EP1124947A2 - Construction de souches de production pour la fabrication de phenols substitues par inactivation ciblee de genes du catabolisme de l'eugenol et de l'acide ferulique - Google Patents

Construction de souches de production pour la fabrication de phenols substitues par inactivation ciblee de genes du catabolisme de l'eugenol et de l'acide ferulique

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Publication number
EP1124947A2
EP1124947A2 EP99953892A EP99953892A EP1124947A2 EP 1124947 A2 EP1124947 A2 EP 1124947A2 EP 99953892 A EP99953892 A EP 99953892A EP 99953892 A EP99953892 A EP 99953892A EP 1124947 A2 EP1124947 A2 EP 1124947A2
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EP
European Patent Office
Prior art keywords
gene
inactivated
pseudomonas
ferulic acid
eugenol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99953892A
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German (de)
English (en)
Inventor
Jürgen Rabenhorst
Alexander Steinbüchel
Horst Priefert
Jörg Overhage
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Symrise AG
Original Assignee
Haarmann and Reimer GmbH
Symrise AG
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Publication date
Application filed by Haarmann and Reimer GmbH, Symrise AG filed Critical Haarmann and Reimer GmbH
Publication of EP1124947A2 publication Critical patent/EP1124947A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01016—Acetyl-CoA C-acyltransferase (2.3.1.16)

