Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • 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

Definitions

• the present invention relates to multiple promoters and expression units containing them; their use for the regulation of transcription and expression of genes; Expression cassettes comprising such multiple promoters or expression units; Vectors containing such expression cassettes; genetically modified microorganisms containing such vectors and / or expression units; and methods for producing biosynthetic products by culturing the genetically modified microorganisms.
• biosynthetic products are made through natural metabolic processes in cells and are used in many industries, including the food, feed, cosmetics, feed, food and pharmaceutical industries.
• These substances collectively referred to as fine chemicals / proteins, include, but are not limited to, organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatics, vitamins and cofactors, as well as proteins and enzymes .
• Their production is most conveniently made on a large scale by growing bacterial strains or other microorganisms 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.
• amino acids can be produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of the great importance is constantly working to improve the manufacturing process. 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. For several years, methods of recombinant DNA technology have also been used for strain improvement of fine chemical / protein-producing strains of Corynebacterium by amplifying individual genes and examining the effect on the production of fine chemicals / proteins.
• RNA polymerase holoenzymes also called -35 and -10 regions
• ribosomal 16S RNA also ribosomal binding site (RBS) or Shine-Dalgamo. Called sequence.
• Nucleic acid sequences with promoter activity can affect the production of mRNA in different ways. Promoters whose activity is independent of the physiological growth phase of the organism are called constitutive. In turn, other promoters respond to external chemical and physical stimuli such as oxygen, metabolites, heat, pH, etc. Still others show a strong dependence of their activity at different stages of growth. For example, in the literature promoters are described 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 impact on productivity for production of fine chemicals and proteins, depending on the metabolic pathway.
• 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.
• the presence of a particular factor, known as an inducer can 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 inductors, 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-A-44 40 118. This promoter was preceded by a structural gene coding for a protein. After transformation of such a construct into a coryneform bacterium, the expression of the downstream of the promoter 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 regulatory nucleic acid sequences, in particular promoter constructs and / or expression units with advantageous properties, for example increased or altered transcription activity and / or translational activity compared to the starting promoter.
• n is an integer from 0 to 10;
• a x and A y are the same or different and represent a chemical bond or a linker nucleic acid sequence;
• Pi, P x and Py encode the same or different promoter sequences which comprise at least one RNA polymerase-binding region, such as the core region; and at least P y comprises a 3'-terminal sequence portion mediating ribosome binding.
• a ribosome binding-mediating sequence section may independently of one another also have P 1 and single or all P x .
• P 1 , P x and P y are derived from the same or different eukaryotic or, in particular, prokaryotic organisms. Also artificial promoters are useful.
• P 1 , P x and P y are each derived from a contiguous sequence of 35 to 500 nucleotide residues, preferably 35 to 300 nucleotide residues, more preferably 35 to 210 nucleotide residues, and most particularly preferably 35 to 100 nucleotide residues, which in the genome of the organism 5'-upstream of the coding sequence for a protein, for example, for a participating in a biosynthesis pathway of the organism protein.
• P 1 , P x and P y of the expression unit are derived from a coryneform bacterium.
• P 1 , P x and P y of the expression unit are independently selected from promoter sequences for the coding sequence of a high abundance protein in a eukaryotic or prokaryotic organism, especially in an origenic bacterium, such as in one of the genus Corynebacterium or Brevibacterium such as a species or a strain as set out in Table 3.
• promoter sequences may be selected from promoter sequences for the coding sequence of a protein listed in Table 1 and involved in the amino acid biosynthesis of an organism.
• at least one of the promoters of the expression unit according to the invention should have high abundance as defined herein.
• P 1 , P x and P y of the expression unit are independently selected from the nucleotide sequences shown in FIG. 1, or, as explained in more detail later, derived from SEQ ID NO: 1 (pGRO), SEQ ID NO: 2 (pEFTs), SEQ ID NO: 3 (pEFTu) and SEQ ID NO: 4 (pSOD).
• P 1 , P x and P y of the expression unit are independently selected from strong, constitutive or regulatable promoters.
• the present invention relates to an expression cassette comprising, in the 5'-3 'direction, a sequence module of the following general formula II:
• G is at least one coding nucleic acid sequence which is operatively linked to the 5 'upstream regulatory sequence.
• G is selected from a) nucleic acids encoding a protein from the biosynthetic pathway of proteinogenic and non-proteinogenic amino acids, b) nucleic acids encoding a protein from the biosynthetic pathway of nucleotides and nucleosides, c) nucleic acids encoding a protein from the biosynthetic pathway d) nucleic acids encoding a protein from the biosynthetic pathway of lipids and fatty acids, e) nucleic acids encoding a protein from the biosynthetic pathway of diols, f) nucleic acids encoding a protein from the biosynthetic pathway of carbohydrates, g) nucleic acids encoding a protein from the biosynthetic pathway of carbohydrates, g) nucleic acids encoding a protein from the biosynthetic pathway of carbohydrates, g) nu
• G is selected from nucleic acids for proteins from the biosynthesis pathway of amino acids selected from aspartate kinase, aspartate-semialdehyde dehydrogenase, diamino-imidate 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, cystathionine beta-lyase, serine hydroxymethyltransferase, O-acetylmoserine sulfhydrylase, methylene tetrahydrofolate reductase, phosphoserine aminotransferase, phosphoserine phosphatase, serine Acetyltransferase, homoserine dehydrogenase, homoserine kinase, threon
• the present invention relates to a vector comprising at least one of the above-mentioned expression cassettes.
• the present invention relates to a genetically modified microorganism transformed with at least one of the aforementioned vectors, or containing at least one of the above expression cassettes, preferably in integrated form.
• the genetically modified organism is derived from coryneform bacteria.
• the genetically modified organism is derived from bacteria of the genus Corynebacterium or Brevibacterium.
• the present invention relates to a process for the preparation of a biosynthetic product, wherein one of the above-mentioned genetically modified microorganisms is cultivated and the desired product is isolated from the culture.
• the biosynthetic product is selected from organic acids, proteins, nucleotides and nucleosides, both proteinogenic and non-proteinogenic amino acids, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors, enzymes and proteins.
• Very particularly preferred biosynthesis products are lysine, methionine, threonine and trehalose.
• the present invention relates to the use of an expression unit according to the invention for regulating a product biosynthesis.
• Figure 1 shows specific sequences for four promoters which were used to prepare the multiple promoters described in the embodiments. Shown are the promoter sequences for pEFTu ( Figure 1A); for promoter pGRO (FIG. 1B) 1 for promoter pEFTs (FIG. 1C) and for promoter pSOD (FIG. 1D). For each promoter, two sequences are indicated, the longer (upper sequence) indicates in each case the complete promoter sequence including Ribosomeitatisstelle (RBS), in bold and in italics printed. In the case of a 3'-terminal arrangement of the respective promoter in the multiple promoter construct according to the invention, the longer nucleotide sequence (including RBS) is preferably used in each case.
• RBS Ribosomeitatisstelle
• Promoter sequences 5'-upstream to the 3'-terminal promoter unit do not comprise RBS and were used in the respectively second position, truncated nucleic acid sequence (without RBS). 3'-terminal partial sequences of the shorter promoter sequences given in capital letters and bold letters may additionally be missing. Potential "- 10 regions" are underlined.
• a “biosynthetic product” in the sense of the invention is understood to mean those which are produced in cells by way of natural metabolic processes (biosynthetic pathways) and are used in a variety of applications, in particular in the food, feed, cosmetics, feed, food and pharmaceutical industries.
• These substances collectively referred to as fine chemicals / proteins, include, but are not limited to, organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatics, vitamins and cofactors, as well as proteins and enzymes ,
• a “promoter”, a “nucleic acid with promoter activity” or a “promoter sequence” is understood according to the invention to mean a nucleic acid which, in functional linkage with a nucleic acid to be transcribed, regulates the transcription of this nucleic acid.
• promoter Unless expressly referred to "single promoters", the term “promoter” according to the invention also includes the sequential, depending on the context Arrangement of more than one nucleic acid sequence (ie “multiple promoters”), each of which would be capable of regulating their transcription when operatively linked to a nucleic acid to be transcribed.
• single promoter refers to a single nucleic acid sequence capable of regulating the transcription of a nucleic acid to be transcribed which, when operatively linked to a nucleic acid sequence to be transcribed, can transcribe it.
• multiple promoters at least two identical or different nucleic acid sequences, each of which, when operatively linked to a nucleic acid to be transcribed, would be capable of regulating its transcription (individual promoters), are sequentially linked such that a transcription start of optionally one of the nucleic acid sequences.
• a linker which has, for example, one or more restriction sites.
• two individual promoters can also be linked directly to one another a ribosome binding site (RBS), in particular in the region of the 3'-terminal end of the promoter.
• RBS ribosome binding site
• Promoter sequences for the coding sequence of a "high A-bound" protein are understood to mean in particular "strong promoters". If one wishes to use multiple promoters to amplify genes, preferably those strong promoters are combined. Strong promoters regulate the transcription of genes such that the frequency of readings by the RNA polymerase is greater than in the majority of genes of the organism. In most cases, the presence of a strong promoter results in the presence of a large amount of transcript in the cell.
• transcript When the transcript is larger in percentage relative to the other cellular transcripts, it is called an "abundant transcript.” In bacteria, the amount of transcript and the corresponding protein encoded in it usually correlate, ie, "abundant transcripts" usually also result. The following is an example of a method that allows the identification of "strong promoters" on the Abun- danz of proteins. Details on the methodological procedure as well as other references can be found eg in Proteomics in Practice - A Laboratory Manual of Proteome Analysis (authors: R. Westermeier, T. Naven, publisher: Wiley-VCH Verlag GmbH, 2002). The bacterial cells are disrupted and the proteins of the cell extract are separated by means of 2D gel electrophoresis.
• the proteins stained by common methods such as Coomassie, silver or fluorescent dye staining. In this case, over-coloring of the proteins should be avoided in order to enable later quantification of the individual protein spots.
• the gel is scanned and the image obtained is evaluated using a suitable software (eg Melanie, Amersham Biosciences).
• a suitable software eg Melanie, Amersham Biosciences.
