EP1124947A2

EP1124947A2 - Construction of production strains for producing substituted phenols by specifically inactivating genes of the eugenol and ferulic acid catabolism

Info

Publication number: EP1124947A2
Application number: EP99953892A
Authority: EP
Inventors: Jürgen Rabenhorst; Alexander Steinbüchel; Horst Priefert; Jörg Overhage
Original assignee: Haarmann and Reimer GmbH; Symrise AG
Current assignee: Symrise AG
Priority date: 1998-10-31
Filing date: 1999-10-20
Publication date: 2001-08-22
Also published as: AU1041300A; CN1325444A; AU761093B2; HUP0104772A3; IL142272A0; KR20020022045A; WO2000026355A3; SK5742001A3; CA2348962A1; DE19850242A1; HUP0104772A2; WO2000026355A2; HK1041902A1; BR9914930A; PL348647A1; JP2003533166A

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

Construction of production strains for the production of substituted phenols through targeted inactivation of genes of eugenol and ferulic acid catabolism

The present invention relates to the construction of production strains and a method for the production of substituted methoxyphenols, in particular vanillin.

DE-A 4 227 076 (process for the preparation of substituted methoxyphenols and a suitable microorganism) describes the preparation of substituted methoxyphenols with a new Pseudomonas sp. The starting material here is eugenol and the products are ferulic acid, vanillic acid, coniferyl alcohol and coniferyl aldehyde.

Also in 1995 an extensive review of the biotransformation possibilities with ferulic acid by Rosazza et al. (Biocatalytic transformation of ferulic acid: an abundant aromatic natural product; J. Ind. Microbiol. 15: 457-471).

The genes and enzymes for the synthesis of coniferyl alcohol, coniferyl aldehyde,

Ferulic acid, vanillin and vanillic acid from Pseudomonas sp. have been described in EP-A 0 845 532.

The enzymes for the conversion of tra / w-ferulic acid to trc s-feruloyl-SCoA ester and further to vanillin, as well as the gene for the cleavage of the ester were from

Institute of Food Research, Norwich, GB, described in WO 97/35999. In 1998 the content of the patent also appeared as 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). DE-A 195 32 317 describes the fermentative production of vanillin from ferulic acid with Amycolatopsis sp. in high yields.

The known processes have the disadvantage that either only very low yields of vanillin are achieved or that relatively expensive starting materials are used. In the latter method (DE-A 195 32 317) high yields are achieved, but the use of Pseudomonas sp. HR199 and Amycolatopsis sp. HR167 for the biotransformation of eugenol to vanillin a two-stage fermentation control and therefore a considerable expense and time.

The object of the present invention is therefore to construct organisms which are able to convert the inexpensive raw material eugenol into vanillin in a one-step process.

This object is achieved by the construction of production strains of single or multicellular organisms, which are characterized in that enzymes of eugenol and / or ferulic acid catabolism are inactivated such that an accumulation of the intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or Vanillic acid takes place.

The production strain can be single-cell or multi-cell. Accordingly, the subject of the invention can be microorganisms, plants or animals. In addition, extracts obtained from the production master can also be used. According to the invention, single-celled organisms are preferably used. These can be microorganisms, animal or plant cells. The use of fungi and bacteria is particularly preferred according to the invention. Bacteria are highly preferred. Among the bacteria, Rhodococcus, Pseudomonas and Esche ichia species in particular can be used after changing the eugenol and / or ferulic acid catabolism. In the simplest case, the organisms which can be used according to the invention can be obtained by means of known, conventional microbiological methods. The enzyme activity of the proteins involved in the catabolism of eugenol and / or ferulic acid can be changed by the use of enzyme inhibitors. In addition, the enzyme activity of the proteins involved in the eugenol and / or ferulic acid catabolism can be changed by mutating the genes coding for these proteins. Such mutations can be generated undirected using classic methods, such as, for example, UV radiation or chemicals that trigger mutations.

Genetic engineering methods for obtaining the organisms according to the invention are also suitable, such as deletions, insertions and / or nucleotide exchanges. For example, the genes of the organisms can be inactivated with the help of other DNA elements (Ω elements). Likewise, the intact genes can be exchanged for modified and / or inactivated gene structures by means of suitable vectors. The genes to be inactivated and the DNA elements used for the inactivation can be obtained by classic cloning techniques or by polymerase chain reactions (PCR).

In a possible embodiment of the invention, for example, the eugenol and ferulic acid catabolism can be changed by inserting Ω elements or introducing deletions into corresponding genes. The functions of the genes which code for dehydrogenases, synthetases, hydratase aldolases, thiolases or demethylases can be inactivated using the abovementioned genetic engineering methods, so that the production of the enzymes in question is blocked. These are preferably the genes which code for coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, ferulic acid-CoA synthetases, enoyl-CoA hydratase aldolases, beta-ketothiolases, vanillin dehydrogenases or vanillinic acid demethylases. Genes which encode the amino acid sequences given in EP-A 0845532 and / or their nucleotide sequences encoding allelic variations are very particularly preferred. The invention accordingly also relates to gene structures for producing transformed organisms and mutants.

Gene structures are preferably used to obtain the organisms and mutants in which the nucleotide sequences coding for dehydrogenases, synthetases, hydratase aldolases, thiolases or demethylases are inactivated. Particularly preferred are gene structures in which the nucleotide sequences coding for coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, ferulic acid-CoA synthetases, enoyl-CoA hydratase aldolases, beta-ketothiolases, vanillin dehydrogenases or vanillic acid demethylases are inactive. Gene structures which have the structures given in FIGS. 1a to 1r with the nucleotide sequences shown in FIGS. 2a to 2r and / or their allele variations coding nucleotide sequences are very particularly preferred. Nucleotide sequences from 1 to 18 are particularly preferred.

The invention also includes the partial sequences of these gene structures as well as functional equivalents. Functional equivalents are to be understood as those derivatives of DNA in which individual nucleobases have been exchanged (Wobbe exchanges) without changing their function. Also at the protein level

Amino acids can be exchanged without changing the function.

One or more DNA sequences can be connected upstream and / or downstream of the gene structures. By cloning the gene structures, plasmids or vectors are obtainable which are suitable for the transformation and / or transfection of an organism and / or for the conjugative transfer into an organism.

The invention further relates to plasmids and / or vectors for producing the transformed organisms and mutants according to the invention. Accordingly, these contain the gene structures described. The present invention relates to accordingly also organisms which contain the plasmids and / or vectors mentioned.

The type of plasmids and / or vectors depends on their intended use. To z. For example, to be able to exchange the intact genes of eugenol and / or ferulic acid catabolism in pseudomonas against the genes inactivated by omega elements, vectors are required which can be transferred on the one hand in pseudomonads (conjugatively transferable plasmids) but on the other hand there cannot be replicated and are therefore unstable in pseudomonas (so-called suicide plasmids). DNA sections which are transferred into pseudomonads with the aid of such a plasmid system can only be preserved if they are integrated into the genome of the bacterial cell by homologous recombination.