Definitions

  • the present invention relates to the construction of production strains and a method for the production of substituted methoxyphenols, in particular vanillin.
  • DE-A 4 227 076 (process for the preparation of substituted methoxyphenols and a suitable microorganism) describes the preparation of substituted methoxyphenols with a new Pseudomonas sp.
  • the starting material here is eugenol and the products are ferulic acid, vanillic acid, coniferyl alcohol and coniferyl aldehyde.
  • the enzymes for the conversion of tra / w-ferulic acid to trc s-feruloyl-SCoA ester and further to vanillin, as well as the gene for the cleavage of the ester were from
  • the object of the present invention is therefore to construct organisms which are able to convert the inexpensive raw material eugenol into vanillin in a one-step process.
  • This object is achieved by the construction of production strains of single or multicellular organisms, which are characterized in that enzymes of eugenol and / or ferulic acid catabolism are inactivated such that an accumulation of the intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or Vanillic acid takes place.
  • the production strain can be single-cell or multi-cell. Accordingly, the subject of the invention can be microorganisms, plants or animals. In addition, extracts obtained from the production master can also be used. According to the invention, single-celled organisms are preferably used. These can be microorganisms, animal or plant cells. The use of fungi and bacteria is particularly preferred according to the invention. Bacteria are highly preferred. Among the bacteria, Rhodococcus, Pseudomonas and Esche ichia species in particular can be used after changing the eugenol and / or ferulic acid catabolism. In the simplest case, the organisms which can be used according to the invention can be obtained by means of known, conventional microbiological methods.
  • the enzyme activity of the proteins involved in the catabolism of eugenol and / or ferulic acid can be changed by the use of enzyme inhibitors.
  • the enzyme activity of the proteins involved in the eugenol and / or ferulic acid catabolism can be changed by mutating the genes coding for these proteins.
  • Such mutations can be generated undirected using classic methods, such as, for example, UV radiation or chemicals that trigger mutations.
  • Genetic engineering methods for obtaining the organisms according to the invention are also suitable, such as deletions, insertions and / or nucleotide exchanges.
  • the genes of the organisms can be inactivated with the help of other DNA elements ( ⁇ elements).
  • the intact genes can be exchanged for modified and / or inactivated gene structures by means of suitable vectors.
  • the genes to be inactivated and the DNA elements used for the inactivation can be obtained by classic cloning techniques or by polymerase chain reactions (PCR).
  • the eugenol and ferulic acid catabolism can be changed by inserting ⁇ elements or introducing deletions into corresponding genes.
  • the functions of the genes which code for dehydrogenases, synthetases, hydratase aldolases, thiolases or demethylases can be inactivated using the abovementioned genetic engineering methods, so that the production of the enzymes in question is blocked.
  • genes which code for coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, ferulic acid-CoA synthetases, enoyl-CoA hydratase aldolases, beta-ketothiolases, vanillin dehydrogenases or vanillinic acid demethylases are preferably the genes which code for coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, ferulic acid-CoA synthetases, enoyl-CoA hydratase aldolases, beta-ketothiolases, vanillin dehydrogenases or vanillinic acid demethylases.
  • Genes which encode the amino acid sequences given in EP-A 0845532 and / or their nucleotide sequences encoding allelic variations are very particularly preferred.
  • the invention accordingly also relates to gene structures for producing transformed organisms and mutants.
  • Gene structures are preferably used to obtain the organisms and mutants in which the nucleotide sequences coding for dehydrogenases, synthetases, hydratase aldolases, thiolases or demethylases are inactivated. Particularly preferred are gene structures in which the nucleotide sequences coding for coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, ferulic acid-CoA synthetases, enoyl-CoA hydratase aldolases, beta-ketothiolases, vanillin dehydrogenases or vanillic acid demethylases are inactive. Gene structures which have the structures given in FIGS. 1a to 1r with the nucleotide sequences shown in FIGS. 2a to 2r and / or their allele variations coding nucleotide sequences are very particularly preferred. Nucleotide sequences from 1 to 18 are particularly preferred.
  • the invention also includes the partial sequences of these gene structures as well as functional equivalents.
  • Functional equivalents are to be understood as those derivatives of DNA in which individual nucleobases have been exchanged (Wobbe exchanges) without changing their function. Also at the protein level,
