MXPA00012041A - Process for the fermentative production of l-amino acids employing coryneform bacteria in which the glya gene activity has been decreased - Google Patents

Abstract

Production of L-amino acids (A) by fermenting an (A)-producing coryneform bacterium in which the glyA (serine hydroxymethyltransferase) gene (I) has been suppressed;enriching (A) in cells or medium, then recovering it. Independent claims are also included for the following:(1) coryneform bacteria in which (I) has been suppressed;(2) the vector pK18mobglyA'(map reproduced), deposited in Escherichia coli as DSM 13170;and (3) isolated polynucleotide (II) selected from:(a) the lacI-tac-5'-glyA unit (2000 bp sequence (S2), defined in the specification) or the lacI-tac-glyA unit (2866 bp sequence (3), defined in the specification);(b) an equivalent of (a) within the degeneracy of the genetic code;(c) a polynucleotide that hybridizes to the complement of (a) or (b);or (d) a polynucleotide that is a functionally neutral sense mutant of (S2) or (S3).

Description

PROCEDURE FOR THE PREPARATION BY FERMENTATION OF L-.AMINOACIDES USING CORINEFORM BACTERIA SUMMARY OF THE INVENTION The invention relates to a process for the preparation of L-amino acids, in which the following steps are carried out: a) fermentation of the coryneproducer bacteria producing of L-amino acids, in which at least the glyA gene is weakened, in particular by means of the separation of the natural promoter, b) enrichment of the desired product in the medium or in the cells of the bacteria, and c) isolated from the L -aminoacids, and eventually bacteria are used in which additional genes of the biosynthetic pathway of the desired L-amino acids are reinforced, or bacteria are used in which at least the metabolic pathways that reduce the formation of the L- are partially deactivated desired amino acids, and the nucleotide sequences of lacI-tac-5'glyA or the lacI-tac-glyA unit.

PROCEDURE FOR THE PREPARATION BY FERMENTATION OF L-AMINOACIDES USING CORINEFORM BACTERIA DESCRIPTION OF THE INVENTION The object of the invention is a process for the preparation by fermentation of L-amino acids using coryneform bacteria, in which the glyA gene is weakened. STATE OF THE ART L-amino acids are used in animal feed, in human medicine and in the pharmaceutical industry. It is known that these amino acids are produced by fermentation of strains of coryneform bacteria, especially with Corynebacterium glutamicum. Due to the great importance of this group of products, we continuously work to improve these preparation procedures. Process improvements may consist of technical fermentation measures such as agitation and oxygen feed, or the composition of nutritive media such as sugar concentration during fermentation, or post-processing to the form of a product, by means of, for example, ion exchange chromatography or by intrinsic properties of microorganisms. To improve the performance properties of these microorganisms, methods such as mutagenesis, ref: 124576 selection and selection of mutants are used. In this way strains are obtained which are resistant against antimetabolites such as, for example, the threonine analog o.-amino-β-hydroxylaleric acid (AHV) or auxotropes for the regulatory-important amino acids and which produce L-amino acids, such as, for example, threonine . For several years, methods of DNA recombination technique have been used for the improvement of strains of strains that produce L-amino acids of Corynebacterium glutamicum, in which individual biosynthesis genes are amplified and their effect on the production of the L-amino acids. An article on this is among others that of Konoshita ("Glutamicum Acid Bacteria" in Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Jetten and Sunskey (Critical Reviews in Biotechnology 15, 73-.103 (1995)) and Sahm et al., (Annuals of the New York Academy of Science 782, 23-39 (1996)) . Task of the Invention The inventors set themselves the task of obtaining new measures to improve the preparation by fermentation of amino acids with coryneform bacteria. Description of the Invention Amino acids are used in human medicine, in the pharmaceutical industry and especially in animal feed. There is therefore a general interest in a new and improved process for the production of amino acids. When L-amino acids are mentioned below, they involve L-threonine or L-isoleucine. The object of the invention is therefore a process for the preparation by fermentation of L-amino acids using coryneform bacteria which in particular already produce the corresponding L-amino acids and in which the nucleotide sequence (glyA gene) coding for the product is weakened genetic glyA, especially expressed at a low level, the desired product is enriched in the medium or in the cells and the L-amino acid is isolated. The strains used preferably already produce L-amino acids before the weakening of the glyA gene. Preferred embodiments are found in the claims. The concept "weakening" describes in this respect the increase in the intracellular activity of one or several enzymes (protein) in a microorganism, which are encoded by the corresponding DNA (here the glyA gene), for which a promoter is used for example , gene or weak allele that encodes the corresponding enzyme with a lower activity, or inactivates the corresponding gene or enzyme (protein) and eventually combine those measurements.

