ES2336192T3 - FERMENTATION PROCESS FOR THE PREPARATION OF L-AMINO ACIDS USING ENTEROBACTERIACEAS FAMILY STRAPS. - Google Patents

Abstract

The invention relates to a fermentation process for the preparation of L-amino acids, especially L-threonine, in which the following steps are carried out: a) fermentation of the microorganisms of the family Enterobacteriaceae producing the desired L-amino acid, in which microorganisms at least the pckA gene and/or the open reading frames yjfA and ytfP are, individually or jointly, attenuated and, in particular, switched off, b) enrichment of the L-amino acid in the medium or in the bacterial cells, and c) isolation of the L-amino acid.

Description

Fermentation process for the preparation of L-amino acids using family strains Enterobacteriaceae

Field of the Invention

The present invention relates to a process of fermentation for the preparation of L-amino acids, especially L-threonine, using strains of the Enterobacteriaceae family in which at least the pckA gene is attenuated.

Prior art

L-amino acids are used in animal nutrition, in human medicine and in industry Pharmaceutical

It is known how to prepare L-amino acids by the fermentation of strains of Enterobacteriaceae, especially Escherichia coli and Serratia marcescens . Due to their great importance, attempts are constantly being made to improve the preparation processes. Process improvements may refer to measures that involve the technology of fermentation, eg food and oxygen supply, or the composition of the nutrient media, eg the concentration of sugar during fermentation, or the finishing of the product form, eg by ion exchange chromatography, or the intrinsic productivity characteristics of the microorganism itself
saying.

The productivity characteristics of these Micro-organisms are improved using methods of mutagenesis, selection and choice of mutants to give strains that are resistant to antimetabolites, e.g. the acid threonine analog α-amino-? -hydroxyvaleric (AHV) or auxotrophs for metabolites of regulatory importance, and produce L-amino acids, e.g. L-threonine

Methods of recombinant DNA technology they have also been used for some years to improve L-amino acid producing strains of the family  Enterobacteriaceae by amplification of biosynthesis genes individual amino acids and study of the effect on production.

EP-A-1094111 provides a process for fermentation preparation of L-amino acids, in particular L-lysine and L-threonine, using coryneform materials that already produce in particular L-amino acids and in which the sequence or sequences of nucleotides that encode the product of the pck gene are dimmed

Prost Jean-Francois et al .: "Detection of an extended - 10 element in the promise region of the pckA gene encoding phosphoenolpyruvate carboxykinase in Escherichia Coli ". Biochemie 81: 197-200 (1999) describe the regulation of transcription of the pckA gene coding for phosphoenolpyruvate carboxykinase in Escherichia coli was analyzed by site- directed mutagenesis of the promoter region and measurement of in vitro initiation of transcription.

Kramer R: "Genetic and physiological approaches for the production of amino acids "Journal of Biotechnology, Elsevier Science Publishers, Amsterdam, NL, vol. Four. Five, no. February 1, 12 (1996-02-12), Pages 1-21, XP004036833 ISSN: 0168-1656, describe the relevant paths in biotechnologically relevant microorganisms producing amino acids.

Database Biosis [Online] Biosciences Information Service, Philadelphia, PA, USA: 1990, Medina V. et al . provides "Sequence of the pck - a gene of Escherichia Coli K-12 Relevance to genetic and allosteric regulation and Homology of Escherichia Coli Phospho-enolpiruvate Carboxykinase with the enzymes from Trypanosorna-Brucei and Saccharomyces-Cerevisiae " Accession No. PREVI99191038347 a la Base of Data XP002193203.

In Database Biosis [On line] Biosciences Information Service, Philadelphia, PA, USA; 1990, Goldie H. et al .: "Physical and Genetic Locus from Escherichia coli K12" PREV Access No. 199089092769 to Database XP002193202.

Object of the invention

The object that they proposed themselves inventors was to provide new procedures to improve the L-amino acid preparation, especially L-threonine, by fermentation.

Summary of the invention

The invention provides a process of fermentation for the preparation of L-amino acids, especially L-threonine, using microorganisms of the Enterobacteriaceae family that, in in particular, they already produce L-threonine and in which the nucleotide sequence (pckA gene) that encodes the enzyme phosphoenolpyruvate carboxykinase (PEP-carboxykinase) (EC 4.1.1.49) is attenuated

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Detailed description of the invention

In the cases in which they are mentioned L-amino acids or amino acids onwards, this means one or more amino acids, including their salts, selected from the group consisting of L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-Leucine, L-Tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-homoserine and L-arginine. Be particularly prefers L-threonine.

In this context, the term "attenuation" describes the reduction or deactivation, in a microorganism, of the intracellular activity of one or more enzymes (proteins) that are encoded by the appropriate DNA, for example by use of a weak promoter or a gene or allele that encodes an appropriate enzyme with low activity, or deactivation of the enzyme (protein) appropriate, and optionally combination of these measures.

By mitigation measures, the activity or corresponding protein concentration is generally reduced up to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of wild-type protein or activity or protein concentration in the starting microorganism.

The process is characterized because they lead to perform the following steps:

to)

fermentation of microorganisms of the Enterobacteriaceae family producers of Desired L-amino acid, in whose microorganisms it is attenuated at least the pckA gene or nucleotide sequences that encode the product of the pckA gene;

b)

enrichment of L-amino acid in the medium or in the cells bacterial, and

C)

insulation L-amino acid or

d)

broth constituent insulation of fermentation and biomass as a whole or portions of the same as a solid product along with the L-amino acid

The microorganisms provided by the present invention can produce L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, optionally starch or optionally cellulose, or from glycerol and ethanol. These microorganisms are representative of the Enterobacteriaceae family selected from the genera Escherichia, Erwinia, Providencia and Serratia. The genera Escherichia and Serratia are preferred. The species Escherichia coli and Serratia marcescens can be mentioned in particular between the genera Escherichia and Serratia respectively.

Examples of suitable strains, particularly L-threonine-producing strains, of the Escherichia genus, specifically of the Escherichia coli species are:

one

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Examples of L-threonine producing strains of the genus Serratia, especially of the Serratia marcescens species, are:

2

The producing strains of L-threonine of the Enterobacteriaceae family possess preferably, among other things, one or more features genetic or phenotypic selected from the group comprising acid resistance α-amino-? -hydroxyvaleric, thialisine resistance, ethionine resistance, resistance to α-methylserine, acid resistance diaminosuccinic acid resistance α-aminobutyric acid, borrelidine resistance, rifampicin resistance, resistance to valine analogs such such as valine hydroxamate, resistance to purine analogues such as 6-dimethylaminopurine, need for L-methionine, optionally partial and compensable for L-isoleucine, need acid meso-diaminopimelic, auxotrophy with respect to dipeptides containing threonine, resistance to L-threonine resistance to L-homoserine, L-lysine, resistance to L-methionine, glutamic acid resistance, resistance to L-aspartate, resistance to L-leucine, resistance to L-phenylalanine, resistance to L-serine, resistance to L-cysteine, L-valine resistance, sensitivity to fluoropyruvate, insufficiency of threonine dehydrogenase, optional capacity for use of sucrose, amplification of the threonine operon, amplification of the homoserine dehydrogenase-I-aspartate-kinase-I,  preferably in the form resistant to feedback, homoserine kinase amplification, amplification threonine synthase, amplification of aspartate kinase, optionally as Feedback resistant, amplification of aspartate-semialdehyde dehydrogenase,  amplification of phosphoenol pyruvate carboxylase, optionally in the form resistant to feedback, amplification of phosphoenol pyruvate synthase, transhydrogenase amplification, product amplification of RhtB gene, product amplification of the RhtC gene, amplification of the YfiK gene product, amplification of malate-quinone oxidoreductase and amplification of a pyruvate carboxylase, as well as attenuation of acetic acid formation.