The gene structures, vectors and plasmids described can be used to produce various transformed organisms or mutants. By means of the named gene structures, intact nucleic acid sequences can be exchanged for modified and / or inactivated gene structures. In the cells obtainable by transformation or transfection or conjugation, the intact gene is exchanged for the modified and / or inactivated gene structure by homologous recombination, as a result of which the resulting cells only have the modified and / or inactivated gene structure in the genome. Thus, according to the invention, genes can preferably be changed and / or inactivated such that the organisms in question are able to produce coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillic acid.

Production strains constructed in accordance with the invention are, for example, mutants of the strain Pseudomonas sp. HR199 (DSM 7063), which was described in detail in DE-A 4 227 076 and EP-A 0845532, where, among other things, the corresponding gene structures result from FIGS. 1a to 1r in conjunction with FIGS. 2a to 2r: 1. Pseudomonas sp. HR199cα 4ΩKm, containing the ΩKm inactivated calA gene instead of the intact cα 4 gene coding for coniferyl alcohol dehydrogenase (Fig. La; Fig. 2a).

2. Pseudomonas sp. HR199cα / ylΩGm, containing the ΩGm-inactivated calA gene instead of the intact calA gene coding for coniferyl alcohol

Dehydrogenase (Fig. Lb; Fig. 2b).

3. Pseudomonas sp. HR199cα / _^ 4Δ, containing the deletion inactivated calA gene instead of the intact calA gene coding for coniferyl alcohol dehydrogenase (FIG. 1c; FIG. 2c). 4. Pseudomonas sp. HR199cα / _J δΩKm, containing the ΩKm-inactivated calB gene instead of the intact calB gene coding for coniferylaldehyde dehydrogenase (FIG. 1d; FIG. 2d)

5. Pseudomonas sp. HR199cα / \ BΩGm, containing the ΩGm-inactivated calB gene instead of the intact cα / 5 gene coding for coniferylaldehyde dehydrogenase (Fig. Le; Fig. 2e).

6. Pseudomonas sp. HR199cα / 5Δ, containing the deletion inactivated cα / 5 gene instead of the intact calB gene coding for coniferylaldehyde dehydrogenase (Fig. 1f; Fig. 2f).

7. Pseudomonas sp. HR199 / αsΩKm, containing the fcs gene inactivated by ΩKm instead of the intact «gene coding for ferulic acid-CoA synthase (Fig.lg; Fig. 2g).

8. Pseudomonas sp. HR199 / αsΩGm, containing the fcs gene inactivated by ΩGm instead of the intact fcs gene coding for ferulic acid-CoA synthase (FIG. 1h; FIG. 2h). 9. Pseudomonas sp. HR199 / csΔ containing the deletion inactivated fcs-

Gene in place of the intact ^ cs gene coding for ferulic acid-CoA synthase (Fig.li; Fig. 2i).

10. Pseudomonas sp. HR199ecbΩKm, containing the ΩKm inactivated ecb gene instead of the intact ecb gene coding for enoyl-CoA hydratase aldolase (Fig.lj; Fig. 2j). 11. Pseudomonas sp. HR199ecbΩGm, containing the ecb gene inactivated by ΩGm instead of the intact ecb gene coding for enoyl-CoA hydratase aldolase (FIG. 1k; FIG. 2k).

12. Pseudomonas sp. HR199ecbΔ, containing the deletion inactivated ech gene instead of the intact ecb gene coding for enoyl-CoA hydratase

Aldolase (Fig. 21; Fig. 21).

13. Pseudomonas sp. HR199 αtΩKm, containing the aat gene inactivated by ΩKm instead of the intact aat gene coding for beta-ketothiolase (Fig. Im; Fig. 2m). 14. Pseudomonas sp. HR199 tΩGm, containing the α t gene inactivated by ΩGm instead of the intact ααt gene coding for beta-ketothiolase (Fig. In; Fig. 2n).

15. Pseudomonas sp. HR199ααtΔ, containing the deletion inactivated aat gene instead of the intact ααt gene coding for beta-ketothiolase (Fig.lo; 2o).

16. Pseudomonas sp. HR199vJbΩKm, containing the vdh gene inactivated by ΩKm instead of the intact vdb gene coding for vanillin dehydrogenase (FIG. 1p; FIG. 2p).

17. Pseudomonas sp. HR199vrfbΩGm, containing the vJb gene inactivated by ΩGm instead of the intact vdh gene coding for vanillin dehydrogenase (FIG. 1q; FIG. 2q).

18. Pseudomonas sp. HR199v bΔ, containing the deletion inactivated vdh gene instead of the intact v b gene coding for vanillin dehydrogenase (Fig.lr; Fig. 2r). 19. Pseudomonas sp. HR199ve? B5ΩKm, containing the väfbß gene inactivated by ΩKm instead of the intact vdhB gene coding for vanillin dehydrogenase II.

20. Pseudomonas sp. HR199vtib5ΩGm, containing the vdhB gene inactivated by ΩGm instead of the intact vdhB gene coding for vanillin dehydrogenase II. 21. Pseudomonas sp. HR199v b5Δ, containing the deletion inactivated vdhB gene instead of the intact vJbß gene coding for vanillin dehydrogenase II.

22. Pseudomonas sp. HR199αdbΩKm, containing the ΩKm inactivated adh gene instead of the intact adh gene coding for alcohol dehydrogenase.

23. Pseudomonas sp. HR199α <ibΩGm, containing the adh gene inactivated by ΩGm instead of the intact adh gene coding for alcohol dehydrogenase. 24. Pseudomonas sp. HR199α <ibΔ containing the deletion inactivated adh-

Gene coding for alcohol dehydrogenase in place of the intact Jb gene. 25. Pseudomonas sp. HR199vα «ΩKm, containing the vanA gene inactivated by ΩKm instead of the intact vanA gene coding for the α-subunit of vanillic acid demethylase. 26. Pseudomonas sp. \ Rλ99vanAQ.Gnx, containing the vanA gene inactivated by ΩGm instead of the intact vanA gene coding for the α-subunit of vanillic acid demethylase.

27. Pseudomonas sp. HR199vα « _^ 4Δ, containing the deletion inactivated vanA gene instead of the intact vanA gene coding for the α-subunit of vanillic acid demethylase.

28. Pseudomonas sp. HR199vα «5ΩKm, containing the vanB gene inactivated by ΩKm instead of the intact vαnß gene coding for the β-subunit of vanillic acid demethylase.

29. Pseudomonas sp. HR199wm5ΩGm, containing the vanG gene inactivated by ΩGm instead of the intact vanB gene coding for the β-subunit of vanillic acid demethylase.

30. Pseudomonas sp. HR199vα / ιBΔ, containing the deletion inactivated vanB gene instead of the intact vanB gene coding for the β-subunit of vanillic acid demethylase. The invention also relates to a method for the biotechnical production of organic compounds. In particular, alcohols, aldehydes and organic acids can be produced with this process. These are preferably coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and vanillic acid.

The organisms described above are used in the process according to the invention. The most particularly preferred organisms include bacteria, especially the Pseudomonas species. In particular, the above-mentioned Pseudomonas species can preferably be used for the following processes:

1. Pseudomonas sp. HR199cα // lΩKm, Pseudomonas sp. HR199cύ7 ΩGm and Pseudomonas sp. HR199cα 4Δ for the production of coniferyl alcohol from eugenol.

