novel organisms. Thus, the genes of 1o the organisms can, for example, be inactivated using other DNA elements (S2 elements). Suitable vectors can likewise be used for replacing the intact genes with gene structures which are altered and/or inactivated. In this context, the genes which are to be inactivated, and the DNA elements which are employed for the inactivation, can be obtained by means of classical cloning techniques or by means of polymerase chain reactions (PCR).
For example, in one possible embodiment of the invention, eugenol catabolism and ferulic acid catabolism can be altered by inserting S2 elements, or introducing deletions, into appropriate genes. In this context, the abovementioned recombinant 2o DNA methods can be used to inactivate the functions of the genes, which encode dehydrogenases, synthetases, hydratase-adolases, thiolases or demethylases, such that production of the relevant enzymes is blocked. Preferably, the genes are those which encode coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, feruloyl-CoA synthetases, enoyl-CoA hydratase-aldolases, beta-ketothiolases, vanillin dehdrogenases or vanillic acid demethylases. Very particular preference is given to genes which encode the amino acid sequences specified in EP-A 0845532 and/or nucleotide sequences which encode their allelic variations.
The invention accordingly also relates to gene structures for preparing transformed organisms and mutants.

Preference is given to employing gene structures in which the nucleotide sequences encoding dehydrogenases, synthetases, hydratase-aldolases, thiolases or demethylases are inactivated for isolating the organisms and mutants. Particular preference is given to gene structures in which the nucleotide sequences encoding coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, feruloyl-CoA synthetases, enoyl-CoA hydratase-aldolases, beta-ketothiolases, vanillin dehydrogenases or vanillic acid demethylases are inactivated. Very particular preference is given to gene structures which exhibit the structures given in Figures la to lr having the nucleotide sequences which are depicted in Figures 2a to 2r and/or nucleotide sequences to encoding their allelic variations. In this context, particular preference is given to nucleotide sequences 1 to 18.
The invention also encompasses the part sequences of these gene structures as well as functional equivalents. Functional equivalents are to be understood as meaning those derivatives of the DNA in which individual nucleobases have been exchanged (wobble exchanges) without the function being altered. Amino acids may also be exchanged at the protein level without this resulting in an alteration in function.
One or more DNA sequences can be inserted upstream and/or downstream of the 2o gene structures. By cloning the gene structures, it is possible to obtain plasmids or vectors which are suitable for the transformation and/or transfection of an organism and/or for conjugative transfer into an organism.
The invention furthermore relates to plasmids and/or vectors for preparing the organisms and mutants which are transformed in accordance with the invention.
These organisms and mutants consequently harbour the gene structures which have been described. The present invention accordingly also relates to organisms which harbour the said plasmids and/or vectors.
The nature of the plasmids and/or vectors depends on what they are being used for. In order, for example, to be able to replace the intact genes of eugenol and/or ferulic acid catabolism in pseudomonads with the genes which have been inactivated with omega elements, there is a need for vectors which, on the one hand, can be transferred into pseudomonads (conjugatively transferable plasmids) but which, on the other hand, cannot be replicated in these organisms and are consequently unstable in pseudomonads (so-called suicide plasmids). DNA segments which are transferred into pseudomonads with the aid of such a plasmid system can only be retained if they become integrated by homologous recombination into the genome of the bacterial cell.
1o The described gene structures, vectors and plasmids may be used for preparing different transformed organisms or mutants. The said gene structures can be used for replacing intact nucleic acid sequences with altered and/or inactivated gene structures. In the cells, which can be obtained by transformation or transfection or conjugation, the intact gene is replaced, by homologous recombination, with the altered and/or inactivated gene structure, as a consequence of which the resulting cells now only possess the altered and/or inactivated gene structure in their genome.
In this way, preferably genes can be altered and/or inactivated, in accordance with the invention, such that the relevant organisms are able to produce coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid.
.. 20 Mutants of the strain Pseudomonas sp. HR199 (DSM 7063), which was described in detail in DE-A 4 227 076 and EP-A 0845532, are examples of production strains which have been constructed in this way in accordance with the invention, with the corresponding gene structures ensuing, inter alia, from Figures la to lr, in combination with Figures 2a to 2r:
1. Pseudomonas sp. HR199calAS2Km, which contains the S2Km-inactivated calA gene in place of the intact calA gene encoding coniferyl alcohol dehydrogenase (Fig. la; Fig. 2a).

2. Pseudomonas sp. HR199calASZGm, which contains the 52Gm-inactivated calA gene in place of the intact calA gene encoding coniferyl alcohol dehydrogenase (Fig. lb; Fig. 2b).
3. Pseudomonas sp. HR199ca1A0, which contains the deletion-inactivated calA
gene in place of the intact calA gene encoding coniferyl alcohol dehydrogenase (Fig. lc; Fig. 2c).

4. Pseudomonas sp. HR199calBSZKm, which contains the S2Km-inactivated calB gene in place of the intact calB gene encoding coniferyl aldehyde ", dehydrogenase (Fig. ld; Fig. 2d) 5. Pseudomonas sp. HR199calBS2Gm, which contains the S2Gm-inactivated calB gene in place of the intact calB gene encoding coniferyl aldehyde dehydrogenase (Fig. le; Fig. 2e).

6. Pseudomonas sp. HR199ca1B0, which contains the deletion-inactivated calB

gene in place of the intact calB gene encoding coniferyl aldehyde dehydrogenase (Fig.lf; Fig. 2f).

7. Pseudomonas sp. HR199fcsS2Km, which contains the S2Km-inactivated fcs gene in place of the intact fcs gene encoding feruloyl-CoA
synthetase (Fig.lg;

Fig. 2g).

8. Pseudomonas sp. HR199fcsS2Gm, which contains the S2Gm-inactivated fcs gene in place of the intact fcs gene encoding feruloyl-CoA
synthetase (Fig.lh;

Fig. 2h).

9. Pseudomonas sp. HR199fcs0, which contains the deletion-inactivated fcs gene in place of the intact fcs gene encoding feruloyl-CoA synthetase (Fig.li;
Fig. 2i).

10. Pseudomonas sp. HR199echS2Km, which contains the S2Km-inactivated ech gene in place of the intact ech gene encoding enoyl-CoA hydratase-aldolase (Fig.lj; Fig. 2j).

11. Pseudomonas sp. HR199echS2Gm, which contains the S2Gm-inactivated ech gene in place of the intact ech gene encoding enoyl-CoA hydratase-aldolase 3o (Fig.lk; Fig. 2k).

7 _ 12. Pseudomonas sp. HR199ech0, which contains the deletion-inactivated ech gene in place of the intact ech gene encoding enoyl-CoA hydratase-aldolase (Fig.ll; Fig. 21).

13. Pseudomonas sp. HR199aat52Km, which contains the S2Km-inactivated aat gene in place of the intact aat gene ecnoding beta-ketothiolase (Fig. lm;
Fig. 2m).