The microorganisms that are the object of the present invention can produce amino acids from glucose, sucrose, lactose, fructose, maltose, melase, starches, cellulose or glycerin and ethanol. It may be a representative of coryneform bacteria, especially of the genus Corynebacterium. In the genus Corynebacterium, mention should be made in particular of the type Corynebacterium gl utamicum, which is known in the art for its ability to produce L-amino acids. Suitable especially of the genus Corynebacterium Corynebacterium glutamicum type strains, are for example the known wild type strains Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium melassecola ATCC17965 Corynebacterium thermoaminogenes FERM BP-1539 Brevibacterium flavum ATCC13869 ATCCC14067 Brevibacterium lactofermentum ATCC14020 and Brevibacterium divarica.tum and mutants or strains prepared from them that produce L-amino acids', such as for example the strains producing L-threonine Corynebacterium glutamicum ATCC21649 Brevibacterium flavum BB69 Brevibacterium flavum DSM5399 Brevibacterium lactofermentum FERM-BP 269, Brevibacterium lactofermentum TBB-10 and, for example, the strains producing L-isoleucine Corynebacter? Um glutamicum ATCC 14309 Corynebacterium glutamicum ATCC 14310 Corynebacterium glutamicum ATCC 14311 Corynebacterium glutamicum ATCC 15168 Corynebacterium ammoniagenes ATCC 6871. It was found that coryneform bacteria after weakening of the glyA gene produce L-amino acids in improved form. The glyA gene encodes the enzyme serine hydroxymethyltransferase (EC 2.1.2.1). The nucleotide sequence of the glyA gene was described in Japanese publication JP-A-08107788. He . The glyA gene described in the aforementioned texts can be used according to the invention. Furthermore, alleles of the glyA gene can be used, which is obtained through the degeneration of the genetic code or through sense mutations of neutral function. To obtain a weakening, the expression of the glyA gene or the catalytic properties of the gene product can be reduced or deactivated. Eventually both measures can be combined. The reduction of gene expression can be carried out by means of carrying out suitable culture or by means of genetic modification (mutation) of the signal structures of the gene expression. The signal structures of the gene expression are for example repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and the terminals. Data on this is found by the technician, for example, in patent application WO 96/15246, by Boyd and Murphy (Journal of Bacteriology 170: 5949 (1088), by Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998), by Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), by Patek et al. (Microbiology 142: 1297 (1996) and in well-known texts of genetics and molecular biology, as for example the text of Knippers ("Molecular Genetics", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or Winnacker ("Genes and Clones", VCH Verlagsgesellschaft, Weinheim, Germany, 1990). The mutations that lead to a modification or reduction of the catalytic properties of the enzymatic proteins are known from the state of the art; as examples, the works of Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Mdckel ("Corynebacterium glutamicum threonine hydratase: preservation of the alostric regulation and structure of the enzyme", Jülich Research Center, Jül-2906 , ISSN09442952, Jülich, Germany, 1994). Abstracts can be found in well-known textbooks on genetics and molecular biology, such as von Hagemann's ("General genetics", Gustav Fischer Verlag, Sttutgart, 1986). Transitions, transversions, insertions and deletions are considered as mutations. Depending on the effect of the amino acid exchange on enzymatic activity, there is talk of missense mutations or nonsense mutations. The insertions or deletions of at least one pair of bases in a gene lead to mutations by shift of the labeling (frame shift mutations) that as a consequence false amino acids are formed or interrupt the translation permanently. Deletions of multiple codons typically lead to a complete reduction of enzyme activity. Instructions to achieve such mutations belong to the state of the art and can be obtained from known texts on genetics and molecular biology, such as the text of Knippers ("Molecular Genetics", 6th edition, Georg Thieme Verlag, Stuttgar, Germany, 1995) or by Winnacker ("Genes and Clones", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or "Hagemann's (" General Genetics "; Gustav Fischer Verlag, Stuttgart, 1986). For example, the glyA gene was weakened by the removal of the natural promoters and the binding of an upstream regulating control element. The lacl-tac system was used as a control element. To achieve the introduction of the lacl-tac system upstream of the chromosomal gene glyA, the integration plasmid pKldmobglyA 'was prepared (figure 1). Plasmid pKldmonglyA "contains the tac promoter (Amann et al., Gene 25: 167-178 (1983); de Boer et al., Proceedings of the National Academy of Sciences of the United States of America USA 80: 21-25 ( 1983)) and directly downstream of the promoter tac a 5 'terminal sequence of the glyA gene represented in SEQ ID No. 1. The plasmid also contains the lacl gene encoding the Lac inhibitor (Farabaugh, Nature 274;: 765-769 (1978) Stark et al., Gene 51: 255-267 (1987).) The sequence of the lacI-tac-5'glyA unit is represented in SEQ ID No. 2. Plasmid pKldmobglyA 'can be replicated in Escherichia coli but not in Corynebacterium glutamicum After the transformation and homologous recombination by means of a process of "cross-over" of integration effect an intact copy of the glyA gene is obtained, whose expression can be controlled or regulated by means of the element of lacl-tac control placed upstream and an inactive copy truncated at the 3 'terminal of the gly gene A of the natural promoter. The sequence of the lacI-tac-glyA unit is represented in SEQ. ID. No. 3. SEQ. ID. No. 4 shows the known amino acid sequence of the genetic product glyA. By addition of suitable concentrations of the lactose analogue isopropylthiogalactoside (Fürste et al., Gene 48, 119-131 (1986)) the expression of the glyA gene can be controlled or the cellular content of serine hydroxymethyltransferase can be weakened or adjusted. Other indications and clarifications about integration mutagenesis are given by Schwarzer and Pühler (Bio / Technology 9, 84-87 (991)), Peters-Wendisch et al., (Microbiology 144, 915-927 (1998)) or Fitzpatrick et al.

