Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium

Definitions

• the present invention relates to the use of nucleic acid sequences for the regulation of transcription and expression of genes, the novel promoters and expression units themselves, methods for altering or causing the transcription rate and / or expression rate of genes, expression cassettes containing the expression units, genetically modified microorganisms with altered or induced rate of transcription and / or rate of expression as well as methods for
• fine chemicals such as amino acids, vitamins, but also proteins are produced 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 culturing bacteria that have been developed to produce and secrete large quantities of the desired substance.
• Particularly suitable organisms for this purpose are coryneform bacteria, gram-positive non-pathogenic bacteria.
• Process improvements may include fermentation measures, such as stirring and supply of oxygen, or the composition of the nutrient media, such as the sugar concentration during fermentation, or the processing of the product, for example by ion exchange chromatography but also spray-drying, or the intrinsic performance characteristics of the microorganism concern yourself.
• RNA polymerase holoenzymes also called -35 and -10 regions
• ribosomal 16S RNA also ribosomal binding site or Shine-Dalgarno sequence called.
• polynucleotide sequences are understood, which are up to 20 bases upstream of the initiation codon of translation.
• 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. Still other promoters react to external chemical, such as physical stimuli such as oxygen, metabolites, heat, pH, etc. Still others show a strong dependence of their activity in different growth phases. For example, 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 effect on productivity for the production of fine chemicals and proteins depending on the metabolic pathway.
• promoters that switch off the expression of a gene during growth but turn it on after optimal growth to regulate a gene that controls the production of a metabolite.
• the altered strain then has the same growth parameters as the parent strain but produces more product per cell. This type of modification can increase both the titer (g product / liter) and the C yield (g product / gC source).
• regulated promoters may increase or decrease the rate at which a gene is transcribed, depending on the internal and / or external conditions of the cell.
• an inducer a particular factor, known as an inducer
• Inducers may directly or indirectly affect transcription from the promoter.
• suppressors is capable of reducing or inhibiting transcription from the promoter. Like the inducer, the suppressors can act directly or indirectly.
• promoters known that are regulated by temperature Thus, for example, the level of transcription of such promoters may be increased or decreased by increasing the growth temperature above the normal growth temperature of the cell.
• promoters from C. glutamicum have been described to date.
• the promoter of the malate synthase gene from C. glutamicum was described in DE 4440118. This promoter was preceded by a structural gene coding for a protein. After transformation of such a construct into a coryneform bacterium, the expression of the downstream structural gene is regulated. The expression of the structural gene is induced as soon as a corresponding inducer is added to the medium.
• the object of the invention was to provide further promoters and / or expression units with advantageous properties.
• nucleic acids with promoter activity containing
• transcription refers to the process by which a complementary RNA molecule is produced starting from a DNA template, in which process proteins such as RNA polymerase are involved in so-called sigma factors and transcriptional regulatory proteins as a template in the process of translation, which then leads to the biosynthetically active protein.
• the rate at which a biosynthetic active protein is produced is a product of the rate of transcription and translation. Both rates can be influenced according to the invention and thus influence the rate of formation of products in a microorganism.
• a "promoter” or a “nucleic acid with promoter activity” is understood as meaning a nucleic acid which, in functional linkage with a nucleic acid to be transected, regulates the transcription of this nucleic acid.
• a "functional linkage” is understood as meaning, 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 optionally further regulatory elements, for example nucleic acid sequences which ensure the transcription of nucleic acids, and, for example, a terminator that each of the regulative elements may perform its function in the transcription of the nucleic acid sequence, but does not necessarily require a direct chemical linkage Genetic control sequences, such as enhancer sequences, may also function from more distant locations or even from a more distant position Preference is given to arrangements in which the nucleic acid sequence to be transcribed is positioned behind (ie at the 3 'end) of the promoter sequence according to the invention, so that at de 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.
• promoter activity is understood as meaning the amount of RNA, that is to say the transcription rate, formed by the promoter in a specific time.
• RNA per promoter activity is meant according to the invention the amount of RNA per promoter formed by the promoter in a certain time.
• wild-type is understood according to the invention as the corresponding starting microorganism.
• microorganism may be understood to mean the starting microorganism (wild type) or a genetically modified microorganism according to the invention, or both.
• wild-type is meant for altering or causing promoter activity or rate of transcription, altering or causing expression activity or expression rate and for increasing the level biosynthetic products each have a reference organism Roger that.
• this reference organism is Corynebacterium glutamicum ATCC 13032.
• starting microorganisms are used which are already capable of producing the desired fine chemical.
• particularly preferred microorganisms of the bacteria of the genus Corynebacteria and the particularly preferred fine chemicals L-lysine, L-methionine and L-threonine those starting microorganisms which are already able to produce L-lysine, L-methionine and / or are particularly preferred To produce L-threonine.
• these are particularly preferably corynebacteria in which, for example, the gene coding for an aspartokinase (ask gene) is deregulated or the feedback inhibition is abolished or reduced.
• such bacteria have a mutation in the ask gene which leads to a reduction or abolition of the feedback inhibition, for example the mutation T3111.
• RNA With a "caused promoter activity" or transcription rate with respect to a gene compared to the wild type, the formation of a RNA is thus caused in comparison to the wild type, which was thus not present in the wild type.
• the amount of RNA formed is thus changed in a certain time compared to the wild type.
• the increased promoter activity or transcription rate can be achieved, for example, by regulating the transcription of genes in the microorganism by nucleic acids with promoter activity according to the invention or by nucleic acids having increased specific promoter activity, the genes being heterologous with respect to the nucleic acids having promoter activity.
