KR20020022045A - Construction of Production Strains for Producing Substituted Phenols by Specifically Inactivating Genes of the Eugenol and Ferulic Acid Catabolism - Google Patents

Abstract

Transformation and / or (or) is characterized by the inactivation of enzymes of eugenol and / or ferulic acid metabolism to accumulate intermediate coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillic acid. ) Mutated unicellular or multicellular organisms.

Description

Construction of Production Strains for Producing Substituted Phenols by Specifically Inactivating Genes of the Eugenol and Ferulic Acid Catabolism}

The present invention relates to the construction of a production strain for the production of substituted methoxyphenols, in particular vanillin, and to methods of making the same.

DE-A 4 227 076 (method for preparing substituted methoxyphenols and microorganisms suitable for this purpose) describes the preparation of substituted methoxyphenols using the novel Pseudomonas sp . The starting material in the context is eugenol and the products are ferulic acid, vanillic acid, coniphenyl alcohol and coniphenyl aldehyde.

An extensive review of possible in vivo transformations using ferularic acid, published in 1995 by Rosazza et al, Biocatalytic transformation of ferulic acid: an abundant aromatic natural product; J.Ind. Microbiol. 15: 457-471.

Genes and enzymes for synthesizing coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillic acid and vanillin from Pseudomonas species are described in EP-A 0 845 532.

Enzymes for converting trans-ferulic acid to trans-feruloyl-SCoA esters, and then vanillin, and genes for cleavage of esters are described in WO97 by the Institute of Food Research, Norwich, UK. / 35999. In 1998, the content of this patent was also issued in the form of a scientific publication [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 a novel isolated strain of Pseudomonas fluorescens. Pseudomonas fluorescens). Microbiology 144: 1397-1405.

DE-A 195 32 317 describes the use of Amycolatopsis sp . To obtain vanillin from ferulic acid in high yield by fermentation.

Known methods have the disadvantage of obtaining vanillin only in very low yields or using relatively expensive starting compounds. Although the method mentioned last (DE-A 19532 317) achieves high yields, the use of Pseudomonas spp. HR199 and Amicholatopsis spp. HR167 for in vivo conversion of eugenol to vanillin is carried out in two stages of fermentation. To do, and eventually cost substantially and time consuming.

Accordingly, it is an object of the present invention to construct an organism that can convert eugenol, a relatively cheap raw material, to vanillin in a one-step process.

The object is characterized by inactivating enzymes of eugenol and / or ferulic acid metabolism to accumulate intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillic acid. Achieved by means of building production lines of multicellular organisms.

Production lines may be unicellular or multicellular. Thus, the present invention may relate to microorganisms, plants or animals. Moreover, extracts obtained from the production week may be used. According to the invention, preference is given to using single cell organisms. The latter organisms can be microorganisms or animal or plant cells. According to the invention, particular preference is given to using fungi and bacteria. Bacterial species are most preferred. Eugenol and (or) after the page has been changed Lula acid metabolism decomposition, in particular bacteria which may be used are also co kusu (Rhodococcus), Pseudomonas (Pseudomonas) and Escherichia is Escherichia (Escherichia) species.

In the simplest case, known conventional microbiological methods can be used to isolate organisms that can be used in the present invention.

In other words, the enzymatic activity of proteins involved in eugenol and / or ferulanic acid metabolism can be altered by using enzyme inhibitors. In addition, the enzymatic activity of proteins involved in eugenol and / or ferulanic acid metabolism can be altered by mutating genes encoding such proteins. Such variations can be generated in a random manner by traditional methods, for example by using UV-irradiation or mutation-inducing chemicals.

Recombinant DNA methods such as deletion, insertion and / or nucleotide conversion are suitable for isolating novel organisms. That is, the gene of the microorganism can be inactivated using, for example, another DNA element (Ω element). Appropriate vectors can also be used to replace native genes with altered and / or inactivated gene structures. In this situation, the gene to be inactivated and the DNA element used for inactivation can be obtained by conventional cloning techniques or by polymerase chain reaction (PCR).

For example, in one possible embodiment of the present invention, eugenol and metabolism of metabolism can be altered by inserting an Ω element into the appropriate gene or by introducing a deletion. In such a situation, the above-mentioned recombinant DNA method for inactivating the function of a gene encoding dehydrogenase, synthetase, hydrolase-aldolase, thiolase or demethylase, such that the production of related enzymes is blocked. This can be used. Preferably, the gene is coniferyl alcohol dehydrogenase, coniferyl aldehyde dehydrogenase, feruloyl-CoA synthetase, enoyl-CoA hydrolase-aldolase, beta-ketothiolase, vanillin dehydrogenase or vanillic acid. It encodes a demethylase. Very particularly preferred are genes encoding the amino acid sequences specified in EP-A 0845532 and / or nucleotide sequences encoding allelic variations thereof.

Accordingly, the present invention also relates to the genetic structure for preparing transformed organisms and mutants.

In order to isolate organisms and mutants, it is preferred to use a gene structure in which the nucleotide sequence encoding dehydrogenase, synthetase, hydrolase-aldolase, thiolase or demethylase is inactivated. Encodes Coniferryl Alcohol Dehydrogenase, Coniferyl Aldehyde Dehydrogenase, Feruloyl-CoA Synthetase, Enoyl-CoA Hydrolase-Aldolase, Beta-Ketothiolase, Vanillin Dehydrogenase or Vanillin Demethylase Particular preference is given to gene structures in which the nucleotide sequence is inactivated. Very particular preference is given to the genetic structure exhibiting the structure given in FIGS. 1A-1R with the nucleotide sequence represented in FIGS. 2A-2B and / or the nucleotide sequence encoding allelic variation thereof. In this situation, nucleotide sequences 1-18 are particularly preferred.

The invention also includes functional equivalents as well as partial sequences of such gene structures. Functional equivalents are understood to mean derivatives of DNA in which each nucleobase has been altered (agitated) with its function unaltered. Amino acids can be changed at the protein level without alterations in function.

One or more DNA sequences may be inserted upstream and / or downstream of the gene structure. By cloning the gene structure, appropriate plasmids or vectors can be obtained for transformation and / or transfection of an organism and / or for conjugation transfer into an organism.

The invention also relates to plasmids and / or vectors for preparing the organisms and mutants transformed according to the invention. Such organisms and mutants eventually have the genetic structure described above. Thus, the present invention also relates to organisms having said plasmids and / or vectors.

The nature of the plasmids and / or vectors depends on what they are used for. For example, in order to be able to substitute natural genes of eugenol and / or ferulic acid metabolism in Pseudomonas bacteria with genes inactivated by omega elements, on the one hand they can be transferred into Pseudomonas spp. Transferable plasmids), on the other hand, require a vector (so-called suicide plasmid) that cannot be replicated in these organisms and is eventually unstable in Pseudomonas. If the DNA fragments are integrated into the genome of bacterial cells by homologous recombination, only DNA fragments transferred to Pseudomonas bacteria with the help of this plasmid system can be maintained.