• all detectable spots are identified and the spot volume determined. The sum of all spot volumes corresponds in the first approximation to the total protein.
• the spots with particularly large spot volumes can then be selected. For example, spots with volumes occupying more than 0.1% of the total pot volume can be said to be abundant. Subsequently, spots with particularly abundant proteins are knocked out of the gel.
• the protein present in the gel fragment is then digested proteolytically (for example with trypsin) and the molar mass of the resulting peptides is determined, for example with the aid of MALDI-ToF MS (matrix assisted laser desorption ionization time of flight mass spectrometry).
• MALDI-ToF MS matrix assisted laser desorption ionization time of flight mass spectrometry
• the promoters regulating this gene are usually located directly upstream of the start codon. Therefore, even the "strong” promoters are often located in a range of about 200 nucleotides upstream of the start codon of "abundant" proteins.
• Articles within the meaning of the invention include in particular those sequences which have no transcriptional activity in situ, are transcriptionally active in another sequence context, in particular in the context of an expression unit or expression cassette according to the invention.
• promoter activity is understood to mean the amount of RNA, that is to say the transcription rate, formed by the promoter in a specific time.
• promoter activity is understood as meaning the amount of RNA per promoter formed in a specific time by the promoter.
• RNA formed is thus changed in a certain time compared with the wild type.
• a "functional” or “operative” 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, such as nucleic acid sequences, which facilitate the transcription of Ensuring nucleic acids, as well as, for example, a 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 enhancer sequences, may also exert their function on the target sequence from more distant locations or even from other DNA molecules.
• the nucleic acid sequence to be transcribed is positioned behind (i.e., at the 3 'end) of the promoter sequence of the invention so that both sequences are covalently linked together.
• the distance between the promoter sequence and the nucleic acid sequence to be expressed transgenically is preferably less than 200 base pairs, more preferably less than 100 base pairs, very particularly preferably less than 50 base pairs.
• ribosomal binding site or “ribosome binding site” (RBS) (or Shine-Dalgamo sequence) is understood to mean A / G-rich polynucleotide sequences located up to 30 bases upstream of the initiation codon of translation.
• transcription refers to the process by which a complementary RNA molecule is produced starting from a DNA template, involving proteins such as RNA polymerase, so-called sigma factors and transcriptional regulator proteins synthesized RNA then serves as a template in the process of translation, which then leads to the biosynthetically active protein.
• a “core region” of a promoter is understood according to the invention to mean a contiguous nucleic acid sequence of about 20 to 80, or 30 to 60 nucleotides, which comprises at least one potential so-called "-10-region". Examples of potential -10 regions are described for individual preferred promoter sequences herein; compare in particular FIG. 1. "-10 regions" are also referred to as TATA boxes or Pribnow-Schaller sequences. te at least one of these potential -10 regions, such. B. 1, 2, 3, 4 or 5, included.
• the core region is 5'-upstream of the RBS and may be 1 to 200 or 10 to 150 or 20 to 100 nucleotide residues away from it.
• an "expression unit” is understood as meaning a nucleic acid with expression activity which comprises a multiple promoter, as defined herein, and after functional linkage with a nucleic acid or gene to be expressed which regulates expression, ie transcription and translation, of this nucleic acid or gene
• a nucleic acid with expression activity which comprises a multiple promoter, as defined herein, and after functional linkage with a nucleic acid or gene to be expressed which regulates expression, ie transcription and translation, of this nucleic acid or gene
• regulatory elements e.g. Enhancers
• an "expression cassette” is understood as meaning an expression unit which is functionally linked to the nucleic acid or gene to be expressed
• an expression cassette thus comprises not only nucleic acid sequences which regulate transcription and translation, but also the nucleic acid sequences. which are to be expressed as protein as a consequence of transcription and translation.
• expression activity is understood as meaning the amount of protein formed in a certain time by the expression cassette or its expression unit, ie the expression rate.
• specific expression activity is understood as meaning the amount of protein per expression unit formed by the expression cassette or its expression unit in a specific time.
• an "altered expression activity” or expression rate with respect to a gene compared to the wild type the amount of protein formed is thus changed in a specific time compared to the wild type.
• “Altered” is understood in this connection to be preferably increased or decreased. This can be done, for example, by increasing or reducing the specific activity of the endogenous expression unit, for example by mutation, or by stimulation or inhibition.
• the altered expression activity or rate of expression for example, by regulation of the expression of genes in the microorganism can be achieved by expression units according to the invention, wherein the genes are heterologous with respect to the expression units.
• the "rate of formation" which produces a biosynthetically active protein 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 / fine chemicals in a microorganism.
• wild type is understood according to the invention as the corresponding starting microorganism and does not necessarily correspond to a naturally occurring organism.
• microorganism may be understood to mean 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 alter or cause the expression activity or expression rate, and to increase the
• this "reference organism” is Corynebacterium glutamicum ATCC 13032 or a microorganism derived by targeted or non-directional mutation of ATCC 13032.
• starting microorganisms are used which are already able to produce the desired product (fine chemical / protein).
• a "derived" sequence eg a derived promoter sequence, according to the invention, unless otherwise stated, is meant a sequence which has an identity of at least 80% or at least 90%, in particular 91%, 92%, 93% with the starting sequence. , 94%, 95%, 96%, 97%, 98% and 99%.
• Identity between two nucleic acids is understood to mean the identity of the nucleotides over the entire nucleic acid length, in particular the identity which is determined by comparison with the Vector NTI Suite 7.1 software from Informax (USA) using the Clustal method (Higgins DG, Sharp Computing Appl. Biosci 1989 Apr; 5 (2): 151-1) is calculated by setting the following parameters: Multiple alignment parameters:
• Pairwise alignment parameter FAST algorithm on K-tuple size 1 Gap penalty 3 Window size 5 Number of best diagonals 5
• 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 takes place under stringent conditions
• stringent conditions are described, for example, in Sambrook, J., Fritsch, EF, Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd edition, CoId 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.
• Stringent hybridization conditions are understood in particular to mean:
• nucleic acid sequences having promoter activity are understood as meaning fragments having substantially the same or altered, lower or higher specific promoter activity as those Starting sequence.
• substantially the same is meant a specific promoter activity that has at least 50%, such as at least 60%, 70%, 80%, 90%, or at least 95% of the specific promoter activity of the starting sequence.
• the present invention relates, inter alia, to the provision of a multiple-promoter-containing expression unit comprising, in the 5'-3 'direction, a sequence module of the following general formula (I):
• n is an integer value of 0 to 10, such as.
• B is 1, 2, 3, 4, 5 or 6;
• a x and Ay are identical or different and represent a chemical, in particular covalent, bond or a chemically, in particular covalently bound, linker nucleic acid sequence;
• P 1 , P x and Py encode the same or different promoter sequences comprising at least one RNA polymerase-binding region, such as a core region; and at least, such as only, P y comprises a 3'-terminal sequence portion mediating ribosome binding.
• the promoter sequences P 1 , P x and P y may be derived from genes from organisms encoding proteins involved in a biosynthetic pathway of the organism.
• the promoter sequences are in the genome of the organism 20 to 500 nucleotide residues, or 20 to 300 nucleotide residues, for example 20 to 210 nucleotide residues and in particular 20 to 100 or 35 to 100 nucleotide residues 5'-upstream of the coding sequence of the respective protein.
• promoter sequences P 1 , P x and P y are the genes listed in Table 1 below.
• Nonlimiting examples of specific promoter sequences are derived from the nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and Figure 1, but are not limited thereto.
• SEQ ID NO: 1 corresponds to the sequence located upstream of the coding region of the GroES chaperonin (pGRO), SEQ ID NO: 2 of the sequence located upstream of the coding region of the protein elongation factor TS (pEFTs), SEQ ID NO.3 of the sequence upstream of the protein elongation factor TU coding region (pEFTu) and SEQ ID NO: 4 of the sequence upstream of the superoxide dismutase coding region (pSOD), each from Corynebacterium glutamicum , SEQ. ID. NO. 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 correspond to the wild-type promoter sequences.
• a "derived" sequence according to the invention is understood as meaning a sequence which has an identity of at least 80% or at least 90% with the starting sequence, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% % and in particular 99%
• This degree of identity applies in particular to the above-defined "core region" of the respective promoter. Outside the respective core region, the degree of identity may be lower, and in the range of 0 to 80%, such as 0 to 80%. 10 to 80%, 20 to 70%, 30 to 60% or 40 to 50%.
• Useful core regions are e.g. for pGRO in the range of positions 50 to 80 of SEQ ID NO: 1; for pEFTs ranging from position 130 to 170 of SEQ ID NO: 2; For pEFTu in the range of positions 30 to 110 of SEQ ID NO: 3; for pSOD in the range of positions 50 to 100 of SEQ ID NO: 4.
• the expression units according to the invention contain in particular two or more nucleic acid sequences with promoter activity, a) preferably derived from identical or different sequences, selected from SEQ. ID. NO. 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; or other promoter sequences for genes of comparable or higher abundance; b) optionally one or more of these sequences representing a sequence derived by substitution, insertion, inversion or deletion or addition of nucleotides, in particular in the core region, having an identity of at least 80% at the nucleic acid level, preferably under the sequence SEQ ID NO: 1, C) or where optionally one or more of these nucleic acid sequences hybridizes with a sequence under stringent conditions which corresponds to one of the nucleic acid sequences according to SEQ ID NO: 1, 2, 3 or 4 or other promoter sequences for genes with comparable or higher abundance 2, 3 or 4 and or to other promoter sequences for genes of comparable or higher abundance is complementary, d) or optionally one or more of these nucle
• promoters which are useful as a constituent of a multiple promoter according to the invention can be prepared, for example, from various organisms whose genomic sequence is known by identity comparisons of the nucleic acid sequences from databases with the sequences SEQ ID NO: 1, 2, 3 or 4 described above or easily find in accordance with Figure 1 or other promoter sequences for genes with comparable or higher abundance.
• suitable natural promoters can be prepared starting from the above-described nucleic acid sequences, in particular starting from sequence SEQ ID NO: 1, 2, 3 or 4 and / or promoter sequences for genes with comparable or higher abundance from different organisms whose genomic sequence is not known. easily find by using hybridization techniques in a manner known per se.