• Vorzusgweise is the regulation of the transcription of genes in the microorganism by nucleic acids according to the invention with promoter activity or by nucleic acid. achieved with increased specific promoter activity in that one
• one or more nucleic acids according to the invention having promoter activity, optionally with altered specific promoter activity is introduced into the genome of the microorganism such that the transcription of one or more endogenous genes takes place under the control of the introduced nucleic acid according to the invention having promoter activity, optionally with altered specific promoter activity
• nucleic acid constructs containing a nucleic acid according to the invention having promoter activity, optionally with altered specific promoter activity, and functionally linked to one or more nucleic acids to be transcribed into which microorganism introduces.
• the nucleic acids according to the invention with promoter activity contain A) the nucleic acid sequence SEQ. ID. NO. 1 or B) a sequence derived from this sequence by substitution, insertion or deletion of nucleotides which has an identity of at least 90% at the nucleic acid level with the sequence SEQ. ID. NO. 1 or C) a nucleic acid sequence which coincides with the nucleic acid sequence SEQ. ID. NO. 1 hybridizes under stringent conditions or D) functionally equivalent fragments of the sequences under A), B) or C)
• the nucleic acid sequence SEQ. ID. NO. 1 represents the promoter sequence of GroES chaperonin (Pgro) from Corynebacterium glutamicum. SEQ. ID.NO. 1 corresponds to the wild-type promoter sequence.
• the invention further relates to nucleic acids with promoter activity comprising a sequence derived from this sequence by substitution, insertion or deletion of nucleotides which has an identity of at least 90% at nucleic acid level with the sequence SEQ. ID. NO. 1 has.
• promoters according to the invention can be prepared, for example, from various organisms whose genomic sequence is known by identity comparisons of the nucleic acid sequences from data Banks with the above-described sequences SEQ ID NO: 1 easily find.
• substitution in the description refers to the replacement of one or more nucleotides by one or more nucleotides.
• “Deletion” is the replacement of a nucleotide by a direct bond Insertions are insertions of nucleotides into the nucleic acid sequence which formally replaces a direct bond with one or more nucleotides.
• Identity between two nucleic acids is understood to mean the identity of the nucleotides over the entire nucleic acid length, in particular the identity which was determined by comparison with the Vector NTI Suite 7.1 software from Informax (USA) using the Clustal method (Higgins DG, Sharp PM Biosci 1989 Apr; 5 (2): 151-1) is calculated by setting the following parameters: and sensitive multiple sequence alignments on a microcomputer.
• a nucleic acid sequence which has an identity of at least 90% with the sequence SEQ ID NO: 1 is accordingly understood to mean a nucleic acid sequence which, when comparing its sequence with the sequence SEQ ID NO: 1, in particular special according to the above program logarithm with the above parameter set has an identity of at least 90%.
• Particularly preferred promoters have with the nucleic acid sequence SEQ. ID. NO. 1 has an identity of 91%, more preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, most preferably 99%.
• promoters can furthermore readily be found starting from the above-described nucleic acid sequences, in particular starting from the sequence SEQ ID NO: 1 from various organisms whose genomic sequence is unknown, by hybridization techniques in a manner known per se.
• Another object of the invention therefore relates to nucleic acids with Promotoreducationi- tat, containing a nucleic acid sequence with the nucleic acid sequence SEQ. ID. No. 1 hybridized under stringent conditions.
• This nucleic acid sequence comprises at least 10, more preferably more than 1, 15, 30, 50, or more preferably more than 150 nucleotides.
• the hybridization is carried out according to the invention under stringent conditions.
• Hybridization conditions are described, for example, in Sambrook, J., Fritsch, EF, Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57 or in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6:
• stringent hybridization conditions are meant in particular: The overnight incubation at 42 ° C in a solution consisting of 50% formamide, 5 x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6 ), 5x Denhardt's solution, 10% dextran sulfate and 20 g / ml denatured salmon sperm DNA, followed by washing the filters with 0.1x SSC at 65 ° C.
• a “functionally equivalent fragment” for nucleic acid sequences with promoter activity fragments understood that have substantially the same or higher specific promoter activity as the starting sequence.
• fragments is meant partial sequences of the nucleic acids having promoter activity described by embodiment A), B) or C. Preferably, these fragments have more than 10, more preferably more than 12, 15, 30, 50 or more preferably more than 150 contiguous nucleotides of the nucleic acid sequence SEQ ID NO: 1.
• nucleic acid sequence SEQ. ID. NO. 1 is a promoter, i. for the transcription of genes.
• the SEQ. ID. NO. 1 has been described without function assignment in Genbank entry AP005283. Therefore, the invention further relates to the novel nucleic acid sequences according to the invention having promoter activity.
• the invention relates to a nucleic acid with promoter activity containing
• nucleic acid having the sequence SEQ. ID. NO. 1 is excluded.
• nucleic acids with promoter activity can furthermore be 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, pages 896-897).
• the attachment of synthetic oligonucleotides and filling in of gaps using the Klenow fragment of the DNA polymerase and ligation reactions and general cloning methods are described in Sambrook et al. (1989), Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.
• the invention further relates to the use of an expression unit comprising one of the nucleic acids according to the invention with promoter activity and additionally functional combines a nucleic acid sequence that ensures the translation of ribonucleic acids, for the expression of genes.
• an expression unit is understood as meaning a nucleic acid with expression activity, ie a nucleic acid which, in functional linkage with a nucleic acid or gene to be expressed, regulates the expression, ie the transcription and the translation, of this nucleic acid or of this gene.
