MXPA01004338A

MXPA01004338A - Construction of production strains for producing substituted phenols by specifically inactivating genes of the eugenol and ferulic acid catabolism

Info

Publication number: MXPA01004338A
Application number: MXPA/A/2001/004338A
Authority: MX
Inventors: Jurgen Rabenhorst; Alexander Steinbuchel; Horst Priefert; Jorg Overhage
Original assignee: Symrise Gmbh&Ampco Kg
Priority date: 1998-10-31
Filing date: 2001-04-30
Publication date: 2002-03-05

Abstract

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

Description

- -CONSTRUCTION OF PRODUCTION STATES FOR THE OBTAINING OF PHENOLS SUBSTITUTED BY SPECIFIC INACTIVATION OF GENES OF THE CATABOLISM OF EUGENOL AND OF THE FAERLIC ACID. Field of the invention The present invention relates to the construction of production strains and to a process for the preparation of substituted methoxyphenols, especially vanillin. Background of the Invention DE-A 4 227 076 (procedure for obtaining substituted methoxyphenols and suitable microorganisms for this) describes the preparation of substituted methoxyphenols with a new Pseudomonas sp .. The starting material in this case is eugenol and the products are the acid ferulic, vanillinic acid, coniferyl alcohol and coniferyl aldehyde. In the same way, an extensive compilation on the possibilities of biotransformation with ferulic acid of Rosaza et al. (Biocatalytic transfusion ormation 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 vanillinic acid from Pseudomonas sp. have been described in EP-A 0 845 532. Enzymes for the transformation of trans-phenolic acid into the trans-feruloyl-SCoA ester and, subsequently, to give vanillin, as well as the gene for ester dissociation have been described by the Institute of Food Research, Norwich, GB, in WO 97/35999. In 1998, the content of the patent was also published as a scientific publication (Gasson et al., 1998. Metabolism of ferulic acid to REF: 128476 via vanillin using a novel CoA-dependent pathway in a newly isolated strain of Pseudomonas ßuorescens. Microbiology 144: 1397-1405). DE-A 195 32 317 describes the fermentation of vanillin from phenolic acid with Amycolatopsis sp with high yields. Known processes have the disadvantage that either only very low yields are obtained in vanillin or that they are starting from relatively expensive educts. In the case of the last-mentioned procedure (DE-A 195 32 317), high yields are certainly achieved (but nevertheless the use of Pseudomonas sp. HR199 and of Amycolatopsis sp. HR 167 for the biotransformation of eugenol in vanillin imposes a conduction of the fermentation in two stages and, therefore, a considerable economic and time cost Detailed description of the invention The task of the present invention is therefore to build organisms that are capable of converting the economic raw material, the eugenol, in vanillin in a single stage process, this task is solved by the construction of mono or polycellular production strains, characterized in that the enzymes of eugenol catabolism and / or ferulic acid are inactivated in such a way that check an accumulation of the intermediate products consisting of the coniferyl alcohol, the coniferyl aldehyde, the ferulic acid, the vanillin and / or the Vanilinic acid The production strain can be single cell or polycellular. Therefore, microorganisms, plants or animals can be the object of the invention. In addition, extracts obtained from the production strain can also be used. According to the invention, monocellular organisms are preferably used. In this case it can be microorganisms, animal cells. or vegetables. The use of fungi and bacteria is particularly preferred according to the invention. Bacteria are the most preferred. Among the bacteria, Rhodococcus, Pseudomonas and Escherichia types can be used in particular after modification of the catabolism of eugenol and / or ferulic acid. The obtaining of the organisms, employable according to the invention, can be carried out, in the simplest case, by known, conventional microbiological methods. In this way, the enzymatic activity of the proteins involved in the catabolism of eugenol and / or ferulic acid can be modified, through the use of enzymatic inhibitors. In addition, the enzymatic activity of the proteins involved in the catabolism of eugenol and / or ferulic acid can be modified by mutating the genes that code for these proteins. Such mutations can be generated nonspecifically according to classical methods, such as, for example, by UV irradiation or by chemical generators of mutations. In the same way, genetic engineering methods are suitable for obtaining the organisms according to the invention, such as deletions, insertions and / or nucleotide exchange. In this way, for example, the genes of organisms can be inactivated with the help of other DNA elements (elements O). In the same way, exchanges of intact genes with modified and / or inactivated genetic structures can be carried out by suitable vectors. . The DNA elements, used to provide the inactivating genes and for the inactivation can be obtained in this case by classical cloning techniques or by polymerase chain reactions (PCR). In a possible configuration of the invention, for example, the catabolism of eugenol as well as the catabolism of ferulic acid can be modified by insertion of O elements or introduction of deletions in the corresponding genes. In this case, the functions of the genes that code for dehydrogenases, synthetases, hydratases-aldolases, thiolases or dimethylases are inactivated with the help of the aforementioned genetic engineering methods in such a way that the generation of the corresponding enzymes is blocked. Preferably, they are genes encoding coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, ferulic acid CoA synthetases, enoyl CoA hydratases aldolases, beta-ketothiolases, vanillyl dehydrogenases or vanillynic acid dimethylases. Genes coding for the amino acid sequences given in EP-A 0 845532 and / or their nucleotide sequences encoding allele variations are very particularly preferred. The object of the invention are, therefore, also genetic structures for obtaining transformed and mutant organisms. Preferably, genetic structures will be used to obtain organisms and mutants, in which the nucleotide sequences coding for dehydrogenases, synthetases, hydratases-aldolases, thiolases or demethylases are inactivated. Genetic structures in which the nucleotide sequences coding for coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, ferulic acid CoA synthetases, enoyl CoA hydratases aldolases, beta-ketothiolases, vanillyl dehydrogenases or vanillic acid are inactivated are especially preferred. -demetilasas. Genetic structures that have the structures indicated in the figures are particularly highly preferred until Ir with the nucleotide sequences shown in FIGS. 2a to 2r and / or their nucleotide sequences encoding variations of the alleles. In this case, nucleotide sequences from 1 to 18 are particularly preferred. The invention also covers the partial sequences of these genetic structures as well as functional equivalents. Functional equivalents are those derivatives of DNA, in which individual corebases have been exchanged (Wobbel exchange), without modifying the function. Amino acids can also be exchanged in the plane of the proteins without resulting in a modification of the function. One or more DNA sequences can be connected to and / or behind the genetic structures. By cloning the genetic structures, plasmids or vectors can be obtained which are suitable for the transformation and / or transfection of an organism and / or for the conjugating transfer of an organism. The object of the invention are also plasmids and / or vectors for obtaining microorganisms and mutants transformed according to the invention. These contain, therefore, the genetic structures described. The present invention therefore also relates to organisms that contain the plasmids and / or vectors mentioned. The type of plasmids and / or vectors depends on their application purposes. To exchange, for example, the intact genes of the catabolism of eugenol and / or ferulic acid in pseudomonados by the genes inactivated by O-elements, vectors are required that, on the one hand, can be transferred to pseudomonados (plasmids transferable in a conjugating manner) , but that, on the other hand, can not be replicated in them and, therefore, are unstable to the pseudomonados (the so-called suicide plasmids). The DNA segments, which are transferred with the aid of such a plasmid system in the pseudomonads, can only be maintained if they are integrated by homologous recombination in the genome of the bacterial cells. The genetic structures, vectors and plasmids described can be used to obtain various transformed organisms or mutants.

By means of the cited genetic structures, intact nucleic acid sequences can be exchanged for modified and / or inactivated genetic structures. In the cells, obtainable by transformation or transfection or conjugation, an exchange of the intact gene by the modified and / or inactivated genetic structure is carried out by homologous recombination, whereby the resulting cells have only modified genetic structure and / or inactivated in the genome. Thus, genes according to the invention can be modified and / or inactivated preferably in such a way that the corresponding organisms are capable of generating coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillinic acid. The production strains, constructed in this way, according to the invention, are, for example, mutants of the strain Pseudomonas sp. HR 199 (DSM 7063) which has been described exactly in DE-A 4 227 076 and in EP-A 0845532, obtaining, among others, the corresponding genetic structures from the figures up to Ir in combination with figures 2a until 2r. 1. Pseudomonas sp. HR199ca / 4OKm, which contains the calA inactivadu gene by OKm in the place of the intact calA gene encoding the coniferyl alcohol dehydrogenase (figure 2a). 2. Pseudomonas sp. HR199calAOGm, which contains the calA gene inactivated by GMO in place of the intact calA gene coding for the coniferyl alcohol dehydrogenase (Figure Ib, Figure 2b). 3. Pseudomonas sp. HR199ca / ??, which contains the inactivated calA gene by deletion in place of the intact calA gene encoding the coniferyl alcohol dehydrogenase (FIG. 1, FIG. 2c). 4. Pseudomonas sp. HR199ca / 5OKm, which contains the calB gene inactivated by OKm in place of the intab calB gene coding for coniferylaldehyde dehydrogenase (Figure Id, Figure 2d). Pseudomonas sp. HR199ca / 5OGm, which contains the calB gene inactivated by GMO in place of the intact calB gene coding for coniferylaldehyde dehydrogenase (Figure 1; Figure 2e). Pseudomonas sp. HR199aar / £ ?, which contains the calB gene inactivated by deletion in place of the intab calB gene coding for coniferylaldehyde dehydrogenase (Figure 1, Figure 2f). Pseudomonas sp. HR199 / csOKm, which contains the inactivated / cs gene by OKm in place of the intact cs gene encoding ferulic acid-CoA synthetase (figure lg, figure 2g). Pseudomonas sp. HR199 / csOGm, which contains the gene / cs inactivated by GMO in place of the intact cs gene coding for ferulic acid-CoA synthetase (figure 1, figure 2h). Pseudomonas sp. HR199 / cs ?, which contains the gene / cs inactivated by deletion in place of the intact cs gene coding for the "erulic acid-CoA synthetase" (figure li, figure 2i) Pseudomonas sp. HR199ec? OKm, which contains the gene was inactivated by OKm in place of the intact ech gene coding for enoyl-CoA-hydratase-aldolase (figure lj, figure 2j) Pseudomonas sp.HR199ec? OGm, which contains the ech gene inactivated by GMO in place of the gene ech intact coding for enoyl-CoA-hydratase-aldolase (figure lk; figure 2k): Pseudomonas sp. HR199ec ?, which contains the gene inactivated by ech deletion in place of the intact ech gene encoding enoyl-CoA- hydratase-aldolase (figure 11, figure 21) 13. Pseudomonas sp.Hrl99aatOKm, which contains the inactivated aat gene by OKm in place of the intact aat gene coding for beta-ketothiolase (figure lm, figure 2m) 14. Pseudomonas sp. HR199aatOGm, which contains the aat gene inactivated by GMO instead of the intact aat gene which codes for beta-ketothiolase (figure ln; figure 2n). 15. Pseudomonas sp. HR199aat ?, which contains the inactivated gene by aat deletion in place of the intact aat gene coding for beta-ketothiolase (figure 1; figure 2o). 16. Pseudomonas sp. HR199v # 2OKm, which contains the inactivated vdh gene by OKm in place of the intact vdh gene coding for beta-ketothiolase (Figure lp; figure 2p). 17. Pseudomonas sp. HR199v / 7OGm, which contains the inactivated vdh gene by GmO instead of the intact vdh gene coding for vanillyndehydrogenase (Figure lq, Figure 2q). 18. Pseudomonas sp. K \ 99vdh ?, which contains the gene inactivated by vdh deletion in place of the intact vdh gene coding for vanillynhydrorenase (figure Ir, figure 2r). 19. Pseudomonas sp. HR199v < # j.BOKm, which contains the inactivated vdhB gene by OKm in place of the intact vdhB gene coding for vanillyn dehydrogenase II. 0. Pseudomonas sp. HR199v £ #? OGm, which contains the inactivated vdhB gene by GMO instead of the intact vdhB gene coding for vanillyn dehydrogenase II. 21. Pseudomonas sp. YíR \ 99vdhB ?, which contains the gene inactivated by vdhB deletion in place of the intact vdhB gene coding for vanillynhydrogenase II. 22. Pseudomonas sp. HR199adhOKm, which contains the inactivated adh gene by OKm instead of the intact adh gene encoding the alcohol dehydrogenase. 23. Pseudomonas sp. HRl 99adhOGm, which contains the adh gene inactivated by GMO in place of the intact adh gene encoding the alcohol dehydrogenase. 24. Pseudomonas sp. HR199adh ?, which contains the inactivated gene by adh deletion in place of the intact adh gene encoding the alcohol dehydrogenase. 25. Pseudomonas sp. HRl 99vanAOKm, which contains the vanA gene inactivated by OKm in place of the intact vanA gene encoding the subunit vanilinic demethylase-6. Pseudomonas sp. HR199vanAOGm, which contains the vanA gene inactivated by GMO instead of the intact vanA gene coding for the subunit vanilinic demethylase 7. Pseudomonas sp. HR199vanA ?, which contains the vanA gene inactivated by deletion in place of the intact vanA gene coding for the subunit vanilinic acid demethylase 8. Pseudomonas sp. HR199vanBO ¥ * m, which contains the vanB gene inactivated by OKm in place of the intab vanB gene that codes for the β-subunit of vanillin-demethylase 29. Pseudomonas sp. HRl 99vanBOGm, which contains the vanB gene inactivated by GMO instead of the intab vanB gene coding for the β-subunit of vanillin-demethylase 30 acid. Pseudomonas sp. HR199vanB ?, which contains the vanB gene inactivated by deletion in place of the intact vanB gene coding for the β-subunit of vanillin-demethylase. The object of the invention is also a process for obtaining organic compounds by biotechnology. In particular, alcohols, aldehydes and organic acids can be obtained by this process. Preference is given in this case to coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and vanillinic acid. In the process according to the invention, the organisms described above are used. Bacteria, especially of the Pseudomonas type, belong to the very particularly preferred organisms. In particular, the types of Pseudomonas mentioned preferably for the following procedures can be used: 1. Pseudomonas sp. HR199caZ4OKm, Pseudomonas sp. HR199ca / ^ OGm and Pseudomonas sp. HR199ca / A? for obtaining coniferyl alcohol from eugenol. 2. Pseudomonas sp. HR199ca / i? OKm, Pseudomonas sp. HR199ca / 5OGm and Pseudomonas sp. HR199ca / B? for obtaining coniferyl alcohol from eugenol or from coniferyl alcohol. 3. Pseudomonas sp. HR199 c.?OKm, Pseudomonas sp. HR199fcsOGm, Pseudomonas sp. HR199fcs ?, Pseudomonas sp HR199ec? OKm, Pseudomonas sp. HR199ec / zOGm and Pseudomonas sp. HR199echA for obtaining ferulic acid from eugenol or coniferilalcohol or coniferilaldehyde. 4. Pseudomonas sp. HR199ví #? OKm, Pseudomonas sp. HR199ví # zOGm, Pseudomonas sp. ? R \ 99vdhA, Pseudomonas sp. HR199v £ # 7OGmv £ / 2? OKm, Pseudomonas sp. HR199v £ /? OKmví # 2Í? OGm, Pseudomonas sp. HR199vf # j? V < / SOGm and Pseudomonas sp. HR199 ví /? V? 5OKm for the production of vaniline from eugenol or from coniferyl alcohol or from coniferyl aldehyde or ferulic acid. 5. Pseudomonas sp. HR199v < OKm, Pseudomonas sp. HR199van / 4OGm, Pseudomonas sp. HR199vanA ?, Pseudomonas sp. HR199van5OKm, Pseudomonas sp. HR199VA «5OGm and Pseudomonas sp. HR199v¿wi ?? for the preparation of vanillinic acid from eugenol or from coniferyl alcohol or from coniferyl aldehyde or from ferulic acid or vanillin. The preferred substrate is eugenol. However, the addition of other substrates is possible, including the exchange of eugenol by another substrate. As a nutrient medium for the organisms used according to the invention, synthetic, semi-synthetic or complex culture media are suitable. These may contain carbon and nitrogen compounds, inorganic salts, optionally trace elements as well as vitamins. Carbonated compounds may include carbohydrates, hydrocarbons or basic organic chemicals. Examples of preferably employable compounds are sugars, alcohols or sugar alcohols, organic acids or complex mixtures. Sugars mainly include glucose. As organic acids, preferably citric acid or acetic acid can be used. The complex mixtures include, for example, extracts of malt, yeast extract, casein or casein hydrolyzate. Suitable nitrogenous substrates are inorganic compounds. Examples in this regard are nitrates and ammonium salts. In the same way, organic nitrogen sources can be used. These include yeast extract, soybean meal, casein, casein hydrolyzate and macerated corn water. The inorganic salts which can be used include, for example, sulfates, nitrates, chlorides, carbonates and phosphates. As metals, the aforementioned salts preferably contain sodium, potassium, magnesium, manganese, calcium, zinc and iron. The temperature for the culture is preferably in the range from 5 to 100 ° C. The range from 15 to 60 ° C is especially preferred, with 22 to 37 ° C being the most preferred. The pH value of the medium is preferably between 2 and 12. The range from 4 to 8 is particularly preferred. Basically, all bioreactors known to the person skilled in the art can be used for carrying out the process according to the invention.

