RU2312145C2 - Corynebacterium glutamicum genes encoding proteins involved in membrane synthesis and membrane transport - Google Patents

Abstract

FIELD: biotechnology, molecular biology, genetic engineering, biochemistry.

SUBSTANCE: invention represents nucleic acids isolated from microorganism Corynebacterium glutamicum that encode a polypeptide eliciting CysQ activity. Also, invention relates to recombinant expressing vectors comprising such nucleic acid molecules, and to host-cells comprising these inserted expressing vectors. Also, invention relates to a method for preparing amino acids by culturing indicated cells. Invention provides expanding assortment of enzymes and to obtain amino acids.

EFFECT: enhanced effectiveness in preparing proteins, improved preparing method of amino acids.

36 cl, 4 tbl, 13 ex

Description

Related Applications

This application claims the priority of provisional patent application US No. 60/141031, filed June 25, 1999. This application also claims the priority of German patent application No. 19931454.3, filed July 8, 1999, German patent application No. 19931478.0, filed July 8, 1999, German patent application No. 199931563.9, filed July 8, 1999, German patent application No. 19932122.1 filed July 9, 1999, German patent application No. 19932124.8, filed July 9, 1999, German patent application No. 19932125.6, filed July 9, 1999, German patent application No. 199932128.0, filed July 9, 1999, German patent application No. 19932180.9, filed July 9, 1999, German patent application No. 199932182.5, filed July 9, 1999, German patent application No. 19932190.6, filed July 9, 1999, German patent application No. 199932191.4, filed July 9, 1999, German patent application No. 199932209.0, filed July 9, 1999, German patent application No. 199932212.0 filed July 9, 1999, German patent application No. 19932227.9, filed July 9, 1999, German patent application No. 19932228.7, filed July 9, 1999, German patent application No. 19932229.5, filed July 9, 1999, German patent application No. 19932230.9, filed July 9, 1999, German patent application No. 199932927.3, filed July 14, 1999, applications for German patent No. 19933005.0, filed July 14, 1999, German patent application No. 19933006.9, filed July 14, 1999, German patent application No. 199940764.9, filed August 27, 1999, German patent application No. 199940765.7, filed August 27, 1999, German patent application No. 19940766.5, filed August 27, 1999, German patent application No. 199940830.0, filed August 27, 1999, German patent application No. 199940831.9, filed August 27, 1999, German patent application No. 199940832.7, filed August 27, 1999 of the year, German patent application No. 19940833.5, filed August 27, 1999, application German patent No. 19941378.9, filed August 31, 1999, German patent application No. 19941379.7, filed August 31, 1999, German patent application No. 19941395.9, filed August 31, 1999, German patent application No. 19942077.7, filed September 3, 1999 German Patent Application No. 19942078.5, filed September 3, 1999, German Patent Application No. 19942079.3, filed September 3, 1999, and German Patent Application No. 19942088.2, filed September 3, 1999. The full contents of all the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Some products and by-products of naturally occurring metabolic processes in cells are used in a wide list of industries, including in the feed, food, cosmetic and pharmaceutical industries. These molecules, collectively called "fine chemicals", include organic acids, both protein (proteinogenic) and non-protein (non-proteinogenic) amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates , aromatic compounds, vitamins and cofactors and enzymes. Their preparation is most conveniently accomplished through a large-scale bacterial culture designed to produce and secrete large quantities of one or more desired molecules. One particularly useful organism for this purpose is Corynebacterium glutamicum, a gram-positive, non-pathogenic bacterium. By selecting strains, a number of mutant strains were obtained that produce a number of desired compounds. However, the selection of strains improved with respect to the production of a particular molecule is a time-consuming process.

SUMMARY OF THE INVENTION

The present invention provides novel bacterial nucleic acid molecules that have many uses. These applications include the identification of microorganisms that can be used to produce fine chemicals, modulate the formation of fine chemicals in C. glutamicum or related bacteria, typing or identifying C. glutamicum or related bacteria, as reference points for genome mapping C. glutamicum and as markers for transformation. These new nucleic acid molecules encode proteins called here membrane construction and membrane transport (MCT) proteins.

C. glutamicum is a gram-positive, aerobic bacterium that is commonly used in industry for large-scale production of many fine chemicals, as well as for the breakdown of hydrocarbons (for example, in oil spills) and for the oxidation of terpenoids. Thus, MCT nucleic acid molecules of the present invention can be used to identify microorganisms that can be used to produce fine chemicals, for example, by fermentation processes. Modulating the expression of the MCT nucleic acid of the present invention or modifying the sequence of the MCT nucleic acid molecules of the present invention can be used to modulate the formation of one or more fine chemicals from a microorganism (for example, to improve the yield or obtain one or more fine chemicals from a Corynebacterium species or Brevibacterium).

The MCT nucleic acids of the present invention can also be used to identify an organism as being Corynebacterium glutamicum or a closely related bacterium, or to identify the presence of C. glutamicum or a related bacterium in a mixed population of microorganisms. The present invention provides nucleic acid sequences of a number of C. glutamicum genes; by probing the extracted genomic DNA culture of a unique or mixed population of microorganisms under stringent conditions with a probe covering a portion of the C. glutamicum gene that is unique to this organism, it can be determined whether this organism is present. Although the bacterium Corynebacterium glutamicum itself is non-pathogenic, it is related to species pathogenic in humans, such as Corynebacterium diphtheriae (diphtheria causative agent); the identification of such organisms is of great clinical importance.

The MCT nucleic acid molecules of the present invention can also serve as reference points for mapping the C. glutamicum genome or genomes of related organisms. Similarly, these molecules, or variants or parts thereof, can serve as markers for genetically engineered species of Corynebacterium or Brevibacterium.

MCT proteins encoded by the novel nucleic acid molecules of the invention are capable, for example, of performing a function involved in the metabolism (e.g., biosynthesis or cleavage) of compounds necessary for membrane biosynthesis, or of facilitating the transmembrane transport of one or more compounds to or from a cell. Subject to the availability of cloning vectors for use in Corynebacterium glutamicum, such as the vectors described in Sinskey et al., U.S. Patent No. 4,449,119, and methods for genetic manipulation of C. glutamicum and related Brevibacterium species (e.g., lactofermentum) (Yoshihama et al., J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159 : 306-311 (1984) and Santamaria et al., J. Gen. Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of this invention can be used in the genetic engineering of this organism to make it better or more effective producer of one or more fine chemicals. This improved production or improved production efficiency of a fine fusion chemical product may be due to the direct action of manipulating the gene of the present invention or may be due to the indirect effect of such manipulation.

There are a number of mechanisms by which a change in the MCT protein of the present invention can directly affect the yield, production and / or production efficiency of a fine chemicals from a C. glutamicum strain including such a modified protein. MCT proteins involved in the export of molecules of fine chemicals from the cell can be increased in number or enhanced in activity, so that large amounts of these compounds are secreted into the extracellular medium from which they are more easily extracted. Similarly, MCT proteins involved in the import of nutrients necessary for the biosynthesis of one or more fine chemicals (e.g. phosphate, sulfate, nitrogen-containing compounds, etc.) can be increased in concentration or activity, so these precursors, cofactors or intermediates increase in concentration in the cell. Further, fatty acids and lipids themselves are desirable fine chemicals; by optimizing the activity or increasing the number of one or more MCT proteins of the present invention involved in the biosynthesis of these compounds, or by disrupting the activity of one or more MCT proteins involved in the cleavage of these compounds, one can increase the yield, production and / or production efficiency of fatty acid molecules and lipids from C. glutamicum.

Mutagenesis of one or more MCT genes of the present invention can also lead to MCT proteins having altered activities that indirectly affect the production of one or more desired fine chemicals from C. glutamicum. For example, the MCT proteins of the present invention involved in the export of spent cell products (possibly increased in number due to overproduction of the desired chemical product of fine synthesis) are effectively exported before they can damage nucleotides and proteins in the cell (which would reduce the viability of the cell) or interfere with biosynthetic pathways of fine chemicals (which would reduce the yield, production or production efficiency th thin organic chemical synthesis). Further, relatively large intracellular amounts of the desired fine organic synthesis product may itself be toxic to the cell, so that by increasing the activity or the number of carriers (transporters) capable of exporting this compound from the cell, it is possible to increase the viability of the given cell in culture, which, in its in turn, leads to a greater number of cells in the culture producing the desired chemical product of fine organic synthesis. The MCT proteins of the present invention can be manipulated in such a way that relatively variable amounts of various lipid and fatty acid molecules are produced. This can have a strong effect on the lipid composition of the cell membrane. Since each type of lipid has different physical properties, a change in the lipid composition of the membrane can significantly change the fluidity of the membrane. Changes in membrane fluidity can affect the transport of molecules across the membrane, as well as cell integrity, both of which have a strong effect on the production of fine chemicals from C. glutamicum in a large-scale fermentation culture.

The present invention provides novel nucleic acid molecules that encode proteins called MCT proteins, which are able, for example, to participate in the metabolism of compounds necessary for constructing cell membranes in C. glutamicum or in the transport of molecules through these membranes. Nucleic acid molecules encoding an MCT protein are referred to herein as MCT nucleic acid molecules. In a preferred embodiment, the MCT protein is involved in the metabolism of compounds necessary for the construction (construction) of cell membranes in C. glutamicum or in the transport of molecules through these membranes. Examples of such proteins include proteins encoded by the genes shown in table 1.

Thus, one aspect of the present invention relates to isolated nucleic acid molecules (e.g., cDNA, DNA or RNA) containing the nucleotide sequence encoding the MCT protein or its biologically active parts, as well as nucleic acid fragments suitable as primers or hybridization probes for detecting or amplifying an MCT coding nucleic acid (e.g., DNA or mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule contains one of the nucleotide sequences shown as having odd SEQ ID NOs in the Sequence List (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 5 : 7 ...), or the coding region or its complement of one of these nucleotide sequences. In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence that hybridizes with the nucleotide sequence or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80 % or 90%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more of a homologous nucleotide sequence represented as containing odd numbers of SEQ ID NO in the Sequence List (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 ...), or parts thereof. In other preferred embodiments, the isolated nucleic acid molecule encodes one of the amino acid sequences shown as even-numbered SEQ ID NO in the Sequence List (e.g., SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 6 :8...). Preferred MCT proteins of the present invention also preferably have at least one of the MCT activities described herein.

In another embodiment, the isolated nucleic acid molecule encodes a protein or part thereof, the protein or part thereof comprising an amino acid sequence that is sufficiently homologous to the amino acid sequence of the present invention (for example, a sequence having an even number SEQ ID NO: in the Sequence List), for example, a sufficiently homologous amino acid sequence of the present invention, so that this protein or part thereof retains the activity of MCT. Preferably, the protein or part thereof encoded by this nucleic acid molecule retains the ability to participate in the metabolism of compounds necessary for the construction of cell membranes in C. glutamicum or in the transport of molecules through these membranes. In one embodiment, the protein encoded by the indicated nucleic acid molecule is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80%, or 90%, and most preferably at least approximately 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence of the present invention (for example, a complete amino acid sequence selected from amino acid sequences having even numbers SEQ ID NO: in List p sequences). In another preferred embodiment, the protein is a full-length C. glutamicum protein that is substantially homologous to the complete amino acid sequence of the invention (encoded by the open reading frame shown in the corresponding odd-numbered SEQ ID NO in the Sequence List (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 ...).

In another preferred embodiment, the isolated nucleic acid molecule originates from C. glutamicum and encodes a protein (e.g., an MCT fusion protein) that includes a biologically active domain that is at least about 50% or more homologous to one of the amino acid sequences of the present invention (e.g., sequence of one of the even-numbered SEQ ID NO: in the Sequence List) and is able to participate in the metabolism of compounds necessary for the construction of cell membranes in C. glutamicum or in trans port molecules across these membranes, or has one or more activities set forth in Table 1, and also includes heterologous nucleic acid sequences encoding a heterologous polypeptide or regulatory regions.

In another embodiment, the isolated nucleic acid molecule has a length of at least 15 nucleotides and hybridizes under stringent conditions to a nucleic acid molecule containing the nucleotide sequence of the present invention (for example, a sequence having an odd SEQ ID NO: in the Sequence List). Preferably, the isolated nucleic acid molecule corresponds to a naturally occurring nucleic acid molecule. More preferably, the isolated nucleic acid encodes a naturally occurring MCT protein of C. glutamicum or a biologically active portion thereof.

Another aspect of the invention relates to vectors, for example, recombinant expression vectors containing the nucleic acid molecules of the invention, and to host cells into which such vectors have been introduced. In one embodiment, such a host cell is used to produce MCT protein by culturing the host cell in a suitable medium. The MCT protein can then be isolated from this medium or from a host cell.

Another aspect of the present invention relates to a genetically modified microorganism in which the MCT gene has been introduced or modified. In one embodiment, the genome of the microorganism has been altered by introducing a nucleic acid molecule of the invention encoding a wild-type MCT sequence or a mutated MCT sequence as a transgene. In another embodiment, the endogenous MCT gene in the genome of the microorganism has been altered, for example, functionally disrupted, by homologous recombination with the altered MCT gene. In another embodiment, the endogenous or introduced MCT gene in the microorganism has been altered by one or more point mutations, deletions or inversions, but still encodes a functional MCT protein gene. In yet another embodiment, one or more regulatory regions (e.g., promoter, repressor or inducer) of the MCT gene in the microorganism has been altered (e.g., by deletion, shortening, inversion, or point mutation), so that the expression of the MCT gene is modulated. In a preferred embodiment, the microorganism belongs to the genus Corynebacterium or Brevibacterium, with Corynebacterium glutamicum being particularly preferred. In a preferred embodiment, the microorganism is also used to produce the desired compound, such as an amino acid, with lysine being particularly preferred.

In another aspect, the invention provides a method for identifying the presence or activity of Corynebacterium diphtheriae in a subject. This method comprises detecting one or more nucleic acid sequences or amino acid sequences of the invention (e.g., sequences shown in SEQ ID NOS: 1-676) in a subject, thereby detecting the presence or activity of Corynebacterium diphtheriae in the subject.

Another aspect of the present invention relates to an isolated MCT protein or part thereof, for example, a biologically active part thereof. In a preferred embodiment, the isolated MCT protein or part thereof can be involved in the metabolism of compounds necessary for the construction of cell membranes in C. glutamicum or in the transport of molecules through these membranes. In another preferred embodiment, the isolated MCT protein or part thereof is sufficiently homologous to the amino acid sequence of the present invention (for example, a sequence having an even number SEQ ID NO: in the Sequence List), so that this protein or part thereof retains the ability to participate in the metabolism of compounds necessary for constructing cell membranes in C. glutamicum or in transporting molecules through these membranes.

The present invention also provides an isolated MCT protein preparation. In preferred embodiments, the MCT protein contains the amino acid sequence of the present invention (for example, a sequence having an even number SEQ ID NO: Sequence Listing). In another preferred embodiment, the present invention relates to an isolated full-sized protein that is substantially homologous to the complete amino acid sequence of the present invention (for example, a sequence having an even number SEQ ID NO: Sequence List) (encoded by an open reading frame given in the corresponding odd-numbered SEQs ID NO: Sequence List). In yet another embodiment, the protein is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80%, or 90%, and most preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to the complete amino acid sequence of the present invention (for example, a sequence having an even number SEQ ID NO: Sequence Listing). In other embodiments, the isolated MCT protein contains an amino acid sequence that is at least about 50% or more homologous to one of the amino acid sequences of the present invention (for example, a sequence having an even number SEQ ID NO: Sequence List), and is able to participate in the metabolism of compounds required for the construction of cell membranes in C. glutamicum in the transport of molecules across the cell membrane, or has one or more activities shown in table 1.

Alternatively, the isolated MCT protein may contain an amino acid sequence that is encoded by a nucleotide sequence that hybridizes, for example, hybridizes under stringent conditions, with a nucleotide sequence, or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more of a homologous nucleotide sequence the characteristics of one of the even-numbered SEQ ID NO: represented in the Sequence Listing. It is also preferred that the preferred forms of MCT proteins also have one or more of the biological activities of MCT described herein.

The MCT polypeptide or its biologically active part may be operably linked to a non-MCT polypeptide to form a fusion protein. In preferred embodiments, the fusion protein has an activity that is different from that of a single MCT protein. In other preferred embodiments, this fusion protein is involved in the metabolism of compounds necessary for the construction of cell membranes in C. glutamicum or in the transport of molecules through these membranes. In particularly preferred embodiments, the integration of this fusion protein into the host cell modulates the production of the desired compound from the given cell.

In another aspect, the invention provides methods for screening molecules that modulate the activity of an MCT protein, either by interacting with the protein itself or by the substrate or binding partner of the MCT protein, or by modulating the transcription or translation of the MCT nucleic acid molecule of the present invention.

Another aspect of the present invention relates to a method for producing a fine chemical product. This method involves culturing a cell containing a vector directing the expression of the MCT nucleic acid molecule of the present invention so that a fine organic synthesis product is formed. In a preferred embodiment, this method further comprises the step of producing a cell containing such a vector, wherein the cell is transfected with a vector directing the expression of the MCT nucleic acid. In another preferred embodiment, this method further comprises the step of recovering fine chemicals from the culture. In a particularly preferred embodiment, the cell is a cell of the genus Corynebacterium or Brevibacterium or is selected from the strains shown in Table 3.

Another aspect of the present invention relates to methods for modulating the production of a molecule from a microorganism. Such methods include contacting the cell with an agent that modulates the activity of the MCT protein or expression of the MCT nucleic acid, so that cell-related activity is altered relative to the same activity in the absence of this agent. In a preferred embodiment, the cell is modulated with respect to one or more C. glutamicum metabolic pathways for components of the cell membrane or modulated with respect to the transport of compounds across such membranes, so that the yields or production rate of the desired fine chemicals by this microorganism is improved. An agent that modulates the activity of the MCT protein may be an agent that stimulates the activity of the MCT protein or expression of the MCT nucleic acid. Examples of agents that stimulate the activity of the MCT protein or expression of the MCT nucleic acid include small molecules, active MCT proteins and nucleic acids encoding the MCT proteins that have been introduced into the cell. Examples of agents that inhibit MCT activity or expression include small molecules and antisense molecules of MCT nucleic acids.