• a “functional linkage” is understood to mean, 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 optionally further regulatory elements such as a terminator such that each of the regulatory elements has its function in the transgenic
• Genetic control sequences such as enhancer sequences, can also exert their function on the target sequence from more distant positions or even from other DNA molecules are arrangements in which the nucleic acid sequence to be transgenically expressed is positioned behind (ie at the 3 'end) of the expression unit sequence according to the invention, so that both sequences are covalently linked to one another the expression unit sequence and the nucleic acid sequence to be expressed transgene less than 200 base pairs, more preferably less than 100 base pairs, most preferably less than 50 base pairs.
• expression activity is understood to mean the amount of protein formed by the expression unit over a certain period of time, ie the expression rate.
• specific expression activity is understood to mean the amount of protein per expression unit formed by the expression unit in a specific time.
• the increased expression activity or expression rate can be achieved, for example, by regulating the expression of genes in the microorganism by expression units according to the invention or by expression units with increased specific expression activity, wherein the genes are heterologous with respect to the expression units.
• the regulation of the expression of genes in the microorganism by expression units according to the invention or by expression units with increased specific expression activity according to the invention is achieved by
• one or more expression units according to the invention optionally with altered specific expression activity, introduced into the genome of the microorganism, so that the expression of one or more endogenous genes under the control of the incorporated expression units according to the invention, optionally with altered specific expression activity, takes place or
• 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, into which microorganism introduces.
• the expression units according to the invention contain a nucleic acid according to the invention described above having promoter activity and additionally functionally linked to a nucleic acid sequence which ensures the translation of ribonucleic acids.
• This nucleic acid sequence which ensures the translation of ribonucleic acids, preferably contains the nucleic acid sequence SEQ. ID. NO. 42 as a ribosomal binding site.
• the expression unit according to the invention contains:
• nucleic acid sequence SEQ. ID. NO. 2 or F a sequence derived from this sequence by substitution, insertion or deletion of nucleotides which has an identity of at least 90% at nucleic acid level with the sequence SEQ. ID. NO. 2 or G) has a nucleic acid sequence which corresponds to the nucleic acid sequence SEQ. ID. NO. 2 hybridizes under stringent conditions or H) functionally equivalent fragments of the sequences under E), F) or G).
• the nucleic acid sequence SEQ. ID. NO. 2 represents the nucleic acid sequence of the expression unit of the GroES chaperonin (Pgro) from Corynebacterium glutamicum. SEQ. ID. NO. 2 corresponds to the sequence of the expression unit of the wild-type.
• the invention furthermore relates to expression units comprising a sequence derived from this sequence by substitution, insertion or deletion of nucleotides which has an identity of at least 90% at the nucleic acid level with the sequence SEQ. ID. NO. 2 has.
• a nucleic acid sequence which has an identity of at least 90% with the sequence SEQ ID NO: 2 is accordingly understood to mean a nucleic acid sequence which, when comparing its sequence with the sequence SEQ ID NO: 2, in particular according to the above program logarithm with the above parameter set Identity of at least 90%.
• Particularly preferred expression units have with the nucleic acid sequence SEQ. ID. NO. 2 has an identity of 91%, more preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, most preferably 99%.
• expression units can furthermore easily be found starting from the above-described nucleic acid sequences, in particular starting from the sequence SEQ ID NO: 2 from various organisms whose genomic sequence is unknown, by hybridization techniques in a manner known per se.
• a further subject of the invention therefore relates to expression units comprising a nucleic acid sequence which is linked to the nucleic acid sequence SEQ. ID. No. 2 hybridized under stringent conditions.
• This nucleic acid sequence comprises at least 10, more preferably more than 1, 15, 30, 50, or more preferably more than 150 nucleotides.
• hybridizing is meant the ability of a poly- or oligonucleotide to bind under stringent conditions to a nearly complementary sequence, while under these conditions non-specific binding between non-complementary partners is avoided.
• sequences should preferably be 90-100% complementary.
• the property of complementary sequences to be able to specifically bind to one another for example, in the Northern or Southern Blot technique or in the primer binding in PCR or RT-PCR advantage.
• hybridization is carried out according to the invention under stringent conditions.
• stringent conditions are described, for example, in Sambrook, J., Fritsch, EF, Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57 or in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6:
• stringent hybridization conditions are meant in particular: The overnight incubation at 42 ° C in a solution consisting of 50% formamide, 5 x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6 ), 5x denhardt solution, 10% dextran sulfate and 20 g / ml denatured, seeded
• the nucleotide sequences according to the invention also allow the generation of probes and primers which are 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 hybridizes under stringent conditions to at least about 12, preferably at least about 25, such as about 40, 50 or 75 consecutive nucleotides of a sense strand of a nucleic acid sequence or antisense strand of the invention.
• nucleic acid sequences which comprise so-called silent mutations or are altered according to the codon usage of a specific source or host organism, in comparison with a specifically mentioned sequence, as well as naturally occurring variants, such as e.g. Splice variants or allelic variants, of which
• a “functionally equivalent fragment” is meant for expression units, fragments having substantially the same or a higher specific expression activity as the starting sequence.
• substantially the same is meant a specific expression activity which has at least 50%, preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, most preferably 95% of the specific expression activity of the starting sequence.
• “Fragments” are understood as meaning partial sequences of the expression units described by embodiment E), F) or G. Preferably, these fragments have more than 10, more preferably more than 12, 15, 30, 50 or, even more preferably, more than 150 contiguous nucleotides of the nucleic acid sequence SEQ ID NO: 1.