Preferably, all devices suitable for immersion processes are suitable. This means that containers according to the invention can be used without or with mechanical mixing devices. The former include, for example, shaker devices, bubble column reactors or hoses. The latter preferably belong to all known devices with agitators of any configuration. The process according to the invention can be carried out continuously or discontinuously. The duration of the fermentation until a maximum amount of product is reached depends on the special type of the organism used. Basically the times of the fermentation are included, however, between 2 and 200 hours. The invention will be explained in more detail below with reference to the examples: Specific mutants of the strain Pseudomonas sp. HRl 99 (DSM 7063) which valorises eugenol, genes of eugenol catabolism being inactivated specifically by insertion of omega-elements or by introduction of deletions. As omega-elements they served DNA segments that code for resistance to antibiotics against kanamycin (OKm) and gentamicin (GMO). These resistance genes were isolated from Tn5 and the plasmid pBBRlMCS-5 with the aid of standard methods. The genes calA, calB, fcs, ech, aat, vdh, adh, vdhB, vanA and vanB, which code for coniferyl alcohol dehydrogenase, coniferyl aldehyde dehydrogenase, ferulic acid CoA synthetase, enoyl CoA hydratase aldolase, beta-ketothiolase, vanillin-dehydrogenase, alcohol-dehydrogenase, vanillin-dehydrogenase II and vanillin-demethylase were isolated from genomic DNA of the strain Pseudomonas sp. HRl 99 using standard methods and cloned into pBluescript SK. "From these genes, DNA segments (deletion) were removed by digestion with appropriate restriction endonucleases, or replaced by omega-elements (insertion), with The genes, mutated in this way, were recloned in transferable vectors in a conjugating manner and then introduced into the Pseudomonas sp. HRl 99 strain. By appropriate selection, transconjugants were obtained, which had exchanged the corresponding native-type gene capable of functioning by the new incorporated inactive gene.The insertion and deletion mutants obtained in this way present only the corresponding inactivated gene.In this way, mutants were obtained with only one defective gene as well as multiple mutants, in which several genes had been inactivated in this way, these mutants were used for the biot ansformation of a) Eugenol to give coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillinic acid; b) Coniferyl alcohol to give coniferyl aldehyde, ferulic acid, vanillin and / or vanillinic acid; c) Coniferyl-aldehyde to give ferulic acid, aniline and / or vanillinic acid; d) Ferulic acid to give vanillin and / or vanillinic acid and e) Vanillin to give vanillinic acid. Material and methods. Growth conditions of bacteria. Strains of Escherichia coli were cultured at 37 ° C in Luria-Bertani (LB) or in the mineral medium M9 (Sambrook, J., EF Fritsch und T. Maniatis, 1989, Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.). Strains of Pseudomonas sp. at 30 ° C in Nutrient Broth (NB, 08%, weight / volume) or in mineral medium (MM) (Schlegel, HG et al., 1961, Arch. Mikrobiol. 38: 209-222) or mineral medium HR (HR -MM) (Rabenhorst, J. 1996, Appl. Microbiol. Biotechnol. 46: 470-474). The ferulic acid, vanillin, vanillinic acid and protocatechuanic acid were dissolved in dimethyl sulfoxide and the corresponding medium was added in a final concentration of 0.1% (w / v). Eugenol was added directly to the medium in a final concentration of 0.1% (volume / volume), or was applied to the lid of the MM agar plate on filter paper (round filter 595, Schleicher &Schuell, Dassel, Germany). For the cultivation of transconjugants and mutants of Pseudomonas sp. Tetracycline, kanamycin and gentamicin were used in final concentrations of 25 μg / ml or 100 μg / ml or 7.5 μg / ml.

Qualitative and quantitative determination of metabolism intermediates in culture supernatants. The culture supernatants were analyzed directly or after dilution with bidistilled H2O by high pressure liquid chromatography (Knauer-HPLC). Chromatography was carried out on Nucleosil-100 C18 (7 μm, 250 x 4 mm).

As the solvents, 0.1% (volume / volume) formic acid and acetonitrile were used. The gradient used for the circumvention of the substances proceeds 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 workup was carried out at 4 ° C. Raw extract. Pseudomonas sp. HR199, grown on eugenol, were washed with 10 mM phosphate buffer, pH 6.0, resuspended in the same buffer and disintegrated by two passes through a French press (Amicon, Silver Spring, Maryland, USA). ) at a pressure of 1,000 psi. The cell homogeneate was subjected to an ultracentrifugation (1 hour, 100,000 x g, 4 ° C), whereby the soluble fraction of the crude extract was obtained as a supernatant. Chromatography by anion exchange 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 transferred to a DEAE-Sephacel column (2.6 cm x 35 cm, bed volume [BV]: 186 ml) equilibrated with 10 mM sodium phosphate buffer, pH 6.0, with a flow rate of 0. 8 ml minute The column was rinsed with twice the bed volume of 10 mM sodium phosphate buffer, pH 6.0. The vanillin-dehydrogenase-II (VDH-II) elution 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). Fractions of 10 ml were collected. The fractions with high activity in VDH-II were combined to give a DEAE-Pool. Determination of the activity of vanillin dehydrogenase. The determination of the VDH activity was carried out at 30 ° C by an optical enzymatic assay. The reaction mixture, with a volume of 1 ml, contained 0.1 mmol of potassium phosphate (pH 7.1), 0.125 μmol of vanillin, 0.5 μmol of NAD, 1.2 μmol of pyruvate (sodium salt). Na), lactate dehydrogenase (1 U, from the heart of pork) and enzyme solution. The oxidation of vanillin was carried out at? = 340 nm (evannyin - 11.6 cm / μmol). The enzymatic activity was given in units (U), 1 U corresponding to the enzymatic amount that converts 1 μmol of vanillin per minute. The protein concentrations in the samples were determined according to Lowry et al. (Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193: 265-275). Determination of the activity of coniferilalcohol-dehydrogenase. The determination of the CADH activity was carried out at 30 ° C by an optical enzymatic assay 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 mmoles of tris / HCl (xpH 9.0), 0.4 μmoles of coniferyl alcohol, 2 μmoles of NAD, 0.1 mmol of semicarbazide and enzyme solution. The reduction of the NAD was carried out to? = 340 nm (e = 6.3 cm / μmoles). Enzymatic activity was indicated in units (U), 1 U corresponding to the enzymatic quantity that transforms 1 μmol of substrate per minute. The protein concentration in the samples was determined according to Lowry et al. (Lowry, O.H., N.J. Rosebrough, A. L. Farr and R.J. Randall, 1951. J. Biol. Chem. 193: 265-275). Determination of the coniferyl aldehyde dehydrogenase activity. The determination of the CALDH activity was carried out at 30 ° C by an optical enzymatic assay, the reaction mixture with a volume of 1 ml contained 0.1 mmole of tris / HCl (pH 8.8), 0.08 μmoles of coniferilaldehyde, 2.7 μmoles of NAD and enzyme solution. The oxidation of the coniferyl aldehyde to give the ferulic acid was carried out at? = 400 nm (e = 34 cm2 / μmol). Enzymatic activity was given in units (U), 1 U corresponding to the enzymatic amount that converts 1 μmol of substrate per minute. The protein concentration in the samples was determined according to Lowry et al. (Lowry, O.H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 11951, J. Biol. Chem. 193: 265-275). Determination of ferulic acid-CoA synthetase activity (ferulic acid-thioquinasa). The determination of the FCS activity was carried out at 30 ° C by an optical enzymatic assay, 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 of potassium phosphate (pH 7.0), 2.1 μmol of MgCl2, 0.7 μmol of ferulic acid, 2 μmol of ATP, 0.4 μmoles of coenzyme A and enzymatic solution. The formation of the CoA ester from the ferulic acid was carried out,? = 345 nm (e = 10 cm / μmoles). Enzymatic activity was indicated in units (U), 1 U corresponding to the enzymatic amount that converts 1 μmol of substrate per minute. The protein concentrations in the samples were determined according to Lowry et al. (Lowry, O.H., N. J. Rosebrough, A. L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193: 265-275). Electrophoretic methods. The separation of the protein-containing extracts was carried out in 7.4% (w / v) polyacrylamide gels under native conditions according to the method of Stegemann et al. (Stegemann et al., 1973. Z. Naturforsch, 28c: 722-732) and under denaturing conditions in 11.5% (weight / volume) polyacrylamide gels according to the Laemmli method (Laemmli, UK 1970, Nature (London) 227: 680-685). For the non-specific staining of the protein, Serva Blue R was used. For specific staining of the coniferyl alcohol-, coniferylaldehyde- and vanillin dehydrogenase, the gels were subjected to a buffer exchange for 20 minutes in 100 mM KP buffer (pH 7, 0) and then incubated at 30 ° C in the same buffer to which 0.08% (weight / volume) of NAD, 0.04% (w / v) of p-nitro-blue-tetrazolium chloride, 0.003 had been added. % (weight / volume) of phenazine methosulfate and 1 mM of the corresponding substrate, until the corresponding color bands were visible. Transfer of proteins from polyacrylamide gels onto PVDF membranes. Proteins from the SDS polyacrylamide gels were transferred with the aid of a Semidry-Fastblot device (B32 / 33, Biometra, Gottingen, Germany) with the manufacturer's indications on PVDF membranes (Waters-Milipore, Bedford, Mass. , USES). Determination of the N-terminal amino acid sequences. The determination of the N-terminal amino acid sequence was carried out with the aid of a peptide protein sequencer (type 477 A. Applied Biosystems, Foster City, USA) and a PTH analyzer according to the manufacturer's instructions.

Isolation and manipulation of DNA. The isolation of the genomic DNA was carried out according to the Marmur method (Marmur, J. 1961, J. Mol. Biol .. 3: 208-218). Isolation and analysis of other DNA plasmids or DNA restriction fragments was carried out according to standardized methods (Sambrook, JEF Fritsch and T. Maniatis, 1989. Molecular cloning: a laboratory manual, 2nd edition.) Cold Spring Harbor Laboratory Press, Cold Spring Habor, New York). DNA transfer. The preparation and transformation of competent cells of Escherichia coli was carried out according to the Hanahan method (Hanahan, D. 1983. J. Mol. Biol. 166: 557-580). The transfer of conjugating plasmids between strains of Escherichia coli S17-1, carrying plasmid (donor) and strains of Pseudomonas sp (receptor) was carried out on NB agar plates according to the method of Friedrich et al. (Friedrich, B. et al., 1981, J. Bacteriol., 147: 198-205), or by a "minicomplementation method" on MM agar plates with 0.5% gluconate (weight / volume) as a source of C and 25 μg / ml of tetracycline or 100 μg / ml of kanamycin. In this case, recipient cells were applied in one direction as the inoculation line. After 5 minutes cells of the donor strain were applied as inoculation line, crossing the line of inoculation of the recipient. After incubation for 48 hours at 30 ° C the transconjugants grew directly behind the crossing points, whereas on the contrary neither the donor strain nor the recipient strain were able to develop. Hybridized experiments. Restriction DNA fragments were separated by electrophoresis on a 0.8% (w / v) agarose gel in 50 mM buffer of tris- 50 mM boric acid- 1.25 mM EDTA (pH 8.5) (Sambrook, JEF Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Habor, New York). The transfer of the denatured DNA from the gel to a positively charged nylon membrane (pore size: 0.45 μm, Pall Filtrationstechnik, Dreieich, Germany), the subsequent hybridization with DNA probes biotinylated or labeled with digoxigenin and the The obtaining of these DNA probes was carried out according to standardized methods (Sambrook, JEF Fritsch and T. Maniatis, 1989 Molecular cloning: a laboratory manual, 2nd edition Cold Spring Harbor Laboratory Press, Cold Spring Habor, New York). DNA sequencing. The determination of the nucleotide sequences was carried out according to the chain dideoxy-cleavage method of Sanger et al. (Sanger et al., 1977. Proc. Nati. Acad. Sci. USA 74: 5463-5467) "non-radioactive" with a "LI-COR DNA sequencer, model 4000L" (LI-COR Inc., Biotechnology Division, Lincoln , NE, USA) using "Termo Sequenase fluorescent labelled first cycle sequencing kit with 7-deaza-dGTP" (Amersham Life Science, Amersham International pls, Little Chalfont, Buckinghamshire, England) respectively according to the manufacturer's instructions. With the help of synthetic oligonucleotides, it was sequenced according to the "First hopping" strategy of Strauss et al. (Strauss, E.C. et al., 1986. Anal. Biochem. 154: 353-360). Chemical products, biochemical products and enzymes. Restriction enzymes, T4 DNA ligase, lambda DNA and enzymes or substrates for optical enzymatic assays were purchased from the firm C.F. Boehringer & Sohne (Mannheim, Germany) or GIBCO / BRL (Eggenstein Germany). He [? P] ATP can be purchased from the firm Amersham / Buchler (Braunschweig, Germany), the oligonucleotides were purchased from the firm MWG-Biotech GmbH (Ebersberg, Germany). Agarose of the NA type was purchased from the Pharmacia-LKB Company (Uppsala, Sweden). All other chemicals were from the Haarman & 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, which provide resistance against kanamycin (OKm) or against gentamicin (GMO). For the construction of the OKm element, the 2099 bp Bg? of transposon Tn5 (Auerswald E.A., G. Ludwig and H. Schaller, 1981. Cold Spring Harb. Symp.Quant. Biol. 45: 107-113; Beck E., G. Ludwig, E.A. 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). The fragment was shortened by treatment with BaI-31 nuclease at approximately 990 bp. This fragment, now encompassing only the kanamycin resistance gene (which codes for an aminoglycoside-3'-O-phosphotransferase), was then ligated with pSKsym-DNA cut with Smal (pBluescript SK derivative), containing a multiple cloning point constructed symmetrically [Salí, HindOl, EcoRI, Smal, EcoR, HindlTl, Salí]). From the resulting plasmid, the OKm element could be isolated again in the form of Smal fragment, EcoJU., HindOl or For the construction of the GMO element, the 983 bp Eael fragment of the plasmid pBBRlMCS-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: 1275-176) and then treated with Mung Bean nuclease (degradation of the ends of the DNA molecule of the individual strands). This fragment, which now only contains the gene for resistance to gentamicin (which codes for a gentamicin-3-acetyltransferase), was then ligated with pSKsym-DNA cut with Smal (see above). From the resulting plasmid, the element OGm could again be isolated as Smal, EcoBl, HindRl or Sa fragment. Example 2. Cloning of genes from Pseudomonas sp. HRl 99 (DSM7063), which must be inactivated by insertion of OR elements or by deletions. The separate clones of the genes fes, ech, vdh and aat were carried out from strains of E.coli S17-1 DSM 10439 and DSM 10440 with the plasmids p? 207 and p? 5-l (see? PA 0845532). From these plasmids, the indicated fragments were isolated in a preparative manner and treated as described below: For the cloning of the fes gene, the Sall / Eco l fragment, with a size of 2,350 bp, of the p? 207 plasmid was cloned. and the EcoRI / Sall fragment, with a size of 3,700 bp of the p? 5-l plasmid, together with the pBluescript SK "in such a way that both fragments were linked together by means of the EcoRl ends. preparatively isolated the San fragment of 6,050 bp and shortened to approximately 2,480 bp by treatment with the Bal-31 nuclease, then ligated onto the ends of the stl-Linker fragment and the fragment was cloned, after digestion of Pstl in pBluescript SK (pSKfes) After the transformation of E. coli XL lB, clones were obtained that expressed the gene / s and had an FCS activity of 0.2 U / mg of protein.