• the invention therefore also includes the combination of such promoters, which are not listed here by name. This also applies to the combination of already known single promoters, as e.g. in (Patek et al (2003), J of Biotechnology 104, 311-323, Vasicova et al (1999) J. Bacteriol 181, 6188-6191).
• a further subject of the invention therefore relates to nucleic acids with promoter activity for incorporation into a multiple-promoter-containing expression unit according to the invention, wherein the nucleic acids with promoter activity comprise a nucleic acid sequence which corresponds to the nucleic acid sequence SEQ ID NO: 1, 2, 3 or 4 or promoter sequences for Genes with comparable or higher abundance hybridized under stringent conditions.
• This nucleic acid sequence comprises at least 10, preferably more than 12, 15, 30, 50 or more than 150 nucleotides.
• Artificial promoter sequences for incorporation into a multiple engine-containing expression unit according to the invention can be obtained starting from the sequence SEQ ID NO: 1, 2, 3 or 4 or according to FIG.
• Suitable ribosome binding mediating 3'-terminal sequence sections of a promoter sequence P y are, for example, the RBS of pGRO: GGAGGGA; the RBS of pEFTs: AGGAGGA; the RBS of pEFTu: AGGAGGA; as well as the RBS of pSOD: GGAGGGA (see also accompanying Figure 1).
• the theoretically optimal RBS ie the sequence 100% complementary to the anti-Shine-Dalgarno sequence on the 16S rRNA of C. glutamicum is: 5 'GAAAGGAGG 3'.
• Further suitable RBS sequence segments can be prepared, for example, by artificial variation and mutation, for example by substitution, insertion, inversion, addition or deletion of nucleotides, or can easily be found in databases via homology comparisons with promoter sequences.
• Nonlimiting examples of multiple promoter-containing expression units of the invention are selected from: e) sequences encoding PeftuPsod from position 18 to 390 in SEQ ID NO: 45;
• PgroPsodPefts from position 11 to 538 in SEQ ID NO: 59;
• PeftuPsod Pefts from position 11 to 559 in SEQ ID NO: 60; or f) sequences derived according to e) by substitution, insertion, inversion, addition or deletion of nucleotides, which has an identity of at least 80% or at least 90% at nucleic acid level in comparison with this starting sequence; or g) nucleic acid sequences which hybridize with a sequence to be e) complementary under stringent conditions; or h) "functionally equivalent fragments" of the abovementioned sequences e), f) and g).
• a "functionally equivalent fragment", in particular according to embodiments d) and h), expression units, fragments are understood to have 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 understood as meaning partial sequences of the sequences described by embodiments a) to h). 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 starting sequence.
• the expression units of the invention may preferably comprise one or more of the following genetic elements: a -10 region; a transcription start, an enhancer region; and an operator region.
• these genetic elements are specific for the species Corynebacterium, especially for Corynebacterium glutamicum.
• Bacterial promoters usually consist of three RNA polymerase recognition sites, namely the -10 region, the -35 region and the UP element.
• TGNGNTA C / T AATGG or GNTANAATNG are described hereinbefore (Patek et al (2003), J. of Biotechnology 104, 311-323, Vasicova et al.
• the invention further relates to the multiple promoter-containing expression units of the invention, operably linked to a translatable nucleic acid sequence.
• These constructs capable of expressing genes are also referred to as expression cassettes in the context of the invention.
• a "functional linkage” is understood as meaning, for example, the sequential arrangement of one of the expression units according to the invention and a nucleic acid sequence to be expressed transgenically and, if appropriate, further regulatory elements, such as a terminator, such that each of the regulatory elements has a function
• Genetic control sequences such as enhancer sequences, can also exert their function on more distant sites or even on other DNA molecules on the target sequence Arrangements are preferred 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 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.
• the expression cassettes according to the invention containing multiple promoters offer the possibility of achieving a different expression activity than the original expression activity and thus of fine-tuning the expression of the desired gene.
• the regulation of the expression of genes in the microorganism by expression units of the invention is achieved by
• nucleic acid constructs containing an expression unit according to the invention optionally with altered specific expression activity, and functionally linked to one or more nucleic acids to be expressed, ie introduces an expression cassette in the microorganism.
• 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 allows 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.
• nucleic acids with promoter activity and expression units and expression cassettes can furthermore be produced 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, p. 896-897).
• the attachment of synthetic OM gonucleotides 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, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or free from chemical precursors or other chemicals if it is synthesized chemically.
• the invention further comprises the nucleic acid molecules complementary to the specifically described nucleotide sequences or a portion thereof.
• the nucleotide sequences of the invention also allow the generation of probes and primers useful 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 stringent conditions, is at least about 12, preferably at least about 25, e.g. about 40, 50 or 75 consecutive nucleotides of a sense strand of a nucleic acid sequence of the invention or a corresponding antisense strand hybridizes.
• 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
• the invention further relates to expression vectors containing an expression cassette according to the invention described above.
• Vectors are well known to those skilled in the art and may be inferred, for example, from "Cloning Vectors" (Pouwels PH 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.
• Particularly suitable plasmids are those which are replicated in coryneform bacteria.
• Numerous known plasmid vectors such as PZKE (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExi (Eikmanns et al., Gene 102: 93-98 (1991)) or pHS2-1 (Sonnen et al. Gene 107: 69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1.
• Other plasmid vectors such as.
• PCLiK5MCS see Example 1
• those based on pCG4 US-A 4,489,160
• pNG2 Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)
• pAG1 US-A 5,158,891
• 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 duplication or amplification of the hom-thrB operon.
• the complete gene is cloned into a plasmid vector which 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)), Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173: 4510-4516) or pBGS ⁇ (Spratt et al., 1986, Gene 41: 337-342) , The plasmid vector containing the gene to be amplified is then transformed into the desired strain of C. glutamicum by transformation.
• the change or causation of the transcription rate and / or the translation rate of genes in microorganisms in comparison to the wild type can be carried out by transcribing genes in the microorganism by means of the invention Regulates expression units, wherein the genes are heterologous with respect to the expression units.
• nucleic acids according to the invention with promoter activity in the genome of the microorganism, so that the transcription of one or more endogenous genes under the control of the introduced nucleic acid with promoter activity, optionally with altered specific promoter actor activity occurs, or
• the insertion can be such that the gene or genes integrated into coding or non-coding regions will be.
• the insertion takes place in non-coding regions.
• the insertion of nucleic acid constructs can be carried out chromosomally or extrachromosomally.
• the insertion of the nucleic acid constructs is chromosomal.
• a "chromosomal" integration is the insertion of a DNA fragment into the chromosome of a host cell.
• endogenous is meant genetic information, such as genes, already included in the wild-type genome (as defined above).
• exogenous is meant genetic information such as genes that are not included in the wild-type genome. If exogenous genetic information, such as the multiple promoter-containing expression units of the present invention, is introduced into the genome of a wild-type strain, thereby producing a genetically altered strain this genetic information is endogenous in comparison to the first generated genetic strain with its offspring, but exogenous compared to the original wild-type strain which did not contain this genetic information.
• genes with regard to regulation of transcription by the nucleic acids according to the invention with promoter activity are preferably nucleic acids. understood that a region to be transcribed, so for example, a region which regulates the translation, a coding region, and optionally further regulatory elements, such as a terminator included.
• genes with regard to the regulation of the expression 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 in reference to nucleic acids with promoter activity and genes is meant that the genes used in the wild type are not transcribed under regulation of the nucleic acids according to the invention having promoter activity, but that a new, non-wild-type functional linkage is formed and the functional combination from nucleic acid according to the invention 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 the regulation of expression units of the invention, but a new, not occurring in the wild type functional linkage and the functional combination of inventive expression unit and specific gene does not occur in wild type.
• the genes are selected from the group nucleic acids encoding a protein from the biosynthetic pathway of fine chemicals, which genes may optionally contain further regulatory elements.
• the genes are selected from:
• 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, encoding nucleic acids tein 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 biosynthesis pathway of vitamins, Nucleic acids encoding a protein from the biosynthetic pathway of cofactors and nucleic acids encoding a protein from the biosynthetic pathway of enzymes, nucleic acids encoding a protein from the central metabolism, where
• the proteins are selected from the biosynthetic pathway of amino acids, namely:
• 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 beta-lyase, serine hydroxymethyltransferase, Acetyl homoserine sulfhydrylase, methylene
• Tetrahydrofolate reductase Tetrahydrofolate reductase, phosphoserine aminotransferase, phosphoserine
• Preferred proteins and nucleic acids encoding these proteins 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.
• the target gene G to be regulated according to the invention is preferably selected.
• proteins from the biosynthetic pathway of amino acids have in each case the amino acid sequence given in Table 1 for this protein, the respective protein each having on at least one of the amino acid positions given in Table 2, column 2 for this amino acid sequence a different proteinogenic amino acid than the respective amino acid given in Table 2, column 3 in the same line.
• the proteins have the amino acid indicated in Table 2, column 4 in the same line on at least one of the amino acid position indicated 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 a promoter construct according to the invention and for the production of amino acids.
• the T311 I mutation results in switching off feedback inhibition from ask.
• nucleic acids encoding a mutant protein of 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
• the selection of integrated expression cassettes is preferably carried out by the SacB method.
• SacB method is known to the person skilled in the art and is described, for example, in Schfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A .; Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosomes of Corynebacterium glutamicum, Gene.
• the invention therefore also relates to a genetically modified microorganism which contains an expression cassette according to the invention or a vector comprising the 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 pen delvector or plasmid vector, which carries at least one expression cassette according to the invention.
• Preferred microorganisms or genetically modified microorganisms are bacteria, algae, fungi or yeasts.
• Particularly preferred microorganisms are especially 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 melassecola ATCC 17965, Corynebacterium efficiens DSM 44547, Corynebacterium efficiens DSM 44548.
• the abbreviation KFCC means the Korean Federation of Culture Collection, with the abbreviation ATCC the American type strain culture collection, with the abbreviation DSM (or DSMZ) the German Collection of Microorganisms (German Collection of Microorganisms and Cell Cultures). Further particularly preferred bacteria of the genera Corynebacterium and Brevifobacterium are listed in Table 3:
• DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
• microorganisms of the bacteria of the genus Corynebacterium in particular those which are already able to produce L-lysine, L-methionine and / or L-threonine.