14. Pseudomonas sp. HR199aatSZGm, which contains the S2Gm-inactivated aat gene in place of the intact aat gene encoding beta-ketothiolase (Fig.ln;
..~. Fig. 2n).
l0 15. Pseudomonas sp. HR199aat0, which contains the deletion-inactivated aat gene in place of the intact aat gene encoding beta-ketothiolase (Fig.lo; 20).
16. Pseudomonas sp. HR199vdhSZKm, which contains the S2Km-inactivated vdh gene in place of the intact vdh gene encoding vanillin dehydrogenase (Fig.lp;
Fig. 2p).
17. Pseudomonas sp. HR199vdhS2Gm, which contains the SZGm-inactivated vdh gene in place of the intact vdh gene encoding vanillin dehydrogenase (Fig.lq;
Fig. 2q).
18. Pseudomonas sp. HR199vdh0, which contains the deletion-inactivated vdh gene in place of the intact vdh gene encoding vanillin dehydrogenase (Fig.lr;
Fig. 2r).
19. Pseudomonas sp. HR199vdhBS2Km, which contains the S2Km-inactivated vdhB gene in place of the intact vdhB gene encoding vanillin dehydrogenase II.
20. Pseudomonas sp. HR199vdhBS2,Gm, which contains the S2Gm-inactivated vdhB gene in place of the intact vdhB gene encoding vanillin dehydrogenase II.
21. Pseudomonas sp. HR199vdhB~, which contains the deletion-inactivated vdhB
gene in place of the intact vdhB gene encoding vanillin dehydrogenase II.
22. Pseudomonas sp. HR199adhS2Km, which contains the S2Km-inactivated adh 3o gene in place of the intact adh gene encoding alcohol dehydrogenase.

_ g _ 23. Pseudomonas sp. HR199adh52Gm, which contains the S2Gm-inactivated adh gene in place of the intact adh gene encoding alcohol dehydrogenase.
24. Pseudomonas sp. HR199adh0 which contains the deletion-inactivated adh gene in place of the intact adh gene encoding alcohol dehydrogenase.
25. Pseudomonas sp. HR199vanAS2Km, which contains the SZKm-inactivated vanA gene in place of the intact vanA gene encoding the a-subunit of vanillic acid demethylase.
26. Pseudomonas sp. HR 199vanA52Gm, which contains the S2Gm-inactivated ~.. vanA gene in place of the intact vanA gene encoding the a-subunit of vanillic 1o acid demethylase.
27. Pseudomonas sp. HR 199vanA~, which contains the deletion-inactivated vanA
gene in place of the intact vanA gene encoding the a-subunit of vanillic acid demethylase.
28. Pseudomonas sp. HR199vanBSZKm, which contains the S2Km-inactivated vanB gene in place of the intact vanB gene encoding the (3-subunit of vanillic acid demethylase.
29. Pseudomonas sp. HR199vanBS2Gm, which contains the S2Gm-inactivated vanB gene in place of the intact vanB gene encoding the (3-subunit of vanillic acid demethylase.
30. Pseudomonas sp. HR199vanB0, which contains the deletion-inactivated vanB
gene in place of the intact vanB gene encoding the (3-subunit of vanillic acid demethylase.
The invention additionally relates to a process for the biotechnological preparation of organic compounds. In particular, this process can be used to prepare alcohols, aldehydes and organic acids. The latter are preferably coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and vanillic acid.
The above-described organisms are employed in the novel process. The organisms 3o which are very particularly preferred include bacteria, in particular the Pseudomonas species. Specifically, the abovementioned Pseudomonas species can preferably be employed for the following processes:
1. Pseudomonas sp. HR199calAS2Km, Pseudomonas sp. HR199calAS2Gm and Pseudomonas sp. HR199ca1A0 for preparing coniferyl alcohol from eugenol.
2. Pseudomonas sp. HR 199calBSZKm, Pseudomonas sp. HR 199ca1B52Gm and Pseudomonas sp. HR199ca1B0 for preparing coniferyl aldehyde from eugenol m~~ or coniferyl alcohol.
to 3. Pseudomonas sp. HR199fcsSZKm, Pseudomonas sp. HR199fcs52Gm, Pseu-domonas sp. HR 199fcs0, Pseudomonas sp. HR 199echS2Km, Pseudomonas sp. HR199echSZGm and Pseudomonas sp. HR199ech~ for preparing ferulic acid from eugenol or coniferyl alcohol or coniferyl aldehyde.
IS
4. Pseudomonas sp. HR 199vdhSZKm, Pseudomonas sp. HR 199vdhSZGm, Pseu-domonas sp. HR 199vdhd, Pseudomonas sp. HR 199vdhS2GmvdhBS2Km, Pseudomonas sp. HR 199vdhS2KmvdhBS2Gm, Pseudomonas sp. HR 199vdh0 vdhBS2Gm and Pseudomonas sp. HR199vdhwdhBS2Km for preparing 2o vanillin from eugenol or coniferyl alcohol or coniferyl aldehyde or ferulic acid.
5. Pseudomonas sp. HR199vanAS2,Km, Pseudomonas sp. HR199vanAS2Gm, Pseudomonas sp. HR 199vanA~, Pseudomonas sp. HR 199vanBS2Km, 25 Pseudomonas sp. HR199vanBS2Gm and Pseudomonas sp. HR199vanB0 for preparing vanillic acid from eugenol or coniferyl alcohol or coniferyl aldehyde or ferulic acid or vanillin.
Eugenol is the preferred substrate. However, it is also possible to add further 3o substrates or even to replace the eugenol with another substrate.