  • Amino acids can be exchanged without changing the function.
  • One or more DNA sequences can be connected upstream and / or downstream of the gene structures.
  • plasmids or vectors are obtainable which are suitable for the transformation and / or transfection of an organism and / or for the conjugative transfer into an organism.
  • the invention further relates to plasmids and / or vectors for producing the transformed organisms and mutants according to the invention. Accordingly, these contain the gene structures described.
  • the present invention relates to accordingly also organisms which contain the plasmids and / or vectors mentioned.
  • plasmids and / or vectors depends on their intended use. To z. For example, to be able to exchange the intact genes of eugenol and / or ferulic acid catabolism in pseudomonas against the genes inactivated by omega elements, vectors are required which can be transferred on the one hand in pseudomonads (conjugatively transferable plasmids) but on the other hand there cannot be replicated and are therefore unstable in pseudomonas (so-called suicide plasmids). DNA sections which are transferred into pseudomonads with the aid of such a plasmid system can only be preserved if they are integrated into the genome of the bacterial cell by homologous recombination.
  • genes can preferably be changed and / or inactivated such that the organisms in question are able to produce coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillic acid.
  • Production strains constructed in accordance with the invention are, for example, mutants of the strain Pseudomonas sp. HR199 (DSM 7063), which was described in detail in DE-A 4 227 076 and EP-A 0845532, where, among other things, the corresponding gene structures result from FIGS. 1a to 1r in conjunction with FIGS. 2a to 2r: 1.
  • Pseudomonas sp. HR199c ⁇ 4 ⁇ Km containing the ⁇ Km inactivated calA gene instead of the intact c ⁇ 4 gene coding for coniferyl alcohol dehydrogenase (Fig. La; Fig. 2a).
  • Pseudomonas sp. HR199ecb ⁇ Km containing the ⁇ Km inactivated ecb gene instead of the intact ecb gene coding for enoyl-CoA hydratase aldolase (Fig.lj; Fig. 2j).
  • Pseudomonas sp. HR199ecb ⁇ Gm containing the ecb gene inactivated by ⁇ Gm instead of the intact ecb gene coding for enoyl-CoA hydratase aldolase (FIG. 1k; FIG. 2k).
  • Pseudomonas sp. HR199 ⁇ t ⁇ Km containing the aat gene inactivated by ⁇ Km instead of the intact aat gene coding for beta-ketothiolase (Fig. Im; Fig. 2m).
  • Pseudomonas sp. HR199 t ⁇ Gm containing the ⁇ t gene inactivated by ⁇ Gm instead of the intact ⁇ t gene coding for beta-ketothiolase (Fig. In; Fig. 2n).
  • Pseudomonas sp. HR199v b ⁇ containing the deletion inactivated vdh gene instead of the intact v b gene coding for vanillin dehydrogenase (Fig.lr; Fig. 2r).
  • the invention also relates to a method for the biotechnical production of organic compounds.
  • alcohols, aldehydes and organic acids can be produced with this process. These are preferably coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and vanillic acid.
  • the organisms described above are used in the process according to the invention.
  • the most particularly preferred organisms include bacteria, especially the Pseudomonas species.
  • the above-mentioned Pseudomonas species can preferably be used for the following processes:
  • the preferred substrate is eugenol.
  • the addition of further substrates or even the replacement of the eugenol with another substrate may be possible.
  • Synthetic, semi-synthetic or complex culture media can be used as the nutrient medium for the organisms used according to the invention. These can contain carbon-containing and nitrogen-containing compounds, inorganic salts, optionally trace elements and vitamins.
  • Carbohydrates, hydrocarbons or basic organic chemicals can be considered as carbon-containing compounds.
  • Examples of compounds which can preferably be used are sugars, alcohols or sugar alcohols, organic acids or complex mixtures.
  • the preferred sugar is glucose.
  • Citric acid or acetic acid can preferably be used as organic acids.
  • the complex mixtures include e.g. B. malt extract, yeast extract, casein or casein hydrolyzate.
  • Inorganic compounds are suitable as nitrogen-containing substrates. Examples include nitrates and ammonium salts. Organic nitrogen sources can also be used. These include yeast extract, soy flour, casein, casein hydrolyzate and corn steep liquor.
  • the inorganic salts that can be used include, for example, sulfates, nitrates, chlorides, carbonates and phosphates. The salts mentioned preferably contain sodium, potassium, magnesium, manganese, calcium, zinc and iron as metals.
  • the temperature for cultivation is preferably in the range of 5 to 100 ° C.
  • the range from 15 to 60 ° C. is particularly preferred, and 22 to 60 ° C. is most preferred
  • the pH of the medium is preferably 2 to 12.
  • the range from 4 to 8 is particularly preferred.
  • bioreactors known to the person skilled in the art can be used to carry out the method according to the invention.
  • All devices suitable for submerged processes are preferred.
