Polynucleotide3 encoding polypeptide eliciting activity of glucose-6-phosphate isomerase, expression plasmid vector, method for enhancing metabolism intensity in pentose phosphate cycle and method for preparing l-amino acids

FIELD: biotechnology, biochemistry, amino acids.

SUBSTANCE: invention describes a polynucleotide showing activity of glucose-6-phosphate isomerase and comprising polynucleotide sequence taken among the group including: a) polynucleotide encoding polypeptide that comprises amino acid sequence identical at least by 90% with amino acid sequence represented in SEQ ID NO:2; b) polynucleotide that is complementary with polynucleotides given in sub-paragraph a). Also, invention describes a method for enhancing the metabolism intensity in pentose phosphate cycle by attenuation of pgi gene and a method for preparing L-amino acids. Invention provides preparing L-amino acids with the high effectiveness degree.

EFFECT: improved preparing method, valuable properties of polynucleotide.

16 cl, 7 dwg, 3 tbl, 6 ex

 

The invention relates to an encoding nucleotide sequences of the gene pgi from corynebacteria and to a method of enhancing the intensity of metabolism in pentose-phosphate cycle by loosening the gene pgi.

Background of invention

Nucleotides, vitamins and predpochtitelno L-amino acids, most preferably lysine and tryptophan, used in the food industry, animal feed, medicine and in the pharmaceutical industry.

It is known that these substances get enzymatically by using strains of corynebacteria, primarily using Corynebacterium glutamicum. Because these products are of great importance, we are constantly working on improving the way they are received. Improvements of the method may relate to technical aspects of the process of fermentation, such as stirring and supply of oxygen, or the composition of the nutrient media, such as the concentration of sugar in the fermentation process, or processing of the product, for example, by using ion-exchange chromatography, or intrinsic to the microorganism productivity.

To improve the productivity of these microorganisms used methods of mutagenesis, selection and selection of mutants. In this way receive the strains that are resistant to antimetabolites or are auxotrophy in relation with p the untranslated function important intermediate products and produce nucleotides, vitamins or amino acids.

For a number of years to improve producing nucleotides, vitamins and L-amino acids strains of Corynebacterium also apply the methods of recombinant DNA.

The usual source for obtaining these compounds is glucose, which is generally used in the form of hydrolyzed starch. As the original product also use sucrose.

When the cellular absorption of glucose undergoes phosphorylation uptake phosphoenolpyruvate (phosphotransferase system) (Malin and Bourd, Journal of Applied Bacteriology 71, 517-523 (1991)) and then enters the cell in the form of glucose-6-phosphate. Sucrose is converted into fructose and glucose-6-phosphate using phosphotransferase system (Shio and others, Agricultural and Biological Chemistry 54, 1513-1519 (1990)) and by interaction with invertase (Yamamoto and others, Journal of Fermentation Technology 64, 285-291 (1986)).

In the process of catabolism of glucose by the enzyme glucose-6-phosphatedehydrogenase (KF 1.1.14.9) and glucose-6-phosphate-isomerase (EC 5.3.1.9) compete with each other in relation to the substrate glucose-6-phosphate. The enzyme glucose-6-phosphate-isomerase catalyzes the first step of the reaction path Embden-Meyerhoff-Parnas (Embden-Meyerhof-Parnas) or glycolysis, namely the conversion of fructose-6-phosphate. The enzyme glucose-6-phosphate dehydrogenase catalyzes the first step of the reaction of oxidative part of the pentose-phosphate cycle, namely pre the treatment in 6-phosphogluconolactonase.

Oxidative stage of the pentose-phosphate cycle, glucose-6-phosphate is converted in ribulose-5-phosphate, resulting in formation of redox equivalents in the form reduced nicotinamide-adenine-dinucleotide (NADPH). If pentose-phosphate cycle continues, pentosephosphate, ecosafety and triosephosphate transformed into each other. Pentosephosphate, such as 5-phosphoribosyl-1-pyrophosphate, actively involved, for example, in the biosynthesis of nucleotides. In addition, 5-phosphoribosyl-1-pyrophosphate is a precursor to aromatic amino acids and amino acids such as L-histidine. NADPH acts as a restorative equivalent in many ways biosynthesis of steroids. For the biosynthesis of one molecule of L-lysine from octoxynol acid requires four molecules of NADPH.

It is known that the pentose-phosphate cycle is important for the biosynthesis and production of amino acids, especially L-lysine, corynebacteria, and therefore it is of interest to many professionals.

In particular, Oishi and Aida (Agricultural and Biological Chemistry 29, 83-89 (1965)described hexosaminidase shunt" Brevibacterium ammoniagenes. Studies of glucose metabolism in the fermentation of glutamic acid and lysine, held Ishino and others (Journal of General and Applied Microbiology 37, 157-165 (1991)) using methods based on the use of isotope13With, detect is whether the correlation between producyrovaniem lysine and intensity of metabolism in pentose-phosphate cycle.

The objective of the invention

The objective of the invention is to develop a method of increasing the rate of metabolism in pentose-phosphate cycle.

Description of the invention

Nucleotides, vitamins, and above all L-amino acids, most preferably L-lysine and L-tryptophan, used in the food industry, animal feed, medicine and in the pharmaceutical industry. Therefore, a common interest to develop new and improved methods of obtaining these products.

The object of the invention is selected polynucleotide containing a polynucleotide sequence selected from the group including

a) polynucleotide identical to at least 70% of polynucleotide, codereuse polypeptide that contains the amino acid sequence represented in SEQ ID NO:2,

b) polynucleotide that encodes a polypeptide that contains an amino acid sequence identical to at least 70% amino acid sequence represented in SEQ ID NO:2,

C) polynucleotide, complementary polynucleotides specified in subparagraph (a) or (b), and

d) polynucleotide containing at least 15 consecutive base pairs polynucleotide sequence specified in subparagraph (a), b) or C).

The object of the invention is also polynucleotide, preferably with is capable of containing a series of DNA replication which includes:

(I) the nucleotide sequence presented in SEQ ID NO:1, or

(II) at least one sequence that corresponds to the sequence specified in subparagraph (I), within the degeneracy of the genetic code, or

(III) at least one sequence that hybridizes with a sequence complementary to the sequence specified in subparagraph (I) or (II), and optional

(IV) functionally neutral sense mutations in the sequence specified in subparagraph (I).

The objects of the invention are:

polynucleotide comprising the nucleotide sequence represented in SEQ ID NO:1,

polynucleotide that encodes a polypeptide that contains the amino acid sequence represented in SEQ ID NO:2,

a vector containing specified above in subparagraph (d) polynucleotide primarily RMS, deposited in E. coli strain DSM 12969, and

Corynebacterium, serving as host cells containing the above-mentioned vector.

The term "isolated" in the context of the present invention refers to any element that is isolated from its natural environment.