2. Pseudomonas sp. HR199cα / 5ΩKm, Pseudomonas sp. HR199cα / £ ΩGm and Pseudomonas sp. HR199cα / 5Δ for the production of coniferyl aldehyde from eugenol or coniferyl alcohol.

3. Pseudomonas sp. HR199 / csΩKm, Pseudomonas sp. HR199 / csΩGm, Pseudomonas sp. HR199fcsΔ, Pseudomonas sp. HR199ecbΩKm, Pseudomonas sp. HR199ecbΩGm and Pseudomonas sp. HR199ecbΔ for the production of ferulic acid from eugenol or coniferyl alcohol or coniferyl aldehyde.

4. Pseudomonas sp. HR199v bΩKm, Pseudomonas sp. HR199v bΩGm, Pseudomonas sp. WRl99vdhA, Pseudomonas sp. HR199vJbΩGmvc b5ΩKm, Pseudomonas sp. HR199v <ibΩKmvJb5ΩGm, Pseudomonas sp. HR199vc / bΔvdbßΩGm and Pseudomonas sp. HR199vJbΔvJb £ ΩKm for the production of vanillin from eugenol or coniferyl alcohol or coniferyl aldehyde or ferulic acid. 5. Pseudomonas sp. HR199vα «y4ΩKm, Pseudomonas sp. HR199v ΩGm,

Pseudomonas sp. HR199vα «y4Δ, Pseudomonas sp. HR199v <ßΩKm, Pseudomonas sp. HR199v n5ΩGm and Pseudomonas sp. HR199v «Z? Δ for the production of vanillic acid from eugenol or coniferyl alcohol or coniferylaldehyde or ferulic acid or vanillin.

The preferred substrate is eugenol. However, the addition of further substrates or even the replacement of the eugenol with another substrate may be possible.

Synthetic, semi-synthetic or complex culture media can be used as the nutrient medium for the organisms used according to the invention. These can contain carbon-containing and nitrogen-containing compounds, inorganic salts, optionally trace elements and vitamins.

Carbohydrates, hydrocarbons or basic organic chemicals can be considered as carbon-containing compounds. Examples of compounds which can preferably be used are sugars, alcohols or sugar alcohols, organic acids or complex mixtures.

The preferred sugar is glucose. Citric acid or acetic acid can preferably be used as organic acids. The complex mixtures include e.g. B. malt extract, yeast extract, casein or casein hydrolyzate.

Inorganic compounds are suitable as nitrogen-containing substrates. Examples include nitrates and ammonium salts. Organic nitrogen sources can also be used. These include yeast extract, soy flour, casein, casein hydrolyzate and corn steep liquor. The inorganic salts that can be used include, for example, sulfates, nitrates, chlorides, carbonates and phosphates. The salts mentioned preferably contain sodium, potassium, magnesium, manganese, calcium, zinc and iron as metals.

The temperature for cultivation is preferably in the range of 5 to 100 ° C.

The range from 15 to 60 ° C. is particularly preferred, and 22 to 60 ° C. is most preferred

37 ° C.

The pH of the medium is preferably 2 to 12. The range from 4 to 8 is particularly preferred.

In principle, all bioreactors known to the person skilled in the art can be used to carry out the method according to the invention. All devices suitable for submerged processes are preferred. This means that vessels with or without a mechanical mixing device can be used according to the invention. The former include e.g. B. shakers, bubble column or loop reactors. The latter preferably include all known devices with stirrers in any configuration.

The process according to the invention can be carried out continuously or batchwise. The duration of the fermentation until a maximum amount of product is reached depends on the particular type of organism used. Basically, however, the times of fermentation are between 2 and 200 hours.

The invention is explained in more detail below with reference to examples:

From the Eugenol utilizing strain Pseudomonas sp. HR199 (DSM 7063), targeted mutants were generated, in which genes of eugenol catabolism were specifically inactivated by inserting omega elements or by introducing deletions. DNA segments that code for antibiotic resistance to kanamycin (ΩKm) and gentamycin (ΩGm) served as omega elements. This Resistance genes were isolated from Tn5 and the plasmid pBBRlMCS-5 using standard methods. The genes calA, calB, fcs, ech, aat, vdh, adh, vdhB, vanA and vanB, which are for coniferyl alcohol dehydrogenase, coniferylaldehyde dehydrogenase, ferulic acid-CoA synthetase, enoyl-CoA hydratase-aldolase, beta-ketothiolase, vanillin Dehydrogenase, alcohol dehydrogenase, vanillin dehydrogenase II and vanillic acid demethylase were encoded starting from genomic DNA of the strain Pseudomonas sp. HR199 isolated using standard methods and cloned into pBluescript SK ^" . DNA sections were removed from these genes by digestion with suitable restriction endonucleases (deletion) or substituted by Ω elements (insertion), whereby the respective gene was inactivated Genes mutated in this way were cloned into conjugatively transferable vectors and then introduced into the strain Pseudomonas sp HR199. By means of suitable selection, transconjugants were obtained which had replaced the functional wild-type gene in question with the newly introduced inactivated gene. and deletion mutants had only the inactivated gene, so that mutants with only one defective gene and multiple mutants in which several genes were inactivated in this way were obtained.These mutants were used for the biotransformation of a) eugenol to coniferyl alcohol , Coniferyl aldehyde, ferulic acid, Va nillin and / or

Vanillic acid; b) coniferyl alcohol to coniferyl aldehyde, ferulic acid, vanillin and / or vanillic acid; c) coniferyl aldehyde to ferulic acid, vanillin and / or vanillic acid; d) ferulic acid to vanillin and / or vanillic acid and e) vanillin to vanillic acid. material and methods

Bacterial growth conditions.

Strains of Escher ichia coli were grown at 37 ° C in Luria-Bertani (LB) or M9 mineral medium (Sambrook, J., EF Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.) Strains of Pseudomonas sp. were at 30 ° C in nutrient broth (NB, 0.8%, wt / vol) or in mineral medium (MM) (Schlegel, HG et al. 1961. Arch. Mikrobiol. 38: 209-222) or HR mineral medium (HR-MM) (Rabenhorst, J. 1996. Appl. Microbiol. Biotechnol. 46: 470-474.). Ferulic acid, vanillin, vanillic acid and protocatechic acid were ^"dissolved in dimethyl sulfoxide and added to the respective medium in a final concentration of 0.1% (wt / vol). Eugenol was added directly to the medium in a final concentration of 0.1% (vol / vol), or applied to the lid of MM agar plates on filter paper (circular filter 595, Schleicher & Schuell, Dassel, Germany)

Cultivation of transconjugants and mutants of Pseudomonas sp. tetracycline, kanamycin and gentamycin were used in final concentrations of 25 μg / ml, 100 μg / ml and 7.5 μg / ml, respectively.

Qualitative and quantitative detection of metabolic intermediates in culture supernatants.

Culture supernatants were immediately, or after dilution with H2θ-bidest. analyzed by high pressure liquid chromatography (Knauer HPLC). Chromatography was carried out on Nucleosil-100 C18 (7 μm, 250 x 4 mm). 0.1% (vol / vol) formic acid and acetonitrile were used as solvents. The gradient used for

Elution of 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 took place at 4 ° C.