For the cloning of the ech gene, the HindbSUEcóKL fragment, with a size of 3,800 bp, was isolated from the plasmid pE207 and shortened to approximately 1470 bp by treatment with the Bal-31 nuclease. They were then ligated onto the ends of the EcoRI-Linker fragment and the fragment was cloned, after EcoRI digestion in pBluescript SK "(pSKech) .For the cloning of the vdh gene, the SallJEcoRl fragment, with a size of 2.350, was isolated by preparation. bp of plasmid p 207. After cloning into pBluescript SK, the fragment was shortened, with the help of an exonuclease III / Mung Bean nuclease system, on one side approximately to 1,530 bp. An EcoRI / Linker was then ligated onto the fragment ends and the fragment was cloned, after EcoRI digestion in p-Bluescript SK "(pSKvdh) After the transformation of E. coli XLl-Blue, clones expressing the gene were obtained. vdh and having a VDH activity of 0.01 U / mg of protein.For the cloning of the aat gene, the Eco JSail fragment, with a size of 3,700 bp, was isolated from the p? 5-l plasmid in a preparative manner. It was shortened to approximately 1590 bp by treatment with the Bal-31 nuclease, then ligated onto the ends of the EcoRI-Linker fragment and the fragment was cloned after EcoRI digestion in pBluescript SK "(pSKaat). Example 3. Inactivation of the previously described genes by insertion of O-elements, or by deletion of partial zones of these genes. The plasmid pSKfcs, which contains the fes gene, was digested with Bss? L, whereby a fragment, with a size of 1,290 bp, of the fes gene was removed by slicing. After religation, the deletion derivative of the fes gene (fcsA) (see figures li and 2i) cloned in pBluescript SK "(pSK / as?) Was obtained.In addition, after removal by extraction of the fragment, the omega elements were ligated, OKm and OGm for these points, in this way the O-inactivated derivatives of the fes gene were formed (fcsOKm, see figures lg and 2g) and (fcsOGm, see figures lh and 2h) cloned in pBluescript SK "(pSK sOKm and pSK / sOGm) In the crude extracts of the obtained E. coli clones, whose hybrid plasmids present a gene fes inactivated by deletion or insertion of O-elements, no FCS activity could be detected The plasmid pSKech, which contains the ech gene, was digested with Nrul, whereby a fragment with a size of 53 was cut out. bp and a fragment with a size of 430 bp from the ech gene. After religation, the deletion derivative of the ech gene was cloned (echA, see figures 11 and 21) (cloned into pBluescript SK "(pSKechA). The separation by fragmenting of the fragments, the omega, OKm and OGm elements were ligated by these points, in this way we obtained the Oactivated derivatives of the ech gene (ec iOKm and echOGm) cloned in pBluescript SK "(pSKec? OKm and pSKec ? OGm.) Plasmid pSKvdh, which contains the vdh gene, was subjected to dig BssHII, with which a fragment of the vdh gene with a size of 210 bp was cleaved. After religation, the vdh deletion derivative (vdh A, see figures 1 and 2) cloned in pBluescript SK "(pSKvdhA) was obtained, and after the fragment separation, the elements omega, OKm and OGm for these points Thus, O-inactivated vdh (vdhOKm and vdhOGm) gene clones were formed in pBluescript SK "(pSKvdhOKm, see figures lm and 2m) and pSKvdhOGm, see figures ln and 2n). In the crude extracts of the obtained E. coli clones, whose hibridoplásmidos present a vdh gene inactivated by deletion or by insertion of O-elements, VDH activity could not be detected.

Plasmid pSKaat, which contains the aat gene, was digested with ifasHH, whereby a fragment with a size of 59 bp was cleaved off by the aat gene. After relining, the deletion derivative of the aat gene (aat A, see figures Ir and 2r) cloned in pBluescript SK "(pSKaatA) was obtained, and after separation by fragmenting the fragments, the bound omega, OKm and GMO elements were obtained. In this way the O-inactivated derivatives of the aat gene (aat OKm, see figures lp and 2p) and (aatOGm, see figures lq and 2q) cloned in pBluescript SK "(pSKaatOKm and pSKaatOGm) are formed. Example 4. Genes modified by cloning or inactivated by O-elements in the "suicide plasmid" transferable in a conjugant manner pSUP202. In order to be able to exchange the inactivated genes by O-elements in Pseudomonas sp. HRl 99 for intact genes, a vector is needed, which, on the one hand, can be transferred in pseudomonads (plasmid transferable in a conjugating manner), but which, on the other hand, can not be replicated in it and, therefore, is unstable in pseudomonados ("suicide plasmid"). The DNA segments, which are transferred with the aid of such a plasmid system in pseudomonads, can only be maintained when they are integrated into the genome of the bacterial cell by homologous recombination (RecA-dependent recombination). In the present case we used the "suicide plasmid" pSUP202 (Simón et al., 1983. In: A. Pühler, Molecular genetics of the bacteria-plant interaction, Springer Verlag, Berlin, Heidelberg, New York, pages 98-106). The inactivated genes fcsOKm and fcsOGm were isolated, following PstI digestion from the plasmids pSK / csOKm and pSKfcsOGm and ligated with pSUP202 DNA cleaved with Pstl. The ligation preparations were transformed according to E. coli S17-1. The selection was carried out on LB medium, which contained tetracycline, with kanamycin or with gentamicin. Transformants resistant to kanamycin were obtained, whose hybrid plasmid (pSUP c-fOKm) contained the fcsOKm inactivated gene. The corresponding hybrid plasmid (pSUP csOGm) of the gentamicin-resistant transformants contained the fcsOGm inactivated gene. The inactivated genes echOKm and echOGm were isolated after EcoRI digestion from the plasmids pSKechOKm and pSKechOGm and ligated with pSUP202 DNA cleaved with EcoR1. The ligation fillers were transformed according to E. coli S17-1. The selection was carried out on LB medium, which contains tetracycline, with kanamycin or with gentamicin. Transformants resistant to kanamycin were obtained, whose hybrid plasmid (pSUPec? OKm) contains the inactivated gene echOKm. The corresponding hybrid plasmid (pSUPec / íOGm) of the gentamicin-resistant transformants contain the inactivated gene echOGm. The inactivated genes vdhOKm and vdhOGmO were isolated after EcoRI digestion from the plasmids pSKvc OKm and pSKvdhOGm and ligated with pSUP202 DNA cleaved with EcsRI. The ligation fillers were transformed according to E. coli S17-1. The selection was carried out on medium L-B, which contains tetracycline, with kanamycin or with gentamicin. Transformants resistant to kanamycin were obtained, whose hybrid plasmid (pSUPvc? OKmO) contains the inactivated vdhOKm gene. The corresponding hybrid plasmid (pSUPvl /? OGm) of the gentamicin-resistant transformants contains the inactivated vdhOG gene. The inactivated genes Gene aatOKm and aatOGm are isolated, after EcoRI digestion from the plasmids pSKaatOKm and pSKaatOGm and ligated with pSUP202 DNA cleaved with EcoRl. The ligation fillers were transformed according to E. coli S 17-1. The selection was carried out on LB medium, which contains tetracycline concamycin or gentamicin. Transformants resistant to kanamycin were obtained, whose hybrid plasmid (pSUPaatOKm) contains the inactivated aatOKm gene. The corresponding hybrid plasmid (pSUPaatOGm) of the gentamicin-resistant transformants contains the inactivated aatOGm gene. Example 5. Transcloning of inactivated genes by deletion in the "transferable plasmid suicide" of conjugating wood with "sacBv selection system pHE55.In order to exchange genes, inactivated by deletion, in Pseudomonas sp. HRl 99 for intact genes, we need a vector that already has the properties described for pSUP202. Since, in contrast to what happens with the genes inactivated by O-elements, in the genes inactivated by deletion, there is no possibility of selection (absence of resistance to antibiotics) for the exchange of the genes in Pseudomonas sp. HR 99, another selection system had to be used. In the case of the "sacB selection system", it is cloned in an inactivated gene plasmid by deletion, exchanged, which also has an antibiotic-resistant gene, also of the sacB gene. After the conjugate transfer of the hybrid plasmid in a pseudomonate, the plasmid is integrated into the genome by homologous recombination at the point where the intact gene is found (first "Cross over"). In this way, a "heterogenoter" strain is formed, which has both an intact gene and also a deletion-inactivated gene, which are separated from one another by means of pHE55-DNA. These strains have the resistance encoded by the vector and also have an active sacB gene. By means of a second homologous recombination event (second "Cross over"), it must now be detached from the genomic DNA of pHE55-DNA together with the intact gene. By means of this recombination event, a strain is formed that now only has the inactivated gene. In addition, the loss of antibiotic resistance encoded by pHE55 and the sacB gene is followed. If strains are spread over media containing sucrose, growth strains expressing the sacB gene will be inhibited, since the gene product transforms sucrose into a polymer that accumulates in the periplasm of the cells. Cells that no longer carry the sacB gene due to the second recombination event are thus inhibited in growth. To have a possibility of phenotypic selection on the integration of the inactivated gene by deletion, this is not exchanged for an intact gene but a strain is used in which the gene to be exchanged is already "marked" by insertion of an O- element. When the exchange is successful, the resulting strain loses antibiotic resistance encoded by the O-element. The fcsA inactivated gene was isolated after PstI digestion from plasmid pSKfcsA and ligated with pHE55 DNA cleaved with PstI. The ligation loading was transformed according to E. coli S17-1. The selection was carried out on LB medium containing tetracycline. Transformants resistant to tetracycline were obtained, whose hybrid plasmid (p EfcsA) contained the inactivated gene fes A. The inactivated gene ech? was isolated, after EcoRI digestion from the pSKech plasmid? and was treated with Mung Bean nuclease ("blunt ends"). The fragment was ligated with pH 55 DNA cleaved with BamHI and treated with Mung Bean nuclease. The ligation load is transformed according to E. coli S17-1. The selection was carried out on LB medium containing tetracycline. Transformants resistant to tetracycline were obtained, whose hybrid plasmid (pHEech?) Contained the inactive echA gene. The inactivated vdhA gene was isolated after EcoRl digestion. from plasmid pSKvdhA and treated with Mung Bean nuclease. The fragment was ligated with pHE55 DNA cleaved with BamYQ. and treated with Mung Bean nuclease. The ligation load was transformed according to E. coli S17-1. The selection was carried out on LB medium containing tetracycline. Transformants resistant to tetracycline were obtained, whose hybrid plasmid (pHEv) contained the inactivated gene vdh A. The inactivated gene aatA was isolated, after EcoRI digestion from the plasmid pSKa t? and was treated with Mung Bean nuclease. The fragment was ligated with pH 55 DNA cleaved with BamHI and treated with Mung Bean nuclease. The ligation loading was transformed according to E. Coli S17-1. The selection was carried out on LB medium, which contained tetracycline. Transformants resistant to tetracycline were obtained, whose hybrid plasmid (pUEaatA) contained the inactivated aatA gene. Example 6. Obtaining mutants of the strain Pseudomonas sp. HR199, in which genes of eugenol catabolism were specifically inactivated by insertof an O-element. The strain Pseudomonas sp. HRl 99 was used as a receptor in conjugatexperiments, in which strains of E. coli S17-1 were used as donors, containing the hybrid plasmids of pSUP202, indicated below. mineral medium containing gluconate, which contained the antibiotic corresponding to the O-element, the "homogenote" transfigantes (exchange of the intact gene for the inactivated gene by insertof the O-element by double "Cross over") and the transconjugants "heterogenotes" ( integratof the hybrid plasmid into the genome by simple "Cross over") could be differentiated by resistance to tetracycline encoded by pSUP202.The mutants Pseudomonas sp HR199./c.sOKm and Pseudomonas sp. HRl 99 fesOGm were obtained after conjugatof Pseudomonas sp. HRl 99 with E.coli S17-1 (pSUP csOKm) or E.coli S17-1 (pSUP / 8OGm). The exchange of the gene fes intact for the inactivated gene by OKm or either GMO (fcsOKmO or fesOGm) was verified by DNA sequencing. The mutants Pseudomonas sp. HRl 99 echOKm and Pseudomonas sp. HRl 99 echOGm were obtained after conjugatof Pseudomonas sp. HRl 99 with E. coli S17-1 (pSUPec / zOKm) or E. coli S17-1 (pSUPec? OGm). The exchange of the intact gene ech by the inactivated gene by OKm or OGm (echOKm or echOGm) was verified by means of DNA sequencing. The mutants Pseudomonas sp. HRl 99 vdhOKm and Pseudomonas sp. HRl 99 vdhOGm were obtained after conjugation of Pseudomonas sp. HRl 99 with E. coli S17-1 (pSUPvc iKm) or with E. coli S17-1 (pSUP / iOGm). The exchange of the intact vdh gene by the inactivated gene by OKm or by GmO (vdhOKm or vdhOGm) was verified by DNA sequencing. The mutants Pseudomonas sp. HRl 99 aatOKm and Pseudomonas sp. HRl 99 aatOGm were obtained after conjugation of Pseudomonas sp. HRl 99 with E. coli S17-1 (pSUPaatOKm) or E. coli S17-1 (pSUPaatOGm). The exchange of the intact aat gene by the inactivated gene by OKm or OGm (aatOKm or aatOGm) was verified by DNA sequencing. The mutants Pseudomonas sp. HR199 / asOKnra // 2OGm were obtained after conjugation of Pseudomonas sp. HRl 99 fcsOKm with E. coli S17-1 (pSUPvl /? OGm). The exchange of the intact vdh gene by the gene inactivated by GMO (vdhOGm) was verified by DNA sequencing. The mutants Pseudomonas sp. HRl 99 vdhOKmaatOGm were obtained after conjugation of Pseudomonas sp. HRl 99 vdhOKm with E.coli S 17- i (pSUPaatOGm). The exchange of the intact aat gene by the gene inactivated by GMO (aatOGm) was verified by DNA sequencing. Mutants Pseudomonas sp HRl 99 vdhOKmechOGm were obtained after conjugation of Pseudomonas sp. HR199 vdhOKm with E. coli S17-1 (pSUPec / zOGm). The exchange of the intact ech gene by the gene inactivated by GMO (echOGm) was verified by DNA sequencing. Example 7. Obtaining mutants of the strain Pseudomonas sp HR199, in which the eugenol catabolism genes have been specifically inactivated by deletion of a part. Strains Pseudomonas sp. HRl 99 fcsOKm, Pseudomonas sp. HRl 99 echOKm, Pseudomonas sp. HRl 99 vdhOKm and Pseudomonas sp HRl 99 aatOKm were used as receptors in the conjugation experiments, in which strains of E. coli S17-1 were used as donors, containing the hybrid plasmids of pHE55 indicated below. The transconjugantes "heterogenotes" were selected on general medium containing gluconate, which contained, in addition to tetracycline (resistance encoded by pHE55) the antibiotic corresponding to the O-element. After spreading on mineral medium containing sucrose, transconjugants were obtained, which had eliminated the DNA vector by means of a second recombination process (second "Cross over"). By spreading on mineral medium without antibiotics or with the antibiotic corresponding to the O-element, the mutants could be recognized, in which the inactivated gene was exchanged by the O-element for the inactivated gene by deletion (absence of resistance to antibiotics). ). The mutant Pseudomonas sp. HRl 99 fe A was obtained after conjugation of Pseudomonas sp. HR199 /c.sOKm with E. coli S17-1 pHEfcsA). The exchange of the inactivated gene by OKm (fcsOKm) by the gene inactivated by deletion (fcsA) was verified by DNA sequencing. The mutant Pseudomonas sp HRl 99 echA was obtained after conjugation Pseudomonas sp. HRl 99 echOKm with E. coli S17-1 (p EechA). The exchange of the inactivated gene by OKm (echOKm) by the deletion-inactivated gene (echA) was verified by DNA sequencing. The mutant Pseudomonas sp. HRl 99 vdhA was obtained after conjugation of Pseudomonas sp. HRl 99 vdhOKm with E.coli S17-1 (pHEvdhA). The exchange of the inactivated gene by OKm (vdhOKm) by the inactivated gene by deletion (vdh A) was verified by DNA sequencing. The mutant Pseudomonas sp. HRl 99 aatA was obtained after conjugation of Pseudomonas sp. HRl 99 aatOKm with E. coli S17-1 (pHEarat?). The exchange of the inactivated gene by OKm (aatOKm) by the inactivated gene by deletion (aatA) was verified by DNA sequencing. Example 8. Biotransformation of eugenol to give vanilin with the mutant Pseudomonas sp. HR199 vrfAOKm. The Pseudomonas sp. Strain was adsorbed. HRl 99 vdhOKm in 50 ml of HR-MN with 6 mM eugenol up to an optical density of approximately OD600nm = 0, 6. After 17 hours, 2.9 mM vanillin, 1.4 mM ferulic acid and 0.4 mM vanillin acid were detected in the culture supernatant. Example 9. Biotransformation of eugenol to give ferulic acid with the mutant Pseudomonas sp. HR199 saw /? OGmaa / OKm. The Pseudomonas sp. Strain was adsorbed. HRl 99 vdhO-GmaatOKm in 50 ml of HR-MN with 6 mM of eugenol up to an optical density of approximately OD500 nm = 0.6. 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. Biotransformation of eugenol in coniferyl alcohol with the mutant Pseudomonas sp. HR199 vrfAOGmflfl / OKm. The Pseudomonas sp. Strain was adsorbed. HRl 99 vdhOGmaatOKm in 50 ml of HR-MM with 6 mM eugenol up to an optical density of approximately OD600nm = 0.4. After 15 hours, 17 mM of coniferyl alcohol could be detected. 1.4 mM vanillin, 1.4 mM ferulic acid and 0.2 mM vanillin acid in the culture supernatant. Example 11. Production by fermentation of natural vanilin from Eugenol in the 10 liter fermenter with the mutant Pseudomonas sp. HR 199 vrf? OKm. The production fermentor was inoculated with 100 ml of a last previous one, with an age of 24 hours, which had been cultivated in a shaker machine (120 revolutions per minute) at 32 ° C in a medium, adjusted to a pH of 7. , 0, constituted by 12.5 g / 1 of glycerin, 10 g / 1 of yeast extract and 0.37 g / 1 of acetic acid. The fermentor contained 9.9 liters of medium with the following composition: 1.5 g / 1 of yeast extract, 1.6 g / 1 of KH2PO4, 0.2 g / 1 of NaCl, 0.2 g / 1 of MgSO4. The pH value was adjusted to pH 7.0 with sodium hydroxide solution. After sterilization, 4 g of eugenol were added to the medium. The temperature was 32 ° C, the ventilation was 3 Nl / minute and the number of revolutions was 600 revolutions per minute. The pH value was maintained at pH 6.5 with sodium hydroxide solution. After 4 hours from the inoculation, the continuous supply of eugenol was started in such a way that at the end of the fermentation, after 255 hours, 255 g of eugenol had been added to the culture. In addition, during the fermentation, 40 g of yeast extract was added. The concentration in eugenol was at the end of the fermentation of 0.2 g / 1. The vanillin content was 2.6 g / 1. Additionally, 3.4 g / 1 of ferulic acid were also present. The vanillin, obtained in this way, can be isolated by known physical methods, such as chromatography, distillation and / or extraction and can be used for the manufacture of natural flavors. Brief description of the Figures. Figures up to Go: Genetic structure for obtaining organisms and mutants. calA *: Part of the inactivated gene of the coniferilalcohol-dehydrogenase. calB *: Part of the inactivated gene of coniferylaldehyde dehydrogenase. fes *: Inactivated part of the ferulic acid-CoA synthetase gene. ech *: Part of the inactivated gene of enoyl-CoA hydratase-aldolase. vdh *: Part of the inactivated vanillin-dehydrogenase gene. aat *: Part of the inactivated beta-ketothiolase gene. The restriction enzymatic cut-off points marked with "*" were used for construction, but are no longer able to function in the resulting duct. FIGURE 2a: Nucleotide sequence of the calAOKm genetic structure.