• corynebacteria in which, for example, the gene coding for an aspartate kinase (ask gene) is deregulated or the feedback inhibition is abolished or reduced.
• such bacteria in the ask gene have a mutation that leads to a reduction or abolition of the feedback inhibition, such as the mutation T311 I.
• the expression units according to the invention make it possible to regulate the metabolic pathways to specific biosynthetic products in the above-described genetically modified microorganisms according to the invention.
• metabolic pathways that lead to a specific biosynthetic see product by causing or increasing the transcription rate or expression rate of genes of this biosynthetic pathway in which the increased amount of protein to increased total activity of these proteins of the desired biosynthetic pathway and thus to an increased metabolic flux the desired biosynthetic product.
• metabolic pathways leading away from a specific biosynthetic product can be attenuated by reducing the transcription rate of genes of this pathway by reducing the amount of protein involved in reduced total activity of these proteins of the undesired biosynthetic pathway and thus in addition to increased metabolic flux 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 various 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 a promoter activity or an 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 of the invention.
• the invention therefore further relates to a process for the preparation of biosynthetic see products by culturing genetically modified microorganisms according to the invention.
• the multi-promoter-containing expression units, expression cassettes and vectors according to the invention can be used particularly advantageously, for example, in improved processes for the fermentative production of biosynthetic products as described below.
• the multiple promoter-containing expression units or expression cassettes according to the invention have the particular advantage that they allow a modulation of the expression of the functionally linked structural gene.
• the expression units according to the invention or expression cassettes comprising them can be used for altering, ie increasing or reducing, or causing the expression rate of genes in microorganisms in comparison to the wild type. This provides the possibility of maximizing the expression of the gene in question in the event of an increase in the expression rate.
• the expression can also take place in a starch which is below the maximum value obtainable by another expression unit, but at the same time supplies the expression product in an amount which is more compatible with the expressing microorganism.
• the expression units according to the invention thus make it possible, by selection among expression units each having different expression levels, to fine-tune the expression to a value which is optimized, in particular in the case of long-term expression.
• expression units or expression units according to the invention can be used 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 the use of a multiple-engine-containing expression unit according to the invention for regulating a product biosynthesis.
• the gene of a protein involved in the regulation of a product biosynthesis is placed under the control of such an expression unit.
• Product biosynthesis is affected depending on the transcription rate of the selected multiple promoter-containing expression unit.
• the gene of a protein involved in the regulation of a product biosynthesis can be expressed as part of an expression cassette according to the invention in a microorganism and the regulation of the product biosynthesis by the protein expressed thereby. If the activity of a multiple promoter is weaker than that of the endogenous promoter, a multiple promoter can also be used to selectively attenuate unwanted biosynthetic pathways.
• the regulation of the transcription rate of an expression unit according to the invention containing multiple promoters can be effected by targeted mutation of one or more individual promoters from which the multiple promoter is composed. Increased or decreased promoter activity can be achieved by using nucleotides in the binding site of the RNA polymerase holoenzyme. Binding sites (also known as the -10 region and -35 region) are exchanged. Furthermore, an influencing can take place in that the distance of the described RNA polymerase holoenzyme binding sites from each other is reduced or increased by deletions of nucleotides or insertions of nucleotides.
• 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
• binding sites are brought into spatial proximity to the binding sites of the RNA polymerase holoenzyme such that these regulators, after binding to a promoter Sequence attenuate or enhance the binding and transcriptional activity of the RNA polymerase holoenzyme, or under a new regulatory influence.
• the translation activity can also be influenced by mutation of the ribosomal binding mediating, 3'-terminal sequence section of a single promoter Py within the multiple promoter-containing expression unit according to the invention.
• Preferred biosynthetic products prepared according to the invention are fine chemicals.
• fine chemical is well known to those skilled in the art and includes compounds produced by an organism that find applications in various industries such as, but not limited to, the pharmaceutical, agricultural, cosmetics, food and feed industries , These compounds include organic acids such as lactic acid, succinic acid, 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 Kuninaka, A. (1996) Nucleotides and 6, Rehm et al., ed.
• organic acids such as lactic acid, succinic acid, tartaric acid, itaconic acid and diaminopimelic acid
• proteinogenic and non-proteinogenic amino acids purine and pyrimidine bases
• nucleosides and nucleotides as described, for example, in Kuninaka, A. (1996) Nucleotides and 6, Rehm et al., ed.
• VCH Weinheim and the citations contained therein
• lipids saturated and unsaturated fatty acids (eg arachidonic acid), diols (eg. Propanediol 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, A27, "Vitamins", Vol. Pp. 443-613 (1996) VCH: Weinheim and the citations therein; and Ong, AS, Niki, E. and Packer, L.
• 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, aromatic compounds, vitamins and cofactors, enzymes and proteins.
• Preferred organic acids are lactic acid, succinic acid, 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 as described, for example 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.
• 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, L-proline, L
• an amino acid such as lysine, methionine and threonine
• 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 understood.
• the lysine, methionine and threonine production which are particularly preferred, are described in more detail.
• the invention relates to a process for the production of lysine by culturing genetically modified microorganisms with an increased or induced expression rate of at least one gene compared to the wild type, wherein
• the expression of at least one gene in the microorganism is caused or changed by introduction of an expression cassette according to the invention comprising the gene into the microorganism.
• the genes are in particular selected from 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, nucleic acids encoding 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 cell Regulator
• a further 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 additionally an increased activity, at least one of the activities selected from: aspartate kinase activity, aspartate-semialdehyde dehydrogenase activity, Diaminopilate 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, transcriptional regulator activity LysR1, transcriptional regulator activity LysR2, malate-quinone-oxo-reductase activity, glucose-6-phosphate de-dehydrogenase activity, 6-phosphogluconate dehydrognea
• 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 additionally a reduced 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 genetically modified microorganisms in addition to the wild type additionally a reduced activity, at least one of the activities selected from the group threonine dehydratase activity, homoserine O-acet
• the invention further relates to a method for the production of methionine by culturing genetically modified microorganisms with increased or caused expression rate of at least one gene compared to the wild type, wherein
• the expression of at least one gene in the microorganism is caused or changed by introduction of an expression cassette according to the invention into the microorganism.
• the genes are in particular selected from 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, nucleic acids encoding a pyruvate carboxylase , Nucleic acids encoding a triosephosphate isomerase, nucleic acids encoding a homoserine O-acetyltransferase, nucleic acids encoding a cystahionin gamma synthase, nucleic acids encoding a cystahionine beta-lyase, nucleic acids encoding a serine hydroxymethyltransferase, nucleic acids encoding an
• Nucleic acids encoding a 6-phosphogluconate dehydrogenase, glucose phosphate isomerase, phosphoglycerate mutase, enolase, pyruvate kinase, aspartate transaminase or malate enzyme are included in Nucleic acids encoding a 6-phosphogluconate dehydrogenase, glucose phosphate isomerase, phosphoglycerate mutase, enolase, pyruvate kinase, aspartate transaminase or malate enzyme.
• a further preferred embodiment of the method for the production of methionine 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 , Homoserine dehydrogenase activity, glyceraldehyde-3-phosphate dehydrogenase activity, 3-phosphoglycerate kinase activity, pyruvate carboxylase activity, triosephosphate isomerase activity, homoserine O-acetyltransferase activity, cystahionin-gamma synthase activity, cystahionine beta Lyase Activity Serine hydroxymethyltransferase activity, O-acetyl homoserine sulfhydrylase activity, methylene tetrahydrofolate reductase activity, phosphoserine aminotransferase activity
• a further particularly preferred embodiment of the method for the production of methionine described above is characterized in that the genetically modified microorganisms in addition to the wild type additionally 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.
• the invention further relates to a process for the production of threonine by culturing genetically modified microorganisms with increased or induced expression rate of at least one gene compared to the wild type, wherein
• the genes are in particular selected from nucleic acids encoding an aspartate kinase, nucleic acids encoding an aspartate semialdehyde dehydrogenase, 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 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 malate Quinone
• 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 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 activity Export carriers, transaldolase activity, transketolase activity, glucose-6-phosphate dehydrogenase activity, malate-quinone
• a further particularly preferred embodiment of the method for producing threonine described above is characterized in that the genetically modified microorganisms in addition to the wild type additionally 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 synthetase activity, dihydrodipolinate reductase activity, asparaginase Activity, aspartate decarboxylase activity, lysine exporter activity, acetolactate synthase activity, ketol-A reductoisomerase activity, branched chain aminotransferase activity, coen
• activity of a protein is understood as meaning the enzyme activity of the corresponding protein in the case of enzymes, and the physiological activity of the proteins in the case of other proteins, such as, for example, structure or transport proteins.
• the enzymes are usually able to convert a substrate into a product or to catalyze this conversion step. Accordingly, the "activity" of an enzyme is understood to mean 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 “activity” is at least 1 to 5%, such as at least 20%, at least 50%, at least 100%, at least 300%, or at least 500%, or at least 600% of the " Activity of the wild-type ".
• 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.
• Reduced activity is preferably understood to mean the partial or substantially complete suppression or blocking of the functionality of this enzyme in a microorganism, based on different cell biological mechanisms.
• Reduction of activity includes a reduction in the amount of an enzyme, to a substantially complete absence of the enzyme (i.e., lack of detectability of the corresponding activity or lack of immunological detectability of the enzyme).
• the activity in the microorganism is reduced by at least 5% as compared to the wild type, such as at least 20%, at least 50%, or by about 100%.
• “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.
• An additional increase of activities can take place in various ways, for example by switching off inhibitory regulation mechanisms on the expression and protein level or by increasing the gene expression of nucleic acids encoding the proteins described above in comparison to the wild type.
• Increasing the gene expression of the nucleic acids encoding the proteins described above with respect to the wild type can also be done in various ways, 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 in the microorganism.
• the manipulation of the expression of the microorganism itself is also affected. understood endogenous proteins. This can be achieved, for example, as described above by changing the promoter sequences of the genes, introducing expression unit according to the invention for regulatory control of the genes and altering the incorporated expression units according to the invention. Such a change, which results in an increased expression rate of the gene, can be done for example by deletion or insertion of DNA sequences.