The gene structures, vectors, and plasmids described above may also be used to prepare different transformed organisms or mutants. The gene construct can be used to replace the native nucleic acid sequence with altered and / or inactivated gene constructs. In cells obtainable by transformation or transfection or conjugation, native genes are replaced with genetic structures altered and / or inactivated by homologous recombination, and the resulting cells are altered in their genome and ( Or) only inactivated gene structure. In this way, genes can be preferably altered and / or inactivated in accordance with the invention, such that related organisms can produce coniferyl alcohol, coniferyl aldehyde, ferulaic acid, vanillin and / or vanillic acid. have.

Mutants of strain Pseudomonas species HR199 (DSM7063) described in detail in DE-A 4 227 076 and EP-A 0845532, in particular, have a corresponding genetic structure obtained from FIGS. 1A-1R in combination with FIGS. An example of a production strain constructed in this way according to the invention is:

1. Pseudomonas spp. HR199calAΩKm (Fig. 1a: Figs. 2aa to 2ac), containing ΩKm-inactivated calA gene instead of the native calA gene encoding coniferryl alcohol dehydrogenase.

2. Pseudomonas spp. HR199calAΩGm, containing ΩGm-inactivated calA gene in place of the native calA gene encoding coniferryl alcohol dehydrogenase (FIG. 1B: FIGS. 2B-2BB).

3. Pseudomonas spp. HR199calAΔ, containing a deletion-inactivated calA gene instead of the native calA gene encoding coniferryl alcohol dehydrogenase (FIG. 1C: FIG. 2C).

4. Pseudomonas spp. HR199calBΩKm, which contains the ΩKm-inactivated calB gene in place of the native calB gene encoding coniferryl aldehyde dehydrogenase (FIG. 1D: FIGS. 2D-2dd).

5. Pseudomonas spp. HR199calBΩGm (Fig. 1E: Figs. 2ea to 2ec), containing ΩGm-inactivated calB gene in place of the native calB gene encoding coniferryl aldehyde dehydrogenase.

6. Pseudomonas spp. HR199calBΔ (FIG. 1F: FIGS. 2F-2FB), which contains a deletion-inactivated calB gene in place of the native calB gene encoding coniferryl aldehyde dehydrogenase.

7. Pseudomonas spp. HR199fcsΩKm, containing the ΩKm-inactivated fcs gene instead of the native fcs gene encoding feruloyl-CoA synthase (FIG. 1G: FIGS. 2G-2GC)

8. Pseudomonas spp. HR199fcsΩGm (Fig. 1H: Figs. 2ha to 2hc), which contains the ΩGm-inactivating fcs gene instead of the native fcs gene encoding feruloyl-CoA synthase.

9. Pseudomonas spp. HR199fcsΔ, which contains a deletion-inactivated fcs gene instead of the native fcs gene encoding feruloyl-CoA synthetase (FIG. 1i: FIGS. 2ia to 2ib).

10. Pseudomonas spp. HR199echΩKm (FIG. 1J; Figs. 2J-2JC), containing the ΩKm-inactivated ech gene in place of the native ech gene encoding the anoyl-CoA hydrolase-aldolase.

11. Pseudomonas spp. HR199echΩGm (FIG. 1K; Figs. 2K to 2kc), containing ΩGm-inactivated ech gene instead of the native ech gene encoding enoyl-CoA hydrolase-aldolase.

12. Pseudomonas spp. HR199echΔ (FIG. 11; FIG. 21), which contains a deletion-inactivating ech gene instead of the native ech gene encoding enoyl-CoA hydrolase-aldolase.

13. Pseudomonas species HR199aatΩKm (FIG. 1M; Figs. 2MA to 2MD), containing the ΩKm-inactivated aat gene instead of the native aat gene encoding beta-ketothiolase.

14. Pseudomonas spp. HR199aatΩGm (FIG. 1n; Figs. 2na to 2nd), containing ΩGm-inactivated aat gene instead of native aat gene encoding beta-ketothiolase.

15. Pseudomonas spp. HR199aatΔ (FIG. 1O; Figs. 2Oa-2Oc), which contains a deletion-inactivating aat gene in place of the native aat gene encoding beta-ketothiolase.

16. Pseudomonas spp. HR199vdhΩKm (Fig. 1p; Figs. 2pa to 2pd), containing the ΩKm-inactivated vdh gene in place of the native vdh gene encoding vanillin dehydrogenase.

17. Pseudomonas spp. HR199vdhΩGm (Fig. 1q; Figs. 2qa to 2qd), containing ΩGm-inactivated vdh gene instead of the native vdh gene encoding vanillin dehydrogenase.

18. Pseudomonas spp. HR199vdhΔ, containing the deletion-inactivating vdh gene instead of the native vdh gene encoding vanillin dehydrogenase (FIG. 1R; FIGS. 2ra to 2rb).

19. Pseudomonas spp. HR199vdhBΩKm, containing ΩKm-inactivated vdhB gene instead of the native vdhB gene encoding vanillin dehydrogenase II.

20. Pseudomonas spp. HR199vdhBΩGm, containing ΩGm-inactivated vdhB gene instead of the native vdhB gene encoding vanillin dehydrogenase II.

21. Pseudomonas spp. HR199vdhBΔ, containing the deletion-inactivating vdhB gene instead of the native vdhB gene encoding vanillin dehydrogenase II.

22. Pseudomonas spp. HR199adhΩKm, which contains the ΩKm-inactivated adh gene instead of the native adh gene encoding alcohol dehydrogenase.

23. Pseudomonas species HR199adhΩGm, which contains the ΩGm-inactivated adh gene in place of the native adh gene encoding the alcohol dehydrogenase.

24. Pseudomonas spp. HR199adhΔ containing a deletion-inactivated adh gene in place of the native adh gene encoding alcohol dehydrogenase.

25. Pseudomonas spp. HR199vanAΩKm, which contains the ΩKm-inactivated vanA gene instead of the native vanA gene encoding the α-subunit of vanillin demethylase.

26. Pseudomonas spp. HR199vanAΩGm, which contains the ΩGm-inactivated vanA gene instead of the native vanA gene encoding the α-subunit of vanillin demethylase.

27. Pseudomonas spp. HR199vanAΔ, which contains a deletion-inactivated vanA gene in place of the native vanA gene encoding the α-subunit of vanillin demethylase.

28. Pseudomonas spp. HR199vanBΩKm, which contains the ΩKm-inactivated vanB gene in place of the native vanB gene encoding the β-subunit of vanillin demethylase.

29. Pseudomonas spp. HR199vanBΩGm, which contains the ΩGm-inactivated vanB gene instead of the native vanB gene encoding the β-subunit of vanillin demethylase.

30. Pseudomonas spp. HR199vanBΔ containing a deletion-inactivated vanB gene in place of the native vanB gene encoding the β-subunit of vanillin demethylase.

The invention further relates to a process for the biotechnological preparation of organic compounds. In particular, this method can be used to prepare alcohols, aldehydes and organic acids. The latter are preferably coniferyl alcohol, coniferyl aldehyde, ferulaic acid, vanillin and vanillic acid.