- -FIGURE 2b: Nucleotide sequence of the calAOGm genetic structure. FIGURE 2c: Nucleotide sequence of the calAA genetic structure FIGURE 2d: Nucleotide sequence of the calBOKm genetic structure FIGURE 2e: Nucleotide sequence of the calBOGm genetic structure FIGURE 2f: Nucleotide sequence of the calBA genetic structure FIGURE 2g: Nucleotide sequence of the fcsOKm genetic structure FIGURE 2h: Nucleotide sequence of the fesOGm genetic structure FIGURE 2i: Nucleotide sequence of the fes genetic structure A FIGURE 2j: Nucleotide sequence of the echOKm genetic structure FIGURE 2k: Nucleotide sequence of the echOGm genetic structure FIGURE 21: Nucleotide sequence of the genetic structure echA FIGURE 2m: Nucleotide sequence of the genetic structure vdhOKm FIGURE 2n: Nucleotide sequence of the genetic structure vdhOGm FIGURE 2o: Nucleotide sequence of the genetic structure vdhA FIGURE 2p: Nucleotide sequence of the aatOKm genetic structure FIGURE 2q: Nucleotide sequence of the genetic structure ATOGM FIGURE 2r: Nucleotide sequence of the aatA genetic structure. It is "stated that, in relation to this date, the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Sequence 1 CTGCAGCCAG GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60 GGCTCCAATT GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120 GCTAGGGAGA TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGGA TGATTTTCTG 180 CATTCTGCAT CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG CACGGGATTG 240 AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300 GCATGGAAAT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT TTTGGCGGAA 360 TGGAAGCACG ATTCCTCGCC CGGTAGAGCG GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420 AAAGAGCATG CAACTGACCA ACAAGAAAAT CGTCGTCACC GGAGTGTCCT CCGGTATCGG 480 TGCCGAAACT GCCCGCGTTC TGCGCTCTCA CGGCGCCACA GTGATTGGCG TAGATCGCAA 540 CATGCCGAGC CTGACTCTGG ATGCTTTCGT TCAGGCTGAC CTGAGCCATC CTGAAGGCAT 600 CGATAAGGCC ATCGGGACAG CAAGCGAACC GGAATTGCCA GCTGGGGCGC CCTCTGGTAA 660 GGTTGGGAAG CCCTGCAAAG TAAACTGGAT GGCTTTCTTG CCGCCAAGGA TCTGATGGCG 720 AGGGGATCA AGATCTGATC AAGAGACAGG ATGAGGATCG TTTCGCATGA TTGAACAAGA • 780 GGGATTGCAC GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG CTATTCGGCT ATGACTGGGC 840 ACAACA GACA ATCGGCTGCT CTGATGCCGC CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC 900 GGTTCTTTTT GTCAAGACCG ACCTGTCCGG TGCCCTGAAT GAACTGCAGG ACGAGGCAGC 960 GCGGCTATCG TGGCTGGCCA CGACGGGCGT TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC 1020 TGAAGCGGGA AGGGACTGGC TGCTATTGGG CGAAGTGCCG GGGCAGGATC TCCTGTCATC 1080 TCACCTTGCT CCTGCCGAGA AAGTATCCAT CATGGCTGAT GCAATGCGGC GGCTGCATAC 1140 GCTTGATCCG GCTACCTGCC CATTCGACCA CCAAGCGAAA CATCGCATCG AGCGAGCACG 1200 TACTCGGATG GAAGCCGGTC TTGTCGATCA GGATGATCTG GACGAAGAGC ATCAGGGGCT 1260 CGCGCCAGCC GAACTGTTCG CCAGGCTCAA GGCGCGCATG CCCGACGGCG AGGATCTCGT 1320 CGTGACCCAT GGCGATGCCT GCTTGCCGAA TATCATGGTG GAAAATGGCC GCTTTTCTGG 1380 ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GGACCGCTAT CAGGACATAG CGTTGGCTAC 1440 CCGTGATATT GCTGAAGAGC TTGGCGGCGA ATGGGCTGAC CGCTTCCTCG TGCTTTACGG 1500 TATCGCCGCT CCCGATTCGC AGCGCATCGC CTTCTATCGC CTTCTTGACG AGTTCTTCTG 1560 AGCGGGACTC TGGGGTTCGA AATGACCGAC CAAGCGACGC CCTGGCCGCG GTGATTGCAT 1620 TCATGTGTGC TGAGGAGTCA CGTTGGATCA ACGGCATAAA TATTCCAGTG GACGGAGGTT 1680 TGGCATCGAC CTACGTGTAA GTTCGTGGAC GCCCTTTGCA CGCGCACTAT ATCTCTATGC 1740 AGCAGCT GAA AGCAGCTTTG GTTTTGATCG GAGGTAGCGG GCGGAAAGGT GCAGAATGTC 1800 TAAATAATAA AGGATTCTTG TGAAGCTTTA GTTGTCCGTA AACGAAAATA AAAATAAAGA 1860 GGAATGATAT GAAAGCAAGT AGATCAGTCT GCACTTTCAA AATAGCTACC CTGGCAGGCG 1920 CCATTTATGC AGCGCTGCCA ATGTCAGCTG CAAACTCGAT GCAGCTGGAT GTAGGTAGCT 1980 CGGATTG € AC GGTGCGTTGG GGACAACACC CTCAAGTATA GCCTTGCCTC TCGCCTGAAT 2040 GAGCAAGACT- CAAGTCTGAC AAATGCGCCG ACTGTCAATG GTTATATCCG GATATTCAAA 2100 GTCAGGGTGA TCGTAACTTT GACCGGGGGC TTGGTATCCA ATCGTCTCGA TATTCTGGCT 2 * 160 GCAG 2164 Sequence 2 CTGCAGCCAG GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60 GGCTCCAATT GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120 GCTAGGGAGA TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGG? TGATTTTCTG 180 CATTCTGCAT CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG CACGGGATTG 240 AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300 GCATGGAAAT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT TTTGGCGGAA 360 TGGAAGCACG ATTCCTCGCC CGGTAGAGCG GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420 AAAGAGCATG CAACTGACCA ACAAGAAAAT CGTCGTCACC GGAGTGTCCT CCGGTATCGG 480 TGCCGAAACT GCCCGCGTTC TGCGCTCTCA CGGCGCCACA GTGATTGGCG TAGATCGCAA 540 CATGCCGAGC CTGACTCTGG ATGCTTTCGT TCAGGCTGAC CTGAGCCATC CTGAGGGGAG 600 AGGCGGTTTG CGTATTGGGC GCATGCATAA AAACTGTTGT AATTCATTAA GCATTCTGCC 660 GACATGGAAG CCATCACAAA CGGCATGATG AACCTGAATC GCCAGCGGCA TCAGCACCTT 720 GTCGCCTTGC GTATAATATT TGCCCATGGA CGCACACCGT GGAAACGGAT GAAGGCACGA 780 ACCCAGTTGA CATAAGCCTG TTCGGTTCGT AAACTGTAAT GCAAGTAGCG TATGCGCTCA 840 CGCAACTGGT CCAGAACCTT GACCGAACGC AGCGGTGGTA ACGGCGCAGT GGCGGTTTTC 900 ATGGCTTGTT ATGACTGTTT TTTTGTACAG TCTATGCCTC GGGCATCCAA GCAGCAAGCG 960 CGTTACGCCG TGGGTCGATG TTTGATGTTA TGGAGCAGCA ACGATGTTAC GCAGCAGCAA 1020 CGAT GTTACG CAGCAGGGCA GTCGCCCTAA AACAAAGTTA GGTGGCTCAA GTATGGGCAT 1080 CATTCGCACA TGTAGGCTCG GCCCTGACCA AGTCAAATCC ATGCGGGCTG CTCTTGATCT 1140 TTTCGGTCGT GAGTTCGGAG ACGTAGCCAC CTACTCCCAA CATCAGCCGG ACTCCGATTA 1200 CCTCGGGAAC TTGCTCCGTACATCGCGCTT GCTGCCTTCG ACCAAGAAGC 1260 GGTTGTTGGC GCTCTCGCGG CTTACGTTCT GCCCAGGTTT GAGCAGCCGC GTAGTGAGAT 1320 CTATATCTAT GATCTCGCAG TCTCCGGCGA GCACCGGAGG CAGGGCATTG CCACCGCGCT 1380 CATCAATCTC CTCAAGCATG AGGCCAACGC GCTTGGTGCT TATGTGATCT ACGTGCAAGC 1440 AGATTACGGT GACGATCCCG CAGTGGCTCT CTATACAAAG TTGGGCATAC GGGAAGAAGT 1500 GATGCACTTT GATATCGACC CAAGTACCGC CACCTAACAA TTCGTTCAAG CCGAGATCGG 1560 CTTCCCTGAT TGCATTCATG TGTGCTGAGG AGTCACGTTG GATCAACGGC ATAAATATTC 1620 CAGTGGACGG AGGTTTGGCA TCGACCTACG TGTAAGTTCG TGGACGCCCT TTGCACGCGC 1680 ACTATATCTC TATGCAGCAG CTGAAAGCAG CTTTGGTTTT GATCGGAGGT AGCGGGCGGA 1740 AAGGTGCAGA ATGTCTAAAT AATAAAGGAT TCTTGTGAAG CTTTAGTTGT CCGTAAACGA 1800 AAATAAAAAT AAAGAGGAAT GATATGAAAG CAAGTAGATC AGTCTGCACT TTCAAAATAG_1860_CTACCCTGGC AGGCGCCATT TATGCAGCGC TGCCAATGTC AGCTGCAAAC TCGATGCAGC 1920 TGGATGTAGG TAGCTCGGAT TGGACGGTGC GTTGGGGACA ACACCCTCAA GTATAGCCTT 1980 GCCTCTCGCC TGAATGAGCA AGACTCAAGT CTGACAAATG CGCCGACTGT CAATGGTTAT 2040 ATCCGGATAT TCAAAGTCAG GGTGAT CGTA ACTTTGACCG GGGGCTCTGGT ATCCAATCGT 2100 CTCGATATTC TGGCTGCAG 2119 Sequence 4 GAATTCCGCG TATCGCCCGG TTCTATCAGC GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC 60 GGTAGGGTCT TTTTCTTGGC CATGCTTGTT GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120 TGCGTTTGCC GCTTCGCTTC GCGATGAACC GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180 TTAACTCGCG TAAGCATTCT GTCATTTTTT TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240 GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300 CGATTAAGAT AATTAAAATA AGGAAACCGC ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360 CTCCAGCTCA AGGGCAATTT TTGGGCTATT GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420 AGAATAACAA TTGACTCCTC AGGAGGTCAG CGATGAGCAT TCTTGGTTTG AATGGTGCCC. 480 CGGTCGGAGC TGAGCAGCTG GGCTCGGCTC TTGATCGCAT GAAGAAGGCG CACCTGGAGC 540 AGGGGCCTGC AAACTTGGAG CTGCGTCTGA GTAGGCTGGA TCGTGCGATT GCAATGCTTC 600 TGGAAAATCG TGAAGCAATT GCCGACGCGG TTTCTGCTGA CTTTGGCAAT CGCAGCCGTG 660 AGCAAACACT GCTTTGCGAC ATTGCTGGCT CGGTGGCAAG CCTGAAGGAT AGCCGCGAGC 720 ACGTGGCCAA ATGGATGGAG CCCGAACATC ACAAGGCGAT GTTTCCAGGG GCGGAGGCAC 780 GCGTTGAGTT TCAGCCGCTG GGTGTCGTTG GGGTCATTAG TCCCTGGAAC TTCCCTATCG 840 TACTGGCCTT TGGGCCGCTG GCCGGCATAT TCGCAGCAGG TAATCGCGCC ATGCTCAAGC 900 CGTCCGAGCT TACCCCGCGG ACTTCTGCCC TGCTTGCGGA GCTAATTGCT CGTTACTTCG 960 ATGAAACTGA GCTGACTACA GTGCTGGGCG ACGCTGAAGT CGGTGCGCTG TTCAGTGCTC 1020 AGCCTTTCGA TCATCTGATC TTCACCGGCG GCACTGCCGT GGCCAAGCAC ATCATGCGTG 1080 CCGCGGCGGA TAACCTAGTG CCCGTTACCC TGGAATTGGG TGGCAAATCG CCGGTGATCG 1140 TTTCCCGCAG TGCAGATATG GCGGACGTTG CACAACGGGT GTTGACGGTG AAAACCTTCA 1200 ATGCCGGGCA AATCTGTCTG GCACCGGACT ATGTGCTGCT GCCGGAAGGG ACAGCAAGCG 1260 AACCGGAATT GCCAGCTGGG GCGCCCTCTG GTAAGGTTGG GAAGCCCTGC AAAGTAAACT 1320 GGATGGCTTT CTTGCCGCCA AGGATCTGAT GGCGCAGGGG ATCAAGATCT GATCAAGAGA 1380 CAGGATGAGG ATCGTTTCGC ATGATTGAAC AAGATGGATT GCACGCAGGT TCTCCGGCCG 1440 CTTGGGTGGA GAGGCTATTC GGCTATGACT GGGCACAACA GACAATCGGC TGCTCTGATG 1500 CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC GCCCGGTTCT TTTTGTCAAG ACCGACCTGT 1560 CCGGTGCCCT GAATGAACTG CAGGACGAGG CAGCGCGGCT ATCGTGGCTG GCCACGACGG 1620 GCGTTCCTTG CGCAGCTGTG CTCGACGTTG TCACTGAAGC GGGAAGGGAC TGGCTGCTAT 1680 TGGGCGAAGT GCCGGGGCAG GATCTCCTGT CATCTCACCT TGCTCCTGCC GAGAAAGTAT 1740 CCATCATGGC TGATGCAATG CGGCGGCTGC ATACGCTTGA TCCGGCTACC TGCCCATTCG 1800 ACCACCAAGC GAAACATCGC ATCGAGCGAG CACGTACTCG GATGGAAGCC GGTCTTGTCG 1860 ATCAGGATGA TCTGGACGAA GAGCATCAGG GGCTCGCGCC AGCCGAACTG TTCGCCAGGC 1920 TCAAGQCGCG CATGCCCGAC GGCGAGGATC TCGTCGTGAC CCATGGCGAT GCCTGCTTGC 1980 CGAATATCAT GGTGGAAAAT GGCCGCTTTT CTGGATTCAT CGACTGTGGC CGGCTGGGTG 2040 TGGCGGACCG CTATCAGGAC ATAGCGTTGG CTACCCGTGA TATTGCTGAA GAGCTTGGCG GCGAATGGGC 0.2100 TGACCGCTTC CTCGTGCTTT ACGGTATCGC CGCTCCCGAT TCGCAGCGCA 2160 Sequence 5 GAATTCCGCG TATCGCCCGG TTCTATCAGC GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC 60 GGTAGGGTCT TTTTCTTGGC CATGCTTGTT GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120 TGCGTTTGCC GCTTCGCTTC GCGATGAACC GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180 TTAACTCGCG TAAGC? TTCT GTCATTTTTT TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240 GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300 CGATTAAGAT AATTAAAATA AGGAAACCGC ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360 CTCCAGCTCA AGGGCAATTT TTGGGCTATT GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420 AGAATAACAA TTGACTCCTC AGGAGGTCAG CGATGAGCAT TCTTGGTTTG AATGGTGCCC 480 CGGTCGGAGC TGAGCAGCTG GGCTCGGCTC TTGATCGCAT GAAGAAGGCG CACCTGGAGC 540 AGGGGCCTGC AAACTTGGAG CTGCGTCTGA GTAGGCTGGA TCGTGCGATT GCAATGCTTC 600 TGGAAAATCG TGAAGCAATT GCCGACGCGG TTTCTGCTGA CTTTGGCAAT CGCAGCCGTG 660 AGCAAACACT GCTTTGCGAC ATTGCTGGCT CGGTGGCAAG CCTGAAGGAT AGCCGCGAGC 720 ACGTGGCCAA ATGGATGGAG CCCGAACATC ACAAGGCGAT GTTTCCAGGG GCGGAGGCAC 780 GCGTTGAGTT TCAGCCGCTG GGTGTCGTTG GGGTCATTAG TCCCTGGAAC TTCCCTATCG 840 TACTGGCC TT TGGGCCGCTG GCCGGCATAT TCGCAGCAGG TAATCGCGCC ATGCTCAAGC 900 CGTCCGAGCT TACCCCGCGG ACTTCTGCCC TGCTTGCGGA GCTAATTGCT CGTTACTTCG 960 ATGAAACTGA GCTGACTACA GTGCTGGGCG ACGCTGAAGT CGGTGCGCTG TTCAGTGCTC 1020 