  • the former include e.g. B. shakers, bubble column or loop reactors.
  • the latter preferably include all known devices with stirrers in any configuration.
  • the process according to the invention can be carried out continuously or batchwise.
  • the duration of the fermentation until a maximum amount of product is reached depends on the particular type of organism used. Basically, however, the times of fermentation are between 2 and 200 hours.
  • genes calA, calB, fcs, ech, aat, vdh, adh, vdhB, vanA and vanB which are for coniferyl alcohol dehydrogenase, coniferylaldehyde dehydrogenase, ferulic acid-CoA synthetase, enoyl-CoA hydratase-aldolase, beta-ketothiolase, vanillin Dehydrogenase, alcohol dehydrogenase, vanillin dehydrogenase II and vanillic acid demethylase were encoded starting from genomic DNA of the strain Pseudomonas sp.
  • mutants had only the inactivated gene, so that mutants with only one defective gene and multiple mutants in which several genes were inactivated in this way were obtained.
  • These mutants were used for the biotransformation of a) eugenol to coniferyl alcohol , Coniferyl aldehyde, ferulic acid, Va nillin and / or
  • Vanillic acid b) coniferyl alcohol to coniferyl aldehyde, ferulic acid, vanillin and / or vanillic acid; c) coniferyl aldehyde to ferulic acid, vanillin and / or vanillic acid; d) ferulic acid to vanillin and / or vanillic acid and e) vanillin to vanillic acid. material and methods
  • Pseudomonas sp Cells grown on eugenol. HR199 were washed in 10 mM sodium phosphate buffer, pH 6.0, resuspended in the same buffer and disrupted by passing twice through a French press (Amicon, Silver Spring, Maryland, USA) at a pressure of 1000 psi. The cell homogenate was subjected to ultracentrifugation (1 h, 100,000 x g, 4 ° C.), whereby the soluble
  • the soluble fraction of the crude extract was dialyzed overnight against 10 mM sodium phosphate buffer, pH 6.0.
  • the dialysate was made up to a 10 mM
  • the column was rinsed with two BV 10 mM sodium phosphate buffers, pH 6.0.
  • VDH-II vanillin dehydrogenase-II
  • VDH activity was determined at 30 ° C. by means of an optically enzymatic test.
  • the reaction mixture with a volume of 1 ml contained 0.1 mmol potassium phosphate (pH 7.1), 0.125 ⁇ mol vanillin, 0.5 ⁇ mol NAD, 1.2 ⁇ mol pyruvate (Na salt), lactate dehydrogenase (1 U; from pig heart) and enzyme solution.
  • the CADH activity was determined at 30 ° C. using an optical enzymatic test according to Jaeger et al. (Jaeger, E., L. Eggeling and H. Sahm. 1981. Current Microbiology. 6: 333-336).
  • the reaction mixture with a volume of 1 ml contained 0.2 mmol Tris / HCl (pH 9.0), 0.4 ⁇ mol coniferyl alcohol, 2 ⁇ mol NAD, 0.1 mmol semicarbazide and enzyme solution.
  • the enzyme activity was given in units (U), where 1 U corresponds to the amount of enzyme that converts 1 ⁇ mol 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).
  • the CALDH activity was determined at 30 ° C. by means of an optically enzymatic test.
  • the reaction mixture with a volume of 1 ml contained 0.1 mmol Tris / HCl (pH 8.8), 0.08 ⁇ mol coniferylaldehyde, 2.7 ⁇ mol NAD and enzyme solution.
  • FCS activity was determined at 30 ° C. by an optically enzymatic test, modified according to Zenk et al. (Zenk et al. 1980. Anal. Biochem. 101: 182-
  • the reaction mixture with a volume of 1 ml contained 0.09 mmol potassium Phosphate (pH 7.0), 2.1 ⁇ mol MgCl2, 0.7 ⁇ mol ferulic acid, 2 ⁇ mol ATP, 0.4 ⁇ mol coenzyme A and enzyme solution.
  • the enzyme activity was given in units (U), where 1 U corresponds to the amount of enzyme that converts 1 ⁇ mol substrate per minute.
  • the protein concentrations in the samples were checked
  • the gels were buffered for 20 min in 100 mM KP buffer (pH 7.0) and then at 30 ° C in the same buffer the 0.08% (wt / vol) NAD, 0.04 % (wt / vol) p-nitroblue tetrazolium chloride, 0.003% »(wt / vol) phenazine methosulfate and 1 mM of the respective substrate had been added until appropriate color bands became visible.
  • N-terminal amino acid sequences were determined using a protein peptide sequencer (type 477 A, Applied Biosystems, Foster City, USA) and a PTH analyzer according to the manufacturer's instructions. Isolation and manipulation of DNA.
  • Genomic DNA was isolated using the method of Marmur (Marmur, J. 1961. J. Mol. Biol. 3: 208-218). The isolation and analysis of other plasmid DNA or of DNA restriction fragments was carried out according to standard methods (Sambrook, JEF Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Habor, New York.).
  • Cells of the recipient were applied in one direction as an inoculation line. After 5 minutes, cells from the donor strains were then applied as inoculation lines, the recipient line being crossed. After incubation for 48 h at 30 ° C, the transconjugants grew directly behind the
  • Nucleotide sequences were determined using the dideoxy chain termination method of Sanger et al. (Sanger et al. 1977. Proc. Natl. Acad. Sci. USA 74: 5463-5467) "non-radioactive" with a "LI-COR DNA Sequencer Model 4000L” (LI-COR Inc., Biotechnology Division, Lincoln , NE, .USA) using a "Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP" (Amersham Life Science, Amersham International pls, Little Chalfont, Buckinghamshire, England) each according to the manufacturer's instructions.
