DE19850242A1 - Construction of production strains for the production of substituted phenols by targeted inactivation of genes of 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

The present invention relates to the construction of production strains and a process for the preparation of substituted methoxyphenols, in particular Vanillin.

DE-A 42 27 076 (Process for the preparation of substituted methoxyphenols and suitable microorganism) describes the production of substituted Methoxyphenols with a new Pseudomonas sp. The starting material here is Euge nol and the products are ferulic acid, vanillic acid, coniferyl alcohol and coni ferylaldehyde.

A comprehensive review of the biotransformation is also published in 1995 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. in EP-A 0 845 532 described.

The enzymes for converting trans-ferulic acid to trans-feruloyl-SCoA ester and on to vanillin and the gene for the cleavage of the ester were from Institute of Food Research, Norwich, GB, described in WO 97/35999. 1998 the content of the patent also appears 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. Micro biology 144: 1397-1405).  

DE-A 195 32 317 describes the fermentative production of vanillin Ferulic acid with Amycolatopsis sp. in high yields.

The known methods have the disadvantage that either only very little can be obtained on vanillin, or it is assumed that the starting materials are relatively expensive. In the latter process (DE-A 195 32 317), high yields are achieved achieved, but the use of Pseudomonas sp. HR199 and Amycolatopsis sp. HR167 for the biotransformation of eugenol to vanillin a two-stage Fermentation management and thus a considerable cost and time expenditure.

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

This task is done by designing production masters multicellular organisms, which are characterized in that enzymes of Eugenol and / or ferulic acid catabolism are inactivated such that a battery mulation of the intermediate coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillic acid.

The production strain can be single-cell or multi-cell. Accordingly, you can The invention relates to microorganisms, plants or animals. About that In addition, extracts from the production master can also be used Come into play. According to the invention, single-celled organisms are preferred used. These can be microorganisms, animal or vegetable Act cells. The use of mushrooms and Bacteria. Bacteria are highly preferred. Among the bacteria in particular special Rhodococcus, Pseudomonas and Escherichia species after changing the Eugenol and / or ferulic acid catabolism are used.  

The simplest way of obtaining the organisms which can be used according to the invention is Case using known, conventional microbiological methods. So can show the enzyme activity of the eugenol and / or ferulic acid catabolism proteins are changed by the use of enzyme inhibitors. In addition, the enzyme activity of the eugenol and / or ferulic acid Proteins involved in catabolism by mutating the coding for these proteins Genes are changed. Such mutations can be done using classic methods generated undirected, such as by UV radiation or muta tion-inducing chemicals.

There are also genetic engineering methods for obtaining the organ of the invention nisms suitable, such as deletions, insertions and / or nucleotide exchanges. So can, for example, the genes of the organisms with the help of other DNA ele elements (Ω elements) can be deactivated. Likewise, using suitable vectors Exchange of intact genes for modified and / or inactivated gene structures be performed. The genes to be inactivated and those for inactivation DNA elements can be placed using classic cloning techniques or can be obtained by polymerase chain reactions (PCR).

In one possible embodiment of the invention, the eugenol as well as ferulic acid catabolism by Ω element insertion or insertion of Deletions can be changed into corresponding genes. The functions of genes for dehydrogenases, synthetases, hydratase aldolases, thiolases or encode demethylases using the genetic engineering methods mentioned above be inactivated so that the production of the enzymes in question is blocked. Before preferably the genes are responsible for coniferyl alcohol dehydrogenases, Coniferylaldehyde dehydrogenases, ferulic acid-CoA synthetases, enoyl-CoA- Hydratase aldolases, beta-ketothiolases, vanillin dehydrogenases or vanillin encode acid demethylases. Genes are particularly preferred which are those in the EP-A 0845532 encode specified amino acid sequences and / or their alleles Variations encoding nucleotide sequences.  

The invention accordingly also relates to gene structures for production 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, coniferylaldehyde 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 indicated in FIGS. 1a to 1r with the nucleotide sequences shown in FIGS. 2a to 2r and / or their allele variations encoding 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 functio equivalents. Such functional derivatives are those of To understand DNA in which individual nucleobases have been exchanged (Wobble exchange) without changing the function. Also at the protein level Amino acids can be exchanged without changing the function of the Episode is.

The gene structures can be preceded and / or followed by one or more DNA sequences be switched. By cloning the gene structures are plasmids or vectors available for the transformation and / or transfection of an organism and / or are suitable for conjugative transmission into an organism.

The invention also relates to plasmids and / or vectors for the production of the transformed organisms and mutants according to the invention. These contain accordingly the gene structures described. The present invention relates to  accordingly also organisms which ent the plasmids and / or vectors mentioned hold.

The type of plasmids and / or vectors depends on their intended use. To z. B. the intact genes of eugenol and / or ferulic acid catabolism in Pseudomona to be able to exchange the genes inactivated by omega elements, you need vectors that can be transferred in pseudomonas (Conjugatively transferable plasmids), on the other hand, however, not to be replicated there can and are therefore unstable in pseudomonas (so-called suicide plasmids). Sections of DNA generated with the help of such a plasmid system in Pseudomonads can only be retained if they are transferred by homologous recom be integrated into the genome of the bacterial cell.