• nucleic acid sequence SEQ. ID. NO. 2 as expression unit, i. for the expression of genes.
• the SEQ. ID. NO. 2 has been described without function assignment in Genbank entry AP005283. Therefore, the invention furthermore relates to the new expression units according to the invention.
• the invention relates to an expression unit comprising a nucleic acid according to the invention with promoter activity additionally functionally linked to a nucleic acid sequence which ensures the translation of ribonucleic acids.
• the invention particularly preferably relates to an expression unit containing E) the nucleic acid sequence SEQ. ID. NO. 2 or F) a sequence derived from this sequence by substitution, insertion or deletion of nucleotides which has an identity of at least 90% at nucleic acid level with the sequence SEQ. ID. NO. 2 or G) has a nucleic acid sequence which corresponds to the nucleic acid sequence SEQ. ID. NO. 2 hybridizes under stringent conditions or H) functionally equivalent fragments of the sequences under E), F) or G),
• nucleic acid having the sequence SEQ. ID. NO. 2 is excluded.
• the expression units of the invention comprise one or more of the following genetic elements: a minus 10 ("-10") sequence; a minus 35 (“-35”) sequence; a transcription start, an enhancer region; and an operator region.
• these genetic elements are specific for the species Corynebacteria, especially for Corynebacterium glutamicum.
• All of the abovementioned expression units can furthermore be prepared in a manner known per se by chemical synthesis from the nucleotide units, for example by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix.
• the chemical synthesis of oligonucleotides can be carried out, for example, in a known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, p. 896-897).
• the attachment of synthetic oligonucleotides and filling in of gaps using the Klenow fragment of the DNA polymerase and ligation reactions and general cloning methods are described in Sambrook et al. (1989), Molecular cloning: A laboratory manual, Cold 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 of 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 promoters and / or expression units according to the invention can be used for example particularly advantageously in improved processes for the fermentative production of biosynthetic products as described below.
• the promoters and / or expression units according to the invention have in particular the advantage that they are induced in microorganisms by stress.
• this stress induction can be rationally controlled to increase the rate of expression / expression of desired genes.
• this stress phase is reached very early, so that here very early an increased transcription / expression rate of desired genes can be achieved.
• nucleic acids according to the invention with promoter activity can be used to alter, ie to increase or reduce, or to cause the transcription rate of genes in microorganisms in comparison to the wild type.
• the expression units according to the invention can be used to alter, ie to increase or reduce, or to cause the expression rate of genes in microorganisms in comparison to the wild type.
• the nucleic acids according to the invention with promoter activity and the expression units according to the invention can serve to regulate and enhance the formation of various biosynthetic products, such as, for example, fine chemicals, proteins, in particular amino acids, in microorganisms, in particular in Corynebacterium species.
• the invention therefore relates to a method for altering or causing the transcription rate of genes in microorganisms in comparison to the wild type by a) altering the specific promoter activity in the microorganism of endogenous nucleic acids according to the invention having promoter activity, which regulate the transcription of endogenous genes, compared to the wild-type or b) Regulation of the transcription of genes in the microorganism by nucleic acids according to the invention with promoter activity or by nucleic acids with altered specific promoter activity according to embodiment a), wherein the genes are heterologous with respect to the nucleic acids with promoter activity.
• the change or causation of the transcription rate of genes in microorganisms in comparison to the wild type can be achieved by modifying, ie increasing or decreasing, the specific promoter activity in the microorganism. This can be done for example by targeted mutation of the nucleic acid sequence according to the invention with promoter activity, ie by targeted substitution, deletion or insertion of nucleotides. Increased or decreased promoter activity can be achieved by exchanging the nucleotides in the binding site of the RNA polymerase holoenzyme binding sites (also known to the person skilled in the art as the -10 region and the region known).
• binding sites also known to those skilled in the art as
• Regulatiosproteine (known to the skilled worker as repressors and activators) are brought into spatial proximity to the binding sites of the RNA polymerase holoenzyme that these regulators after binding to a promoter sequence binding and transcription activity of the RNA polymerase holoenzyme weaken or intensify, or even put under a new regulatory influence.
• the nucleic acid sequence SEQ. ID. NO. 53 preferably represents the ribosomal binding site of the expression units according to the invention, the sequence SEQ. ID. NO. 52 represents the -10 region of the expression units according to the invention. Changes The nucleic acid sequence in these regions leads to a change in the specific expression activity.
• the invention therefore relates to the use of the nucleic acid sequence SEQ. ID. NO. 53 as a ribosomal binding site in expression units that allow the expression of genes in bacteria of the genus Corynebacterium or Brevibacterium.
• the invention relates to the use of the nucleic acid sequence SEQ. ID. NO 52 as -10 region in expression units enabling the expression of genes in bacteria of the genus Corynebacterium or Brevibacterium.
• the invention relates to an expression unit which enables the expression of genes in bacteria of the genus Corynebacterium or Brevibacterium containing the nucleic acid sequence SEQ. ID. NO. 53.
• the nucleic acid sequence SEQ. ID. NO. 53 used as a ribosomal binding site.
• the invention relates to an expression unit which enables the expression of genes in bacteria of the genus Corynebacterium or Brevibacterium, containing the nucleic acid sequence SEQ. ID. NO. 52.
• the nucleic acid sequence SEQ. ID. NO. 52 used as -10 region.
• increasing or reducing in comparison with the wild type means an increase or reduction of the specific activity with respect to the nucleic acid according to the invention having promoter activity of the wild-type, that is to say, for example, with reference to SEQ ID NO.