Suitable nutrient media for the organisms which are employed in accordance with the invention are synthetic, semisynthetic or complex culture media. These media may comprise carbon-containing and nitrogen-containing compounds, inorganic salts, where appropriate trace elements, and vitamins.
Carbon-containing compounds which may be suitable are carbohydrates, hydrocarbons or organic standard chemicals. Examples of compounds which may preferably be used are sugars, alcohols or sugar alcohols, organic acids or complex mixtures.
to The sugar is preferably glucose. The organic acids which may preferably be employed are citric or acetic acid. Examples of the complex mixtures are malt extract, yeast extract, casein or casein hydrolysate.
Inorganic compounds are suitable nitrogen-containing substrates. Examples of these are nitrates and ammonium salts. Organic nitrogen sources can also be used.
These sources include yeast extract, Soya bean meal, casein, casein hydrolysate and corn steep liquor.
2o Examples of the inorganic salts which may be employed are sulphates, nitrates, chlorides, carbonates and phosphates. The metals which the said salts contain are preferably sodium, potassium, magnesium, manganese, calcium, zinc and iron.
The temperature for the culture is preferably in the range from 5 to 100°C. The range from 15 to 60°C is particularly preferred, with 22 to 37°C being most preferred.
The pH of the medium is preferably 2 to 12. The range from 4 to 8 is particularly preferred.
3o In principle, any bioreactor known to the skilled person can be employed for carrying out the novel process. Preferential consideration is given to any appliance which is suitable for submerged processes. This means that vessels which do or do not possess a mechanical mixing device may be employed in accordance with the invention.
Examples of the latter are shaking apparatuses, and bubble column reactors or loop reactors. The former preferably include all the known appliances which are fitted with stirrers of any design.
The novel process can be carned out continuously or batchwise. The fermentation time required for achieving a maximum quantity of product depends on the specific nature of the organism employed. However, in principle, the fermentation times are between 2 and 200 hours.
The invention is explained in more detail below while refernng to examples:
Mutants of the eugenol-utilizing strain Pseudomonas sp. HR199 (DSM 7063) were generated in a targeted manner by specifically inactivating genes of eugenol catabolism by means of inserting omega elements or introducing deletions. The omega elements employed were DNA segments which encoded resistances to the antibiotics kanamycin (S2Km) and gentamycin (S2Gm). These resistance genes were isolated from Tn5 and the plasmid pBBRIMCS-5 using standard methods. The genes . 2o calA, calB, fcs, ech, aat, vdh, adh, vdhB, vanA and vanB, which encode coniferyl alcohol dehydrogenase, coniferyl aldehyde dehydrogenase, feruloyl-CoA
synthetase, enoyl-CoA hydratase-aldolase, beta-ketothiolase, vanillin dehdrogenase, alcohol dehydrogenase, vanillin dehdrogenase II and vanillic acid demethylase, were isolated from genomic DNA of the strain Pseudomonas sp. HR199 using standard methods and cloned into pBluescript SK . By means of digesting with suitable restriction endonucleases, DNA segments were removed from these genes (deletion) or substituted with S2 elements (insertion), resulting in the respective gene being inactivated. The genes which had been mutated in this manner were recloned into conjugatively transferable vectors and subsequently introduced into the strain 3o Pseudomonas sp. HR199. Suitable selection was used to obtain transconjugants which had replaced the respective functional wild-type gene with the newly introduced inactivated gene. The insertion and deletion mutants which were obtained in this way now only possessed the respective inactivated gene. This procedure was used to obtain both mutants possessing only one defective gene and multiple mutants, in which several genes had been inactivated in this manner. These mutants were employed for biotransforming a) eugenol into coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid;
b) coniferyl alcohol into coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid;
c) coniferyl aldehyde into ferulic acid, vanillin and/or vanillic acid;
to d) ferulic acid into vanillin and/or vanillic acid, and e) vanillin into vanillic acid.

Materials and Methods Conditions for growing the bacteria.
Strains of Escherichia coli were propagated at 37°C in Luria-Bertani (LB) or M9 mineral medium (J. Sambrook, E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd Edition., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Strains of Pseudomonas sp. were propagated at 30°C in Nutrient Broth (NB, 0.8%, wt/vol) or in mineral medium (MM) (H.
G.
Schlegel, et al. 1961. Arch. Mikrobiol. 38:209-222) or HR mineral medium (HR-1o MM) (J. Rabenhorst, 1996. Appl. Microbiol. Biotechnol. 46:470-474.).
Ferulic acid, vanillin, vanillic acid and protocatechuic acid were dissolved in dimethyl sulphoxide and added to the respective medium to give a final concentration of 0.1%
(wt/vol).
Eugenol was either added directly to the medium to give a final concentration of 0.1% (vol/vol) or applied to filter paper (circular filter 595, Schleicher &
Schuell, Dassel, Germany) in the lids of MM agar plates. When transconjugants and mutants of Pseudomonas sp. were being propagated, tetracycline, kanamycin and gentamycin were employed in final concentrations of 25 pg/ml, 100 p.~ml and 7.5 p.g/ml, respectively.
2o Qualitative and quantitative detection of metabolic intermediates in culture supernatants.
Culture supernatants were analysed by high pressure liquid chromatography (Knauer HPLC) either directly or after dilution with doubly distilled H20. The chromatography was carried out on Nucleosil 100 C18 (7 Vim, 250 x 4 mm). 0.1%
(vol/vol) formic acid and acetonitrile was used as the solvent. The course of the gradient employed for eluting the substances was as follows:
00:00 - 06:30 -~ 26% acetonitrile 06:30 - 08:00 -~ 100% acetonitrile 08:00 - 12:00 -~ 100% acetonitrile 12:00 - 13:00 -~ 26% acetonitrile 13:00 - 18:00 -~ 26% acetonitrile Purification of vanillin dehydrogenase II.
The purification was carried out at 4°C.
Crude extract.
Pseudomonas sp. HR199 cells which had been propagated on eugenol were washed in 10 mM sodium phosphate buffer, pH 6.0, then resuspended in the same buffer and disrupted by being passed twice through a French press (Amicon, Silver Spring, Maryland, USA) at a pressure of 1000 psi. The cell homogenate was subjected to an 1o ultracentrifugation (1 h, 100,000 x g, 4°C), resulting in the soluble fraction of crude extract being obtained as the supernatant.
Anion exchange chromatography on DEAE Sephacel.
The soluble fraction of the crude extract was dialysed overnight against 10 mM
sodium phosphate buffer, pH 6Ø The dialysate was loaded onto a DEAE-Sephacel column (2.6 cm x 35 cm, bed volume[BV]: 186 ml) which had been equilibrated with 10 mM sodium phosphate buffer, pH 6.0, and which had a flow rate of 0.8 ml/min. The column was rinsed with two BV of 10 mM sodium phosphate buffer, pH 6Ø The vanillin dehydrogenase II (VDH II) was eluted with a linear salt 2o gradient of from 0 to 400 mM NaCI in 10 mM sodium phosphate buffer, pH 6.0 (750 ml). 10 ml fractions were collected. Fractions having a high VDH II activity were combined to form the DEAE pool.
Determining the vanillin dehydrogenase activity.
The VDH activity was determined at 30°C using an optical enzymic test. The reaction mixture, whose volume was 1 ml, contained 0.1 mmol of potassium phosphate (pH 7.1), 0.125 ~.mol of vanillin, 0.5 p.mol of NAD, 1.2 ~Cmol of pyruvate (Na salt), lactate dehydrogenase (1 U; from pig heart) and enzyme solution.
The oxidation of vanillin was monitored at ~, = 340 nm (~~anil~in = 11.6 cm2/~mol). The 3o enzyme activity was given in units (U), with 1 U corresponding to the quantity of enzyme which converts 1 p.mol of vanillin per minute. The protein concentrations in the samples were determined using the method of Lowry et al. (O. H. Lowry, N.
J.
Rosebrough, A. L. Farr and R. J. Randall. 1951. J. Biol. Chem. 193:265-275).
Determining the coniferyl alcohol dehydrogenase activity.
The CADH activity was determined at 30°C using an optical enzymic test in accordance with Jaeger et al. (E. L. Jaeger, Eggeling and H. Sahm. 1981.
Current Microbiology. 6:333-336). The reaction mixture, whose volume was 1 ml, contained 0.2 mmol of tris/HCl (pH 9.0), 0.4 ~mol of coniferyl alcohol, 2 ~Cmol of NAD, 0.1 mmol of semicarbazide and enzyme solution. The reduction of NAD was to monitored at ~. = 340 nm (~ = 6.3 cm2/~mol). The enzyme activity was given units (U), with 1 U corresponding to the quantity of enzyme which converts 1 ~,mol of substrate per minute. The protein concentrations in the samples were determined by the method of Lowry et al. (O. H. Lowry, N. J. Rosebrough, A. L. Farr and R.
J. Randall. 1951. J. Biol. Chem. 193:265-275).
Determining the coniferyl aldehyde dehydrogenase activity.
The CALDH activity was determined at 30°C using an optical enzymic test. The reaction mixture, whose volume was 1 ml, contained 0.1 mmol of tris/HCl (pH
8.8), 0.08 ~mol of coniferyl aldehyde, 2.7 ~,mol of NAD and enzyme solution. The oxidation of coniferyl aldehyde to ferulic acid was monitored at ~. = 400 nm (E =
34 cm2/p,mol). The enzymic activity was given in units (U) with 1 U
corresponding to the quantity of enzyme which converts 1 ~mol of substrate per minute. The protein concentrations in the samples were determined by the method of Lowry et al.
(O. H.
Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. J. Biol. Chem.
193:265-275).
Determining the feruloyl-CoA synthetase (ferulic acid thiokinase) activity.
The FCS activity was determined at 30°C using an optical enzymic test which was a modification of that of Zenk et al. (Zenk et al. 1980. Anal. Biochem. 101:182-187).
3o The reaction mixture, whose volume was 1 ml, contained 0.09 mmol of potassium phosphate (pH 7.0), 2.1 ~,mol of MgCl2, 0.7 ~mol of ferulic acid, 2 ~mol of ATP, 0.4 ~mol of coenzyme A and enzyme solution. The formation of the CoA ester from ferulic acid was monitored at ~, = 345 nm (~ = 10 cm2/~Cmol). The enzymic activity was given in units (U), with 1 U corresponding to the quantity of enzyme which converts 1 ~.mol of substrate per minute. The protein concentrations in the samples were determined using the method of Lowry et al. (O. H. Lowry, N. J.
Rosebrough, A. L. Farr and R. J. Randall. 1951. J. Biol. Chem. 193:265-275).
Electrophoretic methods.
Protein-containing extracts were fractionated under native conditions in 7.4%
(wt/vol) polyacrylamide gels using the method of Stegemann et al. (Stegemann et al.
1973. Z. Naturforsch. 28c:722-732) and under denaturing conditions in 11.5°Io (wt/vol) polyacrylamide gels using the method of Laemmli (Laemmli, U. K. 1970.
Nature (London) 227:680-685). Serva Blue R was used for non-specific protein staining. For specifically staining the coniferyl alcohol dehydrogenase, coniferyl aldehyde dehydrogenase and vanillin dehydrogenase, the gels were rebuffered for min in 100 mM KP buffer (pH 7.0) and subequently incubated at 30°C in the same buffer to which 0.08% (wtlvol) NAD, 0.04% (wtlvol) p-nitro blue tetrazolium chloride, 0.003% (wt/vol) phenazine methosulphate and 1 mM of the respective substrate had been added until corresponding colour bands became visible.