Another aspect of the present invention relates to methods for modulating the yields of a desired compound from a cell, comprising introducing a wild-type or mutant MCT MCT gene into a cell, either stored on a separate plasmid or integrated into the genome of the host cell. When integrated into the genome, such integration may be random or it may occur through homologous recombination, so that the native gene is replaced by the introduced copy, causing modulation of the production of the desired compound from this cell. In a preferred embodiment, said yields are increased. In another preferred embodiment, the specified chemical product is a chemical product of fine organic synthesis. In a particular preferred embodiment, said fine fusion chemical is an amino acid. In a particularly preferred embodiment, said amino acid is L-lysine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides nucleic acid and MCT protein molecules that are involved in the metabolism of compounds necessary for the construction of cell membranes in C. glutamicum or in the transport of compounds through these membranes. The molecules of this invention can be used to modulate the production of fine chemicals from microorganisms, such as C. glutamicum, directly (for example, when overexpression or optimization of a protein involved in the biosynthesis of fatty acids has a direct effect on output, productivity and / or efficiency production of fatty acids from modified C. glutamicum) or may have an indirect effect, which nevertheless leads to an increase in the yield, production and / or effectiveness of odutsirovaniya desired compound (e.g., where modulation of the metabolism of cell membrane components results in changes in yield, production and / or efficiency of production or the composition of the cell membrane, which in turn affects the production of one or more fine chemicals products). Aspects of the present invention are further explained below.

I. Fine chemicals

The term "fine chemicals" is recognized in this field and includes molecules produced by the body, which have applications in various industries, such as, but not limited to, pharmaceutical, agricultural and cosmetic industries. Such compounds include organic acids such as tartaric acid, itaconic acid and diaminopimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides and nucleotides (described, for example, in Kuninaka, A. (1996) Nucleotides and related compounds , p. 561-612, in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, and the references therein), lipids, saturated and unsaturated fatty acids (e.g., arachidonic acid), diols (e.g., propanediol and butanediol), carbohydrates (e.g. hyaluronic acid and trehalose), and aromatic compounds (e.g. aromatic amines, vanillin and indigo), vitamins and cofactors (described in Ullmann's Encyclopedia of Industrial Chemistry, vol. A27, "Vitamins", p.443-613 (1996) VCH: Weinheim and references therein; and Ong, AS, Niki, E. and Packer, L. (1995) "Nutrition, Lipids, Health, and Disease" Proceedings of the UNESCO / Confederation of Scientific and Technological Associations in Malaysia, and Society for Free Radical Research-Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes, polyketides (Cane et al., (1998) Science 282: 63-68) and all other chemical products described in Gutcho (1983) ) Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and references cited therein. The metabolism and uses of some of these fine chemicals are further disclosed below.

A. Metabolism and use of amino acids

Amino acids form the basic structural units of all proteins and, as such, are important for normal cellular functioning in all organisms. The term “amino acid” is recognized in the art. Proteinogenic amino acids, which are 20 species, serve as structural units for proteins in which they are linked by peptide bonds, while non-proteinogenic amino acids (hundreds of which are known) are usually not found in proteins (see Ullmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH: Weinheim (1985)). Amino acids can be in the D- or L-configuration, although L-amino acids are usually the only type found in naturally occurring proteins. Biosynthetic and degradation pathways of each of the 20 proteinogenic amino acids were well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3 ^rd edition, pages 578-590 (1988)). “Essential” amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine), named so, because they are usually necessary in the diet due to the complexity of their biosynthesis, are easily converted through the simple biosynthetic pathways into the remaining 11 "not “Essential” amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine). Higher animals do retain the ability to synthesize some of these amino acids, but essential amino acids must be provided from the diet in order for normal protein synthesis to take place.

In addition to their function in protein biosynthesis, these amino acids are of interest in themselves, and it has been found that many of them have various uses in the feed, food, chemical, cosmetic, agricultural and pharmaceutical industries. Lysine is an important amino acid in the diet of not only humans, but also monogastric animals (animals with a single chamber stomach), such as poultry and pigs. Glutamate is most often used as a flavoring agent (monosodium glutamate, MSG) and is widely used in the food industry, as well as aspartate, phenylalanine, glycine and cysteine. Glycine, L-methionine and tryptophan are used in the pharmaceutical industry. Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are used in both the pharmaceutical and cosmetic industries. Threonine, tryptophan and D / L-methionine are common dietary supplements (Leuchtenberger, W. (1996) Amino acids - technical production and use, p. 466-502 in Rehm et al., (Eds.) Biotechnology vol. 6, chapter 14a, VCH: Weinheim). In addition, it has been found that these amino acids are useful as precursors for the synthesis of synthetic amino acids and proteins, such as N-acetylcysteine, S-carboxymethyl-L-cysteine, (S) -5-hydroxytryptophan and others described in Ullmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH: Weinheim, 1985.

The biosynthesis of these natural amino acids in organisms capable of producing them, such as bacteria, has been well characterized (for a review of bacterial amino acid biosynthesis and its regulation see, for example, Umbarger, HE (1978) Ann. Rev. Biochem. 47: 533-606 ) Glutamate is synthesized by reductive amination of α-ketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline and arginine, each, are then formed from glutamate. Serine biosynthesis is a three-step process starting with 3-phosphoglycerate (an intermediate in glycolysis) and leading to this amino acid after the stages of oxidation, transamination and hydrolysis. Both cysteine and glycine are formed from serine; the former by condensation of homocysteine with serine, and the latter by transfer of the β-carbon atom of the side chain to tetrahydrofolate, in the reaction catalyzed by serine transhydroxymethylase. Phenylalanine and tyrosine are synthesized from the precursors of the glycolytic and pentose phosphate pathways of erythrose-4-phosphate and phosphoenolpyruvate in the 9-stage biosynthetic pathway, which differs only in the final two stages after the synthesis of prefenate. Tryptophan is also formed from these two starting molecules, but its synthesis is an 11-step route. Tyrosine can also be synthesized from phenylalanine, in a reaction catalyzed by phenylalanine hydroxylase. Alanine, valine, and leucine are all biosynthetic products of pyruvate, the end product of glycolysis. Aspartate is formed from oxaloacetate, an intermediate in the citric acid cycle. Asparagine, methionine, threonine, and lysine are each formed by the conversion of aspartate. Isoleucine is formed from threonine. A complex 9-stage pathway leads to the formation of histidine from 5-phosphoribosyl-1-pyrophosphate, activated sugar.

Amino acids in excess of the needs of the cell protein synthesis amount can not be stored, and instead are destroyed to form intermediates for the major metabolic pathways cells (for review see. Stryer, L. Biochemistry 3 ^{rd ed. Ch.21 "Amino Acid Degradation} and. The Urea Cycle "p. 495-516 (1998)). Although the cell is capable of converting unwanted amino acids into useful metabolic intermediates, the production of amino acids is expensive in terms of energy, precursor molecules and enzymes necessary for their synthesis. Thus, it is not surprising that amino acid biosynthesis is regulated by feedback inhibition, in which the presence of a particular amino acid serves to slow down or completely stop its own formation (for a review of feedback mechanisms in amino acid biosynthesis pathways, see Stryer, L. Biochemistry 3 ^rd ed. Ch.24: "Biosynthesis of Amino Acids and Heme" p. 575-600 (1988)). Thus, the yield of any particular amino acid is limited by the amount of this amino acid present in the cell.

B. Metabolism and the use of vitamins, cofactors and nutraceuticals (food additives)

Vitamins, cofactors and nutraceuticals (food additives) constitute another group of molecules that higher animals do not synthesize due to the loss of their synthesis ability and which, therefore, must be taken by animals with food, although they are easily synthesized by other organisms, such as bacteria. These molecules are either biologically active substances themselves, or they are precursors of biologically active substances that can serve as carriers of electrons or intermediates in numerous metabolic pathways. In addition to their nutritional value, these compounds also have significant industrial value as coloring agents, antioxidants and catalysts or other processing aids. (For a review of the structure, activity, and industrial uses of these compounds, see, for example, Ullmann's Encyclopedia of Industrial Chemistry, "Vitamins" vol. A27, p. 433-613 VCH: Weinheim, 1996). The term "vitamin" is recognized in this field and includes nutrients that the body needs for normal functioning, but which this body cannot synthesize itself. A group of vitamins may include cofactors and additional nutritional (nutraceutical) compounds (nutritional supplements). The term "cofactor" includes non-protein compounds required for normal enzymatic activity to take place. Such compounds may be organic or inorganic; The cofactor molecules of the present invention are preferably organic. The term "nutraceutical substance" includes nutritional supplements useful for the health of plants and animals, in particular humans. Examples of such molecules are vitamins, antioxidants, and also certain lipids (e.g. polyunsaturated fatty acids).

The biosynthesis of these molecules in organisms capable of producing them, such as bacteria, has been largely characterized (Ullmann's Encyclopedia of Industrial Chemistry, Vitamins vol. A27, p. 433-613, VCH: Weinheim, 1996; Michal, G. ( 1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Ong, AS, Niki, E and Packer, L. (1995) "Nutrition, Lipids, Health, and Disease" Proceedings of the UNESCO / Confederation of Scientific and Technological Associations in Malaysia, and Society for Free Radical Research-Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press: Champaign, IL X, 374 S).

Thiamine (Vitamin B ₁ ) is formed by chemical bonding of the pyrimidine and thiazole moieties. Riboflavin (vitamin B ₂ ) is synthesized from guanosine 5'-triphosphate (GTP) and ribose-5'-phosphate. Riboflavin, in turn, is used to synthesize flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The family of compounds collectively referred to as “Vitamin Be” (eg, pyridoxine, pyridoxamine, pyridoxal-5′-phosphate and commercially used pyridoxine hydrochloride) includes compounds that are derivatives of a common structural unit, 5-hydroxy-6-methylpyridine. Pantothenate (pantothenic acid, (R) - (+) - N- (2,4-dihydroxy-3,3-dimethyl-1-oxobutyl) -β-alanine) can be obtained by chemical synthesis or fermentation. The final stages in pantothenate biosynthesis consist of ATP-controlled condensation of β-alanine and pantoic acid. Enzymes responsible for the biosynthesis steps for conversion to pantoic acid, β-alanine and for condensation to pantothenic acid are known. The metabolically active form of pantothenate is Coenzyme A, the biosynthesis of which proceeds in the form of 5 enzymatic stages. Pantothenate, pyridoxal-5'-phosphate, cysteine and ATP are the precursors of Coenzyme A. These enzymes catalyze not only the formation of pantothenate, but also the formation of (R) -pantoic acid, (R) -pantolactone, (R) -panthenol (provitamin B ₅ ) (and its derivatives) and coenzyme A.

The biosynthesis of biotin from the pimeloyl-CoA precursor molecule in microorganisms has been studied in detail, and several genes involved have been identified. It was found that many of the corresponding proteins are also involved in the synthesis of the Fe cluster and are members of the nifS class proteins. Lipoic acid is obtained from octanoic acid and it serves as a coenzyme in energy metabolism, where it becomes part of the pyruvate dehydrogenase complex and α-ketoglutarate-dehydrogenase complex. Folates are a group of substances that are all derivatives of folic acid, which, in turn, is formed from L-glutamic acid, p-aminobenzoic acid and 6-methylopterin. The biosynthesis of folic acid and its derivatives, starting with the intermediate products of metabolism of guanosine 5'-triphosphate (GTP), L-glutamic acid and p-aminobenzoic acid, has been studied in detail in some microorganisms.

Corrinoids (such as cobalamins and, in particular, vitamin B ₁₂ ) and porphyrins belong to the group of chemical products characterized by the tetrapyrrole ring system. The biosynthesis of vitamin B ₁₂ is quite complex, so that it has not yet been fully characterized, but many of the enzymes and substrates involved in it are currently known. Nicotinic acid (nicotinate) and nicotinamide are pyridine derivatives, which are also called "niacin." Niacin is a precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms.

The large-scale production of these compounds is largely based on cell-free chemical synthesis, although some of these chemical products have also been obtained by large-scale cultivation of microorganisms such as riboflavin, vitamin B ₆ , pantothenate and biotin. Only vitamin B _{12 is} obtained exclusively by fermentation due to the complexity of its synthesis. In vitro methodologies require significant material and time, often at high cost.

C. Metabolism and uses of purines, pyrimidines, nucleosides and nucleotides

Purine and pyrimidine metabolism genes and their corresponding proteins are important targets for the treatment of tumor diseases and viral infections. The terms “purine” or “pyrimidine” include nitrogenous bases, which are constituents of nucleic acids, coenzymes, and nucleotides. The term "nucleotide" includes the basic structural units of nucleic acid molecules, which consist of a nitrogenous base, pentose sugar (in the case of RNA, this sugar is ribose; in the case of DNA, this sugar is D-deoxyribose) and phosphoric acid. The term “nucleoside” includes molecules that serve as precursors for nucleotides, but which do not contain a phosphoric acid moiety that the nucleotides have. By inhibiting the biosynthesis of these molecules or their mobilization for the formation of nucleic acid molecules, one can inhibit the synthesis of RNA and DNA; by inhibiting this activity in a cancer cell-targeted manner, it is possible to inhibit the ability of tumor cells to divide and multiply. In addition, there are nucleotides that do not form nucleic acid molecules, but serve as energy stores (i.e., AMP) or as coenzymes (i.e., FAD and NAD).

Several publications have described the use of these chemical products for medical purposes by affecting the metabolism of purines and / or pyrimidines (e.g. Christopherson, RI and Lyons, SD (1990) "Potent inhibitors of de novo pyrimidine and purine biosynthesis as chemotherapeutic agents." Med. Res. Reviews 10: 505-548). Studies of enzymes involved in the metabolism of purines and pyrimidines have focused on the development of new drugs that can be used, for example, as immunosuppressants or antiproliferants (Smith, JL, (1995) "Enzymes in nucleotide synthesis." Curr. Opin. Struct Biol. 5: 752-757; (1995) Biochem. Soc. Transact. 23: 877-902). However, purine and pyrimidine bases, nucleosides and nucleotides have other uses: as intermediates in the biosynthesis of certain fine chemicals (e.g. thiamine, S-adenosylmethionine, folates or riboflavin), as energy carriers for a cell (e.g. ATP or GTP) and for the chemical products themselves, usually used as flavor enhancers (e.g., IMP or GMP), or for some medical applications (see, e.g., Kuninaka, A. (1996) Nucleotides and Related Compounds in Biotechnology vol. 6, Rehm et al ., eds. VCH: Weinheim, p. 561-612). In addition, the enzymes involved in the metabolism of purines, pyrimidines, nucleosides or nucleotides are increasingly being used as targets against which chemical products are being developed to protect crop yields, including fungicides, herbicides and insecticides.

The metabolism of these compounds in bacteria has been characterized (for a review see, for example, Zaikin, H. and Dixon, JE (1992) "de novo purine nucleotide biosynthesis", in: Progress in Nucleic Acid Research and Molecular Biology, vol. 42, Academic Press: p. 259-287 and Michal, G. (1999) "Nucleotides and Nucleosides", Chapter 8 in: Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York). Purine metabolism has been the subject of intensive research and is essential for the normal functioning of the cell. Impaired purine metabolism in higher animals can cause serious illness such as gout. Purine nucleotides are synthesized from ribose-5-phosphate, in a number of stages through the inosine-5'-phosphate (IMP) intermediate, leading to the formation of guanosine-5'-monophosphate (GMP) or adenosine-5'-monophosphate (AMP), from which triphosphate forms are easily formed, used as nucleotides. These compounds are also used as energy reserves, and thus their decomposition provides energy for many different biochemical processes in the cell. The biosynthesis of pyrimidines proceeds through the formation of uridine-5'-monophosphate (UMF) from ribose-5-phosphate. UMF, in turn, is converted to cytidine-5'-triphosphate (CTF). The deoxy forms of all these nucleotides are formed in a one-step reduction reaction from the diphosphatribose form of the nucleotide to the diphosphate deoxyribose form of this nucleotide. When phosphorylated, these molecules are able to participate in DNA synthesis.

D. Metabolism and the use of trehalose

Trehalose consists of two glucose molecules linked in an α, α-1,1-bond. It is usually used in the food industry as a sweetener, additives for dried or frozen foods, and in drinks. However, it is also used in the pharmaceutical, cosmetic, and biotechnological industries (see, for example, Nishimoto et al., (1998) US Patent No. 5759610; Singer, MA and Lindquist, S. (1998) Trends Biotech. 16: 460- 467; Paiva, CLA and Panek, AD (1996) Biotech. Ann. Rev.2: 293-314; and Shiosaka, M. (1997) J. Japan. 172: 97-102). Trehalose is produced by enzymes from many microorganisms and is naturally released into the environment from which it can be collected using methods known in the art.

II. Membrane biosynthesis and transmembrane transport

Cell membranes serve various functions in the cell. First and foremost, the membrane differentiates the contents of the cell from the environment, thereby creating cell integrity. Membranes can also serve as barriers to entry of harmful or undesirable compounds, as well as the release of desired compounds. Cell membranes are, by their nature, impermeable to the uncomplicated diffusion of hydrophilic compounds, such as proteins, water molecules and ions, due to their structure: a bilayer of lipid molecules in which groups of polar heads are facing outward (in the direction of the outer and inner space of the cell, respectively), and non-polar tails face inward at the center of the bilayer, forming a hydrophobic base (for a general overview of membrane structure and function, see Gennis, RB (1989) Biomembranes, Molecular Structure and Function, Springer: Heidelberg). This barrier allows cells to maintain a relatively higher concentration of the desired compounds and a relatively lower concentration of the undesirable compounds, compared to the environment, since the diffusion of these compounds is effectively blocked by the membrane. However, the membrane also provides an effective barrier to the import of desired compounds and the export of spent molecules. To overcome this difficulty, cell membranes include numerous types of carrier proteins (transporters) that are able to facilitate transmembrane transport of various types of compounds. There are two main classes of these transport proteins: pores or channels and carriers (transporters). The former are integral membrane proteins, sometimes complexes of proteins that form an adjustable hole through the membrane. This regulation, or “gate mechanism,” is usually specific for molecules transported by the pore or channel, which makes these transmembrane structures selectively permeable to a specific class of substrates; for example, the potassium channel is designed in such a way that only ions having a charge and size similar to the charge and size of potassium can pass through it. Channel and pore proteins tend to have discrete hydrophobic and hydrophilic domains, so that the hydrophobic surface of the protein can bind to the inside of the membrane, while the hydrophilic surface lines the interior of the channel, thereby providing a protected hydrophilic environment through which the selected hydrophilic molecule can pass. Many such pores / channels are known in the art, including channels for potassium, calcium, sodium, and chloride ions.