The organisms described above are used in novel methods. Very particularly preferred organisms include bacteria, in particular Pseudomonas species. Specifically, the aforementioned Pseudomonas species can preferably be used for the following method.

1. Pseudomonas species HR199calAΩKm, Pseudomonas species HR199calAΩGm, and Pseudomonas species HR199calAΔ, for producing coniferyl alcohol from eugenol.

2. Pseudomonas species HR199calBΩKm, Pseudomonas species HR199calBΩGm, and Pseudomonas species HR199calBΔ for preparing coniferyl aldehyde from eugenol or coniferyl alcohol.

3. Pseudomonas species HR199fcsΩKm, Pseudomonas species HR199fcsΩGm, Pseudomonas species HR199fcsΔ, Pseudomonas species HR199echΩKm, Pseudomonas species HR199echΔGm and Pseudomonas species HR199echΩGm and Pseudomonas species HR199echΩGm and

4. Pseudomonas species HR199vdhΩKm, Pseudomonas species HR199vdhΩGm, Pseudomonas species HR199vdhΔ, Pseudomonas species HR199vdhΩGmvdhBΩm199, Pseudomonas ssgdh ΩKm, Pseudomonas ΔMhsdhdh monass Pseudomonas species HR199vdhΔvdhBΩKm.

5. Pseudomonas species HR199vanAΩKm, Pseudomonas species HR199vanAΩGm, Pseudomonas species HR199vanAΩGm, Pseudomonas species HR199vanBΩKm species, Pseudomonas species, and Pseudomonas species .

Eugenol is a preferred substrate. However, it is also possible to add additional substrates or even replace eugenol with another substrate.

Suitable nutrient media for the organisms used according to the invention are synthetic, semisynthetic or complex culture media. Such media may also include carbon-containing and nitrogen-containing compounds, inorganic salts, where appropriate, trace elements and vitamins.

Carbon-containing compounds that may be suitable are carbohydrates, hydrocarbons or organic general compounds. Examples of compounds which may preferably be used are sugars, alcohols or sugar alcohols, organic acids or complex mixtures.

The sugar is preferably glucose. Organic acids which can preferably be used are citric acid or acetic acid. Examples of complex mixtures are malt extract, yeast extract, casein or casein hydrolysate.

Inorganic compounds are suitable nitrogen-containing substrates. Examples of these are nitrates and ammonium salts. Organic nitrogen sources can also be used. Such sources include yeast extract, soy flour, casein, casein hydrolysate and corn steep liquor.

Examples of inorganic salts that can be used are sulfates, nitrates, hydrochlorides, carbonates and phosphates. The metals contained in these salts are preferably sodium, potassium, magnesium, manganese, calcium, zinc and iron.

The temperature for the culture is preferably in the range of 5 to 100 ° C. The range of 15-60 degreeC is especially preferable, and 22-37 degreeC is the most preferable.

The pH of the medium is preferably 2 to 12. The range of 4-8 is especially preferable.

In principle, bioreactors known to those skilled in the art can be used to carry out the novel methods. Any apparatus suitable for the immersion method is first considered. This means that containers with or without mechanical mixing devices can be used according to the invention. Examples of the latter are shaking devices, and bubble column reactors or loop reactors. The former preferably includes all known instruments equipped with a stirrer of any design.

The new method can be carried out continuously or batchwise. The fermentation time required to achieve the maximum amount of product depends on the specific nature of the organism used. In principle, however, the fermentation time is 2 to 200 hours.

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

Mutants of the eugenol-using strain Pseudomonas species HR199 (DSM 7063) were generated in a targeted manner by specifically inactivating the genes of eugenol degradation metabolism by inserting omega elements or introducing deletions. The omega element used was a DNA fragment encoding resistance to the antibiotics kanamycin (ΩKm) and gentamicin (ΩGm). These resistance genes were isolated from Tn5 and plasmid pBBR1MCS-5 using standard methods. Coniferyl Alcohol Dehydrogenase, Coniferyl Aldehyde Dehydrogenase, Feruloyl-CoA Synthetase, Enoyl-CoA Hydrolase-Aldolase, Beta-Ketothiolase, Vanillin Dehydrogenase, Alcohol Dehydrogenase, Vanillin Dehydrogenase II And genes calA, calB, fcs, ech, aat, vdh, adh, vdhB, vanA and vanB encoding vanillin demethylases were isolated from genomic DNA of strain Pseudomonas species HR199 using standard methods, pBluescript) SK ^- cloned into. By digesting with appropriate restriction endonucleases, DNA fragments were removed (deleted) or substituted (inserted) with Ω elements from each of these genes to inactivate each gene. In this way the mutated gene is recloned into a vector transmissible by conjugation and then introduced into strain Pseudomonas species HR199. Appropriate screening methods were used to obtain conjugates in which each functional wild-type gene was replaced with a newly introduced inactivated gene. Insertion and deletion mutants obtained in this manner now have only their respective inactivated genes. Using this procedure, both mutants with only one missing gene and multiple mutants in which several genes were inactivated in this manner were obtained. These mutants

a) eugenol to coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillin acid,

b) coniferyl alcohol to coniferyl aldehyde, ferulic acid, vanillin and / or vanillin acid,

c) coniferyl aldehyde to ferulaic acid, vanillin and / or vanillic acid,

d) ferulic acid to vanillin and / or vanillic acid,

e) vanillin to vanillic acid

It was used to convert in vivo.

Materials and methods

Bacterial growth conditions

Strains of Escherichia coli were grown at 37 ° C. in Luria-Bertani (LB) or M9 mineral medium (J. Sambrook, EFFritsch and T. Maniatis. 1989. Molecular cloning: Experiment Guide, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Strains of Pseudomonas spp. May be grown in nutrient broth (NB, 0.8%, wt / vol) or in mineral medium (MM) (HGSchlegal et al., 1961, Arch. Mikrobiol, 38: 209-222) or HR mineral medium (HR-MM (J. Rabenhorst, 1996. Appl. Microbiol. Biotechnol. 46: 470-474) at 30 ° C. Ferulic acid, vanillin, vanillic acid and protocatecuic acid were dissolved in dimethyl sulfoxide and added to each medium to obtain a final concentration of 0.1% (wt / vol). Eugenol is added directly to the medium to obtain a final concentration of 0.1% (vol / vol), or applied to filter paper (round filter 595, Schleicher & Schuell, Dassel, Germany) on the lid of the MM agar plate It was. When the conjugates and mutants of Pseudomonas species were propagated, tetracycline, kanamycin and gentamycin were used at 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 analyzed either directly or by dilution with double distilled H ₂ O by high pressure liquid chromatography (Knauer HPLC). Chromatography was performed on Nucleosil 100 C18 (7 μm, 250 × 4 mm). 0.1% (vol / vol) formic acid and acetonitrile were used as solvent. The gradient route used to elute the material was as follows:

00:00-06:60 → 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

Purification was performed at 4 ° C.