Transfer of proteins from polyacrylamide gels to PVDF membranes.
Proteins were transferred from SDS-polyacrylamide gels to PVDF membranes (Waters-Millipore, Bedford, Mass., USA) using a Semidry Fastblot appliance (B32/33, Biometra, Gottingen, Germany) in accordance with the manufacturer's instructions.
Determining N-terminal amino acid sequences.
N-terminal amino acid sequences were determined using a Protein Peptide Sequencer (Type 477 A, Applied Biosystems, Foster City, USA) and a PTH analyser in 3o accordance with the manufacturer's instructions.

Isolating and manipulating DNA
Genomic DNA was isolated using the method of Marmur (J. Marmur, 1961. J. Mol.
Biol. 3:208-218). Other plasmid DNA and/or DNA restriction fragments was/were isolated and analysed using standard methods (J. E. Sambrook, F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd Edition., Cold Spring Harbor Laboratory Press, Cold Spring Habor, New York).
Transferring DNA.
Competent Escherichia coli cells were prepared and transformed using the method of 1o Hanahan (D. Hanahan, 1983. J. Mol. Biol. 166:557-580). Conjugative plasmid transfer between plasmid-harbouring Escherichia coli S 17-1 strains (donor) and Pseudomonas sp.strains (recipient) was performed on NB agar plates in accordance with the method of Friedrich et al. (B. Friedrich et al. 1981. J. Bacteriol.
147:198-205), or by means of a "minicomplementation method" on MM agar plates containing 0.5% (wt/vol) gluconate as the C source and 25 p,g of tetracycline/ml or 100 ~.g of kanamycin/ml. In this case, cells of the recipient were applied in one direction as an inoculation streak. After 5 min, cells of the donor strains were then applied as inoculation streaks, with these streaks crossing the recipient inoculation streak. After incubating at 30°C for 48 h, the transconjugants grew directly 2o downstream of the crossing site whereas neither the donor strain nor the recipient strain was able to grow.
Hybridization experiments.
DNA restriction fragments were fractionated electrophoretically in a 0.8%
(wt/vol) agarose gel in 50 mM tris- 50 mM boric acid- 1.25 mM EDTA buffer (pH 8.5) (J.
E.
Sambrook, F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual.
2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.).
The transfer of the denatured DNA out of the gel onto a positively charged nylon membrane (pore size: 0.45 ~,m, Pall Filtrationstechnik, Dreieich, Germany), the 3o subsequent hybridization with biotinylated or digoxigenin-labelled DNA
probes, and the preparation of these DNA probes, were all performed using standard methods (J. E. Sambrook, F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
DNA sequencing.
Nucleotide sequences were determined "non-radioactively" in accordance with the Sanger et al. (Sanger et al. 1977. Proc. Natl. Acad. Sci. USA 74:5463-5467) dideoxy chain termination method using a "LI-COR" DNA Sequencer Model 4000L"
~y (LI-COR Inc., Biotechnology Division, Lincoln, NE, USA) and using a "thermo 1o sequenase fluorescent labelled primer cycle sequencing kit with 7-deaza-dGTP"
(Amersham Life Science, Amersham International plc., Little Chalfont, Buckinghamshire, England), in each case in accordance with the manufacturer's instructions.
Synthetic oligonucleotides were used to carry out sequencing in accordance with the "primer-hopping strategy" of Strauss et al. (E. C. Strauss et al. 1986. Anal.
Biochem.
154:353-360).
Chemicals, biochemicals and enzymes.
2o Restriction enzymes, T4 DNA ligase, lambda DNA and enzymes and substrates for the optical enzymic tests were obtained from C.F. Boehringer & Sohne (Mannheim, Germany) or from GIBCO/BRL (Eggenstein, Germany). [y-32P]ATP was from Amersham/Buchler (Braunschweig, Germany). Oligonucleotides were obtained from MWG-Biotech GmbH (Ebersberg, Germany). Type NA agarose was obtained from Pharmacia-LKB (Uppsala, Sweden). All other chemicals were from Haarmann &
Reimer (Holzminden, Germany), E. Merck AG (Darmstadt, Germany), Fluka Chemie (Bucks, Switzerland), Serva Feinbiochemica (Heidelberg, Germany) or Sigma Chemie (Deisenhofen, Germany).