• 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.
• enzyme activity is also enhanced.
• 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 to eliminate unwanted side reactions in addition to the expression or amplification of a gene
• the gene expression of a nucleic acid encoding one of the proteins described above is increased by introducing at least one nucleic acid encoding a corresponding protein into the microorganism.
• the introduction of the nucleic acid can be chromosomal or extrachromosomal, ie by increasing the copy number on the chromosome and or a copy of the gene on a replicating plasmid in the Western microorganism.
• nucleic acid for example in the form of an expression cassette according to the invention containing the nucleic acid, preferably takes place chromosomally, in particular by the SacB method described above.
• SacB method any gene which codes for 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 to use.
• the reduction of the above-described activities in microorganisms is carried out by at least one of the following processes:
• knockout mutants can be generated by targeted insertion into the desired target gene by homologous recombination or introduction of sequence-specific nucleases against the target gene.
• RNA sense RNA
• Each of these methods can cause a reduction in the amount of protein, mRNA amount and / or activity of a protein. Even a combined application is conceivable.
• Other methods are known to those of skill in the art and may include inhibiting or inhibiting processing of the protein, transport of the protein or its mRNA, inhibition of ribosome attachment, inhibition of RNA splicing, induction of an RNA degrading enzyme and / or inhibition of translation elongation or termination ,
• 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 batchwise in the batch process or in the fed batch process or repeated fed batch 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 is in the textbook of Chemiel (bioprocess 1. Introduction to bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)) Find.
• 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 of Bacteriology (Washington D.C. 1 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.
• Very good sources of carbon are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
• Sugar can also be added to the media via complex compounds such as molasses or other by-products of sugar refining. It may also be advantageous to add mixtures of different carbon sources.
• 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
• inorganic compounds such as, for example, sulfates, sulfites, dithionites, Tetrathionates, thiosulfates, sulfides but also organic sulfur compounds, such as mercaptans and thiols can be used.
• 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 media 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 on the media optimization is available from the textbook "Applied Microbiol Physiology, A Practical Approach” (ed. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3).
• Growth media may also be obtained from commercial suppliers, such as standard 1 (Merck) or BHI (Brain Heart Infusion, DIFCO) and the like.
• All media components are sterilized either by heat (20 min at 1, 5 bar and 121 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 added randomly or in batches as desired.
• the temperature of the culture is usually between 15 ° C and 45 ° 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 e.g. B. Antibiotics, to be added.
• oxygen or oxygen-containing gas mixtures such as B. ambient air
• the temperature of the culture is usually from 20 0 C to 45 ° 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 especially over at least 30% of the fermentation period. 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 physico-chemical properties of the biosynthetic desired product and the biosynthetic by-products.
• the fermentation broth can then be further processed, for example.
• the biomass can be wholly or partly by separation methods, such. As centrifugation, filtration, decantation or a combination of these methods are removed from the fermentation broth or 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, concentrated or concentrated.
• 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.
• chromatographic steps may be repeated if necessary, 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 can 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 pressure liquid chromatography (HPLC), spectroscopic methods, staining methods, thin-layer chromatography, NIRS, enzyme testing or microbiological tests. 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.
• HPLC high pressure liquid chromatography
• NIRS enzyme testing or microbiological tests.
• ampicillin resistance and replication origin of the vector pBR322 with the oligonucleotide primers SEQ ID NO: 5 and SEQ ID NO: 6 were amplified by means of the polymerase chain reaction (PCR).
• SEQ ID NO: 5 ⁇ '-CCCGGGATCCGCTAGCGGCGCGCCGGCCGGCCCGGTGTGAAATACCGCACA G-3 '
• SEQ ID NO: 6 5'-TCTAGACTCGAGCGGCCGCGCGGCCGGCCTTTAAATTGAAGACGAAAGGGCCTC G-3 '
• telomere primer In addition to the pBR322 complementary sequences, contains the oligonucleotide primer
• the resulting DNA fragment was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the blunt ends of the DNA fragment were ligated together with the Rapid DNA Ligatio ⁇ kit (Roche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation mixture according to standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, ColD Spring Harbor, described (1989)), transformed into competent E. coli XL-1 Blue (Stratagene, La JoIIa, USA). Selection for plasmid-carrying cells was achieved by plating on ampicillin (50 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the plasmid DNA of an individual clone was isolated with the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer and checked by restriction digests.
• the resulting plasmid is named pCLiKI.
• a kanamycin resistance cassette was amplified using the oligonucleotide primers SEQ ID NO: 7 and SEQ ID NO.8.
• the oligonucleotide primer SEQ ID NO: 7 in 5'-3 'direction contains the cleavage sites for the restriction endonucleases Xbal, Smal, BamHI, NheI and the oligonucleotide primer SEQ ID NO: 8 in 5'-3' direction Interfaces for the restriction endonucleases Ascl and Nhel.
• the PCR reaction was carried out by standard method as Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press (1990)) with PfuTurbo polymerase (Stratagene, La JoIIa, USA).
• the resulting DNA fragment was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the DNA fragment was cut with the restriction endonucleases XbaI and AscI (New England Biolabs, Beverly, USA), and then again with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions cleaned.
• the vector pCLiKI was also cut with the restriction endonucleases XbaI and AscI and dephosphorylated with alkaline phosphatase (I (Roche-Diagnostics, Mannheim)) according to the manufacturer's instructions.
• the linearized vector (approximately 2.1 kb) was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• This vector fragment was ligated with the aid of the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer with the cut PCR fragment and the ligation mixture by standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, ColD Spring Harbor, described (1989)), transformed into competent E. coli XL-1 Blue (Stratagene, La JoIIa, USA).
• plasmid-bearing cells were achieved by plating on ampicillin (50 ⁇ g / ml) and kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the plasmid DNA of an individual clone was isolated with the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer and checked by restriction digests.
• the resulting plasmid is named pCLiK2.
• the vector pCLiK2 was cut with the restriction endonuclease Dral (New England Biolabs, Beverly, USA). After electrophoresis in a 0.8% agarose gel, a ca. 2.3 kb vector fragment was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions. This vector fragment was religated using the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation mixture was purified by standard methods as described in Sambrook et al. (Molecular Cloning, A Laboratory Manual, ColD Spring Harbor, described (1989)), transformed into competent E.
• coli XL-1 Blue (Stratagene, La JoIIa, USA). Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the plasmid DNA of an individual clone was isolated with the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer and checked by restriction digests.
• the resulting plasmid is named pCLiK3.
• the replication origin pHM1519 was amplified using the oligonucleotide primers SEQ ID NO: 9 and SEQ ID NO: 10.
• SEQ ID NO: 9 5'-GAGAGGGCGGCCGCGCAAAGTCCCGCTTCGTGAA-S '
• SEQ ID NO: 10 5'-GAGAGGGCGGCCGCTCAAGTCGGTCAAGCCACGC-3 '
• the oligonucleotide primers SEQ ID NO: 9 and SEQ ID NO: 10 contain cleavage sites for the restriction endonuclease NotI.
• the PCR reaction was carried out by standard method, such as Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press (1990)) with PfuTurbo polymerase (Stratagene, La JoIIa, USA).
• the resulting 2.7 kb DNA fragment was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the DNA fragment was cut with the restriction endonuclease Notl (New England Biolabs, Beverly, USA) and subsequently purified again with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the vector pCLiK3 was also cut with the restriction endonuclease NotI and dephosphorylated with alkaline phosphatase (I (Roche Diagnostics, Mannheim)) according to the manufacturer's instructions.
• the linearized vector (approximately 2.3 kb) was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• This vector fragment was ligated with the aid of the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer with the cut PCR fragment and the ligation mixture by standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, ColD Spring Harbor, described (1989)), transformed into competent E. coli XL-1 Blue (Stratagene, La JoIIa, USA). Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the plasmid DNA of an individual clone was isolated with the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer and checked by restriction digests.
• the resulting plasmid is named pCLiK5.
• SEQ ID NO: 11 5'-TCGAATTTAAATCTCGAGAGGCCTGACGTCGGGCCCGGTACCACGCGTCATAT
• SEQ ID NO: 12 5'-GATCATTTAAATCCCGGGTCTAGAGGATCCCAATTGTTAATTAACGCAGAAGAG
• the vector pCUK5 was cut with the restriction endonuclease Xhol and BamHI (New England Biolabs, Beverly, USA) and dephosphorylated with alkaline phosphatase (I (Roche Diagnostics, Mannheim)) according to the manufacturer's instructions. After electrophoresis in a 0.8% agarose gel, the linearized vector (approximately 5.0 kb) was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• This vector fragment was ligated with the help of the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer with the synthetic double-stranded DNA fragment and the ligation onsinsatz according to standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, ColD Spring Harbor, described (1989)), transformed into competent E. coli XL-1 Blue (Stratagene, La JoIIa, USA). Selection for plasmid-carrying cells was achieved by plating on kaamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the plasmid DNA of an individual clone was isolated with the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer and checked by restriction digests.
• the resulting plasmid is named pCLiK5MCS.
• Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt).
• the resulting plasmid pCLiK5MCS is listed as SEQ ID NO: 13.
• the obtained DNA fragment of about 1.3 kb size was purified with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions. Subsequently, it was cleaved with the restriction enzymes Asp718 and Spei (Roche Diagnostics, Mannheim) and the DNA fragment was purified with GFX TM PCR, DNA and Gel Band Purification Kit.
• the vector pClik ⁇ MCS SEQ ID NO: 13 was cut with the restriction enzymes Asp718 and Spei and a 5 kb fragment after electrophoretic separation with GFX TM PCR, DNA and Gel Band Purification Kit isolated.
• the vector fragment was ligated together with the PCR fragment using the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation mixture according to standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, CoId Spring Harbor, described (1989)), transformed into competent E. coli XMBIue (Stratagene, La JoIIa, USA). Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the preparation of the plasmid DNA was carried out by methods and materials of Fa. Quiagen. Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt).
• Chromosomal DNA from C. glutamicum ATCC 13032 was isolated according to Tauch et al. (1995) Plasmid 33: 168-179 or Eikmanns et al. (1994) Microbiology 140: 1817-1828.