The concept of "polynucleotide" generally refers to polyribonucleotides and polyethoxylated, which can each be unmodified RNA or the NC, or modified RNA or DNA.

The term "polypeptide" refers to peptides or proteins containing two or more amino acids that are linked by peptide bonds.

The polypeptides according to the invention include a polypeptide, the sequence of which is presented in SEQ ID NO:2, primarily with the biological activity of glucose-6-phosphate isomerase, and a polypeptide identical to the polypeptide sequence of which is presented in SEQ ID NO:2, at least 70%, preferably at least 80% and most preferably identical to at least 90-95% of the polypeptide sequence of which is presented in SEQ ID NO:2, and having the specified activity.

The object of the invention is also a method of enzymatic get nucleotides, vitamins, and above all L-amino acids, most preferably lysine and tryptophan, using Corynebacterium, especially those who already have the ability to produce these materials and in which weaken, primarily to provide a low level of expression of a coding nucleotide sequences of the gene pgi.

The term "attenuation" in the context of the present description means the reduction or suppression of the microorganism intracellular activity of one or more enzymes (proteins)encoded correspond to what her DNA, due to, for example, the use of a weaker promoter or a gene or allele that encodes the corresponding enzyme with a low activity or inactivating the corresponding enzyme (protein), and not necessarily due to the combination of these measures.

The microorganisms, which is the object of the present invention, can produce nucleotides, vitamins, and above all L-amino acids, most preferably lysine and tryptophan, glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. These microorganisms may include representatives of corynebacteria, especially of the genus Corynebacterium. In the genus Corynebacterium is particularly noteworthy species Corynebacterium glutamicum, which, as we all know has the ability to produce L-amino acids.

Suitable strains of bacteria R. Corynebacterium, primarily species Corynebacterium glutamicum, are, for example, the following well-known wild-type strains:

Corynebacterium glutamicum ATCC 13032,

Corynebacterium acetoglutamicum ATCC 15806,

Corynebacterium acetoacidophilum ATCC 13870,

Corynebacterium thermoaminogenes FERM BP-1539,

Brevibacterium flavum ATCC 14067,

Brevibacterium lactofermentum ATCC 13869 and

Brevibacterium divaricatum ATCC 14020, and derived mutants, respectively strains producing nucleotides, vitamins or L-amino acids, for example, the following producing 5'-inozinovu acid strains:

Corynebacterium ammoniagenes ATCC 15190,

Corynebacterium ammoniagenes ATCC 15454 and

Corynebacterium glutamicum ATCC 14998, or for example, the following producing 5'-voinilovo acid strains:

Corynebacterium glutamicum ATCC and 21171

Corynebacterium ammoniagenes ATCC 19216, or for example, the following producing D-Pantothenic acid strains:

Corynebacterium glutamicum ATCC 13032/pECM3ilvBNCD,pEKEx2panBC and

Corynebacterium glutamicum ATCC 13032/pND-D2, or, for example, the following producing L-lysine strains:

Corynebacterium glutamicum FERM-P 1709,

Brevibacterium flavum FERM-P 1708,

Brevibacterium lactofermentum FERM-P 1712,

Corynebacterium glutamicum FERM-P 6463,

Corynebacterium glutamicum FERM-P 6464

Corynebacterium glutamicum DSM 5714, or, for example, the following producing L-tryptophan strains:

Corynebacterium glutamicum ATCC and 21850

Corynebacterium glutamicum KY9218(pKW9901).

When creating inventions failed to allocate new gene pgi from .glutamicum encoding the enzyme glucose-6-phosphate-isomerase (EC 5.3.1.9).

To highlight the pgi gene, and other genes from .glutamicum first create a library of genes of this organism in E. coli. Creating a library of genes described in well-known textbooks and reference books. As examples tutorial Winnacker: Gene und Klone, Einein die Gentechnologie (Izd-vo Chemie, Weinheim, Germany (1990)) or reference Sambrook and others: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press (1989)). One of the well-known libraries of genes is a library of genes of strain E. coli K-12 W3110, created Kohara and others (Cell 50, 495-508 (1987)) λ-vectors. We Bathe and others (olecular and General Genetics, 252: 255-265 (1996)) describes a library of genes of strain .glutamicum ATCC 13032, obtained using kosmidou vector SuperCos I (Wahl and others, Proceedings of the National Academy of Sciences USA, 84:2160-2164 (1987)) in the strain E. coli K-12 NM554 (Raleigh and others, Nucleic Acids Research 16, 1563-1575 (1988)). In turn, theand others (Molecular Microbiology 6(3), 317-326) described a library of genes of strain .glutamicum ATCC 13032, obtained using Comedy RNs (clear Hohn and Collins, Gene 11, 291-298 (1980)). The O Donohue (The Cloning and Molecular Analysis of Four Common Aromatic Amino Acid Biosynthetic Genes from Corynebacterium glutamicum, doctoral thesis, national University of Ireland, Galway, 1997) described the cloning of genes .glutamicum using the expression system λ Zap described Short and others (Nucleic Acids Research, 16:7583).

To create a library of genes .glutamicum in E. coli can also be used such plasmids pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Viera and others, Gene, 19:259-268 (1982)). As hosts primarily can be used such strains of E. coli, which have defects resulting from restriction and recombination, for example strain DH5α (Jeffrey H. Miller: "A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria", Cold Spring Harbor Laboratory Press (1992)).

Then the library of genes inserted into the indicator strain by transformation (Hanahan, Journal of Molecular Biology 166, 557-580 (1983)) or by electroporation (Tauch and others, FEMS Microbiological Letters, 123:343-347 (1994)). The indicator strain is characterized by the presence of m is to be treated in this gene which leads to a detectable phenotype. For the purposes of the present invention is of interest mutant E. coli DF1311 described Kupor and Fraenkel (Journal of Bacteriology 100: 1296-1301 (1969)). This strain carries a mutation in the genes pgi and pgl, resulting in growth on glucose inhibited to a significant extent. After transformation with a vector containing the gene pgi, growth on glucose is restored. An example of such a vector containing the gene pgi is rams (figure 1).

Then cloned using cosmid or other λ-vectors of long DNA fragments can be subcloned in normal suitable for DNA sequencing vectors.

Methods for DNA sequencing are described inter alia in Sanger and others (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467 (1977)).

After that, the obtained DNA sequences can be studied using well-known algorithms or programs of the sequence analysis, such as program created Staden (Nucleic Acids Research 14, 217-232 (1986)), the GCG program created by Butler (Methods of Biochemical Analysis 39, 74-97 (1998)), the FASTA algorithm, developed by Pearson and Lipman (Proceedings of the National Academy of Sciences of the USA, 85, 2444-2448 (1988)), or the BLAST algorithm developed by Altschul and others (Nature Genetics 6, 119-129 (1994))and compared with data sequences that can be derived from the available scientific community databases. Available societies is nasty databases of nucleotide sequences are for example, the database of the European molecular biology laboratory the European Molecular Biology Laboratory (EMBL), Heidelberg, Germany) or the database of the National center for biotechnology information (NCBI, Bethesda, Maryland, Columbia, Maryland, USA).

