WO2001073038A2

WO2001073038A2 - Method for the biotechnological production of l-alaninol

Info

Publication number: WO2001073038A2
Application number: PCT/EP2001/003651
Authority: WO
Inventors: Thomas Leisinger; Jan Van Der Ploeg; Andreas M. Kiener; Susana Ivone DE AZEVEDO WÄSCH; Tere Maire
Original assignee: Lonza Ag
Priority date: 2000-03-31
Filing date: 2001-03-30
Publication date: 2001-10-04
Also published as: AU4652101A; WO2001073038A3

Abstract

Disclosed are novel micro-organisms which are capable of transforming isopropylamine into L-alaninol and wherein the genes ipuH and ipul coding for enzymes involved in the metabolization of L-alaninol are deactivated. The invention also relates to a method for the production of L-alaninol or theanine using said novel micro-organisms.

Description

Process for the biotechnological production of L-alaninol

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 γ-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 γ-glutamylamides, in particular also for the production of theanine based on ethylamine ,

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. However, 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 γ-L-glutamylamides, with the high yields of L-alaninol or N -5-substituted γ-L-glutamylamides can be achieved.

This object is achieved with the microorganisms, nucleic acids and polypeptides according to the invention according to claims 1, 4, 10, 11, 12, 13 and 17 and with the methods according to claims 16 and 20. 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. Furthermore, 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. In the plasmids shown in FIGS. 4, 5 and 6, 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. In the plasmids shown in FIGS. 7, 8 and 9, 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.

2 shows the transposon insertions and the arrangement of the ipu genes from Pseudomonas sp. KIE171-BI (DSM 11629) and Pseudomonas sp. KIE171-BII (DSM 13389).

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 ρME4255. 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

11629) and Pseudomonas sp. KIE171-BIII (DSM 13177).

13 shows the substrate specificity of the γ-glutamylamide synthase.

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).

As described in WO 99/07199 by the same applicant, 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 ipuA, ipuB, ipuC, ipuD, ipuE, ipuF, ipuG, ipuH, ipul were isolated and the corresponding possible enzyme functions were assigned to them according to conventional comparison methods with known sequences, and these possible functions of the enzymes involved in the metabolism of IPA and L-alaninol (see Table 2) become a new degradation route for IPA proposed (Figure 1).

Depending on whether the starting MilαOorganisms as described above, 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. After transfer of the vector e.g. the conjugation 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.

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).

The 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. As already described above, 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. Herein, 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

6533 to 7960 the protein coding region of the ipuG gene and the nucleotide sequence from 8051 to 9571 the protein coding region of the ipuH gene.

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.

The genes ipuD, ipuE, ipuF, ipuG and ipuH are particularly preferred by the genes in SEQ ID No. 8 includes nucleotide sequence shown.

These 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,

Insertions and substitutions of bases or codons insofar as the biological function of the gene products of genes modified in this way remains essentially unchanged. 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.

At the level of the translation product, i.e. of the polypeptide or protein, the homology is preferably 95%.

The 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.

Depending on the type of vectors chosen, 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. Examples of 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). PBLUESCRIPT II KS + ®, pPDl 11 or its derivatives or pET28a (+) applied. All vectors which are suitable for Gram-negative bacteria can be used as “broad host rank” vectors.

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.

Based on such a vector, for example, the plasmid pME4755 was obtained.

A vector with a specific host spectrum is preferably used, particularly preferably pPDl 11, pBLUESCRIPT II KS + ^® or pET28a (+).

In this way, for example, the plasmids pME4254, pME4255, pME4256, pME4257, pME4259, pME4267, pME4275 and pME4277 were obtained.

Appropriate 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 DH5α and Escherichia coli XLl-Blue®.

Particularly preferred vectors are vectors such as plasmid pME4255 as deposited in E. coli DH5α (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).