Crude extract.

Pseudomonas sp. Cells grown on eugenol. HR199 were washed in 10 mM sodium phosphate buffer, pH 6.0, resuspended in the same buffer and disrupted by passing twice through a French press (Amicon, Silver Spring, Maryland, USA) at a pressure of 1000 psi. The cell homogenate was subjected to ultracentrifugation (1 h, 100,000 x g, 4 ° C.), whereby the soluble

Fraction of the crude extract was obtained as a supernatant.

Anion exchange chromatography on DEAE-Sephacel.

The soluble fraction of the crude extract was dialyzed overnight against 10 mM sodium phosphate buffer, pH 6.0. The dialysate was made up to a 10 mM

Sodium phosphate buffer, pH 6.0 equilibrated DEAE-Sephacel column (2.6 cm x 35 cm, bed volume [BV]: 186 ml) applied at a flow rate of 0.8 ml / min.

The column was rinsed with two BV 10 mM sodium phosphate buffers, pH 6.0. The

The vanillin dehydrogenase-II (VDH-II) was eluted with a linear salt gradient from 0 to 400 mM NaCl in 10 mM sodium phosphate buffer, pH 6.0 (750 ml). 10 ml fractions were collected. Fractions with high VDH II activity were pooled into the DEAE pool.

Determination of vanillin dehydrogenase activity. The VDH activity was determined at 30 ° C. by means of an optically enzymatic test. The reaction mixture with a volume of 1 ml contained 0.1 mmol potassium phosphate (pH 7.1), 0.125 μmol vanillin, 0.5 μmol NAD, 1.2 μmol pyruvate (Na salt), lactate dehydrogenase (1 U; from pig heart) and enzyme solution. The oxidation of vanillin was monitored at λ = 340 nm (εvanillin ⁼ 1.6 cm ^ / μmol). The enzyme activity was given in units (U), where 1 U corresponds to the amount of enzyme that converts 1 μmol vanillin per minute. The protein concentrations in according to Lowry et al. (Lowry, OH, NJ Rosebrough, AL Farr and RJ Randall. 1951. J. Biol. Chem. 193: 265-275).

Determination of coniferyl alcohol dehydrogenase activity.

The CADH activity was determined at 30 ° C. using an optical enzymatic test according to Jaeger et al. (Jaeger, E., L. Eggeling and H. Sahm. 1981. Current Microbiology. 6: 333-336). The reaction mixture with a volume of 1 ml contained 0.2 mmol Tris / HCl (pH 9.0), 0.4 μmol coniferyl alcohol, 2 μmol NAD, 0.1 mmol semicarbazide and enzyme solution. The reduction in NAD was followed at λ = 340 nm (ε = 6.3 cm ^ / μmol). The enzyme activity was given in units (U), where 1 U corresponds to the amount of enzyme that converts 1 μmol substrate per minute. The protein concentrations in the samples were determined according to Lowry et al. (Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall. 1951. J. Biol. Chem. 193: 265-275).

Determination of coniferylaldehyde dehydrogenase activity.

The CALDH activity was determined at 30 ° C. by means of an optically enzymatic test. The reaction mixture with a volume of 1 ml contained 0.1 mmol Tris / HCl (pH 8.8), 0.08 μmol coniferylaldehyde, 2.7 μmol NAD and enzyme solution. The oxidation of coniferyl aldehyde to ferulic acid was followed at λ = 400 nm

(ε = 34 cm.2 / μmol). The enzyme activity was given in units (U), where 1 U corresponds to the amount of enzyme that converts 1 μmol substrate per minute. The protein concentrations in the samples were determined according to Lowry et al. (Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall. 1951. J. Biol. Chem. 193: 265-275).

Determination of ferulic acid-CoA synthetase (ferulic acid thiokinase) activity.

The FCS activity was determined at 30 ° C. by an optically enzymatic test, modified according to Zenk et al. (Zenk et al. 1980. Anal. Biochem. 101: 182-

187). The reaction mixture with a volume of 1 ml contained 0.09 mmol potassium Phosphate (pH 7.0), 2.1 μmol MgCl2, 0.7 μmol ferulic acid, 2 μmol ATP, 0.4 μmol coenzyme A and enzyme solution. The formation of the CoA ester from ferulic acid was followed at λ = 345 nm (ε = 10 cmNμmol). The enzyme activity was given in units (U), where 1 U corresponds to the amount of enzyme that converts 1 μmol substrate per minute. The protein concentrations in the samples were checked

Lowry et al. (Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall. 1951. J. Biol. Chem. 193: 265-275).

Electrophoretic methods. Protein-containing extracts were separated in 7.4% (wt / vol) polyacrylamide gels under native conditions by the method of Stegemann et al. (Stegemann et al. 1973. Z. Naturforsch. 28c: 722-732) and under denaturing conditions in 11.5% (wt / vol) polyacrylamide gels according to the method of Laemmli (Laemmli, UK 1970. Nature (London) 227: 680-685 ). Serva Blue R was used for non-specific protein staining. For specific staining of coniferyl alcohol, coniferyl aldehyde and vanillin dehydrogenase, the gels were buffered for 20 min in 100 mM KP buffer (pH 7.0) and then at 30 ° C in the same buffer the 0.08% (wt / vol) NAD, 0.04 % (wt / vol) p-nitroblue tetrazolium chloride, 0.003% »(wt / vol) phenazine methosulfate and 1 mM of the respective substrate had been added until appropriate color bands became visible.

Transfer of proteins from polyacrylamide gels to PVDF membranes. Proteins were transferred from SDS-polyacrylamide gels using a Semidry-Fasfblot device (B32 / 33, Biometra, Göttingen, Germany) according to the manufacturer's instructions on PVDF membranes (Waters-Milipore, Bedford, Mass., USA).

Determination of 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 analyzer according to the manufacturer's instructions. Isolation and manipulation of DNA.

Genomic DNA was isolated using the method of Marmur (Marmur, J. 1961. J. Mol. Biol. 3: 208-218). The isolation and analysis of other plasmid DNA or of DNA restriction fragments was carried out according to standard methods (Sambrook, JEF Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Habor, New York.).

Transfer of DNA. The preparation and transformation of competent Escherichia co / cells was carried out using the Hanahan method (Hanahan, D. 1983. J. Mol. Biol. 166: 557-580). Conjugative plasmid transfer between plasmid-bearing Escherichia coli S 17-1 strains (donor) and Pseudomonas sp. Strains (recipient) were carried out on NB agar plates by the method of Friedrich et al. (Friedrich, B. et al. 1981. J. Bacteriol. 147: 198-205), or by a "mini-complementation method"

MM agar plates with 0.5%> (wt / vol) gluconate as a C source and 25 μg / ml tetracycline or 100 μg / ml kanamycin. Cells of the recipient were applied in one direction as an inoculation line. After 5 minutes, cells from the donor strains were then applied as inoculation lines, the recipient line being crossed. After incubation for 48 h at 30 ° C, the transconjugants grew directly behind the

Crossing point, whereas neither donor nor recipient strain was able to grow.