Crude extract

Pseudomonas spp. HR199 cells grown in eugenol were washed in 10 mM sodium phosphate buffer (pH 6.0) and then resuspended in the same buffer and French press at a pressure of 1000 psi (Amicon, USA Collapsed by passing two passes through Silver Spring, Maryland. Cell homogenates were ultracentrifuged (1 h, 100,000 × g, 4 ° C.) to obtain a soluble fraction of the crude extract as 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 loaded on a DEAE-Sephacel column (2.6 cm × 35 cm, bed volume [BV]: 186 ml) equilibrated with 10 mM sodium phosphate buffer (pH 6.0) and with a flow rate of 0.8 ml / min. The column was rinsed with 2 BV of 10 mM sodium phosphate buffer, pH 6.0. Vanillin dehydrogenase II (VDH II) was eluted with a linear salt gradient of 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 combined to form a DEAE pool.

Determination of Vanillin Dehydrogenase Activity

The optical enzyme test was used to determine VDH activity at 30 ° C. The reaction mixture, 1 ml in volume, contains 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; pig) From the heart) and the enzyme solution. The oxidation of _vanillin was monitored at λ = 340 nm (ε _vanillin = 11.6 cm 2 / μmol). Enzyme activity is expressed in units (U), where 1U corresponds to the amount of enzyme converting 1 μmol of vanillin per minute. Protein concentration in the samples was determined using the method of Lowry et al. (0. H. Lowry, NJ Rosebrough, ALFarr and RJRandall, 1951, J. Biol. Chem. 193: 265-275).

Determination of Coniferyl Alcohol Dehydrogenase Activity

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

Determination of Coniferyl Aldehyde Dehydrogenase Activity

The optical enzyme test was used to determine CALDH activity at 30 ° C. The reaction mixture, 1 ml in volume, contained 0.1 millimoles of Tris / HCl (pH 8.8), 0.08 μmol of coniferyl aldehyde, 2.7 μmol of NAD and an enzyme solution. The oxidation of coniferyl aldehyde to ferulic acid was monitored at λ = 400 nm (ε = 34 cm 2 / μmol). Enzyme activity is expressed in units (U), where 1 U corresponds to the amount of enzyme converting 1 μmol substrate per minute. Protein concentration in the samples was determined using the method of Lowry et al. (O.H.Lowry, N.J.Rosebrough, A.L.Farr and R.J.Randall, 1951, J.Biol.Chem. 193: 265-275).

Determination of Feruloyl-CoA Synthetase (ferulic Acid Thiokinase) Activity

FCS activity was determined at 30 ° C. using an optical enzyme test which is a variation of the method of Genk et al. (Zenk et al., 1980. Anal. Biochem. 101: 182-187). The 1 ml volume reaction mixture 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 an enzyme solution. . The formation of CoA esters from ferulic acid was monitored at λ = 345 nm (ε = 10 cm 2 / μmol). Enzyme activity is expressed in units (U), where 1 U corresponds to the amount of enzyme converting 1 μmol substrate per minute. Protein concentration in the samples was determined using the method of Lowry et al. (OHLowry, NJ Rosebrough, ALFarr and RJRandall, 1951, J. Biol. Chem. 193: 265-275).

Electrophoresis method

Method of Ramley under native conditions and in a 7.4% (wt / vol) polyacrylamide gel using the method of Stegemann et al. (Stegemann et al., 1973. Z. Naturalforsch. 28c: 722-732) (Laemli, UK 1970 Nature (London) 227: 680-685) was used to fractionate protein-containing extracts under denaturing conditions on a 11.5% (wt / vol) polyacrylamide gel. Serva Blue R was used for non-specific protein staining. To specifically stain coniferyl alcohol dehydrogenase, coniferyl aldehyde dehydrogenase and vanillin dehydrogenase, the gel was rebuffered in 100 mM KP buffer (pH 7.0) for 20 minutes and then 0.08% (wt / vol). NAD, 0.04% (wt / vol) p-nitro blue tetrazolium chloride, 0.03% (wt / vol) phenazine methosulfate and 1 mM of each substrate were added until the corresponding color band was seen. Incubated at 30 ° C.

Protein Transfer from Polyacrylamide Gel to PVDF Membrane

According to the manufacturer's instructions, the protein was transferred from the SDS-polyacrylamide gel using a semidry Fastblot apparatus (B32 / 33, Biuttra, Kutingen, Germany) (Waters-Millipore). (Waters-Millipore), Bedford, Massachusetts).

Determination of the N-terminal Amino Acid Sequence

N-terminal amino acid sequences were determined using a protein peptide sequencer (type 477A, Applied Biosystems, Foster City, USA) and PTH analyzer according to the manufacturer's instructions.

Isolation and Manipulation of DNA

Genomic DNA was isolated using the method of Marmur (J. Marmur, 1961. J. Mol. Biol. 3: 208-218). Other plasmid DNA and / or DNA restriction enzyme fragments were isolated and analyzed using standard methods (JESambrook, F. Frisch and T. Maniatis. 1989. Molecular cloning: Experiment Guide. Second Edition, Cold Spring Harbor Laboratory) Press, Cold Spring Habor, New York).

DNA transfer

Competent Escherichia coli cells were prepared and transformed using one method (D. Hanahan, 1983. J. Mol. Biol. 166: 557-580). Conjugated plasmid transfer between plasmid-containing Escherichia coli S17-1 strain (donor) and Pseudomonas species strain (receptor) was performed by Friedrich et al. (B. Friedrich et al., 1981. J. Bacteriol. 147: 198-205). According to the "minicomplementation method" in NB agar plates or in MM agar plates containing 0.5% (wt / vol) gluconate and 25 μg tetracycline / ml or 100 μg kanamycin / ml as carbon source. Was performed. In this case, the cells of the receptor were applied in one direction as inoculation smears. After 5 minutes, the cells of the donor strain were applied as inoculation smears while crossing the receptor inoculation smears. After 48 hours of incubation at 30 ° C., the conjugate was grown directly downstream of the crossover site, whereas neither the donor strain nor the receptor strain could grow.

Hybrid Formation Experiment

DNA restriction enzyme fractions were fractionated by electrophoresis in 50 mM Tris-50 mM boric acid-1.25 mM EDTA buffer (pH 8.5) on 0.8% (wt / vol) agarose gel (JESambrook, F.Fritsch and T.Maniatis). 1989. Molecular cloning: Experiment Guide, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Denatured DNA is transferred from the gel to a positive charge nylon membrane (pore size: 0.45 μm, Pall Filtrationstechnik, Dray, Germany), followed by hybridization with biotinylated or digoxygenin-labeled DNA probes And the preparation of these DNA probes was all performed using standard methods (JESambrook, F. Frisch and T. Manatis. 1989. Molecular cloning: Experiment Guide. Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor , New York).

DNA sequencing

According to the dideoxy chain termination method of Sanger et al. (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463-5467), “LI-COR” DNA sequencer model 4000L ”(LI-COR Inc.) , Department of Biotechnology, Lincoln, Nebraska, USA, and a 'thermo sequenase fluorescently labeled primer cycle sequencing kit using 7-deaza-dGTP' (Amersham Life Science, Armor) A sham sequences were determined "non-radioactive" in each case according to manufacturer's instructions using Amersham International plc., Buckinghamshire Little Challenge, UK.