This pore and channel mediated facilitated diffusion system is limited to very small molecules such as ions, since pores or channels large enough to allow whole proteins to pass through facilitated diffusion would be unable to prevent the passage of smaller hydrophilic molecules as well. Molecular transport through this process is sometimes called “facilitated diffusion”, since this transport requires a driving force in the concentration gradient. Permeases also allow facilitated diffusion of larger molecules, such as glucose or other sugars, into the cell when the concentration of these molecules on one side of the membrane is higher than the concentration on the other side (also called "uniport", i.e. transport of one solute through the membrane). In contrast to pores or channels, these integral membrane proteins (often having 6-14 α-helices extending through the membrane) do not form open channels through the membrane, but bind to the target molecule on the membrane surface and then undergo a conformational shift, so that the target molecule released on the opposite side of the membrane.

However, cells need to import or export molecules against an existing concentration gradient (“active transport”), a situation in which facilitated diffusion cannot occur. There are two main mechanisms used by cells for such membrane transport: symport or antiport, and energy-coupled transport, for example, mediated by ABC transporters. The symport and antiport systems conjugate the movement of two different molecules through the membrane (through permeases having two separate binding sites for two different molecules); in simport, both molecules are transported in the same direction, while in the antiport one molecule is imported, while the other is exported. This is energetically possible, since one of the two molecules moves in accordance with the concentration gradient, and this energetically favorable event is allowed only with the concomitant movement of the desired compound against the prevailing concentration gradient. Individual molecules can be transported across the membrane against a concentration gradient in an energy-using process, for example, a process used by ABC transporters. In this system, a transport protein located in the membrane has an ATP-binding cassette; upon binding of the target molecule, ATP is converted to ADP + Pi, and the resulting energy release is used to move the target molecule to the opposite side of the membrane, facilitated by this transporter. For more detailed descriptions of all these transport systems, see: Bamberg, E. et al., (1993) "Charge transport of ion pumps on lipid bilayer membranes", Q. Rev. Biophys. 26: 1-25; Findlay, J.B.C. (1991) "Structure and function in membrane transport systems", Curr. Opin. Struct. Biol. 1: 804-810; Higgins, C.F. (1992) "ABC transporters from microorganisms to man", Ann. Rev. Cell Biol. 8: 67-113; Gennis, R.B. (1989) "Pores, Channels and Transporters", in: Biomembranes, Molecular Structure and Function, Springer: Heidelberg, p. 270-322; and Nikaido, H. and Saier, H. (1992) "Transport proteins in bacteria: common themes in their design", Science 258: 936-942, and the references contained in each of these works.

Membrane synthesis is a well-characterized process in which a number of components are involved, the most important of which are lipid molecules. The synthesis of lipids can be divided into two parts: the synthesis of fatty acids and their addition to sn-glycerol-3-phosphate and the addition or modification of the polar group of the head. Typical lipids used in bacterial membranes include phospholipids, glycolipids, sphingolipids and phosphoglycerides. The synthesis of fatty acids begins with the conversion of acetyl-CoA either to malonyl-CoA by acetyl-CoA-carboxylase, or to acetyl-ACP by acetyltransacylase. After the condensation reaction, these two molecules obtained form together acetoacetyl-ACP, which is converted through a series of condensation, reduction and dehydration reactions to form a fatty acid molecule with the desired chain length. The formation of unsaturated fatty acids from such molecules is catalyzed by specific desaturases either aerobically using molecular oxygen or anaerobically (for a link on the synthesis of fatty acids see FC Neidhardt et al. (1996) E. coli and Salmonella. ASM Press: Washington, DC , p.612-636 and the references contained in this work; Lengeler et al. (eds) (1999) Biology of Procaryotes. Tieme: Stuttgart, New York and the references contained in this work; and Magnuson, K. et al. ( 1993) Microbiological Reviews 57: 522-542, and references in these works). Cyclopropane fatty acids (CFA) are synthesized by specific CFA synthase using SAM as a co-substrate. Branched chain fatty acids are synthesized from branched chain amino acids which are deaminated to give branched chain 2-oxo acids (see Lengeler et al. (Eds) (1999) Biology of Procaryotes. Thieme: Stuttgart, New York, and contained therein link work). Another essential step in lipid synthesis is the transfer of fatty acids to groups of the polar head using, for example, glycerophosphate acyltransferases. The combination of various precursor molecules and biosynthetic enzymes leads to the production of various fatty acid molecules that have a strong effect on the composition of the membrane.

III. Elements and methods of the invention

The present invention is based, at least in part, on the discovery of new molecules, called here nucleic acid molecules and MCT protein molecules, which regulate the production of cell membranes in C. glutamicum and control the movement of molecules through such membranes. In one embodiment, MCT molecules are involved in the metabolism of compounds necessary for the construction of cell membranes in C. glutamicum or in the transport of molecules through these membranes. In a preferred embodiment, the activity of the MCT molecules of the present invention in regulating the production of membrane components and membrane transport affects the production of the desired fine organic synthesis product by this organism. In a particularly preferred embodiment, the MCT molecules of the present invention are modulated in activity such that the C. glutamicum metabolic pathways that regulate the MCT proteins of the present invention are modulated with respect to yield, production and / or production efficiency, and the transport of compounds across membranes changes in efficiency, which either directly or indirectly modulates the yield, production, and / or production efficiency of the desired fine chemical C. glutamicum.

The expression "MCT protein" or "MCT polypeptide" includes proteins that are involved in the metabolism of compounds necessary for the construction of cell membranes in C. glutamicum or in the transport of molecules through these membranes. Examples of MCT proteins include proteins encoded by the MCT genes shown in Table 1 and having odd numbers SEQ ID NO :. The terms “MCT gene” or “MCT nucleic acid sequence” include nucleic acid sequences encoding the MCT protein, which consist of the coding region as well as the corresponding untranslated 5 ′ and 3 ′ regions of the sequence. Examples of MCT genes include the genes shown in table 1. The terms "production" or "productivity" are recognized in this field and include the concentration of the fermentation product (for example, the desired chemical product of fine synthesis) formed in a specific time and in a specific volume fermentation (for example, kg of product per hour per liter). The term “production efficiency” includes the time required for a particular level of production to be achieved (for example, the time it takes for a cell to achieve a specific yield of a fine organic chemical product). The term “yield” or “product / carbon yield” is recognized in the art and includes the efficiency of converting a carbon source into a product (ie, a fine organic chemical product). This is usually expressed as, for example, kg of product per kg of carbon source. By increasing the yield or production of a compound, the number of recovered molecules of this compound in a specific amount of culture over a specific period of time increases. The terms “biosynthesis” or “biosynthetic pathway” are recognized in the art and include the synthesis of a compound, preferably an organic compound, by a cell from intermediates, which may include a multi-step and highly regulated process. The terms “degradation” or “degradation pathway” are recognized in the art and include decomposition of a compound, preferably an organic compound, by a cell into degradation products (generally speaking, smaller or less complex molecules), which may include multi-step and highly regulated process. The term "metabolism" is recognized in this field and includes the entire amount of biochemical reactions that take place in the body. Then the metabolism of a particular compound (for example, the metabolism of an amino acid, for example, glycine) includes common biosynthetic pathways, modification and decomposition pathways in the cell associated with this compound.

In another embodiment, the MCT molecules of the present invention are capable of modulating the production of the desired molecule, such as a fine chemical product, in a microorganism such as C. glutamicum. There are a number of mechanisms by which a change in the MCT protein of the present invention can directly affect the yield, production and / or production efficiency of a fine organic chemical product from C. glutamicum including such a modified protein. MCT proteins involved in the export of fine chemicals from cells can be increased in number or activity, so that large amounts of these compounds are secreted into the extracellular environment from which they can be more easily extracted. Similarly, MCT proteins involved in the import of nutrients necessary for the biosynthesis of one or more fine chemicals (e.g. phosphate, sulfate or nitrogen compounds, etc.) can be increased in number or activity, so these precursor compounds, cofactor compounds or intermediates increase in concentration in the cell. Further, fatty acids and lipids themselves are desirable fine chemicals; by optimizing the activity or increasing the number of one or more MCT proteins of the present invention that are involved in the biosynthesis of these compounds, or by disrupting the activity of one or more MCT proteins that are involved in the decomposition of these compounds, one can increase the yield, production and / or production efficiency of fatty acid molecules and lipids from C. glutamicum.

Mutagenesis of one or more MCT genes of the present invention may also lead to the presence of MCT proteins having altered activities that indirectly affect the production of one or more desired fine chemicals from C. glutamicum. For example, MCT proteins of the present invention involved in the export of waste cell products can be increased in quantity or activated in such a way that the waste products of cell metabolism (possibly increased in number due to overproduction of the desired fine chemicals) are exported efficiently before they can damage nucleotides and proteins in the cell (which would reduce the viability of this cell) or interfere with the biosynthetic pathways of chemical products fine chemicals (which would reduce the yield, production or efficiency of production of the desired fine chemical organic synthesis). Further, relatively large intracellular amounts of the desired fine chemicals can itself be toxic to the cell, so that by increasing the activity or the number of transporters capable of exporting this compound from the cell, it is possible to increase the viability of the cell in culture, which in turn leads to to a larger number of cells in the culture producing the desired chemical product of fine organic synthesis. The MCT proteins of the present invention can be manipulated in such a way that relative amounts of different lipid and fatty acid molecules are produced. This can have a strong effect on the lipid composition of the cell membrane. Since each type of lipid has different physical properties, a change in the lipid composition of the membrane can significantly change the fluidity of the membrane. Changes in membrane fluidity can affect the transport of molecules across the membrane, as well as cell integrity, both of which have a strong effect on the production of fine chemicals from C. glutamicum in a large-scale fermentation culture.

The isolated nucleic acid sequences of the present invention are contained in the genome of the Corynebacterium glutamicum strain, available from the American Type Culture Collection, with an accession number of ATCC 13032. The nucleotide sequence of the isolated C. glutamicum MCT DNAs and the predicted amino acid sequences of C. glutamicum MCT proteins are shown in the List of sequences as having odd numbers SEQ ID NO: and even numbers SEQ ID NO :, respectively. Computer analyzes were performed that classified and / or identified these nucleotide sequences as sequences encoding proteins involved in the metabolism of cell membrane components, or proteins involved in the transport of compounds across such membranes.

The present invention also relates to proteins having an amino acid sequence that is substantially homologous to the amino acid sequence of the present invention (for example, a sequence having an even number SEQ ID NO: Sequence Listing). As used herein, a protein having an amino acid sequence that is substantially homologous to a selected amino acid sequence is at least about 50% homologous to a selected amino acid sequence, for example, a complete selected amino acid sequence. A protein having an amino acid sequence that is substantially homologous to the selected amino acid sequence may also be at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80% , 80-90% or 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to the selected amino acid sequence.

The MCT protein or its biologically active part or biologically active fragment of the present invention may participate in the metabolism of compounds necessary for the construction of cell membranes in C. glutamicum, or in the transport of molecules through these membranes, or may have one or more activities shown in table 1 .

Various aspects of the present invention are described in further detail in the following subsections:

A. Isolated nucleic acid molecules

One aspect of the present invention relates to isolated nucleic acid molecules that encode MCT polypeptides or their biologically active parts, as well as nucleic acid fragments sufficient for use as hybridization probes or primers to identify or amplify MCT-encoding nucleic acid (e.g., MCT DNA) . As used herein, the term “nucleic acid molecule” includes DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and DNA or RNA analogs obtained using nucleotide analogs. This term also includes an untranslated sequence located at both 3 ′ and 5 ′ ends of the coding region of this gene: at least about 100 nucleotides of the sequence above (against the transcription) from the 5 ′ end of the coding region and at least approximately 20 nucleotides lower (transcription) from the 3 ′ end of the coding region of this gene. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. An “isolated” nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that are present in the natural source of that nucleic acid. Preferably, the "isolated" nucleic acid does not contain sequences that naturally flank this nucleic acid (i.e., sequences localized at the 5'- and 3'-ends of this nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, an isolated MCT nucleic acid molecule may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 t bp, 0.5 bp or 0.1 kb nucleotide sequences that naturally flank this nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived (e.g., C. glutamicum cells). In addition, an “isolated” nucleic acid molecule, for example, a DNA molecule, may essentially not contain other cellular material or culture medium when produced by recombinant methods or in the chemical synthesis of chemical precursors or other chemical products.

A nucleic acid molecule of the present invention, for example, a nucleic acid molecule having a nucleotide sequence having odd numbers SEQ ID NO: A list of sequences, or a part thereof, can be isolated using standard molecular biology methods and sequence information provided herein. For example, C. glutamicum MCT DNA can be isolated from a C. glutamicum library using all or part of one of the odd-numbered SEQ ID NO: Sequence List sequences as a hybridization probe and standard hybridization methods (e.g., those described in Sambrook J., Fritsh , EF, and Maniatis, T. Molecular Cloning. A Laboratory Manual, 2 ^nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In addition, a nucleic acid molecule encompassing the entire sequence or part of one of the nucleic acid sequences of the present invention (e.g. having odd numbers SEQ ID NO: Sequence Listing) can be isolated by polymerase chain reaction using oligonucleotide primers designed based on this sequence ( for example, a nucleic acid molecule encompassing the entire sequence or part of one of the nucleic acid sequences of the present invention (n For example, having odd numbers SEQ ID NO: Sequence Listing) can be isolated by polymerase chain reaction using oligonucleotide primers designed on the basis of the same sequence). For example, mRNA can be isolated from normal endothelial cells (for example, Chirgwin et al. (1979) Biochemistry 18: 5294-5299 guanidinium thiocyanate extraction procedure), and DNA can be obtained using reverse transcriptase (e.g. Moloney MLV reverse transcriptase available from Gibco / BRL, Bethesda, MD; or AMV reverse transcriptase available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for amplification by polymerase chain reaction can be designed based on one of the nucleotide sequences shown in the List of sequences. The nucleic acid of the present invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and suitable oligonucleotide primers in accordance with standard PCR amplification methods. The nucleic acid thus amplified can be cloned into a suitable vector and characterized by DNA sequence analysis. In addition, oligonucleotides corresponding to the nucleotide sequence of the MCT can be obtained by standard synthetic methods, for example, using an automatic DNA synthesizer.

In a preferred embodiment, the isolated nucleic acid molecule of the present invention contains one of the nucleotide sequences shown in the Sequence Listing. The nucleic acid sequences of the present invention shown in the Sequence List correspond to the MCT DNA of Corynebacterium glutamicum of the present invention. This DNA contains sequences encoding MCT proteins (i.e., the "coding region" shown in each odd-numbered SEQ ID NO: sequences in the Sequence List), as well as 5'-untranslated sequences and 3'-untranslated sequences also shown in each odd-numbered SEQ ID NO: sequence in the Sequence List. Alternatively, the nucleic acid molecule may contain only the coding region of any of the nucleic acid sequences of the Sequence List.

For the purposes of this application, it should be understood that each of the nucleic acid sequences or amino acid sequences listed in the Sequence List has an RXA, RXN, RXS or RXC identification number, designated "RXA", "RXN", "RXS" or " RXC ", followed by 5 digits (i.e. RXA02099, RXN03097, RXS00148 or RXC01748). Each of these nucleic acid sequences contains up to three parts: the upper 5'-region (against the course of transcription), the coding region and the lower region (during the transcription). Each of these three sections is identified by the same designation as RXA, RXN, RXS or RXC to avoid confusion. Then the reference to “one of the odd-numbered Sequence List sequences” refers to any of the nucleic acid sequences in the Sequence List, which can also be distinguished by different designations RXA, RXN, RXS or RXC. The coding region of each of these sequences is translated into the corresponding amino acid sequence, which is also presented in the Sequence List in the form of even numbers SEQ ID NO: immediately after the corresponding nucleic acid sequence. For example, the coding region for RXA03097 is represented in SEQ ID NO: 1, while the amino acid sequence that it encodes is represented as SEQ ID NO: 2. The nucleic acid molecule sequences of this invention are identified by the same designations RXA, RXN, RXS or RXC as the amino acid molecules that they encode, so that they can be easily correlated with each other. For example, the amino acid sequences designated RXA02099, RXN03097, RXS00148 or RXC01748 are translations of the coding region of the nucleotide sequences of nucleic acid molecules RXA02099, RXN03097, RXS00148 or RXC01748, respectively. The correspondence between the nucleotide and amino acid sequences RXA, RXN, RXS and RXC of the present invention and the corresponding SEQ ID NO: are shown in table 1. For example, as shown in table 1, the nucleotide sequence of RXA00104 is SEQ ID NO: 5, and the amino acid sequence RXA00104 is SEQ ID NO: 6.

Several genes of the present invention are “F-labeled genes.” The F-labeled gene includes genes shown in Table 1 that have an “F” before RXA, RXN, RXS or RXC. For example, the sequence SEQ ID NO: 11, indicated by as indicated in table 1, as "F RXA02581", is an F-designated gene, as well as SEQ ID NO: 31, 33 and 43 (indicated in table 1 as "F RXA02487", "F RXA02490" and "F RXA02809", respectively).