(Applied Microbiology Biotechnology 42, 575-580 (1994)). An example of an amino acid-producing strain of coryneform bacteria with the weakened glyaA gene is that of Corynebacterium gl utamicum DM368-2:: pK18mobglyA 'producing threonine. Additionally, it may be advantageous for the production of amino acids, in addition to the weakening of the glyA gene, to reinforce one or more enzymes of the biosynthetic pathway, of glycolysis, the anapleoric, the citric acid cycle or the export of amino acids.

Thus for example for the preparation of L-threonine, the homing gene can be simultaneously over-expressed to homoserine dihydrogenase (Peoples et al., Molecular Microbiology 2, 63-72 (1988)) or the homofer allele encoding the dihydrogenase to "feedback-resistant" homoserin (Archer et al., Gene 107, 53-59 (1991)) and / or • the gap gene encoding glyceraldehyde 3-phosphate dihydrogenase (Eikmanns (1992). Bacteriology 174: 6076-6086), or • the pyc gene encoding pyruvate carboxylase (Peters-Wendisch et al., Microbiology 144: 915-927 (1998)), or • the mqo gene encoding malate oxidoreductase: quinone (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)), or • the thrE gene coding for the export of threonine (DE 199 41 478.5, DSM 12840). It can also be advantageous for the production of amino acids, in addition to the glyA gene, simultaneously weaken • the pck gene encoding phosphoenolpyruvate carboquinase (DE 199 50 409.1; DSM 13047) and / or • the poxB gene coding for pyruvate oxidase (DE 199 51 975.7; DSM 13114) can also be advantageous for the production of amino acids corresponding to the weakening of the glyA gene, avoid unwanted side reactions (Nakayama: "Breeding of .Amino Acid Producing Micro-organisms", in: Overpoduction of Microbial Products, Kru phanzl, Sikytam Vanek (eds.); Academic Press, London, UK, 1982). The culture medium used must suitably be sufficient for the requirements of the microorganisms in question. The description of culture media of different microorganisms can be found in the manual "Manual of Methods for General Bacteriology" of the American Society of Bacteriology (Washington D.C., US, 1981). As a carbon source, sugars and carbohydrates can be used, such as glucose, sucrose, lactose, fructose, maltose, melase, starches and cellulose, oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil. fatty acids such as palmitic acid, stearinic acid and linoleic acid, alcohols such as for example glycerin and ethanol and organic acids such as acetic acid. These substances can be used individually or in mixtures. Nitrogen-containing organic compounds such as peptone, yeast extract, meat extract, malt extract, corn steep water, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, phosphate can be used as nitrogen source. of ammonium, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used alone or as mixtures. Phosphoric acid, potassium dihydrophosphate or dipotassium hydrophosphate or the corresponding sodium-containing salts can be used as phosphorus sources. The culture medium must also contain salts of cerium metals, for example magnesium sulfate or iron, which are necessary for growth. Finally, growth promoters such as amino acids and vitamins can be used in addition to the mentioned substances. Suitable pre-stages can be added to the culture medium. The additives mentioned can be added to the culture in the form of a single charge or in a suitable form added during cultivation. To control the pH of the crop, basic compounds such as sodium hydroxide are used, potassium hydroxide, ammonia or acidic compounds such as phosphoric acid or sulfuric acid in suitable form. To control foaming, anti-foaming agents such as fatty acid