  • the 2099 bp 5g / l fragment of the transposon Tn5 (Auerswald EA, G. Ludwig and H. Schaller. 1981. Cold Spring Harb. Symp. Quant. Biol. 45: 107-113; Beck E., G. Ludwig, EA Auerswald, B. Reiss and H. Schaller. 1982. Gene 19: 327-336; Mazodier P., P. Cossart, E. Giraud and F. Gasser. 1985. Nucleic Acids Res. 13 : 195-205.) Preparatively isolated. The fragment was shortened to approximately 990 bp by treatment with the nuclease Bal-31.
  • This fragment which only comprised the kanamycin resistance gene (coding for an aminoglycoside-3'-O-phosphotransferase), was then cut with Smally cut pSKsym-DNA (pBluescript SK derivative, which has a symmetrically constructed multiple cloning site [Sall, Hindlll, EcoRI, Sm ⁇ l, EcoRI, Hindl ⁇ l, Sall] contains) ligated.
  • the ⁇ Km element could be reisolated from the resulting plasmid as a Smal, EcoRI, HwdIII or So / I fragment.
  • Plasmids pBBRlMCS-5 (Kovach M. ⁇ ., P. ⁇ . Elzer, DS Hill, GT Robertson, MA Farris, RM Roop and KM Peterson. 1995. Gene 166: 175-176.) Were preparatively isolated and then with mung bean nuclease (Digestion of single-stranded DNA molecule ends) treated. This fragment, which only comprised the gentamycin resistance gene (coding for a gentamycin-3-acetyltransferase), was then ligated with Smal cut pSKsym-DNA (see above). The ⁇ Gm element could be re-isolated from the resulting plasmid as a Smal, EcoRI, H dlll or Sall fragment.
  • Example 2 Example 2
  • Plasmid p ⁇ 207 and the 3700 bp EcoRI / S ⁇ / I fragment of plasmid p ⁇ 5-l were cloned together in pBluescript SK in such a way that both fragments were connected to one another via the EcoRI ends.
  • the 6050 bp S ⁇ I fragment was isolated and shortened to about 2480 bp by treatment with the nuclease Bal-31.
  • ligated to the fragment ends / sfl linker and the fragment after Pstl digestion was cloned into pBluescript SK " (pSK ⁇ s).
  • pSK ⁇ s After transformation of E. coli XLl-Blue, clones were obtained which expressed the fcs gene and one FCS activity of 0.2 U / mg protein.
  • the 3800 bp Hmdlll / EcoRI fragment of the plasmid p ⁇ 207 was preparatively isolated and shortened to approximately 1470 bp by treatment with the nuclease Bal-31. Then EcoRI linkers were ligated to the fragment ends and the fragment was cloned into pBluescript SK " after EcoRI digestion (pSKecb).
  • Plasmids p ⁇ 207 isolated preparatively. After cloning in pBluescript SK " that was
  • the 3700 bp EcoRI / S ⁇ / I fragment of the plasmid p ⁇ 5-l was preparatively isolated and shortened to approximately 1590 bp by treatment with the nuclease Bal-31. Then EcoRI linkers were ligated to the fragment ends and the fragment was cloned into pBluescript SK " (pSKaat) after EcoRI digestion.
  • the plasmid pSKfcs which contained the fcs gene, was included digested, whereby a 1290 bp fragment was cut out of the ⁇ cs gene.
  • the deletion derivative of the fcs gene (fcsA) (see Figs. Li and 2i) cloned in pBluescript SK " (pSK / ⁇ sA) was obtained.
  • the omega elements ⁇ Km and ⁇ Gm on the fragment were cut out
  • the plasmid pSKecb which contained the ecb gene, was digested with NrwI, whereby a 53 bp and a 430 bp fragment was excised from the ecb gene. After religation, the deletion derivative of the ecb gene (ecb ⁇ , see FIGS. 11 and 21) was obtained cloned in pBluescript SK " (pSKecb ⁇ ) after cutting out the fragments, the omega elements ⁇ Km and ⁇ Gm were inserted in their place.
  • the plasmid pSKvJb which contained the vJb gene, was digested with ⁇ ssHII, whereby a 210 bp fragment was excised from the vdh gene. After religation, the deletion derivative of the vdh gene (vdhA, see Figs. Lo and 2o) was obtained cloned in pBluescript SK (pSKvdh ⁇ ). In addition, after cutting out the fragment, the omega elements _ ⁇ Km and ⁇ Gm were inserted in its place.
  • the plasmid pSK ⁇ t which contained the aat gene, was digested with ⁇ wHII, whereby a 59 bp fragment was cut out of the aat gene. After religation, the deletion derivative of the ⁇ ⁇ t gene (aatA, see Fig. Lr and
  • the "suicide plasmid" was pSUP202 (Simon et al. 1983. In: A. Pühler. Molecular genetics of the bacteria-plant interaction. Springer Verlag, Berlin, Heidelberg, New York, pp. 98-106.) used.
  • the inactivated genes fcs ⁇ Km and c ⁇ Gm were isolated from the plasmids pSK / ⁇ s ⁇ Km and pSK / c ⁇ ⁇ Gm after stl digestion and ligated with PstL cut pSUP202 DNA. The ligation batches were transformed to E. coli S17-1. The selection was made on LB medium containing tetracycline with kanamycin or gentamycin. Kanamycin-resistant transformants were obtained whose hybrid plasmid (pSUP / cs ⁇ Km) contained the inactivated yes ⁇ Km gene. The corresponding hybrid plasmid (pSUP / ⁇ s ⁇ Gm) of the gentamycin-resistant transformants contained the inactivated Genevacs ⁇ Gm.
  • the inactivated genes ecb ⁇ Km and ecb ⁇ Gm were isolated from the plasmids pSKecb ⁇ Km and pSKecb ⁇ Gm after EcoRI digestion and ligated with EcoRI cut pSUP202 DNA.