It has been found that the production of L-amino acids, especially L-threonine, by family microorganisms Enterobacteriaceae is improved after attenuation and, in particular, deactivation of the pckA gene that encodes PEP-carboxykinase (EC 4.1.1.49).

The nucleotide sequence of the Esckichia coli pckA gene has been published by Medina et al . (Journal of Bacteriology 172, 7151-7156 (1990)) and can also be taken from the genome sequence of Escherichia coli published by Blattner et al . (Science 277, 1453-1462 (1997)). The nucleotide sequence of the Escherichia coli pckA gene is represented in SEQ ID No. 1 and the amino acid sequence of the product of the corresponding gene is represented in SEQ ID No. 2.

The pckA genes described in the references Previous bibliographies can be used according to the invention. It is also possible to use alleles of the pckA gene that are result of degeneracy of the genetic code or mutations neutral sense.

The attenuation can be achieved for example by reduction or deactivation of the expression of the pckA gene or of the catalytic properties of the enzymatic protein. Both measures can be combined optionally.

Gene expression can be reduced by a appropriate culture technique, by genetic modification (mutation) of the signal structures of gene expression, or by means of antisense DNA technology. Examples of signal structures from gene expression are repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, The initial codon and terminators. Those skilled in the art you will find relevant information among other places in, e.g. Jensen and Hammer ((Biotechnology and Bioengineering 58, 191-195 (1998)), Carrier and Keasling (Biotechnology Progress 15, 58-64 (1999)), Franch and Gerdes (Current Opinion in Microbiology 3, 159-164 (2000)) and books well-known text about genetics and molecular biology, for example the textbook of Knippers ("Molekulare Genetik" ("Molecular Genetics"), 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or the Winnacker textbook ("Gene und Klone "(" From Genes to Clones "), VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

Mutations that cause a change or reduction in the catalytic properties of enzyme proteins are known from the prior art. Examples that may be mentioned are the studies by Qiu and Goodman (Journal of Biological Chemistry 272, 8611-8617 (1997)), Yano et al . (Proceedings of the National Academy of Sciences USA 95, 5511-5515 (1998)) and Wente and Schachmann (Journal of Biological Chemistry 266; 20833-20839 (1991)). Examinations can be found in well-known textbooks on genetics and molecular biology, eg the Hagemann textbook ("Allgemeine Genetik"("GeneralGenetics"), Gustav Fischer Verlag, Stuttgart, 1986).

Mutations to consider are transitions, transversions, insertions and deletions. Depending  of the effect of the change of amino acids on the enzymatic activity, the term sense mutations or nonsense mutations are used. Insertions or deletions of at least one base pair in a gene they cause frame shift mutations, the result of which is that false amino acids are incorporated or prematurely terminated translation. Deletions of several codons typically lead to a complete loss of enzyme activity. Instructions for the production of such mutations are part of the technique above and can be found in well-known textbooks on genetics and molecular biology, e.g. the textbook of Knippers ("Molekulare Genetik" (6th Molecular Genetics)) edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), the book of Winnacker text ("Gene und Klone" ("From Genes to Clones "), VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or Hagemann's textbook ("Allgemeine Genetik" ("General Genetics "), Gustav Fischer Verlag, Stuttgart, 1986).

An example of a plasmid by which the pckA gene of Escherichia coli can be attenuated and, in particular, deactivated by position-specific mutagenesis is plasmid pMAK705 ΔpckA (Figure 1). It contains only part of region 51 and part of the 3 'region of the pckA gene. A 349 bp segment of the coding region (deletion) has been omitted. The sequence of this DNA, which can be used for mutagenesis of the pckA gene, is depicted in SEQ ID No. 3.

PckA gene deletion mutation can incorporated into suitable strains by changing genes or alleles.

A common method is the gene change method using a conditionally replicating derivative of pSC101, pMAK705, as described by Hamilton et al . (Journal of Bacteriology 174, 4617-4622 (1989)). Other methods described in the prior art can also be used, for example that of Martínez-Morales et al . (Journal of Bacteriology, 7143-7148 (1999)) or that of Boyd et al . (Journal of Bacteriology 182, 842-847 (2000)).

When the change has been made, the way of the Δplock allele represented in SEQ ID No. 4, which is a additional object of the invention, is present in the strain in question.

Mutations in the pckA gene or mutations that involve the expression of the pckA gene can also be transferred to different strains by conjugation or transduction.

Additionally, for the production of L-amino acids, especially L-threonine, with family strains Enterobacteriaceae, it can be advantageous not only to attenuate the gene pckA, but also amplify one or more enzymes of the way known biosynthetic of threonine, or metabolism enzymes anaplerotic, or enzymes for the production of nicotinamide adenine dinucleotide phosphate reduced.

In this context, the term "amplification" describes the increase in activity intracellular, in a micro-organism, of one or more enzymes or proteins that are encoded by the appropriate DNA, by example by increasing the number of copies of the gene (s), using a strong promoter or using a gene that encodes a appropriate enzyme or protein with high activity, and optionally  combination of these measures.

By amplification measures, in particular over + expression, protein activity or concentration corresponding is generally increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on that of the wild-type protein or activity or protein concentration in the starting microorganism.

Thus, for example, one or more selected genes of the group that includes:

?

the thrABC operon coding aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase (US-A-4,278,765),

?

the pyc gene that encodes pyruvate carboxylase (DE-A-19831609),

?

the pps gene that encodes phosphoenolpyruvate synthase (Molecular and General Genetics 231, 332 (1992)),

?

the ppc gene that encodes phosphoenolpyruvate carboxylase (Gene 31, 279-283 (1984)),

?

the pntA and pntB genes that encode transhydrogenase (European Journal of Biochemistry 158, 647-653 (1986)),

?

the rhtB gene for resistance to homoserine (EP-A-0994190), Y

?

the rhtC gene for resistance to threonine (EP-A-1013765),

?

the gdhA gene that encodes glutamate dehydrogenase (Gene 27: 193-199 (1984))

can be amplified simultaneously and, in particular overexpress.

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Additionally, for the production of L-amino acids, especially L-threonine, it can be advantageous not only to attenuate the pckA gene but also attenuate and in particular disable one or more genes selected from the group comprising:

?

the tdh gene that encodes threonine dehydrogenase (Ravnikar and Somerville, Journal of Bacteriology 169, 4716-4721 (1987)),

?

the mdh gene encoding malate dehydrogenase (EC 1.1.1.37) (Vogel et al ., Archives in Microbiology 14 9, 36-42 (1987)),

?

the gene product of the framework of open reading (orf) yjfA (National Access Number AAC 77180 Center for Biotechnology Information (NCBI, Bethesda, MD, USA) and SEQ ID No. 5), and

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?

the gene product of the framework of open reading (orf) ytfP (National Access Number AAC 77179 Center for Biotechnology Information (NCBI, Bethesda, MD, USA) and SEQ ID No. 5),

or reduce their expression.

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It is preferred to dim the open reading frame yjfA and / or the open reading frame ytfP.