The described gene structures, vectors and plasmids can be used for the production various transformed organisms or mutants can be used. Means of the named gene structures can intact nucleic acid sequences against changed and / or inactivated gene structures are exchanged. In the by Trans formation or transfection or conjugation is carried out by cells homologous recombination an exchange of the intact gene for the changed one and / or inactivated gene structure, whereby the resulting cells only over have the modified and / or inactivated gene structure in the genome. So can According to the invention, genes are preferably changed and / or inactivated in this way, that the organisms concerned coniferyl alcohol, coniferyl aldehyde, ferulic acid, Can produce 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 42 27 076 and EP-A 0845532, the corresponding gene structures resulting from FIGS . 1a to 1r in connection with FIGS. 2a to 2r, among others:

• 1. Pseudomonas sp. HR199caAlΩKm, containing the ΩKm inactivated calA gene instead of the intact calA gene coding for coniferyl alcohol dehydrogenase ( Fig. 1a; Fig. 2a).
• 2. Pseudomonas sp. HR1999calAΩGm, containing the ΩOm-inactivated calA gene instead of the intact calA gene coding for coniferyl alcohol dehydrogenase ( FIG. 1b; FIG. 2b).
• 3. Pseudomonas sp. HR199ca1AΔ, containing the deletion inactivated calA gene instead of the intact calA gene coding for coniferyl alcohol dehydrogenase ( FIG. 1c; FIG. 2c).
• 4. Pseudomonas sp. HR199calBΩKm, containing the ΩKm-inactivated calB gene instead of the intact calB gene coding for coniferylaldehyde dehydrogenase ( FIG. 1d; FIG. 2d).
• 5. Pseudomonas sp. HR199calBΩGm, containing the ΩGm-inactivated calB gene instead of the intact calB gene coding for coniferylaldehyde dehydrogenase ( Fig. 1e; Fig. 2e).
• 6. Pseudomonas sp. HR199caLBΔ, containing the deletion inactivated calB gene instead of the intact calB gene coding for coniferylaldehyde dehydrogenase ( FIG. 1f; FIG. 2f).
• 7. Pseudomonas sp. HR199fcsΩKm, containing the fcs gene inactivated by ΩKm instead of the intact fcs gene coding for ferulic acid-CoA synthesis ( FIG. 1g; FIG. 2g).
• 8. Pseudomonas sp. HR199fcsΩGm, containing the fcs gene inactivated by ΩGm instead of the intact fcs gene coding for ferulic acid-CoA synthesis ( FIG. 1h; FIG. 2h).
• 9. Pseudomonas sp. HR199fcsΔ, containing the deletion-inactivated fcs gene instead of the intact fcs gene coding for ferulic acid-CoA synthesis ( FIG. 1i; FIG. 2i).
• 10. Pseudomonas sp. HR199echΩKm, containing the ΩKm-inactivated ech gene instead of the intact ech gene coding for enoyl-CoA hydratase aldolase ( Fig. 1j; Fig. 2j).
• 11. Pseudomonas sp. HR199echΩGm, containing the ΩGm inactivated ech gene instead of the intact ech gene coding for enoyl-CoA hydra tase aldolase ( Fig. 1k; Fig. 2k).
• 12. Pseudomonas sp. HR199echΔ, containing the deletion inactivated ech gene instead of the intact ech gene coding for enoyl-CoA hydratase aldolase ( FIG. 11; FIG. 21).
• 13. Pseudomonas sp. HR199aatΩKm, containing the aat gene inactivated by ΩKm instead of the intact aat gene coding for beta-ketothiolase ( FIG. 1m; FIG. 2m).
• 14. Pseudomonas sp. HR199aatΩGm, containing the aat gene inactivated by ΩGm instead of the intact aat gene coding for beta-ketothiolase ( FIG. 1n; FIG. 2n).
• 15. Pseudomonas sp. HR199aatΔ, containing the deletion inactivated aat gene instead of the intact aat gene coding for beta-ketothiolase ( Fig. 1o; 2o).
• 16. Pseudomonas sp. HR199vdhΩKm, containing the ΩKm inactivated vdh gene instead of the intact vdh gene coding for vanillin dehydrogenase ( Fig. 1p; Fig. 2p).
• 17. Pseudomonas sp. HR199vdhΩGm, containing the ΩGm inactivated vdh gene instead of the intact vdh gene coding for vanillin dehydrogenase ( Fig. 1q; Fig. 2q).
• 18. Pseudomonas sp. HR199vdhA, containing the deletion inactivated vdh gene instead of the intact vdh gene coding for vanillin dehydrogenase ( FIG. 1r; FIG. 2r).
• 19. Pseudomonas sp. HR199vdhBΩKm, containing that inactivated by ΩKm vdhB gene instead of the intact vdhB gene coding for vanillin dehydro genase II.
• 20. Pseudomonas sp. HR199vdhBΩGm, containing that inactivated by ΩGm vdhB gene instead of the intact vdhB gene coding for vanillin dehydro genase II.  
• 21. Pseudomonas sp. HR199vdhBΔ containing the deletion inactivated vdhB gene instead of the intact vdhB gene coding for vanillin dehydro genase II.
• 22. Pseudomonas sp. HR199adhΩKm, containing that inactivated by ΩKm adh gene instead of the intact adh gene coding for alcohol dehydrogen nose.
• 23. Pseudomonas sp. HR199adhΩGm, containing that inactivated by ΩGm adh gene instead of the intact adh gene coding for alcohol dehydrogen nose.
• 24. Pseudomonas sp. HR199adhΔ containing the deletion inactivated adh Gene in place of the intact adh gene coding for alcohol dehydrogenase.
• 25. Pseudomonas sp. HR199vanAΩKm, containing that inactivated by ΩKm vanA gene instead of the intact vanA gene coding for the α subunit of vanillic acid demethylase.
• 26. Pseudomonas sp. HR199vanAΩGm, containing that inactivated by ΩGm vanA gene instead of the intact vanA gene coding for the α subunit of vanillic acid demethylase.
• 27. Pseudomonas sp. HR199vanAΔ containing the deletion inactivated vanA gene instead of the intact vanA gene coding for the α subunit of vanillic acid demethylase.
• 28. Pseudomonas sp. HR199vanBΩKm, containing that inactivated by ΩKm vanB gene instead of the intact vanB gene coding for the β subunit of vanillic acid demethylase.
• 29. Pseudomonas sp. HR199vanBΩGm, containing that inactivated by ΩGm vanB gene instead of the intact vanB gene coding for the β subunit of vanillic acid demethylase.
• 30. Pseudomonas sp. HR199vanBΔ 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 producing biotechnology development of organic compounds. In particular, using this method Alcohols, aldehydes and organic acids are produced. Preferably han it is 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 Pseu domonas species can preferably be used for the following processes:

• 1. Pseudomonas sp. HR199calAΩKm, Pseudomonas sp. HR199calAΩGm and Pseudomonas sp. HR199calAΔ for the production of coniferyl alcohol Eugenol.
• 2. Pseudomonas sp. HR199calBΩKm, Pseudomonas sp. HR199calBΩGm and Pseudomonas sp. HR199calBΔ for the production of coniferylaldehyde Eugenol or coniferyl alcohol.
• 3. Pseudomonas sp. HR199fcsΩKm, Pseudomonas sp. HR199fcsΩGm, pseu domonas sp. HR199fcsΔ, Pseudomonas sp. HR199echΩKm, Pseudomonas sp. HR199echΩGm and Pseudomonas sp. HR199echΔ for the production of Ferulic acid from eugenol or coniferyl alcohol or coniferyl aldehyde.
• 4. Pseudomonas sp. HR199vdhΩKm, Pseudomonas sp. HR199vdhΩGm, pseu domonas sp. HR199vdhD, Pseudomonas sp. HR199vdhΩGmvdhBΩKm, Pseudomonas sp. HR199vdhΩKmvdhBΩGm, Pseudomonas sp. HR199vdhΔvdhBΩGm and Pseudomonas sp. HR199vdhΔvdhBΩKm for Production of vanillin from eugenol or coniferyl alcohol or coni ferylaldehyde or ferulic acid.  
• 5. Pseudomonas sp. HR199vanAΩKm, Pseudomonas sp. HR199vanAΩGm, Pseudomonas sp. HR199vanAΔ, Pseudomonas sp. HR199vanBΩKm, Pseudomonas sp. HR199vanBΩGm and Pseudomonas sp. HR199vanBΔ for Production of vanillic acid from eugenol or coniferyl alcohol or Coniferyl aldehyde or ferulic acid or vanillin.

The preferred substrate is eugenol. However, the addition of further substrates or even exchanging the eugenol for another substrate.

The nutrient medium for the organisms used according to the invention are syn synthetic, semi-synthetic or complex culture media. these can carbon and nitrogen compounds, inorganic salts, given contain trace elements and vitamins.

Carbohydrates, hydrocarbons can be used as carbon-containing compounds or organic basic chemicals. Examples of preferred usable compounds are sugar, alcohols or sugar alcohols, organic Acids or complex mixtures.

The preferred sugar is glucose. As organic acids can preferably citric acid or acetic acid are used. To the com plex mixtures include z. B. malt extract, yeast extract, casein or casein hydro lysate.

Inorganic compounds are suitable as nitrogen-containing substrates. At games for this are nitrates and ammonium salts. Likewise, organic stick material sources are used. These include yeast extract, soy flour, casein, Casein hydrolyzate and corn steep liquor.  

The inorganic salts which can be used include, for example, sulfates, nitrates, Chlorides, carbonates and phosphates. The salts mentioned contain as metals preferably sodium, potassium, magnesium, manganese, calcium, zinc and iron.

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 is particularly preferred Range from 4 to 8.

In principle, everyone can carry out the method according to the invention bioreactors known to the person skilled in the art can be used. Preferably come all devices suitable for submerged processes. That is, it can, according to the invention, vessels with or without a mechanical mixing device be set. The former include e.g. B. shakers, bubble or Loop reactors. The latter preferably include all known devices with stirrers of any design.

The process according to the invention can be carried out continuously or batchwise be performed. The duration of the fermentation until a maximum is reached The amount of product depends on the specific type of organism used. Reason in addition, however, the fermentation times 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) the targeted mutants were generated, specifically genes of eugenol catabolism were 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. These resistance genes were isolated from Tn5 and the plasmid pBBR1MCS-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 on the basis of genomic DNA from the strain Pseudomonas sp. Strain HR199 using standard methods and isolated in pBluescript SK ^- cloned. DNA sections were removed from these genes by digestion with suitable restriction endonucleases (deletion), or substituted by Ω elements (insertion), as a result of which the respective gene was inactivated. The genes mutated in this way were cloned into conjugatively transferable vectors and then into the strain Pseudomonas sp. HR199 introduced. By suitable selection, transconjugants were obtained which had exchanged the functional wild-type gene for the newly introduced inactivated gene. The insertion and deletion mutants obtained in this way only had the inactivated gene. In this way, 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, vanillin and / or Vanillic acid;
• b) coniferyl alcohol to coniferylaldehyde, 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 used to vanillic acid.

material and methods Bacterial growth conditions

Strains of Escherichia coli were grown at 37 ° C in Luria-Bertani (LB) or M9 mine ralmedium (Sambrook, J., E.F. 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 in at 30 ° C Nutrient Broth (NB, 0.8%, wt / vol) or in mineral medium (mm) (Schlegel, H. G. et al. 1961. Arch. Microbiol. 38: 209-222) or HR mineral medium (HR-MM) (Rabenhorst, J. 1996. Appl. Microbiol. Biotechnol. 46: 470-474). Ferula Acid, vanillin, vanillic acid and protocatechic acid were in dimethyl sulfoxide dissolved, and the respective medium in a final concentration of 0.1% (wt / vol) added. Eugenol was added to the medium directly in a final concentration of 0.1% (vol / vol) added, or in the lid of MM agar plates on filter paper (Circular filter 595, Schleicher & Schuell, Dassel, Germany). In the Cultivation of transconjugants and mutants of Pseudomonas sp. became Tetra cyclin, kanamycin, and gentamycin in final concentrations of 25 µg / ml and 100 µg / ml, respectively or 7.5 µg / ml.