In one embodiment, the nucleic acid molecules of the present invention do not include the nucleic acid molecules shown in Table 2. In the case of the dapD gene, the sequence for this gene was published by Wehrmann, A., et al., (1998) J. Bacteriol. 180 (12): 3159-3165. However, the sequence obtained by the authors of this application is significantly longer than the published version. The authors believe that the published version is based on an incorrect starting (initiating) codon and, therefore, represents only a fragment of the true coding region.

In another preferred embodiment, the isolated nucleic acid molecule of the present invention comprises a nucleic acid molecule that is a complement of one of the nucleotide sequences of the present invention (for example, a sequence having an odd number SEQ ID NO: Sequence Listing), or part thereof. A nucleic acid molecule that is complementary to one of the nucleotide sequences of the present invention is a sequence that is sufficiently complementary to one of the nucleotide sequences in the Sequence List (for example, a sequence having an odd number SEQ ID NO :) so that it can hybridize with one of the nucleotide sequences of the present invention with the formation of a stable duplex.

In another preferred embodiment, the isolated nucleic acid molecule of the present invention contains a nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59 % or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71% , 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 84%, 85%, 86%, 87%, 88%, 89 % or 90% or 91%, 92%, 94%, or even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous eotidnoy sequence of the invention (e.g., a sequence having odd number of SEQ ID NO: Sequence Listing), or a portion thereof. It is understood that ranges and values of identity intermediate between the above ranges (e.g., 70-90% identical or 80-95% identical) are also encompassed by this invention. For example, it is understood that ranges of identity values are also included using a combination of any of the values cited above as upper and / or lower limits. In a further preferred embodiment, the isolated nucleic acid molecule of the present invention contains a nucleotide sequence that hybridizes, for example, hybridizes under stringent conditions, with one of the nucleotide sequences of the present invention or with a part thereof.

In addition, the nucleic acid molecule of the present invention may contain only part of the coding region of one of the odd numbers SEQ ID NO: Sequence List, for example, a fragment that can be used as a probe or primer, or a fragment encoding a biologically active part of the MCT protein . These nucleotide sequences, determined from cloning MCT genes from C. glutamicum, provide probes and primers designed for use in identifying and / or cloning MCT homologs in other cell types and organisms, as well as MCT homologs from other Corynebacteria or related species . The probe / primer typically contains a substantially purified oligonucleotide. An oligonucleotide typically contains a portion of a nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of the sense strand of one of the nucleotide sequences of the invention (e.g., sequences having odd SEQ numbers ID NO: Sequence List), the antisense sequence of one of these sequences or their naturally occurring sequences utantines. The nucleotide sequence primers of this invention can be used in PCR reactions for cloning MCT homologs. MCT nucleotide sequence probes can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, such a probe further comprises a label group attached to it, for example, this label group can be a radioactive isotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used as part of a diagnostic test kit to identify cells that have impaired expression of the MCT protein, for example, by measuring the level of the MCT coding nucleic acid in the cell sample, for example, by detecting the levels of MCT mRNA, or to determine if the genomic MCT gene has been mutated or deleted.

In one embodiment, the nucleic acid molecule of the present invention encodes a protein or part thereof that includes an amino acid sequence that is sufficiently homologous to the amino acid sequence of the present invention (for example, sequences having even numbers SEQ ID NO: Sequence List), so that the protein or part thereof retains the ability to participate in the metabolism of compounds necessary for the construction of cell membranes in C. glutamicum or in the transport of molecules through these membranes. As used herein, the term “sufficiently homologous” refers to proteins or parts thereof that have amino acid sequences that include a minimum number of identical or equivalent (for example, an amino acid residue that has the same side chain as the amino acid residue in the sequence of one of even numbers SEQ ID NO: Sequence Listing) of amino acid residues relative to the amino acid sequence of the present invention, so that this protein or part thereof is able to participate in the tabolism of compounds necessary for the construction of cell membranes in C. glutamicum or in the transport of molecules through these membranes. Protein members of such metabolic pathways of membrane components or membrane transport systems, as described herein, may play a role in the production and secretion of one or more fine chemicals. Examples of such activities are also described here. Thus, the "function of the MCT protein" contributes, directly or indirectly, to the yield, productivity and / or efficiency of the production of one or more chemical products of fine organic synthesis. Examples of MCT protein activities are shown in table 1.

In another embodiment, the protein is at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%, 80-90%, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to the complete amino acid sequence of the present invention (for example, sequences from even numbers SEQ ID NO: Sequence Listing).

The portions of the proteins encoded by the MCT nucleic acid molecules of the present invention are preferably biologically active parts of one of the MCT proteins. As used herein, the term “biologically active portion of the MCT protein” includes a portion, for example, a domain / motif, of the MCT protein, which is involved in the metabolism of compounds necessary for constructing cell membranes in C. glutamicum or in the transport of molecules through these membranes, or has activity shown in table 1. To determine whether the MCT protein or its biologically active part can participate in the metabolism of compounds necessary for the construction of cell membranes in C. glutamicum or in the transport of molecules through these membranes, Enzymatic activity assay. Such assay methods are well known in the art to those of ordinary skill in the art, as described in detail in Example 8 of the Examples section.

Additional nucleic acid fragments encoding the biologically active portions of the MCT protein can be obtained by isolating a portion of one of the amino acid sequences of the present invention (e.g., a sequence having an even number SEQ ID NO: Sequence Listing), expression of the encoded portion of the protein or MCT peptide (e.g., recombinant by in vitro expression) and evaluation of the activity of the encoded portion of this protein or MCT peptide.

Further, the present invention encompasses nucleic acid molecules that differ from the nucleotide sequences of the present invention (for example, a sequence having an odd number SEQ ID NO: Sequence List) (or parts thereof) due to the degeneracy of the genetic code and, therefore, encode the same MCT protein as encoded by the nucleotide sequences of the present invention. In another embodiment, the isolated nucleic acid molecule of the present invention has a nucleotide sequence encoding a protein having the amino acid sequence shown in the Sequence List (for example, having an even number SEQ ID NO :). In yet another embodiment, the nucleic acid molecule of the invention encodes a full-length C. glutamicum protein that is substantially homologous to the amino acid sequence of the invention (encoded by the open reading frame shown in odd numbers SEQ ID NO: Sequence Listing).

The person skilled in the art should understand that in one embodiment, the sequences of the present invention should not include sequences of the prior art, such as the Genbank sequences shown in tables 2 or 4, which were known prior to this invention. In one embodiment, the invention includes nucleotide and amino acid sequences having a percent identity with respect to the nucleotide or amino acid sequence of the present invention that is higher than the sequences of the prior art (for example, the Genbank sequence (or the protein encoded by such a sequence) shown in tables 2 or four). For example, the invention includes a nucleotide sequence that is more than 38% and / or at least 38% identical to the nucleotide sequence designated RXA01420 (SEQ ID NO: 7), a nucleotide sequence that is more than 43 %, and / or at least 43% of the identical nucleotide sequence designated RXA00104 (SEQ ID NO: 5), and a nucleotide sequence that is more than 45% and / or at least 45% identical to the nucleotide sequence designated RXA02173 (SEQ ID NO: 25). One of ordinary skill in the art will be able to calculate the lowest percent identity limit for any particular sequence of the invention by examining the percent identity estimates calculated using the GAP program given in Table 4 for each of the three highest matches for a particular sequence and subtracting the highest calculated using the program GAP percent identity of 100 percent. One skilled in the art will also understand that nucleic acid and amino acid sequences having percent identities are larger than the lower limit thus calculated (e.g., at least 50%, 51%, 52%, 53%, 54% , 55%, 56%, 57%, 58%, 59% or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69 % or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 84% , 85%, 86%, 87%, 88%, 89% or 90%, or 91%, 92%, 94%, or even more preferably at least about 95%, 96%, 97%, 98%, 99% or more identical) are also encompassed by this invention.

Specialists in this field should be clear that, in addition to the nucleotide sequences of MCT C. glutamicum, shown in the List of sequences in the form of odd numbers SEQ ID NO: in the population (for example, in the C. glutamicum population), DNA sequence polymorphisms may exist, which lead to substitutions in the amino acid sequences of MCT proteins. Such genetic polymorphism in the MCT gene may exist among individuals in a population due to natural variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules containing an open reading frame encoding an MCT protein, preferably C. glutamicum MCT protein. Such natural variations can usually result in 1-5% dispersion in the nucleotide sequence of the MCT gene. It is understood that any and all such nucleotide variations and the resulting amino acid polymorphisms in MCTs that are the result of natural variation and that do not alter the functional activity of MCT proteins are included in the scope of this invention.

Nucleic acid molecules corresponding to natural variants and not related to C. glutamicum homologs of MCT C. glutamicum MCT DNA of the present invention can be isolated on the basis of their homology to the C. glutamicum MCT nucleic acid described herein using C. glutamicum DNA or part thereof as a hybridization probe according to standard hybridization methods under stringent hybridization conditions. Thus, in another embodiment, the isolated nucleic acid molecule of the present invention has a length of at least 15 nucleotides and hybridizes under stringent conditions to a nucleic acid molecule containing a nucleotide sequence having an odd SEQ ID NO: Sequence Number. In other embodiments, the nucleic acid has a length of at least 30, 50, 100, 250, or more nucleotides. As used herein, the term “hybridizes under stringent conditions” refers to hybridization and washing conditions in which nucleotide sequences that are at least 60% homologous to each other typically remain hybridized to each other. Preferably, these conditions are such that the sequences of at least about 65%, more preferably at least about 70% and even more preferably at least about 75% or more homologous to each other, usually remain hybridized to each other . Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-restrictive example of stringent hybridization conditions is hybridization conditions in a 6X sodium chloride / sodium citrate (SSC) mixture at approximately 45 ° C followed by one or more washes in 0.2 × SSC, 0.1 SDS at 50-65 ° C. Preferably, the isolated nucleic acid molecule of the invention, which hybridizes under stringent conditions to the nucleotide sequence of the invention, corresponds to a naturally occurring nucleic acid molecule. As used herein, a “naturally occurring” nucleic acid molecule is an RNA or DNA molecule having a nucleotide sequence that is found in nature (eg, encodes a natural protein). In one embodiment, this nucleic acid encodes a natural C. glutamicum MCT protein.

One skilled in the art will also understand that, in addition to naturally occurring variants of the MCT sequence that may exist in a population, changes by mutation can be introduced into the nucleotide sequence of the present invention, which will lead to changes in the amino acid sequence of the encoded MCT protein, without changing the functional ability of the protein MST For example, nucleotide substitutions can be made in the nucleotide sequence of the present invention, resulting in amino acid substitutions in the “non-essential” amino acid residues. An “irreplaceable” amino acid residue is a residue that can be changed in comparison with the wild-type sequence of one of the MCT proteins (for example, having even numbers of SEQ ID NO: Sequence List) without changing the activity of the indicated MCT protein, while the “irreplaceable” amino acid residue is necessary for the activity of the MCT protein. However, other amino acid residues (for example, residues that are non-conservative or only semi-conservative in a domain having MCT activity) may not be indispensable for activity and, therefore, will likely be available for change without changing the activity of MCT.

Thus, another aspect of the present invention relates to nucleic acid molecules encoding MCT proteins that contain changes in amino acid residues that are not indispensable for MCT activity. Such MCT proteins differ in amino acid sequence from sequences having even numbers SEQ ID NO: Sequence List, but still retain at least one of the MCT activities described herein. In one embodiment, the isolated nucleic acid molecule contains a nucleotide sequence encoding a protein that contains at least about 50% amino acid sequence homologous to the amino acid sequence of the present invention and is able to participate in the metabolism of compounds necessary for constructing cell membranes in C. glutamicum or in transporting molecules through these membranes, or has one or more of the activities shown in table 1. Preferably, the protein encoded by this a nucleic acid molecule is at least about 50-60% homologous to the amino acid sequence of one of the odd numbers SEQ ID NO: Sequence List, more preferably at least about 60-70% homologous to one of these sequences, even more preferably at least about 70-80%, 80-90%, 90-95% homologous to one of these sequences, and most preferably at least about 96%, 97%, 98% or 99% homologous to one of the amino acid sequences the sequences of this invention.

To determine the percent homology of two amino acid sequences (for example, one of the amino acid sequences of the present invention and its mutant form) or two nucleic acids, these sequences are compared for optimal comparison (for example, gaps (gaps) can be introduced into the sequence of a single protein or nucleic acid for optimal comparison with another protein or other nucleic acid). Then compare amino acid residues or nucleotides at the corresponding positions of amino acids or nucleotides. When a position in one sequence (for example, in one of the amino acid sequences of the present invention) is occupied by the same amino acid residue or nucleotide as the corresponding position in another sequence (for example, in a mutant form of this amino acid sequence), then these molecules are homologous in this position (ie, as used herein, the “homology” of an amino acid or nucleic acid is equivalent to the “identity” of an amino acid or nucleic acid). Percent homology between two sequences is a function of the number of identical positions common to two sequences (i.e.,% homology = number of identical positions / total number of positions × 100).

An isolated nucleic acid molecule encoding an MCT protein homologous to a protein sequence of the invention (for example, sequences having even numbers SEQ ID NO: Sequence Listing) can be created by introducing one or more nucleotide substitutions, additions or deletions to the nucleotide sequence of the invention, so that one or more substitutions, additions or deletions of amino acids are introduced into the encoded protein. Mutations can be introduced into one of the nucleotide sequences of the present invention by standard methods, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid changes are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with major side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenyl alanine, tryptophan, histidine). Thus, the predicted non-essential amino acid residue in the MCT protein is preferably replaced by another amino acid residue from the same family of side chains. Alternatively, in another embodiment, mutations may be introduced randomly along all or part of the MCT coding sequence, for example, saturating mutagenesis, and the resulting mutants may be screened for MCT activity described herein to identify mutants that retain MCT activity. After mutagenesis of one of the nucleotide sequences having an odd number SEQ ID NO: Sequence List, the encoded protein can be expressed recombinantly, and the activity of this protein can be determined using, for example, the assays described here (see Example 8 of the Examples section) .

In addition to the nucleic acid molecules encoding the MCT proteins described above, another aspect of the present invention relates to isolated nucleic acid molecules that are antisense to them. An "antisense" nucleic acid contains a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, for example, complementary to the coding chain of a double-stranded cDNA molecule or complementary to an mRNA sequence. Thus, an antisense nucleic acid can be hydrogenated to a sense nucleic acid. The antisense nucleic acid may be complementary to the entire MCT coding chain, or only part thereof. In one embodiment, the antisense nucleic acid molecule is antisense with respect to the “coding region” of the coding strand of the nucleotide sequence encoding the MCT protein.

The term "coding region" refers to a region of a nucleotide sequence containing codons that are translated into amino acid residues (for example, the complete coding region of SEQ ID NO: 5 (RXA00104) contains nucleotides 1-756). In another embodiment, the antisense nucleic acid molecule is antisense with respect to the "non-coding region" of the coding strand of the nucleotide sequence encoding MCT. The term "non-coding region" refers to 5'- and 3'-sequences that flank the coding region that are not translated into amino acids (i.e., they are also called 5'- and 3'-untranslated regions).

If there are MCT coding chain sequences described herein (for example, sequences shown in odd numbers SEQ ID NO: in the Sequence Listing), the antisense nucleic acids of this invention can be constructed according to Watson and Crick base pairing rules. The antisense nucleic acid molecule may be complementary to the complete coding region of the MCT mRNA, but more preferably it is an oligonucleotide that is antisense relative to only part of the coding or non-coding region of the MCT mRNA. For example, the antisense oligonucleotide may be complementary to the region surrounding the start site of translation of the MCT mRNA. The antisense oligonucleotide may have a length of, for example, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides. The antisense nucleic acid of the present invention can be constructed by chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of these molecules or to increase the physical stability of the duplex formed between antisense and sense nucleic acids, for example, phosphorothioate derivatives and acridine substituted nucleotides should be used. Examples of modified nucleotides that can be used to produce an antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlororacil, 5-iodouracil, hypoxanthin, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethyl 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylguanine, 3 methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyl sludge, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid (v), pivutuoxinosine, 2, vibutoxinosine, -thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of uracil-5-hydroxyacetic acid, uracil-5-hydroxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w and 2,6-diaminopurine. Alternatively, an antisense nucleic acid can be biologically produced using an expression vector into which the nucleic acid has been subcloned in the antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will have an antisense orientation relative to the target nucleic acid of interest, described below in the next subsection).

The antisense nucleic acid molecules of the invention are typically introduced into a cell or generated in situ such that they hybridize or bind to cellular mRNA and / or genomic DNA encoding the MCT protein to inhibit protein expression through this, for example, by transcription inhibition and / or broadcasts. Hybridization can occur through universally accepted complementarity of nucleotides to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes through specific interactions in a large groove in the double helix. An antisense molecule can be modified so that it specifically binds to a receptor or antigen expressed on the surface of a selected cell, for example, by binding an antisense nucleic acid molecule to a peptide or antibody that binds to a cell surface receptor or antigen. An antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To obtain sufficient intracellular concentrations of antisense molecules, vector constructs are preferred in which the antisense nucleic acid molecule is placed under the control of a strong, preferably prokaryotic, viral or eukaryotic promoter.

In yet another embodiment, the antisense nucleic acid molecule of the present invention is an α-anomeric nucleic acid molecule. The α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNAs, in which, in contrast to the usual β-units, the chains run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res. 15: 6625-6641). An antisense nucleic acid molecule may also contain a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15: 6131-6148) or a chimeric RNA DNA analog (Inoue et al. (1987) FEBS Lett. 215: 327-330).

