WO2001073038A2 - Procede de production de l-alaninol par voie biotechnologique - Google Patents

Procede de production de l-alaninol par voie biotechnologique Download PDF

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WO2001073038A2
WO2001073038A2 PCT/EP2001/003651 EP0103651W WO0173038A2 WO 2001073038 A2 WO2001073038 A2 WO 2001073038A2 EP 0103651 W EP0103651 W EP 0103651W WO 0173038 A2 WO0173038 A2 WO 0173038A2
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microorganisms
alaninol
dsm
genes
pseudomonas
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WO2001073038A3 (fr
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Thomas Leisinger
Jan Van Der Ploeg
Andreas M. Kiener
Susana Ivone DE AZEVEDO WÄSCH
Tere Maire
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Lonza Ag
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas

Definitions

  • the invention relates to new microorganisms which are capable of converting isopropylamine to L-alaninol and their genes ipuH and ipul which code for enzymes which are involved in the metabolism of L-alaninol (S - (+) - 2-amino-l -propanol) are involved, are inactivated.
  • the invention also relates to the genes required for biosynthesis, or DNA fragments and vectors relating thereto, and polypeptides capable of biosynthesis of ⁇ -glutamylamides.
  • the new microorganisms or the related DNA fragments or polypeptides are used for a new process for the production of L-alaninol based on isopropylamine (IPA) and for a new process for the production of ⁇ -glutamylamides, in particular also for the production of theanine based on ethylamine ,
  • IPA isopropylamine
  • L-alaninol is an important intermediate for the production of pharmaceuticals, for example for the production of ofioxacin (J. Med. Chem 1997, 30, 2883-2286).
  • a biotechnological process for the production of L-alaninol is described in WO 99/07199.
  • the mutants Pseudomonas sp. KIE171-B and -BI are capable of producing L-alaninol from IPA.
  • both mutants break down the resulting L-alaninol, which is disadvantageous for an industrial application of this process.
  • the object of the present invention was to provide an industrially viable process for the production of L-alaninol and, as a sub-step, for the production of N-5-substituted ⁇ -L-glutamylamides, with the high yields of L-alaninol or N -5-substituted ⁇ -L-glutamylamides can be achieved.
  • the present invention accordingly relates to microorganisms which are capable of transforming IPA to L-alaninol and in which the genes ipuH and ipul which code for enzymes which are involved in the metabolism of L-alaninol are inactivated, and cell-free enzyme extracts it.
  • Genes that are in inactive form are understood to mean genes that e.g. have been changed by insertion, mutation or deletion in such a way that either no product is formed or the product formed is no longer functionally active.
  • the ipuH and ipul genes which code for enzymes which are involved in the metabolism of L-alaninol, can be inactivated by known methods.
  • Appropriate methods for inactivation so-called “knock out” methods, are, for example, mutation methods such as the point mutation method, frame shift method, deletion method or the transposon insertion method.
  • methods for site-specific recombination of the corresponding genes with a previously inactivated gene can be used, as described, for example, in Hoang et al., 1998, Gene 212, 77-86, which are preferably used.
  • the invention is illustrated by the following figures.
  • the numbers in brackets show the position of the nucleotides within the sequence shown in SEQ ID No. 1 described nucleotide sequence of the ipul gene.
  • the numbers in brackets show the position of the nucleotides within the nucleotide sequence of the ipu operon (FIG. 3).
  • Figure 1 shows the enzymes involved in the metabolism of IPA and L-alaninol and a new pathway for IPA.
  • 3A to 3N show the nucleotide and amino acid sequence of the genes ipuA, ipuB, ipuC, ipuD, ipuE, ipuF, ipuG and ipuH of the ipu operon.
  • Figure 4 shows plasmid pME4254.
  • Figure 5 shows plasmid ⁇ ME4255.
  • Figure 6 shows plasmid pME4256
  • Figure 7 shows plasmid pME4257.
  • Figure 8 shows plasmid pME4259.
  • Figure 9 shows plasmid pME4267.
  • Figure 10 shows plasmid pME4275.
  • Figure 11 shows plasmid pME4277.
  • Figure 12 shows the production of L-alaninol by Pseudomonas sp. KIE171-BI (DSM
  • Microorganisms which are capable of converting IPA to L-alaninol and which contain the genes ipuH and ipul, which code for enzymes of the metabolism of L-alaninol, in active form can be used as starting microorganisms for the production of the microorganisms according to the invention which have one of these genes in inactive form.
  • the starting microorganisms are expediently cultivated in an aqueous nutrient medium which contains a carbon and nitrogen source, mineral salts and a vitamin source, and at a temperature of 20 to 40 ° C., preferably 30 to 37 ° C. , and cultivated at a pH of 5 to 9, preferably at a pH of 6 to 8.
  • Preferred starting microorganisms are microorganisms of the genus Pseudomonas, particularly preferably the Pseudomonas sp. Described in WO 99/07199 by the same applicant. KIE171-BI (DSM 11629), KIE171-B (DSM 11521) or KIE171 (DSM 12360).