Qualitative and quantitative evidence of metabolic intermediates in Kul overhangs

Culture supernatants were immediately or after dilution with H ₂ O bidist. analyzed by high pressure liquid chromatography (Knauer HPLC). Chromatography was carried out on Nucleosil-100 C18 (7 µm, 250 × 4 mm). 0.1% (vol / vol) formic acid and acetonitrile were used as solvents. The gradient used to elute 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 in 10 mM Sodium phosphate buffer, pH 6.0 washed, resuspended in the same buffer and through two passages of 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 overnight against 10 mM sodium phosphate buffer, pH 6.0 dialyzed. The dialysate was made up to a 10 mM Sodium phosphate buffer, pH 6.0 equilibrated DEAE-Sephacel column (2.6 cm × 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 Elution of vanillin dehydrogenase-II (VDH-II) was carried out 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 shares vity were combined to form the DEAE pool.

Determination of vanillin dehydrogenase activity

The VDH activity was determined at 30 ° C. by 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 (ε _vanillin = 11.6 cm ² / 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 the samples were determined 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. by 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 of NAD was followed at λ = 340 nm (ε = 6.3 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, OH, NJ Rosebrough, AL Farr and RJ 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 monitored 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, OH, NJ Rosebrough, AL Farr and RJ Randall. 1951. J. Biol. Chem. 193: 265-275).

Determination of ferulic acid-CoA synthetase (ferulic acid thiokinase) activi act

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 MgCl ₂ , 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 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, OH, NJ Rosebrough, AL Farr and RJ Randall. 1951. J. Biol. Chem. 193: 265-275).

Electrophoretic methods

Protein-containing extracts were separated into 7.4% (wt / vol) poly acrylamide gels under native conditions using the method of Stegemann et al. (Stegemann et al. 1973. Z. Naturforsch. 28c: 722-732) and among denaturing Conditions in 11.5% (wt / vol) polyacrylamide gels according to the Laemmli method (Laemmli, U.K. 1970. Nature (London) 227: 680-685). For non-specific protein Serva Blue R was used for the coloring. For the specific coloring of the Conife Ryl alcohol, coniferyl aldehyde and vanillin dehydrogenase, the gels were for 20 min buffered in 100 mM KP buffer (pH 7.0) and then at 30 ° C in the same Chen the 0.08% (wt / vol) NAD, 0.04% (wt / vol) p-nitro blue 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 made from SDS polyacrylamide gels using a Semidry Fastblot device (B32 / 33, Biometra, Göttingen, Germany) according to the manufacturer's instructions on PVDF- Membranes (Waters-Milipore, Bedford, Mass., USA) transferred.

Determination of N-terminal amino acid sequences

N-terminal amino acid sequences were determined using a Protein peptide sequencers (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 Marmur method (Marmur, J. 1961. J. Mol. Biol. 3: 208-218). Isolation and analysis from others Plasmid DNA or DNA restriction fragments were carried out according to the standard method den (Sambrook, J.E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a labora tory manual. 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Habor, New York).

Transfer of DNA

The preparation and transformation of competent Escherichia coli cells was carried out according to the Hanahan method (Hanahan, D. 1983. J. Mol. Biol. 166: 557-580). Conjugative plasmid transfer between plasmid-bearing Escherichia coli S17-1 strains (donor) and Pseudomonas sp. Strains (recipient) followed NB agar plates using the method of Friedrich et al. (Friedrich, B. et al. 1981. J. Bacteriol. 147: 198-205), or by a "mini-complement method" MM agar plates with 0.5% (wt / vol) gluconate as a C source and 25 µg / ml tetracycline or 100 µg / ml kanamycin. The cells of the recipient were unidirectional applied as an inoculation line. After 5 min, cells from the donor strains were then removed as Vaccination lines are applied, the recipient vaccination line being crossed. To After incubation for 48 h at 30 ° C, the transconjugants grew directly behind the Crossing point, whereas neither donor nor recipient strain for wax was able to.

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 separates (Sambrook, J.E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd ed., 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, Drei eich, Germany), the subsequent hybridization with biotinylated or  Digoxigenin-labeled DNA probes and the preparation of these DNA probes was carried out according to standard methods (Sambrook, J.E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Habor, New York.).

DNA sequencing

Nucleotide sequences were determined according to the dideoxy chain ab break method by 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) under use a "Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP "(Amersham Life Science, Amersham International pls, Little Chalfont, Buckinghamshire, England) 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) sequenced.

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 & Sons (Mannheim, Germany) or from GIBCO / BRL (Eggenstein, Germany). [γ- ³² P] ATP came from 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, resistance to kanamycin (ΩKm) or gentamycin (ΩGm) mediate

The 2099 bp BglI 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 Bal-31 nuclease. This fragment, which only comprised the kanamycin resistance gene (coding for an aminoglycoside-3'-O-phosphotransferase), was then cut with SmaI-cut pSKsym-DNA (pBluescript SK ^- derivative, which has a symmetrically constructed multiple cloning site [SalI, HindIII, EcoRI, SmaI, EcoRI, HindIII, SalI] contains) ligated. The Ωkm element could be re-isolated from the resulting plasmid as a SmaI, EcoRI, HindIII or SalI fragment.