Using these methods was granted a new coding DNA sequence of the gene pgi from .glutamicum, which is presented in the present description as SEQ ID NO:1. In addition, this DNA sequence using the methods described above, was deduced amino acid sequence of the corresponding protein. In the sequence of SEQ ID NO:2 shows the deduced amino acid sequence of the pgi gene product.

Encoding DNA sequence is derived from SEQ ID NO:1 based on the degeneracy of the genetic code, are also part of the invention. Likewise part of the invention are DNA sequences which hybridize with SEQ ID NO:1 or parts of SEQ ID NO:1. Finally, the DNA sequences obtained by polymerase chain reaction (PCR) using primers derived from SEQ ID NO:1, are also part of the present invention.

Recommendations for identifying DNA sequences by means of hybridization can be found, in particular, in the Handbook "The DIG System Users Guide for Filter Hybridization" is Irma Boehringer Mannheim GmbH (Mannheim, Germany (1993)and in Liebl and other (International Journal of Systematic Bacteriology 41:255-260 (1991)). Recommendations for amplification of DNA sequences using the polymerase chain reaction (PCR) can be found, inter alia, in the Handbook Gait: Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (published in Spectrum Akademischer Verlag, Heidelberg, Germany,1994).

In the claimed invention, it was found that the relaxation of pgi gene in Corynebacterium can be enhanced intensity of road metabolism in pentose-phosphate cycle and the efficiency of the synthesis of nucleotides, vitamins, and above all L-amino acids, most preferably L-lysine and L-tryptophan.

The weakening can be achieved by reducing or suppressing any expression of pgi gene or the catalytic properties of the protein enzyme. Both methods are optional can be used together.

To reduce gene expression by the corresponding control culture or by genetic modification (mutation) of the signal structures that control gene expression. Signal structures that control gene expression are, for example, genes repressor substances, genes promoters, operators, promoters, attenuators, the binding sites of the ribosome, the initiating codon and a terminator. Relevant information can be found, for example, in WO 96/15246, Boyd and Murphy (Journal of Bacterology 170:5949 (1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998)), in Jensen and Hammer (Biotechnology and Bioengineering 58:191 (1998)), Patek and others (Microbiology 142:1297 (1996)) and in known textbooks on genetics and molecular biology, as, for example, the textbook Knippers ("Moleculare Genetik", 6th ed., published by Georg Thieme Verlag, Stuttgart, Germany, 1995) or Winnacker ("Gene und Klone", published by VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

Mutations that can lead to modification or loss of catalytic properties of proteins, enzymes, known from the prior art; as examples should be called articles Qiu and Goodman (Journal of Biological Chemistry 272:8611-8617 (1997)), Sugimoto and others (Bioscience Biotechnology and Biochemistry 61:1760-1762 (1997)) and("Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der allosterischen Regulation und Struktur des Enzyms", reports of the Research centre jülich (Forschungszentrum), Jul-2906, ISSN 09442952, jülich, Germany, 1994). A review of publications can be found in known textbooks on genetics and molecular biology such as, for example, the textbook Hagemann ("Allgemeine Genetik", published by Gustav Fischer Vertag, Stuttgart, 1986).

Mutations that can be used for this purpose are the transitions, insertions, deletions and transverse. Depending on the impact of the replacement of amino acids on enzyme activity mutation is divided into missense mutations and nonsense mutations. Insertions or deletions of at least one base pair in a gene lead to mutations shift of the frame, resulting in the incorporation of incorrect amino acids or premature termination of translation. Deletions of two or more codons, as a rule, lead to a complete disruption of the enzymatic activity. Methods of creating such mutations are known, and recommendations can be found in known textbooks on genetics and molecular biology such as, for example, the textbook Knippers ("Moleculare Genetik", 6th ed., published by Georg Thieme Verlag, Stuttgart, Germany, 1995), Winnacker ("Gene und Klone", published by VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or Hagemann ("Allgemeine Genetik", published by Gustav Fischer Verlag, Stuttgart, 1986).

Example of the implementation of mutagenesis by insertion plasmid is RMS (figure 2), which may be subjected to mutation of the gene pgi. Plasmid RMS is a plasmid pBGS8 described Spratt and others (Gene 41:337 (1986)), in which the integrated internal fragment of the gene pgi, presented as SEQ ID NO:3. After transformation and homologous recombination of the gene pgi (insertion) this plasmid leads to a complete loss of function of the enzyme. Relevant data and explanations about the mutagenesis by insertion can be found in Schwarzer and(Bio/Technology 9, 84-87 (1991)) or in Fitzpatrick and others (Applied Microbiology and Biotechnology 42, 575-580 (1994)).

For the production of nucleotides, vitamins, and above all L-amino acids, most preferably L-lysine and L-tryptophan, m which can be appropriate along with the weakening of the gene pgi to add, first of all sverkhekspressiya, one or more enzymes involved in the particular biosynthesis pathway.

For example, for the production of nucleotides can

at the same time sverkhekspressiya purF gene encoding glutamine-PRPP (phosphoribosylpyrophosphate)amidotransferase and/or

at the same time sverkhekspressiya gene carAB encoding carbamoyltransferase.

For producing L-lysine, for example,

at the same time sverkhekspressiya dapA gene encoding digidrive-colinet synthase (EP-0197335), and/or

at the same time sverkhekspressiya gdh gene encoding glutaraldehydes (Bormann and other Molecular Microbiology 6, 317-326 (1992)), and/or

- at the same time to amplify the DNA fragment, causing the resistance to S-(2-amino-ethyl)cysteine (EP-A 0088166).

For producing L-tryptophan, for example,

at the same time sverkhekspressiya tkt gene encoding transketolase, and/or

at the same time sverkhekspressiya prs gene encoding phosphoribosyl-pyrophosphatase.

In addition, for the production of nucleotides, vitamins, and above all L-amino acids, most preferably L-lysine and L-tryptophan, it may be appropriate along with the weakening of the gene pgi to eliminate undesirable side reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms", Overproduction of Microbial Products, Ed. by Krumphanzl, Sikyta, Vanek, ed-what about Academic Press, London, UK, 1982).

Microorganisms containing the proposed polynucleotide, are also an object of the invention, and for the production of nucleotides, vitamins, and above all L-amino acids, most preferably L-lysine and L-tryptophan, they can be cultivated continuously or periodically using a periodic process (cultivation of the parties) or periodic process with water or periodic process with repeated injections. An overview of known cultivation methods is presented in the textbook Chmiel (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (published by Gustav Fischer, Stuttgart, 1991)) or in the textbook Storhas (Bioreaktoren und periphere Einrichtungen (publishing house Vieweg, Braunschweig/Wiesbaden, 1994)).