It is also possible, according to the description, dim the open reading frames yjfA and / or ytfP regardless of the pckA gene, in order to achieve an improvement in amino acid production in particular L-threonine

The description also provides agreement with it a process characterized because the steps are carried out following:

d)

fermentation of microorganisms of the Enterobacteriaceae family in which at least the framework of open reading yjfA and / or ytfP is grayed out,

and)

enrichment of L-amino acid in the medium or in the cells of the Enterobacteriaceae family microorganisms, and

F)

isolation of the constituents of L-threonine from fermentation broth and biomass in all or portions thereof that are optionally isolated as a solid product along with the L-amino acid.

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An example of a plasmid by means of which the open reading frames yjfA and ytfP of Escherichia coli can be attenuated and, in particular, deactivated by position-specific mutagenesis is the plasmid
pMAK705 ΔyjfA (Figure 2). It contains only the 5 'and 3' edges of the ytfP-yjfA region, including very short residues of the open reading frames yjfA and ytfP. A part of 337 bp in length of the ytfP-yjfA region is absent (deletion). The sequence of this DNA, which can be used for mutagenesis of the ytfP-yjfA region, is depicted in SEQ ID No. 6.

A further example of a plasmid by means of which the open reading frames yjfA and ytfP of Escherichia coli can be attenuated and, in particular, deactivated by position-specific mutagenesis is plasmid pMAK705 Δ90bp (Figure 5). It also contains only the 5 'and 3' flanks of the ytfP-yjfA region, including very short residues of the open reading frames yjfA and ytfP. A 90 bp long part of the ytfP-yjfA region is absent (deletion). The sequence of this DNA, which can be used for mutagenesis of the ytfP-yjfA region, is depicted in SEQ ID No. 7.

This deletion mutation can be incorporated into suitable strains by replacement of genes or alleles. It is like that possible to transfer mutations in open reading frames yjfA and / or ytfP or mutations that affect the expression of these frames of open reading in various strains by conjugation or transduction

When the replacement has been carried out, the shape of the ΔytfP and ΔyjfA allele represented in SEQ ID No. 6 or SEQ ID No. 7, which are an additional object of the description, is present in the strain in question.

Additionally, for the production of L-amino acids, especially L-threonine, can be advantageous, in addition to the individual or joint attenuation of the pckA gene or of the frames of open reading yjfA e / o ytfP, disable react secondary undesirable (Nakayama: "Breeding of Amino Acid Producing Microorganisms ", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (compilers), Acadernic Press, London, United Kingdom, 1982).

The microorganisms prepared according to the invention can be cultivated by the batch process or the process of feedback. A summary of known culture methods is provided in Chmiel's textbook (Bioprozesstechnik 1. Einführung in die Bioverfah-renstechnik (Bioprocess Technology 1. Introduction to Bioengineering) (Gustav Fischer Verlag, Stuttgart, 1991)) or in the Storhas textbook (Bioreaktoren und periphere Einrichtungen (Bioreactors and Peripheral Equipment) (Vieweg Verlag, Brunswick / Wiesbaden, 1994)).

The culture medium to be used must satisfy adequately the demands of the particular strains. Descriptions  of culture media for various microorganisms can found in the manual "Manual of Methods for General Bacteriology "of the American Society for Bacteriology (Washington DC, USA, 1981).

Carbon sources that can be used are sugars and carbohydrates, e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and optionally cellulose, oils and fats, e.g. soybean oil, sunflower oil, peanut, and coconut oil, fatty acids, e.g. palmitic acid, stearic acid and linoleic acid, alcohols, e.g. glycerol and ethanol, and organic acids, e.g. acetic acid. These substances they can be used individually or in the form of mixtures.

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Sources of nitrogen that can be used are organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, liquor maceration of corn, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources they can be used individually or in the form of mixtures.

Phosphorus sources that can be used are phosphoric acid, potassium dihydrogen phosphate or hydrogen phosphate dipotassium, or the corresponding sodium salts. The middle of crop must also contain metal salts, e.g. sulfate magnesium or iron sulfate, which are necessary for the increase. Finally, essential substances promoting growth such as amino acids and vitamins can be used in addition to the substances mentioned above. Can also be added to the culture medium suitable precursors. Such materials of feed can be added to the crop at once or fed properly during cultivation.

The pH of the culture is controlled by use appropriate of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or compounds acids such as phosphoric acid or sulfuric acid. The formation foam can be controlled using antifoaming agents such as polyglycol esters of fatty acids. The stability of the Plasmids can be maintained by adding suitable substances from selective action, e.g. antibiotics, to the medium. Remain aerobic conditions by introduction of oxygen or gaseous mixtures containing oxygen, e.g. air, in the crop. The temperature of culture is normally 25 ° C to 4 5 ° C and preferably 30 ° C to 40 ° C. The cultivation is continued until the formation of L-amino acids or L-threonine has reached a maximum. This goal is normally achieved within from 10 hours to 160 hours.

L-amino acids can be analyzed by anion exchange chromatography followed by ninhydrin derivation, as described by Spackman et al . et al . (Analytical Chemistry 30, 1190 (1958)), or by reverse phase HPLC, as described by Lindroth et al . (Analytical Chemistry 51, 1167-1174. (1979)).

A pure culture of the Escherichia coli strain K-12 DH5α / pMAK705 was deposited on September 8, 2000 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty as DSM 13720.

A pure culture of the Escherichia coli strain K-12 MG442 \ DeltapckA was deposited on October 2, 2000 in the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) according to the Budapest Treaty as DSM 13761.

A pure culture of the K-12 strain of Escherichia coli B-3996kur \ Deltatdh \ DeltapckA / pVIC40 was deposited on March 9, 001 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) in accordance with the Budapest Treaty as DSM 14150.

A pure culture of the K-12 strain of Escherichia coli MG442 \ Delta90yjfA was deposited on May 9, 2001 in the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) according to the Budapest Treaty as DSM 14289.

It is also possible according to the description, individually dim open reading frames ytfP and yjfA in order to improve the production of L-amino acids

The process according to the invention is used for the preparation of L-amino acids, e.g. L-threonine, L-isoleucine, L-methionine, L-homoserine and L-lysine, especially L-threonine, by fermentation.

The present invention is further illustrated detail below with examples.

Isolation of Escherichia coli plasmid DNA and all techniques for restriction, Klenow treatment and alkaline phosphatase treatment were carried out as described by Sambrook et al . (Molecular Cloning - A Laboratory Manual (1989), Cold Spring Harbor Laboratory Press). Unless otherwise indicated, the Escherichia coli transformation was carried out as described by Chung et al . (Proceedings of the National Academy of Sciences USA 86, 2172-2175 (1989)).

The incubation temperature for the preparation of strains and transformants was 37 ° C. Temperatures of 30 ° C and 44 ° C were used in the gene exchange process of Hamilton et al .