For the construction of the ΩGm element, the 983 bp EaeI fragment of the Plasmids pBBR1MCS-5 (Kovach M.E., P.H. Elzer, D.S. Hill, G.T. Robertson, M.A. Farris, R. M. Roop and K. M. Peterson. 1995. Gene 166: 175-176.) Preparative iso and then with mung bean nuclease (digesting single-stranded DNA molecule ends) treated. This fragment, which is now only the gentamycin Resistance gene (coding for a gentamycin-3-acetyltransferase) was included then ligated with SmaI-cut pSKsym DNA (see above). From the result plasmid, the ΩGm element could be used as SmaI, EcoRI, HindIII or SalI Fragment to be reisolated.  

Example 2 Cloning of the genes from Pseudomonas sp. HR199 (DSM7063) by Inser tion of 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 plasmids pE207 and pE5-1 (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 SalI / EcoRI fragment of plasmid pE207 and the 3700 bp EcoRI / SalI fragment of plasmid pE5-1 were cloned together in pBluescript SK ^- in such a way that both fragments were cloned via the EcoRI ends were joined together. Starting from the resulting hybrid plasmid, the 6050 bp SalI fragment was isolated preparatively and shortened to approximately 2480 bp by treatment with the nuclease Bal-31. Pst I linkers were then ligated to the fragment ends, and the fragment after PstI digestion in pBluescript SK ^- cloned (pSKfcs). After transformation of E. coli XL1-Blue, clones were obtained which expressed the fcs gene and had an FCS activity of 0.2 U / mg protein.

For the cloning of the ech gene, the 3800 bp HindIII / EcoRI fragment of the plasmid pE207 was preparatively isolated and shortened to approximately 1470 bp by treatment with the nuclease Bal-31. Linkers were then ligated to the fragment ends of EcoRI and the fragment after EcoRI digestion in pBluescript SK ^- cloned (pSKech).

For the cloning of the vdh gene, the 2350 bp SalI / EcoRI fragment of the plasmid pE207 was isolated. After cloning in pBluescript SK ^- the fragment was shortened on one side by approx. 1530 bp using an exonuclease III / Mung Bean nuclease system. Linker was then ligated to the fragment end an EcoRI and the fragment after EcoRI digestion in pßluescript SK ^- cloned (pSKvdh). After transformation of E. coli XL1-Blue, clones were obtained which expressed the vdh gene and had a VDH activity of 0.01 U / mg protein.

For the cloning of the aat gene, the 3700 bp EcoRI / SalI fragment of the plasmid pE5-1 was preparatively isolated and shortened to approximately 1590 bp by treatment with the nuclease Bal-31. Linkers were then ligated to the fragment ends of EcoRI and the fragment after EcoRI digestion in pBluescript SK ^- cloned (pSKaat).

Example 3 Inactivation of the genes described above by inserting Ω elements, or by deleting parts of these genes

The plasmid pSKfcs, which contained the fcs gene, was digested with BssHII, whereby a 1290 bp fragment was cut out of the fcs gene. After religation, the deletion derivative of the fcs gene (fcsΔ) (see FIGS. 1i and 2i) cloned in pBluescript SK ^- (pSKfcsΔ) was 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 fcs gene (fcsΩKm, see Fig. 1g and 2g) and (fcsΩGm, see Fig. 1h and 2h) cloned in pBluescript SK ^- (pSKfcsΩKm and pSKfcsΩGm). No FCS activity could be detected in crude extracts of the E. coli clones obtained, whose hybrid plasmids had an fcs gene inactivated by deletion or Ω element insertion.

The plasmid pSKech, which contained the ech gene, was digested with NruI, whereby a 53 bp and a 430 bp fragment was cut out of the ech gene. After religation, the deletion derivative of the ech gene (echΔ, see FIGS. 1l and 2l) was obtained cloned in pBluescript SK ^- (pSKechΔ). In addition, 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 ech gene (echΩKm and echΩGm) cloned in pBluescript SK ^- (pSKechΩKm and pSKechΩGm).

The plasmid pSKvdh, which contained the vdh gene, was digested with BssHII, whereby a 210 bp fragment was cut out of the vdh gene. After religation, the deletion derivative of the vdh gene (vdhΔ, see Fig. 1o and 2o) cloned in pBluescript SK ^- (pSKvdhΔ) was 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 vdh gene (vdhΩKm and vdhΩGm) cloned in pBluescript SK ^- (pSKvdhΩKm, see Fig. 1m and 2m) and (pSKvdhΩGm, see Fig. 1n and 2n). No VDH activity could be detected in crude extracts of the E. coli clones obtained, whose hybrid plasmids had a vdh gene inactivated by deletion or Ω-element insertion.

The plasmid pSKaat, which contained the aat gene, was digested with BssHII, whereby a 59 bp fragment was cut out of the aat gene. After religation, the deletion derivative of the aat gene (aatΔ, see Figs. 1r and 2r) was obtained cloned in pBluescript SK ^- (pSKaatΔ). 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 aat gene (aat ΩKm, see Fig. 1p and 2p) and (aatΩGm, see Fig. 1q and 2q) cloned in pBluescript SK ^- (pSKaatZΩKm and pSKaatΩGm).