Hybridization experiments. DNA restriction fragments were in a 0.8% (wt / vol) agarose gel in 50 mM

Tris-50 mM boric acid-1.25 mM EDTA buffer (pH 8.5) electrophoresed (Sambrook, JEF Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Habor , New York.). The transfer of the denatured DNA from the gel to a positively charged nylon membrane (pore size: 0.45 μm, Pall Filtrationstechnik, Dreieich, Germany), the subsequent hybridization with biotinylated or Digoxigenin-labeled DNA probes and the production of these DNA probes were carried out according to standard methods (Sambrook, JEF Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Laboratory, New York.).

DNA sequencing.

Nucleotide sequences were determined using the dideoxy chain termination method of Sanger et al. (Sanger et al. 1977. Proc. Natl. Acad. Sci. USA 74: 5463-5467) "non-radioactive" with a "LI-COR DNA Sequencer Model 4000L" (LI-COR Inc., Biotechnology Division, Lincoln , NE, .USA) using a "Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP" (Amersham Life Science, Amersham International pls, Little Chalfont, Buckinghamshire, England) each according to the manufacturer's instructions.

With the help of synthetic oligonucleotides the "primer hopping

Strategy "by Strauss et al. (Strauss, E.C. et al. 1986. Anal. Biochem. 154: 353-360).

Chemicals, biochemicals and enzymes. Restriction enzymes, T4 DNA ligase, lambda DNA and enzymes or substrates for the optical enzymatic tests were obtained from CF Boehringer & Söhne (Mannheim, Germany) or from GIBCO / BRL (Eggenstein, Germany), [γ- 32p] ATP came by Amersham / Buchler (Braunschweig, Germany). Oligonucleotides were obtained from MWG-Biotech GmbH (Ebersberg, Germany). NA-type agarose was purchased from Pharmacia-LKB (Uppsala, Sweden).

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). Examples

example 1

Construction of omega elements that are resistant to kanamycin (Ω

Km) or gentamycin (ΩGm).

For the construction of the ΩKm element, the 2099 bp 5g / l fragment of the transposon Tn5 (Auerswald EA, G. Ludwig and H. Schaller. 1981. Cold Spring Harb. Symp. Quant. Biol. 45: 107-113; Beck E., G. Ludwig, EA Auerswald, B. Reiss and H. Schaller. 1982. Gene 19: 327-336; Mazodier P., P. Cossart, E. Giraud and F. Gasser. 1985. Nucleic Acids Res. 13 : 195-205.) Preparatively isolated. The fragment was shortened to approximately 990 bp by treatment with the nuclease Bal-31. This fragment, which only comprised the kanamycin resistance gene (coding for an aminoglycoside-3'-O-phosphotransferase), was then cut with Smally cut pSKsym-DNA (pBluescript SK derivative, which has a symmetrically constructed multiple cloning site [Sall, Hindlll, EcoRI, Smαl, EcoRI, Hindlϊl, Sall] contains) ligated. The ΩKm element could be reisolated from the resulting plasmid as a Smal, EcoRI, HwdIII or So / I fragment.

For the construction of the ΩGm element, the 983 bp Eαel fragment of the

Plasmids pBBRlMCS-5 (Kovach M.Ε., P. Η. Elzer, DS Hill, GT Robertson, MA Farris, RM Roop and KM Peterson. 1995. Gene 166: 175-176.) Were preparatively isolated and then with mung bean nuclease (Digestion of single-stranded DNA molecule ends) treated. This fragment, which only comprised the gentamycin resistance gene (coding for a gentamycin-3-acetyltransferase), was then ligated with Smal cut pSKsym-DNA (see above). The ΩGm element could be re-isolated from the resulting plasmid as a Smal, EcoRI, H dlll or Sall fragment. Example 2

Cloning of the genes from Pseudomonas sp. HR199 (DSM7063), which should be inactivated by inserting Ω elements or by deletions. The separate cloning of the genes fcs, ech, vdh and aat was carried out starting from the E. coli S17-1 strains DSM 10439 and DSM 10440 with the plasmids pE207 and pE5-l (see EP-A 0845532). The fragments indicated were preparatively isolated from these plasmids and treated as described below:

For the cloning of the fcs gene, the 2350 bp Sα / I / EcoRI fragment of the

Plasmid pΕ207 and the 3700 bp EcoRI / Sα / I fragment of plasmid pΕ5-l were cloned together in pBluescript SK in such a way that both fragments were connected to one another via the EcoRI ends. Starting from the resulting hybrid plasmid, the 6050 bp Sα I fragment was isolated and shortened to about 2480 bp by treatment with the nuclease Bal-31. Subsequently, ligated to the fragment ends / sfl linker and the fragment after Pstl digestion was cloned into pBluescript SK ^" (pSK αs). After transformation of E. coli XLl-Blue, clones were obtained which expressed the fcs gene and one FCS activity of 0.2 U / mg protein.

For the cloning of the ecb gene, the 3800 bp Hmdlll / EcoRI fragment of the plasmid pΕ207 was preparatively isolated and shortened to approximately 1470 bp by treatment with the nuclease Bal-31. Then EcoRI linkers were ligated to the fragment ends and the fragment was cloned into pBluescript SK ^" after EcoRI digestion (pSKecb).

For the cloning of the vJb gene, the 2350 bp Sα / I / EcoRI fragment of the

Plasmids pΕ207 isolated preparatively. After cloning in pBluescript SK ^" that was

Fragment shortened on one side by approximately 1530 bp using an exonuclease 111 / Mung Bean nuclease system. Subsequently, an EcoRI- Linker ligated and the fragment cloned into pBluescript SK after EcoRI digestion (pSKvdh). After transformation of E. coli XLl-Blue, clones were obtained which expressed the vJb gene and had a VDH activity of 0.01 U / mg protein.

For the cloning of the aat gene, the 3700 bp EcoRI / Sα / I fragment of the plasmid pΕ5-l was preparatively isolated and shortened to approximately 1590 bp by treatment with the nuclease Bal-31. Then EcoRI linkers were ligated to the fragment ends and the fragment was cloned into pBluescript SK ^" (pSKaat) after EcoRI digestion.

Example 3

Inactivation of the genes described above by inserting Ω elements or deleting partial areas of these genes.

The plasmid pSKfcs, which contained the fcs gene, was included digested, whereby a 1290 bp fragment was cut out of the ^ cs gene. After religation, the deletion derivative of the fcs gene (fcsA) (see Figs. Li and 2i) cloned in pBluescript SK ^" (pSK / αsA) was obtained. In addition, the omega elements ΩKm and ΩGm on the fragment were cut out

Position registered. This resulted in the Ω-inactivated derivatives of the / c gene (fcsΩ Km, see Fig. Lg and 2g) and (fcsΩ.Gm, see Fig. Lh and 2h) cloned in pBluescript SK ^" (pSK csΩKm and pSK / αsΩGm). No FCS activity could be detected in crude extracts of the E. coli clones obtained, whose hybrid plasmids had a / cs gene inactivated by deletion or Ω-element insertion.