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Example 1 Construction of the pckA gene deletion mutation

Parts of the 5 'and 3' regions of the Escherichia coli K12 pckA gene were amplified using the polymerase chain region (PCR) and synthetic oligonucleotides. The nucleotide sequence of the pckA gene in E. coli K12 MG1655 (SEQ ID No. 1) was used to synthesize the following PCR primers (MWG Biotech, Ebersberg, Germany):

3

The chromosomal DNA of E. coli K12 MG1655 used for PCR was isolated with "Qiagen Genomics-tips 100 / G" (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. A DNA fragment of approx. 500 bp of the 5 'region of the pckA gene (designated as pck1) and a DNA fragment of approx. 600 bp of the 3 'region of the pckA gene (designated as pck2) could be amplified with the specific primers under standard PCR conditions (Innis et al . (1990), PCR Protocols. A Guide to Methods and Applications, Academic Press) using DNA- Taq polymerase (Gibco-BRL, Eggenstein, Germany). PCR products were ligated with each vector pCR2.1TOPO (TOPO TA Cloning Kit, Invitrogen, Groningen, The Netherlands) according to the manufacturer 's instructions and transformed into the strain TOP10F 'E. coli. Plasmid carrier cells were selected on LB agar containing 50 µg / ml ampicillin. After isolation of the plasmid DNA, the vector pCR2.1TOPOpck2 was cleaved with the restriction enzymes StuI and XbaI and, after separation in 0.8% agarose gel, the pck2 fragment was isolated with the aid of the Gel Extraction Kit QIAquick (QIAGEN, Hilden, Germany). After isolation of the plasmid DNA, the vector pCR2.1TOPOpck1 was cleaved with the EcoRV and XbaI enzymes and ligated to the isolated pck2 fragment. E. coli strain DH5α was transformed with the ligation mixture and plasmid carrier cells were selected on LB agar containing 50 µg / ml ampicillin. After isolation of the plasmid DNA, control cleavage with the SpeI and XbaI enzymes was used to detect plasmids containing, in cloned form, the mutagenic DNA sequence depicted in SEQ ID No. 3. One of the plasmids was designated as pCR2. 1 TOP \ DeltapckA.

Example 2 Construction of the exchange vector pMAK705 \ DeltapckA

After restriction with the SpeI and XbaI enzymes and 0.8% agarose gel separation, the pckA allele described in Example 1 was isolated from the vector pCR2.1TOPO? And was bound to plasmid pMAK705 (Hamilton et al ., Journal of Bacteriology 174, 4617-4622 (1989)) that had been digested with the enzyme XbaI. DH5α was transformed with the ligation mixture and plasmid carrier cells were selected on LB agar containing 20 µg / ml chloramphenicol. After plasmid DNA isolation and cleavage with the enzymes HpaI, KpnI, HindIII, SalI and PstI, cloning was detected successfully. The exchange vector formed, pMAK705 ΔpckA (= pMAK705deltapckA), is shown in Figure 1.

Example 3 Specific mutagenesis of the position of the pckA gene in E. coli strain MG442

The E. coli strain MG442 producing L-threonine is described in US-A-4,278,765 and has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM, Moscow, Russia) as CMIM B-1628.

The MG442 strain exhibits acid resistance α-amino-? -hydroxyvaleric  and has optionally partial and compensable need of L-Isoleucine

For the exchange of the chromosomal pckA gene for the deletion construct encoded by the plasmid, MG442 was transformed with the plasmid pMAK705 Δpck Δ. Gene exchange was carried out by the selection method described by Hamilton et al . (Journal of Bacteriology 174, 4617-4622 (1989)) and was checked by standard PCR methods (Innis et al . (1990), PCR Protocols. A Guide to Methods and Applications, Academic Press) using the following oligonucleotide primers:

4

The strain obtained was designated as MG442 ΔpckA.

Example 4 Preparation of L-threonine with the strain MG442 \ DeltapckA

MG442 \ DeltapckA was grown in minimum medium of The following composition: 3.5 g / l Na2HPO4.2H2O, 1.5 g / l of KH 2 PO 4, 1 g / l of NH 4 Cl, 0.1 g / l of MgSO 4 .7H 2 O, 2 g / l glucose and 20 g / l agar. The L-threonine formation was checked in cultures by 10 ml batches contained in 100 ml Erlenmeyer flasks. Be they inoculated these with 10 ml of a preculture medium of the following composition: 2 g / l yeast extract, 10 g / l of (NH 4) 2 SO 4, 1 g / l of KH 2 PO 4, 0.5 g / l MgSO 4 .7H 2 O, 15 g / l CaCO 3 and 20 g / l glucose, and it was incubated for 16 hours at 37 ° C and 180 rpm in an ESR incubator of Kühner AG (Birsfelden, Switzerland). 250 µl of this was transferred preculture to 10 ml of a production medium (25 g / l of (NH 4) 2 SO 4, 2 g / l of KH 2 PO 4, 1 g / l of MgSO 4 • 7H 2 O, 0.03 g / l FeSO 4 • 7 O 2 O, 0.018 g / l of MgSO 4 • 1H 2 O, 30 g / l of CaCO 3, 20 g / l glucose) and incubated for 48 hours at 37 ° C. After incubation, the optical density (OD) of the suspension was determined culture with an LP2W photometer from Dr. Lange (Berlin, Germany) to a measurement wavelength of 660 nm.

L-threonine concentration formed was then determined in the culture supernatant filtered in sterile conditions with an amino acid analyzer of Eppendorf-BioTronik (Hamburg, Germany) through of ion exchange chromatography and reaction in post-column with ninhydrin detection.

The result of the experiment is shown in the Table 1.

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TABLE 1

5

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Example 5 Preparation of L-threonine with the strain MG442 \ DeltapckA / pMW218gdhA 5.1 Amplification and cloning of the gdhA gene

The Escherichia coli K12 glutamate dehydrogenase gene is amplified using the polymerase chain reaction (PCR) and synthetic oligonucleotides. Starting from the nucleotide sequence for the gdhA gene in E. coli K12 MG1655 (gene library: Accession No. AE000270 and No. AE000271) the PCR primers (MWG Biotech, Ebersberg, Germany) are synthesized:

6

The chromosomal DNA of E. coli K12 MG1655 used for PCR is isolated according to the manufacturers instructions with "QIAGEN Genomic-tips 100 / G" (QIAGEN, Hilden, Germany). A DNA fragment with a size of approximately 2150 bp, which comprises the coding region of gdhA and approx. 350 bp of the 5 'flanking sequence and approx. 450 bp of the 3 'flanking sequence can be amplified with specific primers under standard PCR conditions (Innis et al .: PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) with Pfu DNA polymerase (Promega Corporation : Madison, USA). The PCR product is cloned into plasmid pCR2.1TOPO and transformed into E. coli TOP10 strain (Invitrogen, Leek, The Netherlands, Product Description TOPO TA Cloning Kit, Cat. No. K4500-01). Successful cloning is demonstrated by cleavage of plasmid pCR2.1TOPOgdhA with restriction enzymes EcoRI and EcoRV. For this, the plasmid DNA is isolated by means of the "QIAprep Spin Plasmid Kits" (QIAGEN, Hilden, Germany) and, after excision, it is separated on a 0.8% agarose gel.

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5.2 Cloning of the gdhA gene in the plasmid vector pMW218

Plasmid pCR2.1TOPOgdhA is cleaved with the EcoRI enzyme, the 0.8% agarose gel cleavage batch is separated and the 2.1 kbp size gdhA fragment is isolated with the help of the "QIAquick Gel Extraction Kit" ( QIAGEN, Hilden, Germany). Plasmid pMW218 (Nippon Gene, Toyama, Japan) is cleaved with the EcoRI enzyme and ligated with the gdhA fragment. The E. coli strain DH5α is transformed with the ligation batch and the pMW218 carrier cells are selected by extension on plates on LB agar (Lennox, Virology 1955, 1: 190), to which 20 µg / ml of Kanamycin

Successful cloning of the gdhA gene can be demonstrated after plasmid DNA isolation and excision of control with EcoRI and EcoRV. The plasmid is designated pMW218gdhA (Figure 3).

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5.3 Preparation of the strain MG442 \ DeltapckA / pMW218gdhA

The strain MG442 ΔpckA obtained in the Example 3 and strain MG442 are transformed with plasmid pMW218gdhA, and the Transformants are selected on LB agar, which is complemented with 20 mug / ml of kanamycin. In this way the strains are formed MG442 ΔpckA / pMW218gdhA and MG442 / pMW218gdhA.