AGCCTTTCGA TCATCTGATC TTCACCGGCG GCACTGCCGT GGCCAAGCAC ATCATGCGTG 1080 CCGCGGCGGA TAACCTAGTG CCCGTTACCC TGGAATTGGG TGGCAAATCG CCGGTGATCG 1140 TTTCCCGCAG TGCAGATATG GCGGACGTTG CACAACGGGT GTTGACGGTG AAAACCTTCA 1200 ATGCCGGGCA AATCTGTCTG GCACCGGACT ATGTGCTGGG GGAGAGGCGG TTTGCGTATT 1260 GGGCGCATGC ATAAAAACTG TTGTAATTCA TTAAGCATTC TGCCGACATG GAAGCCATCA 1320 CAAACGGCAT GATGAACCTG AATCGCCAGC GGCATCAGCA CCTTGTCGCC TTGCGTATAA 1380 TATTTGCCCA TGGACGCACA CCGTGGAAAC GGATGAAGGC ACGAACCCAG TTGACATAAG 1440 CCTGTTCGGT TCGTAAACTG TAATGCAAGT AGCGTATGCG CTCACGCAAC TGGTCCAGAA 1500 CCTTGACCGA ACGCAGCGGT GGTAACGGCG CAGTGGCGGT TTTCATGGCT TGTTATGACT 1560 GTTTTTTTGT ACAGTCTATG CCTCGGGCAT CCAAGCAGCA AGCGCGTTAC GCCGTGGGTC 1620 GATGTTTGAT GTTATGGAGC AGCAACGATG TTACGCAGCA GCAACGATGT TACGCAGCAG 1680 GGCAGTCGCC CTAAAACAAA GTTAGGTGGC TCAAGTATGG GCATCATTCG CACATGTAGG 1740 CTCGGCCCTG ACCAAGTCAA ATCCATGCGG GCTGCTCTTG ATCTTTTCGG TCGTGAGTTC 1800 GGAGACGTAG CCACCTACTC CCAACATCAG CCGGACTCCG ATTACCTCGG GAACTTGCTC 1860 CGTAGTAAGA CATTCATCGC GCTTGCTGCC TTCGACCAAG AAGCGGTTGT TGGCGCTCTC 1920 GCGGCTTACG TTCTGCCCAG GTTTGAGCAG CCGCGTAGTG AGATCTATAT CTATGATCTC 1980 GCAGTCTCCG GCGAGCACCG GAGGCAGGGC ATTGCCACCG CGCTCATCAA TCTCCTCAAG 2040 CATGAGGC A ACGCGCTTGG TGCTTA TGTG ATCTACGTGC AAGCAGATTA CGGTGACGAT 2100 CCCGCAGTGG CTCTCTATAC AAAGTTGGGC ATACGGGAAG AAGTGATGCA CTTTGATATC 2160 GACCC? AGTA CCGCCACCTA ACAATTCGTT CAAGCCGAGA TCGGCTTCCC TGCAAAGTCC 2220 BGTGGGTGAG TCGAACTTGG CGATGCGCGC ACCCTACGGA GAAGCGATCC ACGGACTGCT 2280 CTCTGTCCTC CTTTCAACGG AGTGTTAGAA CCGTTGGTAG TGGTTTTGGA CGGGCCCAGG 2340 AGCATGCGCT TCTGGGCCCG TTTCTTGAGT ATTCATTGGA TAGTCACGCG TGGTAGCTTC 2400 GAGCCTGCAC AGCTGATGAG CACCCTGGAA GGCGCGCTGT ACGCGGACGA CTGGGTTCAT 2460 CTTCGCCATT CATGACGGAA CTCCGTTCCC CAGTACCGCG ATGACTATTT TGCCTCTTCC 2520 GATGTCCGAT TCCACGCCGC CTGACGCTAA GCGGGGGCGG GGGCGCCCGC ATCCCAGCCC 2580 AGACAGCAAC AAATGAGTAG GCTCTTGGAT GCCGCGGCGG CTGAGATTGG TAACGGCAAT 2640 TTCGTCAATG TGACGATGGA TTCGATTGCC CGTGCTGCCG GCGTCTCAAA AAAAACGCTG 2700 TACGTCTTGG TGGCGAGCAA GGAAGAACTC ATTTCCCGGT TAGTGGCTCG AGACATGTCC 2760 AACCTTGAGG AATTC 2775 Sequence 6 FAATTCCGCG TATCGCCCGG TTCTATCAGC GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC 60 GGTAGGGTCT TTTTCTTGGC CATGCTTGTT GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120 TGCGTTTGCC GCTTCGCTTC GCGATGAACC GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180 TTAACTCGCG TAAGCATTCT GTCATTTTTT TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240 GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300 CGATTAAGAT AATTAAAATA AGGAAACCGC ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360 CTCCAGCTCA AGGGCAATTT TTGGGCTATT GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420 AGAATAACAA TTGACTCCTC AGGAGGTCAG CGATGAGCAT TCTTGGTTTG AATGGTGCCC 480 CGGTCGGAGC TGAGCAGCTG GGCTCGGCTC TTGATCGCAT GAAGAAGGCG CACCTGGAGC 540 AGGGGCCTGC AAACTTGGAG CTGCGTCTGA GTAGGCTGGA TCGTGCGATT GCAATGCTTC 600 TGGAAAATCG TGAAGCAATT GCCGACGCGG TTTCTGCTGA CTTTGGCAAT CGCAGCCGTG 660 AGCAAACACT GCTTTGCGAC ATTGCTGGCT CGGTGGCAAG CCTGAAGGAT AGCCGCGAGC 720 ACGTGGCCAA ATGGATGGAG CCCGAACATC ACAAGGCGAT GTTTCCAGGG GCGGAGGCAC 780 GCGTTGAGTT TCAGCCGCTG GGTGTCGTTG GGGTCATTAG TCCCTGGAAC TTCCCTATCG 840 TACTGGCC TT TGGGCCGCTG GCCGGCATAT TCGCAGCAGG TAATCGCGCC ATGCTCAAGC 900 CGTCCGAGCT TACCCCGCGG ACTTCTGCCC TGCTTGCGGA GCTAATTGCT CGTTACTTCG 960 ATGAAACTGA GCTGACTACA GTGCTGGGCG ACGCTGAAGT CGGTGCGCTG TTCAGTGCTC 1020 AGCCTTTCGA TC? TCTGATC TTCACCGGCG GCACTGCCGT GGCCAAGCAC ATCATGCGTG 1080 CCGCGGCGGA TAACCTAGTG CCCGTTACCC TGGAATTGGG TGGCAAATCG CCGGTGATCG 1140 TTTCCCGCAG TGCAGATATG GCGGACGTTG CACAACGGGT GTTGACGGTG AAAACCTTCA 1200 ATGCCGGGCA AATCTGTCTG GCACCGTGGG TGAGTCGAAC TTGGCGATGC GCGCACCCTA 1260 CGGAGAAGCG ATCCACGGAC TGCTCTCTGT CCTCCTTTCA ACGGAGTGTT AGAACCGTTG 1320 GTAGTGGTTT TGGACGGGCC CAGGAGCATG CGCTTCTGGG CCCGTTTCTT GAGTATTCAT 1380 TGGATAGTCA CGCGTGGTAG CTTCGAGCCT GCACAGCTGA TGAGCACCCT GGAAGGCGCG 1440 CTGTACGCGG ACGACTGGGT TCATCTTCGC CATTCATGAC GGAACTCCGT TCCCCAGTAC 1500 CGCGATGACT ATTTTGCCTC TTCCGATGTC CGATTCCACG CCGCCTGACG CTAAGCGGGG 1560 GCGGGGGCGC CCGCATCCCA GCCCAGACAG CAACAAATGA GTAGGCTCTT GGATGCCGCG 1620 GCGGCTGAGA TTGGTAACGG CAATTTCGTC AATGTGACGA TGGATTCGAT TGCCCGTGCT 1680 GCCGGCGTCT CAAAA AAAAC GCTGTACGTC TTGGTGGCGA GCAAGGAAGA ACTCATTTCC 1740 CGGTTAGTGG CTCGAGACAT GTCCAACCTT GAGGAATTC 1779 Sequence 7 CTGCAGCCGA GCATCGATTG AGCACTTTAC CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60 CCCGCGGCAC TATCCAATCT AAATCGATCT TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120 CTCGCCTCAT TTCAATCTCT AACTTGATAA AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180 CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AGAGTACAGT GAACGCCGAG TCCACATTGC 240 AACCGCAGGC ATCATCATGC TCTGCTCAGC CACGCTACCG CAGTGTGTCG ATTGGTCATC 300 CTCCGGTTGA GGTTACGCAA GACGCTGGAG GTATTGTCCG GATGCGTTCT CTCGAGGCGC 360 TTCTTCCCTT CCCGGGTCGA ATTCTTGAGC GTCTCGAGCA TTGGGCTAAG ACCCGTCCAG 420 AACAAACCTG CGTTGCTGCC AGGGCGGCAA ATGGGGAATG GCGTCGTATC AGCTACGCGG 480 AAATGTTCCA CAACGTCCGC GCCATCGCAC AGAGCTTGCT TCCTTACGGA CTATCGGCAG 540 AGCGTCCGCT GCTTATCGTC TCTGGAAATG ACCTGGAACA TCTTCAGCTG GCATTTGGGG 600 CTATGTATGC GGGCATTCCC TATTGCCCGG TGTCTCCTGC TTATTCACTG CTGTCGCAAG 660 ATTTGGCGAA GCTGCGTCAC ATCGTAGGTC TTCTGCAACC GGGACTGGTC TTTGCTGCCG 720 ATGCAGCACC TTTCCAGGGG ACAGCAAGCG AACCGGAATT GCCAGCTGGG GCGCCCTCTG 780 GTAAGGTTGG GAAGCCCTGC AAAGTAAACT GGATGGCTTT CTTGCCGCCA AGGATCTGAT 840 GGCGCAGGGG ATCAAGATCT GATCAAGAGA CAGGATGAGG ATCGTTTCGC ATGATTGAAC 900 AAGATGGATT GCACGCAGGT TCTCCGGCCG CTTGGGTGGA GAGGCTATTC GGCTATGACT 960 GGGCACAACA GACAATCGGC TGCTCTGATG CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC 1020 GCCCGGTTCT TTTTGTCAAG ACCGACCTGT CCGGTGCCCT GAATGAACTG CAGGACGAGG 1080 CAGCGCGGCT ATCGTGGCTG GCCACGACGG GCGTTCCTTG CGCAGCTGTG CTCGACGTTG 1140 TCACTGAAGC GGGAAGGGAC TGGCTGCTAT TGGGCGAAGT GCCGGGGCAG GATCTCCTGT 1200 CATCTCACCT TGCTCCTGCC GAGAAAGTAT CCATCATGGC TGATGCAATG CGGCGGCTGC 1260 ATACGCTTGA TCCGGCTACC TGCCCATTCG ACCACCAAGC GAAACATCGC ATCGAGCGAG 1320 CACGTACTCG GATGGAAGCC GGTCTTGTCG ATCAGGATGA TCTGGACGAA GAGCATCAGG 1380 GGCTCGCGCC AGCCGAACTG TTCGCCAGGC TCAAGGCGCG CATGCCCGAC GGCGAGGATC 1440 TCGTCGTGAC CCATGGCGAT GCCTGCTTGC CGAATATCAT GGTGGAAAAT GGCCGCTTTT 1500 CTGGATTCAT CGACTGTGGC CGGCTGGGTG TGGCGGACCG CTATCAGGAC ATAGCGTTGG 1560 CTACCCGTGA TATTGCTGAA GAGCTTGGCG GCGAATGGGC TGACCGCTTC CTCGTGCTTT 1620 ACGGTATCGC CGCTCCCGAT TCGCAGCGCA TCGCCTTCTA TCGCCTTCTT GACGAGTTCT 1680 TCTGAGCGGG ACTCTGGGGT TCGAAATGAC CGACCAAGCG ACGCCCCTGT TTTGCAATGG 1740 CGGTCGGCGA AAGTTGATGC GCTGTATCGT GGTGAAGATC AATCCATGCT GCGTGACGAG 1800 GCCACACTGT GAGTTGGTCA GGGGGGGCTT ACTCGGCGTT TTCCGACACT GCGTTGGTTG 1860 CGGCAGTGCG CACCCCCTGG ATTGATTGCG GGGGTGCCCT GTCGCTGGTG TCGCCTATCG 1920 ACTTAGGGGT AAAGGTCGCT CGCGAAGTTC TGATGCGTGC GTCGCTTGAA CCACAAATGG 1980 TCGATAG € GT ACTCGCAGGC TCTATGGCTC AAGCAAGCTT TGATGCTTAC CTGCTCCCGC 2040 GGCACATTGG- CTTGTACAGC GGTGTTCCCA AGTCGGTTCC GGCCTTGGGG GTGCAGCGCA 2JL00 TTTGCGGCAC AGGCTTCGAA CTGCTTCGGC AGGCCGGCGA GCAGATTTCC CAAGGCGCTG 2160 ATCACGTGCT GTGTGTCGCG GGCTGCAG 2188 Sequence 8 tTGCAGCCGA GCATCGATTG AGCACTTTAC CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60 CCCGCGGCAC TATCCAATCT AAATCGATCT TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120 CTCGCCTCAT TTCAATCTCT AACTTGATAA AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180 CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AGAGTACAGT GAACGCCGAG TCCACATTGC 240 AACCGCAGGC ATCATCATGC TCTGCTCAGC CACGCTACCG CAGTGTGTCG ATTGGTCATC 300 CTCCGGTTGA GGTTACGCAA GACGCTGGAG GTATTGTCCG GATGCGTTCT CTCGAGGCGC 360 TTCTTCCCTT CCCGGGTCGA ATTCTTGAGC GTCTCGAGCA TTGGGCTAAG ACCCGTCCAG 420 AACAAACCTG CGTTGCTGCC AGGGCGGCAA ATGGGGAATG GCGTCGTATC AGCTACGCGG 480 AAATGTTCCA CAACGTCCGC GCCATCGCAC AGAGCTTGCT TCCTTACGGA CTATCGGCAG 540 AGCGTCCGCT GCTTATCGTC TCTGGAAATG ACCTGGAACA TCTTCAGCTG GCATTTGGGG 600 CTATGTATGC GGGCATTCCC TATTGCCCGG TGTCTCCTGC TTATTCACTG CTGTCGCAAG 660 ATTTGGCGAA GCTGCGTCAC ATCGTAGGTC TTCTGCAACC GGGACTGGTC TTTGCTGCCG 720 ATGCAGCACC TTTCCAGGGG GAGAGGCGGT TTGCGTATTG GGCGCATGCA TAAAAACTGT 780 TGTAATTCAT TAAGCATTCT GCCGACATGG AAGCCATCAC AAACGGCATG ATGAACCTGA 840 ATCGCCAGC G GCATCAGCAC CTTGTCGCCT TGCGTATAAT ATTTGCCCAT GGACGCACAC 900 CGTGGAAACG GATGAAGGCA CGAACCCAGT TGACATAAGC CTGTTCGGTT CGTAAACTGT 960 AATGCAAGTA GCGTATGCGC TCACGCAACT GGTCCAGAAC CTTGACCGAA CGCAGCGGTG 1020 GTAACGGCGC AGTGGCGGTT TTCATGGCTT GTTATGACTG TTTTTTTGTA CAGTCTATGC 1080 CTCGGGCATC CAAGCAGCAA GCGCGTTACG CCGTGGGTCG ATGTTTGATG TTATGGAGCA 1140 GCAACGATGT TACGCAGCAG CAACGATGTT ACGCAGCAGG GCAGTCGCCC TAAAACAAAG 1200 TTAGGTGGCT CAAGTATGGG CATCATTCGC ACATGTAGGC TCGGCCCTGA CCAAGTCAAA 1260 TCCATGCGGG CTGCTCTTGA TCTTTTCGGT CGTGAGTTCG GAGACGTAGC CACCTACTCC 1320 CAACATCAGC CGGACTCCGA TTACCTCGGG AACTTGCTCC GTAGTAAGAC ATTCATCGCG 1380 CTTGCTGCCT TCGACCAAGA AGCGGTTGTT GGCGCTCTCG CGGCTTACGT TCTGCCCAGG 1440 TTTGAGCAGC CGCGTAGTGA GATCTATATC TATGATCTCG CAGTCTCCGG CGAGCACCGG 1500 AGGCAGGGCA TTGCCACCGC GCTCATCAAT CTCCTCAAGC ATGAGGCCAA CGCGCTTGGT 1560 GCTTATGTGA TCTACGTGCA AGCAGATTAC GGTGACGATC CCGCAGTGGC TCTCTATACA 1620 AAGTTGGGCA TACGGGAAGA AGTGATGCAC TTTGATATCG ACCCAAGTAC CGCCACCTAA 1680 CAATTCGTTC AAGCCG AGAT CGGCTTCCCC TGTTTTGCAA TGGCGGTCGG CGAAAGTTGA 1740 TGCGCTGTAT CGTGGTGAAG ATCAATCCAT GCTGCGTGAC GAGGCCACAC TGTGAGTTGG 1800 TCAGGGGGGG CTTACTCGGC GTTTTCCGAC ACTGCGTTGG TTGCGGCAGT GCGCACCCCC 1860 TGGATTGATT GCGGGGGTGC CCTGTCGCTG GTGTCGCCTA TCGACTTAGG GGTAAAGGTC 1920 GCTCGCGAAG TTCTGATGCG TGCGTCGCTT GAACCACAAA TGGTCGATAG CGTACTCGCA 1980 GGCTCTATGG CTCAAGCAAG CTTTGATGCT TACCTGCTCC CGCGGCACAT TGGCTTGTAC 2040 AGCGGTGTTC CCAAGTCGGT TCCGGCCTTG GGGGTGCAGC GCATTTGCGG CACAGGCTTC 2100 GAACTGCTTC " GGCAGGCCGG CGAGCAGATT TCCCAAGGCG CTGATCACGT GCTGTGTGTC 2160 GCGGGCTGCA G 2171 eleven Sequence 9 60 120 240 300 360 480 600 660 720 780 900 960 1020 1200 12 Sequence 10 GGCGACGAAA GGGCGGCAGG CCGCATGGCC ACGGCTGGGC GGTAACTGAT 60 GCTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG AGAATCATGC 120 GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT 180 AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC ACAGCACTGG 240 GGTGCACGAT GAATAGCTAC GATGGCCGTT GGTCTACCGT TGATGTGAAG GTTGAAGAAG 300 GTATCGCTTG GGTCACGCTG AACCGCCCGG AGAAGCGCAA CGCAATGAGC CCAACTCTCA 360 ATCGAGAGAT GGTCGAGGTT CTGGAGGTGC TGGAGCAGGA CGCAGATGCT CGCGTGCTTG 420 TTCTGACTGG TGCAGGCGAA TCCTGGACCG CGGGCATGGA CCTGAAGGAG TATTTCCGCG 480 AGACCGATGC TGGCCCCGAA ATTCTGCAAG AGAAGATTCG TCGGGGACAG CAAGCGAACC 540 GGAATTGCCA GCTGGGGCGC CCTCTGGTAA GGTTGGGAAG CCCTGCAAAG TAAACTGGAT 600 