The plasmid pSKecb, which contained the ecb gene, was digested with NrwI, whereby a 53 bp and a 430 bp fragment was excised from the ecb gene. After religation, the deletion derivative of the ecb gene (ecbΔ, see FIGS. 11 and 21) was obtained cloned in pBluescript SK ^" (pSKecbΔ) after cutting out the fragments, the omega elements ΩKm and ΩGm were inserted in their place. This resulted in the Ω-inactivated derivatives of the ecb gene (ecbΩKm and ecbΩGm) cloned in pBluescript SK (pSKecbΩKm and pSKecbΩGm).

The plasmid pSKvJb, which contained the vJb gene, was digested with ÄssHII, whereby a 210 bp fragment was excised from the vdh gene. After religation, the deletion derivative of the vdh gene (vdhA, see Figs. Lo and 2o) was obtained cloned in pBluescript SK (pSKvdhΔ). In addition, after cutting out the fragment, the omega elements _ ΩKm and ΩGm were inserted in its place. This resulted in the Ω-inactivated derivatives of the vdh gene (vdhΩKm and vdhΩGm) cloned in pBluescript SK (pSKvdhΩKm, see Fig. Im and 2m) and (pSKvöfbΩGm, see Fig. In and 2n). No VDH activity could be detected in crude extracts of the E. coli clones obtained, whose hybrid plasmids had a vtib gene inactivated by deletion or Ω element insertion.

The plasmid pSKααt, which contained the aat gene, was digested with ÄwHII, whereby a 59 bp fragment was cut out of the aat gene. After religation, the deletion derivative of the α ^ t gene (aatA, see Fig. Lr and

2r) cloned in pBluescript SK (pSKααtΔ) obtained. In addition, after cutting out the fragment, the omega elements ΩKm and ΩGm were inserted in its place. This resulted in the Ω-inactivated derivatives of the ααt gene (aat ΩKm, see Fig. Lp and 2p) and (ααtΩGm, see Fig. Iq and 2q) cloned in pBluescript SK ^" (pSKααtΩKm and pSKααtΩGm). Example 4

Cloning of the genes inactivated by Ω elements into the conjugatively transferable "suicide plasmid" pSUP202. The genes inactivated by Ω elements in Pseudomonas sp. To be able to exchange HR199 for the intact genes, you need a vector that can be transferred in pseudomonas (conjugatively transferable plasmids) but cannot be replicated there and is therefore unstable in pseudomonas ("suicide plasmid"). DNA sections which are transferred into pseudomonads with the aid of such a plasmid system can only be preserved if they are integrated into the genome of the bacterial cell by homologous recombination (RecA-dependent recombination). In the present case, the "suicide plasmid" was pSUP202 (Simon et al. 1983. In: A. Pühler. Molecular genetics of the bacteria-plant interaction. Springer Verlag, Berlin, Heidelberg, New York, pp. 98-106.) used.

The inactivated genes fcsΩKm and cΛΩGm were isolated from the plasmids pSK / αsΩKm and pSK / c ^ ΩGm after stl digestion and ligated with PstL cut pSUP202 DNA. The ligation batches were transformed to E. coli S17-1. The selection was made on LB medium containing tetracycline with kanamycin or gentamycin. Kanamycin-resistant transformants were obtained whose hybrid plasmid (pSUP / csΩKm) contained the inactivated yesΩKm gene. The corresponding hybrid plasmid (pSUP / αsΩGm) of the gentamycin-resistant transformants contained the inactivated GenevacsΩGm.

The inactivated genes ecbΩKm and ecbΩGm were isolated from the plasmids pSKecbΩKm and pSKecbΩGm after EcoRI digestion and ligated with EcoRI cut pSUP202 DNA. The ligation batches were transformed according to E. coli S17-1. The selection was made on LB medium containing tetracycline with kanamycin or gentamycin. Kanamycin-resistant transformants were obtained, whose Hybrid plasmid (pSUPecbΩKm) containing the inactivated ecbΩKm gene. The corresponding hybrid plasmid (pSUPecbΩGm) of the gentamycin-resistant transformants contained the inactivated gene ecbΩGm.

The inactivated genes vJbΩKm and vJbΩGm were isolated from the plasmids pSKv ^ bΩKm and pSKvtibΩGm after EcoRI digestion and ligated with EcoRI cut pSUP202 DNA. The ligation batches were transformed according to E. coli S17-1. The selection was made on LB medium containing tetracycline with kanamycin or gentamycin. Kanamycin-resistant transformants were obtained whose hybrid plasmid (pSUPvdbΩKm) contained the inactivated gene v bΩKm. The corresponding hybrid plasmid (pSUPvJbΩGm) of the gentamycin-resistant transformants contained the inactivated gene vJbΩGm.

The inactivated genes aatΩKm and ααtΩGm were isolated from the plasmids pSKααtΩKm and pSKααtΩGm after EcoRI digestion and ligated with EcoRI cut pSUP202 DNA. The ligation batches were transformed according to E. coli S17-1. The selection was made on LB medium containing tetracycline with kanamycin or gentamycin. Kanamycin-resistant transformants were obtained whose hybrid plasmid (pSUP αtΩKm) contained the inactivated gene aatΩKm. The corresponding hybrid plasmid (pSUPααtΩGm) of the gentamycin-resistant transformants contained the inactivated gene αtΩGm.

Example 5

Cloning of the genes inactivated by deletion into the conjugatively transferable "suicide plasmid" with "sαcß selection system" pHE55.

The genes in Pseudomonas sp. To be able to exchange HR199 for the intact genes, you need a vector that has the properties already described for pSUP202. In contrast to the genes inactivated by Ω element, no deletion-inactivated genes possibility (no antibiotic resistance) for the exchange of genes in Pseudomonas sp. HR199 exists, a different selection system had to be used. In the "raαß selection system", the gene to be exchanged and deactivated by deletion is cloned in a plasmid which, in addition to an antibiotic resistance gene, also has the sacB gene. After conjugative transfer of this hybrid plasmid into a pseudomonad, the plasmid is integrated into the genome by homologous recombination at the point at which the intact gene is located (first "cross over"). In this way, a "heterogeneous" strain is created which has both an intact and a deletion-inactivated gene, which are separated from one another by the pHE55 DNA. This

Strains have the resistance encoded by the vector and also have an active sacB gene. Through a second homologous recombination event (second "cross over"), the pHE55 DNA together with the intact gene is now to be separated from the genomic DNA. This recombination event creates a strain that only has the inactivated gene. In addition, there is a loss of the pHE55-encoded antibiotic resistance and the .sαcS gene. If strains are streaked on sucrose-containing media, strains which express the sacB gene are inhibited in growth because the gene product converts sucrose into a polymer which is accumulated in the periplasm of the cells. Cells that the sacB-

No longer carrying genes are therefore not inhibited in growth. In order to have a phenotypic selection option for the integration of the gene inactivated by deletion, this is not exchanged for an intact gene, but a strain is used in which the gene to be exchanged is already “marked” by insertion of an Ω element. If the exchange is successful, the resulting strain loses the antibiotic resistance encoded by the Ω element.

The inactivated gene fcs A was isolated from the plasmid pSK / csΔ after stl digestion and ligated with Pstl cut pHE55 DNA. The ligation mixture was transformed into E. coli S17-1. The selection was made on LB containing tetracycline Medium. Tetracycline-resistant transformants were obtained whose hybrid plasmid (pHEfcsA) contained the inactivated GenevacsA.