The microorganisms of the species Escherichia coli DH5α (DSM 13178) and Escherichia coli XLl-Blue (DSM 13179), each containing plasmid pME4255 and pME4267, were released on December 3, 1999 at the German Collection for Microorganisms and

Zellkulturen GmbH, Mascheroderweg lb, D-38124 Braunschweig, deposited in accordance with the Budapest Treaty. The microorganism of the species Escherichia XLl-Blue (DSM 13417), containing plasmid pME4259, was deposited on March 31, 2000 with the German Collection for Microorganisms and Cell Cultures GmbH, Mascheroderweg lb, D-38124 Braunschweig, in accordance with the Budapest Treaty. The 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, which is a further subject of the present application, 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.

In the case of the use of the microorganisms according to the invention described above, in which the ipuH and ipul genes which code for enzymes which are involved in the metabolism of L-alaninol or which are generally not capable, 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. The 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.

After a customary reaction time of 1 to 100 h, preferably at most 30 h, 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 γ-glutamylamide synthetase activity which is capable of γ-glutamylamides of the general formula

or the general formula

wherein 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. Preferably 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.

An 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. Useful substituents on the alkyl group 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.

Examples of alkoxyalkyl 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.

As mentioned above, 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. Alternatively, 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 (+)

(Novagen) ligated and then transformed into competent cells of 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 γ-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 γ-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). In this way, plasmid pME4275 was obtained which contains the ipuC gene adjacent to a DNA sequence which codes for six histidines and which for the polypeptide γ-glutamylamide synthetase to which six histidine residues are attached (His ₆ day - γ- 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.

Microorganisms of the species E. coli BL21 (DE 3) (DSM 13180) containing plasmid pME4275 were deposited on December 3, 1999 at the German Collection for Microorganisms and Cell Cultures GmbH, Mascheroderweg lb, D-38124 Braunschweig, in accordance with the Budapest Treaty.

The polypeptide according to the invention with γ-glutamylamide synthetase activity which is capable of synthesizing γ-glutamylamides of the general formula I or II is preferably characterized by the following properties:

a) Substrate specificity for methylamine, ethylamine, ethanolamine, glycine methyl ester, propylamine, l-amino-2-propanol, 3-amino-l-propanol, isopropylamine, L-alaninol, D-alanol, 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 / or pentylamine.

b) Molecular weight of the monomer: 52478 Da

Another object of the invention is a process for the preparation of γ-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

NFL,

III l ¹

or the general formula

wherein 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.

Examples of 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.

In a preferred embodiment, 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.

After a customary reaction time of 1 to 20 h, γ-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. When growing the biomass or when using growing cells of a microorganism according to the invention as an additive to the biotransformation medium, 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. At the most preferred is 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).

In a further preferred embodiment, 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. Cells with low cell density, expediently with a cell density of OD650 = 5 to 20, most preferably with a cell density of approximately OD650 = 10, are preferably used for the production of theanine. In a particularly preferred embodiment, the concentration of ethylamine is 5-60 mM and is kept approximately constant during the biotransformation by repeated or continuous addition of ethylamine. For the purposes of the present application, theanine is N-5-ethyl-L-glutamine corresponding to formula I.

Examples:

example 1

Clarification of the ipu operon

Cloning of the ipu operon

Genes coding for proteins which are involved in IPA degradation were able to be determined thanks to the transposon mutants BI (Pseudomonas sp. KIE171-BI (DSM 11629)) and 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 genes of the mutants BI and BII, which were interrupted by the transposon insertion, were cloned by the 'transposon rescue' technique, and their sequence could be determined. For this purpose, the DNA fragments containing the inactivated gene were cloned into a suitable vector. The 'upstream' and 'downstream' regions of this insertion were sequenced. 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.

Construction of the plasmids The genes of the mutants BI and BII interrupted by the transposon insertion were cloned according to the 'transposon rescue' technique. 25 ml overnight cultures of mutants BI and BII were grown in Mimmal medium (MM) (WO 99/07199, Example 1, Table 1) in the presence of L-glutamate (20 mM) and with kanamycin (Km) (50 μg / ml) in a 50 ml bottle. 10 ml of this was centrifuged at 4000 g for 10 minutes. After removing the supernatant, the genomic DNA was isolated from the sediment. The precipitated genomic DNA was dissolved in 100 ul sterile water. The genomic DNA of the mutants BI and BII and the Vector pBluescript KS + and vector pPDl 11 with the restriction enzymes Xhol,

Sacl, or Notl and Xhol digested according to the usual protocol.