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5.4 Preparation of L-threonine

The preparation of L-threonine by The MG442 \ DeltapckA / pMW218gdhA and MG442 / pMW218gdhA strains are tested as described in Example 4. The minimum medium and the medium of preculture are further supplemented with 20 µg / ml of Kanamycin

The result of the experiment is summarized in the Table 2.

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TABLE 2

7

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Example 6 Preparation of L-threonine with the strain MG442 \ DeltapckA / pMW219rhtC 6.1 Amplification of the rhtC gene

The rhtC gene of Escherichia coli K12 is amplified using the polymerase chain reaction (PCR) and synthetic oligonucleotides. Starting from the nucleotide sequence for the rhtC gene in E. coli K12 MG1655 (gene library: Accession No. AE000458, Zakataeva et al . (FEBS Letter 452, 228-232 (1999)), PCR primers are synthesized ( MWG Biotech, Ebersberg, Germany):

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8

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The chromosomal DNA of E. coli K12 MG1655 used for PCR is isolated according to the manufacturers instructions with "QIAGEN Genomic-tips 100 / G" (QIAGEN, Hilden, Germany). A DNA fragment of a size approx. 800 bp can be amplified with specific primers under standard PCR conditions (Innis et al .: PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) with Pfu DNA polymerase. (Promega Corporation, Madison, USA).

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6.2 Cloning of the rhtC gene in the plasmid vector pMW219

Plasmid pMW219 (Nippon Gene, Toyama, Japan) is cleaved with the SamI enzyme and ligated with the rhtC-PCR fragment. The E. coli strain DH5α is transformed with the ligation batch and the pMW219 carrier cells are selected on LB agar, which is supplemented with 20 µg / ml kanamycin. Successful cloning can be demonstrated after plasmid DNA isolation and control cleavage with KpnI, HindIII and NcoI. Plasmid pMW219rhtC is shown in Figure 4.

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6.3 Preparation of the strain MG442 \ DeltapckA / pMW219rhtC

The strain MG442 ΔpckA obtained in the Example 3 and strain MG442 are transformed with plasmid pMW219rhtC and the Transformants are selected on LB agar, which is complemented with 20 mug / ml of kanamycin. In this way the strains are formed MG442 \ DeltapckA / pMW219rhtC and MG442 / pMW219rhtC.

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6.4 Preparation of L-threonine

The preparation of L-threonine by Strains MG442 \ DeltapckA / pMW219rhtC and MG442 / pMW219rhtC are tested as described in Example 4. The minimum medium and the medium of culture are further supplemented with 20 µg / ml of Kanamycin

The result of the experiment is summarized in the Table 3.

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TABLE 3

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Example 7 Preparation of L-threonine with the strain B-3996kur \ Deltatdh \ DeltapckA / pVIC40

The strain of E. coli B-3996 producing L-threonine is described in US-A-5,175,107 and has been deposited in the Russian National Collection for Industrial Microorganisms (VKPM, Moscow, Russia).

The strain B-3996 has, between other things, acid resistance α-amino-? -hydroxyvaleric, have an attenuated threonine dehydrogenase, in particular deactivated, or deficient, has homoserine dehydrogenase I-aspartate kinase I intensified in the form resistant to feedback, it has a need optionally partial and compensable for L-isoleucine and has the ability to use sucrose.

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7.1 Preparation of the strain B-3996kur \ Deltatdh \ DeltapckA / pVIC40

After cultivation in complete medium free of antibiotics for about 10 generations, a derived from strain B-3996 that no longer contains the plasmid pVIC40. The strain formed is streptomycin sensitive and is has designated as B-3996kur.

To incorporate a deletion into the tdh gene, the method described by Hamilton et al . (Journal of Bacteriology (1989) 171: 4617-4622), which is based on the use of plasmid pMAK705 with a temperature sensitive replicon. Plasmid pDR121 (Ravnikar and Somerville, Journal of Bacteriology (1987) 169: 4716-4721) contains a fragment of E. coli DNA of 3.7 kilograms of bases (kpb) in size, in which the tdh gene is encoded . To generate a deletion of the region of the tdh gene, pDR121 is cleaved with the restriction enzymes ClaI and EcoRV, and the isolated 5 kbp DNA fragment is ligated, after treatment with the Klenow enzyme. The ligation batch is transformed into the E. coli strain DH5α and the plasmid carrying cells are selected on LB agar, to which 50 µg / ml ampicillin is added.

Successful deletion of the tdh gene can be demonstrated after plasmid DNA isolation and excision of control with EcoRI. The 1.7 kbp EcoRI fragment is isolated, and binds with plasmid pMAK705, which is partially digested with EcoRI The ligation batch is transformed into DH5α and the plasmid carrying cells are selected on LB agar, to which add 20 µg / ml chloramphenicol. Successful cloning is demonstrates after plasmid DNA isolation and excision with EcoRI The derivative pMAK705 formed is designated pDM32.

For the replacement of the gene, B-3996kur is transformed with the plasmid pDM32. The replacement of the tdh chromosomal gene with the deletion construct encoded by the plasmid is carried out by the selection process described by Hamilton et al . and is checked by standard PCR methods (Innis et al . (1990), PCR Protocols. A Guide to Methods and Applications, Academic Press) with the following oligonucleotide primers:

10

The strain formed is tested for Kanamycin sensitivity and is designated B-3996kur \ Deltatdh.

For position-specific mutagenesis of pckA gene, B-3996kur \ Deltatdh is transformed with the replacement vector pMAK705? described in the Example 2. The replacement of the chromosomal pckA gene by deletion construct encoded by the plasmid is carried out as described in Example 3. The strain obtained is designated B-3996kur \ Deltatdh \ DeltapckA.

B-3996kur \ Deltatdh and B-3996kur \ Deltatdh \ DeltapckA are transformed with plasmid pVIC40 isolated from B-3996 and cells plasmid carriers are selected on LB agar with 20 µg / ml of streptomycin. In each case, an individual colony selected is designated B-3996kur \ Deltatdh / pVIC40 and B-3996kur \ Deltatdh \ DeltapckA / pVIC40.

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7.2 Preparation of L-threonine

The preparation of L-threonine by strains B-3996kur \ Deltatdh / pVIC40 and B-3996kur \ Deltatdh \ DeltapckA / pVIC40 is tested as described in Example 4. The minimum medium, the medium of preculture and the means of production are further complemented with 20 mg / ml streptomycin.

The result of the experiment is summarized in the Table 4

TABLE 4

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Example 8 Preparation of L-lysine with the strain TOC21R \ DeltapckA

The E. coli strain pDA1 / TOC21R is described in patent application FA-2511032 and has been deposited in the National Microorganism Culture Collection (CNCM = National Microorganism Culture Collection, Pasteur Institute, Paris, France) under number 1 -167. The strain and the plasmid-free host have also been described by Deuce-Le Reverend et al . (European Journal of Applied Microbiology and Biotechnology 15: 227-231 (1982)) under the name TOC21R / pDA1.

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8.1 Position specific mutagenesis of the pckA gene in the E. coli TOC21R strain

After cultivation in LB medium free of antibiotics for about six generations, it is isolated a derivative of strain pDA1 / TOC21R that no longer contains the plasmid pDA1. The strain formed is tetracycline sensitive and is designated TOC21R.