In yet another embodiment, the antisense nucleic acid of the present invention is a ribozyme. Ribozymes are catalytic ribonuclease RNA molecules that are capable of cleaving a single-stranded nucleic acid, such as mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammer-headed ribozymes (described in Haselhoff and Gerlach (1988) Nature 334: 585-591)) can be used for catalytic cleavage of MCT mRNA transcripts, thereby inhibiting translation of MCT mRNA. A ribozyme having specificity for the MCT encoding nucleic acid can be constructed based on the nucleotide sequence of the MCT DNA described herein (i.e., SEQ ID NO: 5 (RXA00104)). For example, a Tetrahymena L-19 IVS RNA derivative can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence that must be cleaved in the MCT coding mRNA. See, for example, Cech et al. U.S. Patent No. 4987071 and Cech et al. U.S. Patent No. 5116742. Alternatively, MCT mRNA can be used to select catalytic RNA having specific ribonuclease activity from a pool of RNA molecules. See, for example, Bartel, D. and Szostak, J.W. (1993) Science 261: 1411-1418.

Alternatively, expression of the MCT gene can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the MCT nucleotide sequence (e.g., the promoter and / or enhancers of the MCT) with the formation of triple helical structures that inhibit transcription of the MCT gene in target cells. See, in general, Helene, C. (1991) Anticancer Drug Des. 6 (6): 569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660: 27-36; and Maher, L.J. (1992) Bioassays 14 (12): 807-15.

B. Recombinant Expression Vectors and Host Cells

Another aspect of the present invention relates to vectors, preferably expressing vectors containing a nucleic acid encoding an MCT protein (or part thereof). As used herein, the term “vector” refers to a nucleic acid molecule capable of transferring another nucleic acid to which it has been linked. One type of vector is a "plasmid", which is a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (for example, bacterial vectors having a bacterial replication origin and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of the host cell when introduced into the host cell and therefore replicate along with the host genome. In addition, some vectors are able to control the expression of genes with which they are functionally linked. Such vectors are referred to herein as “expression vectors”. Typically, expression vectors used in recombinant DNA methods are often in the form of plasmids. As used herein, the terms “plasmid” and “vector” are used interchangeably since the plasmid is the most commonly used form of the vector. However, the invention contemplates the inclusion of other forms of expression vectors such as viral vectors (e.g., defective replication retroviruses, adenoviruses and adeno-associated viruses) that perform equivalent functions.

The recombinant expression vectors of the present invention contain the nucleic acid of the present invention in a form suitable for expression of the nucleic acid in the host cell, which means that these recombinant expression vectors include one or more regulatory sequences selected based on the expression of the host cells, which functionally linked to an expressed nucleic acid sequence. In a recombinant expression vector, “operably linked” means that the nucleotide sequence of interest is linked to a regulatory sequence (regulatory sequences) such that expression of the nucleotide sequence is possible (for example, in an in vitro transcription / translation system or in a host cell, when introducing this vector into the host cell). The term "regulatory sequence" includes promoters, enhancers and other elements of regulation of expression (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include sequences that control the constitutive expression of the nucleotide sequence in many types of host cells and which control the expression of the nucleotide sequence only in specific host cells. Preferred regulatory sequences are, for example, promoters such as cos-, tac-, trp-, tet-, trp-tet, lpp-, lac-, lpp-lac-, lacI ^q -, T7-, T5-, T3- , gal-, trc-, ara-, SP6-, arny, SPO2, λ-P _R - or λ P _L , which are preferably used in bacteria. Additional regulatory sequences are, for example, yeast and fungal promoters, such as ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, plant promoters, such as CaMV / 35S, SSU, OCS, lib4 , usp, STLS1, B33, nos or ubiquitin or phaseolin promoters. Synthetic promoters may also be used. One skilled in the art will understand that the design of the expression vector may depend on factors such as the choice of the transformed host cell, the level of expression of the desired protein, etc. The expression vectors of the present invention can be introduced into host cells to produce proteins or peptides, including fusion proteins or peptides encoded by the nucleic acids described herein (e.g., MCT proteins, mutant forms of MCT proteins, fusion proteins, etc.). d.).

Recombinant expression vectors of the present invention can be designed to express MCT proteins in prokaryotic or eukaryotic cells. For example, MCT genes can be expressed in bacterial cells such as C. glutamicum, insect cells (using baculovirus expression vectors), yeast, and other fungal cells (see Romanes, MA et al. (1992) "Foreign gene expression in yeast : a review ". Yeast 8: 423-488; van den Hondel, CAMJJ et al. (1991)" Heterologous gene expression in filamentous fungi "in: More Gene Manipulations in Fungi, JW Bennet and LLLasure, eds., p. 396-428: Academic Press: San Diego; and van den Hondel, CAMJJ and Punt, PJ (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, JF et al., Eds ., p.1-28, Cambridge University Press: Cambridge), algae and multicellular plant cells (c . Schmidt, R. and Willmitzer, L. (1988) High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants "Plant Cell Rep .: 583-586), or mammalian cells. Suitable host cells are discussed further in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector may be transcribed and translated in vitro, for example, using the T7 promoter regulatory sequences and T7 polymerase.

The expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters that direct the expression of fused or non-fused proteins. Fusion vectors add a number of amino acids to the protein encoded therein, usually to the amino terminus of the recombinant protein, but also to the C-terminus, or fuse with suitable sites in the protein. Such fusion vectors typically serve three purposes: 1) to increase the expression of a recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) in order to facilitate the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion portion of the molecule and the recombinant protein to allow separation of the recombinant protein from the fusion portion of the molecule after purification of the fusion protein. Such enzymes and their related recognition sequences include factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, DB and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), which fusion of glutathione S-transferase (GST), maltose E-binding protein, or protein A, respectively, with the recombinant target protein. In one embodiment, the MCT protein coding sequence is cloned into a pGEX expression vector to create a vector encoding a fusion protein containing, from the N-terminal to the C-terminal, a GST thrombin-X protein cleavage site. This fusion protein can be purified by affinity chromatography using glutathione agarose resin. Recombinant MCT protein not fused to GST can be recovered by cleaving the fusion protein with thrombin.

Examples of suitable inducible non-fused E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69: 301-315), pLG338, pACYC184, pBR322, pUC18, pUC19, pKS30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11, pBdC1 and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; and Pouwels et al. , (1985) Cloning Vectors, Elsevier: New York IBSN 0 444 904018). The expression of the target gene from the pTrc vector is based on transcription of the host RNA polymerase from the hybrid trp-lac fusion promoter. The expression of the target gene from the vector pET 11d is based on transcription from the gn10-lac T7 fusion promoter mediated by coexpressed viral RNA polymerase (gnl T7). This viral polymerase is supplied by host strains BL21 (DE3) or HMS174 (DE3) from resident prophage λ carrying the T7 gnl gene under the transcriptional control of the lacUV 5 promoter. Appropriate vectors can be selected to transform other bacterial species. For example, it is known that plasmids pIJ101, pIJ364, pIJ702 and pIJ361 are applicable in Streptomyces transformation, while plasmids pUB110, pC194 or pBD214 are suitable for transformation of Bacillus species. Several plasmids useful in transferring genetic information to Corynebacterium include pHM1519, pBL1, p3A77, or pAJ667 (Pouwels et al., (1985) Cloning Vectors, Elsevier: New York IBSN 0 444 904018).

One strategy for maximizing the expression of a recombinant protein is to express the protein in a host bacterium with impaired proteolytic cleavage of the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128 ) Another strategy is to change the nucleic acid sequence that must be inserted into the expression vector so that the individual codons for each amino acid are codons primarily used in the bacteria selected for expression, such as C. glutamicum (Wada et al. (1992 ) Nucleic Acids Res. 20: 2111-2118). Such a change in the nucleic acid sequences of the present invention can be carried out by standard DNA synthesis methods.

In another embodiment, the MCT protein expression vector is a yeast expression vector. Examples of vectors for expression in S. cerevisiae yeast include pYepSec1 (Baldari, et al., (1987) EMBO J. 6: 229-234), 2 μ, pAG-1, Yep6, Yep13, pEMBLYe23, pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 (Schuiz et al., (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods for constructing vectors suitable for use in other fungi, such as filamentous fungi, include methods and vectors described in detail in van den Hondel, C.A.M.J.J. and Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, JF Peberdy, et al., Eds., P. 1-28, Cambridge University Press: Cambridge, and Pouwels et al., (1985) Cloning Vectors, Elsevier: New York IBSN 0 444 904018).

Alternatively, MCT proteins can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors suitable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAC series (Smith et al., (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers ( 1989) Virology 170: 31-39).

In another embodiment, the MCT proteins of the present invention can be expressed in cells of unicellular plants (such as algae) and in plant cells from higher plants (e.g., seed plants, such as crops). Examples of plant expression vectors include vectors described in detail in Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol . Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acid Res. 12: 8711-8721, and include pLGV23, pGHlac +, pBIN19, pAK2004 and pDH51 (Pouwels et al., (1985) Cloning Vectors, Elsevier: New York IBSN 0 444 904018).

In yet another embodiment, the nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al., (1987) EMBO J. 6: 187-195). When used in mammalian cells, the regulatory functions of this expression vector are often provided by viral regulatory elements. For example, commonly used promoters come from polyoma, adenovirus 2, cytomegalovirus, and monkey virus 40. For suitable expression systems for prokaryotic and eukaryotic cells, see chapters 16 and 17 of Sambrook J., Fritsh, EF, and Maniatis, T. Molecular Cloning. A Laboratory Manual, 2 ^nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

In another embodiment, the mammalian recombinant expression vector is capable of directing nucleic acid expression predominantly in a particular type of cell (e.g., tissue-specific regulatory elements are used to express nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., (1987) Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43: 235- 275), in particular, promoters of T-cell receptors (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and immunoglobulins (Banerji et al., (1983) Cell 33: 729-740; Queen and Baltimore (1983 ) Cell 33: 741-748), neuron-specific promoters (e.g., neurofilament promoter; Byrne and Ruddle (1989) PNAS 86: 5473-5477), pancreatic-specific promoters (Ed-lund et al. (1985) Science 230 : 912-916) and specific for milk oh gland promoters (e.g., milk whey promoter; U.S. Patent №4 873 316 and European Application Publication №264 166). Stage-regulated promoters are also included, for example, hox mouse promoters (Kessel and Gruss (1990) Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546).

Further, the present invention provides a recombinant expression vector comprising a DNA molecule of the invention cloned into this expression vector in an antisense orientation. That is, this DNA molecule is functionally linked to a regulatory sequence in such a way that expression (transcription of this DNA molecule) of an RNA molecule is possible, which is antisense with respect to MCT mRNA. Regulatory sequences functionally linked to an antisense cloned nucleic acid that directs the continuous expression of this antisense RNA molecule in multiple cell types can be selected, for example, viral promoters and / or enhancers, or regulatory sequences can be selected that direct the constitutive, tissue-specific or specific for a particular type of cell expression of antisense RNA. The antisense expression vector may be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a highly efficient regulatory region, the activity of which can be determined by the type of cells into which this vector is introduced. For a discussion of the regulation of gene expression using antisense genes, see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis. Reviews - Trends in Genetics, Vol. 1 (1) 1986.

Another aspect of the invention relates to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is clear that such terms refer not only to the particular cell under consideration, but also to the offspring or potential offspring of such a cell. Since some modifications may occur in subsequent generations due to mutations or environmental influences, such offspring may not actually be identical to the parent cell, but it is nevertheless included in the scope of this term, as used here.

The host cell may be any prokaryotic or eukaryotic cell. For example, MCT protein can be expressed in bacterial cells such as C. glutamicum, insect cells, yeast or mammalian cells (such as Chinese hamster ovary (CHO) cells or COS cells). Other suitable host cells are known to those skilled in the art. Microorganisms related to Corynebacterium glutamicum, which can be easily used as host cells for the nucleic acid molecules and proteins of the present invention, are shown in table 3.

The DNA vector can be introduced into prokaryotic or eukaryotic cells by conventional methods of transformation or transfection. As used herein, the terms "transformation" and "transfection", "conjugation" and "transduction" refer to various recognized methods in the art for introducing foreign nucleic acid (e.g., linear DNA or RNA (e.g., linearized vector or gene construct separately without a vector) ) or a nucleic acid in the form of a vector (e.g., plasmid, phage, phasmid, phagemid, transposon or other DNA) into the host cell, including co-precipitation of calcium phosphate or calcium chloride mediated by DEAE-dextran transfec iey, lipofection, natural competition chemically mediated transfer, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al., (Molecular Cloning. A Laboratory Manual, 2 ^{nd ed., Cold Spring Harbor Laboratory} , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory reference manuals.

With respect to stable transfection of mammalian cells, it is known that, depending on the expression vector used and the transfection method, only a small fraction of cells can integrate foreign DNA into their genome. To identify and select these integrants, a gene that encodes a selectable marker (e.g., an antibiotic resistance gene) is usually introduced into the host cells along with the gene of interest. Preferred breeding markers include markers that confer resistance to drugs such as G418, hygromycin and methotrexate. A nucleic acid encoding a selectable marker may be introduced into the host cell on the same vector as the vector encoding the MCT protein, or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (for example, cells that included the selectable marker gene survive while other cells die).

To create a homologous recombinant microorganism, a vector is obtained that contains at least a portion of the MCT gene into which a deletion, addition or substitution has been introduced, thereby changing, for example, a functional disorder, the MCT gene. Preferably, this gene is the Corynebacterium glutamicum MCT gene, but it can be a homolog from a related bacterium or even from a mammal, yeast or insect. In a preferred embodiment, this vector is designed in such a way that, upon homologous recombination, the endogenous MCT gene is functionally destroyed (that is, it no longer encodes a functional protein; this vector is also called a knockout vector). Alternatively, the vector can be designed in such a way that, by homologous recombination, the endogenous MCT gene mutates or otherwise changes, but still encodes a functional protein (for example, the regulatory region against the course of transcription can be changed to change through this expression of the endogenous MCT protein) . In the homologous recombination vector, the variable part of the MCT gene is flanked at its 5'- and 3'-ends with additional nucleic acid of the MCT gene to enable homologous recombination between the exogenous MCT gene carried by this vector and the endogenous MCT gene in the microorganism. The additional flanking MCT nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Usually, a few kb flanking DNA (at both the 5'- and 3'-ends) are included in this vector (see, for example, Thomas, K.R. and Capecchi, M.R. (1987) Cell 51: 503 for a description of homologous recombination vectors). The vector is introduced into the microorganism (for example, by electroporation), and cells in which the introduced MCT gene has been homologously recombined with the endogenous MCT gene are selected using methods known in the art.

In another embodiment, recombinant microorganisms can be obtained that contain selected systems that enable controlled expression of the introduced gene. For example, the inclusion of the MCT gene on a vector, placing it under the control of the lac operon, makes it possible to express the MCT gene only in the presence of IPTG. Such a regulatory system is well known in the art.

In another embodiment, the endogenous MCT gene in the host cell is destroyed (for example, by homologous recombination or other genetic methods known in the art), so that expression of its protein product does not occur. In another embodiment, the endogenous or introduced MCT gene in the host cell has been altered by one or more point mutations, deletions or inversions, but still encodes a functional MCT protein. In yet another embodiment, one or more regulatory regions (e.g., promoter, repressor or inducer) of the MCT gene in the microorganism has been altered (e.g., by deletion, shortening, inversion, or point mutation), so that the expression of the MCT gene is modulated. One skilled in the art will recognize that host cells containing more than one of the described modifications of the MCT gene and protein can be easily obtained using the methods of the invention, and it is intended that they be included in the invention.

A host cell of the invention, for example, a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e. express) an MCT protein. Thus, the invention further provides methods for producing MCT proteins using host cells of the invention. In one embodiment, the method comprises culturing a host cell of the invention (into which a recombinant expression vector encoding an MCT protein has been introduced, or into the genome of which a gene encoding a wild-type MCT protein or an altered MCT protein has been introduced) in a suitable medium until until MCT protein is produced. In another embodiment, this method further comprises isolating MCT proteins from the medium or from the host cell.

C. Isolated MST Proteins

Another aspect of the invention relates to isolated MCT proteins and their biologically active parts. The "isolated" or "purified" protein or its biologically active part essentially does not contain cellular material when produced by recombinant DNA methods or in the chemical synthesis of chemical precursors or other chemical products. The expression “substantially free of cellular material” includes MCT protein preparations in which the protein is separated from the cellular components of cells in which it is naturally or recombinantly produced. In one embodiment, the term “substantially free of cellular material” includes MCT protein preparations having less than about 30% (dry weight) of a non-MCT protein (also referred to as a “contaminant (impurity) protein”), more preferably less than about 20% non-MCT protein, even more preferably less than about 10% non-MCT protein, and most preferably less than about 5% non-MCT protein. In the recombinant preparation of the MCT protein or its biologically active part, it is preferably substantially free of culture medium, i.e. the culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The expression "substantially free of chemical precursors or other chemical products" includes MCT protein preparations in which the protein is separated from chemical precursors or other chemical products that are involved in the synthesis of this protein. In one embodiment, the term “substantially free of chemical precursors or other chemical products” includes MCT protein preparations having less than about 30% (dry weight) non-MCT chemical precursors or chemical products, more preferably less than about 20% chemical precursors or non-MCT chemical products, even more preferably less than about 10% of non-MCT chemical precursors or chemical products, and most preferably less than about 5% of chemical precursors or non-MCT chemical products. In preferred embodiments, the isolated proteins or their biologically active parts do not contain contaminating (impurity) proteins from the same organism from which the MCT protein is obtained. Typically, such proteins are obtained by recombinant expression, for example, C. glutamicum MCT protein, in a microorganism, for example C. glutamicum.