Used cultural environment must be appropriately adapted to the requirements of the particular strain. Descriptions of culture media for various microorganisms are contained in the Handbook "Manual of Methods for General Bacteriology" of the American society for bacteriology (Washington D.C., USA, 1981). As a carbon source can be used sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycera and ethanol, and organic acids such as acetic acid. These substances can be used individually or as mixtures. As the source of nitrogen can be applied organic nitrogen-containing compounds such as gelatin, yeast extract, meat extract, malt extract, liquid formed after soaking grains of corn to swell, soybean flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as mixtures. As a source of phosphorus can be applied phosphoric acid, acidic potassium phosphate or declivities, or the corresponding sodium salt. In addition, the culture medium should contain metal salts such as magnesium sulfate or iron sulfate, which are necessary for growth. In addition, in addition to the above compounds can be used such important for growth substances such as amino acids and vitamins. In addition, the culture medium can be added to the appropriate precursors. The above additives can be introduced into the culture medium in the form of a single additive or added accordingly in the process of cultivation.

To control the pH of the culture medium used with therefore, its no reason, such as sodium hydroxide, potassium hydroxide, ammonia, respectively, ammonia water, or acids, such as phosphoric acid or sulfuric acid. To control foaming add antispyware, such as polyglycolide esters of fatty acids. To maintain the stability of plasmids can be added specific environment of substances having a selective action, such as antibiotics. To maintain aerobic conditions in a culture introducing oxygen or oxygen-containing gas mixture such as air. The temperature of the culture medium is usually in the range from 20°C to 45°C, preferably from 25°C to 40°C. the Cultivation is continued until, until yields the maximum amount of lysine. Typically, this goal is achieved 10-160 PM

The intensity of metabolism in pentose-phosphate cycle determine, for example, using a culture containing as a carbon source glucose labeled with13In position C-1. This analytical method is based on the well-known fact that when glucose catabolized in pentose-phosphate cycle, the carbon atom at position C-1 is converted into carbon dioxide, whereas if it catabolized by glycolysis,13With out the s-1 is moved to position C-3 of pyruvate. To study vneck mocnych metabolites, such as lactate and especially lysine, after a certain period of time is determined using nuclear magnetic resonance or mass spectroscopy contents13In position C-3 of pyruvate. Alternatively, from biomass by acid hydrolysis can be obtained from amino acids and can then be used to determine the content of the13With certain provisions of carbon atoms in a particular amino acid. Appropriate instructions concerning, in particular, assessment using computer content13In different positions of the carbon atoms in the studied metabolites, can be found at Sonntag and others (European Journal of Biochemistry 213, 1325-1331 (1993)), Sonntag and others (Applied Microbiology and Biotechnology 44, 489-495 (1995)), Marx and others (Biotechnology and Bioengineering 49, 111-129 (1996)) and Marx and others (Biotechnology and Bioengineering 56, 168-180 (1997)).

Methods for the determination of nucleotides, vitamins and L-amino acids are known. For example, analysis of L-amino acids can be performed using anion exchange chromatography, followed by derivatization with detection by ninhydrin method described in Spackman and others (Analytical Chemistry, 30, 1190 (1958)), or they can be analyzed using GHUR with reversed phase, as described by Lindroth and others (Analytical Chemistry 51: 1167-1174 (1979)).

In accordance with the Budapest Treaty in the German collection of microorganisms and cell cultures (Deutsche SammlungMikroorganismen und Zelkulturen (DSMZ), Braunschweig, Germany) was deposited the following microorganisms:

a strain of Escherichia coli DH5α/pMC1 under the registration number DSM 12969.

Examples

Below the present invention is illustrated by examples of its implementation. The methods used in molecular biology, such as isolation of plasmid DNA processing restrictase, ligation, a standard transformation of Escherichia coli, etc. (unless otherwise specified), described in Sambrook and others (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor (1989)).

Example 1

Create a gene library of strain Corvnebacterium elutamicum AS019

Library DNA of strain Corinebacterium glutamicum AS019 (Yoshihama and others, Journal of Bacteryology 162, 591-597 (1985)) were designed using gene-expression system λ Zap Express™ (Short and others, Nucleic Acids Research, 16: 7583-7600 (1988)) using the method described by O Donohue (O Donohue M., The Cloning and Molecular Analysis of Four Common Aromatic Amino Acid Biosynthetic Genes from Corynebacterium glutamicum. doctoral dissertation, national University of Ireland, Galway, 1997). Set λ Zap Express™ was acquired by the company Stratagene (Stratagene, 11011 North Torrey Pines Rd., La Jolla, California 92037) and it was used according to manufacturer's instructions. DNA strain AS019 were digested with restriction enzyme Sau3A and by ligating embedded in the cleaved with BamHI and dephosphorylated "shoulders" λ Zap Express™.

Example 2

Cloning and sequencing of the gene pgi

1. Cloning

The strain Escherichia coli DF1311 carrying mutations in the city the fuck pgi and pgl, as described in Kupor and Fraenkel (Journal of Bacteriology 100: 1296-1301 (1969)), transformed using approximately 500 ng of library AS019 plasmid λ Zap Express™described in example 1. Selection of transformants was carried out on minimal M9 medium (Sambrook and other, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, USA (1989))containing kanamycin at a concentration of 50 mg/l, and incubation was carried out for 48 h at 37°C. one transformant were isolated plasmid DNA according to the method described by Birnboim and Doly (Nucleic Acids Research 7:1513-1523 (1979)), and this plasmid was designated as rams (figure 1).

2. Sequencing

For sequence analysis of the cloned insert rams used the method described in Sanger and others (Proceedings of the National Academy of Sciences of the USA, 74:5463-5467 (1977)), using primers are differentially labeled stained with a fluorescent label. The analysis was performed using a genetic analyzer type ABI prism 310 firm Perkin Elmer Applied Biosystems (Perkin Elmer Corporation, Norwalk, standartised, USA) and sequencing type AV prism 310 Big Dye™ Terminator Cycle Sequencing Ready Reaction also firm Perkin Elmer.

Primary sequence analysis was performed using a universal direct primer and reverse primer M13 derived from the firm Pharmacia Biotech (St. Albans, Herts, AL1 3AW, UK):

universal direct primer: one HUNDRED ATA CGA CTC ACT ATA GGG C;

reverse primer M13: GGA AAC AGC TAT GAC CAT g

p> Then from the obtained sequence was designed internal primers, which allowed for complete gene pgi. Internal primers had the following sequences:

internal primer 1: GGA AAC AGG GGA GCC GTC;

internal primer 2: TGC TGA GAT ACC AGC GGT.