  • the ligation batches were transformed according to E. coli S17-1. The selection was made on LB medium containing tetracycline with kanamycin or gentamycin. Kanamycin-resistant transformants were obtained, whose Hybrid plasmid (pSUPecb ⁇ Km) containing the inactivated ecb ⁇ Km gene.
  • the corresponding hybrid plasmid (pSUPecb ⁇ Gm) of the gentamycin-resistant transformants contained the inactivated gene ecb ⁇ Gm.
  • the inactivated genes vJb ⁇ Km and vJb ⁇ Gm were isolated from the plasmids pSKv ⁇ b ⁇ Km and pSKvtib ⁇ Gm after EcoRI digestion and ligated with EcoRI cut pSUP202 DNA.
  • the ligation batches were transformed according to E. coli S17-1. The selection was made on LB medium containing tetracycline with kanamycin or gentamycin. Kanamycin-resistant transformants were obtained whose hybrid plasmid (pSUPvdb ⁇ Km) contained the inactivated gene v b ⁇ Km.
  • the corresponding hybrid plasmid (pSUPvJb ⁇ Gm) of the gentamycin-resistant transformants contained the inactivated gene vJb ⁇ Gm.
  • the inactivated genes aat ⁇ Km and ⁇ t ⁇ Gm were isolated from the plasmids pSK ⁇ t ⁇ Km and pSK ⁇ t ⁇ Gm after EcoRI digestion and ligated with EcoRI cut pSUP202 DNA. The ligation batches were transformed according to E. coli S17-1. The selection was made on LB medium containing tetracycline with kanamycin or gentamycin. Kanamycin-resistant transformants were obtained whose hybrid plasmid (pSUP ⁇ t ⁇ Km) contained the inactivated gene aat ⁇ Km. The corresponding hybrid plasmid (pSUP ⁇ t ⁇ Gm) of the gentamycin-resistant transformants contained the inactivated gene ⁇ t ⁇ Gm.
  • the plasmid After conjugative transfer of this hybrid plasmid into a pseudomonad, the plasmid is integrated into the genome by homologous recombination at the point at which the intact gene is located (first "cross over”). In this way, a "heterogeneous" strain is created which has both an intact and a deletion-inactivated gene, which are separated from one another by the pHE55 DNA.
  • Strains have the resistance encoded by the vector and also have an active sacB gene.
  • second homologous recombination event second "cross over"
  • the pHE55 DNA together with the intact gene is now to be separated from the genomic DNA.
  • This recombination event creates a strain that only has the inactivated gene.
  • the inactivated gene fcs A was isolated from the plasmid pSK / cs ⁇ after stl digestion and ligated with Pstl cut pHE55 DNA. The ligation mixture was transformed into E. coli S17-1. The selection was made on LB containing tetracycline Medium. Tetracycline-resistant transformants were obtained whose hybrid plasmid (pHEfcsA) contained the inactivated GenevacsA.
  • the inactivated echA gene was isolated from the plasmid pSKecb ⁇ after EcoRI digestion and treated with mung bean nuclease (generation of smooth
  • the fragment was ligated with BamHl cut and mung bean nuclease treated pHE55 DNA.
  • the ligation mixture was transformed into E. coli S17-1.
  • the selection was made on LB medium containing tetracycline. Tetracycline-resistant transformants were obtained whose hybrid plasmid (pHEecb ⁇ ) contained the inactivated gene echA.
  • the inactivated gene vdhA was isolated from the plasmid pSKvüfb ⁇ after EcoRI digestion and treated with mung bean nuclease. The fragment was ligated with BamHl cut and Mung Bean nuclease treated pH ⁇ 55 DNA. The ligation mixture was transformed into E. coli S17-1. The selection was made on
  • Tetracycline-resistant transformants were obtained whose hybrid plasmid (pHEv b ⁇ ) contained the inactivated gene vJb ⁇ .
  • the inactivated gene aatA was isolated from the plasmid pSK ⁇ t ⁇ after EcoRI digestion and treated with mung bean nuclease. The fragment was made with
  • the Pseudomonas sp. HR199 was used as a recipient in conjugation experiments in which strains of E. coli S17-1 were used as donors, which contained the hybrid plasmids of pSUP202 listed below.
  • the transconjugants were selected on mineral medium containing gluconate, which contained the antibiotic corresponding to the ⁇ element. "Homogenote” (exchange of the intact gene for the gene inactivated by ⁇ -element insertion by double “cross over”) and "heterogenote” (integration of the hybrid plasmid into the genome by simple "cross over”) transconjugants could be coded using pSUP202 Tetracycline resistance can be distinguished.
  • the mutants Pseudomonas sp. HR199 c.s ⁇ Km and Pseudomonas sp. HR199 fcs ⁇ Gm were conjugated by Pseudomonas sp. Get HR199 with E. coli S17-1 (pSUP / cs ⁇ Km) or E. coli S17-1 (pSUP / ⁇ Gm).
  • the exchange of the intact fcs gene for the gene inactivated by ⁇ Km or ⁇ Gm (fcs ⁇ Km or fcs ⁇ Gm) was verified by means of DNA sequencing.
  • the mutants Pseudomonas sp. HR199 ecb ⁇ Km and Pseudomonas sp. HR199 ech ⁇ Gm were conjugated from Pseudomonas sp. Receive HR199 with E. coli S17-1 (pSUPecb ⁇ Km) or E. coli S17-1 (pSUPecb ⁇ Gm).
  • the exchange of the intact ecb gene for the gene inactivated by ⁇ Km or ⁇ Gm (ecb ⁇ Km or ecb ⁇ Gm) was verified by means of DNA sequencing.
  • ⁇ Gm were obtained after conjugation of Pseudomonas sp. Receive HR199 with E. coli S17-1 (pSUPv b ⁇ Km) or E. coli S17-1 (pSUPvJb ⁇ Gm). The exchange of the intact vJb gene against the gene inactivated by ⁇ Km or ⁇ Gm (wtTz ⁇ Km or vJb ⁇ Gm) was verified by means of DNA sequencing.