  • these microorganisms are advantageously grown from soil samples, sludge or waste water with the aid of customary microbiological techniques and selected with regard to the transformation of IPA to L-alaninol.
  • the genes involved in the metabolism of IPA and L-alaninol can be labeled using conventional techniques such as, for example, the transposon insertion method and then identified, isolated, sequenced and cloned by the so-called "transposon rescue" technique as described, for example, in De Lorenzo V., Timmis KN, Methods Enzymol., 1994, 235, 386-405
  • DNA fragments are isolated from the starting microorganisms and the enzymes encoded by the DNA fragments can be assigned to the metabolism of IPA and L-alaninol in the usual way.
  • the genes ipuH and ipul which code for enzymes involved in the metabolism of L-alaninol, either contain both in an active form or one of these genes is in an inactive form, one or both are expediently the ipuH and ipul genes into a suitable vector, for example a plasmid, cloned and inactivated, e.g. by inserting a marker gene cassette, such as a gene coding for antibiotic resistance.
  • the construct obtained is conveniently placed in a Velctor, e.g. cloned a plasmid which is in a suitable host organism such as e.g. E. coli, but cannot be replicated in the starting microorganism.
  • a Velctor e.g. cloned a plasmid which is in a suitable host organism such as e.g. E. coli, but cannot be replicated in the starting microorganism.
  • selection for the product of the corresponding marker gene and subsequent deletion of the plasmid integrated into the chromosome and of the active ipuH and / or ipul gene can be carried out according to the invention
  • Microorganism are obtained in which the genes ipuH and ipul, which code for enzymes of the metabolism of L-alaninol, are inactivated.
  • the microorganisms according to the invention in which the ipuH and ipul genes coding for enzymes for the metabolism of L-alaninol are inactivated preferably belong to the genus Pseudomonas, particularly preferably to the species Pseudomonas citronellolis, Pseudomonas azalaica, Pseudomonas nitroreducens, Pseudomonas alcaligenes, Pseudomonas aeruginosa or Pseudomonas putida, most preferred to the species Pseudomonas sp. corresponding to the species of the deposited strain Pseudomonas sp. KIE171-BIII (DSM 13177). The functionally equivalent variants or mutants are also included.
  • the deposited strain DSM 13177 represents such a preferred embodiment of the present invention.
  • the microorganism Pseudomonas sp. KIE171-BIII (DSM 13177) was deposited on December 3, 1999, with the German Collection for Microorganisms and Cell Cultures GmbH, Mascheroderweg lb, D-38124 Braunschweig, in accordance with the Budapest Treaty.
  • the strain KIE171-B (DSMl 1521), from which the strain mutants of the B series are derived, was examined taxonomically.
  • the phylogenetic analysis showed the classification as a separate new species of the genus Pseudomonas, with the greatest similarity to the known species Pseudomonas citronellolis.
  • Chemotaxonomic analyzes (detailed in WO 99/07199) confirmed the belonging of the new species to the RNA group I of the Pseudomonas (including, inter alia, Pseudomonas citronellolis and Pseudomonas aeruginosa).
  • “Functionally equivalent mutants” are understood to mean microorganisms which have essentially the same properties and functions of the original microorganisms. Such genetic variants or mutants can be obtained by sufficiently known methods, including random mutagenesis, for example with UV radiation or alkylating reagents (described in Miller, J., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory 1972), 'error prone' PCR or gene 'shuffling' of polymorphic DNA sequences in vitro and subsequent retransfer of these gene fragments into an organism, or spontaneously through the natural mutation rate of microorganisms be generated.
  • random mutagenesis for example with UV radiation or alkylating reagents (described in Miller, J., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory 1972), 'error prone' PCR or gene 'shuffling' of polymorphic DNA sequences in vitro and subsequent retransfer of these gene fragments into an organism, or spontaneously through the natural mutation rate of microorganis
  • the enzymes or enzyme extracts according to the invention for the cell-free system can be obtained by disrupting the microorganisms by customary methods. For example, the ultrasonic, French press or lysozyme method can be used for this.
  • the cell-free enzymes can also be immobilized on a suitable carrier material, as is well known to the person skilled in the art.
  • the microorganisms according to the invention are expediently cultivated in an aqueous nutrient medium which contains a carbon, nitrogen source, mineral salts and a vitamin source in a customary manner, advantageously as described in WO 99/07199.
  • the invention further relates to isolated DNA fragments which comprise one or more of the genes ipuA, ipuB, ipuC, ipuD, ipuE, ipuF, ipuG, ipuH, ipul, which code for enzymes which are involved in the metabolism of IPA to L- Alaninol and the metabolism of L-Alaninol are involved.
  • These DNA fragments according to the invention are preferably derived from microorganisms of the genus Pseudomonas such as e.g.
  • Pseudomonas putida Pseudomonasas citronellolis, Pseudomonas aeruginosa, Pseuodomonas alcaligenes, Pseudomonas nitroreducens, Pseudomonas azalaica isolated, particularly preferably from microorganisms of the species Pseudomonas citronellolis or the species Pseudomonas sp. corresponding to the species of one of the deposited strains or mutants Pseudomonas sp.