Sequencing was performed using synthetic oligonucleotides according to the "primer-hopping method" of Strauss et al. (E. C. Strauss et al., 1986. Anal. Biochem. 154: 353-360).

Chemicals, Biochemicals and Enzymes

Restriction enzymes, T4 DNA ligase, lambda DNA and enzymes and substrates for optical enzyme testing. Purchased from Boehringer & Sohne (Mannheim, Germany) or GIBCO / BRL (Egenstein, Germany). [γ- ³² P] ATP was purchased from Amersham / Buchler (Braunschwig, Germany). Oligonucleotides were purchased from MWG-Biotech GmbH (Ebersberg, Germany). NA type agarose was purchased from Pharmacia-LKB (Sweden Uppsala). All other chemicals include Haarmann & Reimer (Holzminden, Germany), E.Merck AG (Damstadt, Germany), Fluka Chemie (Switzerland Bush), It was purchased from Serva Feinbiochemica (Heidelberg, Germany) or Sigma Chemie (Deissenhofen, Germany).

Example 1

Construction of Omega Elements Mediating Resistance to Kanamycin (ΩKm) or Gentamicin (ΩGm)

To construct the ΩKm element, Transposons Tn5 (EA Auerswald, G. Ludwig and H. Schaller. 1981. Cold Spring Harb. Symp. Quant. Biol. 45: 107-113; E. Beck. G. Ludwig, 1982. Genes 19: 327-336; P. Mazodier, P. Cossart, E. Giraud and F. Gasser. 1985. Nucleic Acids Res. 13: 195-205). 2099 bp BglI Fragments were isolated on a preparation scale. The fragment was shortened to about 990 bp by treatment with Bal 31 nuclease. Fragments containing only kanamycin resistance gene (encoding aminoglycoside-3′-0-phosphotransferase) now contain SmaI-cleaved pSKsym DNA (symmetrically constructed multiple cloning sites [SalI, HindIII, EcoRI, SmaI, EcoRI was ligated to the derivative) ^-, p blue script SK containing the HindIII, SalI]. From the resulting plasmids, ΩKm elements could be cut out as SmaI fragments, EcoRI fragments, HindIII fragments or SalI fragments.

To construct the ΩGm element, a 983 bp EaeI fragment of plasmid pBR1MCS-5 (MEKovach, PHElzer, DSHill, GTRobertson, MAFarris, RMRoop and KMPeterson 1995. Genes 166: 175-176) was prepared on a preparation scale. Isolated and then treated with mung bean nuclease (gradual digestion of single stranded DNA molecule ends). This fragment, which now contains only the gentamicin resistance gene (coding for gentamicin-3-acetyltransferase), was ligated to the SmaI-cleaved pSKsym DNA (see above). From the obtained plasmid, OMEGA Gm elements could be cut out as SmaI fragments, EcoRI fragments, HindIII fragments or SalI fragments.

Example 2

Gene Cloning from Pseudomonas Species HR199 (DSM7063) Inactivated by Inserting a Urea Element or by Deletion

The fcs, ech, vdh and aat genes from E. coli S17-1 strains DSM 10439 and DSM 10440 were cloned separately using plasmids pE207 and pE5-1 (see EP-A 0845532). Fragments determined from these plasmids were isolated on a preparative scale and treated as described below:

To clone the fcs gene, 2350 bp SalI / EcoRI fragments from plasmid pE207 and 3700 bp EcoRI / SalI fragments from plasmid pE5-1 were cloned together into pBluescript SK ⁻ , so that the two fragments together by EcoRI ends Combined. The 6050 bp SalI fragment was isolated from the obtained hybrid plasmid on the preparation scale, and shortened to about 2480 bp by treatment with Bal 31 nuclease. The PstI linker was then ligated to the ends of the fragment, digested with PstI, and then the fragment was cloned into pBluescript SK ⁻ (pSKfcs). After transformation of E. coli XL1 blue, a clone was obtained that expressed the fcs gene and exhibited the FCS activity of 0.2 U / mg of protein.

To clone the ech gene, the 3800 bp HindIII / EcoRI fragment from plasmid pE207 was isolated on the preparation scale and shortened to about 1470 bp by treatment with Bal31 nuclease. EcoRI linkers were then ligated to the ends of the fragments, digested with EcoRI, and then fragments cloned into pBluescript SK ⁻ (pSKech).

To clone the vdh gene, 2350 bp SalI / EcoRI fragments from plasmid pE207 were isolated on the preparation scale. After cloning in pBlueScript SK ⁻ , fragments were truncated at one end at about 1530 bp using the Exonuclease III / Mung bean nuclease system. EcoRI linkers were then ligated to the ends of the fragments, digested with EcoRI, and then the fragments were cloned into pBlueScript SK ⁻ (pSKvdh). After transformation of E. coli XL1 blue, clones were expressed that express the VDH gene and exhibit VDH activity of 0.01 U / mg of protein.

To clone the aat gene, 3700 bp EcoRI / SalI fragment from plasmid pE5-1 was isolated on the preparation scale and shortened to about 1590 bp by treatment with Bal 31 nuclease. EcoRI linkers were then ligated to the ends of the fragments, digested with EcoRI, and then fragments cloned into pBluescript SK ⁻ (pSKaat).

Example 3

Inactivation of the above-described genes by inserting Ω elements or by deleting the constituent regions of the genes

Plasmid pSKfcs containing the fcs gene was digested with BssHII to obtain a 1290 bp fragment cleaved from the fcs gene. After re ligation, deletion derivatives of the fcs gene (fcs Δ) (see FIGS. 1I and 2I) were obtained in cloned form in pBluescript SK ⁻ (pSKfcsΔ). In addition, after the fragment was cut out, the omega elements ΩKm and ΩGm were ligated in place. Thus, Ω-inactivating derivatives of the fcs gene (fcsΩKm, see FIGS. 1G and 2G) and (fcsΩGm, see FIGS. 1H and 2H) were obtained in the form cloned in pBluescript SK ⁻ (pSKfcsΩKm and pSKfcsΩGm). FCS activity could not be detected in the crude extract of the obtained E. coli clone, in which the hybrid plasmid had the fcs gene inactivated by deletion or Ω element insertion.

Plasmid pSKech containing ech gene was digested with NruI to obtain 53 bp fragment and 430 bp fragment cut out from ech gene. After religation, deletion derivatives of the ech gene (echΔ, see FIGS. 1L and 2L) were obtained in the form cloned in pBluescript SK ⁻ (pSKechΔ). In addition, after the fragment was cut out, the omega elements ΩKm and ΩGm were ligated in place. Thereby, Ω-inactivated derivatives of the ech gene (echΩKm and echΩGm) were obtained in the form cloned into pBlueScript SK ⁻ (pSKechΩKm and pSKechΩGm).