The isolated MCT protein or part of this invention can participate in the metabolism of compounds necessary for constructing cell membranes in C. glutamicum or in the transport of molecules through these membranes, or has one or more activities shown in Table 1. In preferred embodiments, this protein or its the portion contains an amino acid sequence that is sufficiently homologous to the amino acid sequence of the present invention (e.g., sequences having even numbers SEQ ID NO: Sequence List functions), so that this protein or its part retains the ability to participate in the metabolism of compounds necessary for the construction of cell membranes in C. glutamicum or in the transport of molecules through these membranes. A portion of this protein is preferably a biologically active portion, as described herein. In another preferred embodiment, the MCT protein of the present invention has an amino acid sequence represented as even-numbered SEQ ID NO: Sequence Listing. In another preferred embodiment, the MCT protein has an amino acid sequence that is encoded by a nucleotide sequence that hybridizes, for example, hybridizes under stringent conditions, to the nucleotide sequence of the present invention (for example, sequences having odd numbers SEQ ID NO: Sequence Listing). In another preferred embodiment, the MCT protein has an amino acid sequence that is encoded by a nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, or 91%, 92%, 94%, or even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to the cleotide sequence of the present invention, or part thereof. It is understood that ranges and values of identity intermediate between the above ranges (e.g., 70-90% identical or 80-95% identical) are also encompassed by this invention. For example, it is understood that ranges of identity values are also included using a combination of any of the values cited above as upper and / or lower limits. Preferred MCT proteins of the present invention also preferably possess at least one of the MCT activities described herein. For example, a preferred MCT protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence that hybridizes, for example, hybridizes under stringent conditions, with the nucleotide sequence of the present invention, and can be involved in the metabolism of compounds necessary for constructing cell membranes in C. glutamicum or in transport molecules through these membranes, or has one or more activities shown in table 1.

In other embodiments, the MCT protein is substantially homologous to the amino acid sequence of the present invention (e.g., sequences having even numbers SEQ ID NO: Sequence List) and retains the functional activity of the protein of one of the amino acid sequences of the present invention, despite the fact that it differs in amino acid sequence due to natural variation or mutagenesis, as described in detail in subsection I above. Thus, in another embodiment, the MCT protein is a protein that contains an amino acid sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, or 91%, 92%, 94%, or even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to the complete amino acid sequence the identity of the present invention, and which has at least one of the MCT activities described herein. It is understood that ranges and values of identity intermediate between the above ranges (e.g., 70-90% identical or 80-95% identical) are also encompassed by this invention. For example, it is understood that ranges of identity values are also included using a combination of any of the values cited above as upper and / or lower limits. In another embodiment, this invention relates to a full-sized protein of C. glutamicum, which is essentially homologous to the complete amino acid sequence of the present invention.

The biologically active portions of the MCT protein include peptides containing amino acid sequences derived from the amino acid sequence of the MCT protein, for example, the amino acid sequence having an even number SEQ ID NO: Sequence List, or the amino acid sequence of a protein homologous to the MCT protein that contains fewer amino acids than full length MCT protein or a full-sized protein that is homologous to the MCT protein and exhibit at least one activity of the MCT protein. Typically, biologically active parts (peptides, for example, peptides that are 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100, or more amino acids in length) contain a domain or a motive with at least one activity of the MCT protein. In addition, other biologically active parts in which other regions of this protein are deleted can be obtained by recombinant methods and evaluated for one or more of the activities described herein. Preferably, the biologically active portions of the MCT protein include one or more domains / motifs or portions thereof having biological activity.

MCT proteins are preferably obtained by recombinant DNA methods. For example, a nucleic acid molecule encoding this protein is cloned into an expression vector (as described above), the expression vector is introduced into the host cell (as described above) and the MCT protein is expressed in the host cell. MCT protein can then be isolated from cells using an appropriate purification scheme using standard protein purification methods. Alternatively, by recombinant expression, the MCT protein, polypeptide or peptide can be chemically synthesized using standard peptide synthesis techniques. In addition, native MCT protein can be isolated from cells (eg, endothelial cells), for example, using an anti-MCT antibody, which can be obtained by standard methods using the MCT protein or a fragment of this invention.

The invention also provides chimeric or fused MCT proteins. As used herein, an “MCA chimeric protein” or “fusion protein” comprises an MCT polypeptide operably linked to a non-MCT polypeptide. "MCT polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an MCT protein, while "non-MCT polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to an MCT protein, for example, a protein that is different from a protein MCT and which is derived from the same or another organism. In a fusion protein, the term “operably linked” means that the MCT polypeptide and the non-MCT polypeptide are fused in reading frame to each other. The non-MCT polypeptide may be fused to the N-terminus or C-terminus of the MCT polypeptide. For example, in one embodiment, the fusion protein is a GST-MCT fusion protein in which the MCT sequences are fused to the C-terminus of the GST sequences. Such fusion proteins may facilitate the purification of recombinant MCT proteins. In another embodiment, the fusion protein is an MCT protein containing a heterologous signal sequence at its N-terminus. In some host cells (e.g., mammalian host cells), expression and / or secretion of the MCT protein can be increased by using a heterologous signal sequence.

Preferably, the chimeric or fusion protein of the MCT of the present invention is obtained by standard recombinant DNA methods. For example, DNA fragments encoding different polypeptide sequences are ligated together in a reading frame in accordance with generally accepted methods, for example, by using blunt ends or step ends for ligation, cleavage with restrictase enzymes to provide suitable ends, completion of sticky ends, if necessary, treatment with alkaline phosphatase to avoid unwanted binding and enzymatic ligation. In another embodiment, the fused gene can be synthesized by conventional methods, including using automatic DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that cause protrusions between two consecutive gene fragments, which can then be annealed and re-amplified to form a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology , eds. Ausubel et al., John Wiley and Sons: 1992). In addition, many expression vectors that already encode the part necessary for fusion (for example, the GST polypeptide) are commercially available. The MCT-encoding nucleic acid can be cloned into such an expression vector such that the fusion portion binds to the reading frame in the MCT protein.

Homologs of the MCT protein can be obtained by mutagenesis, for example, discrete point mutation or shortening of the MCT protein. As used herein, the term “homologue” refers to a variant form of the MCT protein that acts as an agonist or antagonist of the activity of the MCT protein. An MCT protein agonist can retain essentially the same biological activities, or a subpopulation of biological activities of the MCT protein. An MCT protein antagonist can inhibit one or more activities of a naturally occurring form of MCT protein, for example, by competitively linking to a lower or higher member of the metabolic cascade of cell membrane components, which includes the MCT protein, or by binding to the MCT protein, which mediates the transport of compounds through such membranes, thereby preventing translocation.

Alternatively, homologs of the MCT protein can be identified by screening combinatorial libraries of mutants, for example, truncated mutants, of the MCT protein for agonistic or antagonistic activity against the MCT protein. In one embodiment, a mixed library of MCT variants is generated by combinatorial mutagenesis at the nucleic acid level and encoded by a library of mixed (diverse) genes. A mixed library of MCT variants can be obtained, for example, by enzymatically ligating a mixture of synthetic oligonucleotides in a gene sequence so that a degenerate set of potential MCT sequences can be expressed as individual polypeptides, or alternatively as a set of larger fusion proteins (e.g., for phage display (phage representation)) containing a number of MCT sequences in them. There are various methods that can be used to obtain libraries of potential homologues of MCT from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic gene is then ligated into a suitable expression vector. The use of a degenerate set of genes makes it possible to provide, in one mixture, all sequences encoding the desired set of potential MCT sequences. Methods for the synthesis of degenerate oligonucleotides are known in the art (see, e.g., Narang, SA (1983) Tetrahedron 39: 3; Itakura et al., (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., (1984 ) Science 198: 1056; Ike et al., (1983) Nucleic Acid Res. 11: 477.

In addition, libraries of fragments of the MCT protein coding sequence can be used to generate a mixed population of MCT fragments for screening and subsequent selection of homologues of the MCT protein. In one embodiment, a library of fragments of the coding sequence can be obtained by processing a double-stranded PCR fragment of the MCT coding sequence with a nuclease under conditions in which the formation of single-strand breaks occurs only approximately once per molecule, denaturation of the double-stranded DNA, renaturation of this DNA with the formation of double-stranded DNA, which may include semantic / antisense pairs from various torn products, by removing single-chain parts from re-formed dupes eksov treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. Using this method, an expression library can be obtained that encodes the N-terminal, C-terminal and internal fragments of various sizes of the MCT protein.

Several methods are known in the art for screening gene products of combinatorial libraries prepared using point mutations or shortening, and for screening cDNA libraries for gene products having a selected property. Such methods can be adapted for rapid screening of gene libraries generated by combinatorial mutagenesis of MCT homologs. The most common high-throughput assay methods for screening large gene libraries typically include cloning a gene library into replicable expression vectors, transforming suitable cells with a resulting vector library, and expressing combinatorial genes under conditions in which the detection of the desired activity facilitates the isolation of the vector encoding the gene whose product has been detected. Recursive complex mutagenesis (REM), a new method that enhances the frequency of occurrence of functional mutants in libraries, can be used in combination with screening assays to identify homologs of MCTs (Arkin and Yourvan (1992) PNAS 89: 7811-7815; Delgrave et al., (1993) Protein Engineeribg 6 (3): 327-331).

In another embodiment, cell-based assays can be used to analyze a mixed MCT library using methods well known in the art.

D. Applications and methods of the invention

Nucleic acid molecules, proteins, protein homologs, fusion proteins, primers, vectors and host cells described herein can be used in one or more of the following methods: identification of C. glutamicum and related organisms; mapping of the genomes of organisms related to C. glutamicum; identification and localization of sequences of interest C. glutamicum; evolutionary research; identification of sections of the MCT protein necessary for function; modulating the activity of MCT protein; modulating the metabolism of one or more components of cell membranes; modulating the transmembrane transport of one or more compounds; and modulating the cell production of the desired compound, such as a fine chemicals.

MCT nucleic acid molecules of the present invention have many uses. Firstly, they can be used to identify the organism as being Corynebacterium glutamicum or a closely related organism. They can also be used to identify the presence of C. glutamicum or a related organism in a mixed population of microorganisms. The present invention provides nucleic acid sequences of a number of C. glutamicum genes; by probing the extracted genomic DNA culture of a unique or mixed population of microorganisms under stringent conditions with a probe covering a portion of the C. glutamicum gene that is unique to this organism, it can be determined whether this organism is present.

Although the bacterium Corynebacterium glutamicum is non-pathogenic, it is related to pathogenic species such as Corynebacterium diphtheriae. Corynebacterium diphtheriae is a causative agent of diphtheria, a rapidly developing, acute, proceeding with increasing temperature of infection, which includes both local and systemic pathology. In this disease, local damage develops in the upper respiratory tract and includes necrotic damage to epithelial cells; Bacilli secrete a toxin that is disseminated through this damage to the distal sensitive tissues of the body. Degenerative changes caused by inhibition of protein synthesis in these tissues, which include the heart, muscles, peripheral nerves, adrenal glands, kidneys, liver and spleen, lead to systemic pathology of this disease. Diphtheria continues to occur with high frequency in many parts of the world, including Africa, Asia, Eastern Europe and the independent states of the former Soviet Union. Ongoing diphtheria epidemics in the last two areas have resulted in at least 5,000 fatalities since 1990.

In one embodiment, the invention provides a method for identifying the presence or activity of Corynebacterium glutamicum in a subject. This method comprises detecting one or more nucleic acid or amino acid sequences of the invention (e.g., sequences shown in odd or even numbers of SEQ ID NO: respectively in the List of Sequences) in a subject, thereby detecting the presence or activity of Corynebacterium diphtheriae in this subject. C. glutamicum and C. diphtheriae are related bacteria, and many of the nucleic acid molecules and protein molecules in C. glutamicum are homologous to nucleic acid molecules and protein molecules of Corynebacterium diphtheriae and therefore can be used to detect Corynebacterium diphtheriae in a subject.

The nucleic acid molecules and protein molecules of the present invention may also serve as markers for specific regions of the genome. This finds application not only in genome mapping, but also for functional studies of C. glutamicum proteins. For example, to identify the portion of the genome to which a particular C. glutamicum DNA binding protein binds, the C. glutamicum genome could be cleaved and fragments could be incubated with the DNA binding protein. Fragments that bind this protein can be further probed with the nucleic acid molecules of the present invention, preferably with easily detectable labels; the binding of such a nucleic acid molecule to a fragment of the genome allows you to determine the location of this fragment on the map of the C. glutamicum genome and, when carried out many times with various enzymes, facilitates the rapid determination of the nucleic acid sequence to which this protein binds. Further, the nucleic acid molecules of the present invention can be sufficiently homologous to sequences of related species, so that these nucleic acid molecules can serve as markers for constructing a genomic map in related bacteria, such as Brevibacterium lactofermentum.

MCT nucleic acid molecules of the invention are also applicable to evolutionary studies and protein structure studies. The metabolic and transport processes in which the molecules of this invention are involved are used in a wide variety of prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention with nucleic acid molecules encoding similar enzymes from other organisms, the evolutionary affinity of these organisms can be estimated. Similarly, such a comparison allows us to evaluate which parts of this sequence are conservative and which are not conservative, which can help to determine those parts of this protein that are indispensable for the functioning of this enzyme. This type of determination is valuable for protein engineering research and may provide an indication of which protein can tolerate mutagenesis conditions without loss of function.

Manipulation of MCT nucleic acid molecules of the present invention can lead to the production of MCT proteins having functional differences compared to wild-type MCT proteins. These proteins may be improved in efficiency or activity, may be present in higher amounts in the cell than usual, or may have reduced efficiency or activity.

The present invention provides methods for screening molecules that modulate the activity of an MCT protein, either by interacting with the protein itself or the substrate or the MCT protein binding partner, or by modulating the transcription or translation of the MCT nucleic acid molecule of the present invention. In such methods, a microorganism expressing one or more MCT proteins of the present invention is contacted with one or more test compounds and the effect of each test compound on the activity or expression level of the given MCT protein is evaluated.

There are a number of mechanisms by which the MCT protein of the present invention can directly affect the yield, production and / or production efficiency of a fine chemicals from a C. glutamicum strain carrying such an altered protein. Extraction of fine chemicals from large-scale cultures of C. glutamicum is significantly improved if C. glutamicum secretes these desired compounds, since such compounds can be easily purified from the culture medium (as opposed to extraction from the mass of C. glutamicum cells). By increasing either the number or activity of transporter molecules that export fine chemicals from the cell, it is possible to increase the amount of fine chemicals that are present in the extracellular medium, providing easier collection and purification. In contrast, effective overproduction of one or more fine chemicals may require increased amounts of cofactors, precursor molecules, and intermediates for suitable biosynthetic pathways. Thus, by increasing the number and / or activity of transporter proteins involved in the import of nutrients such as carbon sources (i.e. sugars), nitrogen sources (i.e. amino acids, ammonium salts), phosphate and sulfur, it is possible to improve the production of a chemical product of fine organic synthesis due to the elimination of any limiting factors in nutrients in the biosynthetic process. Further, fatty acids and lipids themselves are desirable fine chemicals, so optimizing the activity or increasing the number of one or more MCT proteins of this invention that are involved in the biosynthesis of these compounds, or disrupting the activity of one or more MCT proteins that are involved in decomposition of these compounds, it is possible to increase the yield, production and / or production efficiency of fatty acid and lipid molecules from C. glutamicum.

The construction of one or more MCT genes of the present invention can also lead to MCT proteins having altered activities that indirectly affect the production of one or more fine chemicals from C. qlutamicum. For example, normal biochemical metabolic processes lead to the production of various waste products (for example, hydrogen peroxide and other reactive oxygen species) that can actively interact with the same metabolic processes (for example, peroxynitrite is known to nitrate side chains of tyrosine, inactivating some enzymes having tyrosine in the active site (Groves, JT (1999) Curr. Opin. Chem. Biol. 3 (2): 226-235). Although these waste products are usually excreted, C. glutamicum s used for large-scale enzymatic production are optimized for overproduction of one or more fine chemicals and therefore can produce more waste products than is typical for wild-type C. glutamicum. more MCT proteins of the present invention involved in the export of waste molecules can improve cell viability and maintain effective metabolic activity. The presence of high intracellular levels of the desired fine chemicals can also be virtually toxic to the cell, so increasing cell viability can be improved by increasing the ability of this cell to secrete these compounds.

Further, the MCT proteins of the present invention can be manipulated such that the relative amounts of different lipid and fatty acid molecules change. This can have a strong effect on the lipid composition of the cell membrane. Since each type of lipid has different physical properties, a change in the lipid composition of the membrane can significantly change the fluidity of the membrane. Changes in membrane fluidity can affect the transport of molecules across the membrane, which, as explained earlier, can modify the export of waste products or produced fine chemicals or the import of essential nutrients. Such changes in membrane fluidity can also greatly affect cell integrity; cells with relatively weaker membranes are more sensitive in the environment of a large-scale fermenter to mechanical stresses that can damage or kill the cell. When manipulating MCT proteins involved in the production of fatty acids and lipids for constructing membranes, so that the resulting membrane has a membrane composition that is more adaptable to environmental conditions that currently exist in cultures used for the production of fine chemicals the proportion of C. glutamicum cells will survive and multiply. Higher numbers of C. glutamicum cells in the culture will give higher yields, production or production efficiency of a fine chemical product from a given culture.

The aforementioned mutagenesis strategies for MCT proteins in order to obtain enhanced yields of fine chemicals from C. glutamicum should not be considered organic; variations in such strategies will be readily apparent to those of ordinary skill in the art. Using such strategies and by incorporating the mechanisms described here, the nucleic acid and protein molecules of the present invention can be used to generate C. glutamicum or related bacterial strains expressing mutated nucleic acid and MCT proteins, so that the desired production, production and / or production efficiency is desired connection is improving. This desired compound can be any natural product produced by C. glutamicum, which includes end products of biosynthesis pathways and intermediates of naturally occurring metabolic pathways, as well as molecules that are not found naturally in the metabolism of C. glutamicum, but which are produced by the C. glutamicum strain of this inventions.

The invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patent applications, patents, published patent applications, tables and the List of sequences cited in this application are hereby incorporated by reference.