For the replacement of the chromosomal gene of pckA by the deletion construct encoded by the plasmid, TOC21R is transformed with the plasmid pMAK705? (Example 2). Gene replacement is carried out by the selection method described by Hamilton et al . (1989), Journal of Bacteriology 174, 4617-4622) and is checked by standard PCR methods (Innis et al . (1990) PCR Protocols. A guide to Methods and Applications, Academic Press) with the following oligonucleotide primers:

12

The strain obtained is designated TOC21R \ DeltapckA.

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8.2 Preparation of L-lysine with the strain TOC21R \ DeltapckA

The formation of L-lysine with the strain TOC21R \ DeltapckA and TOC21R is checked in batch cultures 10 ml contained in 100 ml conical flasks. For this, I know inoculate 10 ml of preculture medium of the following composition: 2 g / l yeast extract, 10 g / l of (NH 4) 2 SO 4, 1 g / l of KH 2 PO 4, 0.5 g / l of MgSO 4 * 7H 2 O, 15 g / l of CaCO 3, 2 0 g / l of glucose and the batch is incubated for 16 hours at 37 ° C and 180 rpm in a ESR incubator from Kühner AG (Birsfelden, Switzerland). They transinoculate 250 µl of this preculture in 10 ml of production medium (25 g / l (NH 4) 2 SO 4, 2 g / l KH 2 PO 4, 1 g / l MgSO 4 * 7H 2 O, 0.03 g / l FeSO 4 * 7H 2 O, 0.018 g / l MnSO 4 • 1 H 2 O, 30 g / l CaCO 3, 20 g / l glucose, 25 mg / l L-isoleucine and 5 mg / l thiamine) and incubate the batch for 72 hours at 37 ° C. After incubation, it determines the optical density (OD) of the culture suspension with a LP2W photometer of Dr. Lange (Berlin, Germany) at a length of 660 nm measurement wave.

The concentration of L-lysine formed in the culture supernatant filtered under sterile conditions with an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange and reaction chromatography post-column with ninhydrin detection.

The results of the experiment are shown in the Table 5.

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TABLE 5

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Example 9 Formation of L-isoleucine with the strain B-3996kur \ DeltatdhilvA + \ DeltapckA / pVIC40 9.1 Preparation of the strain B-3996kur \ DeltatdhilvA + \ DeltapckA / pVIC40

The strain B-3996kur \ Deltatdh, which needs L-isoleucine, obtained in the Example 7.1 is transduced with the help of phage Plkc (Lennox, Virology 1, 190-206 (1955); Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory 1972) and the transductants L-isoleucine-prototrophic.

For this, the phage Plkc is multiplied in strain MG1655 (Guyer et al ., Cold Spring Harbor Symposium Quantitative Biology 45, 135-140 (1981) and Blattner et al ., Science: 277, 1453-1462 (1997)) and phage lysate is used for the transduction of strain B-3996kur \ Deltatdh. The multiplicity of the infection is approximately 0.2. The selection for prototrophic transducers of L-isoleucine is carried out on a minimum agar, which contains 2 g / l of glucose and 10 mg / l of L-threonine. A prototrophic transducer of L-isoleucine is isolated, spread on LB agar for purification or isolation and is designated B-3996kur ΔtdhilvA +.

The pckA strain gene B-3996kur \ DeltatdhilvA + is then replaced, as described in Example 3, by the ΔpckA allele prepared in Examples 1 and 2. The strain obtained is designated B-3996kur \ DeltatdhilvA + \ DeltapckA.

Strains B-3996kur \ DeltatdhilvA + and B-3996kur \ DeltatdhilvA + \ DeltapckA se transformed with plasmid pVIC40 isolated from strain B-3996 and plasmid carrying cells are selected on LB agar, which is complemented with 20 µg / ml of streptomycin. In each case, a selected individual colony will be designates B-3996kur \ DeltatdhilvA + \ DeltapckA / pVIC40  and B-3996kur \ DeltatdhilvA + / pVIC40.

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9.2 Preparation of L-isoleucine

The preparation of L-isoleucine by strains B-3996kur \ DeltatdhilvA + / pVIC40 and B3996kur \ DeltatdhilvA + \ DeltapckA / pVIC40 is tested in the test conditions described in Example 4. The medium minimum, the preculture medium and the production medium are additionally supplement with 20 µg / ml streptomycin.

The result of the experiment is shown in the Table 6.

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TABLE 6

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Example 10 Preparation of L-valine with the strain B-12288 \ DeltapckA

The AJ 11502 strain of E. coli producing L-valine is described in patent specification US-A-4,391,907 and has been deposited with the National Center for Agricultural Use Research (Peoria, Illinois, USA). .) as NRRL B-12288.

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10.1 Specific mutagenesis of the position of the pckA gene in E. coli strain B-1288

After cultivation in LB medium free of antibiotics for about 6 generations, a plasmid-free derivative of strain AJ 11502. The strain formed is sensitive to ampicillin and is designated AJ 11502kur.

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To replace the pckA chromosomal gene with the deletion construct encoded by the plasmid, AJ 11502kur is transformed with the plasmid pMAK705? (See Example 2). Gene replacement is carried out by the selection method described by Hamilton et al . (1989) Journal of Bacteriology 174, 4617-4622) and is checked by standard PCR methods (Innis et al . (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press) with the following oligonucleotide primers:

fifteen

The strain obtained is designated AJ11502kur \ DeltapckA. The plasmid described in memory patent description US-A-4,391,907, which bears the genetic information regarding the production of valine, it isolates strain NRRL B-12288. Strain AJll502kur \ DeltapckA is transformed with this plasmid. One of the obtained transformants is designated B-12288 ΔpckA.

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10.2 Preparation of L-valine with the strain B-12288 \ DeltapckA

The formation of L-valine by strains B-12288 \ DeltapckA and NRRL B-12288 is checked in 10 ml batch cultures contents in conical flasks of 100 ml. For this, 10 are inoculated ml of preculture medium of the following composition: 2 g / l yeast extract, 10 g / l (NH 4) 2 SO 4, 1 g / l KH 2 PO 4, 0.5 g / l MgSO 4 * 7H 2 O, 15 g / l CaCO 3, 20 g / l glucose and 50 mg / l ampicillin, and the batch is incubated for 16 hours at 37 ° C and 180 rpm in an ESR incubator from Kühner AG (Birsfelden, Switzerland). 250 µl of this preculture is transinoculated in 10 ml of production medium (25 g / l (NH 4) 2 SO 4, 2 g / l KH 2 PO 4, 1 g / l MgSO 4 * 7H 2 O, 0.03 g / l FeSO 4 * 7H 2 O, 0.018 g / l MnSO 4 • 1H 2 O, 30 g / l CaCO 3, 20 g / l glucose, 5 mg / l thiamine and 50 mg / ml ampicillin) and the batch is incubated for 72 hours at 37 ° C. After incubation, the density is determined Optics (OD) of the culture suspension with an LP2W photometer Dr. Lange (Berlin, Germany) at a measured wavelength of 660 nm

L-valine concentration formed is then determined in the culture supernatant filtered in sterile conditions with an amino acid analyzer of Eppendorf-BioTronik (Hamburg, Germany) by ion exchange and reaction chromatography post-column with ninhydrin detection.

The result of the experiment is shown in the Table 7.