Then the obtained sequence was analyzed using the program for DNA Strider (Marck, Nucleic Acids Research 16:1829-1836 (1988)), version 1.0 for computer Apple Macintosh. This program allows to carry out such tests as the detection of the most common restriction site, analysis of the open reading frame and identifying the most frequent codon. The comparison of the obtained DNA sequences with sequences from databases EMBL and Genbank was performed using the program BLAST (Altschul et al. Nucleic Acids Research, 25:3389-3402 (1997)). Comparative analysis of DNA sequences and protein was performed using the program Clustal V and Clustal W (Higgins and Sharp, Gene 73:237-244 (1988)).

Thus obtained sequence presented as SEQ ID NO:1. Analysis of the obtained nucleotide sequences allowed us to identify an open reading frame length 1650 base pairs, which was identified as the gene pgi. This gene encodes a protein consisting of 550 amino acids, and are provided as SEQ ID NO:2.

Example 3

Introduction mutatie in the gene pgi

1. Designed for the study of dezintegriruetsja vector pgi

The internal segment of the gene pgi amplified using polymerase chain reaction (PCR) using as template genomic DNA isolated from strain Corynebacterium glutamicum AS019 (Neagu and Dunican, Applied and Environmental Microbiology 59:791-799 (1993)). Used the following primers for pgi:

direct primer: ATG GAR WCC AAY GGH HH;

reverse primer: YTC CAC GCC CCA YTG RTC,

where R=A+G, Y=C+T, W=A+T, H=A+T+C.

The PCR parameters were as follows:

35 cycles

when 94°C for 1 min,

at 47°C for 1 min,

at 72°C for 30 s,

1.5 mm MgCl2,

approximately 150-200 ng DNA template.

The obtained PCR product was cloned into the commercially available vector pGEM-T provided by the company Promega Corp.(Promega UK, Southampton), using E. coli strain JM109 (Yanisch-Perron and others, Gene, 33:103-119 (1985)) as the owner. The sequence of the PCR product is presented in SEQ ID NO:3. Then the cloned insert was cut in the form of an EcoRI fragment and ligated with plasmid pBGS8 (Spratt and others, Gene 41:337-342 (1986)), pre-treated EcoRI. Used restrictase received from the firm Boehringer Mannheim UK Ltd. (Bell Lane, Lewes, East Sussex BN7 1LG, UK) and were used according to manufacturer's instructions. Then strain E. coli JM109 transformed this the resulting ligation mixture and transformants obtained by electroporation, were selected on agar, Luria, supplemented IPTG (isopropyl-β -D-thiogalactopyranoside), XGAL (5-bromo-4-chloro-3-indolyl-galactopyranoside) and kanamycin at concentrations of 1 mm, 0.02% and 50 mg/l, respectively. Agar plates were incubated for 12 h at 37°C. one transformant were isolated plasmid DNA was determined by its characteristics using restriction analysis using EcoRI, BamHI and Sall, and this plasmid was designated as RMS (figure 2).

2. Mutagenesis by insertion of the gene pgi in strain DSM 5715

After that, strain DSM 5715 transformed with plasmid RMS using the electroporation method described in Liebl and others (FEMS Microbiology Letters, 53:299-303 (1989)). Selection of transformants was performed on LBHIS-agar containing 18.5 g/l of broth used for infusion into the brain and heart, 0.5 m sorbitol, 5 g/l bacto-tryptone, 2.5 g/l extract, bacto-yeast, 5 g/l NaCl and 18 g/l bacto-agar and supplemented with 15 mg/l kanamycin and 1% fructose. Incubation was performed for 2 days at 33°C. Thus was obtained transformants 1, 2 and 3.

The resulting transformants were tested using polymerase chain reaction (PCR). For this purpose, from the obtained transformants and from strain DSM 5715 were isolated chromosomal DNA according to the method described in Eikmanns, etc. (Microbiology 140:1817-1828 (1994)). On the basis of the pgi gene sequence presented in SEQ ID NO:1, as primers were selected following oligonucleotides:

pgi-1: 5'-ACC CAC GCT GTC HUNDRED CST T is -3';

pgi-2: 5'-TGT CCC AAA TCA CGC CCT AG-3';

pgi-3: 5'-gat gat age ggc cag tgc at-3'.

These primers were synthesized by the company MWG Biotech (Ebersberg, Germany)and PCR was performed using a standard method described by Innis and others (PCR Protocols. A guide to methods and applications, Academic Press (1990)). As the matrix used chromosomal DNA of the transformants and the control used chromosomal DNA of strain DSM 5715. Each matrix used in two PCR reactions, one with a pair of primers pgi-1/pgi-2, and another pair of primers pgi-1/pgi-3.

PCR products were separated by electrophoresis on 0.8%agarose gel. Using a pair of primers pgi-1/pgi-2 in each of the four PCR reactions were obtained DNA fragment with a length of 0.5 TPN using pairs of primers pgi-1/pgi-3 only control using DNA of strain DSM 5715 was obtained amplificatory product length 0,7 TPN In experiments with chromosomal DNA from transformants was not detected PCR product.

The transformant No. 3, characterized in this way was designated as strain DSM5715::pMC1.

Example 4

Receiving lysine

Strain .glutamicum DSM5715::pMC1 obtained in example 3 was cultured in a nutrient medium suitable for production of lysine, and determined the content of lysine in the supernatant of the culture.

To do this, first strain were incubated for 24 h at 33°agar plate containing the her the appropriate antibiotic (agar environment used for infusion into the brain and heart, and containing kanamycin (25 mg/l)). This grown on agar plate culture was inoculable pre-culture (10 ml medium in Erlenmeyer flask 100 ml).

As the environment for the pre-culture was used full medium CgIII (Kase and Nakayama, Agricultural and Biological Chemistry 36 (9) 1611-1621 (1972)). In this pre-culture was added kanamycin (25 mg/l). Pre-cultures were incubated for 24 h at 33°on the shaker at 240 rpm This pre-culture was inoculable main culture so that the initial optical density (OD) measured at a wavelength of 660 nm) of the main culture was 0.1. For the main crop used environment CGC.

The composition of the medium CGC:

(NH4)2SO45 g/l
urea5 g/l
the liquid formed when soaking grains of corn to swelling (GSK)5 g/l
glucose (autoclaved separately)36 g/l
KN2RHO4/K2NRA40.5 g/l each
MgSO4·7H2O0.25 g/l
CaCl2·2H2O10 mg/l
Biotin (sterilize the p filter) 0.2 mg/l
FeSO4·7H2O10 mg/l
MnSO4·H2O10.0 mg/l
CuSO40.2 mg/l
ZnSO4·7H2O1 mg/l
NiCl2·6H2O0.02 mg/l
leucine0.15 g/l

The pH values for JSC and salt solution was brought to 7 with ammonia water and autoclaved. Then add sterile substrate and the solutions of vitamins.