polyglycol ester or silicone oils can be used. To maintain the stability of the plasmids, selective substances, for example antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or gaseous mixtures containing oxygen, such as air, are introduced into the culture. The culture temperature is usually at 20 ° C to 45 ° C and preferably at 25 ° C to 40 ° C. The culture is continued until a maximum amount of L-amino acid has been formed. This objective is normally reached in the course of 10 to 160 hours. Methods for the determination of L-amino acids are known in the state of the art. The analysis can be performed as described by Spackman et al. (Analytical Chemistry, 30, 1190 (1958), 1190), by means of anion exchange chromatography with the subsequent derivatization or can be performed by reverse phase CLAP, as described by Lindroth et al. (Analytical Cemistry (1979) 51: 1167-1174). The following microorganisms were deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty: • Strain DH5oí / pK18mobglyA 'of Escherichia coli as DSM 13170. Examples The present invention will be explained in more detail with the help of the examples of realization. The isolate of the Escherichia coli plasmid DNA as well as all the techniques on restriction treatment with Klenow phosphatase and alkaline were performed according to Sambrook et al. (Molecular Cloning, A laboratory manual (1989) Cold Spring Harbor Laboratory Press). The transformation of Escherichia coli, when not described-something else, was done according to Chung et al. (Proceedings of the National Academy of Sciences of the United States of America USA (1989) 86: 2172-2175). Example 1 Cloning and sequencing of the glyA gene from Corynebacterium glutamicum ATCC13032 The glyA gene was cloned into the cloning vector of E. coli pUC18 (Norrander et al., Gene (1983) 26: 101-106, Roche diagnostics, Mannheim , Germany) . The cloning was done in two steps. First by means of a polymerase chain reaction (PCR) the gene was amplified Corynebacterium glutamicum ATCC13032 by means of the following oligonucleotide primer derived from the Japanese publication JP-A-0810778d. glyAl-direct: 5 '-GCT TGC AGC GTT TTG CTC TGC C-3' glyAl-inverse: 5 '-ACC CGT AAC CTC TTC CAC ATA GG-3"The PCR reaction was performed in 30 cycles in the presence of 200LTM of deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP), each 1 Dm of the corresponding oligonucleotide, 100 ng of chromosomal DNA of Corynebacterium glutamicum ATCC13032, 1/10 volume of reaction buffer lOx, and 2.6 units of a heat stable mixture of Tag / Pwo DNA polymerase (Expand High Fidelity PCTR System from Roche Diagnostics, Mannheim, Germany) in a thermocycle (PTC-100, MJ Research, Inc., Watertown, US) under the following conditions: 94 ° C for 30 seconds, 64 ° C for 1 minutes and 68 ° C for 3 minutes The amplified fragment of approximately 1.7 kb was then ligated with the help of the SureClone Ligation Kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's data at the cut point Smal of the pUCld vector. The E. coli strain DH5amcr (Grant et al., Proceedings of the National Academy of Sciences of the United States of America, US (1990) 87: 4645-4649) was transformed. The transformants were identified with the aid of their resistance to carbencillin on LB agar plates containing 50 μg / mL carbencillin. Plasmids were prepared from the 7 transformants and the presence of the 1.7 kb PCR fragment as an insert was verified by restriction analysis. The recombinant plasmid thus formed is referred to below as pUCldglyA. The nucleotide sequence of the 1.7 kb PCR fragment in the pUCldglyA plasmid was determined according to the dideoxy chain interruption method of Sanger et al.