• the oligonucleotide primers SEQ ID NO 17 and SEQ ID NO 18, the chromosomal DNA as template and Pfu Turbo Polymerase (Stratagene) were amplified by the polymerase chain reaction (PCR) according to standard methods such as Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press amplified a DNA fragment of about 200 base pairs from the non-coding 5 'region (region of the expression unit) of superoxide dismutase (Psod).
• the resulting DNA fragment was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the obtained DNA fragment of approximately 470 base pairs was purified with the GFX TM PCR, DNA and Gel Band Purification Kit according to the manufacturer's instructions.
• the two fragments obtained above were used together as a template.
• the standard method was modified in such a way that the oligonucleotide primers SEQ ID NO: 17 and SEQ ID NO: 20 used were added to the reaction mixture only at the beginning of the second cycle.
• the amplified DNA fragment of approximately 675 base pairs was purified using the GFX TM PCR, DNA and Gel Band Purification Kit according to the manufacturer's instructions. Subsequently, it was cleaved with the restriction enzymes Xhol and Ncol (Roche Diagnostics, Mannheim) and separated by gel electrophoresis. Subsequently, the ca. 620 base pair DNA fragment was purified from the agarose using GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg).
• the plasmid PmetA metA SEQ ID NO 16: was cleaved with the restriction enzymes Ncol and Spei (Roche Diagnostics, Mannheim). After gel electrophoretic separation, a ca. 0.7 kb metA fragment was purified from the agarose using GFX TM PCR, DNA and Gel Band Purification Kit.
• the vector pClik5MCS SEQ ID NO: 13 was cut with the restriction enzymes Xhol and Spei (Roche Diagnostics, Mannheim) and a 5 kb fragment after electrophoretic separation with GFX TM PCR, DNA and Gel Band Purification Kit isolated.
• the vector fragment was ligated together with the PCR fragment and the metA fragment using the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation mixture according to standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, CoId Spring Harbor, supra, 1989)), transformed into competent E. coli XL-1Blue (Stratagene, La JoIIa, USA). Selection for plasmid-bearing cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the preparation of the plasmid DNA was carried out by methods and materials of Fa. Quiagen. Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt).
• Chromosomal DNA from C. glutamicum ATCC 13032 was isolated according to Tauch et al. (1995) Plasmid 33: 168-179 or Eikmanns et al. (1994) Microbiology 140: 1817-1828.
• the oligonucleotide primers SEQ ID NO 22 and SEQ ID NO 23, the chromosomal DNA as template and Pfu Turbo Polymerase (Stratagene) were amplified by polymerase chain reaction (PCR) according to standard methods such as Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press, amplified a DNA fragment of about 200 base pairs from the noncoding 5 'region (promoter region) of superoxide dismutase (Psod).
• the DNA fragment obtained was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the resulting DNA fragment of approximately 470 base pairs was purified using the GFX TM PCR, DNA and Gel Band Purification Kit according to the manufacturer's instructions.
• the two fragments obtained above were used together as a template.
• the standard This method was modified such that the oligonucleotide primers SEQ ID NO: 22 and SEQ ID NO: 25 used were added to the reaction mixture only at the beginning of the second cycle.
• the amplified DNA fragment of approximately 675 base pairs was purified using the GFX TM PCR, DNA and Gel Band Purification Kit according to the manufacturer's instructions. Subsequently, it was cleaved with the restriction enzymes Xhol and Ncol (Roche Diagnostics, Mannheim) and separated by gel electrophoresis. Subsequently, the ca. 620 base pair DNA fragment was purified from the agarose using GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg).
• the plasmid PmetA metA SEQ ID NO: 16 was cleaved with the restriction enzymes Ncol and Spei (Roche Diagnostics, Mannheim). After gel electrophoresis, a ca. 0.7 kb metA fragment was purified from the agarose using GFX TM PCR, DNA and Gel Band Purification Kit.
• the vector pClik ⁇ MCS SEQ ID NO: 13 was cut with the restriction enzymes Xhol and Spei (Roche Diagnostics, Mannheim) and a 5 kb fragment after electrophoretic separation with GFX TM PCR, DNA and Gel Band Purification Kit isolated.
• the vector fragment was ligated together with the PCR fragment and the metA fragment using the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation mixture according to standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, ColD Spring Harbor, described (1989)), transformed into competent E. coli XL-1 Blue (Stratagene, La JoIIa, USA). Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the preparation of the plasmid DNA was carried out by methods and materials of Fa. Quiagen. Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Weertadt).
• the resulting plasmid pCLiK5MCS P_EFTUmetA is listed as SEQ ID NO: 26.
• Chromosomal DNA from C. glutamicum ATCC 13032 was isolated according to Tauch et al. (1995) Plasmid 33: 168-179 or Eikmanns et al. (1994) Microbiology 140: 1817-1828.
• the oligonucleotide primers SEQ ID NO 27 and SEQ ID NO 28, the chromosomal DNA as template, and Pfu Turbo Polymerase (Stratagene) were amplified by polymerase chain reaction (PCR) according to standard methods such as Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press amplified a DNA fragment of about 200 base pairs from the noncoding 5 'region (region of the expression unit) of the gene GroES (Pgro).
• the resulting DNA fragment was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the resulting DNA fragment of approximately 470 base pairs was purified using the GFX TM PCR, DNA and Gel Band Purification Kit according to the manufacturer's instructions.
• the two fragments obtained above were used together as a template.
• the standard This method was modified so that the oligonucleotide primers SEQ ID NO: 27 and SEQ ID NO: 30 used were added to the reaction mixture only at the beginning of the second cycle.
• the amplified DNA fragment of approximately 675 base pairs was purified using the GFX TM PCR, DNA and Gel Band Purification Kit according to the manufacturer's instructions. Subsequently, it was cleaved with the restriction enzymes Xhol and Ncol (Roche Diagnostics, Mannheim) and separated by gel electrophoresis. Subsequently, the ca. 620 base pair DNA fragment was purified from the agarose using GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg).
• the plasmid PmetA metA SEQ ID NO: 16 was cleaved with the restriction enzymes Ncol and Spei (Roche Diagnostics, Mannheim). After gel electrophoresis, a ca. 0.7 kb metA fragment was purified from the agarose using GFX TM PCR, DNA and Gel Band Purification Kit.
• the vector pClik5MCS SEQ ID NO: 13 was cut with the restriction enzymes Xhol and Spei (Roche Diagnostics, Mannheim) and a 5 kb fragment after electrophoretic separation with GFX TM PCR, DNA and Gel Band Purification Kit isolated.
• the vector fragment was ligated together with the PCR fragment and the metA fragment using the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation mixture according to standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, ColD Spring Harbor, described (1989)), transformed into competent E. coli XL-1 Blue (Stratagene, La JoIIa, USA). Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the preparation of the plasmid DNA was carried out by methods and materials of Fa. Quiagen. Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Weertadt).
• Chromosomal DNA from C. glutamicum ATCC 13032 was isolated according to Tauch et al. (1995) Plasmid 33: 168-179 or Eikmanns et al. (1994) Microbiology 140: 1817-1828. With the oligonucleotide primer BK 1849 (SEQ ID NO: 32) and BK 1862 (SEQ ID NO: 33), the chromosomal DNA as template and Pfu Turbo Polymerase (Stragene) was detected by the polymerase chain reaction (PCR) Standard methods, as described in Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press, amplified the metA gene encoding homoserine O-acetyltransferase.
• PCR polymerase chain reaction
• BK 1862 (SEQ ID NO: 33) 5'-ATGCCCACCCTCGCGCC -3 '
• the obtained DNA fragment of about 1134 bp size was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• oligonucleotide primers Haf 26 (SEQ ID NO: 34) and Haf 27 (SEQ ID NO: 35), the chromosomal DNA as template, and Pfu Turbo Polymerase (Stratagene) were amplified by polymerase chain reaction (PCR) according to standard methods, as in
• Haf 26 (SEQ ID NO: 34) ⁇ '-GAGAGGATCCCCCCCACGACAATGGAAC-S 'and
• Haf 27 (SEQ ID NO: 35) 5'-CCTGAAGGCGCGAGGGTGGGCATTACGGGGCGATCCTCCTTATG -3 '
• the obtained DNA fragment of about 195 bp size was purified with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the primers Haf 27 and BK I862 contain an overlapping sequence and are homologous to each other at their 5 'ends.
• PCR products obtained above were used as templates for a further PCR in which the primers BK 1849 (SEQ ID NO: 32) and Haf 26 (SEQ ID NO: 34) were used.
• the vector fragment was ligated together with the PCR fragment using the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation mixture according to standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, CoId Spring Harbor, supra; 989)), transformed into competent E. coli XL-1 Blue (Stratagene, La JoIIa, USA). Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the preparation of the plasmid DNA was carried out by methods and materials of Fa. Quiagen. Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Weertadt).
• the resulting plasmid was designated pClik 5a MCS P EF-TS metA (SEQ ID NO: 36).
• BK 1754 (SEQ ID NO: 38) GCGACTAGTGCCCCACAAATAAAAACAC
• the obtained DNA fragment of about 77 bp size was purified with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer, cut with the restriction enzymes Xba I and Bcu I and purified again.
• the vector pClik ⁇ MCS (SEQ ID NO: 13) was linearized with the restriction enzyme Xbal and purified with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions,
• the linearized vector was ligated together with the PCR fragment using the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation mixture according to standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, ColD Spring Harbor, described (1989)), transformed into competent E. coli XL-1 Blue (Stratagene, La JoIIa 1 USA). Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the preparation of the plasmid DNA was carried out by methods and materials of Fa. Quiagen. Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Weertadt).
• the resulting plasmid was designated H247 (SEQ ID NO: 39).
• This plasmid was treated with the restriction enzymes Beul and Sali and purified with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions,
• the plasmid pClik ⁇ MCS Psod metA (SEQ ID NO: 21) as template and Pfu Turbo Polymerase (Stratagene) was prepared by standard methods as described in Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press.
• the amplified fragment had an expected length of about 1350 bp and was purified with the GFX TM PCR, DNA and Gel Band Purification Kit (Amerersham Pharmacia, Freiburg) according to the manufacturer's instructions, cut with the restriction enzymes Beul and Sali and again cleaned.