The inactivated echA gene was isolated from the plasmid pSKecbΔ after EcoRI digestion and treated with mung bean nuclease (generation of smooth

Blunt ends). The fragment was ligated with BamHl cut and mung bean nuclease treated pHE55 DNA. The ligation mixture was transformed into E. coli S17-1. The selection was made on LB medium containing tetracycline. Tetracycline-resistant transformants were obtained whose hybrid plasmid (pHEecbΔ) contained the inactivated gene echA.

The inactivated gene vdhA was isolated from the plasmid pSKvüfbΔ after EcoRI digestion and treated with mung bean nuclease. The fragment was ligated with BamHl cut and Mung Bean nuclease treated pHΕ55 DNA. The ligation mixture was transformed into E. coli S17-1. The selection was made on

LB medium containing tetracycline. Tetracycline-resistant transformants were obtained whose hybrid plasmid (pHEv bΔ) contained the inactivated gene vJbΔ.

The inactivated gene aatA was isolated from the plasmid pSKααtΔ after EcoRI digestion and treated with mung bean nuclease. The fragment was made with

BamHl cut and mung bean nuclease treated pHΕ55 DNA ligated. The ligation mixture was transformed into E. coli S17-1. The selection was made on LB medium containing tetracycline. Tetracycline-resistant transformants were obtained whose hybrid plasmid (pHEααtΔ) contained the inactivated gene aatA. Example 6

Generation of mutants of the Pseudomonas sp. HR199, in which genes of eugenol catabolism were specifically inactivated by inserting an Ω element.

The Pseudomonas sp. HR199 was used as a recipient in conjugation experiments in which strains of E. coli S17-1 were used as donors, which contained the hybrid plasmids of pSUP202 listed below. The transconjugants were selected on mineral medium containing gluconate, which contained the antibiotic corresponding to the Ω element. "Homogenote" (exchange of the intact gene for the gene inactivated by Ω-element insertion by double "cross over") and "heterogenote" (integration of the hybrid plasmid into the genome by simple "cross over") transconjugants could be coded using pSUP202 Tetracycline resistance can be distinguished.

The mutants Pseudomonas sp. HR199 c.sΩKm and Pseudomonas sp. HR199 fcsΩ Gm were conjugated by Pseudomonas sp. Get HR199 with E. coli S17-1 (pSUP / csΩKm) or E. coli S17-1 (pSUP / ∞ΩGm). The exchange of the intact fcs gene for the gene inactivated by ΩKm or ΩGm (fcsΩKm or fcsΩGm) was verified by means of DNA sequencing.

The mutants Pseudomonas sp. HR199 ecbΩKm and Pseudomonas sp. HR199 echΩ Gm were conjugated from Pseudomonas sp. Receive HR199 with E. coli S17-1 (pSUPecbΩKm) or E. coli S17-1 (pSUPecbΩGm). The exchange of the intact ecb gene for the gene inactivated by ΩKm or ΩGm (ecbΩKm or ecbΩGm) was verified by means of DNA sequencing.

The mutants Pseudomonas sp. HR199 vJbΩKm and Pseudomonas sp. HR199 vdh

ΩGm were obtained after conjugation of Pseudomonas sp. Receive HR199 with E. coli S17-1 (pSUPv bΩKm) or E. coli S17-1 (pSUPvJbΩGm). The exchange of the intact vJb gene against the gene inactivated by ΩKm or ΩGm (wtTzΩKm or vJbΩGm) was verified by means of DNA sequencing.

The mutants Pseudomonas sp. HR199 αötΩKm and Pseudomonas sp. HR199 aatΩ Gm were conjugated from Pseudomonas sp. HR199 with E. coli S17-1

(pSUPαα / ΩKm) or E. coli S17-1 (pSUPααtΩGm) obtained. The exchange of the intact aat gene for the gene inactivated by ΩKm or ΩGm (ααtΩKm or ααtΩGm) was verified by means of DNA sequencing.

The mutant Pseudomonas sp. HR199 csΩKmvJbΩGm were conjugated by Pseudomonas sp. HR199 / csΩKm obtained with E. coli S17-1 (pSUPvJbΩGm). The exchange of the intact vdh gene for the gene inactivated by ΩGm (vdhΩ Gm) was verified by DNA sequencing.

The mutant Pseudomonas sp. HR199 vJbΩKmααtΩGm were after conjugation of Pseudomonas sp. HR199 viibΩKm with E. coli S17-1 (pSUPααtΩGm) obtained. The exchange of the intact ααt gene for the gene inactivated by ΩGm (aatΩ Gm) was verified by means of DNA sequencing.

The mutant Pseudomonas sp. HR199 vJbΩKmecbΩGm were conjugated to Pseudomonas sp. HR199 vtibΩKm obtained with E. coli S17-1 (pSUPecbΩGm). The exchange of the intact ecb gene for the gene inactivated by ΩGm (echΩ Gm) was verified by DNA sequencing.

Example 7

Generation of mutants of the Pseudomonas sp. HR199, in which genes of eugenol catabolism were specifically inactivated by deletion of a partial area.

The Pseudomonas sp. HR199 αsΩKm, Pseudomonas sp. HR199 ecbΩKm, Pseudomonas sp. HR199 vdhΩK and Pseudomonas sp. HR199 aatΩKm were used as a recipient in conjugation experiments in which strains of E. coli S17-1 were used as donors, which contained the hybrid plasmids of pHE55 listed below. The "heterogeneous" transconjugants were selected on mineral medium containing gluconate, which contained the antibiotic corresponding to the Ω element in addition to tetracycline (resistance coded pHE55). After streaking on sucrose-containing mineral medium, transconjugants were obtained which had eliminated the vector DNA by a second recombination event (second "cross over"). By spreading on mineral medium without antibiotics or with the antibiotic corresponding to the Ω element, it was possible to identify the mutants in which the gene inactivated by Ω element had been replaced by the gene inactivated by deletion (no antibiotic resistance).

The mutant Pseudomonas sp. HR199 ^ c.sA was obtained after conjugation of Pseudomonas sp. HR199 csΩKm obtained with E. coli S17-1 (pHEfcsA). The exchange of the gene inactivated by ΩKm (fcsΩK) for the gene inactivated by deletion (fcsA) was verified by means of DNA sequencing.

The mutants Pseudomonas sp. HR199 echA was conjugated to Pseudomonas sp. HR199 ecbΩKm obtained with E. coli S17-1 (pHEecbΔ). The exchange of the gene inactivated by ΩKm (ecbΩKm) for the gene inactivated by deletion (echA) was verified by means of DNA sequencing. The mutants Pseudomonas sp. HR199 vdhA was conjugated to Pseudomonas sp. HR199 vdhΩKm. obtained with E. coli S17-1 (pHEvJbΔ). The exchange of the gene inactivated by ΩKm (vdhΩKm) for the gene inactivated by deletion (vdhA) was verified by means of DNA sequencing.