• the alteration or causation of the transcription rate of genes in microorganisms compared to the wild type can be carried out by regulating the transcription of genes in the microorganism by nucleic acids according to the invention with promoter activity or by nucleic acids with altered specific promoter activity according to embodiment a) Genes are heterologous with respect to the nucleic acids having promoter activity.
• one or more nucleic acids according to the invention having promoter activity, optionally with altered specific promoter activity are introduced into the genome of the microorganism such that the transcription of one or more endogenous genes under the control of the introduced nucleic acid with promoter activity, optionally with altered specific promoter activity , or
• embodiment b2) introduces one or more endogenous genes into the genome of the microorganism such that transcription of one or more of the introduced endogenous genes is under the control of the endogenous nucleic acids of the invention having promoter activity, optionally with altered specific promoter activity, or
• nucleic acid constructs comprising a nucleic acid according to the invention with promoter activity, optionally with altered specific promoter activity, and functionally linked one or more endogenous nucleic acids to be transcribed, into which microorganism introduces.
• embodiment b2) introduces one or more exogenous genes into the genome of the microorganism such that the transcription of one or more of the introduced exogenous genes under the control of the endogenous nucleic acids according to the invention having promoter activity, optionally with modified specific pro motor activity, or occurs
• nucleic acid constructs comprising a nucleic acid according to the invention with promoter activity, optionally with altered specific promoter activity, and functionally linked one or more exogenous nucleic acids to be transcribed, into which microorganism introduces.
• the insertion of genes according to embodiment b2) can be carried out so that the gene is integrated into coding regions or non-coding regions. Preferably, the insertion takes place in non-coding regions.
• the insertion of nucleic acid constructs according to embodiment b3) can be carried out chromosomally or extrachromosomally.
• the insertion of the nucleic acid constructs is chromosomal.
• a "chromosomal" integration is the insertion of an exogenous DNA fragment into the host cell's chromosome, a term also used for homologous recombination between an exogenous DNA fragment and the corresponding region on the host cell's chromosome.
• nucleic acids according to the invention with modified specific promoter activity according to embodiment a). These can be present in the microorganism and prepared in embodiment b), as described in embodiment a), or introduced into the microorganism in isolated form.
• endogenous is meant genetic information such as genes already contained in the wild-type genome.
• exogenous is meant genetic information such as genes that are not included in the wild-type genome.
• genes with regard to regulation of transcription by the nucleic acids with promoter activity according to the invention are preferably understood as meaning nucleic acids which have a region to be transcribed, for example a region of translation, a coding region, and optionally further regulatory elements, such as, for example Terminator, included.
• genes with regard to the regulation of expression by the expression units according to the invention described below is preferably understood to mean nucleic acids which have a coding region and optionally also tere regulatory elements, such as a terminator included.
• coding region is meant a nucleic acid sequence encoding a protein.
• heterologous with respect to nucleic acids with promoter activity and genes is meant that the genes used are not transcribed in wild-type under regulation of the nucleic acids according to the invention with promoter activity, but that a new, not occurring in wild-type functional linkage is formed and the functional combination of inventive Nucleic acid with promoter activity and specific gene does not occur in the wild type.
• heterologous in terms of expression units and genes is meant that the genes used are not expressed in wild-type under regulation of expression units of the invention, but that a new, does not occur in the wild-type functional linkage and the functional combination of inventive expression unit and specific gene does not occur in the wild type.
• the invention further relates, in a preferred embodiment, to a method for increasing or causing the transcription rate of genes in microorganisms in comparison to the wild type by comparing ah) the specific promoter activity in the microorganism of endogenous nucleic acids according to the invention with promoter activity which regulate the transcription of endogenous genes increased to the wild type or bh) the transcription of genes in the microorganism by nucleic acids according to the invention with promoter activity or by nucleic acids with increased specific promoter activity according to embodiment a) regulated, wherein the genes are heterologous with respect to the nucleic acids with promoter activity.
• the regulation of the transcription of genes in the microorganism by nucleic acids according to the invention with promoter activity or by inventive nucleic acids with increased specific promoter activity according to embodiment ah) is achieved by bh1) one or more nucleic acids according to the invention having promoter activity, optionally with increased specific promoter activity into the genome of the microorganism so that the transcription of one or more enzymes or genes bh2) introduces one or more genes into the genome of the microorganism, so that the transcription of one or more of the genes introduced under the control of the endogenous invention, under the control of introduced nucleic acid with promoter activity, optionally with increased specific promoter activity Nucleic acids with promoter activity, optionally with increased specific promoter activity, or bh3) one or more nucleic acid constructs containing a nucleic acid according to the invention with promoter activity, optionally with increased specific promoter activity, and functionally linked one or more nucleic acids to be transcribed into the microorganism.
• the invention further relates, in a preferred embodiment, to a method for reducing the transcription rate of genes in microorganisms in comparison to the wild type by a) the specific promoter activity in the microorganism of endogenous inventive nucleic acids having promoter activity, which regulate the transcription of the endogenous genes Reduced compared to wild-type or br) nucleic acids with reduced specific promoter activity according to embodiment a) in the genome of the microorganism brings, so that the transcription endogenous genes under the control of the introduced nucleic acid with reduced promoter activity occurs.
• the invention further relates to a method for altering or causing the expression rate of a gene in microorganisms in comparison to the wild type by c) altering the specific expression activity in the microorganism of endogenous expression units according to the invention, which regulate the expression of the endogenous genes, in comparison to the wild type or d) Regulation of the expression of genes in the microorganism by expression units according to the invention or by expression units according to the invention with modified specific expression activity according to embodiment c), wherein the genes are heterologous with respect to the expression units.