• This fragment was ligated with the plasmid H247 (cleavage Beul and Sali) using the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer and the ligation mixture according to standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, ColD Spring Harbor, described (1989)), transformed into competent E. coli XL-1Blue (Stratagene, La JoIIa, USA). Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the preparation of the plasmid DNA was carried out by methods and materials of Fa. Quiagen. Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt).
• the resulting plasmid was named pG A4 (H344) and is listed under SEQ ID NO: 42.
• the plasmid pG A4 (SEQ ID NO: 42) was cut with the restriction enzymes Xhol and Beul and purified with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• Chromosomal DNA from C. glutamicum ATCC 13032 was isolated according to Tauch et al. (1995) Plasmid 33: 168-179 or Eikmanns et al. (1994) Microbiology 140: 1817-1828 prep. riert.
• the oligonucleotide primers BK 1695 (SEQ ID NO: 43) and Haf16 (SEQ ID NO: 44), the chromosomal DNA as template and Pfu Turbo Polymerase (Stratagene) were amplified according to standard methods using polymerase chain reaction (PCR) as described in Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press, a DNA fragment of about 182 base pairs from the non-coding 5 'region (region of the expression unit) of the gene EF Tu (Peftu) amplified.
• SEQ ID NO: 43 (BK1695) GAGACTCGAGGGCCGTTACCCTGCGAATG
• the obtained DNA fragment of about 200 bp size was purified with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer, cut with the restriction enzymes Xho I and Bcu I and purified again.
• This fragment was ligated with the plasmid plasmid pG A4 (H344) (Xhol and Beul cleavage sites) using the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer's instructions, and the ligation mixture was determined by standard methods as described in Sambrook et al. (Molecular Cloning, A Laboratory Manual, ColD Spring Harbor, described (1989)), transformed into competent E. coli XL-1Blue (Stratagene, La JoIIa, USA). Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the resulting plasmid was named pClik ⁇ MCS PeftuPsod metA (H473). It is listed under SEQ ID NO: 45. It contains (from 5 'to 3') a Peftu promoter, a Psod expression unit and immediately afterwards the metA open reading frame.
• Chromosomal DNA from C. glutamicum ATCC 13032 was isolated according to Tauch et al. (1995) Plasmid 33: 168-179 or Eikmanns et al. (1994) Microbiology 140: 1817-1828. With the oligonucleotide primers SEQ ID NO: 46 (Haf64) and SEQ ID NO: 47 (Haf65), the chromosomal DNA as template and Pfu Turbo Polymerase (Fa. days) was determined by the polymerase chain reaction (PCR) according to standard methods, as described in Innis et al. (1990) PCR Protocols. A DNA fragment of about 200 from the non-coding 5 'region (region of the expression unit) of the EF Tu gene (Peftu) was amplified.
• PCR polymerase chain reaction
• the obtained DNA fragment of about 200 bp size was purified with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the metA open reading frame was determined by standard methods as described in Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press. The resulting fragment, approximately 1140 bp in length, was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• SEQ ID NO: 48 (BK1862) ATGCCCACCCTCGCGCC
• the two fragments obtained above were used together as a template.
• the meta homologous sequences introduced with the oligonucleotide primer Haf 65 SEQ ID NO: 47 lead to an attachment of the two fragments to one another in the course of the PCR reaction and to a continuous DNA strand through the polymerase used.
• the standard method was modified in such a way that the oligonucleotide primers Haf 64 and BK 1849 SEQ ID NO: 46 and SEQ ID NO: 49 used were added to the reaction mixture only at the beginning of the second cycle.
• the amplified DNA fragment of approximately 1350 base pairs was purified using the GFX TM PCR, DNA and Gel Band Purification Kit according to the manufacturer's instructions.
• oligonucleotide primers BK 1697 SEQ ID NO: 50
• Haf17 SEQ ID NO: 51
• chromosomal DNA as a template and Pfu Turbo Polymerase (Stratagene) was purified by polymerase chain reaction (PCR) according to standard methods as described in Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press, amplified a DNA fragment (total length: 193 bp, of which 173 pSOD) from the non-coding 5 'region (region of the expression unit) of the gene SOD (Psod).
• PCR polymerase chain reaction
• the plasmid (H344) pG A4 SEQ ID NO: 42 was cleaved with the restriction enzymes Xhol and Sali (Roche Diagnostics, Mannheim) and separated by gel electrophoresis. Subsequently, linearized vector was purified from the agarose using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg).
• the linearized vector was ligated with the two fragments (fragment Peftu-metA and fragment Psod) using the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation mixture according to standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, Colard Spring Harbor, described (1989)) into competent E. coli XL-1Blue (Stratagene, La JoIIa, USA). transformed. Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the preparation of the plasmid DNA was carried out by methods and materials of Fa. Quiagen. Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt).
• the resulting plasmid pClik ⁇ MCS PsodPeftu metA is listed as SEQ ID NO: 52.
• Chromosomal DNA from C. glutamicum ATCC 13032 was isolated according to Tauch et al. (1995) Plasmid 33: 168-179 or Eikmanns et al. (1994) Microbiology 140: 1817-1828.
• the oligonucleotide primers SEQ ID NO: 53 (BK1701) and SEQ ID NO: 54 (Haf18), the chromosomal DNA as template, and Pfu Turbo Polymerase (Stratagene) were purified by polymerase chain reaction (PCR) according to standard methods, as in Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press, amplified a DNA fragment of approximately 175 Nt. It contains 155 base pairs from the non-coding 5 'region (region of the expression unit) of the GRO EL (Pgro) gene and one restriction restriction at the 5' and 3 'ends (Xhol and Beul, respectively).
• the obtained DNA fragment of about 175 bp size was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions. Subsequently, it was cleaved with the restriction enzymes Xhol and Beul (Roche Diagnostics, Mannheim) and the DNA fragment was purified with GFX TM PCR, DNA and Gel Band Purification Kit. Plasmid H344 (SEQ ID NO: 42) was cut with the restriction enzymes Xhol and Beul, and an approximately 6.4 kb fragment was isolated after electrophoretic separation with GFX TM PCR, DNA and Gel Band Purification Kit.
• the PCR product and the cut open plasmid H344 were ligated using the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation mixture according to standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, ColD Spring Harbor, described (1989)), transformed into competent E. coli XL-1 Blue (Stratagene, La JoIIa, USA). Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the preparation of the plasmid DNA was carried out by methods and materials of Fa. Quiagen. Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467.
• the resulting plasmid contains a Pgro promoter directly followed by a Ps
• PCR was performed according to standard methods as described in Innis et al , (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press.
• the amplified fragment comprises about 474 base pairs and contains a Pgro Psod cassette with an Acc65I site at the 3 'end.
• the DNA fragment obtained was purified with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions. Subsequently, it was cleaved with the restriction enzymes Xhol and Acc65I and the DNA fragment (333 base pairs) was purified with GFX TM PCR, DNA and Gel Band Purification Kit.
• SEQ ID NO: 56 (BK1782) TCGAGAGATTGGATTCTTAC
• Plasmid H479 (SEQ ID NO: 58) contains the Pefts expression unit fused to the metA ORF. It was cut with the restriction enzymes Xhol and Acc65I (directly upstream from Pefts) and an approximately 6.4 kb fragment was isolated after electrophoretic separation using GFX TM PCR, DNA and Gel Band Purification Kit.
• the preparation of the plasmid DNA was carried out by methods and materials of Fa. Quiagen. Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt).
• the resulting plasmid contains the 3 promoters Pgro Psod and P EF -Ts fused to the metA ORF. It is listed as pCLiK5MCS Pgro Psod Pefts metA (H501) is shown as SEQ ID NO: 59.
• PCR was carried out according to standard methods as described in Innis et al. (1990) PCR Protocols. A guide to
• the amplified fragment comprises approximately 495 base pairs and contains a Peftu-Psod cassette with an Acc65I site at the 3 'end.
• the resulting DNA fragment was detected by the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) Information from the manufacturer cleaned. Subsequently, it was cleaved with the restriction enzymes Xhol and Acc65I and the DNA fragment (354 base pairs) was purified with GFX TM PCR, DNA and Gel Band Purification Kit.
• Plasmid H479 (SEQ ID NO: 58) contains the Pefts expression unit fused to the metA ORF. It was cut with the restriction enzymes Xhol and Acc65I (directly upstream from Pefts) and an approximately 6.4 kb fragment was isolated after electrophoretic separation using GFX TM PCR, DNA and Gel Band Purification Kit.
• the preparation of the plasmid DNA was carried out by methods and materials of Fa. Quiagen. Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt).
• the resulting plasmid contains the 3 regulatory sequences Peftu Psod and P EF -Ts fused to the metA ORF. It is listed as pCLiK5MCS Peftu Psod Pefts metA (H501) is shown as SEQ ID NO: 60.
• the strain Corynebacterium glutamicum ATCC13032 was in each case labeled with the plasmids pClik ⁇ MCS, pClik MCS Psod metA, pClik MCS Peftu metA, pClik ⁇ MCS Pefts metA, pClik ⁇ MCS Peftu Psod metA, pClik ⁇ MCS Psod Peftu metA, pClik ⁇ MCS Pgro Psod meta, pClik ⁇ MCS Pgro Psod Pefts metA, pClik ⁇ MCS Peftu Psod Pefts metA according to the method described (Liebl, et al.
• the cells were centrifuged at 4 0 C and washed twice with cold Tris-HCl buffer (0.1%, pH 8.0). After another centrifugation, the cells in cold Tris-HCl buffer (0.1 %, pH 8.0) and set an OD 60 O of 160.
• 1 ml of this cell suspension was transferred to 2 ml Ribolyser tubes from Hybaid and to a Ribolyser from Hybaid at a rotation setting of 6.0 three times lysed for each 30 sec. The lysate was clarified by centrifugation at 15,000 rpm at 4 ° C. for 30 minutes in an Eppendorf centrifuge and the supernatant was transferred to a new Eppendorf village.
• the protein content was determined by Bradford, MM (1976) Anal. Biochem. 72: 248-254.