  • KIE171 (DSM 12360), KIE171-B (DSM 11521), KIE171-BI (DSM 11629), Pseudomonas sp. KIE171-BII (DSM 13389) and / or Pseudomonas sp. KIE171-BIII (DSM 13177), most preferably from Pseudomonas sp. KIE171 (DSM 12360), Pseudomonas sp. KIE171-BI (DSM 11629) and / or Pseudomonas sp. KIE171-BII (DSM 13389).
  • microorganism Pseudomonas sp. KIE171-BII (DSM 13389) was deposited on March 27, 2000 with the German Collection for Microorganisms and Cell Cultures GmbH (DSMZ), Mascheroderweg lb, D-38124 Braunschweig, in accordance with the Budapest Treaty.
  • DSMZ German Collection for Microorganisms and Cell Cultures GmbH
  • the other strains mentioned were deposited in the context of the present application or WO 99/07199 by the same applicant, in accordance with the Budapest Treaty and form part of the present description.
  • the genes ipuA, ipuB, ipuC, ipuD, ipuE, ipuF, ipuG, without or together with ipuH, are useful, which code for enzymes which are involved in the metabolism of IPA to L-alaninol or the catabolism of L-alaninol, arranged in a preferred embodiment in the order ipuA, ipuB, ipuC, ipuD, ipuE, ipuF, ipuG, and optionally ipuH, and are available as a single transcription unit (operon).
  • the gene Ipul is preferred by the in SEQ ID No. 1 includes nucleotide sequence shown.
  • the genes ipuA, ipuB, ipuC, ipuD, ipuE, ipuF, ipuG, ipuH are preferably encompassed by the nucleotide sequence shown in FIG. 3.
  • the nucleotide sequence from 1314 to 2339 includes the protein coding region of the ipuA gene, the nucleotide sequence from 2342 to 2677 the protein coding region of the ipuB gene, the nucleotide sequence from 2743 to 4119 the protein coding region of the ipuC gene, the nucleotide sequence from 4194 to 5351 the protein coding region of the ipuD gene, the nucleotide sequence from 5371 to 5562 the protein coding region of the ipuE gene, the nucleotide sequence from 5589 to 6473 the protein coding region of the ipuF gene, the nucleotide sequence from
  • the ipuA and ipuB genes are particularly preferred by the genes in SEQ ID no. 3 comprises nucleotide sequence shown.
  • the ipuC gene is particularly preferred by the gene described in SEQ ID no. 6 nucleotide sequence shown comprises.
  • genes ipuD, ipuE, ipuF, ipuG and ipuH are particularly preferred by the genes in SEQ ID No. 8 includes nucleotide sequence shown.
  • nucleotide sequences according to the present invention also include functionally equivalent genetic variants, also referred to as alleles or mutants, i.e. H. non-identical genes whose base sequence is derived from the genes of the organisms from which the genes were isolated and whose gene products (proteins) can have the same enzymatic function.
  • the functionally equivalent genetic variants or mutants thus include, for example, base exchanges in the context of the known degeneration of the genetic code, for example in order to adapt the gene sequence to the preferred codon use of a particular microorganism in which artificial expression is to take place.
  • the genetic variants and mutants also include deletions,
  • These equivalent genetic variants or mutants preferably include gene sequences which have a high sequence homology to the sequences isolated from the organisms, for example higher than 70%, preferably higher than 80%, particularly preferably higher than 90%, and under stringent hybridization conditions, z. B. at temperatures between 60 and 70 ° C and at 0.5 to 1.5 M salt content, in particular at a temperature of 62 - 66 ° C and at 0.8 - 1.2 M salt content for hybridization with the complement of the isolated Sequences are capable.
  • the homology is preferably 95%.
  • DNA fragments according to the invention can be isolated, for example, as described above with the aid of customary techniques, e.g. by means of the transposon insertion method and subsequent use of the "transposon rescue" technique, or by using a suitable gene probe together with a suitable gene library.
  • the present invention furthermore relates to vectors which contain these DNA fragments and recombinant microorganisms which contain these vectors.
  • Autonomous and self-replicating plasmids or integration vectors can be used as vectors.
  • the ipu genes can be introduced into various microorganisms. Both vectors with a specific host spectrum and vectors with a broad host spectrum (“broad host ranks”) are suitable as vectors.
  • vectors with a specific host spectrum for example for E. coli, are the commercially available pBLUESCRIPT II KS + ®, pBLUESCRIPT SK + ® (Stratagene), pPDl 11 or its derivatives (described in Dersch et al., FEMS Microbiol Lett. 15, 123, 19-26, 1994), pET24a (+) (Novagen) or pET28a (+) (Novagen).
  • Examples of such “broad host rank” vectors are pRK290 (described in Ditta et al., PNAS, 77, 7347-7351, 1980) or its derivatives, pKT240 (described in Bagdasarian et al., Gene, 26, 273-282, 1983) or its derivatives, pVKIOO (described in Knauf and Nester, Plasmid, 8, 45-54, 1982) or its derivatives, pBBRIMCS (described in Kovach et al, Biotechniques 16: 800-802, 1994) or its derivatives
  • the vectors mentioned, in particular the expression vector pBBRIMCS simultaneously represent preferred embodiments of the present invention.