GGCTTTCTTG CCGCCAAGGA TCTGATGGCG CAGGGGATCA AGATCTGATC AAGAGACAGG 660 ATGAGGATCG TTTCGCATGA TTGAACAAGA TGGATTGCAC GCAGGTTCTC CGGCCGCTTG 720 GGTGGAGAGG CTATTCGGCT ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC 780 CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC GGTTCTTTTT GTCAAGACCG ACCTGTCCGG 840 TGCCCTGAAT GAACTGC AGG ACGAGGCAGC GCGGCTATCG TGGCTGGCCA CGACGGGCGT 900 TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC TGAAGCGGGA AGGGACTGGC TGCTATTGGG 960 CGAAGTGCCG GGGCAGGATC TCCTGTCATC TCACCTTGCT CCTGCCGAGA AAGTATCCAT 1020 CATGGCTGAT GCAATGCGGC GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA 1080 CCAAGCGAAA CATCGCATCG AGCGAGCACG TACTCGGATG GAAGCCGGTC TTGTCGATCA 1140 GGATGATCTG GACGAAGAGC ATCAGGGGCT CGCGCCAGCC GAACTGTTCG CCAGGCTCAA 1200 GGCGCGCATG CCCGACGGCG AGGATCTCGT CGTGACCCAT GGCGATGCCT GCTTGCCGAA 1260 TATCATGGTG GAAAATGGCC GCTTTTCTGG ATTCATCGAC TGTGGCCGGC TGGGTGTGGC 1320 GGACCGCTAT CAGGACATAG CGTTGGCTAC CCGTGATATT GCTGAAGAGC TTGGCGGCGA 1380 ATGGGCTGAC CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC AGCGCATCGC 1440 CTTCTATCGC CTTCTTGACG AGTTCTTCTG AGCGGGACTC TGGGGTTCGA AATGACCGAC 1500 CAAGCGACGC CCCGAGCAGG GCATGAAGCA GTTCCTTGAC GAGAAAAGCA TCAAGCCGGG 1560 CTTGCAGACC TACAAGCGCT GATAAATGCG CCGGGGCCCT CGCTGCGCCC CCGGCCTTCC 1620 AATAATGACA ATAATGAGGA GTGCCCAATG TTTCACGTGC CCCTGCTTAT TGGTGGTAAG 1680 CCTTGTTCAG CATCTGATGA GCGC ACCTTC GAGCGTCGTA GCCCGCTGAC CGGAGAAGTG 1740 GTATCGCGCG TCGCTGCTGC CAGTTTGGAA GATGCGGACG CCGCAGTGGC CGCTGCACAG 1800 GCTGCGTTTC CTGAATGGGC GGCGCTTGCT CCGAGCGAAC GCCGTGCCCG ACTGCTGCGA 1860 GCGGCGGATC TTCTAGAGGA CCGTTCTTCC GAGTTCACCG CCGCAGCGAG TGAAACTGGC 1920 GCAGCGGGAA ACTGGTATGG GTTTAACGTT TACCTGGCGG CGGGCATGTT GCGGGGAATT 1980 C 1981 13 Sequence 11 TCCCCT GGCGACGAAA GGGCGGCAGG CCGCATGGCC ACGGCTGGGC GGTAACTGAT 60 ^ CCTTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG AGAATCATGC 120 GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT 180 AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC ACAGCACTGG 240 GGTGCACGAT GAATAGCTAC GATGGCCGTT GGTCTACCGT TGATGTGAAG GTTGAAGAAG 300 GTATCGCTTG GGTCACGCTG AACCGCCCGG AGAAGCGCAA CGCAATGAGC CCAACTCTCA 360 ATCGAGAGAT GGTCGAGGTT CTGGAGGTGC TGGAGCAGGA CGCAGATGCT CGCGTGCTTG 420 TTCTGACTGG TGCAGGCGAA TCCTGGACCG CGGGCATGGA CCTGAAGGAG TATTTCCGCG 480 AGACCGATGC TGGCCCCGAA ATTCTGCAAG AGAAGATTCG TCGGGGGAGA GGCGGTTTGC 540 GTATTGGGCG CATGCATAAA AACTGTTGTA ATTCATTAAG CATTCTGCCG ACATGGAAGC 600 CATCACAAAC GGCATGATGA ACCTGAATCG CCAGCGGCAT CAGCACCTTG TCGCCTTGCG 660 TATAATATTT GCCCATGGAC GCACACCGTG GAAACGGATG AAGGCACGAA CCCAGTTGAC 720 ATAAGCCTGT TCGGTTCGTA AACTGTAATG CAAGTAGCGT ATGCGCTCAC GCAACTGGTC 780 CAGAACCTTG ACCGAACGCA GCGGTGGTAA CGGCGCAGTG GCGGTTTTCA TGGCTTGTTA 840 TGACTGTTTT TTTGTACAGT CTATGCCTCG GGCATCCAAG CAGCAAGCGC GTTACGCCGT 900 GGGTCGATGT TTGATGT TAT GGAGCAGCAA CGATGTTACG CAGCAGCAAC GATGTTACGC 960 AGCAGGGCAG TCGCCCTAAA ACAAAGTTAG GTGGCTCAAG TATGGGCATC ATTCGCACAT 1020 GTAGGCTCGG CCCTG? CCAA GTCAAATCCA TGCGGGCTGC TCTTGATCTT TTCGGTCGTG 1080 AGTTCGGAGA CGTAGCCACC TACTCCCAAC ATCAGCCGGA CTCCGATTAC CTCGGGAACT 1140 TGCTCCGTAG TAAGACATTC ATCGCGCTTG CTGCCTTCGA CCAAGAAGCG GTTGTTGGCG 1200 CTCTCGCGGC TTACGTTCTG CCCAGGTTTG AGCAGCCGCG TAGTGAGATC TATATCTATG 1260 ATCTCGCAGT CTCCGGCGAG CACCGGAGGC AGGGCATTGC CACCGCGCTC ATCAATCTCC 1320 TCAAGCATGA GGCCAACGCG CTTGGTGCTT ATGTGATCTA CGTGCAAGCA GATTACGGTG 1380 ACGATCCCGC AGTGGCTCTC TATACAAAGT TGGGCATACG GGAAGAAGTG ATGCACTTTG 1440 ATATCGACCC AAGTACCGCC ACCTAACAAT TCGTTCAAGC CGAGATCGGC TTCCCCGAGC 1500 AGGGCATGAA GCAGTTCCTT GACGAGAAAA GCATCAAGCC GGGCTTGCAG ACCTACAAGC 1560 GCTGATAAAT GCGCCGGGGC CCTCGCTGCG CCCCCGGCCT TCCAATAATG ACAATAATGA 1620 GGAGTGCCCA ATGTTTCACG TGCCCCTGCT TATTGGTGGT AAGCCTTGTT CAGCATCTGA 1680 TGAGCGCACC TTCGAGCGTC GTAGCCCGCT GACCGGAGAA GTGGTATCGC GCGTCGCTGC 1740 TGCCAGTTTG GAAGATGCGG ACGCCGCAGT GGCCGCTGCA CAGGCTGCGT TTCCTGAATG 1800 GGCGGCGCTT GCTCCGAGCG AACGCCGTGC CCGACTGCTG CGAGCGGCGG ATCTTCTAGA 1860 GGACCGTTCT TCCGAGTTCA CCGCCGCAGC GAGTGAAACT GGCGCAGCGG GAAACTGGTA 1920 TGGGTTTAAC GTTTACCTGG CGGCGGGCAT GTTGCGGGGA ATTC 1964 14 Sequence 12 60 ^ AATTCCCCT GGCGACGAAA GGGCGGCAGG CCGCATGGCC ACGGCTGGGC GGTAACTGAT 120 GCTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG AGAATCATGC 180 GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT 240 AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC ACAGCACTGG 300 GGTGCACGAT GAATAGCTAC GATGGCCGTT GGTCTACCGT TGATGTGAAG GTTGAAGAAG 360 GTATCGCTTG GGTCACGCTG AACCGCCCGG AGAAGCGCAA CGCAATGAGC CCAACTCTCA 420 ATCGAGAGAT GGTCGAGGTT CTGGAGGTGC TGGAGCAGGA CGCAGATGCT CGCGTGCTTG 480 TTCTGACTGG TGCAGGCGAA TCCTGGACCG CGGGCATGGA CCTGAAGGAG TATTTCCGCG 540 AGACCGATGC TGGCCCCGAA ATTCTGCAAG AGAAGATTCG TCGCGAGCAG GGCATGAAGC 600 AGTTCCTTGA CGAGAAAAGC ATCAAGCCGG GCTTGCAGAC CTACAAGCGC TGATAAATGC 660 GCCGGGGCCC TCGCTGCGCC CCCGGCCTTC CAATAATGAC AATAATGAGG AGTGCCCAAT 720 GTTTCACGTG CCCCTGCTTA TTGGTGGTAA GCCTTGTTCA GCATCTGATG AGCGCACCTT 780 CGAGCGTCGT AGCCCGCTGA CCGGAGAAGT GGTATCGCGC GTCGCTGCTG CCAGTTTGGA 840 I AGATGCGGAC GCCGCAGTGG CCGCTGCACA GGCTGCGTTT CCTGAATGGG CGGCGCTTGC 900 'TCCGAGCGAA CGCCG TGCCC GACTGCTGCG AGCGGCGGAT CTTCTAGAGG ACCGTTCTTC 960 CGAGTTCACC GCCGCAGCGA GTGAAACTGG CGCAGCGGGA AACTGGTATG GGTTTAACGT 992 TTACCTGGCG GCGGGCATGT TGCGGGGGAAT TC fifteen Sequence 13 AATGACAATA ATGAGGAGTG CCCAATGTTT CACGTGCCCC TGCTTATTGG 60 TGGTAAGCCT TGTTCAGCAT CTGATGAGCG CACCTTCGAG CGTCGTAGCC CGCTGACCGG 120 AGAAGTGGTA TCGCGCGTCG CTGCTGCCAG TTTGGAAGAT GCGGACGCCG CAGTGGCCGC 180 TGCACAGGCT GCGTTTCCTG AATGGGCGGC GCTTGCTCCG AGCGAACGCC GTGCCCGACT 240 GCTGCGAGCG GCGGATCTTC TAGAGGACCG TTCTTCCGAG TTCACCGCCG CAGCGAGTGA 300 AACTGGCGCA GCGGGAAACT GGTATGGGTT TAACGTTTAC CTGGCGGCGG GCATGTTGCG 360 GGAAGCCGCG GCCATGACCA CACAGATTCA GGGCGATGTC ATTCCGTCCA ATGTGCCCGG 420 TAGCTTTGCC ATGGCGGTTC GACAGCCATG TGGCGTGGTG CTCGGTATTG CGCCTTGGAA 480 TGCTCCGGTA ATCCTTGGCG TACGGGCTGT TGCGATGCCG TTGGCATGCG GCAATACCGT 540 GGTGTTGAAA AGCTCTGAGC TGAGTCCCTT TACCCATCGC CTGATTGGTC AGGTGTTGCA 600 TGATGCTGGT CTGGGGGATG GCGTGGTGAA TGTCATCAGC AATGCCCCGC AAGACGCTCC 660 TGCGGTGGTG GAGCGACTGA TTGCAAATCC TGCGGTACGT CGAGTGAACT TCACCGGTTC 720 ^ GACCCACGTT GGACGGATCA TTGGTGAGCT GTCTGCGCGT CATCTGAAGC CTGCTGTGCT 780 .GAATTAGGT GGTAAGGCTC CGTTCTTGGT CTTGGACGAT GCCGACCTCG ATGCGGCGGT 840"CGAAGCGGCG GCCTT TGGTG CCTACTTCAA TCAGGGTCAA ATCTGCATGT CCACTGAGCG 900 TCTGATTGTG ACAGCAGTCG CAGACGCCTT TGTTGAAAAG CTGGCGAGGA AGGTCGCCAC 960 ACTGCGTGCT GGCGATCCTA ATGATCCGCA ATCGGTCTTG GGTTCGTTGA TTGATGCCAA 1020 TGCAGGTCAA CGCATCCAGG TTCTGGTCGA TGATGCGCTC GGGGACAGCA AGCGAACCGG 1080 AATTGCCAGC TGGGGCGCCC TCTGGTAAGG TTGGGAAGCC CTGCAAAGTA AACTGGATGG 1140 CTTTCTTGCC GCCAAGGATC TGATGGCGCA GGGGATCAAG ATCTGATCAA GAGACAGGAT 1200 GAGGATCGTT TCGCATGATT GAACAAGATG GATTGCACGC AGGTTCTCCG GCCGCTTGGG 1260 TGGAGAGGCT ATTCGGCTAT GACTGGGCAC AACAGACAAT CGGCTGCTCT GATGCCGCCG 1320 TGTTCCGGCT GTCAGCGCAG GGGCGCCCGG TTCTTTTTGT CAAGACCGAC CTGTCCGGTG 1380 CCCTGAATGA ACTGCAGGAC GAGGCAGCGC GGCTATCGTG GCTGGCCACG ACGGGCGTTC 1440 CTTGCGCAGC TGTGCTCGAC GTTGTCACTG AAGCGGGAAG GGACTGGCTG CTATTGGGCG 1500 AAGTGCCGGG GCAGGATCTC CTGTCATCTC ACCTTGCTCC TGCCGAGAAA GTATCCATCA 1560 TGGCTGATGC AATGCGGCGG CTGCATACGC TTGATCCGGC TACCTGCCCA TTCGACCACC 1620 AAGCGAAACA TCGCATCGAG CGAGCACGTA CTCGGATGGA AGCCGGTCTT GTCGATCAGG 1680 ATGATCTGGA CGAAGAGCAT CAGGGGCTCG CGCCAGCCGA ACTGTTCGCC AGGCTCAAGG 1740 CGCGCATGCC CGACGGCGAG GATCTCGTCG TGACCCATGG CGATGCCTGC TTGCCGAATA 1800 TCATGGTGGA AAATGGCCGC TTTTCTGGAT TCATCGACTG TGGCCGGCTG GGTGTGGCGG 1860 ACCGCTATCA GGACATAGCG TTGGCTACCC GTGATATTGC TGAAGAGCTT GGCGGCGAAT 1920 GGGCTGACCG CTTCCTCGTG CTTTACGGTA TCGCCGCTCC CGATTCGCAG CGCATCGCCT 1980 TCTATC GCCT TCTTGACGAG TTCTTCTGAG CGGGACTCTG GGGTTCGAAA TGACCGACCA 2040 AGCGACGCCC GGCCCAGCGC GTCGATTCGG GCATTTGCCA TATCAATGGA CCGACTGTGC 2100 ATGACGAGGC TCAGATGCCA TTCGGTGGGG TGAAGTCCAG CGGCTACGGC AGCTTCGGCA 2160 16 GTCGAGCATC GATTGAGCAC TTTACCCAGC TGCGCTGGCT GACCATTCAG AATGGCCCGC 2220 AATCTAAATC GATCTTCGGG CGCCGCGGGC ATCATGCCCG CGGCGCTCGC 2280 ? ATTTCAA TCTCTAACTT GATAAAAACA GAGCTGTTCT CCGGTCTTGG TGGATCAAGG 2340 CCAGTCGCGG AGAGTCTCGA AGAGGAGAGT ACAGTGAACG CCGAGTCCAC ATTGCAACCG 2400 CAGGCATCAT CATGCTCTGC TCAGCCACGC TACCGCAGTG TGTCGATTGG TCATCCTCCG 2460 GTTGAGGTTA CGCAAGACGC TGGAGGTATT GTCCGGATGC GTTCTCTCGA GGCGCTTCTT 2520 CCCTTCCCGG GTGGAATTC 2539 17 Sequence 14 d ^ tTCCAAT AATGACAATA ATGAGGAGTG CCCAATGTTT CACGTGCCCC TGCTTATTGG 60 TGGTAAGCCT TGTTCAGCAT CTGATGAGCG CACCTTCGAG CGTCGTAGCC CGCTGACCGG 120 AGAAGTGGTA TCGCGCGTCG CTGCTGCCAG TTTGGAAGAT GCGGACGCCG CAGTGGCCGC 180 TGCACAGGCT GCGTTTCCTG AATGGGCGGC GCTTGCTCCG AGCGAACGCC GTGCCCGACT 240 GCTGCGAGCG GCGGATCTTC TAGAGGACCG TTCTTCCGAG TTCACCGCCG CAGCGAGTGA 300 AACTGGCGCA GCGGGAAACT GGTATGGGTT TAACGTTTAC CTGGCGGCGG GCATGTTGCG 360 GGAAGCCGCG GCCATGACCA CACAGATTCA GGGCGATGTC ATTCCGTCCA ATGTGCCCGG 42.0 TAGCTTTGCC ATGGCGGTTC GACAGCCATG TGGCGTGGTG CTCGGTATTG CGCCTTGGAA 480 TGCTCCGGTA ATCCTTGGCG TACGGGCTGT TGCGATGCCG TTGGCATGCG GCAATACCGT 540 GGTGTTGAAA AGCTCTGAGC TGAGTCCCTT TACCCATCGC CTGATTGGTC AGGTGTTGCA 600 TGATGCTGGT CTGGGGGATG GCGTGGTGAA TGTCATCAGC AATGCCCCGC AAGACGCTCC 660 TGCGGTGGTG GAGCGACTGA TTGCAAATCC TGCGGTACGT CGAGTGAACT TCACCGGTTC 720 GACCCACGTT GGACGGATCA TTGGTGAGCT GTCTGCGCGT CATCTGAAGC CTGCTGTGCT 780? jAATTAGGT GGTAAGGCTC CGTTCTTGGT CTTGGACGAT GCCGACCTCG ATGCGGCGGT 840 AGCGGCG GCCTTTGGTG CCTACTTCAA TCAGGGTCAA ATCTGCATGT CCACTGAGCG 900 TCTGATTGTG ACAGCAGTCG CAGACGCCTT TGTTGAAAAG CTGGCGAGGA AGGTCGCCAC 960 ACTGCGTGCT GGCGATCCTA ATGATCCGCA ATCGGTCTTG GGTTCGTTGA TTGATGCCAA 1020 TGCAGGTCAA CGCATCCAGG TGGGGAGAGG CGGTTTGCGT ATTGGGCGCA TGCATAAAAA 1080 CTGTTGTAAT TCATTAAGCA TTCTGCCGAC ATGGAAGCCA TCACAAACGG CATGATGAAC 1140 CTGAATCGCC AGCGGCATCA GCACCTTGTC GCCTTGCGTA TAATATTTGC CCATGGACGC 1200 ACACCGTGGA AACGGATGAA GGCACGAACC CAGTTGACAT AAGCCTGTTC GGTTCGTAAA 1260 CTGTAATGCA AGTAGCGTAT GCGCTCACGC AACTGGTCCA GAACCTTGAC CGAACGCAGC 1320 GGTGGTAACG GCGCAGTGGC GGTTTTCATG GCTTGTTATG ACTGTTTTTT TGTACAGTCT 1380 ATGCCTCGGG CATCCAAGCA GCAAGCGCGT TACGCCGTGG GTCGATGTTT GATGTTATGG 1440 AGCAGCAACG ATGTTACGCA GCAGCAACGA TGTTACGCAG CAGGGCAGTC GCCCTAAAAC 1500 AAAGTTAGGT GGCTCAAGTA TGGGCATCAT TCGCACATGT AGGCTCGGCC CTGACCAAGT 1560 CAAATCCATG CGGGCTGCTC TTGATCTTTT CGGTCGTGAG TTCGGAGACG TAGCCACCTA 1620 CTCCCAACAT CAGCCGGACT CCGATTACCT CGGGAACTTG CTCCGTAGTA AGACATTCAT 1680 CGCGCTTGCT GCCTTCGACC AAGAAGCGGT TGTTGGCGCT CTCGCGGCTT ACGTTCTGCC 1740 CAGGTTT GAG CAGCCGCGTA GTGAGATCTA TATCTATGAT CTCGCAGTCT CCGGCGAGCA 1800 CCGGAGGCAG GGCATTGCCA CCGCGCTCAT CAATCTCCTC AAGCATGAGG CCAACGCGCT 1860 EGGTGCTTAT GTGATCTACG