Plasmid pSKvdh containing the vdh gene was digested with BssHII to cut 210 bp fragments from the vdh gene. After re-ligation, deletion derivatives of the vdh gene (vdh Δ, see FIGS. 1O and 2oa-2oc) were obtained in the form cloned in pBluescript SK ⁻ (pSKvdhΔ). In addition, after the fragment was cut out, the omega elements ΩKm and ΩGm were ligated in place. This gave Ω-inactivating derivatives (vdhΩKm and vdhΩGm) of the vdh gene in the form cloned into pBluescript SK ⁻ (pSKvdhΩ Km, see FIGS. 1m and 2m) and (pSKvdhΩGm, see FIGS. 1n and 2n). It was not possible to detect VDH activity in crude extracts of the obtained E. coli clones in which the hybrid plasmid had the vdh gene inactivated either by deletion or by Ω element insertion.

The plasmid pSKaat containing the aat gene was digested with BssHII to cut 59 bp fragments from the aat gene. After re ligation, deletion derivatives of the aat gene (aatΔ, see FIGS. 1R and 2R) were obtained in the form cloned in pBluescript SK ⁻ (pSKaatΔ). In addition, after the fragment was cut out, the omega elements ΩKm and ΩGm were ligated in place. Thereby, Ω-inactivated derivatives of the aat gene (aatΩKm, see FIGS. 1P and 2P) and (aatΩGm, see FIGS. 1Q and 2Q) were obtained in the form cloned into pBluescript SK ⁻ (pSKaatΩKm and pSKaatΩGm).

Example 4

Subcloning of Ω-Inactivation Genes into a Splicing Transferable "Suicide Plasmid" pSUP202

In order to be able to replace native genes in Pseudomonas species HR199 with Ω-element inactivating genes, on the one hand they are transferable into Pseudomonas genus (plasmids that can be conjugated), but on the other hand they cannot be replicated in bacteria and eventually There is a need for unstable ("suicide plasmid") vectors in Pseudomonas spp. DNA fragments transferred into Pseudomonas bacteria using this plasmid system can only be maintained when integrated into the genome of bacterial cells by homologous recombination (RecA-dependent recombination). In this case, the "suicide plasmid" pSUP202 (Simom et al., 1983. In: A. Puhler. Molecular genetics of the bacteria-plant interaction. Springer Verlag, Berlin, Heidelberg, New York, pp. 98-106) was used.

After digestion with PstI, the inactivated genes fcsΩKm and fcsΩGm were isolated from plasmids pSKfcsΩKm and pSKfcsΩGm and ligated to PstI-cleaved pSUP202 DNA. The ligation mixture was transformed into E. coli S17-1. Selection was performed in tetracycline-containing LB medium, which also contained kanamycin or gentamicin, respectively. A kanamycin-resistant transformant in which the hybrid plasmid (pSUPfcsΩKm) contains the inactivating gene fcsΩKm was obtained. The corresponding hybrid plasmid of gentamicin-resistant transformants (pSUPfcsΩGm) contained the inactivated gene fcsΩGm.

After EcoRI digestion, the inactivated genes echΩKm and echΩGm were isolated from plasmids pSKechΩKm and pSKechΩGm and ligated to EcoRI-cleaved pSUP202 DNA. The ligation mixture was transformed into E. coli S17-1. Screening was performed on tetracycline-containing LB medium, which also contained kanamycin or gentamicin, respectively. Kanamycin-resistant transformants containing the gene echΩKm in which the hybrid plasmid (pSUPechΩKm) was inactivated were obtained. The corresponding hybrid plasmid (pSUPechΩGm) of gentamicin-resistant transformants contained the inactivating gene echΩGm.

After EcoRI digestion, the inactivation genes vdhΩKm and vdhΩGm were isolated from plasmids pSKvdhΩKm and pSKvdhΩGm and ligated to EcoRI-cleaved pSUP202 DNA. The ligation mixture was transformed into E. coli S17-1. Screening was performed on tetracycline-containing LB medium, which also contained kanamycin or gentamicin, respectively. Kanamycin-resistant transformants containing the gene vdhΩKm in which the hybrid plasmid (pSUPvdhΩKm) was inactivated were obtained. The corresponding hybrid plasmid (PSUPvdhΩGm) of gentamicin-resistant transformants contained the inactivated gene vdhΩGm.

After EcoRI digestion, the inactivation genes aatΩKm and aatΩGm were isolated from plasmids pSKaatΩKm and PSKaatΩGm and ligated to EcoRI-cleaved pSUP202 DNA. The ligation mixture was transformed into E. coli S17-1. Selection was performed in tetracycline-containing LB medium, which also contained kanamycin or gentamicin, respectively. Kanamycin-resistant transformants containing the gene aatΩKm in which the hybrid plasmid (pSUPaatΩKm) was inactivated were obtained. The corresponding hybrid plasmid (pSUPaatΩGm) of gentamicin-resistant transformants contained the inactivated gene aatΩGm.

Example 5

Subcloning of a Deletion-Inactivated Gene into a Splicing Transferable "Suicide Plasmid" PHE55 with a "sacB Selection System"

In order to be able to replace the native gene in Pseudomonas species HR199 with a deletion-inactivated gene, a vector with the properties already described in the case of pSUP202 is required. Unlike Ωelement-inactivated genes, there is no possibility of screening for successful substitution of genes in Pseudomonas spp. HR199 for deletion-inactivated genes (no antibiotic resistance), so other screening systems Had to be used. In the "sacB selection system", the deletion-inactivated gene to be replaced is cloned into a plasmid with the sacB gene in addition to the antibiotic resistance gene. Such hybrid plasmids are conjugated and transferred into Pseudomonas bacteria, and then the plasmids are integrated by homologous recombination at the site in the genome where the native gene is located (first crossing). This results in a “heterogene” monarch with both native and deletion-inactivated genes, which are separated from each other by pHE55DNA. These strains are encoded by the vector and also exhibit resistance with the active sacB gene. The next goal is to separate the pHE55 DNA from the genomic DNA by a second homologous recombination event (second crossing), along with the native gene. A strain having only the genes inactivated by this recombination event is obtained. In addition, both pHE55-encoded antibiotic resistance and the sacB gene are lost. If the strain is plated on sucrose-containing medium, the growth of the strain expressing the sacB gene is inhibited because the gene product converts the sucrose into a polymer and accumulates in the periplasm of the cell. As a result, the growth of cells that no longer carry the sacB gene as a result of the second recombination event is not inhibited. In order to be able to select by phenotype for integration of a deletion-inactivated gene, this gene does not replace the native gene; Instead, use strains in which the gene to be replaced has already been "labeled" by insertion of the Ω element. In the event of successful substitution, the resulting strain loses antibiotic resistance encoded by the Ω element.

After digestion with PstI, the inactivated gene fcsΔ was isolated from plasmid pSKfcsΔ and ligated to PstI-cleaved pHE55 DNA. The ligation mixture was transformed into E. coli S17-1. Selection was performed on tetracycline-containing LB medium. Tetracycline-resistant transformants containing the gene fcsΔ in which the hybrid plasmid (pHEfcsΔ) was inactivated were obtained.

After digestion with EcoRI, the inactivated gene echΔ was isolated from plasmid pSKechΔ and treated with mung bean nuclease (generation of blunt ends). The fragment was ligated to BamHI-cleaved and mung bean nuclease-treated pHE55 DNA. The ligation mixture was transformed into E. coli S17-1. Selection was performed on tetracycline-containing LB medium. Tetracycline-resistant transformants containing the gene echΔ in which the hybrid plasmid (pHEechΔ) was inactivated were obtained.