  • the plasmid pME4755 was obtained.
  • a vector with a specific host spectrum is preferably used, particularly preferably pPDl 11, pBLUESCRIPT II KS + ® or pET28a (+).
  • the plasmids pME4254, pME4255, pME4256, pME4257, pME4259, pME4267, pME4275 and pME4277 were obtained.
  • microorganisms which contain the vectors mentioned are microorganisms of the genus Escherichia, preferably of the species Escherichia coli, particularly preferably of the species Escherichia coli DH5 ⁇ and Escherichia coli XLl-Blue®.
  • vectors such as plasmid pME4255 as deposited in E. coli DH5 ⁇ (DSM 13178), plasmid pME4755 as deposited in E. coli XLl-Blue (DSM T3388), plasmid pME4267 as deposited in E. coli XLl-Blue (DSM 13179 ), and plasmid pME4259 as deposited in E. coli XLl-Blue (DSM13417).
  • vectors such as plasmid pME4255 as deposited in E. coli DH5 ⁇ (DSM 13178), plasmid pME4755 as deposited in E. coli XLl-Blue (DSM T3388), plasmid pME4267 as deposited in E. coli XLl-Blue (DSM 13179 ), and plasmid pME4259 as deposited in E. coli XLl-Blue (DSM13417).
  • microorganism of the species Escherichia XLl-Blue (DSM 13388), containing plasmid pME4755, was deposited on March 24, 2000 with the German Collection for Microorganisms and Cell Cultures GmbH, Mascheroderweg lb, D-38124 Braunschweig, in accordance with the Budapest Treaty.
  • the process according to the invention for producing L-alaninol comprises the conversion of isopropylamine (IPA) to L-alaninol by means of the microorganisms according to the invention already described above, in which the ipuH and ipul genes are responsible for enzymes encode which are involved in the metabolism of L-alaninol, are inactivated, or by means of enzyme extracts from these microorganisms.
  • L-alaninol in the sense of the present invention is L-2-amino-l-propanol.
  • the biotransformation can be L-alaninol metabolize, but have the necessary biosynthetic genes, as already described in WO 99/07199.
  • the relevant information in WO 99/07199 on the bio-formation process, the isolation of L-alaninol and the cultivation of the microorganisms according to the invention is an integral part of the present application.
  • a first analysis of the mel-stage biosynthetic pathway and the enzyme activities involved therein, as already mentioned above, is given in FIG. 1.
  • microorganisms according to the invention described above either have the necessary biosynthesis genes as a wild type, or have been recombinantly equipped with the corresponding DNA fragments or protein expression vectors according to the invention such as, for example, pME4755 in E. coli. It is also possible, in the microorganisms according to the invention which are capable of L-alaninol biosynthesis, to additionally recombinantly express individual or multiple ipu genes in a recombinant manner.
  • the biotransformation can be carried out with resting cells (non-growing cells which no longer need a C and energy source or which is no longer available) or with growing cells.
  • a cell suspension with a cell density of OD650 40-60 is preferably used.
  • Suitable media for the biotransformation are the commercially customary, for example low-molecular phosphate buffers, Hepes buffers and full media such as "Nutrient Yeast Broth” (NYB) or mineral salt media as described, for example, by Kulla et al, (Arch. Microbiol. 135, 1 (1983)) , or in WO99 / 07199 (Table 1), preferably mineral salt media as described for example in Kulla et al., (Arch. Microbiol. 135, 1 (1983)), or in WO99 / 07199 (Table 1).
  • the biotransformation is preferably carried out with a single or continuous addition of IPA in such a way that the concentration of IPA does not exceed 10% by weight, preferably 1% by weight.
  • the pH can range from 4 to 10, preferably from 5 to 9.
  • the biotransformation is expediently carried out at a temperature of 10 to 50 ° C., preferably 20 to 40 ° C., most preferably 25-35 ° C.
  • the biotransformation preferably takes place in the presence of 5 to 100 mM glutamate, preferably 10 to 30 mM glutamate.
  • L-alaninol can be isolated by customary work-up methods, such as, for example, by extraction or distillation of the basic, cell-free fermentation broth.
  • the present invention furthermore relates to a polypeptide with ⁇ -glutamylamide synthetase activity which is capable of ⁇ -glutamylamides of the general formula or the general formula
  • R 1 is an optionally substituted alkyl group, an optionally substituted
  • Aralkyl group an optionally substituted alkoxyalkyl group or an optionally substituted aryl group
  • R 2 is hydrogen or an optionally substituted alkyl group and n is one to five.
  • R 1 is substituted or unsubstituted alkyl or aryl group, more preferably a substituted or unsubstituted alkyl group.
  • Optionally substituted is to be understood as being substituted or unsubstituted.