• the alteration or causation of the expression rate of genes in microorganisms in comparison to the wild type can take place in that in the microorganism the specific expression activity is changed, ie increased or decreased.
• the extension of the distance between Shine-Dalgarno sequence and the translational start codon usually leads to a change, a reduction or else an increase in the specific expression activity.
• a change in the specific expression activity can also be achieved by shortening or lengthening the sequence of the Shine-Dalgarno region (ribosomal binding site) in its distance from the translational start codon by deletions or insertions of nucleotides. But also by the fact that the sequence of the Shine-Dalgarno region is changed so that the homology to complementary 3 'Page 16S rRNA either increased or decreased.
• an increase or reduction in comparison with the wild type is understood to mean an increase or reduction of the specific activity compared to the expression unit of the wild-type according to the invention, thus for example with respect to SEQ ID NO.
• the modification or causation of the expression rate of genes in microorganisms compared to the wild type can be carried out by regulating the expression of genes in the microorganism by expression units according to the invention or by expression units with altered specific expression activity according to embodiment c) according to the invention are heterologous with respect to the expression units.
• This is preferably achieved by introducing into the genome of the microorganism one or more expression units according to the invention, optionally with altered specific expression activity, so that the expression of one or more endogenous genes takes place under the control of the introduced expression units or d2) introduces several genes into the genome of the microorganism, so that the expression of one or more of the introduced genes is under the control of the endogenous expression units of the invention, optionally with altered specific expression activity, or d3) one or more 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, into which microorganism introduces.
• one or more expression units according to the invention are introduced into the genome of the microorganism such that expression of one or more endogenous genes takes place under the control of the introduced expression units or
• embodiment d2) introduces one or more genes into the genome of the microorganism so that the expression of one or more of the introduced genes takes place under the control of the endogenous expression units according to the invention, optionally with altered specific expression activity, or
• nucleic acid constructs containing an expression unit according to the invention, optionally mrt altered specific expression activity, and functionally linked one or more nucleic acids to be expressed brings in the microorganism.
• embodiment d2) introduces one or more exogenous genes into the genome of the microorganism, so that the expression of one or more of the introduced genes takes place under the control of the endogenous expression units according to the invention, optionally with altered specific expression activity, or
• nucleic acid constructs containing an expression unit according to the invention, optionally with altered specific expression activity, and functionally linked one or more exogenous nucleic acids to be expressed, into which microorganism introduces.
• the insertion of genes according to embodiment d2) can be carried out so that the gene is integrated into coding regions or non-coding regions. Preferably, the insertion takes place in non-coding regions.
• the insertion of nucleic acid constructs according to embodiment d3) can be effected chromosomally or extrachromosomally. Preferably, the insertion of the
• nucleic acid constructs are also referred to below as expression cassettes.
• embodiment d) it is also preferred to use expression units according to the invention with modified specific expression activity according to embodiment c). These can be present in the microorganism and prepared in embodiment d), as described in embodiment d), or introduced into the microorganism in isolated form.
• ie regulates the expression of genes in the microorganism by expression units according to the invention or by expression units with increased specific expression activity according to embodiment c), wherein the genes are heterologous with respect to the expression units.
• the regulation of the expression of genes in the microorganism by expression units according to the invention or by expression units with increased specific expression activity according to embodiment c) is achieved by introducing one or more expression units according to the invention, optionally with increased specific expression activity, into the genome of the microorganism, so that the expression of one or more endogenous genes under the control of the introduced expression units, optionally with increased specific expression activity occurs, or dh2) introduces one or more genes into the genome of the microorganism, so that the expression of one or more of the genes introduced under the Kon control the endogenous expression units according to the invention, given if with increased specific expression activity, or dh3) one or more nucleic acid constructs containing an expression unit according to the invention, optionally with increased specific expression activity, and functionally linked one or more nucleic acids to be expressed, introduced into the microorganism.
• the invention further relates to a method for reducing the expression rate of genes in microorganisms compared to the wild type by
• dr introduces expression units with reduced specific expression activity according to embodiment (s) into the genome of the microorganism such that the expression of endogenous genes takes place under the control of the introduced expression units with reduced expression activity.
• the genes are selected from the group nucleic acids encoding a protein from the biosynthetic pathway of fine chemicals, which genes may optionally contain further regulatory elements.
• the genes are selected from the group nucleic acids encoding a protein from the biosynthetic pathway of proteinogenic and non-proteinogenic amino acids, nucleic acids encoding a protein from the biosynthetic pathway of nucleotides and nucleosides, nucleic acids encoding a protein from the biosynthetic pathway of organic acids, nucleic acids encoding a protein from the biosynthetic pathway of lipids and fatty acids, nucleic acids encoding a protein from the biosynthetic pathway of diols, nucleic acids encoding a protein from the biosynthetic pathway of conhonomate hydrates , Nucleic acids encoding a protein from the biosynthetic pathway of aromatic compound, nucleic acids encoding a protein from the biosynthetic pathway of vitamins, nucleic acids
• the proteins from the biosynthetic pathway of amino acids selected from the group aspartate kinase, aspartate semialdehyde dehydrogenase, diaminopimelate dehydrogenase, diaminopimelate decarboxylase, dihydrodipicolinate synthetase, dihydrodipicolinate reductase, glyceraldehyde-3-phosphate dehydrogenase , 3-phosphoglycerate kinase, pyruvate carboxylase, triosephosphate isomerase, transcriptional regulator LuxR, transcriptional regulator LysR1, transcriptional regulator LysR2, malate-quinone oxidoreductase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrognease , Transketolase, transaldolase, homoserine O-acetyltransferase, cystahionine gamma synthase,
• Preferred proteins and nucleic acids encoding these proteins of the above-described proteins from the biosynthetic pathway of amino acids are protein sequences or nucleic acid sequences of microbial origin, preferably from bacteria of the genus Corynebacterium or Brevibacterium, preferably from coryneform bacteria, more preferably from Corynebacterium glutamicum.