Examples Example 1 Constructing omega elements which mediate resistances to kanamycin (S2 Km) or gentamycin (S2Gm).
For constructing the S2,Km element, the 2099 by BgII fragment of Transposons Tn5 (E. A. Auerswald, G. Ludwig and H. Schaller. 1981. Cold Spring Harb. Symp.
Quant. Biol. 45:107-113; E. Beck, G. Ludwig, E. A. Auerswald, B. Reiss and H.
Schaller. 1982. Genes 19:327-336; P. Mazodier, P. Cossart, E. Giraud and F.
Gasser.
1985. Nucleic Acids Res. 13:195-205) was isolated on a preparative scale. The fragment was shortened down to approx. 990 by by treating it with Bal 31 nuclease.
This fragment, which now only comprised the kanamycin resistance gene (encoding an aminoglycoside-3'-O-phosphotransferase), was then ligated to SmaI-cut pSKsym DNA (pBluescript SK derivative which contains a symmetrically constructed multiple cloning site [SaII, HindI>Z, EcoRI, SmaI, EcoRI, HindIlI, SaII]). It was possible to reisolate the S2Km element from the resulting plasmid as a SmaI
fragment, an EcoRI fragment, a HindIlZ fragment or a SaII fragment.
2o For constructing the S2Gm element, the 983 by EaeI fragment of the plasmid pBRIMCS-5 (M. E. Kovach, P. H. Elzer, D. S. Hill, G. T. Robertson, M. A.
Farris, R. M. Roop and K. M. Peterson. 1995. Genes 166:175-176) was isolated on a preparative scale and then treated with mung bean nuclease (progressive digestion of single-stranded DNA molecule ends). This fragment, which now only comprised the gentamycin resistance gene (encoding a gentamycin-3-acetyltransferase), was then ligated to SmaI-cleaved pSKsym DNA (see above). It was possible to reisolate the 52Gm element from the resulting plasmid as a SmaI fragment, an EcoRI fragment, a HindIll fragment or a SaII fragment.

Example 2 Cloning the genes from Pseudomonas sp. HR199 (DSM7063) which were to be inactivated by inserting S2 elements or by means of deletions.
The fcs, ech, vdh and aat genes were cloned separately proceeding from the E.
coli S 17-1 strains DSM 10439 and DSM 10440 and using the plasmids pE207 and pE5-1 (see EP-A 0845532). The given fragments were isolated on a preparative scale from these plasmids and treated as described below:
to For cloning the fcs gene, the 2350 by SalIlEcoRI fragment from plasmid pE207 and the 3700 by EcoRIlSaII fragment from plasmid pE5-1 were cloned together in pBluescript SK such that the two fragments were joined together by way of the EcoRI ends. The 6050 by SuII fragment was isolated on a preparative scale from the resulting hybrid plasmid and shortened down to approx. 2480 by by being treated with Bal 31 nuclease. PstI linkers were subsequently ligated to the ends of the fragment and, after digestion with PstI, the fragment was cloned into pBluescript SK
(pSKfcs). After transformation of E. coli XL1 blue, clones were obtained which expressed the fcs gene and exhibited an FCS activity of 0.2 U/mg of protein.
2o For cloning the ech gene, the 3800 by Hind>ZIlEcoRI fragment from plasmid pE207 was isolated on a preparative scale and shortened down to approx. 1470 by by treating it with Bal 31 nuclease. EcoRI linkers were then ligated to the ends of the fragment and, after digestion with EcoRI, the fragment was cloned into pBluescript SK (pSKech).
For cloning the vdh gene, the 2350 by SalIlEcoRI fragment from plasmid pE207 was isolated on a preparative scale. After cloning into pBluescript SK , the fragment was truncated at one end by approx. 1530 by using an exonuclease III/mung bean nuclease system. An EcoRI linker was then ligated to the end of the fragment and, 3o after digestion with EcoRI, the fragment was cloned into pBluescript SK
(pSKvdh).

Following transformation of E. coli XL1 blue, clones were obtained which expressed the VDH gene and exhibited a VDH activity of 0.01 U/mg of protein.
For cloning the aat gene, the 3700 by EcoRIlSaII fragment from plasmid pE5-1 was isolated on a preparative scale and shortened down to approx. 1590 by by treating it with Bal 31 nuclease. EcoRI linkers were then ligated to the ends of the fragment and, after digestion with EcoRI, the fragment was cloned into pBluescript SK
(pSKaat).
1o Example 3 Inactivating the above-described genes by inserting S2 elements or by deleting constituent regions of these genes.
Plasmid pSKfcs, which contained the fcs gene, was digested with BssHII, resulting in ~5 a 1290 by fragment being excised from the fcs gene. Following religation, the deletion derivative of the fcs gene (fcs0) (see Figs. li and 2i) was obtained in cloned form in pBluescript SK (pSKfcsO). In addition, after the fragment had been excised, the omega elements S2Km and S2Gm were ligated in in its stead. This resulted in the S2-inactivated derivatives of the fcs gene (fcsS2Km, see Figs. lg and 2g) and 20 (fcsS2Gm, see Fig. lh and 2h) being obtained in cloned form in pBluescript SK
(pSKfcsS2Km and pSKfcsS2Gm). It was not possible to detect any FCS activity in crude extracts of the resulting E. coli clones, whose hybrid plasmids possessed an fcs gene which was inactivated by deletion or by SZ element insertion.
25 Plasmid pSKech, which contained the ech gene, was digested with NruI, resulting in a 53 by fragment and a 430 by fragment being excised from the ech gene. After religation, the deletion derivative of the ech gene (echo, see Fig. 11 and 21) was obtained in cloned form in pBluescript SK (pSKechO). In addition, after the fragments had been excised, the omega elements S2Km and S2Gm were ligated in in 3o their stead. This resulted in the SZ-inactivated derivatives of the ech gene (echS2Km and echS2Gm) being obtained in cloned form in pBluescript SK (pSKechSZKm and pSKechS2Gm).
Plasmid pSKvdh, which contained the vdh gene, was digested with BssHII, resulting in a 210 by fragment being excised from the vdh gene. After religation, the deletion derivative of the vdh gene (vdh0, see Figs. to and 20) was obtained in cloned form in pBluescript SK (pSKvdhO). In addition, after the fragment had been excised, the omega elements S2Km and S2Gm were ligated in in its stead. This resulted in the S2-inactivated derivatives of the vdh gene (vdhS2,Km and vdhSZGm) being obtained in to cloned form in pBluescript SK (pSKvdh52Km, see Figs. lm and 2m) and (pSKvdhS2Gm, see Figs. In and 2n). It was not possible to detect any VDH
activity in crude extracts of the resulting E. coli clones, whose hybrid plasmids possessed a vdh gene which was inactivated by deletion or by SZ element insertion.
Plasmid pSKaat, which contained the aat gene, was digested with BssHII, resulting in a 59 by fragment being excised from the aat gene. After religation, the deletion derivative of the aat gene (aat0, see Figs. lr and 2r) was obtained in cloned form in pBluescript SK (pSKaatO). In addition, after the fragment had been excised, the omega elements SZKm and S2Gm were ligated in in its stead. This resulted in the SZ-inactivated derivatives of the aat gene (aatS2Km, see Figs. lp and 2p) and (aatS2Gm, see Figs. lq and 2q) being obtained in cloned form in pBluescript SK
(pSKaatS2Km and pSKaat52Gm).