  • alkyl group is to be understood as a straight-chain or branched alkyl group, preferably with 1 to 6 carbon atoms. Mention should be made of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl,
  • Pentyl and its isomers and hexyl and its isomers are, for example, hydroxy, oxo, cyano or amino. preferred
  • Hydroxy is a substituent.
  • An aryl group means phenyl or naphthyl, aralkyl, for example, benzyl.
  • the appropriate substituent for the aryl group is nitro.
  • alkoxyalkyl examples include methoxy-ethyl, ethoxy-ethyl.
  • R 1 preferably has the meaning of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxyethyl, 1-hydroxybutyl, 2,2- dihydroxyisopropyl.
  • R 1 particularly preferably has the meaning of 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxyethyl, 1-hydroxybutyl.
  • R 2 preferably has the meaning of methyl, n preferably has the meaning of one or two.
  • the polypeptide according to the invention can be obtained by using conventional methods for disrupting microorganisms which naturally contain this polypeptide or the ipuC gene coding for the polypeptide.
  • the ipuC gene encoding the polypeptide can be isolated from microorganisms containing an ipuC gene encoding the polypeptide as described above, cloned as described above and e.g. in E. coli BL21 using a suitable expression vector as described above.
  • the polypeptide or the ipuC gene coding for the polypeptide is preferably isolated from microorganisms which are capable of transforming IPA into L-alaninol.
  • Microorganisms of the genus Pseudomonas are preferred, particularly preferably the Pseudomonas sp. Described in WO 99/07199. KIE171 (DSM 12360), KIE171-B (DSM 11521), KIE171-BI (DSM 11629), or the KIE171-BIII (DSM 13177) according to the invention.
  • the ipuC gene obtained is preferably amplified after isolation by customary methods and converted into a vector e.g. Vector pET 28a (+) (Novagen) or pET24a (+)
  • E. coli e.g. E. coli BL21 (DE3).
  • Competent cells are understood to be cells that are able to take up free, even alien DNA.
  • the transformed cells obtained in this way are cultivated in the usual nutrient media which contain a carbon and nitrogen source, mineral salts and a vitamin source, for example in LB medium, the polypeptide according to the invention having ⁇ -glutamylamide synthetase activity as already mentioned above by unlocking the Microorganisms can be obtained using methods familiar to the person skilled in the art, or the whole cells as such, optionally after pretreatment with permeabilizing agents, can be used for the biotransformation with the recombinant protein expressed therein.
  • the polypeptide according to the invention is preferably a protein tagged for simplified purification, in particular His 6- day ⁇ -glutamylamide synthetase, as expressible by pME4275.
  • Plasmid pME4277 was obtained by incorporating the ipuC gene into vector pET24a (+).
  • the ipuC gene is advantageously incorporated into Velctor pET 28a (+), which is transformed into E. coli BL21 (DE3).
  • plasmid pME4275 was obtained which contains the ipuC gene adjacent to a DNA sequence which codes for six histidines and which for the polypeptide ⁇ -glutamylamide synthetase to which six histidine residues are attached (His 6 day - ⁇ - Glutamylamide synthetase).
  • the histidine tag enables simple, rapid purification or purification of the tagged protein, for example for use in an enzyme reactor. It is also possible to use other tag sequences, as is well known to the person skilled in the art, for example protein A fusions or the FLAG tag.
  • polypeptide according to the invention with ⁇ -glutamylamide synthetase activity which is capable of synthesizing ⁇ -glutamylamides of the general formula I or II is preferably characterized by the following properties:
  • Another object of the invention is a process for the preparation of ⁇ -glutamylamides of the general formula I or II, which is carried out in such a way that L-glutamate with an amine of the general formula
  • R 1 and R 2 have the meaning given above, by means of a microorganism according to the invention or by means of a microorganism expressing the IpuC gene according to the invention recombinantly or by means of a polypeptide according to the invention, as described above, to the product of the general formula I or II.
  • the reaction can also be carried out with essentially cell-free enzyme extract from the corresponding microorganisms or with purified polypeptide.
  • Methods for producing an enzyme extract are well known to the person skilled in the art and include, for example, cell disruption by means of French Press, the lysozyme method and ultrasound treatment.
  • the reaction can be carried out, for example, in a buffer such as Tris-HCl together with imidazole with the addition of ATP and magnesium ions.
  • the pH can range from 4 to 9, preferably from 5 to 8.
  • the reaction is conveniently carried out at a temperature of 10 to 50 ° C, preferably 20 to 40 ° C, most preferably 25 to 35 ° C.
  • Suitable amines for the present process are e.g. listed in section 'Other glutamyl compounds formed' on page 23, example 4.
  • the glutamate concentration is preferably 1 to 100 mM, more preferably 10-30 mM.
  • Suitable substrate concentrations for the amine are, for example, 1-200 mM, preferably 10-100 mM.
  • the amine is a primary amine of the general formula III.
  • examples are benzylamine, ethylamine, isopropylamine, butylamine, isobutylamine, hydroxy-butylamine.
  • the definitions and preferred forms of completion of R 1 given above apply accordingly.