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 DH5α or competent E.coli XL-I blue cells are used.

The transformed cells were on LB plates with Km (50 ug / ml) or ampicillin

(Amp) (200 μg / ml) or chloramphenicol (Cm) (30 μg / ml) and incubated at 37 ° C. for 16 h. The colonies then obtained were tested for the presence of plasmids pME4254, pME4255, pME4256, pME4257, pME4259 and pME4267 (Table 1).

Table 1

Plasmid genotype or description

pME4254 8 kb Clal Insert from BI in pBluescript II KS +

pME4255 5.2-kb Notl-Xhol insert from BI in pBluescript II KS + pME4256 3 live Sfil Δ insert in pME4255

pME4257 18-23 leb, Notl-Xhol insert from BII in pBluescript II KS + pME4259 19-23 leb, Xhol insert from BII in pBluescript II KS + pME4267 8 leb Sacl insert from BII in pPDlII pME4755 ipuABCDEFG operon under control T7 promoter

In order to shorten the length of the DNA to be sequenced, 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. In order to place the ipuABCDEFG genes as an operon under the control of the T7 RNA polymerase promoter, they were cloned into pBBRIMCS (Kovach et al., Ibid.). First, 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. In order to add ipuA without the promoter, 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. Finally, 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.

sequencing

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.

Analysis of the DNA and protein sequence

The analysis of DNA sequences and protein sequences was carried out with the 'Genetics Computer Group Package Version 9'. The results are shown in Table 2. Based on these results, the degradation pathway of IPA, as described in Figure 1, is postulated. Table 2

Example 2

Production of the mutant BIII (Pseudomonas sp. KIE171-BIII (DSM 13177))

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. For this, pME4268 was digested with the restriction enzyme BαmHl 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 Smαl as a 855 bp fragment with blunt ends. The gentamycin resistance gene was then ligated into the linear pME4268 so that the SαlVSmαl insert has a length of 3.55 kb. The newly created plasmid is pME4269. After Sαl / Smαl digestion of the plasmid pME4269, the 3.55 kb fragment with the inactive ipuHv ^' α. recloned the vector pEXTCIS, also digested with Sαll / Smαl. Plasmid pEXTCIS cannot replicate in pseudomonads. The newly created plasmid pME4270 carries the resistance genes for Gm and tetracycline (Tc). The plasmid pME4270 was transformed into E. coli S17-λpir according to the CaCl ₂ method. S17-λpir are E.coli cells that are suitable for conjugation with the mutant BI.

E.coli S17-λpir, which contains the plasmid pME4270 with the gentamycin and tetracycline resistance gene, was conjugated with the mutant BI. The procedure as described in WO 99/07199, example 2b, was chosen. The differences are: E. coli S17-λpir was grown on LB at 37 ° C in the presence of antibiotics Gm (25 μg / ml) and Tc (35 μg / ml). The mutant BI was raised in 25 ml MM with L-glutamate (20 mM) and Km (50 μg / ml) at 30 ° C. 300 μl of the cell suspension were plated directly onto MM plates with L-glutamate (20 mM) and Gm (25 μg / ml) and incubated at 30 ° C. The colonies obtained were then tested for growth on MM plates with Tc (35 μg / ml). In the colonies that were able to grow in the presence of both antibiotics, there was a successful first crossover of the plasmid pME4270 into the chromosomal DNA of the mutant BI. One of these colonies was grown on MM liquid culture with L-glutamate (20 mM) and

Antibiotics Gm (25 μg / ml), Tc (35 μg / ml) and Km (50 μg / ml) were grown overnight at 30 ° C at 150 rpm. 300 μl 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 μg / 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 μg / ml) or Km (50 μg / ml) but not with Tc (35 μg / 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.