  • the mutant Pseudomonas sp. HR199 cs ⁇ KmvJb ⁇ Gm were conjugated by Pseudomonas sp. HR199 / cs ⁇ Km obtained with E. coli S17-1 (pSUPvJb ⁇ Gm).
  • the exchange of the intact vdh gene for the gene inactivated by ⁇ Gm (vdh ⁇ Gm) was verified by DNA sequencing.
  • the mutant Pseudomonas sp. HR199 vJb ⁇ Km ⁇ t ⁇ Gm were after conjugation of Pseudomonas sp. HR199 viib ⁇ Km with E. coli S17-1 (pSUP ⁇ t ⁇ Gm) obtained.
  • the exchange of the intact ⁇ t gene for the gene inactivated by ⁇ Gm (aat ⁇ Gm) was verified by means of DNA sequencing.
  • the mutant Pseudomonas sp. HR199 vJb ⁇ Kmecb ⁇ Gm were conjugated to Pseudomonas sp. HR199 vtib ⁇ Km obtained with E. coli S17-1 (pSUPecb ⁇ Gm).
  • the exchange of the intact ecb gene for the gene inactivated by ⁇ Gm (ech ⁇ Gm) was verified by DNA sequencing.
  • the Pseudomonas sp. HR199 ⁇ s ⁇ Km, Pseudomonas sp. HR199 ecb ⁇ Km, Pseudomonas sp. HR199 vdh ⁇ K and Pseudomonas sp. HR199 aat ⁇ Km were used as a recipient in conjugation experiments in which strains of E. coli S17-1 were used as donors, which contained the hybrid plasmids of pHE55 listed below.
  • the "heterogeneous" transconjugants were selected on mineral medium containing gluconate, which contained the antibiotic corresponding to the ⁇ element in addition to tetracycline (resistance coded pHE55).
  • transconjugants After streaking on sucrose-containing mineral medium, transconjugants were obtained which had eliminated the vector DNA by a second recombination event (second "cross over”).
  • second "cross over” By spreading on mineral medium without antibiotics or with the antibiotic corresponding to the ⁇ element, it was possible to identify the mutants in which the gene inactivated by ⁇ element had been replaced by the gene inactivated by deletion (no antibiotic resistance).
  • the mutant Pseudomonas sp. HR199 ⁇ c.sA was obtained after conjugation of Pseudomonas sp. HR199 cs ⁇ Km obtained with E. coli S17-1 (pHEfcsA).
  • the exchange of the gene inactivated by ⁇ Km (fcs ⁇ K) for the gene inactivated by deletion (fcsA) was verified by means of DNA sequencing.
  • the mutants Pseudomonas sp. HR199 echA was conjugated to Pseudomonas sp. HR199 ecb ⁇ Km obtained with E. coli S17-1 (pHEecb ⁇ ).
  • the exchange of the gene inactivated by ⁇ Km (ecb ⁇ Km) for the gene inactivated by deletion (echA) was verified by means of DNA sequencing.
  • the mutants Pseudomonas sp. HR199 vdhA was conjugated to Pseudomonas sp. HR199 vdh ⁇ Km. obtained with E. coli S17-1 (pHEvJb ⁇ ).
  • the exchange of the gene inactivated by ⁇ Km (vdh ⁇ Km) for the gene inactivated by deletion (vdhA) was verified by means of DNA sequencing.
  • the mutants Pseudomonas sp. HR199 aatA was conjugated to Pseudomonas sp. HR199 ⁇ t ⁇ Km obtained with E. coli S17-1 (pHE ⁇ / ⁇ ).
  • the exchange of the gene inactivated by ⁇ Km ( ⁇ t ⁇ Km) for the gene inactivated by deletion (aatA) was verified by means of DNA sequencing.
  • the production fermenter was inoculated with 10 g / 1 yeast extract and 0.37 g / 1 acetic acid.
  • the fermenter contained 9.9 liters of medium with the following composition: 1.5 g / 1 yeast extract, 1.6 g / 1 KH 2 PO 4 , 0.2 g / 1 NaCl, 0.2 g / 1 MgSO 4 .
  • the pH was adjusted to pH 7.0 with sodium hydroxide solution. After sterilization, 4 g of eugenol was added to the medium.
  • the temperature was 32 ° C, the ventilation 3
  • FIG. la to lr
  • calA * part of the inactivated gene of the coniferyl alcohol dehydrogenase calB *: part of the inactivated gene of the coniferylaldehyde dehydrogenase fcs *: part of the inactivated gene of the ferulic acid-CoA synthetase ech *: part of the inactivated gene of the enoyl-CoA hydratase aldolase vdh * : Part of the inactivated gene of vanillin dehydrogenase aat *: Part of the inactivated gene of beta-ketothiolase
  • restriction enzyme interfaces provided with "*" were used for the construction, but are no longer functional in the resulting construct.
  • FIG. 2a nucleotide sequence of the calA ⁇ Km gene structure
  • FIG. 2b nucleotide sequence of the gene structure calA ⁇ Gm
  • FIG. 2c nucleotide sequence of the calAA gene structure
  • FIG. 2d Nucleotide sequence of the gene structure calB ⁇ Km
  • FIG. 2e nucleotide sequence of the calB ⁇ Gm gene structure
  • FIG. 2f Nucleotide sequence of the gene structure calBA
  • FIG. 2g nucleotide sequence of the gene structure fcs ⁇ Km
  • FIG. 2h nucleotide sequence of the gene structure fcs ⁇ Gm
  • FIG. 2i nucleotide sequence of the gene structure fcsA
  • FIG. 2j nucleotide sequence of the gene structure ecb ⁇ Km
  • FIG. 2k nucleotide sequence of the gene structure ecb ⁇ Gm
  • FIG. 21 Nucleotide sequence of the gene structure echA
  • FIG. 2m nucleotide sequence of the gene structure vtib ⁇ Km
  • FIG. 2n nucleotide sequence of the gene structure vJb ⁇ Gm
  • FIG. 2o Nucleotide sequence of the gene structure vdhA
  • FIG. 2p nucleotide sequence of the gene structure aat ⁇ Km
  • FIG. 2q nucleotide sequence of the gene structure aat ⁇ Gm
  • FIG. 2r nucleotide sequence of the gene structure aatA