  • ⁇ -glutamylisopropylamides of the general formula I or II can be isolated by customary working-up methods, for example by isoelectrical focusing or extraction.
  • the process is preferably carried out with whole cells, analogously to the production of L-alaninol.
  • the information given there on the implementation of the procedure applies accordingly.
  • preference is given, in addition to the amine serving as the substrate molecule of the formula III or IV, to a further primary low molecular weight C1-C4 alkyl amine, for example methyl, ethyl , Isopropylamine, was added as an enzyme inducer.
  • IPA suitably in a concentration of 1-20 mM, preferably in a concentration of 1-10 mM.
  • This embodiment is particularly advantageous when using a microorganism according to the invention of the genus Pseudomonas, in particular of the strain KIE-171-BIII (DSM 13177).
  • the process according to the invention is used for the production of theanine starting from ethylamine.
  • the method can be carried out with microorganisms according to the invention, purified polypeptide or enzyme extracts as mentioned above.
  • the concentration of ethylamine is 5-60 mM and is kept approximately constant during the biotransformation by repeated or continuous addition of ethylamine.
  • theanine is N-5-ethyl-L-glutamine corresponding to formula I.
  • transposon mutants BI Pseudomonas sp. KIE171-BI (DSM 11629)
  • BII Pseudomonas sp. KIE171-BII
  • the production of transposon mutants was described in WO 99/07199 (Example 2b) for the production of mutant BI.
  • the mutant BI can produce L-alaninol from IPA, but degrades it again in a biotransformation with a high OD 650 (> 5).
  • Mutant BII was also produced in accordance with this method.
  • the mutant BII grew neither on IPA nor on L-alaninol, but was still able to utilize L-alanine, L-lactate and L-alanine, and L-glutamate.
  • the mutant BII does not produce L-alaninol.
  • the DNA fragments containing the inactivated gene were cloned into a suitable vector.
  • DNA sequences and the derived protein sequences were analyzed with the GCG software package. Based on these results, a new degradation route for IPA was postulated.
  • the DNA fragments of the mutants BI and BII were ligated into the vectors pBluescript or pPDl 11.
  • the ligation mixture was competent for transformation
  • E.coli DH5 ⁇ or competent E.coli XL-I blue cells are used.
  • the transformed cells were on LB plates with Km (50 ug / ml) or ampicillin
  • the DNA fragment of the kanamycin resistance gene which had been introduced by the mini-Tn5 was removed by digestion with the enzyme Sfil and subsequent ligation in the plasmid pME4255.
  • the plasmid pME4256 was formed.
  • the ipuABCDEFG genes were cloned into pBBRIMCS (Kovach et al., Ibid.).
  • pBBRIMCS Kovach et al., Ibid.
  • a 3.8 leb Xhol-Sacl fragment from pME4259 was cloned into the Xhol-Sacl restriction sites of pBluescript II KS (+).
  • the 3 live BglII-Sacl transposon insert in ipuC which still contained this plasmid was replaced by the 0.95 kb BglII-Sacl fragment of the native ipuC sequence; the resulting plasmid comprises ipuB and ipuC.
  • a 1.6 leb Xhol-Pvull fragment was cut out of this vector and cloned together with a further 4.4 kb PvwII-Pstl fragment comprising ipu gons contained in pME4257 in pBBRl-MCS, which had previously been linearized with Xhol-Pstl.
  • the resulting intermediate plasmid comprised the ipuBCDEFG genes.
  • an Xbal interface was created immediately upstream of the ipuA-Gvo with PCR and the primers GCCTTCTAGAATTCTTGTAGG and CACCCAGCCTAATCGTGTCG.
  • the PCR fragment was digested in iXXbal and Xhol and cloned in pBBRIMCS. The absence of an unintended mutation generated by the PCR in the ipuA sequence was confirmed by sequencing.
  • the 11.6 leb plasmid pME4755 was generated by cloning the 0.9 kb Xbal-Xhol ipuA fragment into the intermediate plasmid that had been linearized with Xbal-Xhol.
  • the sequencing was carried out by the company Microsynth.
  • the DNA sequences were double-stranded according to the 'dideoxy chain termination method' according to Sanger et al., Proc. Natl. Acad. Be. USA, 74, 1977, 5463-5467.
  • the plasmids pME4254, pME4256, pME4259, pME4267 and pME4275 were used for sequencing.
  • the plasmid pME4259 was digested with the restriction enzyme Smal and the fragments separated by agarose gel electrophoresis. The 7 kb fragment was isolated and purified. The 7 leb fragment was then digested a second time with Saä and the DNA fragments were separated again. The 2.7 leb Sall / Smal fragment was isolated and purified and cloned into the low copy vector pPDl 11, also digested by Sall / Smal.
  • the newly formed plasmid pME4268 contains the ipuH gene as a 2.7 kb insert. The ipuH gene was interrupted by inserting a gentamycin resistance gene.
  • pME4268 was digested with the restriction enzyme B ⁇ mHl and the 5 'overhanging restriction ends were converted into blunt ends by the Klenow fragment of DNA polymerase I in a' fill-in 'reaction with the addition of the four deoxynucleotides.