The cultivation was carried out using the medium amounts to 10 ml in the Erlenmeyer flask 100 ml with deflectors. Added kanamycin (25 mg/l). Cultivation was carried out at 33°C and 80%humidity.

After 48 h was determined by optical density (OD) at a wavelength of 660 nm using a device type Biomek 1000 (firm Beckmann Instruments GmbH, Munich). The concentration of lysine formed was determined using an amino acid analyzer company Eppendorf-BioTronik (Hamburg, Germany) using ion-exchange chromatography and subsequent derivatization on columns with detection by ninhydrin.

The results of the experiment are presented in table 1.

StrainOD (660)lysine·HCl
DSM515 14,84,8
DSM5715::pMC111,57,2

Example 5

The increase in the intensity of metabolism in pentose-phosphate pathway (PFI)

The cells were pre-cultured in 10 ml of medium CGIII (Menkel and others, Applied and Environmental Microbiology 55:684-688 (1989)). Cultivation, for which used shake flasks with a volume of 100 ml, equipped with baffles, was performed within 24 h when the initial pH value of 7.0 at 33°C on a shaker with a diameter of rotation 50 mm at 250 rpm/min, the Cells were washed in 9 g/l NaCl and used for inoculation of the main culture to an optical density at 660 nm 0.1 (unit-type Biochrom Novaspec 4049, firm LKB Instrument GmbH, Gräfelfing, Germany, the width of the cuvette 10 mm). The main culture was grown in 10 ml of medium CGC (Schrumpf and others, Journal of Bacteriology 173:4510-4516 (1991)), modified by adding 5 g/l liquid formed by soaking the grains of corn to swell. In addition, the environment was added to 30 g/l [1-13With]dextrose (experiment A) or 15 g/l unlabeled dextrose+15 g/l [6-13With] dextrose (experiment B). [1-13With]dextrose (containing 99%) and [6-13With]dextrose (containing 99%) was obtained from Cambridge Isotope Laboratories, Cambridge, strassacker, USA. Cultivation, for which used shake flasks with a volume of 100 ml, equipped with baffles, conducted in accordance with the s at 72 h when the initial pH value of 7.0 at 33° With the shaker with a diameter of rotation 50 mm at 250 rpm/min After completion of fermentation was determined by optical density at 660 nm and the concentration of lysine·HCl (amino acid analyzer, the company Eppendorf-BioTronik, Hamburg, Germany). Then the biomass was removed by centrifugation at 15000 g and not containing cell supernatant was dried by freezing.

The dried powder was re-dissolved in 1 ml of D2O (of 99.98%, the firm Deutero GmbH, Kastellaun, Germany) was added as a standard 3-trimethylsilylpropyne-2,2,3,3,d4(firm MSD Isotopes, Montreal, Canada). Spectroscopic studies of nuclear magnetic resonance using the method of studying the spectrum of spin-echo and differential spectral analysis was performed using a spectrometer type AMX 400-WB (firm Bruker Analytik GmbH, Karlsruhe, Germany) according to the method described by Marx and others (Biotechnology and Bioengineering 49:111-129 (1996)), Marx and others (Metabolic Engineering 1:35-48 (1999)) and Wendisch and other Analytical Biochemistry 245:196-202 (1997)). Determined13C-enrichment in different positions of the carbon atoms in lysine and, as described below, to assess the level of metabolism in the pentose-phosphate pathway (PFI) used the provisions set forth in Marx and others (Biotechnology and Bioengineering 49:111-129(1996)), Marx and others (Biotechnology and Bioengineering 56:168-180 (1997)), Marx and others (Metabolic Engineering 1:35-48 (1999)), Sonntag and others (European Journal of Biochemistry 213:1325-1331 (1993)) and Sonntag and others (Applied Microbiology and Biotechnology 44:48-495 (1995)).

The results of spectroscopic studies by the method of nuclear magnetic resonance is shown in Fig.3-6. On spectogram a) presents the integrals divided13With spectra. Were obtained integrals of differential spectra, which are presented on spectogram b), and the integrals describing13C-enrichment in specific positions of the carbon atoms that was obtained by dividing the value of the integral given in spectogram b), the value of the integral given in pictogramme). To the carbon atom at position C-4 of lysine (marked on figa) and figb) as L-4) the value of the integral of the differential spectrum, equal 38,286, divided by the value of the integral divided13With spectrum equal to 198,867, and the result divided by 1.95, which gave the amount of enrichment, equal to 9.9% (table 2). The effectiveness of experimental studies of the spin-echo spectrum was different for different positions of the carbon atoms in lysine, as noted in Wendisch and others, (Analytical Biochemistry 245:196-202 (1997)), so the separation factor ranged from 1.80 to 1.99. The results presented in figure 3 and 5, were obtained for culturing conditions, when the environment was added [6-13With] dextrose (experiment B). The results presented in figure 4 and 6, were obtained for culturing conditions, when the environment was added [1-13With] Dec is trozo (experiment A).

Table 2

13C-Enrichment in specific positions of the carbon atoms in lysine for the parent strain DSM 5715 and strain DSM5715::pMC1 containing the mutant gene pgi. The position of the carbon atoms in the lysine labeled L-2...L-6 (cf. figure 3-6). In the last column presents the intensity of metabolism in pentose-phosphate path, standardized, taking into account the rate of absorption of dextrose. The eighth column shows the enrichment factor (B-a)/b

4,3
StrainThe code1L-2 %L-3 %L-4 %L-5 %L-6 %C.O4%Intensity %
12[1-13S]the 11.628,0of 17.028,45,6
12[6-13S]11,923,68,424,51,443±460±6
23[1-13S]1,71,514,61,71,4
23[6-13S]5,027,79,928,198±198±2
Notes:

1enriched with dextrose,

2strain 1, representing DSM 5715,

3strain 2, representing DSM5715::pMC1,

4C.O mean enrichment factor (B-a)/b

For cells grown in the experiment and in experiment B were obtained with different values of13C-enrichment in the relevant provisions of the carbon atom in lysine (table 2). In particular, the low level of enrichment in positions C3 and C5 lysine in the experiment And for the strain DSM5715::pMC1 indicates a high intensity of metabolism in the TFG. The intensity of metabolism in the TFG was determined by the ratio (B-a)/B, where a denotes the full13C-enrichment in lysine obtained in experiment a, And B denotes the full13C-enrichment in lysine obtained in experiment B (equation 1, equation 2 and equation 3; table 2). For example, LYS_2_A means13C-enrichment in the position 2 carbon atom of the lysine in experiment A, A GLC_6_B means13C-enrichment in position 6 carbon atoms in the glucose as substrate in experiment A. using equation 3 enrichment was standardized by dividing by the enrichment in position C1 dextrose equal to 99%, and for enrichment in position C6 of dextrose, equal to 49%, EC is periment and in experiment B, respectively. For strain DSM 5715 data13C-enrichment in the trehalose indicate the presence of a complete balance between cytoplasmic pools of glucose-6-phosphate and fructose-6-phosphate, which is important for determining the intensity of metabolism in PFI obtained by the ratio (B-a)/b

equation 1: LYS_A=LYS_2_A+LYS_3_A+LYS_5_A+LYS_6_A - 4,4,

equation 2: LYS_=LYS_2_+LYS_3_+LYS_5_+LYS_6_ - 4,4,

equation 3: (B-a)/B=[LYS_.99/(GLC_6_ - 1,1) -

LYS_A·99/(GLC_1_A - 1,1)]/[LYS_·99/(GLC__6_ - 1,1)].