Example 4 Cloning of the genes inactivated by Ω elements into the conjugative via portable "suicide plasmid" pSUP202

The genes inactivated by Ω elements in Pseudomonas sp. HR199 against the To be able to exchange intact genes, you need a vector that is in one hand Pseudomonads can be transferred (conjugative transferable plasmids), others on the other hand, however, cannot be replicated there and thus insta in Pseudomonas bil is ("suicide plasmid"). DNA sections made using such a plasmid systems in Pseudomonas can only be preserved if by homologous recombination (RecA-dependent recombination) into the genome the bacterial cell can be integrated. In the present case, the "suicide Plasmid "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 fcsΩGm were extracted from the PstI after digestion Plasmids pSKfcsΩKm and pSKfcsΩGm isolated and cut with PstI pSUP202 DNA ligated. The ligation batches were transformed according to E. coli S17-1 lubricated. The selection was made on LB medium containing tetracycline with kanamycin or gentamycin. Kanamycin-resistant transformants were obtained, whose Hybrid plasmid (pSUPfcsΩKm) containing the inactivated gene fcsΩKm. That corresponds hybrid plasmid (pSUPfcsΩGm) of the gentamycin-resistant transformants contained the inactivated gene fcsΩGm.

The inactivated genes echΩKm and echΩGm were identified after EcoRI digestion the plasmids pSKechΩKm and pSKechΩGm isolated and cut with EcoRI pSUP202 DNA ligated. The ligation batches were transformed according to E. coli S17-1 lubricated. The selection was made on LB medium containing tetracycline with kanamycin or gentamycin. Kanamycin-resistant transformants were obtained, whose  Hybrid plasmid (pSUPechΩKm) containing the inactivated echΩKm gene. That corresponds hybrid plasmid (pSUPechΩGm) of the gentamycin-resistant transforman contained the inactivated echΩGm gene.

The inactivated genes vdhΩKm and vdhΩGm were identified after EcoRI digestion the plasmids pSKvdhΩKm and pSKvdhΩGm isolated and cut with EcoRI pSUP202 DNA ligated. The ligation batches were transformed according to E. coli S17-1 lubricated. The selection was made on LB medium containing tetracycline with kanamycin or gentamycin. Kanamycin-resistant transformants were obtained, whose Hybrid plasmid (pSUPvdhΩKm) containing the inactivated gene vdhΩKm. That corresponds hybrid plasmid (pSUPvdhΩGm) of the gentamycin-resistant transforman that contained the inactivated gene vdhΩGm.

The inactivated genes aatΩKm and aatΩGm were eliminated after EcoRI digestion the plasmids pSKaatΩKm and pSKaatΩGm isolated and cut with EcoRI pSUP202 DNA ligated. The ligation batches were transformed according to E. coli S17-1 lubricated. The selection was made on LB medium containing tetracycline with kanamycin or gentamycin. Kanamycin-resistant transformants were obtained, whose Hybrid plasmid (pSUPaatΩKm) containing the inactivated gene aatΩKm. That corresponds hybrid plasmid (pSUPaatΩGm) of the gentamycin-resistant transformants contained the inactivated gene aatΩGm.

Example 5 Cloning of the genes inactivated by deletion into the conjugative portable "suicide plasmid" with "sacB selection system" pHE55

The genes in Pseudomonas sp. HR199 against the To be able to exchange intact genes, you need a vector that is already used for pSUP202 described properties. In contrast to the Ω- Element-inactivated genes with deletion-inactivated genes no selek  possibility (no antibiotic resistance) for the exchange of genes in Pseudomonas sp. HR199, another selection system had to be used Application come. With the "sacB selection system", the Cloned inactivated gene in a plasmid, which next to a Antibiotic resistance gene also has the sacB gene. After conjugative over Carrying this hybrid plasmid in a pseudomonad is the plasmid homologous recombination integrated into the genome at the point at which the intact gene (first "cross over"). In this way, a "heterogeneous noter "strain that has both an intact and a deletion inacti fourth gene, which are separated by the pHE55 DNA. This Strains have the resistance encoded by the vector and possess it an active sacB gene. By a second homologous recombination event (second "cross over"), the pHE55 DNA should now together with the intact gene be extracted from the genomic DNA. Through this recombination event, a strain is created that only has the inactivated gene. About that in addition, there is a loss of the pHE55-encoded antibiotic resistance and the sacB gene. If one strikes out strains on sucrose-containing media, Strains expressing the sacB gene are inhibited in growth because the gene product Sucrose converts to a polymer which is in the periplasm of the cells is accumulated. Cells that the sacB- No longer carrying genes are therefore not inhibited in growth. To one phenotypic selection option for the integration of the deletion inacti fourth gene, you don't exchange it for an intact gene, but one uses a strain in which the gene to be exchanged already exists "marked" by insertion of an Ω element. With a successful exchange the resulting strain loses the antibiotic encoded by the Ω element Resistance.

The inactivated gene fcsΔ were digested from plasmid pSKfcsΔ after PstI digestion isolated and ligated with PstI cut pHE55 DNA. The ligation approach was transformed to E. coli S17-1. The selection was made on LB containing tetracycline  Medium. Tetracycline-resistant transformants, their hybrid, were obtained plasmid (pHEfcsΔ) contained the inactivated gene fcsΔ.

The inactivated echΔ gene was removed from the plasmid after EcoRI digestion pSKechΔ isolated and treated with mung bean nuclease (generation of smooth Blunt ends). The fragment was cut with BamHI and Mung Bean nuclease treated pHE55 DNA ligated. The ligation approach was carried out according to E. coli S17-1 transformed. The selection was made on LB containing tetracycline Medium. Tetracycline-resistant transformants were obtained, the Hybrid plasmid (pHEechΔ) containing the inactivated echΔ gene.

The inactivated vdhΔ gene were removed from the plasmid after EcoRI digestion pSKvdhΔ isolated and treated with mung bean nuclease. The fragment was made with BamHI cut and mung bean nuclease treated pHE55 DNA ligated. The Ligation mix was transformed to E. coli S17-1. The selection was made on LB medium containing tetracycline. There were transformants resistant to tetracycline obtained, whose hybrid plasmid (pHEvdhΔ) contained the inactivated gene vdhΔ.