  • the gentamycin resistance gene was obtained from the plasmid pUCGM by digestion with Sm ⁇ l as a 855 bp fragment with blunt ends. The gentamycin resistance gene was then ligated into the linear pME4268 so that the S ⁇ lVSm ⁇ l insert has a length of 3.55 kb.
  • the newly created plasmid is pME4269.
  • the newly created plasmid pME4270 carries the resistance genes for Gm and tetracycline (Tc).
  • the plasmid pME4270 was transformed into E. coli S17- ⁇ pir according to the CaCl 2 method. S17- ⁇ pir are E.coli cells that are suitable for conjugation with the mutant BI.
  • E.coli S17- ⁇ pir which contains the plasmid pME4270 with the gentamycin and tetracycline resistance gene, was conjugated with the mutant BI.
  • the mutant BI was raised in 25 ml MM with L-glutamate (20 mM) and Km (50 ⁇ g / ml) at 30 ° C.
  • Antibiotics Gm (25 ⁇ g / ml), Tc (35 ⁇ g / ml) and Km (50 ⁇ g / ml) were grown overnight at 30 ° C at 150 rpm. 300 ⁇ l of each were plated out on a fresh LB plate with sucrose (5%) and incubated at 30 ° C. overnight. The colonies subsequently obtained were tested for growth on MM plates with L-glutamate (20 mM) and Tc (35 ⁇ g / ml).
  • the mutant BIII Pseudomonas sp.
  • KIE171-BIII, strain DSM 13177 which arose from this procedure, has the phenotype on MM plates with L-glutamate (20 mM) and Gm (25 ⁇ g / ml) or Km (50 ⁇ g / ml) but not with Tc (35 ⁇ g / ml).
  • a second 'crossover' took place at her. In doing so, she deleted the plasmid integrated into the chromosome with the active ipuH gene. This allows sucrose to be tolerated.
  • Mutant BIII now only has the ipuHGen inactivated by inserting the Gm resistance cassette.
  • mutant BIII strain DSM 13177
  • IPA 10 mM
  • OD 650 of the culture 1-1.3 at 4000 rpm was 15 min. centrifuged and the sediment washed twice with half the amount of culture medium without a C source. The cells could then be taken up in the desired volume of MM medium with L-glutamate (25 mM), so that 3 ml of concentrated cell suspension (OD 650 ⁇ 50) were obtained.
  • the culture was stored at 4 ° C for 16 hours. After adding IPA (20, 50 or 100 mM), this culture was shaken at 150 rpm at 30 ° C. The samples were taken at different times (1 h, 3 h, 5 h, 7 h, 23 h and 58 h).
  • the digested 1.4 leb PCR product was ligated into the vector pET28a (+) (Novagen), which had also been digested with the enzymes Ndel and Hindlll.
  • the newly formed plasmid was named pME4275 and transformed into competent cells from E. coli BL21 (DE3) according to the CaCl 2 method.
  • the digested 1.4 kb PCR product was ligated into the vector pET24a (+) (Novagen), which gave plasmid ⁇ ME4277.
  • Plasmid pME4275 contains the ipuC gene adjacent to a DNA sequence which codes for six histidines. This fusion took place by cloning ipuC into the vector pET28a (+).
  • the newly created DNA sequence codes for the polypeptide Tag- ⁇ -Glutamylamid synthetase.
  • the protein Tag- ⁇ -glutamylamide synthetase was then purified using chelate affinity chromography using 'His * Bind Resin'.
  • the N-terminal histidine end of the recombinant protein interacts with the carrier material 'His »Bind Resin'.
  • E. coli BL21 (DE3) (DSM 13180) containing pME4275 was used for the production of pure day ⁇ -glutamylamide synthetase. This was grown on 5 ml LB medium at 37 ° C with Km (50 ⁇ g / ml) and used to inoculate a culture of 100 ml in a 500 ml bottle of the same medium. After an OD 650 of 1.0 was reached, the culture was induced with 0.4 mM IPTG at 30 ° for 3 hours. The culture was then cooled on ice for 5 minutes and then centrifuged at 4 ° C. and 5000 g. The sediment was washed with cold 50 mM Tris-HC1 with 2 mM EDTA at pH 8.0 and centrifuged again under the same conditions. The sediment could then be stored at -20 ° C.
  • the frozen sediment was resuspended in 4 ml binding buffer (containing 10 ⁇ g / ml DNase I).
  • the cell extract was obtained after two passages through the French press at 5.5 Mpa and subsequent centrifugation at 39000 g for 20 minutes. The supernatant was filtered through a 0.2 ⁇ m filter.
  • Tag- ⁇ -glutamylamide synthetase was purified at 4 ° C. using "His # Bind Resin".
  • the solutions used for the chromatography were as follows:
  • Binding buffer 5 mM imidazole, 0.5 mM NaCl, 20 mM Tris-HCl, pH 7.9
  • Elution buffer 1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl pH 7.9
  • NiSO 4 solution 50 mM NiSO 4 wash buffer: 60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9
  • the bound day ⁇ -glutamylamide synthetase could then be eluted with 15 ml of elution buffer and stored at 4 ° C.