After digestion with EcoRI, the inactivated gene vdhΔ was isolated from plasmid pSKvdhΔ and treated with mung bean nuclease. The fragment was ligated to BamHI-cleaved and mung bean nuclease-treated pHE55 DNA. The ligation mixture was transformed into E. coli S17-1. Selection was performed on tetracycline-containing LB medium. Tetracycline-resistant transformants containing the gene vdhA in which the hybrid plasmid (PHEvdhΔ) was inactivated were obtained.

After digestion with EcoRI, the inactivated gene aatΔ was isolated from plasmid pSKaatΔ and treated with mung bean nuclease. The fragment was ligated to BamHI-cleaved and mung bean nuclease-treated pHE55 DNA. The ligation mixture was transformed into E. coli S17-1. Selection was performed on tetracycline-containing LB medium. A tetracycline-resistant transformant containing the gene aatΔ in which the hybrid plasmid (pHEaatΔ) was inactivated was obtained.

Example 6

Generation of mutants of the strain Pseudomonas spp. H199, in which the gene of eugenol degradation metabolism was specifically inactivated by the insertion of the Ω element

The strain Pseudomonas species HR199 was used as the receptor in conjugation experiments in which an E. coli S17-1 strain with a hybrid plasmid of pSUP202 described below was used as donor. Conjugates were selected in gluconate-containing mineral medium containing antibiotics corresponding to Ωurea. Based on pSUP202-encoded tetracycline resistance, "isogenes" (substituting native genes with Ω element insertion-inactivated genes by double crossover) and "heterogenes" (hybrid plasmids by single crossover genome) Integrated into) the splicing body could be distinguished.

After conjugation of Pseudomonas species HR199 with E. coli S17-1 (pSUPfcsΩKm) and E. coli S17-1 (pSUPfcsΩGm), respectively, mutant Pseudomonas species HR199 fcsΩKm and Pseudomonas species HR199 fcsΩGm were obtained. Substitution of the native fcs gene with ΩKm-inactivation or ΩGm inactivation genes (fcsΩKm and fcsΩGm, respectively) was demonstrated by DNA sequencing.

After conjugation of Pseudomonas species HR199 with E. coli S17-1 (pSUPechΩKm) and E. coli S17-1 (pSUPechΩGm), respectively, mutant Pseudomonas species HR199 echΩKm and Pseudomonas species HR199 echΩGm were obtained. Substitution of the native ech gene by the ΩKm-inactivated or ΩGm-inactivated genes (echΩKm and echΩGm, respectively) was demonstrated by DNA sequencing.

After conjugation of Pseudomonas species HR199 with E. coli S17-1 (pSUPvdhΩKm) and E. coli S17-1 (pSUPvdhΩGm), respectively, mutant Pseudomonas species HR199 vdhΩKm and Pseudomonas species HR199 vdhΩGm were obtained. Substitution of the native vdh gene with the ΩKm-inactivated or ΩGm-inactivated genes (vdhΩKm and vdhΩGm, respectively) was demonstrated by DNA sequencing.

After conjugation of Pseudomonas species HR199 with E. coli S17-1 (pSUPaatΩKm) and E. coli S17-1 (pSUPaatΩGm), respectively, mutant Pseudomonas species HR199 aatΩKm and Pseudomonas species HR199 aatΩGm were obtained. Substitution of the native aat gene with the ΩKm-inactivated or ΩGm-inactivated genes (aatΩKm and aatΩGm, respectively) was demonstrated by DNA sequencing.

After conjugation of Pseudomonas species HR199 fcsΩKm with E. coli S17-1 (pSUPvdhΩGm), the mutant Pseudomonas species HR199 fcsΩKmvdhΩGm was obtained. Substitution of the native vdh gene with the ΩGm-inactivating gene (vdhΩGm) was demonstrated by DNA sequencing.

After conjugation of Pseudomonas species HR199 vdhΩKm with E. coli S17-1 (pSUPaatΩGm), the mutant Pseudomonas species HR199 vdhΩKmaatΩGm was obtained. Substitution of the native aat gene with the ΩGm-inactivated gene (aatΩGm) was demonstrated by DNA sequencing.

After conjugation of Pseudomonas species HR199 vdhΩKm with E. coli S17-1 (pSUPechΩGm), the mutant Pseudomonas species HR199 vdhΩKmechΩGm was obtained. Substitution of the native ech gene by the ΩGm-inactivating gene (echΩGm) was demonstrated by DNA sequencing.

Example 7

Generation of Mutants of Strain Pseudomonas Species HR199 With Specific Deactivation of Eugenol Metabolism Gene by Deletion of Constituent Regions

In the conjugation experiments, strains Pseudomonas species HR199 fcsΩKm, Pseudomonas species HR199 echΩKm, Pseudomonas species HR199 vdhΩKm, and Pseudomonas species HR199 aatΩKm were used as acceptors, and strains of E. coli S17-1 having a hybrid plasmid of pHE55 described below were used as donors. . "Heterogene" conjugates were selected in gluconate-containing mineral medium which also contains antibiotics corresponding to the urea element in addition to tetracycline (pHE55-coding resistance). After plating on sucrose-containing mineral medium, a transformant with vector DNA removed by a second recombination event (second crossover) was obtained. After plating on mineral medium that does not have antibiotics or contains antibiotics corresponding to the Ω element, mutants (without antibiotic resistance) in which the urea-inactivated gene is replaced with a deletion-inactivated gene can be identified. there was.

The mutant Pseudomonas species HR199 fcsΔ was obtained after conjugation of Pseudomonas species HR199 fcsΩKm with E. coli S17-1 (pHEfcsΔ). Substitution of the ΩKm inactivating gene (fcsΩKm) with the deletion-inactivating gene (fcsΔ) was demonstrated by DNA sequencing.

The mutant Pseudomonas species HR199 echΔ was obtained after conjugation of Pseudomonas species HR199 echΩKm with E. coli S17-1 (pHEechΔ). Substitution of the ΩKm inactivating gene (echΩKm) with the deletion-inactivating gene (echΔ) was demonstrated by DHA sequencing.

The mutant Pseudomonas species HR199 vdhΔ was obtained after conjugation of Pseudomonas species HR199 vdhΩKm with E. coli S17-1 (pHEvdhΔ). Substitution of the ΩKm inactivation gene (vdhΩKm) with the deletion-inactivation gene (vdhΔ) was demonstrated by DNA sequencing.

The mutant Pseudomonas species HR199 aatΔ was obtained after conjugation of Pseudomonas species HR199 aatΩKm with E. coli S17-1 (pHEaatΔ). Substitution of the ΩKm inactivating gene (aatΩKm) with the deletion-inactivating gene (aatΔ) was demonstrated by DNA sequencing.