The inactivated gene aatΔ were digested from the plasmid after EcoRI digestion pSKaatΔ isolated and treated with mung bean nuclease. The fragment was made with BamHI cut and mung bean nuclease treated pHE55 DNA ligated. The Ligation mix was transformed to E. coli S17-1. The selection was made on LB medium containing tetracycline. There were transformants resistant to tetracycline obtained, whose hybrid plasmid (pHEaatΔ) contained the inactivated gene aatΔ.  

Example 6 Generation of mutants of the Pseudomonas sp. HR199 where specifically genes of eugenol catabolism by inserting an Ω element have been inactivated

The Pseudomonas sp. HR199 was used as a recipient in conjugation experiments used in which strains of E. coli S17-1 are used as donors containing 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 against the gene inactivated by Ω element insertion double "cross over") and "heterogenote" (integration of the hybrid plasmid into the Genome by simple "cross over") transconjugants could be determined by the pSUP202 encoded tetracycline resistance can be distinguished.

The mutants Pseudomonas sp. HR199 fcsΩKm and Pseudomonas sp. HR199 fcsΩGm were after conjugation of Pseudomonas sp. HR199 with E. coli S17-1 (pSUPfcsΩKm) or E. coli S17-1 (pSUPfcsΩGm). The exchange of the intact fcs gene against the gene inactivated by ΩKm or ΩGm (fcsΩKm or fcsΩGm) was verified using DNA sequencing.

The mutants Pseudomonas sp. HR199 echΩKm and Pseudomonas sp. HR199 echΩGm were after conjugation of Pseudomonas sp. HR199 with E. coli S17-1 (pSUPechΩKm) or E. coli S17-1 (pSUPechΩGm). The exchange of the intact ech gene against the gene inactivated by ΩKm or ΩGm (echΩKm or echΩGm) was verified using DNA sequencing.

The mutants Pseudomonas sp. HR199 vdhΩKm and Pseudomonas sp. HR199 vdh ΩGm were obtained after conjugation of Pseudomonas sp. HR199 with E. coli S17-1 (pSUPvdhΩKm) or E. coli S17-1 (pSUPvdhΩGm). The exchange of the  intact vdh gene against the gene inactivated by ΩKm or ΩGm (vdhΩKm or vdhΩGm) was verified using DNA sequencing.

The mutants Pseudomonas sp. HR199 aatΩKm and Pseudomonas sp. HR199 aatΩGm were after conjugation of Pseudomonas sp. HR199 with E. coli S17-1 (pSUPaatΩKm) or E. coli S17-1 (pSUPaatΩGm). The exchange of the intact aat gene against the gene inactivated by ΩKm or ΩGm (aatΩKm or aatΩGm) was verified using DNA sequencing.

The mutant Pseudomonas sp. HR199 fcsΩKmvdhΩGm were after conjugation from Pseudomonas sp. HR199 fcsΩKm obtained with E. coli S17-1 (pSUPvdhΩGm). Exchange of the intact vdh gene for the gene inactivated by DGm (vdhΩGm) was verified by DNA sequencing.

The mutant Pseudomonas sp. HR199 vdhΩKmaatΩGm were after conjugation from Pseudomonas sp. HR199 vdhΩKm obtained with E. coli S17-1 (pSUPaatΩGm). The exchange of the intact aat gene for the gene inactivated by ΩGm (aatΩGm) was verified by DNA sequencing.

The mutant Pseudomonas sp. HR199 vdhΩKmechΩGm were after conjugation from Pseudomonas sp. HR1999 vdhΩKm obtained with E. coli S17-1 (pSUPechΩGm). The exchange of the intact ech gene for the gene inactivated by ΩGm (echΩGm) was verified by DNA sequencing.  

Example 7 Generation of mutants of the Pseudomonas sp. HR199 where specifically genes of eugenol catabolism by deletion of a part have been inactivated

The Pseudomonas sp. HR199 fcsΩKm, Pseudomonas sp. HR199 echΩKm, Pseudomonas sp. HR199 vdhΩKm 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, the hybrid plasmids listed below of pHE55 contained. The "heterogeneous" transconjugants were on gluconate containing mineral medium, which encoded in addition to tetracycline (pHE55 Resistance) which contained the antibiotic corresponding to the Ω element. After streak transconjugants were obtained on mineral medium containing sucrose, which by a second recombination event (second "cross over") the vector DNA had been eliminated. By spreading on mineral medium without antibiotics or with the antibiotic corresponding to the Ω element, the mutants could are recognized in which the gene inactivated by Ω element against that by Deletion inactivated gene had been exchanged (no antibiotic resistance).

The mutant Pseudomonas sp. HR199 fcsΔ was obtained after conjugation of Pseudomo nas sp. HR199 fcsΩKm obtained with E. coli S17-1 (pHEfcsΔ). The exchange of the by ΩKm inactivated gene (fcsΩKm) against the deletion inactivated gene (fcsΔ) was verified using DNA sequencing.

The mutants Pseudomonas sp. HR199 echΔ was conjugated by pseudo monas sp. HR199 echΩKm obtained with E. coli S17-1 (pHEechΔ). The exchange of the gene inactivated by ΩKm (echΩKm) against that inactivated by deletion Gene (echΔ) was verified using DNA sequencing.  

The mutants Pseudomonas sp. HR199 vdhΔ was obtained after conjugation of Pseu domonas sp. HR199 vdhΩKm obtained with E. coli S17-1 (pHEvdhΔ). The exchange of the gene inactivated by ΩKm (vdhΩKm) against that inactivated by deletion Gene (vdhΔ) was verified using DNA sequencing.

The mutants Pseudomonas sp. HR199 aatΔ was after conjugation of pseudo monas sp. HR199 aatΩKm obtained with E. coli S17-1 (pHEaatΔ). The exchange of the by ΩKm inactivated gene (aatΩKm) against the deletion inactivated gene (aatΔ) was verified using DNA sequencing.