  • the molecular weight of the monomer of day ⁇ -glutamylamide synthetase is 52478 Da.
  • Theanine which had been produced by the biotransformation of the enzyme IpuC, could be detected by HPLC and its identity confirmed by cochromatography with the pure compound.
  • the sample was previously derivatized by HPLC with phenyl isothiocyanate.
  • a Nucleosil-C18 'reverse phase' column at 25 ° C was used for the analysis. The detection took place at 254 nm.
  • Theanine could also be detected using GC-MS.
  • the fragmentation pattern was identical to that of the reference compound.
  • ethylamine other compounds could be used: methylamine, ethanolamine, glycine methyl ester, propylamine, l-amino-2-propanol, 3-amino-l-propanol, isopropylamine, L-alaninol, D-alaninol, 2-amino-l, 3 propanediol, butylamine, 4-aminobutyrate methyl ester, isobutylamine, sec-butylamine, S-2-amino-1-butanol, R-2-amino-1-butanol, tert-butylamine and pentylamine.
  • DSM 13177 KLEI 71 -Bill strain
  • 31.8 mM theanine Yield 63%) were obtained after 24 h with 50 mM ethylamine as substrate.
  • the product yield was determined by HPLC. A low cell density turned out to be essential for achieving high volume yields.
  • the reaction could also be carried out with resting cells with the same cell density, albeit with poorer yields (max. 45% with 20 mM ethylamine as starting material and 18 h reaction time). Higher initial concentrations of ethylamines and higher cell densities reduced the volume yield.
  • E.coli BL21 (DE3) transformed with the expression vector pME4755 were composed of 64 mM potassium phosphate (pH 7.2), 33 mM NH 4 C1 2 , 1 mM MgCl 2 up to the stationary phase at 37 ° C. and 180 rpm in 25 ml medium. 10 mM glucose, 0.5 ⁇ M (NH 4 ) 2 SO 4 , 1% trace elements (Thurnheer et al., J. Gen. Microbiol. 132: 1215 ff, 1986) and 20 ⁇ g / ml chloramphenicol.
  • the preculture was used to inoculate 100 ml of medium with an OD 650 of 0.15 and then to grow to 0.4. After induction with 400 ⁇ M IPTG (thio-beta-D-1-galactoside), the culture was further grown up to an OD 650 of 0.8. The cells were centrifuged at 6000 g / 10 min. at
  • the mioroorganism identified under I. above was accompanied by:
  • This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2 000 - 03 - 31 (Date of the original deposit) 1 .
  • microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).
  • the microorganism identified under I. above was accompanied by:
  • This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2000 - 03 - 24 (Date of the original deposit) 1 .
  • the mioroorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).
  • the mioroorganism identified under I. above was accompanied by:
  • This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2000 - 03 - 24 (Date of the original deposit) 1 .
  • microorganism identified under I above was received by this International Depositaiy Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).
  • the microorganism identified under I. above was accompanied by:
  • This International Depositary Authority accepts the microorganism identified under I. above, which was received b it on 199 9 - 12 - 03 (Date of the original deposit) '.
  • microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).
  • microorganism identified under I above was accompamed by
  • microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion)
  • the microorganism identified under I. above was accompanied by:
  • This International Depositaiy Authority accepts the microorganism identified under I. above, which was received by it on 1999 - 12 - 03 (Date of the original deposit) 1 .
  • microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).
  • Access to the deposited biological material should only be within the scope of the expert solutions, as provided for in R. 28 (4) EPUe or Regulation 3.25 (3) Australian Patents Act, i.e. be made by handing out a sample to an expert.
  • Access to the deposited biological material should only be within the scope of the expert solutions, as provided for in R. 28 (4) EPUe or Regulation 3.25 (3) Australian Patents Act, i.e. be made by handing out a sample to an expert.
  • Access to the deposited biological material should only be within the scope of the expert solutions, as provided for in R. 28 (4) EPUe or Regulation 3.25 (3) Australian Patents Act, i.e. be made by handing out a sample to an expert.
  • Access to the deposited biological material should only be within the scope of the expert solutions, as provided for in R. 28 (4) EPUe or Regulation 3.25 (3) Australian Patents Act, i.e. be made by giving a sample to an expert.

Abstract

L'invention concerne de nouveaux micro-organismes qui peuvent transformer l'isopropylamine en L-alaninol, et dans lesquels les gènes ipuH et ipul, codant pour des enzymes impliquées dans la métabolisation du L-alaninol, sont inactivés. L'invention concerne en outre un procédé de production de L-alaninol ou de théanine au moyen de ces nouveaux micro-organismes.
PCT/EP2001/003651 2000-03-31 2001-03-30 Procede de production de l-alaninol par voie biotechnologique WO2001073038A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8344182B2 (en) 2007-12-20 2013-01-01 Basf Se Process for the preparation of (S)-2-amino-1-propanol (L-alaninol) from (S)-1-methoxy-2-propylamine

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NENP Non-entry into the national phase

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