(Proceedings of the National Academy of Sciences of the United states of .America US (1977) 74: 5463-5467). For this, the entire pUCldglyA insert was sequenced with the help of the next primer. Universal primer: 5 '-GTA AAA CGA CGG CCA-GT-3' Reverse primer: 5 '-GGA AAC AGC TAT GAC CAT G-3' The obtained nucleotide sequences were analyzed with the programming package Lasergene (Biocomputing Software for Windows, DNASTAR, Madison, US). The analysis gave the identification of an open reading marker of 1302 bp in length. The corresponding gene was designated as the glyA gene. The corresponding genetic product covers 434 amino acids and is represented in SEQ. ID. do not. 4. Example 2 Construction of a vector for the reduced expression of glyA The plasmid described in example 1 pUCldglyA was cut with the restriction enzymes EcoRI and Tfil a DNA fragment of 1418 bp in length, which contained the gel glyA without zone own promoter. The 5 'and 3' ends of these fragments were treated with Klenow enzyme. The resulting DNA fragment was thus linearized with BamHI, ligated with the dephosphorylated vector pVWEx2 (Wendisch, "Physiological and NMR spectroscopy investigations on the in vivo activity of the central metabolic pathways in the active strain and in recombinant strains of Corynebacterium glutamicum. ", Jülich Research Center report, Jül-3397, ISSN09442952, Jülich, Germany, 1997), in such a way that the glyA gene is found with the same orientation directly behind the tac promoter inducible with isopropyl-β-D- thiogalactoside (IPTG) of the vector. The E.coli strain DHdoancr (Grant et al., Proceedings of the National Academy of Sciences of the United States of America (1990) 87: 4645-4649) was transformed with the whole binding product. Transformants were identified with the help of their resistance to tetracycline on LB agar plates containing 15 μg / mL tetracycline. Of the 12 transformants plasmids were prepared and by means of restriction analysis the presence of the 1418 bp fragment was verified as an insert in the correct orientation in relation to the tac promoter. The recombinant plasmid thus obtained is referred to below as pVWEx2glyA. From the pVWEx2glyA plasmid then by means of polymerase chain reaction (PCR) by means of the following oligonucleotide primer a DNA fragment was amplified, which contained lacl, the gene for the repressor of the tac promoter, the tac promoter and the first 438 bp of the cloned glyA gene of Corynebacterium glutamicum. glyA-direct (with EcoRI recognition sequence included, marked with underline:) 5 '-CCG GAA TTC CTG CCC GCT TTC CAG TC-3' glyA-inverse (with BamHl recognition sequence included, marked with underline:) 5 ' -CGG GAT CCC AGC TTT CCG GAG AAG TTC AAC-3 'The PCR reaction was performed in 30 cycles in the presence of 200μM of deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP), each 1 μm of the corresponding oligonucleotide, 100 ng plasmid DNA of pVWEx21glyA 1/10 volume of reaction buffer lOx, and 2.6 units of a heat stable mixture of DNA polymerase Tag / Pwo (Expand High Fidelity PCTR System by Roche Diagnostics, Mannheim, Germany) in a thermocycle (PTC-100, MJ Research, Inc., Watertown, US) under the following conditions: 94 ° C for 30 seconds, 58 ° C for 30 seconds and 72 ° C for 2 minutes. The amplified fragment of approximately 2.0 kb was then digested with EcoRI and BamHl, with the help of the NucleoSpin Extract 2 in a Macherey-Nagel signature equipment (Duren, Germany) according to the manufacturer's data and then also ligated in the vector cut with EcoRI and BamHl and dephosphorylated pK18mbo (Scháfer et al., Gene (1994) 145: 69-73). The E.coli strain DH5amcr (Grant et al., Proceedings of the National Academy of Sciences of the United States of America, (1990) 87: 4645-4649) was transformed with the whole binding product. Transformants were identified with the aid of their resistance to kanamycin on LB agar plates containing 50 μg / mL kanamycin. Plasmids were prepared from the 12 transformants and by means of restriction analysis the presence of the 2.0 kb fragment was verified as an insert. The recombinant plasmid thus obtained is referred to below as pK18mobglyAr (see Figure 1). Example 3 Construction of the Corynebacterium glutamicum strain ATCC13032:: pK18mobglyA 'with reduced and adjustable glyA expression By means of electroporation (Haynes 1989, FEMS Microbiology Letters 61: 329-334) the empty vector pZl (Menkel et al. , Applied and Environmental Microbiology 81989) 64: 549-554) and the plasmid pK18mobglyA 'described in Example 2 in the native strain Corynebacterium gl utamicum ATCC 13032 (Abe et al., Journal of General and Applied Microbiology (1967) 13: 279-301). After transformation with pZ1 transformants were identified with the aid of their kanamycin resistance on LBHIS agar plates containing 15 μg / mL kanamycin (Liebl et al., FEMS Microbiology Letters (1989) 65: 299-304) . Plasmids were prepared from the 3 transformants and the presence of the empty pZl vector was verified by means of restriction analysis. In this way, the control strain of Corynebacterium glutamicum ATCC13032 / pZ1 was formed. After transformation with pKldmobglyA 'the plasmid had to be integrated by homologous recombination of the 5' cloned end of glyA into the chromosome of Corynebacterium glutamicum ATCC13032.