Example 4 Subcloning the S2 element-inactivated genes into the conjugatively transferable "suicide plasmid" pSUP202.
In order to be able to replace the intact genes in Pseudomonas sp. HR199 with the S2-element inactivated genes, there is a need for a vector which can, on the one hand, be transferred into pseudomonads (conjugatively transferable plasmids) but which, on the other hand, cannot replicate in these bacteria and is consequently unstable in ~, pseudomonads ("suicide plasmid"). DNA segments which are transferred into 1o pseudomonads using such a plasmid system can only be retained if they are integrated by means of homologous recombination (RecA-dependent recombination) into the genome of the bacterial cell. In the present case, the "suicide plasmid"
pSUP202 (Simon et al. 1983. In: A. Piihler. Molecular genetics of the bacteria-plant interaction. Springer Verlag, Berlin, Heidelberg, New York, pp. 98-106) was used.
Following digestion with PstI, the inactivated genes fcsS2Km and fcsS2Gm were isolated from plasmids pSKfcs52,Km and pSKfcsS2Gm and ligated to PstI-cleaved pSUP202 DNA. The ligation mixtures were transformed into E. coli S 17-1.
Selection took place on tetracycline-containing LB medium which also contained kanamycin or "- 20 gentamycin, respectively. Kanamycin-resistant transformants whose hybrid plasmid (pSUPfcsS2Km) contained the inactivated gene fcsS2Km were obtained. The corresponding hybrid plasmid (pSUPfcsSZGm) of the gentamycin-resistant transformants contained the inactivated gene fcsS2Gm.
Following EcoRI digestion, the inactivated genes echS2Km and echS2Gm were isolated from plasmids pSKechSZKm and pSKechS2Gm and ligated to EcoRI-cleaved pSUP202 DNA. The ligation mixtures were transformed into E. coli S17-1.
Selection took place on tetracycline-containing LB medium which also contained kanamycin or gentamycin, respectively. Kanamycin-resistant transformants whose hybrid plasmid (pSUPechS2Km) contained the inactivated gene echS2Km were obtained. The corresponding hybrid plasmid (pSUPechS2Gm) of the gentamycin-resistant transformants contained the inactivated gene echS2Gm.
Following EcoRI digestion, the inactivated genes vdhS2Km and vdhSZGm were isolated from plasmids pSKvdhS2Km and pSKvdhS2Gm and ligated to EcoRI-cleaved pSUP202 DNA. The ligation mixtures were transformed into E. coli S17-1.
Selection took place on tetracycline-containing LB medium which also contained kanamycin or gentamycin, respectively. Kanamycin-resistant transformants whose hybrid plasmid (pSUPvdhS2Km) contained the inactivated gene vdhS2,Km were obtained. The 1o corresponding hybrid plasmid (pSUPvdhS2Gm) of the gentamycin-resistant transformants contained the inactivated gene vdhS2Gm.
Following EcoRI digestion, the inactivated genes aatS2Km and aatS2Gm were isolated from plasmids pSKaatS2Km and pSKaatS2Gm and ligated to EcoRI-cleaved pSUP202 DNA. The ligation mixtures were transformed into E. coli S17-1.
Selection took place on tetracycline-containing LB medium which also contained kanamycin or gentamycin, respectively. Kanamycin-resistant transformants whose hybrid plasmid (pSUPaatSZKm) contained the inactivated gene aat52Km were obtained. The corresponding hybrid plasmid (pSUPaatS2Gm) of the gentamycin-resistant 2o transformants contained the inactivated gene aatSZGm.
Example 5 Subcloning the deletion-inactivated genes into the conjugatively transferable "suicide plasmid" PHE55, which possesses the "sacB selection system".
In order to be able to replace the intact genes in Pseudomonas sp. 1-IR199 with the deletion-inactivated genes, there is a need for a vector which possesses the properties which have already been described in the case of pSUP202. Since no possibility (no antibiotic resistance) exists of selecting for successful replacement of the genes in 3o Pseudomonas sp. I-IR199 in the case of deletion-inactivated genes, in contrast to the S2 element-inactivated genes, another selection system had to be used. In the "sacB

selection system", the replacing, deletion-inactivated gene is cloned in a plasmid which possesses the sacB gene in addition to an antibiotic resistance gene.
Following the conjugative transfer of this hybrid plasmid into a pseudomonad, the plasmid is integrated by means of homologous recombination at the site in the genome at which the intact gene is located (first crossover). This results in a "heterogenotic" strain which possesses both an intact gene and a deletion-inactivated gene, with these genes being separated from each other by the pHE55 DNA. These strains exhibit the resistance which is encoded by the vector and also possess an active sacB
gene. The intention then is that the pHE55 DNA, together with the intact gene, should then be to separated out of the genomic DNA by means of a second homologous recombination event (second crossover). This recombination event results in a strain which now only possesses the inactivated gene. In addition, the pHE55-coded antibiotic resistance and the sacB gene are both lost. If strains are streaked on sucrose-containing media, the growth of strains which express the sacB gene is inhibited since the gene product converts sucrose into a polymer which is accumulated in the periplasm of the cells. The growth of cells which no longer carry the sacB
gene as a result of the second recombination event having taken place is consequently not inhibited. In order to have a possibility of selecting phenotypically for the integration of the deletion-inactivated gene, this gene is not exchanged for an intact gene;
rva 2o instead, use is made of a strain in which the gene to be replaced is already "labelled"
by the insertion of an S2 element. When successful replacement takes place, the resulting strain loses the antibiotic resistance which is encoded by the S2 element.
Following digestion with PstI, the inactivated gene fcs~ was isolated from plasmid pSKfcsO and ligated to PstI-cleaved pHE55 DNA. The ligation mixture was transformed into E. coli S 17-1. Selection took place on tetracycline-containing LB
medium. Tetracycline-resistant transformants, whose hybrid plasmid (pHEfcsO) contained the inactivated gene fcs0, were obtained.
Following digestion with EcoRI, the inactivated gene echo was isolated from plasmid pSKechO and treated with mung bean nuclease (generation of blunt ends).

The fragment was ligated to BamHI-cleaved and mung bean nuclease-treated pHE55 DNA. The ligation mixture was transformed into E. coli S17-1. Selection took place on tetracycline-containing LB medium. Tetracycline-resistant transformants, whose hybrid plasmid (pHEechO) contained the inactivated gene echo, were obtained Following digestion with EcoRI, the inactivated gene vdh0 was isolated from plasmid pSKvdhO and treated with mung bean nuclease. The fragment was ligated to BamHI-cleaved and mung bean nuclease-treated pHE55 DNA. The ligation mixture was transformed into E. coli S17-1. Selection took place on tetracycline-containing LB medium. Tetracycline-resistant transformants, whose hybrid plasmid (pHEvdhO) contained the inactivated gene vdh~, were obtained.
Following digestion with EcoRI, the inactivated gene aat0 was isolated from plasmid pSKaatO and treated with mung bean nuclease. The fragment was ligated to BamHI-is cleaved and mung bean nuclease-treated pHE55 DNA. The ligation mixture was transformed into E. coli S 17-1. Selection took place on tetracycline-containing LB
medium. Tetracycline-resistant transformants, whose hybrid plasmid (pHEaatO) contained the inactivated gene aat0, were obtained.