EXAMPLES

Example 1: Obtaining the total genomic DNA of Corynebacterium glutamicum ATCC 13032

A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30 ° C with vigorous shaking in BHI medium (Difco). Cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in 5 ml of buffer-I (5% of the initial culture volume — all indicated volumes were calculated for 100 ml of culture volume). The composition of buffer-I: 140.34 g / l sucrose, 2.46 g / l MgSO ₄ × 7H ₂ O, 10 ml / l K ₂ HPO ₄ solution (100 g / l, adjusted to pH 6.7 using KOH ), 50 ml / l of M12 concentrate (10 g / l (NH ₄ ) ₂ SO ₄ , 1 g / l NaCl, 2 g / l MgSO ₄ × 7Н ₂ О, 0.2 g / l CaCl ₂ , 0.5 g / l yeast extract (Difco), 10 ml / l trace element mixture (200 mg / l FeSO ₄ × H ₂ O, 10 mg / l ZnSO ₄ × 7Н ₂ О, 3 mg / l MnCl ₂ × 4Н ₂ О, 30 mg / L H ₃ BO ₃ , 20 mg / L CoCl ₂ × 6H ₂ O, 1 mg / L NiCl ₂ × 6H ₂ O, 3 mg / L Na ₂ MoO ₄ × 2H ₂ O, 500 mg / L complexing agent ( EDTA or citric acid), 100 ml / l of a mixture of vitamins (0.2 mg / l biotin, 0.2 mg / l folic acid, 20 mg / l p-aminobenzoic acid, 20 mg / l riboflavin, 40 mg / l Ca pantothen ata, 140 mg / l nicotinic acid, 40 mg / l pyridoxol hydrochloride, 200 mg / l myoinositol) Lysozyme was added to this suspension to a final concentration of 2.5 mg / ml After approximately 4 hours of incubation at 37 ° C, the cell wall was destroyed and The obtained protoplasts were collected by centrifugation.The pellet was washed once with 5 ml of buffer-I and once with 5 ml of TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The precipitate was resuspended in 4 ml of TE buffer and 0.5 ml of SDS solution (10%) and 0.5 ml of NaCl solution (5 M) were added. After proteinase K was added to a final concentration of 200 μg / ml, the suspension was incubated for approximately 18 hours at 37 ° C. DNA was purified by phenol extraction, a phenol-chloroform-isoamyl alcohol mixture, and a chloroform-isoamyl alcohol mixture using conventional procedures. DNA was then precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by 30 minutes incubation at -20 ° C and 30 minutes centrifugation at 12,000 rpm in a high speed centrifuge using an SS34 rotor (Sorvall). DNA was dissolved in 1 ml TE-buffer containing 20 μg / ml RNase A and dialyzed at 4 ° C against 1000 ml TE-buffer for at least 3 hours. This time, the buffer was replaced 3 times. Aliquots of 0.4 ml of dialyzed DNA solution were added with 0.4 ml of 2 M LiCl and 0.8 ml of ethanol. After 30 min incubation at -20 ° C, DNA was collected by centrifugation (13000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TE buffer. The DNA obtained by this procedure could be used for all purposes, including Southern blotting or the construction of genomic libraries.

Example 2: Construction of genomic libraries of Corynebacterium glutamicum ATCC 13032 in Escherichia coli

Using DNA obtained as described in Example 1, cosmid and plasmid libraries were constructed according to known and well established methods (see, for example, Sambrook et al. (1989) "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel, FM et al. (1994) "Current Protocols in Molecular Biology", John Wiley and Sons).

Any plasmid or cosmid can be used. Plasmids pBR322 (Sutcliffe, J.G. (1979) Proc. Natl. Acad. Sci. USA, 75: 3737-3741) were particularly useful; pACYC177 (Change and Cohen (1978) J. Bacteriol. 134: 1141-1156), pBS series plasmids (pBSSK +, pBSSK- and others; Stratagene, La Jolla, USA) or cosmids as SuperCos1 (Stratagene, La Jolla, USA) or Lorist6 (Gibson, TJ, Rosenthal A. and Waterson, RH (1987) Gene 53: 283-286. Gene libraries specifically for use in C. glutamicum can be constructed using the plasmid pSL109 (Lee, H.-S. and AJ Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).

Example 3: Sequencing and computational functional DNA analysis

The genomic libraries described in Example 2 were used for DNA sequencing in accordance with standard methods, in particular using a chain termination method using an ABI377 sequencing apparatus (see, for example, Fleischman, RD et al., (1995) "Whole -Genom Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science, 269: 496-512) Sequencing primers were used with the following sequences: 5'-GGAAACAGTATGACCATG-3 '(SEQ ID NO: 677) or 5'-GTAAAACGACGGGCCAGT-3' (SEQ ID NO: 678).

Example 4: In vivo Mutagenesis

In vivo mutation of Corynebacterium glutamicum can be performed by passage of plasmid DNA (or another vector) through E. coli or other microorganisms (e.g., Bacillus species or yeast, such as Saccharomyces cerevisiae), which have impaired their ability to maintain the integrity of their genetic information. Typical mutated strains have mutations in genes for a DNA repair system (e.g., mutHLS, mutD, mutt, etc .; for reference, see Rupp, WD (1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-2294, ASM, Washington). Such strains are well known to the person skilled in the art. The use of such strains is illustrated, for example, in Greener, A. and Callahan, M. (1994) Strategies 7: 32-34.

Example 5: DNA Transfer between Escherichia coli and Corynebacterium glutamicum

Some species of Corynebacterium and Brevibacterium contain endogenous plasmids (e.g., pHM1519 or pBL1) that replicate autonomously (for a review see, e.g., Martin, J.F. et al. (1987) Biotechnology, 5: 137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can be easily constructed using standard vectors for E. coli (Sambrook et al. (1989) "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel, FM et al. ( 1994) "Current Protocols in Molecular Biology", John Wiley and Sons), to which a replication initiation site (origin) and a suitable marker from Corynebacterium glutamicum are added. Such replication initiation sites are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibacterium species. Especially applicable as transformation markers for these species are the genes for resistance to kanamycin (such as those obtained from transposons Tn5 or Tn903) or to chloramphenicol (Winnacker, EL (1987) "From Genes to Clones - Introduction to Gene Technology, VCH, Weinheim). There are numerous examples in the literature of constructing a wide variety of shuttle vectors that are replicated both in E. coli and C. glutamicum and which can be used for several purposes, including for overexpression of genes (for reference, see, for example, Yoshihama, M. et al. (1985) J. Bacteriol. 162: 591-597, Martin, JF et al. (1987) Biotechnology, 5: 137-146 and Eikmanns, B. J. et al. (1991) Gene, 102: 93-98).

Using standard methods, it is possible to clone a gene of interest into one of the shuttle vectors described above and introduce such hybrid vectors into Corynebacterium glutamicum strains. Transformation of C. glutamicum can be achieved by transformation of protoplasts (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-311), electroporation (Liebl, E. et al. (1989) FEMS Microbiol. Letters, 53: 399- 403) and in cases where special vectors are used, also by conjugation (as described, for example, in Schafer, A. et al. (1990) J. Bacteriol. 172: 1662-1666). Shuttle vectors for C. glutamicum in E. coli can also be transferred by obtaining plasmid DNA from C. glutamicum (using standard methods well known in the art) and transforming it into E. coli. This transformation step can be carried out using standard methods, but it is preferable to use a Mgg-deficient strain of E. coli, such as NM522 (Gough and Murray (1983) J. Mol. Biol. 166: 1-19).

Genes can be overexpressed in C. glutamicum strains using plasmids containing pCG1 (US Pat. No. 4,617,267) or fragments thereof, and optionally the kanamycin resistance gene from TN903 (Grindley, ND and Joyce, CM (1980) Proc. Natl. Acad. Sci. USA 77 (12): 7176-7180). In addition, genes can be overexpressed in C. glutamicum strains using the plasmid pSL109 (Lee, H.-S. and A.J.Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).

In addition to the use of replicating plasmids, overexpression of genes can also be achieved by integrating them into the genome. Genomic integration in C. glutamicum or other species of Corynebacterium or Brevibacterium can be accomplished by well-known methods, such as homologous recombination with a portion (s) of the genome, mediated by restriction endonuclease integration (REMI) (see, for example, DE Patent 19823834), or use of transposons. You can also modulate the activity of a gene of interest by modifying regulatory regions (e.g., promoter, repressor and / or enhancer) by modifying the sequence, insertion or deletion using site-directed methods (such as homologous recombination) or random-based methods (such as transposon mutagenesis or REMI). Nucleic acid sequences that function as transcription terminators can also be inserted at position 3 ′ from the coding region of one or more genes of the present invention; such terminators are well known in the art and are described, for example, in Winnacker, E.L. (1987) "From Genes to Clones - Introduction to Gene Technology, VCH, Weinheim.

Example 6: Assessment of Expression of Mutant Protein

Observations of the activity of a mutated protein in a transformed host cell are based on the fact that a mutant protein is expressed in a similar manner and in a similar amount compared to a wild-type protein. A suitable method for determining the level of transcription of a mutant gene (an indicator of the amount of mRNA available for translation of a gene product) is Northern blotting (for reference, see, for example, Ausubel, FM et al. (1994) "Current Protocols in Molecular Biology", Wiley; New York), in which a primer designed to bind to the gene of interest is labeled with a detectable label (usually radioactive or chemiluminescent), so that when the total RNA of an organism is extracted, subjected to gel electrophoresis, transferred to a stable mat Rix and incubation with this probe, the binding and amount of binding of this probe indicates the presence, and also shows the amount of mRNA for this gene. This information shows the degree of transcription of the mutant gene. Total cellular RNA can be obtained from Corynebacterium glutamicum using several methods that are well known in the art, such as those described in Bormann, E.R. et al. (1992) Mol. Microbiol. 6: 317-326.

Standard methods such as Western blotting (see, for example, Ausubel, FM et al. (1994) "Current Protocols in Molecular Biology", Wiley: New, can be used to assess the presence or relative amount of protein translated from this mRNA. York). In this method, common cellular proteins are extracted, separated by gel electrophoresis, transferred to a matrix, such as nitrocellulose, and incubated with a probe, such as an antibody that specifically binds to the desired protein. This probe is usually a labeled chemiluminescent or colorimetric label that can be easily detected. The presence and amount of the observed label indicates the presence and amount of the desired mutant protein present in the cell.

Example 7: Cultivation of genetically modified Corynebacterium glutamicum - culture medium and conditions

Genetically modified Corynebacteria are cultured in synthetic or natural growth media. A number of different growth media for Corynebacteria are both well known and readily available (Lieb et al. (1989) Appl. Microbiol. Biotechnol., 32: 205-210; von der Osten et al. (1998) Biotechnology Letters, 11 : 11-16; Patent DE 4120867; Liebl (1992) "The Genus Corynebacterium, in: The Procaryotes, Volume II, Balows, A. et al., Eds. Springer-Verlag). These media consist of one or more carbon sources sources of nitrogen, inorganic salts, vitamins, and trace elements. Preferred carbon sources are sugars such as mono-, di- or polysaccharides. For example, glucose, fructose, manno are very good sources of carbon. for, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar can also be provided to the medium through complex compounds such as molasses or other sugar refining by-products. Preferably, mixtures of various sources can also be provided. other possible carbon sources are alcohols and organic acids such as methanol, ethanol, acetic acid or lactic acid. Sources of nitrogen are usually organic or inorganic nitrogen compounds or materials containing these compounds. Examples of nitrogen sources include gaseous ammonia or ammonium salts, such as NH ₄ Cl, (NH ₄ ) ₂ SO ₄ , NH ₄ OH, nitrates, urea, amino acids or complex nitrogen sources, such as liquid corn extract, soy flour, soy protein, yeast extract, meat extract and others.

Compounds which are inorganic salts that may be included in these media include chloride, phosphorous or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Chelating compounds can be added to the medium to maintain metal ions in solution. Particularly useful chelating compounds include dihydroxyphenols, such as catechin or procathechate, or organic acids, such as citric acid. It is typical of these media that they also contain other growth factors, such as vitamins or growth stimulants, examples of which include biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts often come from complex components for media, such as yeast extract, molasses, liquid corn extract and others. The exact composition of the compounds of the media is highly dependent on the particular experiment, and the composition decision is made for each special case. Information on environmental optimization is available in the Applied Microbiol. Physiology. A Practical Approach (eds. PMRhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). commercial suppliers such as Standard 1 (Merck) or BHI (infusion solution for the brain and heart, Difco) or others.

All components of the medium are sterilized either by heating (20 min at 1/5 bar and 121 ° C), or by sterile filtration. These components may be sterilized at the beginning of cultivation or they may optionally be added continuously or intermittently.

Cultivation conditions are determined separately for each experiment. The temperature should be between 15 ° C and 45 ° C. The temperature may be kept constant or may vary during the experiment. The pH of the medium should be in the range of 5-8.5, preferably about 7.0, and can be maintained by the addition of buffers to the media. An example of a buffer for this purpose is potassium phosphate buffer. Alternatively or simultaneously, synthetic buffers such as MOPS, HEPES, ACES and others can be used. You can also maintain a constant pH of the culture by adding NaOH or NH ₄ OH during cultivation. When using complex components of the environment, such as yeast extract, the need for additional buffers may be reduced due to the fact that many compounds of the complex have high buffering capabilities. When using a fermenter for the cultivation of microorganisms, the pH can be adjusted using gaseous ammonia.

The incubation time is usually in the range from several hours to several days. This time is chosen to allow the accumulation of the maximum amount of product in the broth. The described culture experiments can be carried out in various vessels, such as microtiter plates, glass tubes, glass flasks or metal fermenters of different sizes. For screening a large number of clones, microorganisms should be cultured in microtiter plates, glass tubes or shaken flasks with or without septa. Preferably, 100 ml shaken flasks are used, filled with 10% (by volume) of the required growth medium. The flasks should be shaken on a rotary shaker (amplitude 25 mm) using a speed range of 100-300 rpm. Evaporation losses can be reduced by maintaining a humid atmosphere; alternatively, mathematical correction for evaporation losses should be carried out.

When testing genetically modified clones, an unmodified control clone or control clone containing the main plasmid without any insertion should also be tested. The medium is inoculated to OD ₆₀₀ 0.5-1.5 using cells grown on agar plates, such as CM plates (10 g / l glucose, 2.5 g / l NaCl, 2 g / l urea, 10 g / l polypeptone, 5 g / l yeast extract, 5 g / l meat extract, 22 g / l agar, pH 6.8 with 2 M NaOH), which were incubated at 30 ° C. Inoculation of the media is carried out either by introducing a suspension of C. glutamicum cells in saline from CM plates or by adding a liquid pre-culture of this bacterium.

Example 8 - Analysis of in vitro function of mutant proteins

The determination of the activities and kinetic parameters of enzymes is well developed in the art. Experiments to determine the activity of any particular altered enzyme should be performed relative to the specific activity of the wild-type enzyme, which is well within the capabilities of one skilled in the art. Reviews of enzymes in general, as well as specific details regarding the structure, kinetics, principles, methods, applications, and examples for determining the activity of many enzymes, can be found in the following references: Dixon, M. and Webb, BC, (1979) Enzymes, Longmans : London; Fersht, (1985) Enzyme Structure and Mechanism, Freeman: New York; Walsh, (1979) Enzymatic Reaction Mechanisms. Freeman: San Francisco; Price, NC, Stevens, L. (1982) Fundamentals of Enzymology, Oxford Univ, Press: Oxford; Boyer, PO, ed. (1983) The Enzymes, 3 ^rd ed. Academic Press: New York; Bisswanger, H., (1994) Enzymkinetik, 2 ^nd ed. VCH: Weinheim (IBSN 3527300325); Bergmeyer, HU, Bergmeyer, J., Grassi, M., eds. (1983-1986) Methods of Enzymatic Analysis, 3 ^rd ed., Vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes", VCH: Weinheim, p. 352-363.

The activity of proteins that bind to DNA can be measured by several established methods, such as DNA band shift assays (also called gel retardation assays (gel migration retardation assays)). The effect of such proteins on the expression of other molecules can be measured using assays with reporter genes (for example, described in Kolmar, H. et al. (1995) EMBO J. 14: 3895-3904 and references cited in this work). Test systems with reporter genes are well known and developed for applications in both prokaryotic and eukaryotic cells, using enzymes such as beta-galactosidase, green fluorescent protein and some others.

Determining the activity of membrane transport proteins can be carried out in accordance with methods such as the methods described in Gennis, R.B. (1989) "Pores, Channels and Transporters", in Biomemranes, Molecular Structure and Function, Springer: Heidelberg, p. 85-137; 199-234 and 270-322.

Example 9: Analysis of the effect of mutant proteins on the production of the desired product

The effect of genetic modification in C. glutamicum on the production of the desired compound (such as an amino acid) can be assessed by culturing the modified microorganism under suitable conditions (such as the conditions described above) and analyzing the medium and / or cell component for increased production of the desired product (i.e. amino acids). Such assay methods are well known to those skilled in the art and include spectroscopy, thin layer chromatography, various kinds of staining methods, enzymatic and microbiological methods, and analytical chromatography, for example, high performance liquid chromatography (see, for example, Ullmann, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 433-613, VCH: Weinheim (1985); Fallen, A. et al., (1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, vol .17; Rehm et al. (1993) Biotechnology, vol. 3, Chapter III: "Product recovery and purification", page 469-714, VCH: Weinheim; Belter, PA et al. (1988) Bioseparations: downstream processing for biotechnology, John Wiley and Sons; Kennedy, JF and Cabral, JMS (1992) Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, JA and Henry, JD (1988) Biochemical separations, in: Ullmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).

In addition to measuring the final fermentation product, you can also analyze other components of the metabolic pathways used to produce the desired compound, such as intermediates and by-products, to determine the overall production efficiency of the compound. Analysis methods include measuring the levels of nutrients in the medium (for example, sugars, hydrocarbons, sources of nitrogen, phosphate and other ions), measuring the composition of biomass and growth, analyzing the production of common metabolites of biosynthetic pathways, and measuring the gases produced during fermentation. Standard methods for these measurements are described in Applied Microbiol. Physiology. A Practical Approach (eds. P. M. Rhodes and P. F. Stanbury, eds., IRL Press p. 103-129; and 165-192 (ISBN 0 199635773) and references cited.