- The kanamycin-resistant clones obtained were identified on LBHIS agar plates containing 15 μg / mL kanamycin and 1 mM isopropyl-β-thiogalactosidase (IPTG) (Liebl et al., FEMS Microbiology Letters (1989) 65: 299-304). The correct integration of pK18mobglyA 'into the chromosome was verified in 2 integration mutants obtained by means of a polymerase chain reaction (PCR) by means of the following oligonucleotide primer. reverse primer (RSP): 5 '-GGA AAC AGC TAT GAC CAT G-3' glyA2-inverse: 5 * -CGG GAT CCC AGC TTT CCG GAG AAG TTC AAC-3 'The PCR reaction was performed in 30 cycles in the presence of 200μM of deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP), each 1 μm of the corresponding oligonucleotide, 100 ng of chromosomal DNA of Corynebacterium glutamicum ATCC13032:: pK18mobglyA ', 1/10, 1/10 volume of reaction buffer lOx , and 2.6 units of a heat stable mixture of Tag / Pwo DNA polymerase (Expand High Fidelity PCTR System from Roche Diagnostics, Mannheim, Germany) in a thermocycle (PTC-100, MJ Research, Inc., Watertown, US ) under the following conditions: 94 ° C for 30 seconds, 40 ° C for 30 seconds and 72 ° C for 2 minutes. In this manner, the strain of Corynebacterium glutamicum ATCC13032:: pK18mobglyA 'was formed, in which the glyA gene is under the control of the tac promoter inducible with isopropyl-β-D-thiogalactosidase (IPTG). Example 4 Determination of the serine hydroxymethyltransferase activity encoded by the glyA gene in the strain of Corynebacterium glutamicum ATCC13032:: pK18mobglyA * To obtain the crude extracts for the determination of the serine hydroxymethyltransferase activity encoded by the glyA gene The strains described in example 3 of C. glutamicum ATCC13032 / pZ1 and C. glutamicum ATCC13032:: pK18mobglyA 'in 100 ml of brain heart infusion medium (Difco Laboratories, Detroit, US) with 25 μg / ml kanamycin were precultured. and 100 μM isopropyl-β-D-thiogalactosidase (IPTG) for 14 hours at ° C. The cells were then washed once with 0.9% (w / v) of sodium chloride solution and with this suspension, 100 ml of CgXII medium were inoculated, so that OD600 (optical density at 600 nm) was 0.5. The medium was identical to the medium described by Keilhauer et al. (Journal of Bacteriology (1993) 175: 5593-5603), additionally containing 25 μg kanamycin / ml and 0, 10 or 100 μM isopropyl-β-D-thiogalactosidase (IPTG). The composition of the medium described by Keilhauer et al. it is represented in table 1. Table 1 Composition of the CGXII medium The culture of both strains is reacted C. After 10 hours the cells were washed once with 50 mM of 4- (2-hydroxyethyl) -1-piperazinetansulfonic acid / sodium hydroxide buffer (pH 7.0), centrifuged ( 10 minutes at 5000 revolutions per minute with a Minifuge RF from Heraeus, Osterode, Germany) and resuspended in 200 mM of 4- (2-hydroxyethyl) -1-piperazinetansulfonic acid / sodium hydroxide buffer (pH 7.90), in such a way that the final volume was 5 ml. To this cell suspension was added 50 μl of a 2mM solution of pyridoxal 5-phosphate and 50 μl of 100mM dithiothreitol solution and the cells were opened. The cell opening was performed at 0 ° C by means of an ultrasound disintegrator (Branson Sonifier W-250, Branson Sonic Power CO. Danbury, US; duration of sonification 6 minutes, pulse length 100%, intensity of sonification 2.5). After the ultrasound treatment, the cell debris was separated by means of centrifugation (30 minutes at 4 ° C and 13,000 rpm in a refrigerated Sigma 202 MK centrifuge from Sigma-Aldrich, Deisenhofen, Germany). The residue was used as cell-free crude extract directly to determine the enzymatic activity. The determination of the proteins in cell-free crude extracts was performed photometrically according to Bensadoun and Weinstein (Analytical Biochemistry (1976) 70: 241-250). The protein content was determined through a curve formed with bovine serum albumin as standard. To determine the activity of the serine hydroxymethyltransferase in cell-free crude extracts, a discontinuous enzyme test was used, with which the glycine formed from the threonine substrate was quantified. The reaction product was incubated with the following composition (modified according to Scrimgeour and Huennekens, Methods in Enzymology (1962), vol.V: 83d-843, Academic Press) for 15 minutes at 37 ° C; 20mM threonine, 200μM pyridoxal-5-phosphate, 900μM tetrahydrofolate, 100mM 4- (2-hydroxyethyl) -1-piperazinetansulfonic acid / sodium hydroxide buffer (pH 7.0) and 1.0-1.5 mg protein (from extract crude) in a final volume of 1 ml. The reaction was stopped by the addition of 0.25 volume 25% (w / v) trichloroacetic acid solution. The product was incubated for 15 minutes at 0 ° C and the denatured protein was centrifuged (15 minutes at 4 ° C and 13,000 rpm in a refrigerated Sigma 202 MK centrifuge from Sigma-Aldrich, Deisenhofen, Germany). The quantitative determination of the glycine formed in the enzymatic test as a residue was carried out by reverse phase CLAP (Lindroth et al., Analytical Chemistry (1979) 51: 1167-1174). An apparatus for CLAP of the HP1100 series (Hewlett-Packard, Waldbronn, Germany) equipped with fluorescence detector (G1321A) was used; The control of the system and the evaluation of the data was carried out with an HP-Chem-Station (Hewlett-Packard). 