Example 3

Biotransformation of IPA to L-alaninol with the mutant BIII

A 25 ml overnight pre-culture of mutant BIII (strain DSM 13177) was used for the biotransformation of IPA to L-alaninol. This was grown on MM medium with L-glutamate (20 mM) and used to inoculate a culture of 250 ml in an 11 bottle of the same medium. After culturing BIII up to the beginning of the exponential phase (OD ₆₅₀ from 0.3 to 0.6), IPA (10 mM) was added. To

Reach an 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). With the three initial concentrations of IPA (20, 50 and 100 mM) after 58 h of biotransformation a final L-alaninol concentration of 8, 14.5 and 19 mM was reached. This corresponds to a molar yield of 40, 29 and 19%. There was no longer any degradation of L-alaninol. The course of the biotransformation of BIII at an initial concentration of 20 mM is shown in FIG. 12 compared to the biotransformation of BI.

Example 4

Manufacture of glutamyl isopropyl amides

Cloning the ipuC gene:

25 ml of an overnight culture of Pseudomonas sp. KLEI 71 (DSM 12360) in MM (WO 99/07199, Example 1. Table) in the presence of IPA (20 mM) at 30 ° C in a 50 ml bottle. 10 ml of this was centrifuged at 4000 g for 10 minutes. After removing the supernatant, the genomic DNA was isolated from the sediment. The precipitated genomic DNA was dissolved in 20 μl sterile water and quantified by measuring the absorption at 260 nm. The ipuC gene was generated from the genomic DNA of Pseudomonas sp. KLEI 71 (DSM 12360) amplified. For this, 2 ng / μl of genomic DNA, 0.6 μM of the primers 5'-AACAGGTGATACATATGAGCGAAG-3 'and 5'-TTTGAAGCTTAGGATCTGGGCG-3', 0.2 mM dNTP, 1.75 mM MgCl ₂ , Pfu buffer and 0.015 U / μl Pfu DNA polymerase were combined in one Final volume of 50 μl used. The 1.4 kb PCR product was cleaned and then digested with the enzymes Ndel and Hindlll and cleaned again. 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. Analogously, the digested 1.4 kb PCR product was ligated into the vector pET24a (+) (Novagen), which gave plasmid ρME4277. 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- γ-Glutamylamid synthetase. The protein Tag-γ-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'.

Overexpression and Purification of Tag-γ-Glutamylamide Synthetase (His ₆ -ipuCp)

An overnight preculture of E. coli BL21 (DE3) (DSM 13180) containing pME4275 was used for the production of pure day γ-glutamylamide synthetase. This was grown on 5 ml LB medium at 37 ° C with Km (50μg / 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.

To prepare the cell extract, the frozen sediment was resuspended in 4 ml binding buffer (containing 10 μg / 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 μm filter. Tag-γ-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 purification of day γ-glutamylamide synthetase was carried out in the following manner. 2.5 ml of the carrier material 'His ^» Bind Resin' were introduced into a column and first washed with 7.5 ml of sterile water. Then 12.5 ml of NiSO ₄ solution were run through and washed again with 7.5 ml of sterile water. 7.5 ml of binding buffer was used to set the flow rate to 25 ml per hour. The cell extract was loaded onto the column and this was mixed with 25 ml of binding Buffer and 15 ml wash buffer washed. The bound day γ-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 γ-glutamylamide synthetase is 52478 Da.

Biotransformation of ethylamine and L-glutamate to theanine with the protein Tag- γ-glutamylamide synthetase His ₆ -IpuCp)

For the biotransformation of ethylamine with L-glutamate to theanine, 10 mM ATP (pH 7), 10 mM IPA (pH 7), 10 mM L-glutamate (pH 7), 50 mM MgCl ₂ (pH 7), 50 mM imidazole (pH 7), 3.5 mM NaCl, 0.1 mM Tris-HCl (pH 7) and 57 ng / μl day-γ-glutamylamide synthetase in a final volume of 400 μl and allowed to react at 25 ° C. After a period of five hours, 8 mM theanine was formed. This corresponds to a molar yield of 80%.

Detection of theanine using HPLC

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. For this, 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.

Solvent: solution A 10 mM potassium phosphate buffer (pH 6.5)

Solution B 10 mM potassium phosphate buffer (pH 6.5) with 80% (v / v) methanol

Retention times: theanine 13.3 min

L-glutamate 8.2 min

Ethylamine 16.0 min

Detection of theanine using GC-MS Theanine could also be detected using GC-MS. The

The fragmentation pattern was identical to that of the reference compound.