By computer simulation using models of metabolism, described by Marx and others (Biotechnology and Bioengineering 49:111-129 (1996)), Marx and others (Biotechnology and Bioengineering 56:168-180 (1997)), Marx and others (Metabolic Engineering 1:35-48 (1999)), Sonntag and others (European Journal of Biochemistry 213:1325-1331 (1993)) and Sonntag and others (Applied Microbiology and Biotechnology 44:489-495 (1995)), it was found that there is a hyperbolic or linear dependence the intensity of the TFG from the enrichment factor (B-a)/B for the circuit of metabolism when there is phosphoglucomutase, and is fully balanced or absent, respectively (Fig.7). The comparison of experimental data for the ratio (B-a)/B, presented in table 2, with the data obtained by computer simulation showed that for the parent strain DSM 5715 molar intensity of metabolism in the TFG is approximately 60 moles per 100 moles of dextrose (figure 5). It's good with the fall with the published literature data about the intensity of metabolism for strain DSM 5715 (Marx and others, Biotechnology and Bioengineering 49:111-129 (1996); Marx and others, Biotechnology and Bioengineering 56:168-180 (1997); Marx and others, preprints of the materials of the 7th International conference on application of computers in biotechnology (Preprints of the 7-th International Conference in Computer Applications in Biotechnology), Osaka, Japan, 31 may - 4 June 1998, str-392; Marx and others, Metabolic Engineering 1:35-48 (1999); Sonntag and others, Applied Microbiology and Biotechnology 44:489-495 (1995)). For the strain DSM5715::pMC1 it was found that the molar intensity of metabolism in the TFG is 98 moles per 100 moles of dextrose.

The results clearly indicate that as a result of mutation, the majority ("off") pgi gene, all the intensity of metabolism during absorption of dextrose switches to the TFG.

Example 6

Reducing the formation of by-products

Strains DSM5715 and DSM5715::pMC1 were cultured and analyzed in accordance with example 5. The analysis of the spectra obtained using NMR spectroscopy using the supernatant of the cultures (figure 3-6)showed that the concentration of by-products (such as trehalose, isopropylmalate, lactate, oxoglutarate and valine) for the strain DSM5715::pMC1 were significantly less than that for the parent strain DSM 5715. The data obtained are summarized in table 3.

Table 3
The extracellular concentration of various compounds (mm) after 72 h fer the orientation in shake flask, certain spectroscopic method based on proton nuclear magnetic resonance. The distribution of chemical shifts in the spectra of nuclear magnetic resonance are presented on figure 5 and 6.
ConnectionDSM 5715DSM5715::pMC1
Trehalose2,9<0,2
Oxoglutarate10,70,7
Lactate3,50,3
Valine3,3<0,1
Isopropylmalate5,51,0
Lysine32,254,4

Brief description of drawings

Figure 1 shows the map of plasmid pAMC1.

Figure 2 presents a map of the plasmid RMS.

Abbreviations and designations used in figure 1 and 2, have the following meanings:

Neo r: fragment, causing resistance to neomycin/kanamycin,

ColEl ori: start replication plasmid ColEl,

CMV: cytomegalovirus promoter,

lacP: the promoter of the lactose,

pgi: gene phosphogluconolactonase,

lacZ: the 5'-end of the gene β-galactosidase,

SV40 3' splice: 3'-site of splicing of the monkey virus, SV40,

SV40 polyA: polyadenylation site of the monkey virus, SV40,

fl(-)ori: replication to begin filamentosa phage f1

SV40 ori: replication to begin the monkey virus, SV40,

kan r: fragment impairment is obliviouse resistance to kanamycin,

pgi insert: internal fragment of the gene pgi,

ori: start replication plasmids pBGS8,

AccI: the restriction site of restrictase AccI,

ApaI: restriction site of restrictase ApaI,

BamHI: restriction site of restrictase BamHI,

ClaI: restriction site of restrictase ClaI,

DraI: restriction site of restrictase DraI,

EcoRI: restriction site of restrictase EcoRI,

HindIII: restriction site of restrictase HindIII,

Mlul: restriction site of restrictase Mlul,

MstII: restriction site of restrictase MstII,

NheI: restriction site of restrictase NheI,

NsiI: restriction site of restrictase NsiI,

> PST : restriction site of restrictase > PST,

PvuII: restriction site of restrictase PvuII,

Sacl: restriction site of restrictase SacI,

SalI: the restriction site of restrictase SalI,

SmaI: restriction site of restrictase SmaI,

SpeI: restriction site of restrictase SpeI,

SspI: the restriction site of restrictase SspI.

Figure 3 presents the spectra of nuclear magnetic resonance for the strain DSM5715::pMC1 grown on [6-13With] dextrose. Abbreviations: L-2...L-6 refer to the results obtained by the method of nuclear magnetic resonance for the protons associated with the carbon atoms at positions C-2...C-6 in lysine, while measurements with13With the separation and processing of results of measurements of the spectrum of the spin echo without13With division (b).

Figure 4 presents the spectra of nuclear magnetic resonance for the strain DSM5715::pMC1, verase the nogo [1- 13With] dextrose. Abbreviations: L-2...L-6 refer to the results obtained by the method of nuclear magnetic resonance for the protons associated with the carbon atoms at positions C-2...C-6 in lysine, while measurements with13With the separation and processing of results of measurements of the spectrum of the spin echo without13With division (b).

Figure 5 presents the spectra of nuclear magnetic resonance for the strain DSM5715 grown on [6-13With] dextrose. Abbreviations: L-2...L-6 refer to the results obtained by the method of nuclear magnetic resonance for the protons associated with the carbon atoms at positions C-2...C-6 in lysine, while measurements with13With the separation and processing of results of measurements of the spectrum of the spin echo without13With division (b).