Example 6 Generating mutants of the strain Pseudomonas sp. HR199 in which genes of eugenol catabolism have been specifically inactivated by inserting an S2-element.
The strain Pseudomonas sp. HR199 was employed as the recipient in conjugation experiments in which strains of E. coli S 17-1 harbouring the hybrid plasmids of pSUP202 which are listed below were used as donors. The transconjugants were selected on gluconate-containing mineral medium which contained the antibiotic corresponding to the ~ element. It was possible to distinguish between "homogenotic" (replacement of the intact gene with the S2 element insertion-inactivated gene by means of a double crossover) and "heterogenotic"
(integration of the hybrid plasmid into the genome by means of a single crossover) transconjugants on the basis of the pSUP202-encoded tetracycline resistance.
The mutants Pseudomonas sp. HR199 fcsS2Km and Pseudomonas sp. HR199 fcs52Gm were obtained after conjugating Pseudomonas sp. HR199 with E. coli S17-(pSUPfcsSZKm) and E. coli S 17-1 (pSUPfcsS2Gm), respectively. The replacement of the intact fcs gene with the S2Km-inactivated or S2Gm-inactivated gene (fcsS2Km and fcsSZGm, respectively) was verified by means of DNA sequencing.
The mutants Pseudomonas sp. HR 199 echS2Km and Pseudomonas sp. HR 199 echS2Gm were obtained after conjugating Pseudomonas sp. HR199 with E. coli S17-1 (pSUPechSZKm) and E. coli S17-1 (pSUPech52Gm), respectively. The replacement of the intact ech gene with the SZKm-inactivated or S2Gm-inactivated gene (ech52Km and echS2Gm, respectively) was verified by means of DNA
sequencing.
The mutants Pseudomonas sp. HR 199 vdh52Km and Pseudomonas sp. HR 199 vdhS2Gm were obtained after conjugating Pseudomonas sp. HR199 with E. coli 3o S17-1 (pSUPvdhS2Km) and E. coli S17-I (pSUPvdhSZGm), respectively. The replacement of the-intact vdh gene with the SZKm-inactivated or S2,Gm-inactivated gene (vdhS2Km and vdhSZGm, respectively) was verified by means of DNA
sequencing.
The mutants Pseudomonas sp. HR199 aatS2Km and Pseudomonas sp. HR199 aatS2Gm were obtained after conjugating Pseudomonas sp. HR199 with E. coli S17-1 (pSUPaatSZKm) and E. coli S17-1 (pSUPaatSZGm), respectively. The replacement of the intact aat gene with the S2Km-inactivated or S2Gm-inactivated gene (aatS2Km and aatS2Gm, respectively) was verified by means of DNA
to sequencing.
The mutant Pseudomonas sp. HR 199 fcsS2KmvdhS2Gm was obtained after conjugating Pseudomonas sp. HR199 fcsS2Km with E. coli S17-1 (pSUPvdhS2Gm).
The replacement of the intact vdh gene with the 52Gm-inactivated gene (vdhS2Gm) was verified by means of DNA sequencing.
The mutant Pseudomonas sp. HR199 vdh52KmaatS2Gm was obtained after conjugating Pseudomonas sp. HR199 vdhS2Km with E. coli S17-1 (pSUPaatS2Gm).
The replacement of the intact aat gene with the S2Gm-inactivated gene (aatS2Gm) 2o was verified by means of DNA sequencing.
The mutant Pseudomonas sp. HR 199 vdhS2KmechS2Gm was obtained after conjugating Pseudomonas sp. HR199 vdhS2Km with E. coli S17-1 (pSUPechS2Gm).
The replacement of the intact ech gene with the S2Gm-inactivated gene (echSZGm) was verified by means of DNA sequencing.

Example 7 Generating of mutants of the strain Pseudomonas sp. HR199 in which genes of eugenol catabolism have been specifically inactivated by deleting a constituent region.
The strains Pseudomonas sp. I-18199 fcs52Km, Pseudomonas sp. HR199 echS2Km, Pseudomonas sp. HR 199 vdhS2Km and Pseudomonas sp. HR 199 aatS2Km were employed as recipients in conjugation experiments in which strains of E. coli _,-" harbouring the hybrid plasmids of pHE55 which are listed below were used as to donors. The "heterogenotic" transconjugants were selected on gluconate-containing mineral medium which also contained the antibiotic corresponding to the 52 element in addition to tetracycline (pHE55-encoded resistance). After streaking out on sucrose-containing mineral medium, transconjugants were obtained which had eliminated the vector DNA by means of a second recombination event (second crossover). By streaking out on mineral medium which was without antibiotic or which contained the antibiotic corresponding to the S2 element, it was possible to identify the mutants in which the S2 element-inactivated gene had been replaced with the deletion-inactivated gene (no antibiotic resistance).
'~'~' 2o The mutant Pseudomonas sp. HR 199 fcs0 was obtained after conjugating Pseudomonas sp. HR 199 fcsS2,Km with E. coli S 17-1 (pl-lEfcsO). The replacement of the S2Km inactivated gene (fcs52Km) with the deletion-inactivated gene (fcs0) was verified by means of DNA sequencing.
The mutant Pseudomonas sp. HR 199 echo was obtained after conjugating Pseudomonas sp. HR199 echSZKm with E. coli S17-1 (pHEechO). The replacement of the S2Km-inactivated gene (echS2Km) with the deletion-inactivated gene (echo) was verified by means of DNA sequencing.
3o The mutant Pseudomonas sp. 1-18199 vdh0 was obtained after conjugating Pseudomonas sp. T1R199 vdhS2Km with E. coli S17-1 (pHEvdh~). The replacement of the S2Km-inactivated gene (vdhS2Km) with the deletion-inactivated gene (vdh0) was verified by means of DNA sequencing.
The mutant Pseudomonas sp. HR199 aat0 was obtained after conjugating Pseudomonas sp. HR199 aatS2Km with E. coli S17-1 (pHEaat~). The replacement of the SZKm-inactivated gene (aatS2Km) with the deletion-inactivated gene (aat~) was verified by means of DNA sequencing.
... Example 8 to Biotransforming eugenol into vanillin using the mutant Pseudomonas sp. HR199 vdh S2Km.
The strain Pseudomonas sp. HR199 vdhS2Km was propagated in 50 ml of HR-MM
containing 6 mM eugenol up to an optical density of approx. OD600nm = 0.6.
After 17 h, it was possible to detect 2.9 mM vanillin, 1.4 mM ferulic acid and 0.4 mM
vanillic acid in the culture supernatant.
Example 9 2o Biotransforming eugenol into ferulic acid using the mutant Pseudomonas sp.
HR199 vdhSZGmaatS2Km.
The strain Pseudomonas sp. I-IR199 vdhSZGmaatS2Km was propagated in 50 ml of HR-MM containing 6 mM eugenol up to an optical density of approx.OD600nm =
0.6. After 18 h, it was possible to detect 1.9 mM vanillin, 2.4 mM ferulic acid and 0.6 mM vanillic acid in the culture supernatant.