Other γ-glutamylamide compounds formed

Instead of 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. The reaction of these compounds was carried out analogously to the above-described biotransformation of ethylamine and L-glutamate to theamine at pH 7 (the reaction of pentylamine was carried out at pH 8) and analyzed by measuring the inorganic phosphate formed (see. Fig. 12). The formation of new peaks of the possible γ-glutamylamide compounds was observed with HPLC.

Example 5 Biotransformation of ethylamine to theanine with mutant BIII

The biotransformation was carried out essentially analogously to Example 3 with the KLEI 71 -Bill strain (DSM 13177), with the difference that growing cells with low cell density (OD ₆₅₀ = 10) were used. In the presence of 20 mM glutamate and 5 mM isopropylamine, 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.

Example 6

Biotransformation of IPA to L-alaninol with an expression vector in E. coli.

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 µM (NH ₄ ) ₂ SO ₄ , 1% trace elements (Thurnheer et al., J. Gen. Microbiol. 132: 1215 ff, 1986) and 20 µg / 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 μM 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

Harvested at room temperature and added to a cell suspension with OD ₆₅₀ 50 in medium without chloramphenicol. After storage for 14 h at 4 ° C, 20 mM IPA (Isopropylamine) added and the culture shaken at 30 ° C (150 rpm). Product and starting material concentrations were determined using HPLC. After 22 h, the starting material IPA was found to be 13 mM and the product L-alaninol to 4 mM. After a total of 58 h, these values had changed only slightly. L-alaninol was not degraded by the E. coli expression strain.

BUDAPEST TREATY ON THE INTERNATIONAL

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⁴ Fill in if the infoimation has been requested and if the results of the test were negative.

Form DSMZ-BP / 9 (sole page) 0196 BUDAPEST TREATY ON THE INTERNAΗONAL

RECOGNΓΠON OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Lonza AG

Dept. of Biotechnology

CH-3930 Visp

RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Ruie 7.1 by the

INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

I. IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the DEPOSITOR. Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY.

DH5a / pME4255

DSM 13 178

II. SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I above was accompamed by

(X) a scientifio description

(X) a proposed taxonomic designation

(Mark with a cross where applicable).

III. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 1999 - 12 - 03 (Date of the original deposit) '

IV. RECEIPT OF REQUEST FOR CONVERSION

The 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)

V INTERNATIONAL DEPOSITARY AUTHORITY

MIKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authonzed official (s):

Address Mascheroder Weg lb D-38124 Braunschweig

Date- 1999-12-06

¹ Where Rule 64 (d) applies, such date is the date on which the Status of international depositary authority was acquired Form DSMZ-BP / 4 (sole page) 0196 BUDAPEST TREATY ON THE INTERNAΗONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Lonza AG

Dept. of Biotechnology

CH-3930 Visp

I. IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the DEPOSITOR: Accession numbcr given by the INTERNATIONAL DEPOSITARY AUTHORITY:

KIE171-BIII

DSM 13177

II. SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONO C DESIGNATION

The microorganism identified under I. above was accompanied by:

(X) a scientific description

(X) a proposed taxonomic designation

(Mark with a cross where applicable).

III. RECEIPT AND ACCEPTANCE

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 .

IV. RECEIPT OF REQUEST FOR CONVERSION

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-GERMAN COLLECTION OF Signature (s) of person (s) having the power to represeπt the MIKROORGANISMEN UND ZELLKULTUREN GmbH International Depositaiy Authority or of authorized official (s):

Address: Mascheroder Weg lb D-3812 Braunschweig

Date: 1999-12-06

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Lonza AG

Dept. of Biotechnology

CH-3930 Visp

I. DEPOSITOR II IDENTIFICATION OF THE MICROORGANISM

Name: Lo za AG Accession number given by the

Dept. of Biotechnology INTERNATIONAL DEPOSITARY AUTHORITY:

Address: DSM 13177

CH- 3930 Visp

Date of the deposit or the transfer ': 1999-12-12

III. VIABILITY STATEMENT

The viability of the microorganism identified under II above was tested on 1999 - 12 - 03 On that date, the said microorganism was

(X) ³ viable

(f no longer viable

IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED ⁴

V. INTERNATIONAL DEPOSITARY AUTHORITY

Address: Mascheroder Weg lb D-38124 Braunschweig

Date: 1999-12-06

Mark with a cross the applicable box.