Example 8 Biotransformation of eugenol to vanillin with the mutant Pseudomonas sp. HR199 vdhΩKm

The Pseudomonas sp. HR199 vdhΩKm was in 50 ml HR-MM with 6 mM Eugenol attracted to an optical density of approx. OD600 nm = 0.6. After 17 h were 2.9 mM vanillin, 1.4 mM ferulic acid and 0.4 mM vanillic acid in the culture Detectable overhang.

Example 9 Biotransformation of eugenol to ferulic acid with the mutant Pseudomonas sp. HR199 vdhΩGmnatΩKm

The Pseudomonas sp. HR199 vdhΩGmaatΩKm was in 50 ml HR-MM with 6 mM eugenol attracted to an optical density of approx. OD600 nm = 0.6. After 18 hours, 1.9 mM vanillin, 2.4 mM ferulic acid and 0.6 mM vanillic acid detectable in the culture supernatant.  

Example 10 Biotransformation of eugenol to coniferyl alcohol with the mutant pseudo monas sp. HR199 vdhΩGmaatΩKm

The Pseudomonas sp. HR199 vdhΩGmaatΩKm was in 50 ml HR-mm with 6 mM eugenol attracted to an optical density of approx. OD600 nm = 0.4. After 15 hours there were 1.7 mM coniferyl alcohol, 1.4 mM vanillin, 1.4 mM ferulic acid and 0.2 mM vanillic acid detectable in the culture supernatant.

Example 11 Fermentative production of natural vanillin from eugenol in 10 l Fermenter with the mutant Pseudomonas sp. HR199 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 of 12.5 g / l glycerol, 10 g / l yeast extract and 0.37 g / l Acetic acid was drawn in, the production fermenter was inoculated. The fermenter contained 9.9 l of medium with the following composition: 1.5 g / l yeast extract, 1.6 g / l KH ₂ PO ₄ , 0.2 g / l NaCl, 0.2 g / l 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 aeration 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 was started with the continuous addition of Eugenol started, so that at the end of the fermentation after 255 hours 255 g euge nol had been added to the culture. In addition, during the fermentation 40 g of yeast extract added. The concentration of eugenol was at the end of the ferment mentation at 0.2 g / l. The vanillin content was 2.6 g / l. In addition, there were still 3.4 g / l ferulic acid before.  

The vanillin thus obtained can be obtained by known physical methods such as Chro matography, distillation and / or extraction isolated and for the production of natural Flavors are used.

Explanations to the figures

FIG. 1a to 1r:

Gene structures for the extraction of organisms and mutants

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

The restriction enzyme interfaces marked with "*" came for the construction used, 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 gene structure calAΔ

Fig. 2d: nucleotide sequence of the gene structure calBΩKm

Fig. 2e: nucleotide sequence of the gene structure calBΩGm

Fig. 2f; Nucleotide sequence of the calBΔ gene structure

Fig. 2g: nucleotide sequence of the gene structure fcsΩKm

Fig 2h. Nucleotide sequence of the gene structure csΩGm

Fig. 2i: nucleotide sequence of the gene structure fcsΔ

Fig. 2j: nucleotide sequence of the gene structure echΩKm

Fig. 2k: nucleotide sequence of the gene structure echΩGm

Fig. 2l: Nucleotide sequence of the gene structure echΔ

Fig. 2m: nucleotide sequence of the gene structure vdhΩKm

Figure 2n. Nucleotide sequence of the gene structure vdhΩGm

Fig. 2o: nucleotide sequence of the gene structure vdhΔ

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 aatΔ

Sequences

Sequence 1

Sequence 2

Sequence 3

Sequence 4

Sequence 5

Sequence 6

Sequence 7

Sequence 8

Sequence 9

Sequence 10

Sequence 11

Sequence 12

Sequence 13

Sequence 14

Sequence 15

Sequence 16

Sequence 17

Sequence 18

Claims (16)

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 the intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillinic acid he follows. 2. Organism according to claim 1, characterized in that the eugenol and / or ferulic acid catabolism by Ω element insertion or insertion ren of deletions is changed into corresponding genes. 3. Organism according to one of claims 1 or 2, characterized in that one or more genes responsible for the coniferyl alcohol dehydro recover, coniferylaldehyde dehydrogenases, ferulic acid-CoA synthetases, Enoyl-CoA hydratase aldolases, beta-ketothiolases, vanillin dehydro genase or encode vanillic acid demethylase enzymes, changed and / or are deactivated. 4. Organism according to one of claims 1 to 3, characterized in that he unicellular, preferably a microorganism or a vegetable or is 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 dehydroge for the enzymes nasal, coniferylaldehyde dehydrogenases, ferulic acid-CoA synthetases, Enoyl-CoA hydratase aldolases, beta-ketothiolases, vanillin dehydrogen nasal or vanillic acid demethylases or two or more of these enes zyme-encoding nucleotide sequences 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 the claims 6 to 8. 10. Transformed organism according to one of claims 1 to 5, characterized characterized in that it contains at least one vector according to claim 9. 11. Organism according to one of claims 1 to 5, characterized in that he at least one gene structure according to any one of claims 6 to 8 in place of the respective intact gene integrated in the genome. 12. Process for the biotechnical production of organic compounds, especially of 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 eugenol and / or ferula Acid catabolism using microbiological breeding known per se methods is achieved. 14. A method for producing an organism according to any 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 using genetic engineering methods.   15. Use of the organisms according to one of claims 1 to 5 or 10 to 11 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 of a vector according to claim 9 for the production of transformed and / or mutagenized organisms.