Example 10 Biotransforming eugenol into coniferyl alcohol using the mutant Pseudomonas sp. HR199 vdhS2GmaatSZKm.
The strain Pseudomonas sp. HR199 vdhS2GmaatS2,Km was propagated in 50 ml of HR-MM containing 6 mM eugenol up to an optical density of approx. OD600nm =
0.4. After 15 h, it was possible to detect 1.7 mM coniferyl alcohol, 1.4 mM
vanillin, 1.4 mM ferulic acid and 0.2 mM vanillic acid in the culture supernatant.
to Example 11 Fermentatively producing natural vanillin from eugenol in a 10 1 fermenter using mutant Pseudomonas sp. HR 199 vdhS2Km.
The production fermenter was inoculated with 100 ml of a 24-hour-old preliminary culture which had been propagated at 32°C on a shaking incubator ( 120 rpm) in a medium which was adjusted to pH 7.0 and which consisted of 12.5 g of glycerol/1, 10 g of yeast extract/1 and 0.37 g of acetic acid/l. The fermenter contained 9.9 1 of medium of the following composition: 1.5 g of yeast extract/l, 1.6 g of KH2P04/1, 0.2 g of NaCI/1, 0.2 g of MgS04/1. The pH was adjusted to pH 7.0 with sodium 2o hydroxide solution. After sterilization, 4 g of eugenol were added to the medium. The temperature was 32°C, the aeration was 3 Nl/min and the stirrer speed was 600 rpm.
The pH was maintained at pH 6.5 with sodium hydroxide solution.
At 4 hours after the inoculation, continuous addition of eugenol was begun such that 255 g of eugenol had been added to the culture when fermentation ended after hours. 40 g of yeast extract were also fed in during the fermentation. At the end of the fermentation, the concentration of eugenol was 0.2 g/1. The content of vanillin was 2.6 g/l. 3.4 g of ferulic acid/1 were also present.

The vanillin which is obtained in this way can be isolated by known physical methods such as chromatography, distillation and/or extraction and used for preparing natural flavourings.
E~lanatory notes regardin tgy he fi ug res:
FIG. la to lr:
y Gene struktures for isolating organisms and mutants to calA*: Part of the inactivated gene for coniferyl alcohol dehydrogenase calB*: Part of the inactivated gene for coniferyl aldehyde dehydrogenase fcs*: Part of the inactivated gene for feruloyl-CoA synthetase ech*: Part of the inactivated gene for enoyl-CoA hydratase-aldolase vdh*: Part of the inactivated gene for vanillin dehydrogenase aat*: Part of the inactivated gene for beta-ketothiolase While the restriction enzyme cleavage sites labelled "*" were used for the construction, they are no longer functional in the resulting construct.

FIG 2a~ Nucleotideuence of the gene structure seq calA52Km FIG 2b' Nucleotideuence of the gene structure:
seg calAS2Gm FIG. 2c: Nucleotideuence of the seq calA~ gene structure FIG 2d' Nucleotideuence of the gene structure sea calBS2Km FIG 2e' Nucleotideuence of the gene structure seq caIBSZGm FIG 2f~ Nucleotideuence of the sea calB~ gene structure FIG 2g' Nucleotidequence of the ene structure se fcsSZKm g FIG 2h' Nucleotidequence of the ene structure se fcsS2Gm g FIG 2i: Nucleotide sequence of the fcs~ gene structure to FIG. 2j: Nucleotide sequence of the echSZKm gene structure FIG. Zk: Nucleotide uence of the echS2Gm gene seq structure FIG. 21: Nucleotide uence of the echo glene sea structure FIG. 2m: Nucleotide quence of the vdhSZKm se gene structure FIG. 2n: Nucleotide uence of the vdhS2Gm gene seq structure 15 FIG. 20: Nucleotideuence of the vdh0 gene seq structure FIG 2y Nucleotide se quence of the aatS2Km gene structure FIG 2cL Nucleotide uence of the aatS2Gm gene seq structure FIG. 2r: Nucleotide uence of the aat0 gene seq structure

Claims (16)

claims

1. Transformed and/or mutagenized unicellular or multicellular organism which is characterized in that enzymes of eugenol and/or ferulic acid catabolism are inactivated such that the intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid accumulate.

2. Organism according to Claim 1, characterized in that eugenol and/or ferulic acid catabolism is altered by inserting .OMEGA. elements, or introducing deletions, into corresponding genes.

3. Organism according to either Claim 1 or 2, characterized in that one or more genes encoding the enzymes coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, feruloyl-CoA synthetases, enoyl-CoA hydratase-aldolases, beta-ketothiolases, vanillin dehydrogenases or vanillic acid demethylases is/are altered and/or inactivated.

4. Organism according to one of Claims 1 to 3, characterized in that it is unicellular, preferably a microorganism or a plant or animal cell.

5. Organism according to one of Claims 1 to 4, characterized in that it is a bacterium, preferably a Pseudomonas species.

6. Gene structures in which the nucleotide sequences encoding the enzymes coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, feruloyl-CoA synthetases, enoyl-CoA hydratase-aldolases, beta-ketothiolases, vanillin-dehydrogenases or vanillic acid demethylases, or two or more of these enzymes, are altered and/or inactivated.

7. Gene structures having the sequences given in Figures 1a to 1r.

8. Gene structures having the sequences given in Figures 2a to 2r.

9. Vectors which contain at least one gene structure according to one of Claims 6 to 8.

10. Transformed organism according to one of Claims 1 to 5, characterized in that it harbours at least one vector according to Claim 9.

11. Organism according to one of Claims 1 to 5, characterized in that it contains at least one gene structure according to one of Claims 6 to 8 which is integrated into the genome instead of the respective intact gene.

12. Process for the biotechnological preparation of organic compounds, in particular alcohols, aldehydes and organic acids, characterized in that an organism according to one of Claims 1 to 5 or 10 to 11 is employed.

13. Process for preparing the organisms according to one of Claims 1 to 5, characterized in that the alteration eugenol and/or ferulic acid catabolism is achieved by means of microbiological culturing methods which are known per se.

14. Process for preparing an organism according to one of Claims 1 to 5 or 10 to 11, characterized in that the alteration in eugenol and/or ferulic acid catabolism, and/or the inactivation of the corresponding genes, is achieved by means of recombinant DNA methods.

15. Use of the organisms according to one of Claims 1 to 5 or 10 to 11 for preparing coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid.

16. Use of gene structures according to one of Claims 6 to 8 or of a vector according to Claim 9 for preparing transformed and/or mutagenized organisms.