Form DSMZ-BP / 9 (sole page) 0196 Applicant's or agent's for ^" International appl: inNo. Referencenumber LP. 1886

T DICATIONS RELATI G TO ADEPOSITED MICROORGANISM

(PCT Rule 136 »)

A. The indications made below relate to the microorganism referred to in the description on age §, left 32

B. DDENTIIilCATIONOFDEPOSrr Further deposits are identified on an additional sheet

Name of depositary institution

German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)

Address of depositary Institution (including postal code and country) Mascheroderweg 1b 38124 Braunschweig Germany

Date of deposit AccessionNumber

March 31, 2000 (March 31, 2000) DSM 13417

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This informationiscontinuedon an additional sheet | |

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.

Subject to the precise provisions of the EPUe or Patent Act, this regulation applies even after the patent application has been withdrawn or rejected or until the day on which the reference to the grant of the respective patent is published.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated states)

EP, AU

E. SEPARATE FURNISHING OF INDICATIONS (leaveblankifnotapplicable)

The indications listed below will be submitted to the International Bureau later (specißi the general nahire ofthe indications e.g., "Accession Nϊimber of Deposit")

For reeeiving Office use only For International Bureauuse only öj This sheet was received with the international application | | This sheetwas receivedby the International Bureau on:

Authorizedo ficer Authorizedofficer

•O. G rge

Form PC17RO / 134 (July 1992) 45 PfflEPfH / 0 3 6 5 1

Applicant's or'agent's: International appl> nNo. referencenumber LP. 1886

INDICATIONS RELATING TO ADEPOSITED MICROORGANISM

(PCT rule up)

A. The indications made below relate to the microorganism ref erred to in the description on page 10, line 32

B. TJ) ENTIFICATIONOFDEPOSΓΓ Further deposits are identified on an additional sheet

Name of depositary institution

German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)

Address of depositary institution (including postal code and country) Mascheroderweg 1 b 38124 Braunschweig Germany

Date of deposit AccessionNumber

03/24/2000 (March 24, 2000) DSM 13388

C ADDITIONAL INDICATIONS (leave blank if not applicable) This Information iscontinuedon an additional sheet _]

D. DESIGNATED STATES FOR WHICH INDIC TIONS ARE MADE (if the indications are not for all designated states)

EP, AU

E. SEPARATE FURNISHING OF INDICATIONS (leaveblankif not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature ofthe indications e.g., "Accession Number of Deposit")

For receiving office use only For International Bureau use only

This sheet was received with the international application ti I | This sheetwasreceivedbythelnternationalBureauon:

Authorized ofßcer Authorized officer

Form PCT / RO / 134 (July 1992)

INDICATIONS RE ATTNG TO ADEPOS1TED MICROORGANISM

(PCT Rule 136 «)

A. The indications made below relate to the microorganism referred to in the description on page 9, left 32

B. JBE TIFICATIONOFDEPOSΓΓ Further deposits are identified on an additional sheet ^ ζ

Name of depositary institution

German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)

Date of deposit AccessionNumber

March 31, 2000 (March 31, 2000) DSM 13180

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This Information iscontinuedon an additional sheet | |

EP, AU

E. SEPARATE FURNISHING OF INDICATIONS (leaveblankif not applicable)

The indications listed below will be submitted to the International Bureau later (specdy the general nature ofthe indications e.g., "Accession Number of Deposit")

For receiving Office use only For International Bureauuse only d Thisshe was received with the international application | I ThissheetwasreoeivedbythelnternationalBureauon:

Authorizedofficer Authorized ofScer

O. «worry

Form PCT / RO / 134 (y 1992)

INDICATIONS RELATING TO ADEPOSITED MICROORGANISM

(PCT Rule 13th)