TGCAAGCAGA TTACGGTGAC GATCCCGCAG TGGCTCTCTA 1920 ACAAAGTTG GGCATACGGG AAGAAGTGAT GCACTTTGAT ATCGACCCAA GTACCGCCAC 1980 CTAACAATTC GTTCAAGCCG AGATCGGCTT CCCAATTGGC CCAGCGCGTC GATTCGGGCA 2040 TTTGCCATÁT CAATGGACCG ACTGTGCATG ACGAGGCTCA GATGCCATTC GGTGGGGTGA 21Q0 AGTCCAGCGG CTACGGCAGC TTCGGCAGTC GAGCATCGAT TGAGCACTTT ACCCAGCTGC 2160 18 2220 GCTGGCTGAC CATTCAGAAT GGCCCGCGGC ACTATCCAAT CTAAATCGAT CTTCGGGCGC 2280 ATGCCCGCGG CGCTCGCCTC ATTTCAATCT CTAACTTGAT AAAAACAGAG 2340 GTCTTGGTGG ATCAAGGCCA GTCGCGGAGA GTCTCGAAGA GGAGAGTACA 2400 AGTCCACATT GCAACCGCAG GCATCATCAT GCTCTGCTCA GCCACGCTAC CGATTGGTCA TCCTCCGGTT GAGGTTACGC AAGACGCTGG AGGTATTGTC 2460 CGGATGCGTT CTCTCGAGGC GCTTCTTCCC TTCCCGGGTG GAATTC 2506 19 Sequence 15 ^ iTTCCAAT AATGACAATA ATGAGGAGTG CCCAATGTTT CACGTGCCCC TGCTTATTGG 60 TGGTAAGCCT TGTTCAGCAT CTGATGAGCG CACCTTCGAG CGTCGTAGCC CGCTGACCGG 120 AGAAGTGGTA TCGCGCGTCG CTGCTGCCAG TTTGGAAGAT GCGGACGCCG CAGTGGCCGC 180 TGCACAGGCT GCGTTTCCTG AATGGGCGGC GCTTGCTCCG AGCGAACGCC GTGCCCGACT 240 GCTGCGAGCG GCGGATCTTC TAGAGGACCG TTCTTCCGAG TTCACCGCCG CAGCGAGTGA 300 AACTGGCGCA GCGGGAAACT GGTATGGGTT TAACGTTTAC CTGGCGGCGG GCATGTTGCG 360 GGAAGCCGCG GCCATGACCA CACAGATTCA GGGCGATGTC ATTCCGTCCA ATGTGCCCGG 420 TAGCTTTGCC ATGGCGGTTC GACAGCCATG TGGCGTGGTG CTCGGTATTG CGCCTTGGAA 480 TGCTCCGGTA ATCCTTGGCG TACGGGCTGT TGCGATGCCG TTGGCATGCG GCAATACCGT 540 GGTGTTGAAA AGCTCTGAGC TGAGTCCCTT TACCCATCGC CTGATTGGTC AGGTGTTGCA 600 TGATGCTGGT CTGGGGGATG GCGTGGTGAA TGTCATCAGC AATGCCCCGC AAGACGCTCC 660 TGCGGTGGTG GAGCGACTGA TTGCAAATCC TGCGGTACGT CGAGTGAACT TCACCGGTTC 720 GACCCACGTT GGACGGATCA TTGGTGAGCT GTCTGCGCGT CATCTGAAGC CTGCTGTGCT 780 fc? AATTAGGT GGTAAGGCTC CGTTCTTGGT CTTGGACGAT GCCGACCTCG ATGCGGCGGT 840 KGAAGCGGCG GCCTTTGGTG CCTACTTCAA TCAGGGTCAA ATCTGCATGT CCACTGAGCG 900 TCTGATTGTG ACAGCAGTCG CAGACGCCTT TGTTGAAAAG CTGGCGAGGA AGGTCGCCAC 960 ACTGCGTGCT GGCGATCCTA ATGATCCGCA ATCGGTCTTG GGTTCGTTGA TTGATGCCAA 1020 TGCAGGTCAA CGCATCCAGG TTCTGGTCGA TGATGCGCTC GCAAAAGGCG CGCAATGGAA 1080 TTGGCCCAGC GCGTCGATTC GGGCATTTGC CATATCAATG GACCGACTGT GCATGACGAG 1140 GCTCAGATGC CATTCGGTGG GGTGAAGTCC AGCGGCTACG GCAGCTTCGG CAGTCGAGCA 1200 TCGATTGAGC ACTTTACCCA GCTGCGCTGG CTGACCATTC AGAATGGCCC GCGGCACTAT 1260 CCAATCTAAA TCGATCTTCG GGCGCCGCGG GCATCATGCC CGCGGCGCTC GCCTCATTTC 1320 AATCTCTAAC TTGATAAAAA CAGAGCTGTT CTCCGGTCTT GGTGGATCAA GGCCAGTCGC 1380 GGAGAGTCTC GAAGAGGAGA GTACAGTGAA CGCCGAGTCC ACATTGCAAC CGCAGGCATC 1440 ATCATGCTCT GCTCAGCCAC GCTACCGCAG TGTGTCGATT GGTCATCCTC CGGTTGAGGT 1500 TACGCAAGAC GCTGGAGGTA TTGTCCGGAT GCGTTCTCTC GAGGCGCTTC TTCCCTTCCC 1560 GGGTGGAATT C 1571 twenty Sequence 16 G ^ TCCGCG GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC 60 GTGACGAGGC CACACTGTGA GTTGGTCAGG GGGGGCTTAC TCGGCGTTTT CCGACACTGC 120 GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT TGATTGCGGG GGTGCCCTGT CGCTGGTGTC 180 GCCTATCGAC TTAGGGGTAA AGGTCGCTCG CGAAGTTCTG ATGCGTGCGT CGCTTGAACC 240 ACAAATGGTC GATAGCGTAC TCGCAGGCTC TATGGCTCAA GCAAGCTTTG ATGCTTACCT 300 GCTCCCGCGG CACATTGGCT TGTACAGCGG TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT 360 GCAGCGCATT TGCGGCACAG GCTTCGAACT GCTTCGGCAG GCCGGCGAGC AGATTTCCCA 42.0 AGGCGCTGAT CACGTGCTGT GTGTCGCGGC AGAGTCCATG TCGCGTAACC CCATCGCGTC 480 GTATACACAC CGGGGCGGGT TCCGCCTCGG TGCGCCCGTT GAGTTCAAGG ATTTTTTGTG 540 GGAGGCATTG TTTGATCCTG CTCCAGGACT CGACATGATC GCTACCGCAG AAAACCTGGG 600 GACAGCAAGC GAACCGGAAT TGCCAGCTGG GGCGCCCTCT GGTAAGGTTG GGAAGCCCTG 660 CAAAGTAAAC TGGATGGCTT TCTTGCCGCC AAGGATCTGA TGGCGCAGGG GATCAAGATC 720 TGATCAAGAG ACAGGATGAG GATCGTTTCG CATGATTGAA CAAGATGGAT TGCACGCAGG 780 • CTCCGGCC GCTTGGGTGG AGAGGCTATT CGGCTATGAC TGGGCACAAC AGACAATCGG 840 GCTCTGAT GCCGCCGTGT TCCGGCTGTC AGCGCAGGGG CGCCCGGTTC TTTTTGTCAA 900 GACCGACCTG TCCGGTGCCC TGAATGAACT GCAGGACGAG GCAGCGCGGC TATCGTGGCT 960 GGCCACGACG GGCGTTCCTT GCGCAGCTGT GCTCGACGTT GTCACTGAAG CGGGAAGGGA 1020 CTGGCTGCTA TTGGGCGAAG TGCCGGGGCA GGATCTCCTG TCATCTCACC TTGCTCCTGC 1080 CGAGAAAGTA TCCATCATGG CTGATGCAAT GCGGCGGCTG CATACGCTTG ATCCGGCTAC 1140 CTGCCCATTC GACCACCAAG CGAAACATCG CATCGAGCGA GCACGTACTC GGATGGAAGC 1200 CGGTCTTGTC GATCAGGATG ATCTGGACGA AGAGCATCAG GGGCTCGCGC CAGCCGAACT 1260 GTTCGCCAGG CTCAAGGCGC GCATGCCCGA CGGCGAGGAT CTCGTCGTGA CCCATGGCGA 1320 TGCCTGCTTG CCGAATATCA TGGTGGAAAA TGGCCGCTTT TCTGGATTCA TCGACTGTGG 1380 CCGGCTGGGT GTGGCGGACC GCTATCAGGA CATAGCGTTG GCTACCCGTG ATATTGCTGA 1440 AGAGCTTGGC GGCGAATGGG CTGACCGCTT CCTCGTGCTT TACGGTATCG CCGCTCCCGA 1500 TTCGCAGCGC ATCGCCTTCT ATCGCCTTCT TGACGAGTTC TTCTGAGCGG GACTCTGGGG 1560 TTCGAAATGA CCGACCAAGC GACGCCCATT GAGGGCGCAA GAGGAGAAAT GGATTGACCA 1620 AGAGATCGTG GCTGTTACGG ATGAACAGTT CGATTTAGAG GGCTACAACA GTCGAGCAAT 1680 TGAACTGCCT CGGAAGGCAA AATTGTTGAT CGTGACAGTC ATCCGCGGCC TAGCAGTCTT 1740 TGAAGCCCTT TCCCGATTGA AGCCTGTTCA TTCTGGCGGG GTGCAGACTG CGGGCAACAG 1800 CTGTGCCGTA GTGGACGGCG CCGCGGCGGC TTTGGTGGCT CGAGAGTCGT CTGCGACACA 1860 jGCCGGTCTTG GCTAGGATAC TGGCTACCTC CGTAGTCGGG ATCGAGCCCG AGCATATGGG 1920 'GCTCGGCCCT GCGCCCGCGA TTCGCCTGCT GCTTGCGCGT AGTGATCTTA GTTTGAGGGA 1980 TATCGACCTC TTTGAGATAA ACGAGGCGCA GGCCGCCCAA GTTCTAGCGG TACAGCATGA 2040 ATTGGGTATT GAGCACTCAA AACTTAATAT TTGGGGCGGG GCCATTGCAC TTGGACACCC 210P GCTTGCCGCG ACCGGATTGC GTCTCTGCAT GACCCTCGCT CACCAATTGC AAGCTAATAA 2160 twenty-one CTTTCGATAT GGAATTGCCT CGGCATGCAT TGGTGGGGGA CAGGGGATGG CGGTTCTTTT 2220 CACTTCGGTT CGTCCTCTGC ACGAAGTTCG ATGATTAACA GAGTTGACCA 2280 AGCTAACGGG CATCTCCTTT GTTGCTTTGA GGTGGCGCAC GAAGGAGGGC 2340 TCGAAAATCT CTGCTAAAAA CAAGAAGAAG GAACAGGGAA CATGATTAGT TTCGCTCGTA 2400 TGGCAGAAAG TTTAGGAGTC CAGGCTAAAC TTGCCCTTGC CTTCGCACTC GTATTATGTG 2460 TCGGGCTGAT TGTTACCGGC ACGGGTTTCT ACAGTGTACA TACCTTGTCA GGGTTGGTGG 2520 GAATTC 2526 22 Sequence 17 GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC 60 GTGACGAGGC CACACTGTGA GTTGGTCAGG GGGGGCTTAC TCGGCGTTTT CCGACACTGC 120 GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT TGATTGCGGG GGTGCCCTGT CGCTGGTGTC 180 GCCTATCGAC TTAGGGGTAA AGGTCGCTCG CGAAGTTCTG ATGCGTGCGT CGCTTGAACC 240 ACAAATGGTC GATAGCGTAC TCGCAGGCTC TATGGCTCAA GCAAGCTTTG ATGCTTACCT 300 GCTCCCGCGG CACATTGGCT TGTACAGCGG TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT 360 GCAGCGCATT TGCGGCACAG GCTTCGAACT GCTTCGGCAG GCCGGCGAGC AGATTTCCCA 420 AGGCGCTGAT CACGTGCTGT GTGTCGCGGC AGAGTCCATG TCGCGTAACC CCATCGCGTC 480 GTATACACAC CGGGGCGGGT TCCGCCTCGG TGCGCCCGTT GAGTTCAAGG ATTTTTTGTG 540 GGAGGCATTG TTTGATCCTG CTCCAGGACT CGACATGATC GCTACCGCAG AAAACCTGGG 600 GGAGAGGCGG TTTGCGTATT GGGCGCATGC ATAAAAACTG TTGTAATTCA TTAAGCATTC 660 TGCCGACATG GAAGCCATCA CAAACGGCAT GATGAACCTG AATCGCCAGC GGCATCAGCA 720 TTGCGTATAA TATTTGCCCA TGGACGCACA CCGTGGAAAC GGATGAAGGC 780 TTGACATAAG CCTGTTCGGT TCGTAAACTG TAATGCAAGT AGCGTATGCG 840 TGGTCCAGAA CCTTGACCGA ACGCAGCGGT GGTAACGGCG CAGTGGCGGT 900 TTTCATGGCT TGTTATGACT GTTTTTTTGT ACAGTCTATG CCTCGGGCAT CCAAGCAGCA 960 AGCGCGTTAC GCCGTGGGTC GATGTTTGAT GTTATGGAGC AGCAACGATG TTACGCAGCA 1020 GCAACGATGT TACGCAGCAG GGCAGTCGCC CTAAAACAAA GTTAGGTGGC TCAAGTATGG 1080 GCATCATTCG CACATGTAGG CTCGGCCCTG ACCAAGTCAA ATCCATGCGG GCTGCTCTTG 1140 ATCTTTTCGG TCGTGAGTTC GGAGACGTAG CCACCTACTC CCAACATCAG CCGGACTCCG 1200 ATTACCTCGG GAACTTGCTC CGTAGTAAGA CATTCATCGC GCTTGCTGCC TTCGACCAAG 1260 AAGCGGTTGT TGGCGCTCTC GCGGCTTACG TTCTGCCCAG GTTTGAGCAG CCGCGTAGTG 1320 AGATCTATAT CTATGATCTC GCAGTCTCCG GCGAGCACCG GAGGCAGGGC ATTGCCACCG 1380 CGCTCATCAA TCTCCTCAAG CATGAGGCCA ACGCGCTTGG TGCTTATGTG ATCTACGTGC 1440 AAGCAGATTA CGGTGACGAT CCCGCAGTGG CTCTCTATAC AAAGTTGGGC ATACGGGAAG 1500 AAGTGATGCA CTTTGATATC GACCCAAGTA CCGCCACCTA ACAATTCGTT CAAGCCGAGA 1560 TCGGCTTCCC ATTGAGGGCG CAAGAGGAGA AATGGATTGA CCAAGAGATC GTGGCTGTTA 1620 CGGATGAACA GTTCGATTTA GAGGGCTACA ACAGTCGAGC AATTGAACTG CCTCGGAAGG 1680 CAAAATTGTT GATCGTGACA GTCATCCGCG GCCTAGCAGT CTTTGAAGCC CTTTCCCGAT 1740 TGAAGCCTGT TCATTCTGGC GGGGTGCAGA CTGCGGGCAA CAGCTGTGCC GTAGTGGACG 1800 .GCGCCGCGGC GGCTTTGGTG GCTCGAGAGT CGTCTGCGAC ACAGCCGGTC TTGGCTAGGA 1860 ACTGGCTAC CTCCGTAGTC GGGATCGAGC CCGAGCATAT GGGGCTCGGC CCTGCGCCCG 1920"CGATTCGCCT GCTGCTTGCG CGTAGTGATC TTAGTTTGAG GGATATCGAC CTCTTTGAGA 1980 TAAACGAGGC GCAGGCCGCC CAAGTTCTAG CGGTACAGCA TGAATTGGGT ATTGAGCACT 2040 CAAAA CTTAA "'TATITGGGGC GGGGCCATTG CACTTGGACA CCCGCTTGCC GCGACCGGAT 2100. TGCGTCTCTG- CATGACCCTC GCTCACCAAT TGCAAGCTAA TAACTTTCGA TATGGAATTG 2160 2. 3 CCTCGGCATG CATTGGTGGG GGACAGGGGA TGGCGGTTCT TTTAGAGAAT CCCCACTTCG 2220 rCGTCCTC TGCACGAAGT TCGATGATTA ACAGAGTTGA CC AC ATCC A CTGAGCTAAC 2280 JCATCTCC TTTGTTGCTT TGAGGTGGCG CACGAAGGAG GGCTCGAAAA TCTCTGCTAA 2340 AAACAAGAAG AAGGAACAGG GAACATGATT AGTTTCGCTC GTATGGCAGA AAGTTTAGGA 2400 GTCCAGGCTA AACTTGCCCT TGCCTTCGCA CTCGTATTAT GTGTCGGGCT GATTGTTACC 2460 GGCACGGGTT TCTACAGTGT ACATACCTTG TCAGGGTTGG TGGGAATTC 2509 24 Sequence 18 GTO Í; TTTCCGCG GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC 60 GTGACGAGGC CACACTGTGA GTTGGTCAGG GGGGGCTTAC TCGGCGTTTT CCGACACTGC 120 GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT TGATTGCGGG GGTGCCCTGT CGCTGGTGTC 180 GCCTATCGAC TTAGGGGTAA AGGTCGCTCG CGAAGTTCTG ATGCGTGCGT CGCTTGAACC 240 ACAAATGGTC GATAGCGTAC TCGCAGGCTC TATGGCTCAA GCAAGCTTTG ATGCTTACCT 300 GCTCCCGCGG CACATTGGCT TGTACAGCGG TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT 360 GCAGCGCATT TGCGGCACAG GCTTCGAACT GCTTCGGCAG GCCGGCGAGC AGATTTCCCA 420 AGGCGCTGAT CACGTGCTGT GTGTCGCGGC AGAGTCCATG TCGCGTAACC CCATCGCGTC 480 GTATACACAC CGGGGCGGGT TCCGCCTCGG TGCGCCCGTT GAGTTCAAGG ATTTTTTGTG 540 GGAGGCATTG TTTGATCCTG CTCCAGGACT CGACATGATC GCTACCGCAG AAAACCTGGC 600 GCGCATTGAG GGCGCAAGAG GAGAAATGGA TTGACCAAGA GATCGTGGCT GTTACGGATG 660 AACAGTTCGA TTTAGAGGGC TACAACAGTC GAGCAATTGA ACTGCCTCGG AAGGCAAAAT 720 GACAGTCATC CGCGGCCTAG CAGTCTTTGA AGCCCTTTCC CGATTGAAGC 780 TGGCGGGGTG CAGACTGCGG GCAACAGCTG TGCCGTAGTG GACGGCGCCG 840 GGTGGCTCGA GAGTCGTCTG C GACACAGCC GGTCTTGGCT AGGATACTGG 900 CTACCTCCGT AGTCGGGATC GAGCCCGAGC ATATGGGGCT CGGCCCTGCG CCCGCGATTC 960 GCCTGCTGCT TGCGCGTAGT GATCTTAGTT TGAGGGATAT CGACCTCTTT GAGATAAACG 1020 AGGCGCAGGC CGCCCAAGTT CTAGCGGTAC AGCATGAATT GGGTATTGAG CACTCAAAAC 1080 TTAATATTTG GGGCGGGGCC ATTGCACTTG GACACCCGCT TGCCGCGACC GGATTGCGTC 1140 TCTGCATGAC CCTCGCTCAC CAATTGCAAG CTAATAACTT TCGATATGGA ATTGCCTCGG 1200 CATGCATTGG TGGGGGACAG GGGATGGCGG TTCTTTTAGA GAATCCCCAC TTCGGTTCGT 1260 CCTCTGCACG AAGTTCGATG ATTAACAGAG TTGACCACTA TCCACTGAGC TAACGGGCAT 1320 CTCCTTTGTT GCTTTGAGGT GGCGCACGAA GGAGGGCTCG AAAATCTCTG CTAAAAACAA 1380 GAAGAAGGAA CAGGGAACAT GATTAGTTTC GCTCGTATGG CAGAAAGTTT AGGAGTCCAG 1440 GCTAAACTTG CCCTTGCCTT CGCACTCGTA TTATGTGTCG GGCTGATTGT TACCGGCACG 1500 GGTTTCTACA GTGTACATAC CTTGTCAGGG TTC TTGGTGGGAA 1543