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Abstract

L'invention concerne un organisme unicellulaire ou multicellulaire transformé et/ou mutagénisé, lequel est caractérisé en ce que des enzymes du catabolisme de l'eugénol et/ou de l'acide férulique sont inactivés de telle manière qu'il se produit une accumulation des intermédiaires alcool coniférylique, aldéhyde coniférylique, acide férulique, vanilline et/ou acide vanillique.
EP99953892A 1998-10-31 1999-10-20 Construction de souches de production pour la fabrication de phenols substitues par inactivation ciblee de genes du catabolisme de l'eugenol et de l'acide ferulique Withdrawn EP1124947A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19850242A DE19850242A1 (de) 1998-10-31 1998-10-31 Konstruktion von Produktionsstämmen für die Herstellung von substituierten Phenolen durch gezielte Inaktivierung von Genen des Eugenol- und Ferulasäure-Katabolismus
DE19850242 1998-10-31
PCT/EP1999/007952 WO2000026355A2 (fr) 1998-10-31 1999-10-20 Construction de souches de production pour la fabrication de phenols substitues par inactivation ciblee de genes du catabolisme de l'eugenol et de l'acide ferulique

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EP1124947A2 true EP1124947A2 (fr) 2001-08-22

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EP99953892A Withdrawn EP1124947A2 (fr) 1998-10-31 1999-10-20 Construction de souches de production pour la fabrication de phenols substitues par inactivation ciblee de genes du catabolisme de l'eugenol et de l'acide ferulique

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BR (1) BR9914930A (fr)
CA (1) CA2348962A1 (fr)
DE (1) DE19850242A1 (fr)
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HU (1) HUP0104772A3 (fr)
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Cited By (1)

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EP2721148B1 (fr) 2011-06-17 2018-09-12 Symrise AG Microorganismes et procedes pour la production de phenols substitues

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KR100830691B1 (ko) * 2006-11-21 2008-05-20 광주과학기술원 이소유제놀과 유제놀로부터 천연바닐린과 바닐린 산으로 생전환하는 신규 미생물
JP6509215B2 (ja) 2013-07-22 2019-05-08 ビーエーエスエフ ソシエタス・ヨーロピアＢａｓｆ Ｓｅ フェルラ酸からのバニリンの迅速かつ高収率な製造のためのシュードモナス・プチダｋｔ２４４０の遺伝子操作
CN103805640B (zh) * 2014-01-26 2016-04-06 东华大学 一种利用细菌氧化松伯醛制备阿魏酸的方法
EP3000888B1 (fr) * 2014-09-29 2018-12-05 Symrise AG Procedée pour la conversion d'acide ferulique en vanilline
FR3041655B1 (fr) * 2015-09-29 2017-11-24 Lesaffre & Cie Nouvelles souches bacteriennes pour la production de vanilline
CN111019995B (zh) 2019-12-31 2021-04-27 厦门欧米克生物科技有限公司 一种以丁香酚为底物发酵生成香兰素的方法

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JPH05227980A (ja) * 1992-02-21 1993-09-07 Takasago Internatl Corp 発酵法によるバニリンおよびその関連化合物の製造法
DE4227076A1 (de) * 1992-08-17 1994-02-24 Haarmann & Reimer Gmbh Verfahren zur Herstellung substituierter Methoxyphenole und dafür geeignete Mikroorganismen
GB9606187D0 (en) * 1996-03-23 1996-05-29 Inst Of Food Research Production of vanillin
DE19649655A1 (de) * 1996-11-29 1998-06-04 Haarmann & Reimer Gmbh Syntheseenzyme für die Herstellung von Coniferylalkohol, Coniferylaldehyd, Ferulasäure, Vanillin und Vanillinsäure und deren Verwendung

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Title
See references of WO0026355A2 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2721148B1 (fr) 2011-06-17 2018-09-12 Symrise AG Microorganismes et procedes pour la production de phenols substitues

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HUP0104772A3 (en) 2003-10-28
DE19850242A1 (de) 2000-05-04
BR9914930A (pt) 2001-07-10
AU761093B2 (en) 2003-05-29
JP2003533166A (ja) 2003-11-11
CA2348962A1 (fr) 2000-05-11
SK5742001A3 (en) 2001-12-03
HUP0104772A2 (hu) 2002-03-28
WO2000026355A2 (fr) 2000-05-11
CN1325444A (zh) 2001-12-05
HK1041902A1 (zh) 2002-07-26
IL142272A0 (en) 2002-03-10
PL348647A1 (en) 2002-06-03
WO2000026355A3 (fr) 2000-11-09
AU1041300A (en) 2000-05-22

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