1 μl of the amino acid solution to be analyzed was mixed in an automatic previous column derivatization with 20 μL of ortho-fatalaldehyde / 2-mercaptoethanol ready-to-use reagent (Pierce Europe BV, Uid-Beijerland, The Netherlands). The fluorescent thiosubstituted isoindoles thus formed (Jones et al., Journal of Chro atography (1983) 266: 471-482) were separated through a combined precolumn (40x4 mm Hypersil ODS 5) and a main column (Hypersil ODS 5, both columns of the firm CS-Chromatographie Service Gm, bH, Langerwehe, Germany) with a gradient program with a non-polar increasing phase (methanol). The polar eluent was sodium acetate (0.1 molar, pH 7.2); the flow rate was 0.8 ml per minute. The fluorescence detection of the derivatized amino acids was performed at an excitation wavelength of 230 nm and an emission wavelength of 450 nm. Glycine concentrations were calculated through a comparison with an external standard and asparagine as an additional internal standard. The results of the enzymatic test with threonine as a substrate are given in Table 2. Table 2: Example 5 Construction of strain Brevibacterium flavum DM368-2:: pK18mobglyA 'with reduced and adjustable expression of glyA By means of electroporation (Haynes 1989, FEMS Microbiology Letters 61: 329-334) the empty pZl vector was introduced (Menkel et al. ., Applied and Environmental Microbiology 81989) 64: 549-554) and plasmid pKldmobglyA 'described in Example 2 in the threonine-forming strain Brevibacterium flavum DM368-2. Strain DM368-2 is described in EP-B-0 385 940 and is deposited as DSM5399. After transformation with pZ1 transformants were identified with the aid of their kanamycin resistance on LBHIS agar plates containing 15 μg / mL kanamycin (Liebl et al., FEMS Microbiology Letters (1989) 65: 299-304) . Plasmids were prepared from the 3 transformants and the presence of the empty pZl vector was verified by means of restriction analysis. In this way, the control strain was formed Brevibacterium flavum DM368-2 / pZl. After transformation with pK18mobglyA 'the plasmid had to be integrated by homologous recombination of the 5' cloned end of glyA into the chromosome of Brevibacterium flavum DM368-2 / pZl. The obtained kanamycin-resistant clones were identified on LBHIS agar plates containing 15 μg / mL kanamycin and 1 mM isopropyl-β-thiogalactosidase (IPTG) (Liebl et al., FEMS Microbiology Letters (19d9) 65: 299-304 ). The correct integration of pKldmobglyA 'in the chromosome was verified in 4 integration mutants obtained by means of a polymerase chain reaction (PCR) as already described in example 3, with 100 ng of chromosomal DNA of Brevibacterium flavum DM368- 26 2:: pK18mobglyA 'as a template. In this manner, the strain Brevibacterium flavum DM366-2 was formed:: pK18mobglyA *, in which the glyA gene under the control of the tac promoter is inducible with isopropyl-β-thiogalactosidase Example 6 Determination of the serine hydroxymethyltransferase activity encoded by glyA in strain Brevibacterium flavum DM368-2:: pK18mobflyA 'Obtaining crude extracts for the determination of serine hydroxymethyltransferase activity encoded by glyA in the strains of B. flavum DM36d-2 / pZl and B. flavum DM368-2: : pKldmobglyA 'described in example 5, was carried out as described already in example 4. The determination of the proteins in the cell-free crude extracts obtained and the discontinuous enzymatic test, in which the glycine formed from the substrate of threonine were also performed as described in example 4. The results of these enzymatic tests with threonine as a substrate are shown in table 3. Table 3: 2. 3 Example 7: Preparation of L-threonine with Brevibacterium flavum To study its threonine formation, the strains of B. flavum DM368-2 / pZ1 and DM369-2 were pre-cultured: pKldmobglyA * described in example 5 in 100 ml of infusion medium Brain heart (Difco Laboratories, Detroit, US) with 25 μg / mL kanamycin and 100 μM isopropyl-β-D-thiogalactosidase (IPTG) for 14 hours at 30 ° C. The cells were then washed once with 0.9% (w / v) of sodium chloride solution and with this suspension, 100 ml of CgXII medium were inoculated, so that OD600 (optical density at 600 nm) was 0.5 The medium was identical to the medium described by Keilhauer et al. (Journal of Bacteriology (1993) 175: 5593-5603), additionally containing 25 μg kanamycin / ml and 0, 10 or 100 μM isopropyl-β-D-thiogalactosidase (IPTG). Culture of both strains was carried out at 30 ° C for a period of 72 hours. After 4d and 72 hours samples were taken and the cells were centrifuged briefly (5 minutes at 13000 rpm with a Biofuge peak from the firm Heraeus, Osterode, Germany). The quantitative determination of the extracellular amino acid concentrations of the culture residue was carried out as already described in example 4 by means of reverse phase CLAP (Lindroth et al., Analytical Chemistry (1979) 51: 1167-1174). Threonine concentrations were calculated through a comparison with an external standard and asparagine as an additional internal standard. The results are given in table 4. Table 4: The following figures are included: Figure 1: map of plasmid pKldmobglyA '. The data on the length should be understood as approximate values. The abbreviations and denominations used have the following meanings: • BamHl: restriction endonuclease of Bacillus amyl ol i guefa ci ens • BglII: restriction endonuclease of Bacillus globigii • BstEII: restriction endonuclease of Bacillus stearothermophilus • EcoRI: restriction endonuclease of Escherichia coli • EcoRV: restriction endonuclease of Escherichia coli m HindIII: restriction endonuclease of Haemophilus influezae • Sacl: restriction endonuclease of Streptomyces achromogenes • kan: kanamycin resistance gene • laclq: tac promoter repressor gene Ptac • Ptac: tac • glyA 'promoter: section 5' of the serine hydroxymethyltransferase gene • glyA2-inverse: primer to verify an integration • RSP: reverse standard primer to verify an integration. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (6)

1. CLAIMS Having described the invention as above, it is claimed as property contained in the following claims: 1.- Procedure for the preparation of L-amino acids characterized because the following steps are carried out: a) fermentation of coryne-producing bacteria of L- amino acids, in which at least the glyA gene is weakened, b) enrichment of the desired product in the medium or in the cells of the bacteria, and c) isolated from the L-amino acids.
2. 2. Method according to claim 1, characterized in that bacteria are used in which additional genes of the biosynthetic pathway of the desired L-amino acids are additionally reinforced.
3. 3. Method according to claim 1, characterized in that bacteria are used in which the metabolic pathways that reduce the formation of the desired L-amino acids are at least partially deactivated.
4. 4. Method according to claim 1, characterized in that the expression of the polynucleotide encoding the glyA gene is reduced.
5. 5. Process according to claim 1, characterized in that the catalytic properties of the polypeptide (enzyme protein), which codes for the glyA polynucleotide, are reduced.
6. 6. Method according to claim 1, characterized in that to achieve the weakening the process of integrating mutagenesis of the vector pKldmobglyA ', represented in Figure 1 and deposited in E.coli as DSM 13170, is used. - Process according to claim 1, characterized in that for the preparation of L-threonine bacteria are fermented in which simultaneously they are overexpressed, especially one or more genes, selected from the group formed by: i) the hom gene encoding homoserin dihydrogenase ii) the gap gene encoding the glyceraldehyde 3-phosphate dihydrogenase iii) the pyc gene coding for pyruvate carboxylase iv) the mqo gene coding for malate oxidoreductase: quinone V ) the thrE gene coding for the export of threonine. d.- Method according to claim 1, characterized in that bacteria are fermented for the production of L-threonine, in which simultaneously one or several of the genes, selected from the group of i) the pck gene coding for the carbokinase, are simultaneously weakened of phosphoenolpyruvate ii) the poxB gene coding for pyruvate oxidase. 9. Method according to one or more of the preceding claims, characterized in that microorganisms of the genus Corynebacterium gl utamicum are used. 10.- Coryneform bacteria, characterized in that the glyA gene weakens in them. 11.- Vector pKldmobglyA ', represented in figure 1 and deposited in E. coli as DSM 13170. 12.- Isolated polynucleotide, characterized in that they contain: (i) the nucleotide sequence of the lad-tac-5'glyA unit, shown in SEQ.ID.no. 2, or (ii) at least one sequence corresponding to the sequences (i) within the degeneracy range of the genetic code, or (iii) at least one sequence that hybridizes with the sequences complementary to the sequences (i) or ( ii), and eventually (iv) neutral function sense mutations in (i). 13. Isolated polynucleotide, characterized in that it contains (i) the nucleotide sequence of the lacI-tac-glyA unit, shown in SEQ. ID. do not. 3, or (ii) at least one sequence corresponding to the sequences (i) within the degeneracy range of the genetic code, or (iii) at least one sequence that hybridizes with the sequences complementary to the sequences (i) or ( ii), and eventually (iv) neutral function sense mutations in (i).