• the enzymatic activity of MetA was performed as follows. Reactions of 1 ml contained 100 mM potassium phosphate buffer (pH 7.5), 5 mM MgCl 2, 100 ⁇ M acetyl CoA, 5 mM L-homoserines, 500 ⁇ M DTNB (Ellman's reagent) and cell extract. The test was started by addition of the respective protein lysate and incubated at room temperature. Kinetics were then recorded at 412 nm for 10 min.
• MetA could be modulated by the use of different combinations of promoters / expression units.
• an allelic replacement of the lysC wild-type gene in Corynebacterium glutamicum ATCC13032 was performed.
• a nucleotide exchange was carried out in the lysC gene, so that in the resulting protein, the amino acid Thr at position 311 was replaced by a He.
• Starting from the chromosomal DNA from ATCC13032 as a template for a PCR reaction was amplified with the oligonucleotide primers SEQ ID NO: 61 and SEQ ID NO: 62 lysC using the Pfu-Turbo PCR system (Stratagene USA) according to the manufacturer.
• Chromosomal DNA from C. glutamicum ATCC 13032 was isolated according to Tauch et al. (1995) Plasmid 33: 168-179 or Eikmanns et al. (1994) Microbiology 140: 1817-1828.
• the amplified fragment is flanked at its 5 'end by a Sali restriction cut and at its 3''end by a MIuI restriction digest .. Prior to cloning, the amplified fragment was digested by these two restriction enzymes and incubated with GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg).
• the resulting polynucleotide was cloned via the Sali and MIuI restriction cleavage into pCLIK ⁇ MCS integratively SacB in the following pCIS (SEQ ID NO: 63) and transformed into E. coli XL-1 blue.
• Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190). The plasmid was isolated and confirmed by sequencing the expected nucleotide sequence.
• the preparation of the plasmid DNA was carried out by methods and materials of Fa. Quiagen. Sequencing reactions were performed according to Sanger et al.
• the plasmid pCIS lysC thr311ile was isolated in C. glutamicum ATCC13032 by electroporation as described in Liebl, et al. (1989) FEMS Microbiology Letters 53: 299-303. Modifications of the protocol are described in DE 10046870.
• the chromosomal arrangement of the lysC locus of individual transformants was determined by standard techniques by Southern blot and hybridization as described in Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual, CoId Spring Harbor, checked. This ensured that the transformants are those which have integrated the transformed plasmid by homologous recombination at the lysC locus.
• sucrose CM agar medium (10% sucrose, 10 g / l glucose, 2.5 g / l NaCl, 2 g / l urea, 10 g / l Bacto Peptone (Difco), 10 g / l yeast extract, 22.0 g / L agar (Difco)) and incubated at 30 0 C for 24 hours.
• Recombination can be deleted either the wild-type gene or the mutated gene together with the sacB gene. When the sacB gene is removed along with the wild-type gene, a mutant transformant results.
• Chromosomal DNA from C. glutamicum ATCC 13032 was isolated according to Tauch et al. (1995) Plasmid 33: 168-179 or Eikmanns et al. (1994) Microbiology 140: 1817-1828.
• the oligonucleotide primers SEQ ID NO: 69 (Old38) and SEQ ID NO: 70 (Old 39), the chromosomal DNA as template, and Pfu Turbo Polymerase (Stratagene) were amplified by the polymerase chain reaction (PCR) according to standard methods as described in Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press, amplified a DNA fragment of ca. 200 from the noncoding 5 'region (region of the expression unit) of the gene EF Tu (Peftu).
• the obtained DNA fragment of about 200 bp size was purified with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the plasmid pCIS (SEQ ID NO: 63) was cleaved with the restriction enzymes MIlu and Xhol (Roche Diagnostics, Mannheim) and separated by gel electrophoresis. Subsequently, linearized vector was purified from the agarose using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg).
• the linearized vector was ligated with the two fragments (fragment Peftu-ddh and fragment 5 'ddh) using the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation mixture according to standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, ColD Spring Harbor, described (1989)), transformed into competent E. coli XL-1 Blue (Stratagene, La JoIIa, USA). Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the preparation of the plasmid DNA was prepared by methods and materials of the Fa.
• the resulting plasmid pCIS Peftu ddh is listed as SEQ ID NO: 75.
• Chromosomal DNA from C. glutamicum ATCC 13032 was isolated according to Tauch et al. (1995) Plasmid 33: 168-179 or Eikmanns et al. (1994) Microbiology 140: 1817-1828.
• the oligonucleotide primers SEQ ID NO: 76 and SEQ ID NO: 77, the chromosomal DNA as template and Pfu Turbo Polymerase (Stratagene) were amplified by polymerase chain reaction (PCR) according to standard methods such as Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press amplified a DNA fragment of approximately 677 base pairs from the 5 'region of the ddh gene (fragment 5'ddh).
• SEQ ID NO: 76 (CK461) CCTGACGTCGCAATATAGGCAGCTGAATC
• the DNA fragment obtained was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions. Subsequently, it was cleaved with the restriction enzymes Aatll and Muni (Roche Diagnostics, Mannheim) and separated by gel electrophoresis. Subsequently, the ca. 661 base pair DNA fragment was purified from the agarose using GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg). The resulting fragment contains the 5 'region of the ddh ORF.
• a PeftuPsod cassette was amplified with the oligonucleotide primers SEQ ID NO.-78 and SEQ ID NO: 79.
• SEQ ID NO: 78 (CK426) CGCCAATTGTCGAGGGCCGTTACCCT
• the resulting DNA fragment of approximately 378 base pairs was purified using the GFX TM PCR, DNA and Gel Band Purification Kit according to the manufacturer's instructions.
• the chromosomal DNA as template and Pfu Turbo Polymerase (Stratagene) was determined by the polymerase chain reaction (PCR) according to standard methods such as Innis et al., (1990) PCR Protocols, a DNA fragment of about 992 base pairs (ddh ORF) amplified (fragment ddh) .
• the amplified DNA fragment was amplified with the GFX TM PCR , DNA and Gel Band Purification Kit according to manufacturer's instructions.
• SEQ ID NO: 80 (CK464) TACGAAAGGATTTTTTACCCATGACCAACATCCGCGTAGC
• the two fragments obtained above (PeftuPsod cassette and ddh ORF) were used together as a template.
• the Psod homologous sequences introduced with the oligonucleotide primer SEQ ID NO: 80 (CK464) lead to an attachment of the two fragments to one another and to a continuous DNA strand through the polymerase used.
• the standard method has been modified to indicates that the oligonucleotide primers SEQ ID NO: 79 (CK426) and SEQ ID NO: 81 (CK467) used were added to the reaction mixture only at the beginning of the second cycle.
• the amplified DNA fragment of approximately 1359 base pairs was purified using the GFX TM PCR, DNA and Gel Band Purification Kit according to the manufacturer's instructions.
• the vector pCIS was cut with the restriction endonucleases Aatll and Smal and dephosphorylated with alkaline phosphatase (Roche Diagnostics, Mannheim) according to the manufacturer's instructions. After electrophoresis in a 0.8% agarose gel, the linearized vector (about 4.2 kb) was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• This vector fragment was ligated with the aid of the Rapid DNA Ligation Kit (Roche Diagnostics, Mannheim) according to the manufacturer with the two cut PCR fragments (5'ddh and PeftuPsod ddh) and the ligation mixture according to standard methods as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, ColD Spring Harbor, described (1989)), transformed into competent E. coli XL-1 Blue (Stratagene, La JoIIa, USA). Selection for plasmid-carrying cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190).
• the plasmid DNA of individual clones was isolated with the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions and checked by restriction digests. 2 correct plasmids were sequenced. Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt). The resulting Plasmid pClik Peftu Psod ddh is capable of chromosomally integrating the PeftuPsod cassette into the ddh ORF by means of 2 consecutive recombination events. The plasmid pCIS PeftuPsod dh is listed as SEQ ID NO: 82.
• This methylation increases the stability of the plasmids pCIS Peftu ddh and pCIS PeftuPsod ddh in C. glutamicum cells.
• the plasmid DNA was subsequently isolated from the Mn522 cells by standard methods and introduced by means of electroporation the above-described Corynebacterium glutamicum strain ATCC 13032 ask (fbr). From this electroporation and subsequent selection on CM plates with kanamycin (25 ⁇ g / ml) several transconjugants were obtained.
• these transconjugants were grown in CM medium overnight without kanamycin and then plated out for selection on CM plates containing 10% sucrose.
• the sacB gene present on the vector pCIS codes for the enzyme laevan sucrase and, when grown on sucrose, leads to the synthesis of laevan. Since Laevan is toxic to C. glutamicum, only C. glutamicum cells which have lost the integration plasmid through the second recombination step can grow on sucrose-containing medium (Jäger et al., Journal of Bacteriology 174 (1992) 5462-5466 ). 100 sucrose-resistant clones were checked for their kanamycin sensitivity.
• a sensitivity to kanamycin could also be detected, which is expected in the case of the desired excision of the vector sequences.
• PCR polymerase chain reaction
• PCR conditions were chosen as follows: pre-denaturation: 5 min at 95 0 C; Denaturation for 30 sec at 95 ° C; Hybridization for 30 sec at 55 ° C; Amplification for 2 min at 72 0 C; 30 cycles ,; End extension 5 min at 72 ° C.
• pre-denaturation 5 min at 95 0 C
• Denaturation for 30 sec at 95 ° C Denaturation for 30 sec at 95 ° C
• Hybridization for 30 sec at 55 ° C
• Amplification for 2 min at 72 0 C
• 30 cycles 30 cycles ,
• End extension 5 min at 72 ° C In the batch with the DNA of the starting strain, no PCR product could be produced by choice of the oligonucleotides. Only in clones that have undergone the integration of the Peftu or PeftuPsod expression units directly 5 'of the ddh gene through the second recombination, a band was expected.
• strains were plated on CM plates (10.0 g / L D-glucose, 2.5 g / L NaCl, 2.0 g / L
• vitamin B12 hydroxycobalamin Sigma Chemicals
• the amino acid concentration was determined by Agilent high pressure liquid chromatography on an Agilent 1100 Series LC System HPLC.
• a pre-column derivatization with ortho-pthalaldehyde allows the quantification of the amino acids formed, the separation of the amino acid mixture takes place on a Hypersil AA column (Agilent).
• the result of the study is shown in the following table

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