The mutants Pseudomonas sp. HR199 aatA was conjugated to Pseudomonas sp. HR199 αtΩKm obtained with E. coli S17-1 (pHEαα / Δ). The exchange of the gene inactivated by ΩKm (ααtΩKm) for the gene inactivated by deletion (aatA) was verified by means of DNA sequencing.

Example 8

Biotransformation of eugenol to vanillin with the mutant Pseudomonas sp. HR199 vdhΩKm. The Pseudomonas sp. HR199 bΩKm was in 50 ml HR-MM with 6 mM

Eugenol attracted to an optical density of approx. ODόOOnm = 0.6. After 17 h, 2.9 mM vanillin, 1.4 mM ferulic acid and 0.4 mM vanillic acid were detectable in the culture supernatant.

Example 9

Biotransformation of eugenol to ferulic acid with the mutant Pseudomonas sp. HR199 vrf / iΩGmöαtΩKm.

The Pseudomonas sp. HR199 vtibΩGmααtΩKm was grown in 50 ml HR-MM with 6 mM eugenol to an optical density of approx. ODόOOnm = 0.6.

After 18 h, 1.9 mM vanillin, 2.4 mM ferulic acid and 0.6 mM vanillic acid were detectable in the culture supernatant. Example 10

Biotransformation of eugenol to coniferyl alcohol with the mutant Pseudomonas sp. HR199 vd / iΩGmaatΩKm. The Pseudomonas sp. HR199 vöbΩGmααtΩKm was in 50 ml HR-MM with

6 mM eugenol attracted to an optical density of approx. ODόOOnm = 0.4. After 15 h, 1.7 mM coniferyl alcohol, 1.4 mM vanillin, 1.4 mM ferulic acid and 0.2 mM vanillic acid were detectable in the culture supernatant.

Example 11

Fermentative production of natural vanillin from eugenol in a 10 l fermenter with the mutant Pseudomonas sp. HR 199 vdhΩKm.

With 100 ml of a 24-hour-old preculture, which is made on a shaking machine (120 rpm) at 32 ° C. in a medium made of 12.5 g / 1 glycerol and adjusted to pH 7.0

The production fermenter was inoculated with 10 g / 1 yeast extract and 0.37 g / 1 acetic acid. The fermenter contained 9.9 liters of medium with the following composition: 1.5 g / 1 yeast extract, 1.6 g / 1 KH ₂ PO ₄ , 0.2 g / 1 NaCl, 0.2 g / 1 MgSO ₄ . The pH was adjusted to pH 7.0 with sodium hydroxide solution. After sterilization, 4 g of eugenol was added to the medium. The temperature was 32 ° C, the ventilation 3

Nl / min and the stirrer speed 600 rpm. The pH was kept at pH 6.5 with sodium hydroxide solution.

Four hours after inoculation, the continuous addition of eugenol was started, so that at the end of the fermentation, 255 g of eugenol had been added to the culture after 65 hours. In addition, 40 g of yeast extract were added during the fermentation. The concentration of eugenol at the end of the fermentation was 0.2 g / 1. The vanillin content was 2.6 g / l. In addition, 3.4 g / 1 ferulic acid were still present. The vanillin thus obtained can be isolated by known physical methods such as chromatography, distillation and / or extraction and used to produce natural flavors.

Explanations to the figures:

FIG. la to lr:

Gene structures for the extraction of organisms and mutants

calA *: part of the inactivated gene of the coniferyl alcohol dehydrogenase calB *: part of the inactivated gene of the coniferylaldehyde dehydrogenase fcs *: part of the inactivated gene of the ferulic acid-CoA synthetase ech *: part of the inactivated gene of the enoyl-CoA hydratase aldolase vdh * : Part of the inactivated gene of vanillin dehydrogenase aat *: Part of the inactivated gene of beta-ketothiolase

The restriction enzyme interfaces provided with "*" were used for the construction, but are no longer functional in the resulting construct.

FIG. 2a: nucleotide sequence of the calAΩKm gene structure FIG. 2b: nucleotide sequence of the gene structure calAΩGm: FIG. 2c: nucleotide sequence of the calAA gene structure FIG. 2d: Nucleotide sequence of the gene structure calBΩKm FIG. 2e: nucleotide sequence of the calBΩGm gene structure

FIG. 2f: Nucleotide sequence of the gene structure calBA FIG. 2g: nucleotide sequence of the gene structure fcsΩKm FIG. 2h: nucleotide sequence of the gene structure fcsΩGm FIG. 2i: nucleotide sequence of the gene structure fcsA FIG. 2j: nucleotide sequence of the gene structure ecbΩKm

FIG. 2k: nucleotide sequence of the gene structure ecbΩGm FIG. 21: Nucleotide sequence of the gene structure echA FIG. 2m: nucleotide sequence of the gene structure vtibΩKm FIG. 2n: nucleotide sequence of the gene structure vJbΩGm FIG. 2o: Nucleotide sequence of the gene structure vdhA

FIG. 2p: nucleotide sequence of the gene structure aatΩKm FIG. 2q: nucleotide sequence of the gene structure aatΩGm FIG. 2r: nucleotide sequence of the gene structure aatA

Claims

Patent claims

1. Transformed and / or mutagenized single or multicellular organism, which is characterized in that enzymes of eugenol and / or ferulic acid catabolism are inactivated such that an accumulation of

Intermediate coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and or vanillic acid takes place.

2. Organism according to claim 1, characterized in that the eugenol and / or ferulic acid catabolism is changed by Ω-elemenf insertion or insertion of deletions in corresponding genes.

3. Organism according to one of claims 1 or 2, characterized in that one or more genes which are responsible for the enzymes coniferyl alcohol dehydrogenase, coniferylaldehyde dehydrogenase, ferulic acid-CoA synthetases,

Enoyl-CoA hydratase aldolases, beta-ketothiolases, vanillin dehydrogenases or vanillic acid demethylases encode enzymes, are modified and / or are inactivated.

4. Organism according to one of claims 1 to 3, characterized in that it is single-celled, preferably a microorganism or a plant or an 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 coniferyl alcohol dehydrogenases, coniferylaldehyde dehydrogenases, ferulic acid-CoA synthetases, enoyl-CoA hydratase aldolases, beta-ketothiolases, vanillin dehydrogenases or vanillic acid demethylases or two or more are used for the enzymes nucleotide sequences encoding these enzymes are changed and / or inactivated.

7. Gene structures with the structures indicated in FIGS. 1a to 1r.

8. Gene structures with the sequences given in FIGS. 2a to 2r.

9. Vectors containing 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 contains at least one vector according to claim 9.

1 1. 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 integrated in place of the respective intact gene in the genome.

12. A process for the biotechnical production 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 used.

13. A method for producing the organisms according to one of claims 1 to 5, characterized in that the change in the eugenol and / or ferulic acid catabolism is achieved by means of microbiological breeding methods known per se.

14. A method for producing an organism according to one of claims 1 to 5 or 10 to 11, characterized in that the change in the eugenol and / or ferulic acid catabolism and / or the inactivation of the corresponding genes is achieved by means of genetic engineering methods.

15. Use of the organisms according to one of claims 1 to 5 or 10 to 1 1 for the production of 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 a vector according to claim 9 for the production of transformed and / or mutagenized organisms.