Example 10: Purification of the desired product from a culture of C. glutamicum

The recovery of the desired product from C. glutamicum cells or the supernatant of the culture described above can be accomplished by various methods well known in the art. If the desired product is not secreted from the cells, the cells can be harvested from the culture by low-speed centrifugation, these cells can be lysed by standard methods, such as mechanical methods or sonication. Cell debris is removed by centrifugation, and the supernatant fraction containing soluble proteins is stored for subsequent purification of the desired compound. If the product is secreted from C. glutamicum cells, these cells are removed from the culture by low-speed centrifugation, and the supernatant fraction is stored for subsequent purification.

The supernatant fraction from any purification method is subjected to chromatography with a suitable resin, in which either the desired molecule is retained on the chromatographic resin, while impurities are not retained in the sample, or impurities are retained by this resin, while the sample is not retained. Such chromatography steps may be repeated, if necessary, using the same resin or different resins. One skilled in the art should be well-versed in the selection of suitable chromatographic resins and in their most effective application for the particular molecule to be purified. The purified product can be concentrated by filtration or ultrafiltration and can be stored at a temperature at which the stability of this product is maximized.

There are a large number of cleaning methods known in the art, and the previous cleaning method should not be construed as limiting the invention. Such purification methods are described, for example, in Bailey, J.E. and Oilis, D.F. Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds can be evaluated by methods standard in the art. These include high performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic analysis or microbiological methods. Such analysis methods are discussed in Patek et al. (1994) App. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27-32 and Schmidt et al. (1998) Bioprocess Engineer, 19: 67-70, Ullmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p.89-90, p.521-540, p.540-547, p.559-566, 575-581 and p.581-587; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallen, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17.

Example 11: Analysis of the gene sequences of the invention

Comparison of sequences and determination of percent homology between two sequences are methods known in the art and can be performed using a mathematical algorithm such as Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68, modified as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-77. Such an algorithm is included in the NBLAST and XBLAST (version 2.0) programs of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. blast searches for nucleotides can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the MCT nucleic acid molecules of the present invention, blast searches for proteins can be performed with the XBLAST program, score = 50, word length = 3 , to obtain amino acid sequences homologous to the molecules of the MCT protein of the present invention. To obtain gapped comparisons for comparison purposes, the Gapped BLAST program described in Altschul, et al. Can be used. (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using the BLAST and Gapped BLAST programs, one skilled in the art should know how to optimize the parameters of this program (for example, XBLAST and NBLAST) for a particular sequence being analyzed.

Another example of a mathematical algorithm used to compare sequences is the Meyers and Miller (1998) Comput algorithm. Appl. Biosci. 4: 11-17). Such an algorithm is included in the ALIGN program (version 2.0), which is part of the GCG package of sequence matching programs. When using the ALIGN program, the PAM120 table of weight residues, a gap length penalty of 12, and a gap gap of 4 can be used to compare amino acid sequences. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM described in Torelli and Robotti (1994) Comput . Appl. Biosci. 10: 3-5; and FASTA described in Pearson and Lipman (1988) P.N.A.S. 85: 2444-8.

Determining the percent homology between two amino acid sequences can also be performed using the GAP program of the GCG software package (available at http://www.gcg.com) using either the Blosum 62 matrix or the PAM250 matrix and a gap weight of 12, 10, 8 , 6 or 4 and a weight coefficient of length 1, 3 or 4. The percentage of homology between two nucleic acid sequences can be determined using the GAP program in the GCG software package using standard parameters such as a gap weight of 50 and a weight coefficient Ient of length 3.

A comparative analysis of the gene sequences of this invention with sequences present in Genbank was performed using methods known in the art (see, for example, Bexevanis and Oullette, eds. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins. John Wiley and Sons: New York). The gene sequences of the present invention were compared with genes present in Genbank in a three-step process. In a first step, BLASTN analysis (e.g., local matching analysis) was performed for each of the sequences of the present invention with respect to the nucleotide sequences present in Genbank, and the higher 500 matches were used for further analysis. A subsequent FASTA search (for example, a combined local and general comparison analysis in which limited portions of these sequences were compared) was performed on these 500 matches. Each gene sequence of the present invention was then compared generally to each of the highest three FASTA matches using the GAP program in the GCG software package (using standard parameters). To obtain accurate results, the length of the sequences extracted from Genbank was adjusted relative to the length of the studied sequences using methods well known in the art. The results of this analysis are shown in Table 4. The information obtained is identical to the information that would be obtained if only a GAP analysis (general) were performed on each of the genes of this invention in comparison with each of the reference sequences in Genbank, but significantly reduced calculation time in Comparing with the calculation time of the GAP analysis (general) with a wide database. The sequences of this invention for which no comparisons were obtained above the cutoff values are shown in Table 4 by the absence of matching information. In addition, one of skill in the art will understand that the percentages of GAP matching homology given in Table 4 under the heading “% Homology (GAP)” are listed in European digital format, where “,” (comma) indicates a decimal point. For example, the value “40.345” in this column means “40.345%”.

Example 12: Design and operation of DNA microarrays

The sequences of this invention can be further used in the construction and use of DNA microarrays (the construction, methodology and applications of DNA matrices are well known in the art and are described, for example, in Schena, M. et al. (1995) Science 270: 467-470, Wodicka , L. et al. (1997) Nature Biotechnology 15: 1359-1367; DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48 and DaRisi, JL et al. (1997) Science 278: 680-686 )

DNA microarrays are solid or flexible substrates consisting of nitrocellulose, nylon, glass, silicone or other materials. Nucleic acid molecules can be attached to the surface in an ordered manner. After suitable labeling, other nucleic acids or mixtures of nucleic acids can be hybridized with immobilized nucleic acid molecules, and the label can be used to monitor and measure individual signal intensities of the hybridized molecules in specific areas. This methodology allows the simultaneous determination of the relative or absolute amount of all or selected nucleic acids in an applied sample or mixture of nucleic acids. Thus, DNA microarrays allow analysis of the expression of multiple (up to 6800 or more) nucleic acids in parallel (see, for example, Schena, M. (1996) BioEssays 18 (5): 427-431).

The sequences of the present invention can be used to construct oligonucleotide primers that are capable of amplifying specific regions of one or more C. glutamicum genes through a nucleic acid amplification reaction, such as a polymerase chain reaction. The selection and construction of 5'- or 3'-oligonucleotide PCR primers or suitable linkers makes it possible to covalently attach the obtained products to the surface of the carrier medium described above (and also described, for example, Schena, M. (1995) Science 270: 467- 470).

Nucleic acid microarrays can also be constructed by in situ synthesis of oligonucleotides, as described by Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-1367. Using photolithographic methods, precisely defined areas of the matrix are exposed to light. Protective groups that are photolabile are activated by this and are subject to the addition of nucleotides, while sites that are masked from light do not undergo any modification. Subsequent cycles of protection and light activation make possible the synthesis of various oligonucleotides in certain positions. Small, specific regions of the genes of this invention can be synthesized on microarrays through solid-phase synthesis of oligonucleotides.

The nucleic acid molecules of the invention present in a sample or mixture of nucleotides can be hybridized with microarrays. These nucleic acid molecules can be labeled in accordance with standard methods. Briefly, nucleic acid molecules (e.g., mRNA molecules or DNA molecules) are labeled by incorporation of isotopically or fluorescently labeled nucleotides, for example, during reverse transcription or DNA synthesis. Hybridization of labeled nucleic acids with microarrays is described (e.g., in Schena, M. et al. (1995) supra; Wodicka, L. et al. (1997) supra and DeSaizieu, A. et al. (1998) supra). Detection and quantification of a hybridized molecule is performed according to the specific label included. Radioactive labels can be detected, for example, as described in Schena, M. et al. (1995 supra), and fluorescence labels can be detected, for example, by the method of Shalon et al. (1996) Genome Research 6: 639-645).

The application of the sequences of the present invention to DNA microarray technology, as described above, allows a comparative analysis of different strains of C. glutamicum or other Corynebacteria. For example, studies of variations between strains based on individual transcript profiles and identification of genes that are important for the specific and / or desirable properties of strains, such as pathogenicity, productivity, and stress tolerance, are facilitated by nucleic acid microarray methodologies. Comparisons of the gene expression profile of the present invention during the fermentation reaction are also possible using nucleic acid matrix technology.

Example 13: Analysis of the dynamics of cell populations of proteins (proteomics)

The genes, compositions and methods of the present invention can be used to study the interactions and dynamics of populations of proteins called “proteomics”. Protein populations of interest include, but are not limited to, the general population of C. glutamicum proteins (e.g., compared to populations of proteins from other organisms), proteins that are active under specific environmental or metabolic conditions (e.g., during fermentation, at high or low temperature or at high or low pH), or other proteins that are active during specific phases of growth and development.

Protein populations can be analyzed by a variety of well-known methods, such as gel electrophoresis. Cellular proteins can be obtained, for example, by lysis or extraction, and can be separated from each other using various electrophoretic methods. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE electrophoresis) separates proteins largely based on their molecular weight. Isoelectric focusing polyacrylamide gel electrophoresis (IAG electrophoresis in PAGE) separates proteins by their isoelectric point (which reflects not only the amino acid sequence, but also post-translational modifications of this protein). Another, more preferred method for protein analysis is the sequential combination of IEP-PAGE and SDS-PAGE, known as 2-D gel electrophoresis (described, for example, in Hermann et al. (1998) Electrophoresis 19: 3217-3221 ; Fountoulakis et al. (1998) Electrophoresis 18: 1184-1192; Antelmann et al. (1997) Electrophoresis 19: 1193-1202; Langen et al. (1997) Electrophoresis 18: 1451-1463). Other separation methods may also be used to separate proteins, such as capillary gel electrophoresis; similar methods are well known in the art.

Proteins separated using these methodologies can be visualized by standard methods such as staining or labeling. Suitable dyes are known in the art and include Coomassie brilliant blue, containing silver dye (silver dye) or fluorescent dyes such as Sypro Ruby (Molecular Probes). The inclusion of radioactively labeled amino acids or other protein precursors (e.g., ³⁵ S-methionine, ³⁵ S-cysteine, ¹⁴ C-labeled amino acids, ¹⁵ N-amino acids, ¹⁵ NO ₃ or ¹⁵ NH ₄ ⁺ or ¹³ C-labeled amino acids) in medium C glutamicum allows the labeling of proteins from these cells before separation. Similarly, fluorescent labels can be used. Labeled proteins can be extracted, isolated and separated in accordance with previously described methods.

Proteins visualized by these methods can be further analyzed by measuring the amount of dye or label used. The amount of a particular protein can be quantified using, for example, optical methods and can be compared with the amount of other proteins in the same gel or in other gels. Comparisons of proteins on gels can be made, for example, by optical comparison, spectroscopy, image scanning and gel analysis, or through the use of photographic films and screens. Such methods are well known in the art.

Direct sequencing or other standard methods can be used to determine the identity of any particular protein. For example, sequencing of N- and / or C-terminal amino acids (e.g., Edman decomposition), as well as mass spectrometry (namely, MALDI or ESI methods (see, e.g., Langen et al. (1997) Electrophoresis, can be used). 18: 1184-1192)). The protein sequences provided herein can be used to identify C. glutamicum proteins using these methods.

The information obtained by these methods can be used to compare the parameters of the presence of proteins, activity or modification between different samples from different biological conditions (for example, different organisms, temporary fermentation points, environmental conditions or different biotopes, among others). Data obtained only from such experiments, or in combination with other methods, can be used for various applications, for example, to compare the behavior of different organisms in a specific (e.g., metabolic) situation, to increase the productivity of strains that produce fine chemicals , or to increase the efficiency of the production of fine chemicals.

Equivalents

Many equivalents will be apparent to those skilled in the art or they will be able to obtain, using no more labor than routine experimentation, numerous equivalents relative to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (43)

1. An isolated Corynebacterium glutamicum nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 5, or a sequence complementary to it, wherein the molecule encodes a polypeptide having cysQ activity. 2. The selected nucleic acid molecule that encodes a polypeptide containing the amino acid sequence of SEQ ID NO: 6 and having cysQ activity, or a sequence complementary to it. 3. An isolated nucleic acid molecule that encodes a natural allelic variant of a polypeptide containing the amino acid sequence of SEQ ID NO: 6 and having cysQ activity, or a sequence complementary to it. 4. The selected nucleic acid molecule containing a nucleotide sequence that is at least 50% identical to the entire nucleotide sequence of SEQ ID NO: 5 or its complementary sequence, where the specified molecule encodes a polypeptide having cysQ activity. 5. The selected nucleic acid molecule containing at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 5 or its complementary sequence, where the specified molecule encodes a peptide having cysQ activity. 6. The selected nucleic acid molecule containing the nucleic acid molecule according to any one of claims 1 to 5 and a nucleotide sequence encoding a heterologous polypeptide. 7. Recombinant expression vector containing a nucleic acid molecule according to any one of claims 1 to 6 and one or more regulatory sequences. 8. The recombinant vector according to claim 7, which is a plasmid. 9. A method of obtaining a recombinant host cell, comprising transfection of the host cell with the expression vector according to claim 7 or 8. 10. The method according to claim 9, where the specified cell is a microorganism. 11. The method of claim 10, wherein said cell belongs to the genus Corynebacterium or Brevibacterium. 12. The method according to claim 9, where the expression of the specified nucleic acid molecule modulates the production of amino acids. 13. The method of claim 12, wherein said amino acid is a proteinogenic or non-proteinogenic amino acid. 14. A method of producing a recombinant host cell, comprising transfecting the host cell with a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 5, where the nucleic acid molecule has one or more modifications of the nucleic acid compared to the sequence of SEQ ID NO: 5 and where the molecule The nucleic acid encodes a peptide having cysQ activity. 15. A method of producing a recombinant host cell, comprising transfecting the host cell with a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 5, where the regulatory region of the nucleic acid molecule is modified relative to the regulatory region of this wild-type molecule and where the nucleic acid molecule encodes a polypeptide having cysQ activity. 16. A method for producing a polypeptide comprising culturing a recombinant host cell transfected with the vector of claim 7 in a suitable culture medium to produce a polypeptide having cysQ activity. 17. The selected polypeptide containing the amino acid sequence of SEQ ID NO: 6, where the specified polypeptide has cysQ activity. 18. The selected polypeptide containing a natural allelic variant of the polypeptide containing the specified amino acid sequence of SEQ ID NO: 6, where the specified natural allelic variant has cysQ activity. 19. The selected polypeptide, which is encoded by a nucleic acid molecule containing a nucleotide sequence that is at least 50% identical to the full-length nucleic acid sequence with the sequence SEQ ID NO: 5, where the specified polypeptide has cysQ activity. 20. The selected polypeptide containing an amino acid sequence that is at least 50% identical to the full-sized amino acid sequence of SEQ ID NO: 6, where the specified polypeptide has cysQ activity. 21. The selected polypeptide containing a fragment of a polypeptide containing the amino acid sequence of SEQ ID NO: 6, where the specified polypeptide fragment retains cysQ activity. 22. The selected polypeptide encoded by a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 5, where the specified polypeptide has cysQ activity. 23. An isolated polypeptide according to any one of claims 17 to 22, further comprising heterologous amino acid sequences. 24. A method for producing an amino acid, comprising culturing a recombinant host cell transfected with the vector of claim 7, wherein the amino acid is produced. 25. The method according to paragraph 24, where the specified method further comprises the step of isolating the amino acid from the specified culture. 26. The method according to paragraph 24, where the specified cell belongs to the genus Corynebacterium or Brevibacterium. 27. The method of claim 24, wherein said cell is selected from the group consisting of Corynebacterium glutamicum, Corynebacterium herculis, Corynebacterium lilium, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium acetophilum, Corynebacterium ammoniagenes, Corynebacterium fujiokense, Corynebacterium nitrilophilus, Brevibacterium ammoniagenes, Brevibacterium butanicum, Brevibacterium divaricatum, Brevibacterium flavum, Brevibacterium healii, Brevibacterium ketoglutamicum, Brevibacterium ketosoreductum, Brevibacterium lactofermentum, Brevibacterium linens, Brevibacterium paraffinolyticum and the strains shown in Table 3. 28. The method according to paragraph 24, where the expression of a nucleic acid molecule from the specified vector leads to modulation of the production of the specified amino acids. 29. The method according to paragraph 24, where the specified amino acid is a proteinogenic or non-proteinogenic amino acid. 30. The method according to paragraph 24, where the specified amino acid is selected from the group consisting of lysine, glutamate, glutamine, alanine, aspartate, glycine, serine, threonine, methionine, cysteine, valine, leucine, isoleucine, arginine, proline, histidine, tyrosine , phenylalanine and tryptophan. 31. A method of producing an amino acid comprising culturing a cell whose genomic DNA has been altered by introducing a nucleic acid according to any one of claims 1 to 6. 32. A method of diagnosing Corynebacterium diphtheriae in vitro, comprising detecting at least one nucleic acid molecule according to claims 1-5 or at least one polypeptide molecule according to claims 17-22, that is, diagnosing or determining the activity of Corynebacterium diphtheriae in vitro. 33. The method according to p, where the diagnosis or determination of the activity of Corynebacterium diphtheriae is carried out in a sample taken from a patient. 34. The selected nucleic acid molecule containing a fragment of at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 5 or its complementary sequence, where the specified nucleic acid molecule is used as a probe. 35. The selected nucleic acid molecule containing a fragment of at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 5 or its complementary sequence, where the specified nucleic acid molecule is used as an antisense molecule. 36. The selected nucleic acid molecule containing a fragment of at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 5 or its complementary sequence, where the specified nucleic acid molecule is used as a primer. Priorities for items: 06/25/1999 according to claims 1-36; 07/08/1999 according to claims 1-36; 07/09/1999 according to claims 1-36; 07/14/1999 according to claims 1-36; 08/27/1999 according to claims 1-36; 08/31/1999 according to claims 1-36; 09/03/1999 according to claims 1-36.

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