Figure 6 presents the spectra of nuclear magnetic resonance for the strain DSM5715 grown on [1-13With]dextrose. Abbreviations: L-2...L-6 refer to the results obtained by the method of nuclear magnetic resonance for the protons associated with the carbon atoms at positions C-2...C-6 in lysine, while measurements with13With the separation and processing of results of measurements of the spectrum of the spin echo without13With division (b).

Figure 7 presents the correlation between the intensity of metabolism in pentose-phosphate pathway and the enrichment factor (B-a)/b By computer simulation of IP models of metabolism, described by Marx and others (Biotechnology and Bioengineering 49:111-129 (1996)), Marx and others (Biotechnology and Bioengineehng 56:168-180 (1997)), the Magh and others (Metabolic Engineering 1:35-48 (1999)), Sonntag and others (European Journal of Biochemistry 213:1325-1331) and Sonntag and others (Applied Microbiology and Biotechnology 44:489-495 (1995)), it was found that the correlation between the intensity of the TFG and the enrichment factor (B-a)/(B describes hyperbolic or linear dependency scheme metabolism when there is phosphoglucomutase (Pgi), and is fully balancedor absent ( - ), respectively. The intensity of metabolism in the TFG was determined by the ratio (B-a)/B, where a denotes the full13C-enrichment in lysine obtained in experiment a, And B denotes the full13C-enrichment in lysine obtained in experiment B (table 2). Obtained in the experiment the values of (B-a)/B and the corresponding intensity of metabolism in PFI marked for strain DSM 5715 using the symboland for the strain DSM5715::pMC1 symbol. The intensity of metabolism in PFI expressed in moles per 100 moles of absorption of dextrose, and enrichment factor (B-a)/B is specified in percent.

1. Polynucleotide containing a polynucleotide sequence selected from the group including

a) polynucleotide that encodes a polypeptide that contains amino acids the th sequence, identical, at least 90% amino acid sequence represented in SEQ ID NO:2,

b) polynucleotide, complementary polynucleotides specified in subparagraph (a),

moreover, the polypeptide has the activity of glucose-6-fortismere.

2. Polynucleotide according to claim 1, which is a replicated, preferably recombinant DNA.

3. Polynucleotide according to claim 1, which is an RNA.

4. Polynucleotide according to claim 2, which contains the nucleotide sequence presented in SEQ ID NO:1.

5. Polynucleotide according to claim 2, which encodes a polypeptide that contains the amino acid sequence represented in SEQ ID NO:2.

6. Plasmid expression vector containing polynucleotide with the sequence SEQ ID NO:3, and deposited in E. coli strain DSM 12969.

7. Method of increasing the rate of metabolism in the pentose-phosphate pathway in Corynebacterium, namely, that polynucleotide according to claim 1 weaken, primarily carry out the expression of low or exclude and conduct the fermentation with the use of these bacteria.

8. The method according to claim 7, in which fermentation using corynebacteria that produce nucleotides, vitamins, and above all L-amino acids.

9. The method according to claim 7, in which reduce the expression of polynucleotide according to claim 1.

10. Pic is b according to claim 7, which reduces the catalytic properties of the polypeptide (protein enzyme), encoded by polynucleotide according to claim 1.

11. The method according to claim 7, in which the reach of attenuation using the method of integration mutagenesis using plasmid pMCl presented in figure 2 and deposited under the registration number DSM 12969.

12. The method according to claim 8, in which the nucleotides produced by fermentation using bacteria in which at the same time sverkhekspressiya a) purF gene encoding glutamine-PRPP-amidotransferase, and/or (b) gene carAB encoding carbamoylphosphate.

13. The method according to claim 8, in which L-lysine produced by fermentation using bacteria in which at the same time sverkhekspressiya dapA gene encoding dihydropyrimidinase, and/or simultaneously sverkhekspressiya gdh gene encoding glutaraldehydes, and/or simultaneously amplified DNA fragment, causing the resistance to S-(2-amino-ethyl)cysteine.

14. The method according to claim 8, in which L-tryptophan produced by fermentation using bacteria in which at the same time sverkhekspressiya tkt gene encoding transketolase, and/or simultaneously sverkhekspressiya prs gene encoding phosphoribosylpyrophosphate.

15. The method according to any of claims 7 to 14, characterized in that use bacteria of the species Corynebacterium glutamicum.

16. The method of obtaining L-Amin is acid, characterized in that, perform the following steps: a) fermentation using coryneform bacteria that weaken or shut down at least polynucleotide according to claim 1, optionally in combination with amplification of other genes, b) accumulation of the desired product in the medium or in the cells of these bacteria and in the selection of the product.

17. The method according to item 16, characterized in that use bacteria of the species Corynebacterium glutamicum.



 

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SUBSTANCE: the present innovation deals with an anti-sense oligonucleotide or one of its derivatives which can inhibit expression of human eg5 protein being relative to kinesin of motor proteins. The oligonucleotide has got a sequence being correspondent to that of nucleic acid coding certain part of human eg5. This innovation deals with the way to obtain the above-mentioned oligonucleotides, pharmaceutical composition for inhibiting human eg5 and its application. Advantage of the innovation deals with developing e new preparation to be applied for inhibiting cell proliferation.

EFFECT: higher efficiency of inhibition.

11 cl, 1 dwg, 2 ex, 3 tbl

FIELD: biotechnology, molecular biology, biochemistry.

SUBSTANCE: invention relates to regulatory sequences. Method involves isolation of DNA molecule with nucleotide sequence SEQIDNO:2 or SEQIDNO:3 that is necessary for expression of the required encoding sequence. Then vector comprising any of indicated sequences and the required sequence is constructed followed by transformation a plant with the prepared vector. Invention provides preparing transgenic plants with regulating expression of the required gene.

EFFECT: improved preparing method.

19 cl, 1 tbl, 6 ex

The invention relates to molecular biology and genetic engineering, specifically to the creation of synthetic polyepitope vaccines against HIV-1

The invention relates to biotechnology and can be used for separation of proteins with desired properties from large pools and nucleic acids (NC)
The invention relates to medicine, epidemiology, Microbiology and molecular biology

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: method involves selection of signal sequence suitable for the effective expression of Leu-hirudine in E. coli cells by the polymerase chain reaction-screening method. Method involves construction of a protein as a precursor of hirudine based on the selected signal sequence of surface membrane protein from Serratia marcescens, oprF protein from Pseudomonas fluorescens or fumarate reductase from Shewanella putrifaciens by joining the Leu-hirudine amino acid sequence with C-end of selected signal sequence. Prepared precursor of Leu-hirudine is used in a method for preparing Leu-hirudine. Invention provides preparing Leu-hirudine by the direct secretion in E. coli cells with the high yield. Invention can be used in preparing the hirudine precursor.

EFFECT: improved preparing method.

4 cl, 1 dwg, 2 tbl, 12 ex

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