Example 8

In vivo conversion of eugenol to vanillin using mutant Pseudomonas species HR199 vdhΩKm

Strain Pseudomonas spp. HR199 vdhΩKm was grown to an optical density of about OD600 nm = 0.6 in 50 ml of HR-MM containing 6 mM eugenol. After 17 hours, 2.9 mM vanillin, 1.4 mM ferulic acid and 0.4 mM vanillin acid could be detected in the culture supernatant.

Example 9

In vivo conversion of eugenol to ferulic acid using mutant Pseudomonas species HR199 vdhΩGmaatΩKm

Strain Pseudomonas spp. HR199 vdhΩGmaatΩKm was propagated in an optical density of about OD600 nm = 0.6 in 50 ml of HR-MM containing 6 mM eugenol. After 18 hours, 1.9 mM vanillin, 2.4 mM ferulic acid and 0.6 mM vanillin acid could be detected in the culture supernatant.

Example 10

In vivo conversion of eugenol to coniferyl alcohol using mutant Pseudomonas species HR199 VdhΩGmaatΩKm

Strain Pseudomonas spp. HR199 vdhΩGmaatΩKm was propagated in an optical density of about OD600 nm = 0.4 in 50 ml of HR-MM containing 6 mM eugenol. After 15 hours, 1.7 mM coniferyl alcohol, 1.4 mM vanillin, 1.4 mM ferulic acid and 0.2 mM vanillin acid could be detected in the culture supernatant.

Example 11

Fermentation Production of Natural Vanillin from Eugenol in a 10 L Fermentor Using Mutant Pseudomonas Species HR199 vdhΩKm

24-hour preculture grown at 32 ° C. in a shake incubator (120 rpm) adjusted to pH 7.0 and grown in medium consisting of 12.5 g glycerol / l, 10 g yeast extract / l and 0.37 g acetic acid / l 100 ml were inoculated into the production fermenter. The fermentor contained 9.9 l of medium of the following composition: 1.5 g yeast extract / l, 1.6 g KH ₂ PO ₄ / l, 0.2 g NaCl / l, 0.2 g MgSO ₄ / l. 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 was 3 Nl / min, and the stirring speed was 600 rpm. The pH was maintained at pH 6.5 with sodium hydroxide solution.

At 4 hours post inoculation, the continuous addition of eugenol was started to allow 255 g of eugenol to be added to the culture at the end of fermentation after 65 hours. 40 g yeast extract was also fed during fermentation. At the end of the fermentation, the concentration of eugenol was 0.2 g / l. The content of vanillin was 2.6 g / l. 3.4 g of ferulaic acid / l were also present.

Vanillin obtained in this way can be isolated by known physical methods such as chromatography, distillation and / or extraction and can be used to prepare natural flavorings.

Explanation of symbols concerning drawing

1A-1R:

Genetic structure for isolating organisms and mutants

calA ^* : part of the inactivated gene for coniferyl alcohol dehydrogenase

calB ^* : part of the inactivated gene for coniferyl aldehyde dehydrogenase

fcs ^* : part of inactivated gene for feruloyl-CoA synthase

ech ^* : part of inactivation gene for Enoyl-CoA hydrolase-aldolase

vdh ^* : part of inactivated gene for vanillin dehydrogenase

aat ^* : part of inactivated gene for beta-ketothiolase

Restriction enzyme cleavage sites labeled with "*" were used for construction, while they no longer function in the resulting constructs.

2A-2AC: Nucleotide Sequence of calAΩKm Gene Structure

2B-2B: Nucleotide Sequence of calAΩGm Gene Structure

2C: Nucleotide sequence of calAΔ gene structure

2D-2DD: Nucleotide Sequence of calBΩKm Gene Structure

2ea-2ec: nucleotide sequence of the calBΩGm gene structure

Figure 2fa-2fb: Nucleotide sequence of the calBΔ gene structure

2ga-2gc: Nucleotide sequence of the fcsΩKm gene structure

2ha-2hc: nucleotide sequence of the fcsΩGm gene structure

2ia to 2ib: Nucleotide sequence of the fcsΔ gene structure

2ja to 2jc: Nucleotide sequence of the echΩKm gene structure

2ka-2kc: Nucleotide sequence of the echΩGm gene structure

Figure 2L: Nucleotide sequence of the echΔ gene structure

2M-2MD: Nucleotide sequence of vdhΩKm gene structure

2na-2nd: Nucleotide sequence of vdhΩGm gene structure

2oa-2oc: Nucleotide sequence of vdhΔ gene structure

Figures 2pa-2pd: Nucleotide sequence of the aatΩKm gene structure

Figures 2qa-2qd: Nucleotide sequence of the aatΩGm gene structure

2ra-2rb: Nucleotide sequence of the aatΔ gene structure

Claims (16)

Transformed and / or mutated single cell, characterized by inactivation of enzymes of eugenol and / or ferulic acid metabolism to accumulate intermediate coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillic acid. Or multicellular organisms. The organism of claim 1, wherein the eugenol and / or ferulanic acid metabolism is altered by inserting an Ω element into the corresponding gene or by introducing a deletion. 3. Enzyme Coniferyl alcohol dehydrogenase, Coniferyl aldehyde dehydrogenase, Feruloyl-CoA synthase, Enoyl-CoA hydrolase-aldolase, Beta-ketothiolase, Vanillin according to claim 1 or 2 An organism characterized in that one or more genes encoding dehydrogenase or vanillin demethylase are altered and / or inactivated. 4. An organism according to any of the preceding claims, characterized in that it is a single cell, preferably a microorganism or a plant or animal cell. 5. The organism according to claim 1, which is a bacterium, preferably Pseudomonas species. Enzyme Coniferyl Alcohol Dehydrogenase, Coniferyl Aldehyde Dehydrogenase, Feruloyl-CoA Synthetase, Enoyl-CoA Hydrolase-Aldolase, Beta-Ketothiolase, Vanillin-Dehydrogenase or Vanillic Acid Demethylase, Or a gene structure in which a nucleotide sequence encoding two or more of these enzymes has been altered and / or inactivated. Gene structure with the sequence given in FIGS. 1A-1R. Gene structure with the sequences given in Figures 2aa-2rb. A vector containing at least one gene structure according to any one of claims 6 to 8. A transformed organism according to any one of the preceding claims, characterized in that it has at least one vector according to claim 9. The organism according to any one of claims 1 to 5, which contains one or more gene structures according to any one of claims 6 to 8 integrated into the genome, instead of each native gene. A process for the biotechnological preparation of organic compounds, in particular alcohols, aldehydes and organic acids, characterized in that the organism according to any one of claims 1 to 5 or 10 to 11 is used. A process for producing an organism according to any one of claims 1 to 5, characterized in that alteration of eugenol and / or ferulic acid metabolism is achieved by a microbiological culture method known per se. The method according to claim 1 to 5 or 10 to 11, characterized in that alteration in eugenol and / or ferulic acid metabolism and / or inactivation of the corresponding gene is achieved by recombinant DNA methods. Method for producing an organism according to any one of the preceding. Use of an organism according to any one of claims 1 to 5 or 10 to 11 for producing coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillic acid. Use of the gene structure according to any one of claims 6 to 8 or the vector according to claim 9 for producing a transformed and / or mutated organism.