• a further example of a particularly preferred protein sequence and the corresponding nucleic acid sequence encoding this protein from the biosynthetic pathway of amino acids is the sequence of fructose-1, 6-bisphosphatase 2, or else termed fbr2 (SEQ ID NO: 51). and the corresponding nucleic acid sequence encoding a fructose-1,6-bisphosphatase 2 (SEQ ID NO: 50).
• Another example of a particularly preferred protein sequence and the corresponding nucleic acid sequence encoding this protein from the biosynthetic pathway of amino acids is the sequence of the protein in sulfate reduction, also called RXA077, (SEQ ID NO: 4) and encoding the corresponding nucleic acid sequence a protein in sulfate reduction (SEQ ID NO: 3)
• proteins from the biosynthesis pathway of amino acids have in each case the amino acid sequence given in Table 1 for this protein, the respective protein in each case having a different proteinogenic amino acid on at least one of the amino acid positions indicated for this amino acid sequence in Table 2 / Column 2 the respective amino acid indicated in Table 2 / Column 3 in the same line.
• the proteins have the amino acid indicated in Table 2 / Column 4 in the same row on at least one of the amino acid positions given in Table 2 / Column 2 for the amino acid sequence.
• the proteins given in Table 2 are mutated proteins of the biosynthetic pathway of amino acids which have particularly advantageous properties and are therefore particularly suitable for expression of the corresponding nucleic acids by the promoter according to the invention and for the production of amino acids.
• the mutation T311 leads to I. switching off the 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 strain which is obtainable from the American Type Culture Collection under the name ATCC 13032 or the nucleic acid sequences referred to in Table 1.
• a Corynebacterium glutamicum strain which is obtainable from the American Type Culture Collection under the name ATCC 13032 or the nucleic acid sequences referred to in Table 1.
• Corynebacterium glutamicum it is preferable for Corynebacterium glutamicum to use the codon usage of Corynebacterium glutamicum.
• the codon usage of the particular organism can be determined in a manner known per se from databases or patent applications which describe at least one protein and a gene which codes for this protein from the desired organism.
• the SacB method is known to the person skilled in the art and is described, for example, in Schwarz A, Tauch A, Jäger W, Kalinowski J, 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. 1994 Jul 22; 145 (1): 69-73 and Blomfield IC, Vaughn V, rest RF, Eisenstein Bl .; Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon; Mol Microbiol. 1991 Jun; 5 (6): 1447-57.
• the change or causation of the transcription rate and / or expression rate of genes in microorganisms by introducing nucleic acids according to the invention with promoter activity or expression units according to the invention carried out in the microorganism.
• the change or causation of the transcription rate and / or expression rate of genes in microorganisms by introducing the above-described nucleic acid constructs or expression cassettes into the microorganism.
• the invention therefore further relates to an expression cassette comprising
• At least one further nucleic acid sequence to be expressed ie a gene to be expressed, and optionally other genetic control elements, such as a terminator,
• the nucleic acid sequence to be expressed is at least one nucleic acid encoding a protein from the biosynthetic pathway of fine chemicals.
• the nucleic acid sequence to be expressed is particularly preferably selected from the group of nucleic acids encoding a protein from the biosynthesis pathway of proteinogenic and non-proteinogenic amino acids, nucleic acids encoding a protein from the biosynthetic pathway of nucleotides and nucleosides, nucleic acids encoding a protein from the biosynthetic pathway of organic acids , Nucleic acids encoding a protein from the biosynthetic pathway of lipids and fatty acids, Nucleic acids encoding a protein from the biosynthetic pathway of diols, Nucleic acids encoding a protein from the biosynthetic pathway of Konhienhydraten, Nucleic acids encoding a protein from the biosynthetic pathway of aromatic compound, Nucleic acids encoding a protein from the Biosynthetic pathway of vitamins, nucleic acids encoding a protein from the biosynthetic pathway of cofactors and nucleic acids encoding a protein from the bio
• Preferred proteins from the biosynthetic pathway of amino acids are described above and their examples in Tables 1 and 2.
• the physical position of the expression unit relative to the gene to be expressed is selected so that the expression unit regulates the transcription and preferably also the translation of the gene to be expressed and thus enables the formation of one or more proteins.
• the "enabling education” involves constitutively increasing the formation, weakening or blocking the formation under specific conditions and / or increasing the formation under specific conditions.
• the “conditions” include: (1) adding a component to the culture medium, (2) removing a component from the culture medium, (3) replacing a component in the culture medium with a second component, (4) increasing the temperature of the culture medium, (5) Lowering the temperature of the culture medium, and (6) regulating the atmospheric conditions, such as the oxygen or nitrogen concentration, in which the culture medium is maintained.
• the invention further relates to an expression vector comprising an expression cassette according to the invention described above.
• Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P.H. et al., Eds. Elsevier, Amsterdam-New York-Oxford, 1985). Vectors other than plasmids are also to be understood as meaning all other vectors known to the person skilled in the art, such as, for example, phages, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be autonomously replicated in the host organism or replicated chromosomally.