TABLE 7

16

Example 11

(It is not part of the invention)

Construction of deletion mutations of the gene region ytfP-yjfA

The ytfP-yjfA gene region is amplified from Escherichia coli K12 using the polymerase chain reaction (PCR) and synthetic oligonucleotides. Starting from the nucleotide sequence of the ytfP-yjfA gene region in E. coli K12 strain MG1655 (SEQ ID No. 5), the following PCR primers are synthesized (MWG Biotech, Ebersberg, Germany):

17

The chromosomal DNA of E. coli K12 MG1655 used for PCR is isolated according to the manufacturers instructions with "Qiagen Genomic-tips 100 / G" (QIAGEN, Hilden, Germany). A DNA fragment of a size approx. 1300 bp can be amplified with specific primers under standard PCR conditions (Innis et al ., (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press) with Taq DNA polymerase (Gibco-BRL, Eggenstein, Germany). The PCR product is ligated with the vector pCR2.1TOPO (Cloning Kit TOPO TA, Invitrogen, Groningen, The Netherlands) according to the manufacturers instructions and transformed into E. coli TOP10F 'strain. The selection of plasmid carrier cells takes place in LB agar, to which 50 µg / ml of ampicillin is added. After isolation of the plasmid DNA, the successful cloning of the PCR product is checked with the restriction enzymes EcoRI and NsiI.

To generate a deletion of 337 bp in the region yftP-yjfA, the vector is cleaved pCR2.1TOPOytfP-yjfA with restriction enzymes NdeI and SspI, and the 4.8 kbp DNA fragment is ligated, after Klenow enzyme treatment.

To generate a 90 bp deletion, the vector pCR2.1TOPOytfP-yjfA is cleaved with NdeI enzymes and SplI, and the 5 kbp DNA fragment is ligated, after Klenow enzyme treatment.

E. coli strain DH5α is transformed with the ligation batches, and the plasmid-bearing cells are selected on LB agar, to which 5 0 µg / ml ampicillin is added. After isolation of plasmid DNA, those plasmids in which the mutagenic DNA sequence depicted in SEQ ID No. 6 and SEQ ID No. 7 is cloned are detected by control cleavage with the EcoRI enzyme. Plasmids are designated pCR2.1TOPO? And JfA and pCR2.1TOPO? 90bp.

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Example 12

(It is not part of the invention)

Construction of replacement vectors pMAK705 \ DeltayjfA and pMAK705 \ Delta90bp

The ytfP-yjfA alleles described in Example 11 are isolated from the pCR2.1TOPO? And pCR2.1TOPO? 90bp vectors after restriction with SacI and XbaI enzymes and 0.8% agarose gel separation, and ligated with the plasmid pMAK705 (Hamilton et al . (1989) Journal of Bacteriology 174, 4617-4622), which is digested with the enzymes SacI and XbaI. Ligation batches are transformed into DH5α and plasmid carrying cells are selected on LB agar, to which 20 µg / ml of chloramphenicol are added. Successful cloning is demonstrated after plasmid DNA isolation and excision with SacI and XbaI enzymes. The replacement vectors formed, pMAK705 \ DeltayjfA
(= pMAK705deltayjfA) and pMAK705 \ Delta90bp (= pMAK705delta90bp) are shown in Figure 2 and Figure 5.

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Example 13

(It is not part of the invention)

Specific mutagenesis of the position of the region of the ytfP-yjfA gene in E. coli strain MG442

To replace the region of the ytfP-yjfA chromosomal gene with the 90 bp deletion construct encoded by the plasmid, MG442 is transformed with the plasmid pMAK705 Δ90bp. Gene replacement is carried out by the selection method described by Hamilton et al . (1989) Journal of Bacteriology 174, 4617-4622) and is checked by standard PCR methods (Innis et al ., (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press) with the following oligonucleotide primers:

18

The strain obtained is designated MG442 Δ90yjfA.

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Example 14

(It is not part of the invention)

Preparation of L-threonine with the strain MG442 \ Delta90yjfA

The preparation of L-threonine by strain MG442 Δ90yjfA is tested as described in the Example 4. The result of the experiment is summarized in Table 8.

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TABLE 8

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Example 15 Preparation of L-threonine with the strain MG442 \ Delta90yjfA \ DeltapckA 15.1 Preparation of the strain MG442 \ Delta90yjfA \ DeltapckA

The pckA gene of strain MG442 Δ90yjfA is replace, as described in Example 3, with the allele ΔpckA (see Examples 1 and 2). The strain obtained is designates MG442 \ Delta90yjfA \ DeltapckA.

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15.2 Preparation of L-threonine

The preparation of L-threonine with strain MG442 Δ90yjfA ΔpckA is carried out as described in Example 4. The result is shown in Table 9.

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TABLE 9

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Brief description of the figures

Figure 1: pMAK705 ΔpckA (= pMAK705deltapckA)

Figure 2: pMAK705 ΔyjfA (= pMAK705deltayjfA)

Figure 3: pMW218gdhA

Figure 4: pMW219rhtC

Figure 5: pMAK705 Δ90bp (= pMAK705delta90bp)

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Length data should be understood as approximate data. Abbreviations and designations used They have the following meaning:

cat: Resistance gene to chloramphenicol

Rep-ts: Region of temperature sensitive replication of plasmid pSC101

pck1: Part of the 5 'region of the gene pckA

pck2: Part of the 3 'region of the gene pckA

y ytfP'-yjfA ': Sequence of DNA containing truncated coding regions of ytfP e yjfA

kan: Resistance gene to Kanamycin

gdhA: Gene of glutamate dehydrogenase

rhtC: Gene that imparts resistance to threonine

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Abbreviations for restriction enzymes They have the following meaning:

BamHI: restriction endonuclease of Bacillus amyloliquefaciens

BglII: Bacillus globigii restriction endonuclease

ClaI: Caryphanon latum restriction endonuclease

EcoRI: restriction endonuclease of Escherichia coli

EcoRV: restriction endonuclease of Escherichia coli

HindIII: restriction endonuclease of Haemophilus influenzae

KpnI: restriction endonuclease of Klebsiella pneumoniae

PstI: Providence stuartii restriction endonuclease

PvuI: Proteus vulgaris restriction endonuclease

SacI: restriction endonuclease of Streptomyces achromogenes

SalI: restriction endonuclease from Streptomyces albus

SmaI: restriction endonuclease from Serratia marcescens

XbaI: Xanthomonas badrii restriction endonuclease

XhoI: restriction endonuclease of Xanthomonas holcicola

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<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (3)

         \ vskip0.400000 \ baselineskip
      

<223> Initial cotton of the delta allele pckA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (598)

         \ vskip0.400000 \ baselineskip
      

<223> Region 5 'of the delta pckA allele

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> (599) .. (618)

         \ vskip0.400000 \ baselineskip
      

<223> Technical DNA / sequence residues of the polylinker

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> (619) .. (1291)

         \ vskip0.400000 \ baselineskip
      

<223> Region 3 'of the delta pckA allele

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> (1292) .. (1294)

         \ vskip0.400000 \ baselineskip
      

<223> Delta allele stop cod pckA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 4

         \ vskip1.000000 \ baselineskip
      

28

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 5

         \ vskip0.400000 \ baselineskip
      

<211> 1248

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Escherichia coli

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> gene

         \ vskip0.400000 \ baselineskip
      

<222> (376) .. (714)

         \ vskip0.400000 \ baselineskip
      

<223> ORF ytfP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> gene

         \ vskip0.400000 \ baselineskip
      

<222> Complement ((461) .. (727))

         \ vskip0.400000 \ baselineskip
      

<223> ORF yjfA

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 5

         \ vskip1.000000 \ baselineskip
      

29

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 6

         \ vskip0.400000 \ baselineskip
      

<211> 911

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Escherichia coli

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (911)

