EP1444332A2 - Genes de corynebacterium glutamicum codant des proteines d'homeostase et d'adaptation - Google Patents

Genes de corynebacterium glutamicum codant des proteines d'homeostase et d'adaptation

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Publication number
EP1444332A2
EP1444332A2 EP02798305A EP02798305A EP1444332A2 EP 1444332 A2 EP1444332 A2 EP 1444332A2 EP 02798305 A EP02798305 A EP 02798305A EP 02798305 A EP02798305 A EP 02798305A EP 1444332 A2 EP1444332 A2 EP 1444332A2
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EP
European Patent Office
Prior art keywords
cell
protein
nucleic acid
glutamicum
proteins
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EP02798305A
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German (de)
English (en)
Inventor
Oskar Zelder
Markus Pompejus
Hartwig Schröder
Burkhard Kröger
Corinna Klopprogge
Gregor Haberhauer
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BASF SE
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BASF SE
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • Certain products and by-products of naturally occurring metabolic processes in cells are used in many industries, including the food, feed, cosmetic and pharmaceutical industries. These molecules, collectively referred to as “fine chemicals", include organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors and enzymes. Their production is best accomplished by growing large-scale bacteria that have been developed to produce and secrete large quantities of the desired molecule.
  • a particularly suitable organism for this purpose is Corynebacterium glutamicum, a gram-positive, non-pathogenic bacterium. Through strain selection, a number of mutant strains have been developed that produce a range of desirable compounds. However, selecting strains that are improved in the production of a particular molecule is a time consuming and difficult process.
  • This invention provides novel nucleic acid molecules that can be used to identify or classify Corynebacterium glutamicum or related types of bacteria.
  • C. glutamicum is a gram-positive, aerobic bacterium that is commonly used in industry for the large-scale production of a number of fine chemicals, as well as for the degradation of hydrocarbons (e.g. when crude oil overflows) and for the oxidation of terpenoids.
  • the nucleic acid molecules can therefore be used to identify microorganisms that can be used for the production of fine chemicals, for example by fermentation processes.
  • glutamicum itself is not pathogenic, but it is related to other Corynebacterium species, such as Corynebacterium diphtheriae (the causative agent of diphtheria), which are important pathogens in humans. The ability to identify the presence of Corynebacterium species can therefore also be of significant clinical importance, for example in diagnostic applications.
  • These nucleic acid molecules can also serve as reference points for mapping the C. glufcamicum genome or organisms related to genomes.
  • These new nucleic acid molecules encode proteins that are referred to here as homeostasis and adaptation (HA) proteins. These HA proteins can, for example, perform a function which is involved in maintaining homeostasis in C. glutamicum or in the ability of this microorganism to adapt to different environmental conditions.
  • HA homeostasis and adaptation
  • the nucleic acid molecules according to the invention can be used for the genetic manipulation of this organism in order to make it better and more efficient as a producer of one or more fine chemicals.
  • the improved production or efficiency of the production of a fine chemical can be based on a direct effect of the manipulation of a gene according to the invention or on an indirect effect of such a manipulation.
  • glutamicum one can increase the conversion of these inorganic compounds (or use alternative compounds), which enables improved rates of incorporation of inorganic atoms into these fine chemicals.
  • the genetic manipulation of C. glutamicum enzymes, which are involved in general cellular processes. Can also directly improve fine chemical production because many of these enzymes directly modify fine chemicals (e.g. amino acids) or the enzymes involved in fine chemical synthesis or secretion.
  • the modulation of activity or the number of cellular proteases can also have a direct effect on fine chemical production, since many proteases lien or enzymes that are involved in fine chemical production or its degradation.
  • the above-mentioned enzymes which are involved in the modification and / or the degradation of aromatic or aliphatic compounds, generally biocatalysis, metabolism or proteolysis of inorganic compounds, are themselves fine chemicals which are desirable for their activity in various in vitro industrial applications.
  • C. glutamicum By changing the copy number of the gene for one or more of these enzymes in C. glutamicum, one can increase the number of these proteins that are produced by the cell, thereby increasing the potential yield or efficiency of producing these proteins from large-scale C. glutamicum or related Bacterial cultures are increased.
  • the modification of an HA protein according to the invention can also indirectly influence the yield, production and / or efficiency of the production of a fine chemical from a C. glutamicum strain which contains a modified protein.
  • a C. glutamicum strain which contains a modified protein.
  • modulating the activity and / or the number of such proteins that are involved in the construction or rearrangement of the cell wall one can, for example, modify the structure of the cell wall itself so that the cell is subjected to the mechanical and other stresses involved in large fermenter cultures exists, withstands better.
  • Large-scale growth of C. glutamicum also requires significant cell wall production. Modulation of the activity or number of cell wall biosynthesis or degradation enzymes can enable faster cell wall biosynthesis rates, which in turn allows stronger growth rates of this microorganism in culture and thereby increases the number of cells that produce the desired fine chemical.
  • the yield, production or efficiency of the production of one or more fine chemicals from C. glutamicum can also be influenced indirectly by modifying the HA enzymes according to the invention.
  • many general enzymes in C. glutamicum have a significant impact on global cell processes (e.g. regulatory processes), which in turn has a significant impact on fine chemical metabolism.
  • proteases enzymes that modify or degrade toxic aromatic or aliphatic compounds, increase the viability of C. glutamicum.
  • the proteases support the selective removal of incorrectly folded or incorrectly regulated proteins, such as those which occur, for example, under the relatively stressful environmental conditions in a large fermenter culture. By changing these proteins, you can still change them Increase activity and improve the viability of C. glutamicum in culture.
  • the aromatic / aliphatic modification or degradation proteins not only serve to detoxify these waste compounds (which are present in the culture medium as contaminants or as waste products from the cells themselves), but also allow the cell to use alternative carbon sources if the optimal carbon source in the culture is limited.
  • the survival of C. glutamicum cells in culture can be increased by increasing the number and / or the activity.
  • the inorganic metabolic proteins according to the invention supply the cell with the inorganic molecules which are necessary for all protein and nucleotide syntheses (among other things) and are therefore decisive for the overall viability of the cell.
  • An increase in the number of viable cells that produce one or more fine chemicals in large culture should cause a simultaneous increase in the yield, production, and / or efficiency of production of the fine chemical in the culture.
  • HA proteins proteins that encode the proteins referred to herein as (HA) proteins, which, for example, perform a function involved in the maintenance of homeostasis in C. glutamicum or in the ability of this microorganism to participate adapt to different environmental conditions.
  • Nucleic acid molecules that encode an HA protein are referred to here as HA nucleic acid molecules.
  • an HA protein is involved in C. glutamicum cell wall biosynthesis or in rearrangements, metabolism of inorganic compounds, modification or degradation of aromatic or aliphatic compounds, or has a C. glutamicum enzymatic or proteolytic compound Activity. Examples of such proteins are those encoded by the genes shown in Table 1.
  • isolated nucleic acid molecules for example cDNAs
  • isolated nucleic acid molecules comprising a nucleotide sequence which codes for an HA protein or biologically active sections thereof, and also nucleic acid fragments which act as primers or hybridization probes for detecting or amplifying HA code - Render nucleic acid (e.g. DNA or mRNA) are suitable.
  • the isolated nucleic acid molecule comprises one of the nucleotide sequences listed in Appendix A or the coding region or a complement thereof of one of these nucleotide sequences.
  • the isolated nucleic acid molecule encodes one of the amino acid sequences listed in Appendix B.
  • the preferred ß HA proteins also preferably have at least one of the HA activities described here.
  • nucleic acid sequences of the sequence listing together with the sequence changes at the respective position described in Table 1 are defined as Appendix A.
  • the isolated nucleic acid molecule is at least 15 nucleotides long and hybridizes under stringent conditions to a nucleic acid molecule which comprises a nucleotide sequence from Appendix A.
  • the isolated nucleic acid molecule preferably corresponds to a naturally occurring nucleic acid molecule.
  • the isolated nucleic acid more preferably encodes a naturally occurring C. glutamicum HA protein or a biologically active portion thereof.
  • vectors for example recombinant expression vectors, which contain the nucleic acid molecules according to the invention, and host cells, into which these vectors have been introduced.
  • a host cell that is grown in a suitable medium is used to produce an HA protein.
  • the HA protein can then be isolated from the medium or the host cell.
  • Another aspect of the invention relates to a genetically modified microorganism in which an HA gene has been introduced or modified.
  • the genome of the microorganism has been changed by introducing at least one nucleic acid molecule according to the invention which encodes the mutated HA sequence as a transgene.
  • an endogenous HA gene in the microorganism genome is modified by homologous recombination with an altered HA gene, e.g. functionally disrupted.
  • the microorganism belongs to the genus Corynebacterium or BreviJbacterium, Corynebacterium glutamicum being particularly preferred.
  • the microorganism is also used to produce a desired compound, such as an amino acid, particularly preferably lysine.
  • host cells that have more than one of the nucleic acid molecules described in Appendix A.
  • Such host cells can be produced in various ways known to the person skilled in the art. For example, they can are transfected by vectors which carry several of the nucleic acid molecules according to the invention. However, it is also possible to introduce one nucleic acid molecule according to the invention into the host cell with one vector and therefore to use several vectors either simultaneously or in a staggered manner. Host cells can thus be constructed which carry numerous up to several hundred of the nucleic acid sequences according to the invention. Such an accumulation often leads to superadditive effects on the host cell with regard to fine chemical productivity.
  • an isolated HA protein or a section for example a biologically active section thereof.
  • the isolated HA protein or its section can be involved in maintaining homeostasis in C. glutamicum or can perform a function which is involved in adapting this microorganism to different environmental conditions.
  • the isolated HA protein or a portion thereof is sufficiently homologous to an amino acid sequence of Appendix B so that the protein or its portion can continue to be involved in maintaining homeostasis in C. glutamicum or to perform a function which is involved in adapting this microorganism to different environmental conditions.
  • the invention also relates to an isolated HA protein preparation.
  • the HA protein comprises an amino acid sequence from Appendix B.
  • the invention relates to an isolated full-length protein which forms a complete amino acid sequence from Appendix B (which is encoded by an open reading frame in Appendix A) is essentially homologous.
  • the HA polypeptide or a biologically active portion thereof can be operably linked to a non-HA polypeptide to form a fusion protein.
  • this fusion protein has a different activity than the HA protein alone and, in other preferred embodiments, results in increased yields, increased production and / or efficiency in the production of a desired fine chemical from C. glutamicum.
  • the integration of this fusion protein into a host cell modulates the production of a desired compound from the cell in particularly preferred embodiments.
  • Another aspect of the invention relates to a method for producing a fine chemical. The method provides for the cultivation of a cell which contains a vector which effects the expression of an HA nucleic acid molecule according to the invention, so that a fine chemical is produced.
  • this method also comprises the step of obtaining a cell which contains such a vector, the cell being transfected with a vector which brings about the expression of an HA nucleic acid.
  • this method also comprises the step in which the fine chemical is obtained from the culture.
  • the cell belongs to the genus Corynebacterium or Breviba ct eri.
  • Another aspect of the invention relates to methods for modulating the production of a molecule from a microorganism. These methods involve contacting the cell with a substance that modulates HA protein activity or HA nucleic acid expression, so that a line-associated activity changes compared to the same activity in the absence of the substance.
  • the cell is used for one or more C. glutamicum processes involved in cell wall biosynthesis or rearrangement, the metabolism of inorganic compounds, the modification or degradation of aromatic or aliphatic compounds or in enzymatic or proteolytic activities are involved, modulated.
  • the substance that modulates HA protein activity stimulates HA protein activity or HA nucleic acid expression.
  • substances that stimulate HA protein activity or HA nucleic acid expression include small molecules, active HA proteins and nucleic acids that encode HA proteins and have been introduced into the cell.
  • substances that inhibit HA activity or expression include small molecules and antisense HA nucleic acid molecules.
  • Another aspect of the invention relates to methods for modulating the yields of a desired compound from a cell, comprising introducing into a cell a wild-type or mutant HA gene that either remains on a separate plasmid or is integrated into the genome of the host cell.
  • the integration into the genome can be random or by homologous recombination, so that the native gene is replaced by the integrated copy, which causes the production of the desired compound from the cell to be modulated.
  • these yields are increased.
  • the chemical is a fine chemical, and in a particularly preferred embodiment an amino acid is. In a particularly preferred embodiment, this amino acid is L-lysine.
  • the present invention provides HA nucleic acid and protein molecules that are involved in cell wall biosynthesis or rearrangement, in the metabolism of inorganic compounds, in the modification or degradation of aromatic or aliphatic compounds in C. glutamicum, or one C. have glutamicum-enzymatic or proteolytic activity.
  • the molecules according to the invention can be used to modulate the production of fine chemicals from microorganisms, such as C. glutamicum, either directly (for example where overexpression or the optimization of the activity of a protein which has a direct effect on the production of a fine chemical (for example an enzyme) Yield, production and / or efficiency of production of a fine chemical from the modified C.
  • glutamicum use or have an indirect effect which nevertheless causes an increase in the yield, production and / or efficiency of production of the desired compound (for example where the modulation of the Activity or number of copies of an aromatic or aliphatic modification or degradation protein from C. glutamicum causes an increase in the viability of C. glutamicum cells, which in turn enables increased production in a large-scale culture explained.
  • fine chemical is known in the art and includes molecules that are produced by an organism and have applications in various industries, such as, but not limited to, the pharmaceutical, agricultural, and cosmetic industries.
  • These compounds include organic acids, such as tartaric acid, itaconic acid and diamino-pimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides and nucleotides (as described, for example, in Kuninaka, A. (1996) Nucleotides and related compounds, pp. 561-612, in Biotechnology Vol. 6, Rehm et al .. ed. VCH: Weinheim and the citations contained therein), lipids, saturated and unsaturated fatty acids (e.g.
  • arachidonic acid arachidonic acid
  • diols e.g. propanediol and butanediol
  • Carbohydrates e.g. hyaluronic acid and trehalose
  • aromatic compounds e.g. aromatic amines, vanillin and indigo
  • vitamins and cofactors as described in Ulimann's Encyclopedia of Industrial Chemistry, Vol. A27, "Vitamins ", Pp. 443-613 (1996) VCH: Weinheim and den quotes contained therein; and Ong, AS, Niki, E. and Packer, L.
  • amino acids comprise the basic structural units of all proteins and are therefore essential for normal cell functions.
  • amino acid is known in the art.
  • the proteinogenic amino acids of which there are 20 types, serve as structural units for proteins in which they are linked to one another via peptide bonds, whereas the non-proteinogenic amino acids (of which hundreds are known) are usually not found in proteins (see Ullmann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97 VCH: Weinheim (1985)).
  • the 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 are well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3rd edition, pp. 578-590 (1988)).
  • the "essential" amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine
  • the "essential” amino acids are converted into the remaining 11 "non-essential” amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine).
  • Higher animals have the ability to synthesize some of these amino acids, but the essential amino acids must be ingested with food for normal protein synthesis to take place.
  • Lysine is not only an important amino acid for human nutrition, but also for monogastric animals such as poultry and pigs.
  • Glutamate is most commonly used as a flavor additive (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 all used in the pharmaceutical industry.
  • Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are used in the pharmaceutical and cosmetic industries. Threonine, tryptophan and D- / L-methionine are widespread feed additives (euchtenberger, W. (1996) Amino acids - technical
  • Glutamate is synthesized by reductive amination of ⁇ -ketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline and arginine are used
  • Phenylalanine and tyrosine are derived from the precursors of glycolysis and pentose phosphate.
  • Tyrösin can also be prepared from phenylalanine in a reaction catalyzed by phenylalanine hydroxylase.
  • Alanine, valine and leucine are each biosynthetic products from pyruvate, the end product of glycolysis.
  • Aspartate is formed from oxa acetate, an intermediate of the citrate cycle. aspartic
  • ragin, methionine, threonine and lysine are each produced by converting aspartate.
  • Isoleucine is made from threonine.
  • Histidine is formed from 5-phosphoribosyl-1-pyrophosphate, an activated sugar.
  • Amino acids the amount of which exceeds the cell's protein biosynthesis requirement, cannot be stored and are instead broken down, so that intermediate products are provided for the main metabolic pathways of the cell (for an overview see Stryer, L., Biochemistry, 3rd ed. Chap. 21 "Amino Acid Degradation and the Urea Cycle”; S 495-516 (1988)).
  • the cell is able to convert unwanted amino acids into useful metabolic intermediates, the production of amino acids is expensive in terms of energy, precursor molecules and the enzymes required for their synthesis.
  • amino acid biosynthesis is regulated by feedback inhibition, the presence of a certain amino acid slowing down or completely stopping its own production (for an overview of the feedback mechanism in amino acid biosynthesis pathways, see Stryer, L ., Biochemistry, 3rd ed., Chapter 24, "Biosynthesis of Amino Acids and Heme", pp. 575-600 (1988)).
  • the output of a certain amino acid is therefore restricted by the amount of this amino acid in the cell.
  • Vitamins, cofactors and nutraceuticals comprise another group of molecules. Higher animals have lost the ability to synthesize them and must therefore absorb them, although they are easily synthesized by other organisms such as bacteria. These molecules are either biologically active molecules per se or precursors of biologically active substances that serve as electron carriers or intermediates in a number of metabolic pathways. In addition to their nutritional value, these compounds also have a significant industrial value as dyes, ⁇ antioxidants and catalysts or other processing aids. (For an overview of the structure, activity and the industrial applications of these compounds, see, for example, Ullmann's Encyclopedia of Industrial Chemistry, "Vitamins", Vol. A27, pp. 443-613, VCH: Weinheim, 1996).
  • vitamin is known in the art and encompasses nutrients which are required by an organism for normal function, but which cannot be synthesized by this organism itself.
  • the group of vitamins can include cofactors and nutraceutical compounds.
  • cofactor encompasses non-proteinaceous compounds which are necessary for the occurrence of normal enzyme activity. These compounds can be organic or inorganic; the cofactor molecules according to the invention are preferably organic cally.
  • nutraceutical encompasses food additives which are beneficial to plants and animals, in particular humans. Examples of such molecules are vitamins, antioxidants and also certain lipids (eg polyunsaturated fatty acids).
  • vitamin Bi Thiamine
  • Riboflavin 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 which are collectively referred to as "vitamin B6" are all derivatives of the common structural unit 5-hydroxy-6-methylpyri- din.
  • Panthothenate (pantothenic acid, R - (+) - N- (2,4-dihydroxy-3, 3-dimethyl-1-oxobutyl) -ß-alanine) can be produced either by chemical synthesis or by fermentation. The last
  • pantothenate biosynthesis consist of the ATP-driven condensation of ß-alanine and pantoic acid.
  • the enzymes responsible for the biosynthesis steps for the conversion into pantoic acid, into ⁇ -alanine and for the condensation into pantothenic acid are known.
  • the metabolically active form of pantothenate is coenzyme A, the biosynthesis of which takes place in 5 enzyatic steps.
  • Pantothenate, pyridoxal-5 '-phosphate, cysteine and ATP are the precursors of coenzyme A.
  • the biosynthesis of biotin from the precursor molecule pimeloyl-CoA in microorganisms has been extensively investigated and several of the genes involved have been identified. It has been found that many of the corresponding proteins are involved in the Fe cluster synthesis and belong to the class of the nifS proteins.
  • the lipoic acid is derived from octanoic acid and serves as a coenzyme in energy metabolism, where it becomes part of the pyruvate dehydrogenase complex and the ⁇ -ketoglutarate dehydrogenase complex.
  • the folates are a group of substances that are all derived from folic acid, which in turn is derived from L-glutamic acid, p-aminobenzoic acid and 6-methylpterine.
  • Corrinoids such as the cobalamins and especially vitamin B 3.2
  • the porphyrins belong to a group of chemicals that are characterized by a tetrapyrrole ring system.
  • the Biosynthesis of Vitamin B 3 . 2 is sufficiently complex that it has not been fully characterized, but a large part of the enzymes and substrates involved is now known.
  • Nicotinic acid (nicotinate) and nicotinamide are pyridine derivatives, which are also called "niacin”.
  • Niacin is the precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms.
  • nucleotide includes the basic structural units of the nucleic acid molecules, which comprise a nitrogenous base, a pentose sugar (for RNA the sugar is ribose, for DNA the sugar is D-deoxyribose) and phosphoric acid.
  • nucleoside encompasses molecules which serve as precursors of nucleotides, but which, in contrast to the nucleotides, have no phosphoric acid unit.
  • nucleic acid molecules By inhibiting the biosynthesis of these molecules or their mobilization to form nucleic acid molecules possible to inhibit RNA and DNA synthesis; if this activity is specifically inhibited in carcinogenic cells, the ability of tumor cells to divide and replicate can be inhibited.
  • nucleotides that do not form nucleic acid molecules but serve as energy stores (ie AMP) or as coenzymes (ie FAD and NAD).
  • the purine and pyrimidine bases, nucleosides and nucleotides also have other possible uses: as intermediates in the biosynthesis of various fine chemicals (e.g. thiamine, S-adenosyl methionine, folate or riboflavin), as energy sources for the cell (e.g. ATP or GTP ) and for chemicals themselves, are usually used as flavor enhancers (for example IMP or GMP) or for many medical applications (see for example Kuninaka, A., (1996) "Nucleotides and Related Compounds in Biotechnology Vol. 6, Rehm et al., ed. VCH: Weinheim, pp. 561-612) Enzymes that are involved in the purine, pyrimidine, nucleoside or nucleotide metabolism are also increasingly serving as targets against which chemicals for the
  • Plant protection including fungicides, herbicides and insecticides are being developed.
  • the purine nucleotides are synthesized from ribose 5-phosphate via a series of steps via the intermediate compound inosine 5 'phosphate (IMP), which leads to the production of guanosine 5'-monophosphate (GMP) or adenosine 5' -monophosphate (AMP) leads from which the triphosphate forms used as nucleotides can be easily produced. These compounds are also used as energy stores so that their degradation provides energy for many different biochemical processes in the cell. Pyrimidine biosynthesis takes place via the formation of uridine 5'-onophosphate (UMP) from ribose 5-phosphate. UMP in turn is converted into cytidine 5 'triphosphate (CTP).
  • IMP intermediate compound inosine 5 'phosphate
  • UMP uridine 5'-onophosphate
  • CTP cytidine 5 'triphosphate
  • the deoxy forms of all nucleotides are produced in a one-step reduction reaction from the diphosphate ribose form of the nucleotide to the diphosphate deoxyribose form of the nucleotide. After phosphorylation, these molecules can participate in DNA synthesis.
  • Trehalose consists of two glucose molecules that are linked by an ⁇ , ⁇ -l, l bond. It is commonly used in the food industry as a sweetener, as an additive for dried or frozen foods, and in beverages. However, it is also used in the pharmaceutical, cosmetics and biotechnology industries (see, e.g., Nishimoto et al., (1998) US Patent No. 5,759,610; Singer, MA and Lindquist, S. Trends Biotech. 16 (1998) 460-467; Paiva, CLA and Panek, AD Biotech Ann. Rev. 2 (1996) 293-314; and Shiosaka, MJ Japan 172 (1997) 97-102). Trehalose is produced by enzymes from many microorganisms and is naturally released into the surrounding medium from which it can be obtained by methods known in the art.
  • the metabolic and other biochemical processes through which cells function are sensitive to environmental conditions such as temperature, pressure, concentrations of solutes and the availability of oxygen. If one or more of these environmental conditions are disturbed or changed in a way that is incompatible with the normal functioning of these cell processes, the cell must maintain an intracellular environment that enables it to survive despite the hostile extracellular environment.
  • Gram-positive bacterial cells such as C. glutamicum cells, have a number of mechanisms by which internal homeostasis can be maintained in spite of unfavorable extracellular conditions. These include a cell wall, proteins that can break down possible toxic aromatic and aliphatic compounds, proteolysis mechanisms that can quickly destroy incorrectly folded or incorrectly regulated proteins, and catalysts that enable intracellular reactions. that would not normally take place under the conditions that are optimal for bacterial growth.
  • C. gluta icum cells can often adapt so that they can take advantage of such conditions.
  • cells in an environment that lack the desired carbon sources can adapt to growth on a less suitable carbon source.
  • Cells can also use less desirable inorganic compounds if the commonly used ones are not available.
  • C. glutamicum cells have a number of genes which enable them to adapt so that they use inorganic molecules which they would normally not encounter under optimal growth conditions as nutrients and precursors for metabolism. Aspects of the cellular processes involved in homeostasis and adaptation are described in detail below.
  • Aromatic compounds are organic molecules with a cyclic ring structure, whereas aliphatic compounds are organic molecules with open chain structures instead of ring structures. These compounds can occur as by-products of industrial processes (e.g. benzene or toluene), but can also be produced by certain microorganisms (e.g. alcohols). Many of these compounds are toxic to the cells, especially the aromatic compounds, which are highly reactive due to the high-energy ring structure. As a result, certain bacteria have developed mechanisms by which they can modify or degrade these compounds so that they are no longer dangerous to the cell.
  • Cells can have enzymes that can hydroxylate, isomerize, or methylate aromatic or aliphatic compounds so that they are either made less toxic or so that the modified form can be processed by standard cell waste and degradation routes. Cells can also have enzymes that can specifically degrade one or more of these potentially toxic substances, thereby protecting the cell. Principles and examples of these types of modification and degradation processes in bacteria are described in several publications, for example Sahm, H. (1999) "Procaryotes in Industrial Production” in Lengler, JW et al., Ed. Biology of the Procaryotes, Thieme Published by Stuttgart, and Schlegel, HG (1992)technische Mikrobioogie, Thieme, Stuttgart).
  • Crude oil is a complex mixture of chemicals that contains aliphatic molecules and aromatic compounds.
  • bacteria that can degrade or modify these toxic compounds from spilled oil many environmental damage can be contained with high efficiency and low costs (see, for example, Smith, MR (1990) "The biodegradation of aromatic hydrocarbons by bacteria” Biodegradation 1 (2-3): 191-206; and
  • Cells e.g. bacterial cells
  • the main mass of an ordinary cell consists of 4 types of atoms: carbon, oxygen, hydrogen and nitrogen.
  • These molecules include phosphorus, sulfur, calcium, magnesium, iron, zinc, manganese, copper, molybdenum, tungsten and cobalt. Many of these compounds are important for the construction of important molecules such as nucleotides (phosphorus) and amino acids (nitrogen and sulfur).
  • inorganic ions serve as cofactors for enzyme reactions or contribute to osmotic pressure. All of these molecules must be taken up by the bacterium from the environment. For each of these organic compounds, it is desirable that the bacterium take the form that is most easily used by the cell's standard metabolic machinery. However, the bacterium can encounter environments in which these preferred forms are not readily available. In order to survive under such conditions, it is important that the bacteria have additional biochemical mechanisms that can convert less metabolically active but readily available forms of these inorganic compounds into forms that can be used by cell metabolism. Bacteria often have a number of genes that encode enzymes for this purpose that are only expressed when the desired inorganic species are not available. These genes for the metabolism of various inorganic compounds serve as another tool that bacteria can use to adapt to suboptimal environmental conditions.
  • nitrogen is the most important element.
  • An ordinary bacterial cell contains 12 to 15% nitrogen. It is part of amino acids and nucleotides as well as many other molecules in the cell. Nitrogen can also serve as a replacement for oxygen as a terminal electron acceptor in energy metabolism.
  • Good sources of nitrogen include many organic and inorganic compounds such as ammonia gas or ammonium salts (e.g. NH 4 CI, (NH 4 ) 2 S0 or NH 4 OH), nitrates, urea, amino acids or complex nitrogen sources such as masiquell water, soy flour, soy protein, yeast extract , Meat extract etc.
  • Ammonium nitrogen is fixed by the action of certain enzymes: glutamate dehydrogenase, glutamine synthase and glutamine-2-oxoglutarate minotransferase.
  • Amino nitrogen is transferred from one organic molecule to another by aminotransferases, a class of enzymes that transfer an amino group from an alpha-amino acid to an alpha-keto acid.
  • Nitrate can be reduced by nitrate reductase, nitrite reductase and other redox enzymes until it is converted to molecular nitrogen or ammonia, which can be easily used in standard metabolic pathways by the cell.
  • Phosphorus is found intracellularly in organic and inorganic forms and can be taken up by the cell in both forms, although most microorganisms prefer to take up inorganic phosphate.
  • the conversion of organic phosphate to a form that the cell can use requires the action of phosphatases (eg phytases that hydrolyze phytate-derived phosphate and inositol derivatives).
  • Phosphate is a key component in the synthesis of nucleic acids and thus plays a role significant role in cellular energy metabolism (eg in the synthesis of ATP, ADP and AMP).
  • Sulfur is necessary for the synthesis of amino acids (e.g. methionine and cysteine), vitamins (e.g. thiamine, biotin and lipoic acid) and iron-sulfur proteins.
  • Bacteria obtain sulfur primarily from inorganic sulfate, although thiosulfate, sulfite, and sulfide are also commonly used. Under conditions where these compounds are not readily available, many bacteria express genes that allow them to use sulfonate compounds such as 2-aminosulfonate (taurine) (Kertesz, MA (1993) "Proteins induced by sulfate limitation in Escherichia coli. Pseudomonas putida or Staphyloccocus aureus "J. Bacteriol. 175: 1187-1190).
  • iron plays a key role in redox reactions and is a cofactor of iron-sulfur proteins, heme proteins, and cytochromes. Iron can be absorbed into the bacterial cell by the action of siderophores, chelating agents that bind extracellular iron ions and transport them into the interior of the cell.
  • siderophores chelating agents that bind extracellular iron ions and transport them into the interior of the cell.
  • Stuttfart Neidhardt, F.C., et al., Ed. Escherichia coli and Salmonella.
  • Enzymes are biological protein catalysts that orient the reacting molecules spatially or create a specialized environment so that the energy hurdle for a biochemical reaction is reduced. Different enzymes catalyze different reactions, and each enzyme can be subject to transcriptional, translational or post-translational regulation, so that the reaction can only be suitable conditions and at certain times. Enzymes can contribute to the degradation (eg proteases), synthesis (eg synthases), or modification (eg transferases or isomerases) of compounds, all of which enable the production of necessary compounds in the cell. This, in turn, helps maintain cellular homeostasis. ,
  • Incorrectly folded proteins can be dangerous for the cell for two reasons.
  • the aberrant folded protein can have similar aberrant activity or no activity.
  • misfolded proteins cannot have conformational areas that are necessary for proper regulation by other cellular systems, and thus can still be active, but in an uncontrolled manner.
  • the cell has a mechanism by which misfolded enzymes and regulatory proteins can be quickly destroyed before the cell is damaged: proteolysis.
  • Proteins such as those from the la / lon family and those from the Clp family, specifically recognize and degrade misfolded proteins (see, for example, Sherman, MY, Goldberg, AL (1999) EXS 77: 57-78 and the references cited therein, and Porankiewicz, J. (1999) Molec. Microbiol. 32 (3). 449-58 and references contained therein; Neidhardt, FC et al. (1996) E. coli and Salmonella, ASM Press: Washington, DC and references therein; and Pritchard, GG and Coolbear, T. (1993) FEMS Microbiol.
  • proteases support the hydrolysis of peptide bonds in the catabolism of complex molecules to provide the necessary breakdown products and in protein modification.
  • Secreted proteases play an important role in the catabolism of external nutrients even before these compounds enter the cell.
  • the proteolytic activity itself can also perform regulatory functions; the sporulation in B. subtilis and the course of the cell cycle in Caulobacter spp. Are known to be regulated by key proteolytic events in each of these species (Gottesman, S. (1999) Curr. Opin. Microbiol. 2 (2): 142-147).
  • proteolytic processes are the key to cellular survival under suboptimal and optimal environmental conditions and contribute to the overall maintenance of homeostasis in cells.
  • the cell wall offers such protection, which also plays a role in adhesion, cell growth and cell division, and in the transport of the desired solutes and waste materials.
  • the cells require intracellular concentrations of metabolites and other molecules that are essentially above those of the surrounding media. Since these metabolites are largely prevented from leaving the cell due to the hydrophobic membrane, the system tends to let water molecules from the external medium into the cell so that the internal concentrations of the solutes correspond to the external concentrations. Water molecules can easily pass through the cell membrane, and this membrane cannot withstand the resulting swelling and pressure, which would result in osmotic lysis of the cell. The strength of the cell greatly improves the cell's ability to withstand these pressures and provides another barrier to the undesired diffusion of these metabolites and the desired solubility. most substances from the cell. The cell wall also prevents unwanted material from entering the cell.
  • the cell wall also participates in a number of other cellular processes, such as adhesion, cell growth and division. Due to the fact that the cell wall completely surrounds the cell, any interaction of the cell with its surroundings must be mediated through the cell wall. The cell wall must therefore be involved in every attachment of the cells to other cells and to desired surfaces. In addition, the cell cannot grow or divide without simultaneous changes in the cell wall. Since the protection that the wall offers must also be present during growth, morphogenesis and reproduction, one of the key steps in cell division is cell wall synthesis in the cell, so that a new cell separates from the old one. Cell wall synthesis is thus often regulated in tandem with cell growth and division (see, for example, Sonenshein, A.L. ed. (1993) Bacillus subtilis and Other Gram-Positive Bacteria, ASM: Washington, D.C.).
  • the structure of the cell wall varies between gram-positive and gram-negative bacteria. In both types, the basic structure of the wall remains similar: an overlapping lattice of two polysaccharides, N-acetyl-glucosmain (NAG) and N-acetylmuramic acid (NAM), which have amino acids (mostly L-alanine, D-glutamate, diamino) - pimelic acid and D-alanine) are cross-linked, which is referred to as "peptidoglycan".
  • NAG N-acetyl-glucosmain
  • NAM N-acetylmuramic acid
  • peptidoglycan The processes involved in cell wall synthesis are known (see, for example, Michal, G. Ed. (1999) Bioch eical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York).
  • the inner cell membrane is coated with a single-layer peptidoglycan layer (about 10 nm thick), which is referred to as murein sacculus.
  • the peptidoglycan structure is very solid and its structure determines the shape of the organism.
  • the outer surface of the Murein sacculus is covered with an outer membrane that contains porins and other membrane proteins, phospholipids and lipospolysaccharides.
  • the gram-negative cell wall has lipid molecules scattered therein, which anchor them in the surrounding membrane.
  • the cytoplasmic membrane is covered with a multilayered peptidoglycan, approximately 20 to 80 nm thick (see, for example, Lengeier et al. (1999) Biology of Prokaryotes Theime-Verlag: Stuttgart, p. 913- 918, pp. 875-899 and pp. 88-109 and the literature cited therein len).
  • the gram-positive cell wall also contains teichoic acid, a polymer made from glycerin or ribitol, which are linked together via a phosphate group. Tondonic acid can also associate with amino acid and forms covalent bonds with mura acid. Lipoteichoic and ponduronic acids must also be present in the cell wall. If available, cellular surface structures such as flagella or capsules are also anchored in this layer of the wall.
  • the present invention is based, at least in part, on the discovery of new molecules, referred to herein as HA nucleic acid and protein molecules, which are involved in maintaining homeostasis in C. glutamicum, or have a function in adapting them Microorganism is involved in different environmental conditions.
  • the HA molecules participate in C. glutamicum cell wall synthesis or rearrangements, in the metabolism of inorganic compounds, in the modification or degradation of aromatic or aliphatic compounds, or have an enzymatic or proteolytic activity.
  • the activity of the HA molecules according to the invention with regard to cell wall biosynthesis or rearrangements, • the metabolism of inorganic compounds, the modification or degradation of aromatic or aliphatic compounds or the enzymatic or proteolytic activity has an effect on the production of a desired fine chemical through this organism.
  • HA molecules of the invention have such a modulated activity that the cellular processes in C. glutamicum, in which the HA molecules are involved (eg C.
  • HA protein or "HA polypeptide” includes proteins that are involved in a number of cellular processes associated with C. glutamicum homeostasis or the ability of C. glutamicum cells to adapt to adverse environmental conditions. menache.
  • a HA protein be found on the C. glutamicu cell wall biosynthesis or on rearrangements, on the metabolism of inorganic compounds in C. glutamicum, on the modification or on Degradation of aromatic or aliphatic compounds in C. glutamicum may be involved, or have an enzymatic or proteolytic C. glutamicum activity.
  • HA proteins include those encoded by the HA genes listed in Table 1 and Appendix A.
  • HA gene or "HA nucleic acid sequence” encompass nucleic acid sequences which encode an HA protein which consists of a coding region and corresponding untranslated 5 'and 3' sequence regions. Examples of HA genes are listed in Table 1.
  • production or “productivity” are known in the art and include the concentration of the fermentation product (for example the desired fine chemical which is formed within a defined period of time and a defined fermentation volume (for example kg product per hour per 1)
  • the term “efficiency of production” encompasses the time it takes to achieve a certain production quantity (for example how long it takes the cell to establish a certain throughput rate of a fine chemical).
  • yield or “product / carbon yield”.
  • biosynthesis or “biosynthetic pathway” are known in the art and encompass the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds, for example in a multi-step or highly regulated process.
  • degradation or “degradation path” are known in the art and include the cleavage of a compound, preferably an organic compound, by a cell into degradation products (more generally, smaller or less complex molecules), e.g. in a multi-step or highly regulated Process.
  • the term “metabolism” is known in the art and encompasses all of the biochemical reactions taking place in an organism. The metabolism of a particular compound (eg the metabolism of an amino acid, such as glycine) then encompasses all biosynthesis, modification and degradation pathways of this compound in the cell.
  • homeostasis is known in the art and encompasses all of the mechanisms used by the cell to maintain a constant intracellular environment despite the prevailing extracellular conditions.
  • a non-limiting example of these processes is the use of the cell wall to prevent osmotic lysis due to the high intracellular concentrations of solutes.
  • the term “adaptation” or “adaptation to an environmental gung” is known in the art and includes mechanisms used by the cell to enable the cell to survive in non-preferred environmental conditions (generally speaking, those environmental conditions in which one or more beneficial nutrients are missing or in which an environmental condition such as temperature, pH, osmolarity, percentage oxygen and the like are outside the optimal survival range of the cell.)
  • Many cells including C. glutamicum cells, have genes that encode proteins that are expressed under these environmental conditions, and that enable continuous growth in these suboptimal conditions.
  • the HA molecules according to the invention are capable of modulating a desired molecule, such as a fine chemical, in a microorganism, such as C. glutamicum.
  • a desired molecule such as a fine chemical
  • a microorganism such as C. glutamicum.
  • the modification of an HA protein according to the invention directly influences the yield, production and / or efficiency of the production of a fine chemical from a C. glufcamicum strain which contains such a modified protein.
  • modifying enzymes that modify aromatic or aliphatic compounds such that these enzymes have a higher or lower activity or number, one can modulate the production of one or more fine chemicals that are the modification or degradation products of these compounds.
  • enzymes which are involved in the metabolism of inorganic compounds provide key molecules (for example phosphorus, sulfur and nitrogen molecules) for the biosynthesis of such fine chemicals as amino acids, vitamins and nucleic acids.
  • C. glutamicum By changing the activity or number of these enzymes in C. glutamicum, one can increase the conversion of these inorganic compounds (or use alternative inorganic compounds) in order to enable improved rates of incorporation of inorganic atoms into these fine chemicals.
  • the genetic manipulation of C. glutamicum enzymes, which are involved in general cellular processes can also directly improve fine chemical production, since many of these enzymes improve the fine chemicals (eg amino acids) or the enzymes involved in fine chemical synthesis or secretion are modify directly.
  • the modulation of the activity or the number of cellular proteases can also have a direct effect on the fine chemical production, since many proteases can break down fine chemicals or enzymes which are involved in the production or degradation of fine chemicals.
  • the above-mentioned enzymes, which are involved in the modification or degradation of aromatic or aliphatic compounds, in general biocatalysis, in the metabolism of inorganic compounds or in proteolysis, are each themselves fine chemicals. lien, which are desirable because of their activity in various industrial in vitro applications.
  • the modification of an HA protein according to the invention can also indirectly affect the yield, production and / or efficiency of the production of a fine chemical from a C. glutamicum strain which contains such a modified protein.
  • modulating the activity and / or the number of these proteins that are involved in the construction or rearrangement of the cell wall one can, for example, modify the structure of the cell wall itself, so that the cell is subjected to the mechanical or other stress that prevails in large-scale fermenter cultivation can withstand better.
  • the growth of C. glutamicum on a large scale requires considerable cell wall production. Modulating the activity or number of cell wall biosynthesis or degradation enzymes can enable faster cell wall biosynthesis rates, which in turn enables stronger growth rates of this microorganism in culture, thereby increasing the number of cells that produce the desired fine chemical.
  • the yield, production or efficiency of the production of one or more fine chemicals from C. glutamicum can be influenced indirectly by modifying the HA enzymes according to the invention.
  • many of the general enzymes in C. glutamicum have a significant impact on global cellular processes (e.g. regulatory processes), which in turn has a significant impact on fine chemical metabolism.
  • proteases enzymes that may modify or degrade toxic aromatic or aliphatic compounds accordingly increase the viability of C. glutamicum.
  • the proteases support the selective removal of incorrectly folded or incorrectly regulated proteins, such as those which occur, for example, under the relatively stressful environmental conditions in a large fermenter culture. Altering these proteins can further increase this activity and improve the viability of C. glutamicum in culture.
  • the aromatic / aliphatic modification or degradation proteins not only serve to detoxify these waste compounds (which occur in the culture medium as contaminants or as waste products from the cells themselves), but also allow the cell to use alternative carbon sources if the optimal carbon source in the culture is limited - is. Increasing the number and / or the activity can increase the survival of C. glutamicum cells in culture.
  • the inorganic metabolic proteins according to the invention supply the cell with the inorganic molecules which are necessary for all protein and nucleotide syntheses (among other things) and are therefore decisive for the overall viability of the cell.
  • An increase in the number of viable cells that produce one or more fine chemicals in large culture should cause a simultaneous increase in the yield, production, and / or efficiency of production of the fine chemical in the culture.
  • the genome of a Corynebacterium glutamicum strain which is available from the American Type Culture Collection under the name ATCC 13032, is suitable as a starting point for the production of the nucleic acid sequences according to the invention.
  • nucleic acid sequences according to the invention can be produced by conventional methods using the changes described in Table 1.
  • nucleic acid molecules which encode HA polypeptides or biologically active sections thereof, and to nucleic acid fragments which are sufficient for use as hybridization probes or primers for identifying or amplifying HA-coding nucleic acids (for example HA-DNA)
  • nucleic acid molecule is intended to encompass DNA molecules (eg cDNA or genomic DNA) and RNA molecules (eg mRNA) as well as DNA or RNA analogs which are generated by means of nucleotide analogs.
  • This term also includes the untranslated sequence located at the 3 'and 5' ends of the coding region: at least about 100 nucleotides of the sequence upstream of the 5 'end of the coding region and at least about 20 nucleotides of the sequence downstream of the 3' end of the coding gene region.
  • the nucleic acid molecule can be single-stranded or double-stranded, but is preferably a double-stranded DNA.
  • An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid.
  • an "isolated" nucleic acid preferably has no sequences which naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid originates (for example sequences which are located at the 5 'or 3' end of the nucleus small acid).
  • the isolated HA nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotide sequences that naturally see the nucleic acid molecule in the genome Flank the DNA of the cell from which the nucleic acid originates (for example a C. glutamicum cell).
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be essentially free of another cellular material or culture medium if it is made by recombinant techniques, or free of chemical precursors or other chemicals if it is chemically synthesized.
  • a nucleic acid molecule according to the invention for example a nucleic acid molecule with a nucleotide sequence from Appendix A or a section thereof, can be isolated using standard molecular biological techniques and the sequence information provided here.
  • a C. glutamicum HA cDNA can be isolated from a C. glutamicum library by using a complete sequence from Appendix A or a portion thereof as a hybridization probe and standard hybridization techniques (as described, for example, in Sambrook, J. , Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd to 1st Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • a nucleic acid molecule comprising a complete sequence from Appendix A or a section thereof, can be isolated by polymerase chain reaction, using the oligonucleotide primers which have been created on the basis of this sequence (for example, a nucleic acid molecule comprising a complete sequence can be used Appendix A, or a portion thereof, can be isolated by polymerase chain reaction using oligonucleotide primers made from this same sequence from Appendix A).
  • mRNA can be isolated from normal endothelial cells (for example by the guanidinium thiocyanate extraction method of Chirgwin et al.
  • cDNA can be obtained by means of reverse transcriptase (for example Moloney-MLV reverse transcriptase) Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Louis, FL).
  • reverse transcriptase for example Moloney-MLV reverse transcriptase
  • Gibco / BRL Bethesda
  • MD or AMV reverse transcriptase
  • Synthetic oligonucleotide primers for amplification via polymerase chain reaction can be created on the basis of one of the nucleotide sequences shown in Appendix A.
  • a nucleic acid according to the invention can be amplified using cDNA or alternatively genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid amplified in this way can be cloned into a suitable vector and characterized by DNA sequence analysis.
  • Oligonucleotides that are a HA nucleotide sequence can be produced by standard synthesis methods, for example using an automatic DNA synthesizer.
  • an isolated nucleic acid molecule according to the invention comprises one of the nucleotide sequences listed in Appendix A.
  • the sequences of Appendix A correspond to the Corynebacterium glutamicum HA cDNAs according to the invention. These cDNAs include sequences that contain HA proteins (i.e. the
  • nucleic acid molecule can alternatively comprise only the coding region of one of the sequences in Appendix A.
  • an isolated nucleic acid molecule according to the invention comprises a nucleic acid molecule which is complementary to one of the nucleotide sequences shown in Appendix A or a portion thereof, which is a nucleus
  • an isolated nucleic acid molecule according to the invention comprises a nucleotide sequence which is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90% or 90-95% and even more preferably at least about 95%, 96%, 97%,
  • an isolated nucleic acid molecule according to the invention comprises a nucleotide sequence which, for example under stringent conditions, with one of the nucleic acids shown in Appendix A
  • the nucleic acid molecule according to the invention can moreover comprise only a section of the coding region of one of the sequences in Appendix A, for example a fragment which acts as a probe or primer
  • HA 40 or fragment can be used which encodes a biologically active section of an HA protein.
  • the nucleotide sequences determined from the cloning of the HA genes from C. glutamicum enable the generation of probes and primers which are used to identify and / or clone HA homologs in other cell types
  • the probe or primers usually comprise substantially purified oligonucleotide.
  • the Oligonucleotide usually comprises a nucleotide sequence region which, under stringent conditions, comprises at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand from one of the sequences given in Appendix A, an antisense strand from one of the in Sequences indicated in Appendix A or naturally occurring mutants thereof hybridized.
  • Primers based on a nucleotide sequence from Appendix A can be used in PCR reactions for cloning HA homologs.
  • Probes based on the HA nucleotide sequences can be used to detect transcripts or genomic sequences which code for the same or homologous proteins.
  • the probe also comprises a labeling group attached to it, for example a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor.
  • These probes can be used as part of a diagnostic test kit to identify cells that express an HA protein, such as by measuring an amount of HA coding nucleic acid in a cell sample, for example measuring HA ⁇ mRNA levels or by determining whether a genomic HA gene is mutated or deleted.
  • the nucleic acid molecule according to the invention encodes a protein or a portion thereof which comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence from Appendix B that the protein or a portion thereof further participates in the maintenance of homeostasis in C. glutamicum can be, or can perform a function which is involved in the adaptation of this microorganism to different environmental conditions.
  • the term "sufficiently homologous" refers to proteins or portions thereof whose amino acid sequences have a minimal number of identical or equivalent amino acid residues (e.g.
  • the protein is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90%, 90-95%, and most preferably at least about 96%, 97 %, 98%, 99% or even more homologous to a complete amino acid sequence in Appendix B.
  • Sections of proteins which are encoded by the HA nucleic acid molecules according to the invention are preferably biologically active sections of one of the HA proteins.
  • biologically active portion of an HA protein is used to a portion, eg., A domain or motif consisting of an HA protein, the / at the 'maintenance of homeostasis in C. glutamicum can be involved, or can perform a function which is involved in the adaptation of this microorganism to different environmental conditions, or has an activity given in Table 1.
  • glutamicum cell wall biosynthesis or rearrangements the metabolism of inorganic compounds, the modification or degradation of aromatic or aliphatic compounds or an enzymatic or proteolytic one C. glutamicum activity, a test of the enzymatic activity can be carried out.
  • Additional nucleic acid fragments encoding biologically active portions of an HA protein can be obtained by isolating a portion of one of the sequences in Appendix B, expressing the encoded portion of the HA protein or peptide (e.g., by recombinant expression in vitro) and determining the activi - did the coded portion of the HA protein or peptide.
  • the invention also encompasses nucleic acid molecules which differ from one of the nucleotide sequences shown in Appendix A (and portions thereof) due to the degenerate genetic code and thus encode the same HA protein as that encoded by the nucleotide sequences shown in Appendix A.
  • an isolated nucleic acid molecule according to the invention has a nucleotide sequence which encodes a protein with an amino acid sequence shown in Appendix B.
  • the nucleic acid molecule according to the invention encodes a full-length C. gluta icum protein which forms an amino acid sequence from Appendix B (encoded by an open reading frame shown in Appendix A) is substantially homologous.
  • An isolated nucleic acid molecule encoding an HA protein that is homologous to a protein sequence from Appendix B can be generated by incorporating one or more nucleotide substitutions, additions or deletions into a nucleotide sequence from Appendix A such that one or more amino acid substitutions , additions or deletions are introduced into the encoded protein.
  • the mutations can be introduced into one of the sequences from Appendix A by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are preferably introduced at one or more of the predicted non-essential amino acid residues. In a "conservative amino acid substitution", the amino acid residue is replaced by an amino acid residue with a similar side chain.
  • Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar Side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine).
  • amino acids with basic side chains eg lysine, arginine, histidine
  • acidic side chains eg aspartic acid, glutamic acid
  • uncharged polar side chains
  • a predicted non-essential amino acid residue in an HA protein is therefore preferably replaced by another amino acid residue of the same side chain family.
  • the mutations can alternatively be introduced randomly over all or part of the HA coding sequence, for example by saturation mutagenesis, and the resulting mutants can be examined for the HA activity described here in order to identify mutants. who maintain HA activity.
  • the encoded protein can be expressed recombinantly, and the activity of the protein can be determined, for example, using the tests described here (see Example 8 of the example section).
  • vectors preferably expression vectors, which contain a nucleic acid which encode an HA protein (or a section thereof).
  • vector refers to a nucleic acid molecule that can transport another nucleic acid to which it is bound is.
  • plasmid which stands for a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, whereby additional DNA segments can be ligated into the viral genome.
  • Certain vectors can replicate autonomously in a host cell into which they have been introduced (e.g. bacterial vectors with bacterial origin of replication and episomal mammalian vectors).
  • vectors eg non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell when it is introduced into the host cell and thereby replicated together with the host genome.
  • certain vectors can control the expression of genes to which they are operably linked. These vectors are called "expression vectors".
  • expression vectors usually the expression vectors used in recombinant DNA techniques are in the form of plasmids.
  • plasmid and “vector” can be used interchangeably because the plasmid is the most commonly used vector form.
  • the invention is intended to encompass these other expression vector forms, such as viral vectors (for example replication-deficient retroviruses, adenoviruses and adeno-related viruses), which perform similar functions.
  • the recombinant expression vector according to the invention comprises a nucleic acid according to the invention in a form which is suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vectors one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
  • “operably linked” means that the nucleotide sequence of interest is bound to the regulatory sequence (s) in such a way that expression of the nucleotide sequence is possible (for example in an in vitro transcription / Translation system or in a host cell if the vector is introduced into the host cell).
  • regulatory sequence is intended to encompass promoters, enhancers and other expression control elements (for example polyadenylation signals). These regulatory sequences are described, for example, in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that control the constitutive expression of a nucleotide sequence in many host cell types and those that control the direct expression of the nucleotide sequence only in certain host cells. The person skilled in the art is aware that the design of an expression vector can depend on factors such as the choice of the host cell to be transformed, the extent of expression of the desired protein, etc.
  • the expression vectors according to the invention can be introduced into the host cells, thereby producing proteins or peptides, including fusion proteins or peptides, which are encoded by the nucleic acids as described here (for example HA proteins, mutated forms of HA proteins, Fusion proteins, etc.).
  • the recombinant expression vectors according to the invention can be designed for the expression of HA proteins in prokaryotic or eukaryotic cells.
  • HA genes in bacterial cells such as C. glutamicum, insect cells (with Baculovirus expression vectors), yeast and other fungal cells (see Romanos, MA et al. (1992) "Foreign gene expression in yeast: a review ", Yeast 8: 423-488; van den Hondel, CA; MJJ et al. (1991)” Heterologous gene expression in filamentous fungi "in: More Gene Manipulations in Fungi, JW Bennet & LL Lasure,
  • Plant cells (see 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 further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Proteins are usually expressed in prokaryotes using vectors which contain constitutive or inducible promoters which control the expression of fusion or non-fusion proteins.
  • Fusion vectors contribute a number of amino acids to a protein encoded therein, usually at the amino terminus of the recombinant protein. These fusion vectors usually have three functions: 1) to increase the expression of recombinant protein; 2) increasing the solubility of the recombinant protein; and 3) supporting the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is often introduced at the junction of the fusion unit and the recombinant protein, so that the recombinant protein can be separated from the fusion unit after the fusion protein has been purified.
  • These enzymes and their corresponding Detection sequences include factor Xa, thrombin and enterokinase.
  • Common fusion expression vectors include pGEX (Pharmacia Biotech Ine; Smith, DB and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT 5 (Pharmacia, Piscataway, NJ), in which glutathione-S-transferase (GST), maltose E-binding protein or protein A is fused to the recombinant target protein.
  • GST glutathione-S-transferase
  • the coding sequence of the HA protein is cloned into a pGEX expression vector so that a vector is generated which encodes a fusion protein, comprising from the N-terminus to the C-terminus, GST - thrombin cleavage site - X- Protein.
  • the fusion protein can be purified by affinity chromatography using glutathione-agarose resin.
  • the recombinant HA protein that is not fused to GST can be obtained by cleaving the fusion protein with thrombin.
  • Suitable inducible non-fusion expression vectors from E. coli include pTrc (Amann et al., (1988) Gene 69: 301-315) and pETIld (Studier et al. Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector is based on transcription by host RNA polymerase from a hybrid trp-lac fusion promoter.
  • the target gene expression from the pETlld vector is based on the transcription from a T7-gnl0-lac fusion promoter, which is mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by the BL 21 (DE3) or HMS174 (DE3) host strains from a resident ⁇ prophage which harbors a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize the expression of the recombinant protein is to express the protein in a host bacterium whose ability to proteolytically cleave the recombinant protein is impaired (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 of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those which are preferably used in a bacterium selected for expression, such as C. glutamicum (Wada et al. (1992 ) Nucleic Acids Res. 20: 2111-2118).
  • the HA protein expression vector is a yeast expression vector.
  • yeast expression vectors for expression in the yeast S. cerevisiae include pYepSecl (Baldari et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88 ( Schultz et al. (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA).
  • Vectors and methods of constructing vectors suitable for use in other fungi such as filamentous fungi include those described in detail in: van den Hondel, CAMJJ & Punt, PJ (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, JF Peberdy et al., ed., pp. 1-28, Cambridge University Press: Cambridge.
  • the HA proteins of the invention can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., (1983) Mol. Cell Biol .. 3: 2156-2165) and pVL series (Lucklow and Summers (1989) Virology 170: 31-39).
  • the HA proteins according to the invention can be expressed in single-cell plant cells (such as algae) or in plant cells of higher plants (for example spermatophytes, such as crops).
  • plant expression vectors include those described in detail in: Bekker, D., Ke per, 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. Acids Res. 12: 8711-8721.
  • a nucleic acid according to the invention is expressed in mammalian cells with a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195).
  • the control functions of the expression vector are often provided by viral regulatory elements. Commonly used promoters come, for example, from Polyoma, Adenovirus2, Cytomegalievirus and Simian Virus 40.
  • the recombinant mammalian expression vector can preferably bring about the expression of the nucleic acid in a specific cell type (for example, tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific regulatory elements are known in the art.
  • 10 gnete 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 immunoglobulin
  • milk serum promoter e.g., milk serum promoter; U.S. Patent No. 4,873,316 and European Patent Application Publication No. 264,166
  • Development-regulated promoters are also included, for example the mouse hox promoters (Kessel and Gruss (1990) Science 249: 374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:
  • the invention also provides a recombinant expression vector comprising a DNA molecule according to the invention which is cloned into the expression vector in the antisense direction.
  • the DNA molecule is operatively linked to a regulatory sequence such that expression (by transcription of the DNA. Molecule) of an RNA molecule that is antisense to HA mRNA is possible. Regulatory sequences can be selected that are functional to an antisense rich
  • 35 cloned nucleic acid are bound and which control the continuous expression of the antisense RNA molecule in a variety of cell types, for example.
  • Viral promoters and / or enhancers or regulatory sequences can be selected which the constitutive, tissue-specific or cell type-specific expression
  • the antisense expression vector can 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 effective regulatory region, the activity of which is determined by the cell type in which
  • Another aspect of the invention relates to the host cells into which a recombinant expression vector according to the invention has been introduced.
  • the terms "host cell” and “recombinant host cell” are used interchangeably here. It goes without saying that these terms refer not only to a specific target cell, but also to the descendants or potential descendants of this cell. Since certain modifications may occur in successive generations due to mutation or environmental influences, these offspring are not necessarily identical to the parental cell, but are still included in the scope of the term as used here.
  • a host cell can be a prokaryotic or eukaryotic cell.
  • an HA protein can be expressed in bacterial cells such as C. glutamicum, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • suitable host cells are known to the person skilled in the art.
  • Microorganisms which are related to Corynebacterium glutamicum and which can be suitably used as host cells for the nucleic acid and protein molecules according to the invention are listed in Table 3.
  • vector DNA can be introduced into prokaryotic or eukaryotic cells.
  • transformation transformation
  • transfection conjuggation
  • transduction transduction
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals.
  • a gene that encodes a selectable marker (eg resistance to antibiotics) is usually introduced into the host cells together with the gene of interest.
  • Preferred select Animal markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate.
  • a nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an HA protein, or can be introduced on a separate vector. Cells "that have been stably transfected with the introduced nucleic acid can be identified by drug selection (eg cells that have integrated the selectable marker survive, whereas the other cells die).
  • a vector which contains at least a section of an HA gene into which a deletion, addition or substitution has been introduced in order to change the HA gene, for example to disrupt it functionally.
  • This HA gene is preferably a Corynebacterium glutamicum HA gene, however a homologue from a related bacterium or even from a mammalian, yeast or insect source can be used.
  • the vector is designed such that the endogenous HA gene is functionally disrupted in the case of homologous recombination (ie no longer encodes a functional protein, also referred to as a "knockout" vector).
  • the vector may alternatively be designed so that the endogenous HA gene homologous recombination mutates or otherwise altered but coded nor the functional protein (eg, may be changed, the upstream. Regulatory region in such a manner that thereby the expression of the endogenous HA Protein is changed.).
  • the modified portion of the HA gene is flanked in the homologous recombination vector at its 5 'and 3' ends by additional nucleic acid of the HA gene, which is a homologous recombination between the exogenous HA gene which is carried by the vector , and an endogenous HA gene in a microorganism.
  • the additional flanking HA nucleic acid is long enough for successful homologous recombination with the endogenous gene.
  • the vector usually contains several kilobases flanking DNA (both at the 5 'and 3' ends) (see, for example, Thomas, KR and Capecchi, MR (1987) Cell 51: 503 for a description of homologous recombination vectors).
  • the vector is introduced into a microorganism (eg, by electroporation), and cells in which the introduced HA gene is homologously recombined with the endogenous HA gene are selected using methods known in the art.
  • recombinant microorganisms can be produced which contain selected systems which allow regulated expression of the introduced gene.
  • the inclusion of an HA gene in a vector under the control of the lac operon, for example, enables the expression of the HA gene only in the presence of IPTG.
  • a host cell according to the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) an HA protein.
  • the invention also provides methods for the production of HA proteins using the host cells according to the invention.
  • the method comprises culturing the host cell according to the invention (into which a recombinant expression vector which encodes an HA protein has been introduced, or into whose genome a gene has been introduced which is a wild-type or modified HA protein encoded) in a suitable medium until the HA protein has been produced.
  • the method comprises isolating the HA proteins from the medium or the host cell.
  • nucleic acid molecules, proteins, protein homologs, fusion proteins, primers, vectors and host cells described here can be used in one or more of the following methods: identification of C. glutamicum and related organisms, mapping of genomes of organisms related to C. glutamicum , Identification and localization of C.
  • the HA nucleic acid molecules according to the invention have a multitude of uses. They can initially be used to identify an organism as Corynebacterium glutamicum or close relatives thereof. They can also be used to identify C. glutamicum or a relative thereof in a mixed population of microorganisms.
  • the invention provides the nucleic acid sequences of a number of C. glutamicum genes.
  • a probe comprising a region of a C. glu amicum gene that is unique to that organism one can determine whether this organism is present.
  • Corynebacterium glutamicum itself is not pathogenic, but it is related to pathogenic species such as Corynebacterium diptheriae. The detection of such an organism is of significant clinical importance.
  • the nucleic acid and protein molecules according to the invention can serve as markers for specific regions of the genome. This is useful not only when mapping the genome, but also for functional studies of C. glufcamicu proteins.
  • the C. glu amicum genome can be cleaved, for example, and the fragments incubated with the DNA-binding protein.
  • Those that bind the protein can additionally be probed with the nucleic acid molecules according to the invention, preferably with easily detectable labels; the binding of such a nucleic acid molecule to the genome fragment enables the fragment to be located on the genomic map of C.
  • nucleic acid molecules according to the invention can also be sufficiently homologous to the sequences of related species so that these nucleic acid molecules can serve as markers for the construction of a genomic map in related bacteria, such as Brevibacterium lactofermentum.
  • the HA nucleic acid molecules according to the invention are also suitable for evolution and protein structure studies.
  • the processes involved in the adaptation and maintenance of homeostasis in which the molecules of the invention are involved are exploited by many types;
  • the degree of evolutionary kinship of the organisms can be determined. Accordingly, such a comparison enables the determination of which sequence regions are conserved and which are not, which can be helpful in determining those regions of the protein which are essential for the enzyme function.
  • This type of determination is valuable for protein technology studies and can provide an indication of which protein can tolerate mutagenesis without losing function.
  • HA nucleic acid molecules can bring about the production of HA proteins with functional differences from the wild-type HA proteins. These proteins can be improved in terms of their efficiency or activity may be present in the cell in greater numbers than usual, or may be weakened in their efficiency or activity.
  • Modulation of the activity or number of HA proteins involved in cell wall biosynthesis or rearrangement can affect the production, yield and / or efficiency of production of one or more fine chemicals from C. glutamicum cells.
  • the structure or the thickness of the cell wall can be modulated by changing the cell activity of these proteins.
  • the cell wall serves to a great extent as a protective device against osseous lysis and external sources of injury; by modifying the cell wall, the ability of C. glutamicum to withstand mechanical stress and the stress due to shear forces to which this microorganism is subject during large fermenter cultivation can be increased.
  • Each C. glutamicum cell is also surrounded by a thick cell wall, and thus a significant proportion of the biomass that is present in a large culture consists of the cell wall.
  • an overall increase in cell wall production can be achieved by lowering the activity or the number of proteins involved in the breakdown of the cell wall or by reducing the repression of cell wall biosynthesis.
  • An increase in the number of viable C. glutamicum cells should result in an increased number of cells which produce the desired fine chemical in large fermenter cultures,. which should increase the yields or the efficiency of the production of these compounds from the culture.
  • Modulation of the activity or number of C. glutamicum EA proteins involved in the modification or degradation of aromatic or aliphatic compounds can also have direct or indirect effects on the production of one or more fine chemicals from these cells.
  • Certain aromatic or aliphatic modification or degradation products are desirable fine chemicals (e.g. organic acids or modified aromatic or aliphatic compounds); thus the yields of these desired compounds can be increased by modifying the enzymes which carry out these modifications (for example hydroxylation, methylation or isomerization) or degradation reactions. Accordingly, by lowering the activity or the number of proteins involved in metabolic pathways that the modified breakdown products of the above further reduce the reactions mentioned, to improve the yields of these fine chemicals from C. glutamicum cells in culture.
  • aromatic and aliphatic modification and degradation enzymes are themselves fine chemicals.
  • these enzymes can be used to break down aromatic and aliphatic compounds (for example toxic chemicals, such as crude oil products), to restore contaminated areas biologically, to technically decompose waste or to produce the desired modified aromatic or aliphatic compounds on a large scale or economically their degradation products, some of which can be suitably used as carbon or energy sources for other fine chemical-producing compounds in culture, are used (see, for example, Faber, K. (1995) Biotransformations in Organic Chemistry, Springer: Berlin and those contained therein Quotations; and Roberts, SM ed. (1992 - 1996) Preparative Biotransformations, Wiley: Chichester and the quotes contained therein).
  • Faber, K. (1995) Biotransformations in Organic Chemistry, Springer: Berlin and those contained therein Quotations and Roberts, SM ed. (1992 - 1996) Preparative Biotransformations, Wiley: Chichester and the quotes contained therein.
  • aromatic and aliphatic modifying and degrading enzymes can also have an indirect effect on the production of one or more fine chemicals.
  • Many aromatic and aliphatic compounds (such as those found in culture media as contaminants or from cellular metabolism as waste products) are toxic to the cells; by modifying and / or disassembling these compounds so that they are easily removed or destroyed, cell viability should be increased.
  • These enzymes can also modify or degrade these compounds in such a way that the resulting products can get into the normal carbon metabolism of the cell. This enables the cell to use these compounds as alternative sources of carbon or energy. In large scale cultures where limiting amounts of the optimal carbon source may be present, these enzymes can provide a method by which the cells can continue to grow and divide using aromatic or aliphatic compounds as nutrients.
  • the resulting increase in the number of C. glutamicum cells in the culture which produce the desired fine chemical should in turn result in increased yields or increased efficiency in the production of the fine chemical (s).
  • the modifications in the activity or number of HA proteins involved in the metabolism of inorganic compounds can directly or indirectly affect the production of one or more fine chemicals from cultures of C. glutamicum or related bacteria.
  • many desirable fine chemicals such as nucleic acids, amino acids, cofactors and vitamins (e.g. thiamine, biotin and lipoic acid) cannot be synthesized without inorganic molecules such as phosphorus, nitrate, sulfate and iron.
  • the proteins of inorganic metabolism according to the invention enable the cell to obtain these molecules from a large number of inorganic compounds and to redistribute them into various fine chemical biosynthetic pathways.
  • By increasing the activity or the number of enzymes involved in the metabolism of these organic compounds one can increase the supply of these potentially limiting inorganic molecules, thereby increasing the production or efficiency of the production of various fine chemicals from C. glutamicum cells that produce these modified proteins included, increase directly.
  • the modification of the activity or the number of proteins of the inorganic metabolism according to the invention can also cause C.
  • glutamicum to make better use of a limited range of inorganic compounds, or that non-optimal inorganic compounds for the synthesis of amino acids, vitamins, cofactors or nucleic acids, all for Continuous growth and cell replication are necessary.
  • the number of C. glutamicum cells that produce one or more fine chemicals in the culture can also be increased, which in turn increases the yields or the efficiency of the production of one or more fine chemicals.
  • the C. glutamicum enzymes for general processes are themselves desirable fine chemicals.
  • the specific properties of the enzymes ie regio- and stereospecificity, etc. make them suitable catalysts for chemical reactions in vitro.
  • Either complete C glutamicum cells can be incubated with a suitable substrate so that the desired product is produced by the enzymes in the cell, or the desired enzymes can be over-produced and purified from C. glutamicum cultures (or those of a related bacterium) and then used in in vitro reactions under industrial conditions (either in solution or immobilized on a suitable immobile phase).
  • the enzyme can either be a natural C. glutamicum protein or it can be mutagenized to have an altered activity.
  • the mutagenesis of the proteolytic enzymes according to the invention can likewise directly or indirectly influence the yield, production and / or efficiency of the production of one or more fine chemicals from C. glutamicum.
  • the ability. increase the bacteria's ability to survive in a large culture because the cell can break down proteins more quickly, which are misfolded due to high temperatures, non-optimal pH and other stress factors that can arise during fermenter cultivation.
  • a larger number of cells in these cultures may result in increased yields or an increased efficiency in the production of one or more desired fine chemicals due to the relatively larger number of cells that these Produce connections in culture, result.
  • glutamicum cells also have multiple cell surface proteases that break down external nutrients into molecules that the cell can use more easily than carbon / energy sources or other types of nutrients. An increase in the activity or number of these enzymes can improve this turnover and increase the amount of available nutrients, which improves cell growth or production.
  • the modifications of the proteases according to the invention can thus indirectly influence C. glutamicum fine chemical production.
  • a more direct effect on the fine chemical product in response to the modification of one or more proteases according to the invention can take place if the proteases are involved in the production or in the degradation of a desired fine chemical.
  • the levels of this fine chemical can be increased (due to the decreased degradation or the increased synthesis of the compound).
  • a similar result can be obtained: increased amounts of the desired fine chemical from C. glutamicum Cells that contain these modified proteins.
  • the nucleic acid and protein molecules according to the invention can be used to generate C. glutamicum or related bacterial strains which express mutated HA nucleic acid and protein molecules, so that the yield, production and / or efficiency of the production of a desired compound is improved becomes.
  • the desired compound can be a product of C. glutamicum, which comprises the end products of the biosynthetic pathways and intermediates of naturally occurring metabolic pathways, as well as molecules which do not occur naturally in the metabolism of C. glutamicum, but which come from a C. glutamicum strain according to the invention to be produced.
  • Example 1 Preparation of the entire genomic DNA from Corynebacterium glutamicum ATCC13032
  • a culture of Corynebacterium glutamicum was grown overnight at 30 ° C with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supernatant was discarded, and the cells were resuspended in 5 ml of buffer I (5% of the original volume of the culture - all stated volumes are calculated for 100 ml of culture volume).
  • buffer I 140.34 g / 1 sucrose, 2.46 g / 1 MgS0 4 • 7 H 2 0, 10 ml / 1 KH 2 P0 solution (100 g / l, adjusted to pH 6 with KOH , 7), 50 ml / 1 M12 concentrate (10 g / 1 (NH) 2 S0 4 , 1 g / 1 NaCl, 2 g / 1 MgS0 4 • 7 H 2 0, 0.2 g / 1 CaCl 2 , 0.5 g / 1 yeast extract (Difco), 10 ml / 1 trace element mixture (200 mg / 1 FeS0 4 • H 2 0, 10 mg / 1 ZnS0 4 • 7 H 2 0, 3 mg / 1 MnCl 2 • 4 H 2 0, 30 mg / 1 H 3 B0 3 , 20 mg / 1
  • the cell wall was broken down and the protoplasts obtained were harvested 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 pellet was resuspended in 4 ml TE buffer and 0.5 ml SDS solution (10%) and 0.5 ml NaCl solution (5 M) were added.
  • proteinase K at a final concentration of 200 ⁇ g / ml, the suspension was incubated at 37 ° C. for about 18 hours.
  • the DNA was purified by extraction with phenol, phenol-chloroform-isoamyl alcohol and chloroform-isoamyl alcohol using standard procedures. Then the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by incubation for 30 min at -20 ° C and 30 min centrifugation at 12000 rpm in a high-speed centrifuge with an SS34 rotor (Sorvall) , The DNA was dissolved in 1 ml of TE buffer containing 20 ⁇ g / ml RNase A and dialyzed against 1000 ml of TE buffer at 4 ° C. for at least 3 hours. During this time the buffer was exchanged 3 times.
  • Plasmids pBR322 (Sutcliffe, J.G. (1979) Proc. Natl Acad. Sci. USA, 75: 3737-3741) found particular use; pACYC177 (Change. Cohen (1978) J. Bacteriol. 134: 1141-1156); Plasmids of the pBS series (pBSSK +, pBSSK- and others; Stratagene, LaJolla, USA) or
  • Cosmide such as ⁇ SuperCosl (Stratagene, LaJolla, USA) or Lorist6 (Gibson, TJ Rosenthal, A-, and Waterson, RH (1987) Gene 53: 283-286.
  • Genomic banks as described in Example 2, were used for DNA sequencing according to standard methods, in particular the chain termination method with ABI377 sequencing machines (see, for example, Fleischman, RD et al. (1995) "Whole-genome Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science 269; 496-512)
  • the sequencing primers with the following nucleotide sequences were used: 5 '-GGAAACAGTATGACCATG-3' or 5 '-GTAAAACGACGGCCAGT-3'.
  • In vivo mutagenesis of Corynebacterium glutamicum can be performed by passing a plasmid (or other vector) DNA through E. coli or other microorganisms (e.g. Bacillus spp. Or yeasts such as Saccharomyces cerevisiae) that maintain the integrity of their genetic information cannot maintain.
  • E. coli or other microorganisms e.g. Bacillus spp. Or yeasts such as Saccharomyces cerevisiae
  • Common mutator strains have mutations in the genes for the DNA repair system (eg, mutHLS, utD, utT, etc., for comparison see Rupp, WD (1996) DNA repair mechanisms in Escherichia coli and Salmonella, pp. 2277-2294 , ASM: Washington). These strains are known to the person skilled in the art. The use of these strains is, for example, in Greener, A. and Callahan, M. (1994) Strategies 7; 32-34 illustrates
  • Example 5 DNA transfer between Escherichia coli and Corynehacterium glutamicum
  • Corynebacterium and Brevibacterium species contain endogenous plasmids (such as pHMl519 or pBLl) that replicate autonomously (for an overview see, for example, Martin, J.F. et al. (1987) Biotechnology 5: 137-146).
  • Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can easily be constructed using standard vectors for E. coli (Sambrook, J. et al., (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel , FM et al.
  • origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevi actertium species.
  • transformation markers for these species are genes for kanamycin resistance (such as those derived from the Tn5 or Tn-903 transposon) or for chloramphenicol (Winnacker, EL (1987) "From Genes to Clones - Introduction to Gene Technology, VCH, Weinheim)
  • kanamycin resistance such as those derived from the Tn5 or Tn-903 transposon
  • chloramphenicol Winnacker, EL (1987) "From Genes to Clones - Introduction to Gene Technology, VCH, Weinheim
  • glutamicum and which can be used for various purposes, including gene overexpression (See, e.g., Yoshihama, M. et al. (1985) J. Bacteriol. 162: 591-597, Martin, JF et al., (1987) Biotechnology, 5: 137-146 and Eikmanns, BJ et al. (1992 ) Genes 102: 93-98).
  • C. glutamicum can be carried out by protoplast transformation (Kastsumata, R. et al., (1984) J. Bacteriol. 159, 306-311), electroporation (Liebl, E. et al., (1989) FEMS Microbiol. Letters , 53: 399-303) and, in cases where special vectors are used, can also be achieved by conjugation (as described, for example, in Schaefer, A., et (1990) J. Bacteriol. 172: 1663-1666).
  • a suitable method for determining the amount of transcription of the mutated gene is to carry out a Northern blot (see, for example, Ausubel et al., (1988) Current Protocols in Molecular Biology, Wiley: New York), wherein a primer that is designed to bind to the gene of interest is provided with a detectable (usually radioactive or chemiluminescent) label so that - if the total RNA is one Culture of the organism extracted, separated on a gel, transferred to a stable matrix and incubated with this probe - the binding and the quantity of binding of the probe indicates the presence and also the amount of mRNA for this gene.
  • Total cell RNA can be isolated from Corynebacterium glutamicum by various methods known in the art, as described in Bormann, E.R. et al., (1992) Mol. Microbiol. 6: 317-326.
  • Standard techniques such as Western blot, can be used to determine the presence or the relative amount of protein that is translated from this mRNA (see, for example, Ausubel et al. (1988) "Current Protocols in Molecular Biology", Wiley, New York).
  • total cell proteins are extracted, separated by gel electrophoresis, transferred to a matrix, such as nitrocellulose, and incubated with a probe, such as an antibody, which specifically binds to the desired protein.
  • This probe is usually provided with a chemiluminescent or colorimetric label that is easy to detect. The presence and amount of label observed indicates the presence and amount of the mutant protein sought in the cell.
  • Example 7 Growth of genetically modified Corynebacterium glutamicum media and growing conditions
  • Corynebacteria are grown in synthetic or natural growth media.
  • a number of different growth media for Corynebakterian are known and easy available (Lieb et al. (1989) Appl. Microbiol. Biotechnol. 32: 205-210; von der Osten et al. (1998) Biotechnology Letters 11: 11-16; Patent DE 4 120 867; Liebl (1992) "The Genus Corynebacterium ", in: The Procaryotes, Vol. II, Balows, A., et al., Ed. Springer-Verlag).
  • These media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements.
  • Preferred carbon sources are sugars, such as mono-, di- or polysaccharides.
  • Very good carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
  • Sugar can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. It can also be advantageous to add mixtures of different carbon sources.
  • 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. Exemplary nitrogen sources include ammonia gas or ammonium salts, such as NH 4 C1 or (NH 4 ) S0, NH 4 0H, nitrates,
  • Urea amino acids or complex nitrogen sources such as corn steep liquor, soy flour, soy protein, yeast extracts, meat extracts and others.
  • Inorganic salt compounds that may be included in the media include the chloride, phosphorus, or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • Chelating agents can be added to the medium to keep the metal ions in solution.
  • Particularly suitable chelating agents include dihydroxyphenols such as catechol or protocatechuate or organic acids such as citric acid.
  • the media usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine.
  • Growth factors and salts often come from complex media components such as yeast extract, molasses, corn steep liquor and the like. The exact composition of the media connections depends heavily on the respective experiment and is decided individually for each case. Information about media optimization is available from the textbook "Applied Microbiol. Physiology, A Practical Approach” (Ed. PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like. All media components are sterilized, either by heat (20 min at 1.5 bar and 121 ° C) or by sterile filtration. The components can be sterilized either together or, if necessary, separately. All media components can be present at the beginning of the cultivation or can be added continuously or in batches.
  • the growing conditions are defined separately for each experiment.
  • the temperature should be between 15 ° C and 45 ° C and can be kept constant or changed during the experiment.
  • the pH of the medium should be in the range of 5 to 8.5, preferably around 7.0, and can be maintained by adding buffers to the media.
  • An exemplary buffer for this purpose is a potassium phosphate buffer.
  • Synthetic buffers such as MOPS, HEPES; ACES etc. can be used alternatively or simultaneously.
  • the cultivation pH value can also be kept constant during the cultivation by adding NaOH or NH 4 0H. If complex media components, such as yeast extract, are used, the need for additional buffers is reduced, since many complex compounds have a high buffer capacity.
  • the pH value can also be regulated with gaseous ammonia.
  • the incubation period is usually in the range of several hours to several days. This time is selected so that the maximum amount of product accumulates in the broth.
  • the disclosed growth experiments can be carried out in a variety of containers, such as microtiter plates, glass tubes, glass flasks or glass or metal fermenters of different sizes.
  • the microorganisms should be grown in microtiter plates, glass tubes or shake flasks with or without baffles.
  • 100 ml shake flasks are used, which are filled with 10% (by volume) of the required growth medium.
  • the flasks should be shaken on a rotary shaker (amplitude 25 mm) at a speed in the range of 100-300 rpm. Evaporation losses can be reduced by maintaining a humid atmosphere; alternatively, a mathematical correction should be carried out for the evaporation losses.
  • the medium is seeded to an OD 600 of 0.5-1.5 using cells grown on agar plates, such as CM plates (10 g / 1 glucose, 2.5 g / 1 NaCl, 2 g / 1 urea, 10 g / 1 polypeptone, 5 g / 1 yeast extract, 5 g / 1 meat extract, 22 g / 1 agar pH 6.8 with 2 M NaOH), which have been incubated at 30 ° C.
  • the inoculation of the media is carried out either by introducing a saline solution of C. glutamicum cells from CM plates or by adding a liquid preculture of this bacterium.
  • Example 8 In vitro analysis of the function of mutated proteins
  • DNA band shift assays also referred to as gel retardation assays
  • reporter gene assays as described in Kolmar, H. et al., (1995) EMBO J. 14: 3895-3904 and the references cited therein. Reporter gene test systems are well known and established for use in pro- and eukaryotic cells using enzymes such as beta-galactosidase, green fluorescent protein and several others.
  • membrane transport proteins The activity of membrane transport proteins can be determined according to the techniques as described in Gennis, RB (1989) "Pores, Channels and Transporters", in Biomembranes, Molecular Structure and Function, Springer: Heidelberg, pp. 85-137; 199-234; and 270-322.
  • Example 9 Analysis of the influence of mutated protein on the production of the desired product
  • the effect of the genetic modification in C. glutamicum on the production of a desired compound can be determined by growing the modified microorganisms under suitable conditions (such as those described above) and the medium and / or the cellular Components for the increased production of the desired product (ie an amino acid) is examined.
  • suitable conditions such as those described above
  • Such analysis techniques are well known to the person skilled in the art and include spectroscopy, thin-layer chromatography, staining methods of various types, enzymatic and microbiological methods and analytical chromatography, such as high-performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, vol. A-2, pp. 89-90 and Pp.
  • the analysis methods include measurements of the amount of nutrients in the medium (e.g. sugar, hydrocarbons, nitrogen sources, phosphate and other ions), measurements of the biomass composition and growth, analysis of the production of common metabolites from biosynthetic pathways and measurements of gases that are produced during fermentation , Standard methods for these measurements are in Applied Microbial Physiology; A Practical Approach, PM Rhodes and PF Stanbury, ed. IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and the literature references specified therein.
  • Example 10 Purification of the desired product from C. glutamicum culture
  • the desired product can be obtained from C. glutamicum cells or from the supernatant of the culture described above by various methods known in the art. If the desired product is not secreted by the cells, the cells can be harvested from the culture by slow centrifugation, the cells can be lysed by standard techniques such as mechanical force or ultrasound. The cell debris is removed by centrifugation and the supernatant fraction containing the soluble proteins is obtained for further purification of the desired compound. If the product is secreted by the C. glutamicum cells, the cells are removed from the culture by slow centrifugation and the supernatant fraction is kept for further purification.
  • the supernatant fraction from both purification procedures is subjected to chromatography with an appropriate resin, either with the desired molecule retained on the chromatography resin but not with many contaminants in the sample, or with the contaminants remaining on the resin but not the sample. If necessary, these chromatography steps can be repeated using the same or different chromatography resins.
  • the person skilled in the art is skilled in the selection of the suitable chromatography resins and the most effective application for a particular molecule to be purified.
  • the purified product can be concentrated by filtration or ultrafiltration and kept at a temperature at which the stability of the product is maximum.
  • the identity and purity of the isolated compounds can be determined by standard techniques in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin-layer chromatography, NIRS, enzyme tests or microbiological tests. These analysis methods are summarized in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-5.47, pp.

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Abstract

L'invention concerne de nouvelles molécules d'acide nucléique, leur utilisation pour construire des micro-organismes améliorés en termes de techniques génique et des procédés permettant de produire des substances chimiques fines, notamment des aminoacides, à l'aide de ces micro-organismes améliorés en termes de technique génique.
EP02798305A 2001-11-05 2002-10-31 Genes de corynebacterium glutamicum codant des proteines d'homeostase et d'adaptation Withdrawn EP1444332A2 (fr)

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DE10154175 2001-11-05
DE10154175A DE10154175A1 (de) 2001-11-05 2001-11-05 Gene die für Homeostase-und Adaptions-Proteine codieren
PCT/EP2002/012133 WO2003040290A2 (fr) 2001-11-05 2002-10-31 Genes codant des proteines d'homeostase et d'adaptation

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JP4469568B2 (ja) 2003-07-09 2010-05-26 三菱化学株式会社 有機酸の製造方法
CN100575496C (zh) 2003-08-28 2009-12-30 三菱化学株式会社 产生琥珀酸的方法
WO2005026349A1 (fr) 2003-09-17 2005-03-24 Mitsubishi Chemical Corporation Procede de production d'acide organique non amine
DE10359661A1 (de) 2003-12-18 2005-07-28 Basf Ag Genvarianten die für Proteine aus dem Stoffwechselweg von Feinchemikalien codieren
BRPI0510919A (pt) 2004-05-20 2008-05-20 Ajinomoto Kk bactéria produtora de ácido succìnico e processo para produzir ácido succìnico
CN101061219A (zh) * 2004-09-22 2007-10-24 谷物食品Crc有限公司 通过灭活或减少具有甜菜醛脱氢酶（badh）活性的功能性蛋白质以产生香味的方法
JP4595506B2 (ja) * 2004-11-25 2010-12-08 味の素株式会社 Ｌ−アミノ酸生産菌及びｌ−アミノ酸の製造方法
EP1947190B1 (fr) 2005-10-18 2017-11-29 Ajinomoto Co., Inc. Procédé de production d'acide succinique
DE102012024435A1 (de) * 2012-12-14 2014-07-10 Forschungszentrum Jülich GmbH Verfahren zur Identifizierung einer Zelle mit gegenüber ihrem Wildtyp erhöhten intrazellulären Konzentration eines bestimmten Metaboliten, wobei die Veränderung der Zelle durch Rekombi-neering erreicht wird, sowie ein Verfahren zur Herstellung einer gegenüber ihrem Wildtyp genetisch veränderten Produktionszelle mit optimierter Produktion eines bestimmten Metaboliten, ein Verfahren zur Herstellung dieses Metaboliten, sowie dafür geeignete Nukleinsäuren

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