A. The indications made below relate to the microorganism referred to in the descπption onpage 14, line 29

B. IDENTIFICATIONOFDEPOSΓΓ Further deposits are identified on an additional sheet

Name of depositaiy institution

German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)

Date of deposit AccessionNumber

December 3, 1999 (December 3, 1999) DSM 13180

C. ADDITIONAL INDICATIONS (leaveblankif not applicable) This information iscontinuedon an additional sheet [_j

Access to the deposited one! biological material should only be used within the framework of 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

D. DESIGNATΕD STATES FOR WHICH INDICATIONS ARE MADE

EP, AU

E. SEPARATE FURNISHING OF INDICATIONS (leaveblankif not applicable)

The indications listed below will be submitted to the International Bureau later (speajythegeneialnatureofthewώcationseg, "Accession Number of Deposit")

Forreceiving Office use only For International Bureauuse only

85 This sheet was received with the international apphcation | ] This sheet was recervedby the International Bureau on

Authorized officer Authorized officer

Ay Taorgθ

Form PCT RO / 134 (July 1992) PCDEPO 1/01 6 5 1

Applicant's or it goes International appl ^; No. referencenumber LP. 1886

INDICATIONS RELATING TO ADEPOSITED MICROORGANISM

(PCT Rule 13bis)

Applicant's or agetrt's F International appli riNo. PCT EP0 1 / f) fi reference number LP 1886

INDICATIONS RELATING TO ADEPOSITED MICROORGANISM

(PCT Rule ttbis)

A. The indications made below relate to the microorganism referred to in the descπption onpage? , lme? 8

B. π ENTTFlCATIONOFDEPOSrr Further deposits are identified on an additional sheet \) ζ \

Name of depositary institution

German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)

Date of deposit AccessionNumber

December 3, 1999 (December 3, 1999) DSM 13178

C. ADDITIONAL INDICATIONS (leaveblankif not applicable) This Information iscontinuedon an additional sheet | |

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.

EP, AU

E. SEPARATE FURNISHING OF INDICATIONS (leave blankif not applicable)

The indications listed below will be submitted to the International Bureau later (speay ie general nature ofthe indications eg., "Accession Number of Deposit")

For receivmg office use only For International Bureau use only

\ f \ This sheet was received with the international apphcation | l This sheet was received by the International Bureau on-

Authorized officer <s Authorized officer

Gorge

Form PCT / RO / 134 (y 1992)

INDICATIONS RE AT1NG TO ADEPOSITED MICROORGANISM

(PCT Rule 13όzs)

A. The indications made below relate to the microorganism ref erred to in the descnpbon on age, lme

B. IDENTIFICATIONOFDEPOSΓΓ Further deposits are identified on an additional sheet \) ζ \

Name of depositary institution

German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)

Address of depositary Institution (including postal code and country) Mascheroderweg 1 b 38124 Braunschweig I Deutschland

Date of deposit AccessionNumber

March 24, 2000 (March 24, 2000) DSM 13177

C. ADDITIONAL INDICATIONS (leave blank ifnot applicable) This Information iscontinuedon an additional sheet j

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

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (ifthe indications ai e not for all designated states)

EP, AU

E. SEPARATE FURNISHING OF INDICATIONS (leaveblankif not applicable)

The indications hsted below will be submitted to the International Bureau later (specißi ήe gaieral natwe ofthe indications eg, "Accession Number of Deposit")

Forreceiving Office use only For International Bureau use only

This sheet was received with the international app cation | | This sheet was received by the International Bureau on- tf

Authorized officer Authoπzed officer

O. Gorge

Form PCT RO / 134 (July 1992)

Claims

claims

1. Microorganisms which are capable of converting isopropylamine into L-alaninol, characterized in that the genes ipuH and ipul, which code for enzymes which are involved in the metabolism of L-alaninol, are inactivated and enzyme extracts from these microorganisms ,

2. Microorganisms according to claim 1 of the genus Pseudomonas.

3. Microorganisms according to claim 2 of the species Pseudomonas sp. corresponding to the species of the strain Pseudomonas sp. KIE171-BIII as deposited under DSM 13177 or their functionally equivalent mutants.

4. DNA fragment comprising one or more genes selected from the group comprising the genes ipuA, ipuB, ipuC, ipuD, ipuE, ipuF, ipuG, ipuH and ipul, these genes coding for enzymes which are involved in the metabolism of isopropylamine to L -Alaninol and / or the metabolism of L-alaninol are involved.

5. DNA fragment according to claim 4, characterized in that the genes

Microorganisms of the genus Pseudomonas are isolated.

6. DNA fragment according to claim 5, characterized in that the genes from microorganisms of the species Pseudomonas sp. KIE171 (DSM 12360), Pseudomonas sp. KIE171-BI (DSM 11629) and / or Pseudomonas sp. KIE171-

BII (DSM 13389) are isolated.

7. DNA fragment according to one of claims 4 to 6, characterized in that the genes ipuA, ipuB, ipuC, ipuD, ipuE, IpuF and ipuG are arranged in the order ipuA, ipuB, ipuC, ipuD, ipuE, ipuF, ipuG and as one

Transcription unit are available.

8. DNA fragment according to one of claims 4 to 7, characterized in that the genes present on the fragment ipuA, ipuB, ipuC, ipuD, ipuE, IpuF, ipuG and / or ipuH by the nucleotide sequence shown in Fig. 3 as well as their functionally equivalent genetic variants are included.

9. DNA fragment according to any one of claims 4 to 6, characterized in that the coding gene ipul present on the fragment by the in SEQ ID No. 1 nucleotide sequence shown and their functionally equivalent genetic variants is included.

10. Vector containing a DNA fragment according to one of claims 4 to 9, preferably a vector capable of expressing the ipu genes comprised by the DNA fragment.

11. Vector labeled pME4255 as deposited in E. coli DH5α (DSM

13178), vector with the designation ρME4267 (DSM 13179) as deposited in E. coli XLl-Blue and vector with the designation pME4259.

12. Vector labeled pME4275 as shown in E. coli BL21 (DE3) (DSM 13180).

13. Recombinant microorganism containing a vector according to any one of claims 10 to 12.

14. Recombinant microorganism according to claim 13 of the genus Escherichia.

15. Recombinant microorganism according to claim 14 of the species Escherichia coli.

16. A process for the preparation of L-alaninol, characterized in that isopropylamine by means of a microorganism as defined in the claims

Claims 1 to 3 or as defined in claims 10 to 15 or enzyme extracts from these microorganisms is converted to L-Alariinol. Polypeptide with γ-glutamylamide synthetase activity, which is capable, γ-glutamylamides of the general formula

or the general formula

wherein 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.

Polypeptide according to claim 17, characterized in that it follows

Features: a) substrate specificity for methylamine, ethylamine, ethanolamine, glycine methyl ester, propylamine, 1-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 / or pentylamine. b) Molecular weight of the monomer: 52478 Da

19. Polypeptide according to claim 17 or 18 obtainable from microorganisms of the genus

Pseudomonas, preferably the translation product of the ipuC gene sequence shown by SEQ ID No.6 and at least 95% homologous, functionally equivalent protein sequences.

20. A process for the preparation of γ-glutamylamides of the general formula I or II, characterized in that L-glutamate with an amine of the general formula

or the general formula

wherein R ¹ and R ^{2 have} the meaning given, by means of a microorganism according to Claims 1 to 3 or by means of a microorganism expressing the ipuC gene product according to Claims 10 to 12, by means of enzyme extracts from these microorganisms or by means of a polypeptide according to Claims 17 to 19, is converted to the product of the general formula I or II.

21. The method according to claim 20, characterized in that the reaction is carried out at a pH of 4 to 9 and a temperature of 10 to 50 ° C.

22. The method according to claim 20 or 21, characterized in that the

Implementation is effected by means of a polypeptide and the polypeptide is a tagged protein.

23. The method according to claim 20 or 21, characterized in that the

Implementation is carried out by means of microorganisms of the genus Pseudomonas as defined in claim 1, preferably with microorganisms of the strain KIE 171-BIII as deposited under DSM 13177.

24. The method according to claim 20 or 21, characterized in that the

Implementation is carried out with L-glutamate and ethylamine and the produlct is theanine.