         \ vskip0.400000 \ baselineskip
      

<223> Region ytfP-yjfA deletion carrier

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (383)

         \ vskip0.400000 \ baselineskip
      

<223> 5 'side of the region ytfP-yjfA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> (384) .. (911)

         \ vskip0.400000 \ baselineskip
      

<223> 3 'side of the region ytfP-yjfA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> (376) .. (318)

         \ vskip0.400000 \ baselineskip
      

<223> ATF cotton of the truncated ORF and tfP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> Complement ((388) .. (390))

         \ vskip0.400000 \ baselineskip
      

<223> ATF cotton of the ORF and truncated jjA

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 6

         \ vskip1.000000 \ baselineskip
      

30

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 7

         \ vskip0.400000 \ baselineskip
      

<211> 1158

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Escherichia coli

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (1158)

         \ vskip0.400000 \ baselineskip
      

<223> Region ytfP-yjfA deletion carrier

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> (1) .. (630)

         \ vskip0.400000 \ baselineskip
      

<223> 5 'side of the region ytfP-yjfA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> (631) .. (1158)

         \ vskip0.400000 \ baselineskip
      

<223> Flank 3'of the region ytfP-yjfA

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> (376) .. (378)

         \ vskip0.400000 \ baselineskip
      

<223> ATF cotton of the truncated ORF and tfP

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> Mixed feature

         \ vskip0.400000 \ baselineskip
      

<222> Complement ((635) .. (637))

         \ vskip0.400000 \ baselineskip
      

<223> ATF cotton of the ORF and truncated jjA

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 7

31

         \ vskip0.400000 \ baselineskip
      

<210> 8

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence description artificial: Initiator pckA '5'-1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 8

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

100

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 9

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence description artificial: Initiator pckA '5'-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 9

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

101

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 10

         \ vskip0.400000 \ baselineskip
      

<211> 22

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence description artificial: Initiator pckA '3'-1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 10

         \ vskip1.000000 \ baselineskip
      

         \ hskip1cm
      

102

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 11

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence description artificial: Initiator pckA '3'-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 11

         \ hskip1cm
      

103

         \ vskip0.400000 \ baselineskip
      

<210> 12

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence description artificial: Gdh1 initiator

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 12

         \ hskip1cm
      

104

         \ vskip0.400000 \ baselineskip
      

<210> 13

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence description artificial: Gdh2 initiator

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 13

         \ hskip1cm
      

105

         \ vskip0.400000 \ baselineskip
      

<210> 14

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence description artificial: RhtC1 initiator

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 14

         \ hskip1cm
      

106

         \ vskip0.400000 \ baselineskip
      

<210> 15

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence description artificial: RhtC2 Initiator

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 15

         \ hskip1cm
      

107

         \ vskip0.400000 \ baselineskip
      

<210> 16

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence description artificial: Tdh1 initiator

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 16

         \ hskip1cm
      

108

         \ vskip0.400000 \ baselineskip
      

<210> 17

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence description artificial: Tdh2 initiator

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 17

         \ hskip1cm
      

109

         \ vskip0.400000 \ baselineskip
      

<210> 18

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<223> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<200>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence description artificial: Initiator ytfP-1

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 18

         \ hskip1cm
      

110

         \ vskip0.400000 \ baselineskip
      

<210> 19

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Sequence description artificial: Initiator ytfP-2

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 19

         \ hskip1cm
      

111

Claims (28)

1. Fermentation process for preparation of L-amino acids, where the next steps:

to)

fermentation of microorganisms of the Enterobacteriaceae family that produce the Desired L-amino acid, in whose microorganisms the minus the pckA gene or nucleotide sequences encoding the pckA gene product are attenuated,

b)

enrichment of L-amino acid in the medium or in the cells bacterial, and

C)

treatment of L-amino acid, or d) constituents of the broth of fermentation and isolation of biomass in its entirety or portions of it as a solid product along with the L-amino acid

         \ vskip1.000000 \ baselineskip
      

2. Process according to claim 1, where microorganisms are used in which other genes of the biosynthetic pathway of the desired L-amino acid is amplify further. 3. Process according to claim 1, where microorganisms are used in which the paths metabolic that reduce the formation of L-amino acid desired are deactivated at least partially. 4. Process according to claim 1, wherein the expression of the polynucleotide (s) encoding the pckA gene product is disabled. 5. Process according to claim 1, wherein the regulatory and / or catalytic properties of the polypeptide  (enzymatic protein) encoded by the pckA polynucleotide are reduced 6. Process according to claim 1, where microorganisms of the Enterobacteriaceae family are ferment, in which one or more genes selected from the group which includes:

6.1

he thrABC operon encoding aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase,

6.2

he pyc gene encoding pyruvate carboxylase,

6.3

he pps gene that encodes phosphoenolpyruvate synthase,

6.4

he ppc gene encoding phosphoenolpyruvate carboxylase,

6.5

the pntA and pntB genes encoding transhydrogenase,

6.6

he rhtB gene for homoserine resistance, and

6.7

he rhtC gene for threonine resistance,

6.8

he gdhA gene encoding glutamate dehydrogenase is amplify simultaneously.

         \ vskip1.000000 \ baselineskip
      

7. Process according to claim 1, where family microorganisms are fermented Enterobacteriaceae in which one or more genes selected from the group comprising:

7.1

he tdh gene encoding threonine dehydrogenase,

7.2

he mdh gene encoding malate dehydrogenase,

7.3

he gene product of the open reading frame (orf) yjfA, and

7.4

he gene product of the open reading frame (orf) ytfP,

they dim, or they reduce their expression.

         \ vskip1.000000 \ baselineskip
      

8. Process according to claim 1, where L-isoleucine is prepared, L-valine, L-lysine or L-threonine 9. Plasmid pMAK705 \ DeltapckA, which contains parts of the 5 'and 3' regions of the pckA gene, corresponding to SEQ ID No. 3.

         \ newpage
      

10. Polynucleotide isolated from microorganisms of the Enterobacteriaceae family that contain a sequence of polynucleotides that encode the 5 'and 3' regions of the pckA gene, represented in SEQ ID No. 4, which is particularly suitable as plasmid constituent for the specific mutagenesis of pckA gene position. 11. Producing strains of L-threonine of the Enterobacteriaceae family that contain a deletion mutation in the pckA gene, corresponding to SEQ ID No. 4. 12. Producing strains of L-threonine of the Enterobacteriaceae family of according to claim 11, which additionally contain a deletion mutation in the open reading frame and tfP, corresponding to SEQ ID No. 6 or 7. 13. Producing strains of L-threonine of the Enterobacteriaceae family of according to claim 11, which additionally contain a deletion mutation in the open reading frame yjfA, corresponding to SEQ ID No. 6 or 7. 14. Producing strains of L-threonine of the Enterobacteriaceae family of according to claim 11, wherein they have one or More features selected from the group comprising: acid resistance α-amino-? -hydroxyvaleric,  homoserine dehydrogenase Amplified I-aspartate kinase I in the form resistant to feedback, partial need optionally compensable for L-isoleucine, attenuated threonine dehydrogenase and ability to use sucrose 15. Escherichia coli K-12 strain MG442 \ DeltapckA, deposited under DSM 13761 in the Deutsche Sammlung für Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures). 16. The B3996kur \ DeltatdhpckA / pVIC40 strain of Escherichia coli K12, deposited under the DSM number 14150 in the Deutsche Sammlung für Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures).