EP1771547A1 - Unites d'expression de p1-35 - Google Patents

Unites d'expression de p1-35

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Publication number
EP1771547A1
EP1771547A1 EP05762080A EP05762080A EP1771547A1 EP 1771547 A1 EP1771547 A1 EP 1771547A1 EP 05762080 A EP05762080 A EP 05762080A EP 05762080 A EP05762080 A EP 05762080A EP 1771547 A1 EP1771547 A1 EP 1771547A1
Authority
EP
European Patent Office
Prior art keywords
activity
nucleic acids
expression
acids encoding
genes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05762080A
Other languages
German (de)
English (en)
Inventor
Jong-Soo 136-203 Daewoo Prugio ATP. CHOI
Weol Kyu Jeong
Il Kwon Kim
Seong Han Lim
Heung-Shick Lee
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Original Assignee
BASF SE
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Filing date
Publication date
Application filed by BASF SE filed Critical BASF SE
Publication of EP1771547A1 publication Critical patent/EP1771547A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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

Definitions

  • the present invention relates to the use of nucleic acid sequences for the regulation of the transcription and expression of genes, the novel promoters and expression units themselves, methods for altering or causing the transcription rate and / or expression rate of genes, expression cassettes containing the expression units, genetically modified microorganisms altered or induced rate of transcription and / or rate of expression and methods for
  • biosynthetic products such as, for example, fine chemicals, such as, inter alia, amino acids, vitamins but also proteins
  • fine chemicals such as, inter alia, amino acids, vitamins but also proteins
  • These substances which are collectively referred to as fine chemicals / proteins, include, among others, organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors, as well as proteins and enzymes.
  • Their production is most conveniently made on a large scale by culturing bacteria that have been developed to produce and secrete large quantities of the desired substance.
  • Particularly suitable organisms for this purpose are coryneform bacteria, gram-positive non-pathogenic bacteria.
  • Process improvements may include fermentation measures, such as stirring and supply of oxygen, or the composition of the nutrient media, such as the sugar concentration during fermentation, or the processing of the product, for example by ion exchange chromatography but also spray-drying, or the intrinsic performance characteristics of the microorganism concern yourself.
  • RNA polymerase holoenzymes also called -35 and -10 regions
  • ribosomal 16S RNA also ribosomal binding site or also Shine-Dalgarno Called sequence.
  • sequence of a ribosomal binding site also called Shine-Dalgarno sequence, for the purposes of this invention is understood to mean polynucleotide sequences which are up to 20 bases upstream of the initiation codon of the translation.
  • Nucleic acid sequences with promoter activity can influence the formation of mRNA in different ways. Promoters whose activity is independent of the physiological growth phase of the organism are called constitutive. Again other promoters react to external chemical as well as physical stimuli such as oxygen, metabolites, heat, pH, etc. Still others show a strong dependence of their activity in different growth phases. For example, promoters are described in the literature, which show a particularly pronounced activity during the exponential growth phase of microorganisms, or else exactly in the stationary phase of microbial growth. Both characteristics of promoters can have a favorable effect on productivity for the production of fine chemicals and proteins depending on the metabolic pathway.
  • promoters which, during growth, turn off the expression of a gene, but turn it on after optimal growth, to regulate a gene that controls the production of a metabolite.
  • the altered strain has the same growth parameters as the Monocytes, but produces more product per cell. This type of modification can increase both the titer (g product / liter) and the C yield (g product / gC source).
  • regulated promoters may increase or decrease the rate at which a gene is transcribed, depending on the internal and / or external conditions of the cell.
  • an inducer may stimulate the rate of transcription from the promoter.
  • Inducers may directly or indirectly affect transcription from the promoter.
  • suppressors is capable of reducing or inhibiting transcription from the promoter. Like the inducer, the suppressors can act directly or indirectly.
  • promoters known that are regulated by temperature Thus, for example, the level of transcription of such promoters may be increased or decreased by increasing the growth temperature above the normal growth temperature of the cell.
  • promoters from C. glutamicum have been described to date.
  • the promoter of the malate synthase gene from C. glutamicum was described in DE 4440118. This promoter was preceded by a structural protein coding for a protein. After transformation of such a construct into a coryneform bacterium, the expression of the downstream structural gene is regulated. The expression of the structural gene is induced as soon as a corresponding inducer is added to the medium.
  • the object of the invention was to provide further promoters and / or expression units with advantageous properties.
  • nucleic acids with promoter activity containing
  • transcription is understood to mean the process by which a complementary RNA molecule is produced from a DNA template, in which process proteins such as RNA polymerase are involved in so-called sigma factors and transcriptional regulator proteins then serves as a template in the process of translation, which then leads to the biosynthetically active protein.
  • the rate at which a biosynthetic active protein is produced is a product of the rate of transcription and translation. Both rates can be influenced according to the invention and thus influence the rate of formation of products in a microorganism.
  • a "promoter” or a “nucleic acid with promoter activity” is understood as meaning according to the invention a nucleic acid which, in functional linkage with a nucleic acid to be transected, regulates the transcription of this nucleic acid.
  • a "functional linkage” is understood to mean, for example, the sequential arrangement of one of the nucleic acids according to the invention with promoter activity and a nucleic acid sequence to be transcribed and, if appropriate, further regulatory elements, for example nucleic acid sequences which ensure the transcription of nucleic acids, and for example Terminator such that each of the regulatory elements can fulfill its function in the transcription of the nucleic acid sequence.
  • further regulatory elements for example nucleic acid sequences which ensure the transcription of nucleic acids, and for example Terminator such that each of the regulatory elements can fulfill its function in the transcription of the nucleic acid sequence.
  • Genetic control sequences such as for example enhancer sequences, can also function
  • Preference is given to arrangements in which the nucleic acid sequence to be transcribed is positioned behind the (ie at the 3 'end) of the promoter sequence according to the invention rd, so that both sequences are covalently linked.
  • the distance between the promoter sequence and the nucleic acid sequence to be expressed transgenically is
  • promoter activity is understood as meaning the amount of RNA, that is to say the transcription rate, formed by the promoter in a specific time.
  • RNA per promoter activity is meant according to the invention the amount of RNA per promoter formed by the promoter in a certain time.
  • wild-type is understood according to the invention as the corresponding starting microorganism.
  • microorganism may be understood as meaning the starting microorganism (wild-type) or a genetically modified microorganism according to the invention or both.
  • the term "wild-type" is used to change or cause the promoter activity or transcription rate, to change or cause the expression activity or expression rate, and for the Er ⁇ increase the content of biosynthetic products each understood a reference organism.
  • this reference organism is Corynebacterium glutamicum ATCC 13032.
  • starting microorganisms are used which are already capable of producing the desired fine chemical.
  • particularly preferred microorganisms of the bacteria of the genus Corynebacteria and the particularly preferred fine chemicals L-lysine, L-methionine and L-threonine those starting microorganisms which are already able to produce L-lysine, L-methionine and / or are particularly preferred To produce L-threonine.
  • these are particularly preferably corynebacteria in which, for example, the gene coding for an aspartokinase (ask gene) is deregulated or the feed-back inhibition is stopped or reduced.
  • such bacteria have a mutation in the ask gene which leads to a reduction or abolition of the feedback inhibition, for example the mutation T3111.
  • RNA With a "caused promoter activity" or transcription rate with respect to a gene compared to the wild type, the formation of a RNA is thus caused in comparison to the wild type, which was thus not present in the wild type.
  • the amount of RNA formed is thus changed in a certain time compared to the wild type.
  • the increased promoter activity or transcription rate can be achieved, for example, by regulating the transcription of genes in the microorganism by nucleic acids according to the invention having promoter activity or by nucleic acids having increased specific promoter activity, the genes being heterologous with respect to the nucleic acids having promoter activity.
  • Vorzusgweise is the regulation of the transcription of genes in the microorganism by nucleic acids according to the invention with promoter activity or by nucleic acid. achieved with increased specific promoter activity in that one
  • one or more nucleic acids according to the invention having promoter activity is introduced into the genome of the microorganism so that the transcription of one or more endogenous genes under the control of the introduced nucleic acid according to the invention having promoter activity is optionally altered specific promoter activity, or
  • nucleic acid constructs containing a nucleic acid according to the invention having promoter activity, optionally with altered specific promoter activity, and functionally linking one or more nucleic acids to be transcribed into the microorganism.
  • nucleic acids according to the invention with promoter activity contain A) the nucleic acid sequence SEQ. ID. NO. 1 or
  • the nucleic acid sequence SEQ. ID. NO. 1 represents the promoter sequence of an ABC transporter (P 1 - 35 ) from Corynebacterium glutamicum. SEQ. ID.NO. 1 corresponds to the wild-type promoter sequence.
  • the invention further relates to nucleic acids with promoter activity comprising a sequence derived from this sequence by substitution, insertion or deletion of nucleotides which has an identity of at least 90% at the nucleic acid level with the sequence SEQ. ID. NO. 1 has.
  • promoters according to the invention can be prepared, for example, from various organisms whose genomic sequences is known by identity comparisons of the nucleic acid sequences from databases with the sequences SEQ ID NO: 1 described above.
  • substitution in the description refers to the replacement of one or more nucleotides by one or more nucleotides.
  • “Deletion” is the replacement of a nucleotide by a direct bond Insertions are insertions of nucleotides into the nucleic acid sequence which formally replaces a direct bond with one or more nucleotides.
  • identity between two nucleic acids is meant the identity of the nucleotides over the respective total length of the nucleic acid, in particular the identity of the nucleic acids
  • Gap Separation penalty ranks 8 gap separation penalty off
  • a nucleic acid sequence which has an identity of at least 90% with the sequence SEQ ID NO: 1 is accordingly understood as meaning a nucleic acid sequence which, in a comparison of its sequence with the sequence SEQ ID NO: 1, in particular special according to the above program logarithm with the above parameter set has an identity of at least 90%.
  • Particularly preferred promoters have with the nucleic acid sequence SEQ. ID. NO. 1 has an identity of 91%, more preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, more preferably 99%.
  • promoters can furthermore readily be found starting from the above-described nucleic acid sequences, in particular starting from the sequence SEQ ID NO: 1 from various organisms whose genomic sequence is unknown, by hybridization techniques in a manner known per se.
  • Another object of the invention therefore relates to nucleic acids with Promotoreducationi- tat, containing a nucleic acid sequence with the nucleic acid sequence SEQ. ID. No. 1 hybridized under stringent conditions.
  • This nucleic acid sequence comprises at least 10, more preferably more than 12, 15, 30, 50, or more preferably more than 150 nucleotides.
  • the hybridization is carried out according to the invention under stringent conditions.
  • Hybridization conditions are described, for example, in Sambrook, J., Fritsch, EF, Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd Edition, ColD Spring Harbor Laboratory Press, 1989, pages 9.31-9.57 or in Current Protocols in Molecular Biolgy, John Wiley & Sons, NY (1989), 6.3.1-6.3.6:
  • Under stringent hybridization conditions mean in particular: Overnight incubation at 42 0 C in a solution of 50% formamide, 5 x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6 ), 5x Denhardt's solution, 10% dextran sulfate and 20 g / ml denatured, salmon sperm spiked DNA, followed by washing the filters with 0.1 x SSC at 65 ° C.
  • a “functionally equivalent fragment” for nucleic acid sequences with promoter activity fragments understood that have substantially the same or higher specific promoter activity as the starting sequence.
  • fragments is meant partial sequences of the nucleic acids having promoter activity described by embodiment A), B) or C. Preferably, these fragments have more than 10, more preferably more than 12, 15, 30, 50 or more preferably more than 150 contiguous nucleotides of the nucleic acid sequence SEQ ID NO: 1.
  • nucleic acid sequence SEQ. ID. NO. 1 is a promoter, i. for the transcription of genes.
  • the SEQ. ID. NO. 1 has been described without function assignment in Genbank entry AP005283. Therefore, the invention further relates to the novel nucleic acid sequences according to the invention with promoter activity.
  • the invention relates to a nucleic acid with promoter activity containing
  • nucleic acid having the sequence SEQ. ID. NO. 1 is excluded.
  • nucleic acids with promoter activity can furthermore be prepared in a manner known per se by chemical synthesis from the nucleotide units, for example by fragment condensation of individual overlapping, complementary nucleic acid units of the double helix.
  • the chemical synthesis of oligonucleotides can be carried out, for example, in a known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
  • the An ⁇ storage of synthetic oligonucleotides and filling gaps with the Klenow fragment of the DNA polymerase and ligation reactions and general cloning procedures are in Sambrook et al. (1989), Molecular cloning: A laboratory manual, Col. Spring Harbor Laboratory Press.
  • the invention further relates to the use of an expression unit comprising one of the nucleic acids according to the invention with promoter activity and additionally functional combines a nucleic acid sequence which ensures the translation of ribonucleic acids, for the expression of genes.
  • an expression unit is understood as meaning a nucleic acid with expression activity, ie a nucleic acid which, in functional linkage with a nucleic acid or gene to be expressed, regulates the expression, ie the transcription and the translation of this nucleic acid or gene.
  • a “functional linkage” is understood to mean, for example, the sequential arrangement of one of the expression units according to the invention and of a nucleic acid sequence to be expressed transgenically and, if appropriate, further regulatory elements such as, for example, a terminator such that each of the regulators regulates
  • genetic control sequences such as enhancer sequences, may also function from more distant locations or even from other DNA molecules
  • Preference is given to arrangements in which the nucleic acid sequence to be transgenically expressed is positioned behind (ie at the 3 'end) of the expression unit sequence according to the invention, so that both sequences are covalently linked to one another between the expression unit sequence and the nucleic acid sequence to be expressed transgene less than 200 base pairs, more preferably less than 100 base pairs, most preferably less than 50 base pairs.
  • expression activity is understood to mean the amount of protein formed by the expression unit over a certain period of time, ie the expression rate.
  • specific expression activity is understood to mean the amount of protein per expression unit formed by the expression unit in a specific time.
  • the amount of protein formed is thus changed in a specific time compared to the wild type.
  • “changed” is preferably increased or decreased.
  • the increased expression activity or expression rate can be achieved, for example, by regulating the expression of genes in the microorganism by expression units according to the invention or by expression units with increased specific expression activity, wherein the genes are heterologous with respect to the expression units.
  • the regulation of the expression of genes in the microorganism by expression units according to the invention or by expression units with increased specific expression activity according to the invention is achieved by
  • one or more expression units according to the invention optionally with altered specific expression activity, into the genome of the microorganism, so that the expression of one or more endogenous genes takes place under the control of the introduced expression units according to the invention, optionally with altered specific expression activity, or
  • nucleic acid constructs containing an expression unit according to the invention, optionally with altered specific expression activity, and functionally linked one or more nucleic acids to be expressed into the microorganism.
  • the expression units according to the invention contain a nucleic acid according to the invention which is described above with promoter activity and additionally functionally linked to a nucleic acid sequence which ensures the translation of ribonucleic acids.
  • the expression unit according to the invention contains:
  • Nucleotide-derived sequence having an identity of at least 90% at the nucleic acid level with the sequence SEQ. ID. NO. 2 or G) has a nucleic acid sequence which corresponds to the nucleic acid sequence SEQ. ID. NO. 2 hybridizes under stringent conditions or H) functionally equivalent fragments of the sequences under E), F) or G).
  • SEQ. ID. NO. 2 represents the nucleic acid sequence of the expression unit of an ABC transporter (Pi- 35 ) from Corynebacterium glutamicum.
  • SEQ. ID.NO. 2 corresponds to the sequence of the expression unit of the wild-type.
  • the invention furthermore relates to expression units comprising a sequence derived from this sequence by substitution, insertion or deletion of nucleotides which has an identity of at least 90% at the nucleic acid level with the sequence SEQ. ID. NO. 2 has.
  • a nucleic acid sequence which has an identity of at least 90% with the sequence SEQ ID NO: 2 is correspondingly understood to be a nucleic acid sequence which, in a comparison of its sequence with the sequence SEQ ID NO: 2, in particular according to above program logarithm with the above parameter set has an identity of at least 90%.
  • Particularly preferred expression units have with the nucleic acid sequence SEQ. ID. NO. 2 has an identity of 91%, more preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, most preferably 99%. Further natural examples of expression units can furthermore easily be found starting from the above-described nucleic acid sequences, in particular starting from the sequence SEQ ID NO: 2 from various organisms whose genomic sequence is unknown, by hybridization techniques in a manner known per se.
  • a further subject of the invention therefore relates to expression units comprising a nucleic acid sequence which is linked to the nucleic acid sequence SEQ. ID. No. 2 hybridized under stringent conditions.
  • This nucleic acid sequence comprises at least 10, more preferably more than 12, 15, 30, 50 or particularly preferably more than 150 nucleotides.
  • hybridizing is meant the ability of a poly or oligonucleotide to bind under stringent conditions to a nearly complementary sequence, while under these conditions nonspecific binding between non-complementary partners is avoided.
  • sequences should preferably be 90-100%, complementary.
  • the property of complementary sequences to be able to specifically bind to one another for example, in the Northern or Southern Blot technique or in the primer binding in PCR or RT-PCR advantage.
  • hybridization is carried out according to the invention under stringent conditions.
  • stringent conditions are described, for example, in Sambrook, J., Fritsch, EF, Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd Edition, ColD Spring Harbor Laboratory Press, 1989, pages 9.31-9.57 or in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6:
  • Under stringent hybridization conditions mean in particular: Overnight incubation at 42 0 C in a solution of 50% formamide, 5 x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6 ), 5x Denhardt solution, 10% dextran sulfate, and 20 g / ml denatured, sheared salmon sperm DNA, followed by washing the filters with 0.1 x SSC at 65 0 C.
  • 5 x SSC 750 mM NaCl, 75 mM trisodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5x Denhardt solution 10% dextran sulfate
  • the nucleotide sequences according to the invention also make it possible to generate probes and primers which can be used for the identification and / or cloning of homologous sequences in other cell types and microorganisms.
  • probes or primers usually comprise a nucleotide sequence region which under stringen ⁇ th conditions at least about 12, preferably at least about 25, such as about 40, 50 or 75 consecutive nucleotides of a sense strand of an invention hybridized to the invention nucleic acid sequence or a corresponding antisense strand.
  • nucleic acid sequences which comprise so-called silent mutations or are altered according to the codon usage of a specific source or host organism, in comparison with a specifically mentioned sequence, as well as naturally occurring variants, such as e.g. Splice variants or allelic variants, of which
  • a “functionally equivalent fragment” is meant for expression units, fragments having substantially the same or a higher specific expression activity as the starting sequence.
  • substantially the same is meant a specific expression activity which has at least 50%, preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, most preferably 95% of the specific expression activity of the starting sequence.
  • “Fragments” are to be understood as meaning partial sequences of the expression units described by embodiment E), F) or G.
  • these fragments Preferably, these fragments have more than 10, more preferably more than 12, 15, 30, 50 or, even more preferably, more than 150 contiguous nucleotides of the nucleic acid sequence SEQ ID NO: 1.
  • nucleic acid sequence SEQ. ID. NO. 2 as expression unit, i. for the expression of genes.
  • the invention further relates to the new expression units according to the invention.
  • the invention relates to an expression unit comprising a nucleic acid according to the invention with promoter activity additionally functionally linked to a nucleic acid sequence which ensures the translation of ribonucleic acids.
  • the invention particularly preferably relates to an expression unit containing
  • nucleic acid having the sequence SEQ. ID. NO. 2 is excluded.
  • the expression units according to the invention comprise one or more of the following genetic elements: a minus 10 ("-10") sequence; a minus 35 (“-35”) sequence; a transcription start, an enhancer region; and an operator region.
  • these genetic elements are specific for the species Corynebacteria, especially for Corynebacterium glutamicum.
  • All of the abovementioned expression units can furthermore be prepared in a manner known per se by chemical synthesis from the nucleotide units, for example by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
  • the chemical synthesis of oligonucleotides can be carried out, for example, in a known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, p. 896-897).
  • the addition of synthetic oligonucleotides and filling in of gaps using the Klenow fragment of the DNA polymerase and ligation reactions and general cloning methods are described in Sambrook et al. (1989), Molecular cloning: A laboratory manual, Col. Spring Harbor Laboratory Press.
  • nucleic acid molecules of the present invention are preferably in the form of an isolated nucleic acid molecule.
  • An "isolated" nucleic acid molecule is separated from other nucleic acid molecules present in the natural source of the nucleic acid and, moreover, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or free of chemical precursors or other chemicals when chemically synthesized.
  • the invention further comprises the nucleic acid molecules complementary to the specifically described nucleotide sequences or a portion thereof.
  • the promoters and / or expression units according to the invention can be used, for example, with particular advantage in improved processes for the fermentative preparation of biosynthetic products as described below.
  • the promoters and / or expression units according to the invention have the particular advantage that they are induced in microorganisms by stress.
  • this stress induction can be controlled for an increase in the rate of inflammation / expression of desired genes.
  • this stress phase is reached very early, so that here very early an increased transcription / expression rate of desired genes can be achieved.
  • nucleic acids according to the invention with promoter activity can be used for modification, ie for increasing or reducing, or for causing the transcription rate of genes in microorganisms in comparison to the wild type.
  • the expression units according to the invention can be used for altering, that is to say for increasing or reducing, or for causing the expression rate of genes in microorganisms in comparison to the wild type.
  • nucleic acids according to the invention having promoter activity and the expression units according to the invention can serve to regulate and enhance the formation of different biosynthetic products, such as, for example, fine chemicals, proteins, in particular amino acids, in microorganisms, in particular in Corynebacterium species.
  • the invention therefore relates to a method for altering or causing the transcription rate of genes in microorganisms in comparison to the wild type
  • the change or causation of the transcription rate of genes in microorganisms in comparison to the wild type can be achieved by modifying, ie increasing or decreasing, the specific promoter activity in the microorganism. This can be done, for example, by targeted mutation of the nucleic acid sequence according to the invention with promoter activity, that is to say by targeted substitution, de-insertion or insertion of nucleotides.
  • An increased or decreased promoter activity can be achieved by exchanging the nucleotides in the binding site of the RNA polymerase holoenzyme binding sites (also known to the person skilled in the art as the -10 region and -35 region).
  • binding sites also known to the person skilled in the art as exciters
  • regulatory proteins known to the person skilled in the art as repressors and activators
  • increasing or reducing in comparison with the wild type means an increase or reduction of the specific activity with respect to the nucleic acid according to the invention having promoter activity of the wild-type, that is to say, for example, with reference to SEQ ID NO.
  • the change or causation of the transcription rate of genes in microorganisms in comparison with the wild type can be achieved by carrying out the transcription of genes in the microorganism by means of the invention Nucleic acids with promoter activity or by nucleic acids with modified specific promoter activity according to embodiment a) regulated, wherein the genes are heterologous with respect to the nucleic acids with promoter activity.
  • one or more nucleic acids according to the invention having promoter activity, optionally with altered specific promoter activity are introduced into the genome of the microorganism such that the transcription of one or more endogenous genes under the control of the introduced nucleic acid having promoter activity, optionally with altered specific promoter activity , or
  • embodiment b2) introduces one or more endogenous genes into the genome of the microorganism such that the transcription of one or more of the introduced endogenous genes is under the control of the endogenous nucleic acids according to the invention having promoter activity, optionally with altered specific promoter activity or
  • embodiment b2) introduces one or more exogenous genes into the genome of the microorganism such that the transcription of one or more of the introduced exogenous genes takes place under the control of the endogenous nucleic acids according to the invention having promoter activity, optionally with altered specific promoter activity or
  • nucleic acid constructs comprising a nucleic acid according to the invention with promoter activity, optionally with altered specific promoter activity, and functionally linked one or more exogenous nucleic acids to be transcribed, into which microorganism introduces.
  • the insertion of genes according to embodiment b2) can be carried out so that the gene is integrated into coding regions or non-coding regions. Preferably, the insertion takes place in non-coding regions.
  • the insertion of nucleic acid constructs according to embodiment b3) can be carried out chromosomally or extrachromosomally.
  • the insertion of the nucleic acid constructs is chromosomal.
  • a "chromosomal" integration is the insertion of an exogenous DNA fragment into the chromosome of a host cell, which is also used for homologous recombination between an exogenous DNA fragment and the corresponding region on the chromosome of the host cell.
  • nucleic acids according to the invention with modified specific promoter activity according to embodiment a). These can be present in the microorganism in Example b), as described in embodiment a), and can be prepared or introduced into the microorganism in isolated form.
  • endogenous is meant genetic information, such as genes, that are already contained in the wild-type genome.
  • exogenous are meant genetic information, such as genes, which are not contained in the wild-type genome.
  • genes with regard to regulation of transcription by the nucleic acids having promoter activity according to the invention are preferably understood to be nucleic acids which have a region to be transcribed, for example a region which regulates translation, a coding region and optionally contain further regulatory elements, such as a terminator.
  • genes with respect to the expression regulation described below by the expression units according to the invention are preferably understood as meaning nucleic acids which contain a coding region and optionally further regulatory elements, such as, for example, a terminator.
  • coding region is meant a nucleic acid sequence encoding a protein.
  • heterologous with reference to nucleic acids with promoter activity and genes is understood that the genes used in the wild type are not transcribed under regulation of the nucleic acids according to the invention with promoter activity, but that a new, non-wild-type functional linkage is formed and the functional combination of inventive nucleic acid with promoter activity and specific gene does not occur in the wild type.
  • heterologous in terms of expression units and genes is meant that the genes used are not expressed in the wild-type under regulation of the expression units according to the invention, but that a new, not occurring in the wild type functional linkage is formed and the functional combination of inventive Expression unit and specific gene does not occur in the wild type.
  • the invention further relates, in a preferred embodiment, to a method for increasing or causing the transcription rate of genes in microorganisms in comparison to the wild-type ""
  • the transcription of genes in the microorganism by nucleic acids according to the invention with promoter activity or by nucleic acids with increased specific promoter activity according to embodiment a) regulated, wherein the genes are heterologous with respect to the nucleic acids with promoter activity.
  • the regulation of the transcription of genes in the microorganism is achieved by nucleic acids according to the invention with promoter activity or by nucleic acids according to the invention with increased specific promoter activity according to embodiment (ah) in that
  • bh1 introduces one or more nucleic acids according to the invention with promoter activity, optionally with increased specific promoter activity, into the genome of the microorganism such that the transcription of one or more endogenous genes under the control of the introduced invention
  • Nucleic acid with promoter activity optionally with increased specific promoter activity, takes place or
  • bh2 introduces one or more genes into the genome of the microorganism such that the transcription of one or more of the genes introduced takes place under the control of the endogenous nucleic acids according to the invention with promoter activity, optionally with increased specific promoter activity, or
  • the invention further relates, in a preferred embodiment, to a method for reducing the transcription rate of genes in microorganisms in comparison to the wild type, by
  • nucleic acids with reduced specific promoter activity according to embodiment a) into the genome of the microorganism, so that the transcription of endogenous genes takes place under the control of the incorporated nucleic acid with reduced promoter activity.
  • the invention further relates to a method for altering or causing the expression rate of a gene in microorganisms compared to the wild type c) alteration of the specific expression activity in the microorganism of endogenous expression units according to the invention which regulate the expression of the endogenous genes in comparison to the wild-type or
  • the change or causation of the expression rate of genes in microorganisms in comparison to the wild type can be achieved by altering, ie increasing or decreasing, the specific expression activity in the microorganism.
  • altering ie increasing or decreasing, the specific expression activity in the microorganism.
  • This can be done, for example, by targeted mutation of the nucleic acid sequence according to the invention with promoter activity, ie by targeted substitution, deletion or insertion of nucleotides.
  • the extension of the distance between the Shine-Dalgamo sequence and the translational start codon generally leads to a change, a reduction or else an increase in the specific expression activity.
  • a change in the specific expression activity can also be achieved by shortening or lengthening the sequence of the Shine-Dalgamo region (ribosomal binding site) in its distance from the translational start codon by deletions or insertions of nucleotides. But also by the fact that the sequence of the Shine-Dalgarno region is changed so that the homology to complementary 3 'Page 16S rRNA either increased or decreased.
  • nucleic acid constructs containing an expression unit according to the invention, optionally with modified specific expression activity, and functionally linked one or more nucleic acids to be expressed, into which microorganism introduces.
  • one or more expression units according to the invention are introduced into the genome of the microorganism such that the expression of one or more endogenous genes takes place under the control of the introduced expression units or
  • embodiment d2) introduces one or more genes into the genome of the microorganism so that the expression of one or more of the introduced genes takes place under the control of the endogenous expression units according to the invention, optionally with altered specific expression activity, or
  • nucleic acid constructs containing an expression unit according to the invention, optionally with modified specific expression activity, and functionally linked one or more nucleic acids to be expressed, into which microorganism introduces.
  • embodiment d2) introduces one or more exogenous genes into the genome of the microorganism such that the expression of one or more of the incorporated genes is under the control of the endogenous expression elements according to the invention. takes place, optionally with altered specific expression activity, or
  • nucleic acid constructs containing an expression unit according to the invention, optionally with altered specific expression activity, and functionally linked one or more exogenous nucleic acids to be expressed, into which microorganism introduces.
  • the insertion of genes according to embodiment d2) can be carried out so that the gene is integrated into coding regions or non-coding regions. Preferably, the insertion takes place in non-coding regions.
  • the insertion of nucleic acid constructs according to embodiment d3) can be effected chromosomally or extrachromosomally.
  • the insertion of the nucleic acid constructs is chromosomal.
  • nucleic acid constructs are also referred to below as expression cassettes.
  • embodiment d) it is also preferred to use expression units according to the invention with altered specific expression activity according to embodiment c). These can be present in the microorganism and prepared in embodiment d), as described in embodiment d), or introduced into the microorganism in isolated form.
  • ie regulates the expression of genes in the microorganism by expression units according to the invention or by expression units with increased specific expression activity according to embodiment c), the genes being heterologous with respect to the expression units.
  • dh1 introduces one or more expression units according to the invention, optionally with increased specific expression activity, into the genome of the microorganism such that the expression of one or more endogenous genes takes place under the control of the introduced expression units, if appropriate with increased specific expression activity, or
  • dh2 introduces one or more genes into the genome of the microorganism, so that the expression of one or more of the introduced genes is under the control of the endogenous expression units according to the invention, if appropriate with increased specific expression activity, or
  • Expression unit optionally with increased specific expression activity, and functionally linked one or more nucleic acids to be expressed, into which microorganism introduces.
  • the invention further relates to a method for reducing the expression rate of genes in microorganisms compared to the wild type by
  • dr introduces expression units with reduced specific expression activity according to embodiment (s) into the genome of the microorganism, so that the expression of endogenous genes takes place under the control of the introduced expression units with reduced expression activity.
  • the genes are selected from the group nucleic acids encoding a protein from the biosynthetic pathway of Feinchemi ⁇ kalien, wherein the genes optionally further regulatory elements can contain.
  • the genes are selected from the group nucleic acids encoding a protein from the biosynthetic pathway of proteinogenic and non-proteinogenic amino acids, nucleic acids encoding a protein from the biosynthetic pathway of nucleotides and nucleosides, nucleic acids encoding a protein from the biosynthetic pathway of organic acids, nucleic acids encoding a protein from the biosynthetic pathway of lipids and fatty acids, nucleic acids encoding a protein from the biosynthetic pathway of diols, nucleic acids encoding a protein from the biosynthetic pathway of carbohydrates, nucleic acids encoding a protein from the biosynthetic pathway of aromatic compound, nucleic acids encoding a protein from the biosynthetic pathway of vitamins, Nucleic acids encoding a protein from the biosynthetic pathway of
  • proteins from the biosynthetic pathway of amino acids are selected from the group
  • Preferred proteins and nucleic acids encoding these proteins of the above-described proteins from the biosynthetic pathway of amino acids are protein sequences or nucleic acid sequences of microbial origin, preferably from bacteria of the genus Corynebacterium or Brevibacterium, preferably from coryneform bacteria, more preferably from Corynebacterium glutamicum.
  • Another example of a particularly preferred protein sequence and the corresponding nucleic acid sequence encoding this protein from the biosynthetic pathway of amino acids is the sequence of the fructose-1,6-bisphosphatase 2, or also called fbr2, (SEQ ID NO. 8) and the corresponding nucleic acid sequence encoding a fructose-1, 6-bisphosphatase 2 (SEQ ID NO: 7).
  • Another example of a particularly preferred protein sequence and the corresponding nucleic acid sequence encoding this protein from the biosynthetic pathway of amino acids is the sequence of the protein in sulfate reduction, or also called RXA077, (SEQ ID NO: 10) and the corresponding Nucleic acid sequence encoding a protein in sulfate reduction (SEQ ID NO. 9)
  • proteins from the biosynthesis pathway of amino acids have in each case the amino acid sequence indicated for this protein in Table 1, wherein the respective protein in each case at at least one of the amino acid positions indicated in Table 2 / column 2 for this amino acid sequence another proteinogenic amino acid than the respective amino acid indicated in Table 2 / Column 3 in the same line.
  • the proteins have the amino acid indicated in Table 2 / Column 4 in the same row on at least one of the amino acid positions given in Table 2 / Column 2 for the amino acid sequence.
  • the proteins given in Table 2 are mutated proteins of the biosynthetic pathway of amino acids which have particularly advantageous properties and are therefore particularly suitable for expression of the corresponding nucleic acids by the promoter according to the invention and for the production of amino acids.
  • the T3111 mutation results in switching off the feedback inhibition from ask.
  • nucleic acids which encode a mutated protein from Table 2 described above can be prepared by conventional methods.
  • the starting point for the preparation of the nucleic acid sequences encoding a mutated protein is, for example, the genome of a Corynebacterium glutamicum strain which is obtainable from the American Type Culture Collection under the name ATCC 13032 or the nucleic acid sequences referred to in Table 1.
  • a Corynebacterium glutamicum strain which is obtainable from the American Type Culture Collection under the name ATCC 13032 or the nucleic acid sequences referred to in Table 1.
  • Coryn ⁇ bacterium glutamicum it is preferable for Coryn ⁇ bacterium glutamicum to use the codon usage of Corynebacterium glutamicum.
  • the codon usage of the particular organism can be determined in a manner known per se from databases or patent applications which describe at least one protein and one gene which codes for this protein from the desired organism.
  • Table 2 are to be understood as follows:
  • mutated protein with a certain function (column 5) and a certain initial amino acid sequence (table 1)
  • at least one mutation is described in columns 2, 3 and 4, and several mutations are also described for some sequences. These multiple mutations always refer to the above-mentioned, closest starting amino acid sequence (Table 1).
  • the term "at least one of the amino acid positions" of a particular amino acid sequence is preferably understood to mean at least one of the mutations described for this amino acid sequence in columns 2, 3 and 4.
  • the SacB method is known to the person skilled in the art and is described, for example, in Schwarz A, Tauch A, Jäger W, Kalinowski J 1 Thierbach G, Pühler A .; Small mobilizable multi-purpose cinging vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosomes of Corynebacterium glutamicum, Gene. 1994 JuI 22; 145 (1): 69-73 and Blomfield IC, Vaughn V, rest RF, Eisenstein Bl .; Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC-101 replicon; Mol Microbiol. 1991 Jun; 5 (6): 1447-57.
  • the change or causation of the transcription rate and / or expression rate of genes in microorganisms takes place by introducing nucleic acids according to the invention having promoter activity or inventive expression units into the microorganism.
  • the change or causation of the transcription rate and / or expression rate of genes in microorganisms takes place by introduction of the above-described nucleic acid constructs or expression cassettes into the microorganism.
  • the invention therefore further relates to an expression cassette comprising
  • At least one further nucleic acid sequence to be expressed ie a gene to be expressed
  • genetic control elements such as a terminator
  • the nucleic acid sequence to be expressed is at least one nucleic acid encoding a protein from the biosynthetic pathway of fine chemicals.
  • the nucleic acid sequence to be expressed is particularly preferably selected from the group of nucleic acids encoding a protein from the biosynthesis pathway of proteinogenic and non-proteinogenic amino acids, nucleic acids encoding a protein from the biosynthetic pathway of nucleotides and nucleosides, nucleic acids encoding a protein from the biosynthetic pathway of organic acids , Nucleic acids encoding a protein from the biosynthetic pathway of lipids and fatty acids, Nucleic acids encoding a protein from the biosynthetic pathway of diols, Nucleic acids encoding a protein from the biosynthetic pathway of carbohydrates, Nucleic acids encoding a protein from the biosynthetic pathway of aromatic compound, nucleic acids encoding a protein from the biosynthetic pathway of vitamins, nucleic acids encoding a protein from the biosynthesis pathway of cofactors and nucleic acids encoding a protein from the biosynthetic pathway of enzymes
  • Preferred proteins from the biosynthetic pathway of amino acids are described above and their examples in Tables 1 and 2.
  • the physical position of the expression unit relative to the gene to be expressed is selected so that the expression unit regulates the transcription and preferably also the translation of the gene to be expressed and thus enables the formation of one or more proteins.
  • the "enabling education” involves constitutively increasing the formation, weakening or blocking the formation under specific conditions and / or increasing the formation under specific conditions.
  • the “conditions” include: (1) adding a component to the culture medium, (2) removing a component from the culture medium, (3) replacing a component in the culture medium with a second component, (4) increasing the temperature of the culture medium, (5) Lowering the temperature of the culture medium, and (6) regulating the atmospheric conditions, such as the oxygen or nitrogen concentration in which the culture medium is maintained.
  • the invention furthermore relates to an expression vector comprising an above-described expression cassette according to the invention.
  • Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P.H. et al., Eds. Elsevier, Amsterdam-New York-Oxford, 1985). Vectors other than plasmids are also to be understood as meaning all other vectors known to the person skilled in the art, such as, for example, phages, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be autonomously replicated in the host organism or replicated chromosomally.
  • Plasmid ⁇ vectors such as. B.
  • pCLiK5MCS or those on pCG4 (US-A 4,489,160) or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)) or pAG1 (US-A 5,158,891) can be used equally.
  • those plasmid vectors by means of which one can apply the method of gene amplification by integration into the chromosome, as described for example by Remscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for the duplication or amplification of the hom-thrB operon.
  • the complete gene is cloned into a plasmid vector that can replicate in a host (typically E. coli) but not in C. glutamicum.
  • vectors which are used are pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), Berard et al. , Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173: 4510-4516) or pBGS8 (Spratt et al., 1986, Gene 41: 337-342) Question.
  • the plasmid vector containing the gene to be amplified is then transformed into the desired strain of C. glutamicum by transformation.
  • the invention further relates to a genetically modified microorganism, wherein the genetic modification leads to a change or causation of the transcription rate of at least one gene in comparison to the wild type and is conditioned by
  • the regulation of the transcription of genes in the microorganism by nucleic acids with promoter activity according to claim 1 or by nucleic acids with promoter activity according to claim 1 with altered specific promoter activity according to embodiment a) is achieved by b1) introduces one or more nucleic acids with promoter activity according to claim 1, optionally with modified specific promoter activity, into the genome of the microorganism such that the transcription of one or more endogenous genes under the control of the introduced nucleic acid with promoter activity according to An - Speech 1, optionally with altered specific promoter activity, takes place or
  • the invention further relates to a genetically modified microorganism having an increased or caused transcription rate of at least one gene in comparison to the wild type, wherein
  • the transcription of genes in the microorganism is regulated by nucleic acids with promoter activity according to claim 1 or by nucleic acids with increased specific promoter activity according to embodiment ah), the genes being heterologous with respect to the nucleic acids having promoter activity.
  • nucleic acids with promoter activity according to claim 1 or by nucleic acids with promoter activity according to claim 1 with altered specific promoter activity according to embodiment a) is achieved by
  • bh1 introduces one or more nucleic acids with promoter activity according to claim 1, where appropriate with increased specific promoter activity, into the genome of the microorganism such that the transcription of one or more endogenous genes under control of the introduced nucleic acid with promoter activity, optionally with increased specific promoter activity, or
  • bh2 introduces one or more genes into the genome of the microorganism such that the transcription of one or more of the introduced genes takes place under the control of the endogenous nucleic acids with promoter activity according to claim 1, optionally with increased specific promoter activity, or
  • nucleic acid constructs comprising a nucleic acid with promoter activity according to claim 1, optionally with increased specific promoter activity, and functionally linked one or more nucleic acids to be transcribed into the microorganism.
  • the invention further relates to a genetically modified microorganism having a reduced transcription rate of at least one gene in comparison to the wild type, wherein
  • nucleic acids with reduced promoter activity according to embodiment a) have been introduced into the genome of the microorganism, so that the transcription of at least one endogenous gene takes place under the control of the incorporated nucleic acid with reduced promoter activity.
  • the invention further relates to a genetically modified microorganism, wherein the genetic modification leads to a change or causation of the Expressionsra ⁇ te of at least one gene in comparison to the wild type and is caused by
  • d1) introduces one or more expression units according to claim 2 or 3, optionally with altered specific expression activity, into the genome of the microorganism such that the expression of one or more endogenous genes under the control of the introduced expression units according to claim 2 or 3, optionally with altered specific expression activity, or
  • d2 introduces one or more genes into the genome of the microorganism such that the expression of one or more of the genes introduced takes place under the control of the endogenous expression units according to claim 2 or 3, optionally with altered specific expression activity, or
  • nucleic acid constructs containing an expression unit according to claim 2 or 3, optionally with altered specific expression activity, and functionally linked to one or more nucleic acids to be expressed, into which microorganism introduces.
  • the invention further relates to a genetically modified microorganism with increased or caused expression rate of at least one gene in comparison to Wild ⁇ type, wherein
  • dh1 introduces one or more expression units according to claim 2 or 3, optionally with increased specific expression activity, into the genome of the microorganism, so that the expression of one or more endogenous genes under the control control of the introduced expression units according to claim 2 or 3, if appropriate with increased specific expression activity, takes place or
  • dh2 introduces one or more genes into the genome of the microorganism, so that the expression of one or more of the introduced genes under the control of endo ⁇ gene expression units according to claim 2 or 3, optionally with increased specific expression activity occurs, or
  • nucleic acid constructs containing an expression unit according to claim 2 or 3, optionally with increased specific expression activity, and functionally linked one or more nucleic acids to be expressed, into which microorganism introduces.
  • the invention further relates to a genetically modified microorganism having a reduced expression rate of at least one gene in comparison to the wild type, wherein
  • one or more expression units according to claim 2 or 3 were introduced with reduced expression activity in the genome of the microorganism, so that the expression of at least one gene under the control of the introduced expression unit according to claim 2 or 3 takes place with reduced expression activity.
  • the invention relates to a genetically modified microorganism containing an expression unit according to claim 2 or 3 and functionally linked to a gene to be expressed, wherein the gene is heterologous with respect to the expression unit.
  • This genetically modified microorganism particularly preferably contains an expression cassette according to the invention.
  • the present invention particularly preferably relates to genetically modified microorganisms, in particular coryneform bacteria, which contain a vector, in particular pendulum vector or plasmid vector, which carries at least one recombinant nucleic acid construct according to the definition of the invention.
  • the genes described above are encoding at least one nucleic acid a protein from the biosynthetic pathway of fine chemicals.
  • the genes described above are selected from the group consisting of nucleic acids encoding a protein from the biosynthetic pathway of proteinogenic and non-proteinogenic amino acids, nucleic acids encoding a protein from the biosynthesis pathway of nucleotides and nucleosides, Nucleic acids encoding a protein from the biosynthetic pathway of organic acids, nucleic acids encoding a protein from the biosynthetic pathway of lipids and fatty acids, nucleic acids encoding a protein from the biosynthetic pathway of diols, nucleic acids encoding a protein from the biosynthesis pathway of carbohydrates, nucleic acids encoding a protein the biosynthetic path of aromatic compound, nucleic acids encoding a protein from the biosynthetic pathway of vitamins, nucleic acids encoding a protein from the biosynthetic pathway of cofactors and nucleic acids encoding a protein from the bio
  • Preferred proteins from the biosynthetic pathway of amino acids are selected from the group aspartate kinase, aspartate-semialdehyde dehydrogenase, diaminopimelate dehydrogenase, diaminopimelate decarboxylase, dihydrodipicolinate synthetase, dihydrodipicolinate reductase, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate Kinase, pyruvate carboxylase, triosephosphate isomerase, transcriptional regulator LuxR, transcriptional regulator LysR1, transcriptional regulator LysR2, malate quinone oxidoreductase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrognease, transketolase, transaldolase, homoserine O-acetyltransferase, cystahionine gamma synthase, cystahionine
  • Preferred microorganisms or genetically modified microorganisms are bacteria, algae, fungi or yeasts.
  • microorganisms are, in particular, coryneform bacteria.
  • Preferred coryneform bacteria are bacteria of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium thermoaminogenes, Corynebacterium melassecola and Corynebacterium efficiens or of the genus Brevibacterium, in particular of the species Brevibacterium flavum, Brevibacterium lactofermentum and Brevibacterium divaricatum.
  • Particularly preferred bacteria of the genera Corynebacterium and Brevibacterium are selected from the group Corynebacterium glutamicum ATCC 13032, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium thermoaminogenes FERM BP-1539, Corynebacterium me ⁇ lassecola ATCC 17965, Corynebacterium efficiens DSM 44547, Corynebacterium effi ⁇ ciens DSM 44548.
  • the abbreviation KFCC means the Korean Federation of Culture Collection
  • the abbreviation ATCC means the American strain strain culture collection
  • the abbreviation DSM the German Collection of Microorganisms.
  • ATCC American Type Culture Collection, Rockville, Md., USA.
  • FERM Fermentation Research Institute, Chiba, Japan
  • NRRL ARS Culture Collection, Northern Regional Research Laboratory, Peoria, IL, USA
  • DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
  • inventive nucleic acids with promoter activity and the expression units according to the invention make it possible with the aid of the above-described inventive methods to regulate the metabolic pathways to specific biosynthetic products in the above-described inventive genetically modified microorganisms.
  • metabolic pathways which lead to a specific biosynthetic product by causing or increasing the transcription rate or expression rate of genes of this biosynthetic pathway in which the increased protein amount to increased total activity of these proteins of the desired biosynthetic pathway and thus to an increased metabolic flux to the gewün - leads biosynthetic product.
  • metabolic pathways can lead away from a specific biosynthetic Pro ⁇ by reducing the transcription rate or expression rate of Genes this way leading biosynthetic pathway are attenuated in that the reduced amount of protein leads to a reduced total activity of these proteins of unwanted biosynthetic pathway and thus in addition to an increased Stoffwech ⁇ selfluß to the desired biosynthetic product.
  • the genetically modified microorganisms according to the invention are, for example, able to produce biosynthetic products from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol.
  • the invention therefore relates to a process for the production of biosynthetic products by culturing genetically modified microorganisms according to the invention.
  • the transcription rate or expression rate of different genes must be increased or reduced.
  • At least one altered, that is to say increased or reduced, transcription rate or expression rate of a gene can be attributed to a nucleic acid according to the invention having promoter activity or the expression unit according to the invention.
  • additional modified i. additionally increased or additionally reduced transcription rates or expression rates of further genes in the genetically modified microorganism can, but need not go back to the nucleic acids according to the invention with promoter activity or the expression units according to the invention.
  • the invention therefore furthermore relates to a process for the preparation of biosynthetic products by culturing genetically modified microorganisms according to the invention.
  • Preferred biosynthetic products are fine chemicals.
  • fine chemical is known in the art and includes compounds that are produced by an organism and find applications in various industries, such as, but not limited to, the pharmaceutical Industry, agriculture, cosmetics, food and feed industry. These compounds include organic acids such as tartaric acid, itaconic acid and diaminopimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides and nucleotides (as described for example in Kuinaka, A. (1996) Nucleotides and Related Compounds, pp. 561-612, Biotechnology Vol. 6, Rehm et al., ed.
  • VCH Weinheim and the citations contained therein
  • lipids saturated and unsaturated fatty acids (for example arachidonic acid), diols (for example propane). diol and butanediol), carbohydrates (for example hyaluronic acid and trehalose), aromatic compounds (for example aromatic amines, vanillin and indigo), vitamins and cofactors (as described in Ullmann's Encyclopedia of Industrial Chemistry, Vol.
  • amino acids comprise the basic structural units of all proteins and are therefore essential for normal cell function.
  • amino acid is known in the art.
  • the proteinogenic amino acids of which there are 20 species, serve as structural units for proteins in which they are linked via peptide bonds, whereas the non-proteinogenic amino acids (of which hundreds are known) usually do not occur in proteins (see Ullmann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97 VCH: Weinheim (1985)).
  • the amino acids may be in the D or L configuration, although L-amino acids are usually the only type found in naturally occurring proteins. Biosynthesis 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.
  • the "essential" amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine
  • the "essential" amino acids referred to as having to be taken up with the nutrition due to the complexity of their biosynthesis, are synthesized by simple synthetic routes converted into the remaining 11 "nonessential" amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine).
  • Higher animals have the ability to sync some of these amino acids.
  • the essential amino acids must be taken up with the food so that a normal protein synthesis takes place.
  • Lysine is an important amino acid not only 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) as well as widely used in the food industry, as are aspartate, phenylalanine, glycine and cysteine.
  • Glycine, L-methionine and tryptophan are all used in the pharmaceutical industry.
  • Glutamine, VaNn, leucine, isoleucine, histidine, arginine, proline, serine and alanine are used in the pharmaceutical and cosmetics industries. Threonine, tryptophan and D- / L-methionine are widely used feed additives (Leuchtenberger, W. (1996) Amino acids - technical production and use, pp. 466-502 in Rehm et al., (Ed.) Biotechnology Vol. 6, chapters 14a, VCH: Weinheim).
  • amino acids are also useful as precursors for the synthesis of synthetic amino acids and proteins such as N-acetylcysteine, S-carboxymethyl-L-cysteine, (S) -5-hydroxytryptophan and others in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97, VCH, Weinheim, 1985.
  • Cysteine and glycine are each produced from serine, the former by condensation of homocysteine with serine, and the latter by transfer of the side chain ⁇ -carbon atom to tetrahydrofolate, in a serine transhydroxymethylase catalyzed reaction.
  • Phenylalanine and tyrosine are synthesized from the precursors of the glycolysis and pentose phosphate pathway, erythrose 4-phosphate and phosphoenolpyruvate in a 9-step biosynthetic pathway that differs only in the last two steps after the synthesis of prephenate. Tryptophan is also produced from these two starting molecules, but its synthesis takes place in an 11-step pathway.
  • Tyrosine undergoes reaction catalyzed by phenylalanine hydroxylase also from phenylalanine produce.
  • Alanine, valine and leucine are each biosynthesis products from pyruvate, the end product of glycolysis.
  • Aspartate is formed from oxalacetate, an intermediate of the citrate cycle.
  • Asparagine, methionine, threonine and lysine are each produced by conversion of aspartate.
  • Isoleucine is formed from threonine.
  • histidine is formed from 5-phosphoribosyl-1-pyrophosphate, an activated sugar.
  • Amino acids whose amount exceeds the protein biosynthetic demand of the cell can not be stored and are instead degraded to provide intermediates for the cell's major metabolic pathways (for review, see Stryer, L., Biochemistry, 3rd Ed. Chapter 21 "Amino Acid Degradation and the Urea Cycle", S 495-516 (1988)). While the cell is capable of converting unwanted amino acids into useful metabolic intermediates, amino acid production is expensive in terms of energy, precursor molecules and the enzymes necessary for their synthesis.
  • amino acid biosynthesis is regulated by feedback inhibition, wherein the presence of a certain amino acid slows down or completely stops its own production (for a review of the feedback mechanism in amino acid biosynthetic pathways, see Stryer , L., Biochemistry, 3rd Ed., Chapter 24, "Biosynthesis of Amino Acids and Hen", pp. 575-600 (1988)).
  • the emission of a particular amino acid is therefore limited 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 thus need to ingest them, although they are readily 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 significant industrial value as dyes, antioxidants and catalysts or other processing aids. (For an overview of the structure, activity and 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 includes nutrients that are needed by an organism for normal function, but can not be synthesized by that organism itself.
  • the group of vitamins may include cofactors and nutraceutical compounds.
  • cofactor includes non-proteinaceous compounds that are necessary for the occurrence of normal enzyme activity. These compounds can be organic or be inorganic; the cofactor molecules according to the invention are preferably organic.
  • nutraceutical includes food additives that are beneficial to the health of plants and animals, especially humans. Examples of such molecules are vitamins, antioxidants and also certain lipids (eg polyunsaturated fatty acids).
  • Thiamine (vitamin B 1 ) is formed by chemical coupling of pyrimidine and thiazole units.
  • Riboflavin (vitamin B 2 ) is synthesized from guanosine 5'-triphosphate (GTP) and ribose 5'-phosphate.
  • riboflavin is used to synthesize flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).
  • the family of compounds collectively referred to as "vitamin B6" eg, pyridoxine, pyridoxamine, pyridoxal-5'-phosphate and the commercially used pyridoxine hydrochloride
  • vitamin B6 eg, pyridoxine, pyridoxamine, pyridoxal-5'-phosphate and the commercially used pyridoxine hydrochloride
  • Panthothenate (pantothenic acid, R - (+) - N- (2,4-dihydroxy-3,3-dimethyl-1-oxobutyl) - ⁇ -alanine) can be prepared either by chemical synthesis or by fermentation.
  • the last steps in pantothenate biosynthesis consist of the ATP-driven condensation of ⁇ -alanine and pantoic acid.
  • the enzymes responsible for the biosynthesis steps for the conversion into pantoic acid, into ⁇ -alanine and for the condensation in pantothenic acid are known.
  • the metabolically active form of pantothenate is coenzyme A, whose biosynthesis proceeds through 5 enzymatic steps.
  • Pantothenate pyridoxal-5'-phosphate, cysteine and ATP are the precursors of coenzyme A. These enzymes not only catalyze the formation of pantothenate, but also the production of (R) -pantoic acid, (R) -pantolactone (R ) Panthenol (provitamin B 5 ), pantethein (and its derivatives) and coenzyme A.
  • the biosynthesis of biotin from the precursor molecule pimeloyl-CoA in microorganisms has been extensively studied, and several of the genes involved have been identified. It has been found that many of the corresponding proteins are involved in Fe cluster synthesis and belong to the class of nifS proteins.
  • the lipoic acid is derived from octanoic acid and serves as a coenzyme in energy metabolism, where it is part of the Pyruvatdehydrogenasekomplexes and the ⁇ -ketoglutarate dehydrogenase complex.
  • the folates are a group of substances which are all derived from folic acid, which in turn is derived from L-glutamic acid, p-aminobenzoic acid and 6-methylpterin.
  • the biosynthesis of folic acid and its derivatives starting from the metabolic intermediates guanosine 5'-triphosphate (GTP), L-glutamic acid and p-
  • Aminobenzoic acid has been extensively studied in certain microorganisms.
  • Corrinoids such as the cobalamins and especially vitamin B 12
  • the porphyrins belong to a group of chemicals that are characterized by a tetrapyrrole ring system.
  • the biosynthesis of vitamin Bi 2 is sufficiently complex that it has not yet been fully characterized, however, a large part of the participating enzymes and substrates is now known.
  • Nicotinic acid (nicotinate) and nicotinamide are pyridine derivatives, also referred to as "niacin”.
  • Niacin is the precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adrenine dinucleotide phosphate) and their reduced forms.
  • purine and pyrimidine metabolism and their corresponding proteins are important targets for the treatment of tumors and viral infections.
  • purine or pyrimidine includes nitrogen-containing bases which are part of the nucleic acids, coenzymes and nucleotides.
  • nucleotide includes the basic structural units of the nucleic acid molecules which comprise a nitrogen-containing base, a pentose sugar (in the case of RNA, the sugar is ribose, in the case of DNA is the sugar D-deoxyribose) and phosphoric acid.
  • nucleoside includes molecules which serve as precursors of nucleotides, but unlike the nucleotides have no phosphoric acid moiety.
  • nucleic acid molecules By inhibiting the biosynthesis of these moieties or mobilizing them to form nucleic acid molecules, it is possible to inhibit RNA and DNA synthesis; If this activity is purposefully inhibited in cancerous cells, the ability to divide and replicate tumor cells can be inhibited.
  • the purine and pyrimidine bases, nucleosides and nucleotides also have other ⁇ possibilities: as intermediates in the biosynthesis of various fine chemicals (eg thiamine, S-adenosyl-methionine, folates or riboflavin), as an energy source for the cell (eg. ATP or GTP) and chemicals themselves are commonly used as flavor enhancers (eg, IMP or GMP) or for many medical applications (see, for example, Kuninaka, A. (1996) Nucleotides and Related Compounds in Biotechnology Vol. Weinheim, pages 561-612).
  • Enzymes involved in purine, pyrimidine, nucleoside or nucleotide metabolism are also increasingly serving as targets against which chemicals are used plant protection, including fungicides, herbicides and insecticides.
  • the purine nucleotides are synthesized via a series of steps via the lnosine 5'-phosphate (IMP) intermediate from ribose-5-phosphate, resulting in the production of guanosine 5'-monophosphate (GMP) or adenosine 5'-monophosphate (AMP ), from which the triphosphate forms used as nucleotides can be easily prepared. These compounds are also used as energy stores, so that their degradation provides energy for many different biochemical processes in the cell.
  • the Pyrimidinbiosynthe- se via the formation of uridine 5'-monophosphate (UMP) from ribose-5-phosphate. In turn, UMP is converted to cytidine 5'-triphosphate (CTP).
  • the deoxy forms of all nucleotides are isolated in a one-step reduction reaction from 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 molecules of glucose linked together by ⁇ , ⁇ -1, 1 bonding. 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, for example, 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 naturally released into the surrounding medium from which it can be recovered by methods known in the art.
  • biosynthetic products are selected from the group of organic acids, proteins, nucleotides and nucleosides, both proteinogenic and non-proteinogenic amino acids, lipids and fatty acids, diols, carbohydrates, aromati cal compounds, vitamins and cofactors, enzymes and proteins.
  • Preferred organic acids are tartaric acid, itaconic acid and diaminopimelic acid
  • nucleosides and nucleotides are described, for example, in Kuninaka, A. (1996) Nucleotides and Related Compounds, pp. 561-612, Biotechnology Vol. 6, Rehm et al., Ed. VCH: Weinheim and the citations contained therein.
  • Preferred biosynthetic products are also lipids, saturated and unsaturated fatty acids, such as arachidonic acid, diols such as propanediol and butanediol, carbohydrates such as hyaluronic acid and trehalose, aromatic compounds such as aromatic amines, vanillin and indigo, vitamins and cofactors, such as for example, described in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A27, "Vitamins", pp. 443-613 (1996) VCH: Weinheim and the citations contained therein; and Ong, AS, Niki, E. and Packer, L.
  • saturated and unsaturated fatty acids such as arachidonic acid, diols such as propanediol and butanediol
  • carbohydrates such as hyaluronic acid and trehalose
  • aromatic compounds such as aromatic amines, vanillin and indigo
  • vitamins and cofactors such as for example, described in Ullmann's Encyclopedia of Industrial Chemistry,
  • biosynthetic products are amino acids, more preferably essential amino acids, in particular L-glycine, L-alanine, L-leucine, L-methionine, L-phenylalanine, L-tryptophan, L-lysine, L-glutamine, L-glutamic acid , L-serine, L-proline, L-valine, L-isoleucine, L-cysteine, L-tyrosine, L-histidine, L-arginine, L-asparagine, L-aspartic acid and L-threonine, L-homoserine, in particular L-lysine, L-methionine and L-threonine.
  • essential amino acids in particular L-glycine, L-alanine, L-leucine, L-methionine, L-phenylalanine, L-tryptophan, L-lysine, L-glutamine, L-glutamic acid , L-serine,
  • both the L and the D form of the amino acid preferably the L-form, that is, for example, L-lysine, L-methionine and L-threonine stood.
  • the invention relates to a process for the production of lysine by cultivation of genetically modified microorganisms with an increased or induced expression rate of at least one gene compared to the wild type, wherein
  • genes are selected from the group of nucleic acids encoding an aspartate kinase, nucleic acids encoding an aspartate semialdehyde dehydrogenase, nucleic acids encoding a diaminopimelate dehydrogenase, nucleic acids encoding a diaminopimelate decarboxylase, nucleic acids encoding a dihydrodipicolinate synthetase, encoding nucleic acids a dihydridipicolinate reductase, nucleic acids encoding a glyceraldehyde-3-phosphate dehydrogenase, nucleic acids encoding a 3-phosphoglycerate kinase, nucleic acids encoding a pyruvate carboxylase, nucleic acids encoding a triosephosphate isomerase, nucleic acids encoding a transcriptional regulator LuxR, Nucleic acids encoding a transcriptional regulator LysR
  • dh1 brings one or more expression units according to the invention, optionally with increased specific expression activity, into the genome of the microorganism such that the expression of one or more of these endogenous genes under the control of the introduced expression units according to the invention, if appropriate with increased specific expression activity, done or
  • dh2 introduces one or more of these genes into the genome of the microorganism, so that the expression of one or more of the introduced genes takes place under the control of the endogenous expression units according to the invention, optionally with increased specific expression activity, or
  • nucleic acid constructs containing an expression unit according to the invention, optionally with increased specific expression activity, and functionally linked one or more nucleic acids to be expressed, into which microorganisms are introduced.
  • a further preferred embodiment of the above-described method for the production of lysine is characterized in that the genetically modified microorganisms in addition to the wild type additionally an increased activity, at least one of the activities selected from the group
  • Aspartate kinase activity Aspartate kinase activity, aspartate-semialdehyde dehydrogenase activity, diaminoperilate dehydrogenase activity, diaminopimelate decarboxylase activity, dihydrodipicolinate synthetase activity, dihydridipicolinate reductase activity, glyceraldehyde-3-phosphate dehydrogenase activity, 3 Phosphoglycerate kinase activity, pyruvate carboxylase activity, triosephosphate isomerase activity, transcriptional regulator activity LuxR, activity of transcriptional regulator LysR1, activity of transcriptional regulator LysR2, malate-quinone oxo-reductase activity, glucose-6 Phosphate de-dehydrogenase activity, 6-phosphogluconate dehydrognease activity, transketolase activity, transaldolase activity, lysine exporter activity, Argi nyl-t
  • a further particularly preferred embodiment of the process for the preparation of lysine described above is characterized in that the genetically modified microorganisms in addition to the wild type in addition a reduced Ak ⁇ activity, at least one of the activities selected from the group threonine dehydratase activity, homoserine O-acetyltransferase activity, O-acetyl homoserine sulfhydrylase activity, phosphoenolpyruvate carboxykinase activity, pyruvate oxidase activity, homoserine kinase activity, homoserine dehydrogenase activity, threonine exporter activity, threonine efflux protein Activity, asparaginase activity, aspartate decarboxylase activity and threonine synthase activity.
  • the activities selected from the group threonine dehydratase activity, homoserine O-acetyltransferase activity, O-acetyl homoserine sul
  • nucleic acid according to the invention having promoter activity and / or an expression unit according to the invention.
  • the invention further relates to a process for the production of methionine by cultivating genetically modified microorganisms having an increased or caused expression rate of at least one gene in comparison to the wild type, wherein
  • genes are selected from the group of nucleic acids encoding an aspartate kinase, nucleic acids encoding an aspartate semialdehyde dehydrogenase, nucleic acids encoding a homoserine dehydrogenase, nucleic acids encoding a glyceraldehyde-3-phosphate dehydrogenase, nucleic acids encoding a 3-phosphoglycerate kinase, encoding nucleic acids Pyruvate carboxylase, nucleic acids encoding a triosephosphate isomerase, nucleic acids encoding a homologous O-acetyltransferase, nucleic acids encoding a cystahionin gamma synthase, nucleic acids encoding a cystahionin beta-lyase, nucleic acids encoding a serine hydroxymethyltransferase, nucleic acids encoding an O
  • Cysteine synthase activity I nucleic acids encoding a cysteine synthase activity II, nucleic acids encoding a coenzyme B12-dependent methionine synthase activity, nucleic acids encoding a coenzyme B12-independent methionine synthase activity, nucleic acids encoding a sulfate adenylyltransferase activity, Nucleic acids encoding a phosphoadenosine phosphosulfate reductase activity, nucleic acids encoding a ferredoxin sulfite reductase activity, nucleic acids encoding a ferredoxin NADPH reductase activity, nucleic acids encoding a ferredoxin activity, nucleic acids encoding a protein of sulfate reduction RXA077, encoding nucleic acids a protein of sulfate reduction RXA248, nucleic acids encoding
  • dh1 introduces one or more expression units according to the invention, optionally with increased specific expression activity, into the genome of the microorganism, so that the expression of one or more of these endogenous genes under the control of the introduced expression units according to the invention, if appropriate with increased specific expression activity, done or
  • dh2 introduces one or more of these genes into the genome of the microorganism, so that the expression of one or more of the introduced genes takes place under the control of the endogenous expression units according to the invention, optionally with increased specific expression activity, or
  • nucleic acid constructs containing an expression unit according to the invention, optionally with increased specific expression activity, and functionally linked one or more nucleic acids to be expressed, into which microorganisms are introduced.
  • a further preferred embodiment of the method for the production of methionine described above is characterized in that the genetically modified in addition to the wild type, an increased activity, at least one of the activities selected from the group aspartate kinase activity, aspartate-semialdehyde dehydrogenase activity, homoserine dehydrogenase activity, glyceraldehyde-3-phosphate dehydrogenase activity, 3-phosphoglycerate kinase Activity, pyruvate carboxylase activity, triosephosphate isomerase activity, homosein O-acetyltransferase activity, cystahionine gamma synthase activity, cystahionine beta-lyase activity serine hydroxymethyltransferase activity, O-acetylhomoserine sulfhydrylase activity , Methylene tetrahydrofolate reductase activity, phosphoserine aminotransferase activity, phosphoser
  • a further particularly preferred embodiment of the method for producing methionine described above is characterized in that the genetically modified microorganisms in addition to the wild type in addition a reduced activity, at least one of the activities selected from the homoserine kinase activity, threonine Dehydratase activity, threonine synthase activity, meso-diaminopimelate D-dehydrogenase activity, phosphoenolpyruvate carboxykinase activity, pyruvate oxidase activity, dihydrodipicolinate synthase activity, dihydrodipicolinate reductase activity, and diaminopicolinate decarboxylase activity.
  • nucleic acid according to the invention having promoter activity and / or an expression unit according to the invention.
  • the invention further relates to a process for the production of threonine by cultivating genetically modified microorganisms having an increased or caused expression rate of at least one gene in comparison to the wild type, wherein
  • the specific expression activity in the microorganism of at least one endogenous expression unit according to the invention which regulates the expression of the endogenous genes is increased in comparison to the wild type or ie) the expression of genes in the microorganism by expression units according to the invention or by expression units according to the invention with increased specific expression activity according to embodiment ch), the genes being heterologous with respect to the expression units,
  • genes are selected from the group of nucleic acids encoding an aspartate kinase, nucleic acids encoding an aspartate semialdehyde dehydrogenase, nucleic acids encoding a GlycerinaIdehyd-3-phosphate dehydrogenase, nucleic acids encoding a 3-phosphoglycerate kinase, nucleic acids encoding a pyruvate carboxylase , Nucleic acids encoding a triosephosphate isomerase, nucleic acids encoding a homoserine kinase, nucleic acids encoding a threonine synthase, nucleic acids encoding a threonine exporter carrier, nucleic acids encoding a glucose-6-phosphate dehydrogenase, nucleic acids encoding a transaldolase, nucleic acids encoding a transketolase, Nucleic acids encoding a
  • Phosphogluconate dehydrogenase nucleic acids encoding a lysine exporter, nucleic acids encoding a biotin ligase, nucleic acids encoding a Phosphoe- nolpyruvat carboxylase, nucleic acids encoding a threonine efflux protein, nucleic acids encoding a fructose-1, 6-bisphosphatase, nucleic acids encoding an OpcA protein, nucleic acids encoding a 1-phosphofructokinase, nucleic acids encoding a 6-phosphofructokinase, and nucleic acids encoding a homoserine dehydrogenase
  • dh1 brings one or more expression units according to the invention, optionally with increased specific expression activity, into the genome of the microorganism such that the expression of one or more of these endogenous genes under the control of the introduced expression units according to the invention, if appropriate with increased specific expression activity, done or
  • dh2 introduces one or more of these genes into the genome of the microorganism, so that the expression of one or more of the introduced genes takes place under the control of the endogenous expression units according to the invention, optionally with increased specific expression activity, or ie3) one or more nucleic acid constructs containing an expression unit according to the invention, optionally with increased specific expression activity, and functionally linked one or more nucleic acids to be expressed, into which microorganisms are introduced.
  • a further preferred embodiment of the method for the production of threonine described above is characterized in that the genetically modified microorganisms in addition to the wild type additionally an increased activity, at least one of the activities selected from the group aspartate kinase activity, aspartate semialdehyde dehydrogenase activity , Glyceraldehyde-3-phosphate dehydrogenase activity, 3-phosphoglycerate kinase activity, pyruvate carboxylase activity, triosephosphate isomerase activity, threonine synthase activity, threonine export carrier activity, transaldolase activity, transketolase Activity, glucose-6-phosphate dehydrogenase activity, malate-quinone oxidoreductase activity, homoserine kinase activity, biotin ligase activity,
  • Phosphoenolpyruvate carboxylase activity threonine efflux protein activity, protein OpcA activity, 1-phosphofructokinase activity, 6-phosphofructokinase activity, fructose 1,6-phosphatase activity, 6-phosphogluconate dehydrogenase and homoserine dehydrogenase Activity.
  • a further particularly preferred embodiment of the above-described method for producing threonine is characterized in that the genetically modified microorganisms in addition to the wild type in addition a reduced activity, at least one of the activities selected from the group threonine dehydratase activity, homoserine O-acetyltransferase activity, serine
  • Hydroxymethyltransferase activity O-acetylhomoserine sulfhydrylase activity, meso-diaminopimelate D-dehydrogenase activity, phosphoenolpyruvate carboxykinase activity, pyruvate oxidase activity, dihydrodipicolinate synthase activity, dihydrodipolinate reductase activity, asparaginase activity, Aspartate decarboxylase activity, lysine exporter activity, acetolactate synthase activity, ketol-Aid reductoisomerase activity, branched chain aminotransferase activity, coenzyme B12-dependent methionine synthase activity, coenzyme B12-independent methion synthase activity having dihydroxy-acid dehydratase activity and diaminopicolinate decarboxylase activity.
  • a nucleic acid according to the invention having promoter activity and / or an expression unit according to the invention.
  • activity of a protein in enzymes means the enzyme activity of the corresponding protein, in other proteins, for example structure or transport proteins, the physiological activity of the proteins.
  • the enzymes are usually able to convert a substrate into a product or to catalyze this conversion step.
  • the "activity" of an enzyme means the amount of substrate or amount of product converted by the enzyme in a certain time.
  • the amount of substrate or the amount of product formed is thus increased by the enzyme compared to the wild type in a certain time.
  • this increase in the "activity" in all the activities described above and below is at least 5%, more preferably at least 20%, more preferably at least 50%, more preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the "wild-type activity".
  • the amount of substrate or the amount of product formed is thus reduced by the enzyme compared to the wild type in a certain time.
  • a reduced activity is preferably understood to mean the partial or substantially complete interruption or blocking of the functionality of this enzyme in a microorganism based on different cell biological mechanisms.
  • a reduction of the activity comprises a quantitative reduction of an enzyme up to an essentially complete absence of the enzyme (ie lacking detectability of the corresponding activity or lacking immunological detectability of the enzyme).
  • the activity in the microorganism is reduced by at least 5%, more preferably by at least 20%, more preferably by at least 50%, more preferably by 100%, compared to the wild type.
  • “reduction” also means the complete absence of the corresponding activity.
  • the activity of certain enzymes in genetically modified microorganisms and in the wild type and thus the increase or reduction of the enzyme activity can be determined by known methods, such as enzyme assays.
  • a pyruvate carboxylase is understood as meaning a protein which has the enzymatic activity of converting pyruvate into oxaloacetate.
  • a pyruvate carboxylase activity is understood as meaning the amount of pyruvate reacted or amount of oxaloacetate converted by the protein pyruvate carboxylase in a specific time.
  • the amount of pyruvate reacted or the amount of oxaloacetate formed is thus increased by the protein pyruvate carboxylase compared to the wild type in a specific time.
  • this increase in pyruvate carboxylase activity is at least 5%, more preferably at least 20%, more preferably at least 50%, even more preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, especially at least 600% of the pyruvate carboxylase. Activity of the wild type.
  • a phosphoenolpyruvate carboxykinase activity as the enzyme activity of a phosphoenolpyruvate carboxykinase.
  • a phosphoenolpyruvate carboxykinase is meant a protein having the enzymatic activity of converting oxaloacetate to phosphoenolpyruvate.
  • phosphoenolpyruvate carboxykinase activity is understood as meaning the amount of oxaloacetate or the amount of phosphoenolpyruvate reacted in a certain time by the protein phosphoenolpyruvate.
  • the amount of oxaloacetate or the amount of phosphoenolpyruvate formed is thus reduced in a certain time by the protein phosphoenolpyruvate carboxykinase in comparison to the wild type.
  • a reduction in phosphoenolpyruvate carboxykinase activity includes a quantitative reduction of a phosphoenolpyruvate carboxykinase to a substantially complete absence of the phosphoenolpyruvate carboxykinase (ie, lack of detectability of phosphoenolpyruvate carboxykinase activity or lack of immunological detectability of phosphoenolpyruvate carboxykinase).
  • the phosphoenolpyruvate carboxykinase activity is reduced by at least 5%, more preferably by at least 20%, more preferably by at least 50%, more preferably by 100% as compared to the wild-type.
  • “reduction” also means the complete absence of phosphoenolpyruvate carboxykinase activity.
  • the additional increase of activities can take place by different ways, for example by switching off inhibitory regulation mechanisms on expression and protein level or by increasing the gene expression of nucleic acids coding the proteins described above against the wild type.
  • the increase in gene expression of the nucleic acids encoding the above-described proteins relative to the wild type can also be effected by different routes, for example by inducing the gene by activators or as described above by increasing the promoter activity or increasing the expression activity or by introducing one or more gene copies into the microorganism.
  • the person skilled in the art can take further different measures individually or in combination.
  • the copy number of the respective genes can be increased, or the promoter and regulatory region or ribosome binding site located upstream of the structural gene can be mutated.
  • inducible promoters it is additionally possible to increase the expression in the course of fermentative production. Measures to extend the lifetime of mRNA also improve expression.
  • by preventing the degradation of the enzyme protein also enhances the enzyme activity.
  • the genes or gene constructs may either be present in different copy number plasmids or be integrated and amplified in the chromosome. Alternatively, overexpression of the genes in question can be achieved by changing the composition of the medium and culture.
  • biosynthetic products in particular L-lysine, L-methionine and L-threonine
  • L-lysine in particular L-lysine
  • L-methionine in particular L-methionine
  • L-threonine in particular L-threonine
  • the gene expression of a nucleic acid encoding one of the proteins described above is increased by incorporating at least one nucleic acid encoding a corresponding protein into the microorganism.
  • Introduction of the nucleic acid can be chromosomally or extrachromosomally, ie by increasing the copy number on the chromosome and / or a copy of the gene on a replicating plasmid in the host microorganism.
  • the introduction of the nucleic acid preferably takes place chromosomally, in particular by the SacB method described above.
  • any gene encoding one of the proteins described above can be used for this purpose.
  • genomic nucleic acid sequences from eukaryotic sources containing introns in the event that the host microorganism is unable or unable to express the corresponding proteins, preferably already processed nucleic acid sequences such as the corresponding cDNAs are available use.
  • the reduction of the above-described activities in microorganisms is carried out by at least one of the following processes:
  • Knockout mutants can preferably be generated by targeted insertion into the desired target gene by homologous recombination or introduction of sequence-specific nucleases against the target gene.
  • the step of cultivating the genetically modified microorganisms is preferably followed by isolating biosynthetic products from the microorganisms or from the fermentation broth. These steps may take place simultaneously and / or preferably after the culturing step.
  • the genetically modified microorganisms according to the invention can be used continuously or discontinuously in the batch process (batch culturing) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the production of biosynthetic products, in particular L-lysine, L-methionine and L-threonine, to be cultured.
  • biosynthetic products in particular L-lysine, L-methionine and L-threonine, to be cultured.
  • a summary of known cultivation methods can be found in the textbook by Chmiel (Bioreatechnik 1. Introduction to bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden) 1994)).
  • the culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are contained in the Manual of Methods for General Bacteriology of the American Society for Bacteriology (Washington D.C, USA, 1981).
  • These media which can be used according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.
  • Preferred carbon sources are sugars, such as mono-, di- or polysaccharides.
  • examples of very good sources of carbon are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
  • sugars such as mono-, di- or polysaccharides.
  • very good sources of carbon are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
  • complex compounds such as molasses, or other sugar refining by-products.
  • Other possible sources of carbon are oils and fats such.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds.
  • Exemplary nitrogen sources include ammonia gas or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soybean meal, soy protein, yeast extract, meat extract and others.
  • the nitrogen sources can be used singly or as a mixture.
  • Inorganic salt compounds which 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
  • sulfur source for the production of fine chemicals in particular of methionine
  • inorganic compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, but also organic sulfur compounds, such as mercaptans and thiols.
  • Phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.
  • 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 fermentation media used according to the invention 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 are often derived from complex drug components such as yeast extract, molasses, corn steep liquor, and the like.
  • suitable precursors can be added to the culture medium.
  • the exact composition of the media compounds will depend heavily on the particular experiment and will be decided on a case by case basis. Information about the media optimization is obtainable 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 Such as standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and der ⁇ same.
  • All media components are sterilized either by heat (20 min at 1, 5 bar and 121 0 C) or by sterile filtration.
  • the components can either be sterilized together or, if necessary, sterilized separately. All media components may be present at the beginning of the culture or optionally added continuously or in batches.
  • the temperature of the culture is normally between 15 0 C and 45 0 C, preferably at 25 ° C to 40 0 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.
  • the pH for cultivation can be controlled during cultivation by addition of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid.
  • foam anti-foaming agents such as.
  • As fatty acid polyglycol are used.
  • suitable selective substances such as, for example, can be added to the medium.
  • antibiotics are added.
  • oxygen or oxygen-containing gas mixtures such as B. ambient air
  • the temperature of the culture is normally 2O 0 C to 45 0 C.
  • the culture is continued until a maximum of the desired product has formed. This goal is usually reached within 10 hours to 160 hours.
  • the fermentation broths thus obtained usually have a dry matter content of 7.5 to 25% by weight.
  • the fermentation is driven sugar-limited at least at the end, but in particular over at least 30% of the fermentation time. This means that during this time the concentration of utilizable sugar in the fermentation medium is maintained at 0 to 3 g / l, or lowered.
  • biosynthetic products from the fermentation broth and / or the microorganisms takes place in a manner known per se in accordance with the physicochemical properties of the biosynthetic desired product and the biosynthetic by-products.
  • the fermentation broth can then be further processed, for example.
  • the biomass may be completely or partially separated by separation methods. the, such. As centrifugation, filtration, decantation or a combination of these methods from the fermentation broth or be completely left in it.
  • the fermentation broth with known methods, such as. B. with the aid of a rotary evaporator, thin film evaporator, falling film evaporator, by reverse osmosis, or by nanofiltration, thickened or aufkon ⁇ centered.
  • This concentrated fermentation broth can then be worked up by freeze drying, spray drying, spray granulation or by other methods.
  • the product-containing broth is subjected to chromatography with a suitable resin, the desired product or impurities being wholly or partially retained on the chromatography resin. If necessary, these chromatographic steps can be repeated using the same or different chromatography resins.
  • the person skilled in the art is familiar with the choice of suitable chromatography resins and their most effective use.
  • the purified product may be concentrated by filtration or ultrafiltration and stored at a temperature at which the stability of the product is maximized.
  • biosynthetic products can be obtained in different forms, for example in the form of their salts or esters.
  • the identity and purity of the isolated compound (s) can be determined by techniques of the prior art. These include high performance liquid chromatography (HPLC), spectroscopic methods, staining procedures, thin layer chromatography, NIRS, enzyme assay or microbiological assays. These analytical 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. 540-547, pp.
  • the shuttle vector pMT1 (Follettie et al., (1993) J. Bacteriol 175: 4096-4103) was digested with the restriction enzymes Xhol and BamHI, then treated with the Klenow fragments and religated. The resulting plasmid was designated pMT1 -del.
  • the vector pMT1-del was digested with the restriction enzymes Bgl II and XbaI.
  • the 2.5 kb fragment contains the pSR1 ori from Corynebacterium glutamicum and was introduced into the 2 kb Plasperson pTnMod-Okm also cut with BglII and XbaI (Dennis and Zylstra (1998) Appl. Environ.
  • the vector thus obtained was designated pSK1.
  • the fragment of the plasposon pTnMod-Okm carries the pMB1 replication origin for Escherichia coli and a kanamycin resistance marker (Tn903).
  • the cat gene without promoter was identified by means of oligonucleotide primer A (SEQ ID NO: 4) and B (SEQ ID NO: 5) with the vector pKK232-8 (SEQ ID NO: 3) as template the polymerase chain reaction (PCR) by standard methods, as described in Innis et al. (1990) PCR Protocol, A Guide to Methods and Applications, Academic Press.
  • This PCR product was ligated into the vector pSK1 after vector and insert were digested with the restriction enzymes BglII and KpnI.
  • the plasmid was named pSKICat ( Figure 1).
  • Oligonucleotide primer A SEQ. ID. NO. 4 ⁇ '-GGAAGATCTTTCAAGAATTCCCAGGCA-S 'oligonucleotide primer B SEQ. ID. NO. 5 ⁇ '-GGGGTACCTACCGTATCTGTGGGGGG-S '
  • the plasmid pKK223-3 SEQ. ID. NO. 6 contains the tac promoter (P tac ). This promoter was isolated by digestion with the restriction enzyme BamHI and the fragment was cloned in the BamHI linearized vector pSKICat SEQ ID. The plasmid was named pSK1 Ptao ( Figure 2).
  • the chromosomal DNA of Corynebacterium glutamicum ASOI 9E12 was isolated from cells of the late exponential phase by the method of Eikmanns et al. (1994) Microbiology 140: 1817-1828 and then partially digested with the restriction enzyme Sau3AI. The resulting fragments of 0.4-1.0 kb in size were ligated into the vector pSKICat linearized with the restriction enzyme BamHI. The ligation mixture was prepared by electroporation by the method of Follettie et al. (1993) J. Bacteriol. 175: 4096-4103 trans ⁇ formed in Corynebacterium glutamicum AS019E12.
  • the cells were spread on plates containing 5 .mu.g / ml chloramphenicol. Plasmids from single colonies growing on these plates were isolated and analyzed.
  • One such plasmid was pSKI Cat P 1-35 containing the promoter P 1-35 (SEQ ID NO: 1). This promoter is located in the upstream region of the gene encoding an ABC transporter.
  • the insert has a size of 280 bp.
  • Corynebacterium glutamicum cells containing only the plasmid pSK1 Cat are not capable of MB (Follettie et al. (1993) J. Bacteriol. 175: 4096-4103) and MCGC plates (available from the East et al (1989) Biotechnol Lett 11:... to grow 11-16) with a Chloramphenicolkonzentraion of 5 // g / ml at 3O 0 C. The cat gene is not expressed.
  • Corynebacterium glutamicum cells containing the plasmid pSK1 CatP tac Figure 2 grow on MB and MCGC plates with a chloramphenicol concentration of 40 // g / ml.
  • the CAT activities of Corynebacterium glutamicum AS019E12 were determined to determine a relative potency of promoter P 1-35 (SEQ ID NO: 1). For this raw extracts were by the method of Jetten and Sinsky (1993) FEMS Microbiol. Lett. 111: 183-188.
  • the activity of chloramphenicol acetyltransferase (CAT) was determined by the method of Shaw et al (1993) Methods Enzymol. 43: 737-755 certainly.
  • the reaction contained 100 mM Tris * HCl pH 7.5, 1 mM DTNB, 0.1 mM acetyl CoA, 0.25 mM chloramphenicol, and a suitable amount of enzyme. The changes in the optical density at a wavelength of 412 nm were measured. Protein concentration was determined by the Bradford method (1976) Anal. Biochem. 72: 248-254 analyzed.
  • Figure 1 shows a plasmid map of pSK1 Cat (A) and the nucleotide sequence of the BamHI cloning site (B).
  • the underlined sequences in B give the regions which were used for the preparation of sequencing oligonucleotides.
  • the start codon and the BamHI cloning site are indicated.
  • FIG. 2 shows a part of the nucleotide sequence of pSK1 P 130 .
  • the promoter P t30 is shown in italics. Furthermore, the -35 and -10 regions, the RBS and the start code of the cat gene are identified.

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Abstract

La présente invention concerne l'utilisation de séquences d'acides nucléiques pour réguler la transcription et l'expression de gènes, les nouveaux promoteurs et unités d'expression eux-mêmes, des procédés pour modifier ou induire le taux de transcription et/ou le taux d'expression de gènes, des cassettes d'expression contenant lesdites unités d'expression, des micro-organismes génétiquement modifiés présentant un taux de transcription et/ou un taux d'expression modifié ou induit, ainsi que des procédés permettant d'obtenir des produits biosynthétiques par culture des micro-organismes génétiquement modifiés.
EP05762080A 2004-07-20 2005-07-16 Unites d'expression de p1-35 Withdrawn EP1771547A1 (fr)

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DE102004035067A DE102004035067A1 (de) 2004-07-20 2004-07-20 P1-35-Expressionseinheiten
PCT/EP2005/007758 WO2006008103A1 (fr) 2004-07-20 2005-07-16 Unites d'expression de p1-35

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