Claims

- - CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. Transformed and / or mutagenized mono or polycellular organisms characterized by the inactivation of enzymes of the catabolism of eugenol and / or ferulic acid. Thus, an accumulation of the intermediates coniferilalcohol, coniferilaldehyde, ferulic acid, vanillin and / or vanillinic acid is verified. 2. - Organism according to claim 1, characterized in that the catabolism of eugenol and / or ferulic acid is modified by insertion of O-elements or by incorporation of deletions in the corresponding genes. 3. The organism according to claim 1, characterized in that one or more genes encoding the enzymes coniferyl alcohol dehydrogenase, coniferyl aldehyde dehydrogenase, ferulic acid CoA synthetase, enoyl CoA hydratase are modified and / or inactivated. aldolase, beta-ketothiolase, vanillin-dehydrogenase or for the vanillin-demethylase acid enzyme. 4. Organism according to one of claims 1 to 3, characterized in that it is monocellular, preferably a microorganism or a plant cell or an animal cell. 5. Organism according to one of claims 1 to 4, characterized in that it is a bacterium preferably a type of Pseudomonas, 6.- Genetic structures, characterized in that the nucleotide sequences coding for the coniferyl alcohol-dehydrogenase enzymes are modified and / or inactivated, coniferyl aldehyde dehydrogenase, ferulic acid CoA synthetase, enoyl CoA hydratase aldolase, beta-ketothiolase, vanillin dehydrogenase or vanillin demethylase or two or more of these enzymes. -37- 1.- Genetic structures with the structures indicated in the figures up to Ir. 8.- Genetic structures with the sequences indicated in figures 2a to 2r. 9.- Vectors containing at least one genetic structure according to one of claims 6 to 8. 10. Transformed organism according to one of claims 1 to 5, characterized in that it contains at least one vector according to claim 9. 11.- Organism according to one of claims 1 to 5, characterized in that it contains at least one genetic structure integrated in the genome according to one of claims 6 to 8 in points of the corresponding intact gene. 12. Process for the biotechnological preparation of organic compounds, especially alcohols, aldehydes and organic acids, characterized in that an organism according to one of claims I to 5, or 10 to 11 is used. 13. - Process for obtaining organisms according to one of claims 1 to 5, characterized in that the modification of the catabolism of eugenol and / or ferulic acid is achieved with the aid of known microbiological culture methods. 14. Process for obtaining an organism according to one of claims 1 to 5 or 10 to 11, characterized in that the modification of the catabolism of eugenol and / or ferulic acid and / or the inactivation of the corresponding genes is achieved with the help of of genetic engineering methods. 15. Use of the microorganisms according to one of claims 1 to 5 or 10 to 11, characterized in that they are for the preparation of coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillinic acid. 16. Use of the genetic structures according to one of claims 6-a 8, or of a vector according to claim 9, characterized in that for obtaining transformed and / or mutagenized organisms.