• Particularly suitable plasmids are those which are replicated in coryneform bacteria.
• Numerous known plasmid vectors such. PZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1 (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 pGAL.
• Other plasmid vectors e.g. B.
• pCLiK5MCS or those based on pCG4 (US-A 4,489,160) or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)) or pAG1 (US-A 5,158,891), can be in be used in the same way.
• 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 pBGS8 (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 invention further relates to a genetically modified microorganism, wherein the genetic alteration leads to a change or causation of the transcription rate of at least one gene compared to the wild type leads and is conditioned by
• one or more nucleic acids with promoter activity according to claim 1, optionally with altered specific promoter Aktiv2011 brings into 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 according to claim 1, optionally with modified specific promoter Aktiv2011, takes place or
• the invention further relates to a genetically modified microorganism having an increased or caused transcription rate of at least one gene compared to the wild type, wherein
• the transcription of genes in the microorganism is regulated by promoter-activated nucleic acids according to claim 1 or by nucleic acids with increased specific promoter activity according to embodiment ah), wherein the genes are heterologous with respect to the promoter-active nucleic acids.
• bh1 introduces one or more nucleic acids with promoter activity according to claim 1, optionally with increased specific promoter activity, into the genome of the microorganism such that the transcription of one or more endogenous genes under the control of the introduced nucleic acid with promoter activity, optionally with increased specific promoter, is active , or
• bh2 introduces one or more genes into the genome of the microorganism, so that the transcription of one or more of the introduced genes under the control of endogenous nucleic acids with promoter activity according to claim 1, optionally with increased specific promoter Aktiv expedijan takes place, or
• Promoter Akt.2011 according to claim 1, optionally with increased specific promoter Aktivreli, and functionally linked to one or more, to be transcribed nucleic acids, brings in the microorganism.
• the invention further relates to a genetically modified microorganism having a reduced transcription rate of at least one gene compared to the wild type, wherein
• one or more reduced-promoter nucleic acids according to embodiment a) have been introduced into the genome of the microorganism such that the transcription of at least one endogenous gene is under the control of the microorganism Nucleic acid with reduced promoter Aktivrt2011 takes place.
• the invention further relates to a genetically modified microorganism, wherein the genetic modification leads to a change or causation of the expression rate of at least one gene in comparison with the wild type and is caused by
• d1) introduces one or more expression units according to claim 2 or 3, optionally with altered specific expression activity, into the genome of the microorganism such that the expression of one or more endogenous genes under the control of the incorporated expression units according to claim 2 or 3, optionally with altered specific expression activity, takes place or
• d2 introduces one or more genes into the genome of the microorganism such that the expression of one or more of the introduced genes takes place under the control of the endogenous expression units according to claim 2 or 3, optionally with altered specific expression activity, or
• nucleic acid constructs containing an expression unit according to claim 2 or 3, optionally with altered specific expression activity, and functionally linked to one or more nucleic acids to be expressed, into which microorganism introduces.
• the invention further relates to a genetically modified microorganism with increased or caused expression rate of at least one gene compared to the wild type, wherein ch) the specific expression activity in the microorganism of at least one endogenous expression units according to claim 2 or 3, which regulates the expression of the endogenous genes, compared to the wild type increased or
• dh2 introduces one or more genes into the genome of the microorganism, so that the expression of one or more of the introduced genes under the control of the endogenous expression units according to claim 2 or 3, optionally with increased specific expression activity occurs, or
• nucleic acid constructs containing an expression unit according to claim 2 or 3 optionally mrt increased specific expression activity, and functionally linked one or more to be expressed nucleic acids, brings in the microorganism.
• the invention further relates to a genetically modified microorganism having a reduced expression rate of at least one gene in comparison with the wild type, wherein
• the invention relates to a genetically modified microorganism comprising an expression unit according to claim 2 or 3 and functionally linked to a gene to be expressed, wherein the gene is heterologous with respect to the expression unit.
• This genetically modified microorganism particularly preferably contains an expression cassette according to the invention.
• the present invention particularly preferably relates to genetically modified microorganisms, in particular coryneform bacteria, which contain a vector, in particular pendulum vector or plasmid vector, which carries at least one recombinant nucleic acid construct according to the definition of the invention.
• the genes described above are at least one nucleic acid encoding a protein from the biosynthetic pathway of fine chemicals.
• the genes described above are selected from the group of nucleic acids encoding a protein from the biosynthetic pathway of proteinogenic and non-proteinogenic amino acids, encoding nucleic acids encoding a protein from the biosynthesis pathway of nucleotides and nucleosides, encoding nucleic acids a protein from the biosynthetic pathway of organic acids, nucleic acids encoding a protein from the biosynthetic pathway of lipids and fatty acids, nucleic acids encoding a protein from the biosynthetic pathway of diols, nucleic acids encoding a protein from the conhydrated biosynthetic pathway, nucleic acids encoding a protein from the aromatic biosynthesis pathway Compound, nucleic acids encoding a protein from the biosynthetic pathway of vitamins, nucleic acids encoding a protein from the biosynthetic pathway of cofactors and nucleic acids encoding a protein from
• Preferred proteins from the biosynthetic pathway of amino acids are selected from the group aspartate kinase, aspartate-semialdehyde dehydrogenase, diaminopimelate dehydrogenase, diaminopimeate decarboxylase, dihydrodipicolinate synthetase, dihydrodipicolinate reductase, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate Kinase, pyruvate carboxylase, triosephosphate isomerase, transcript the regulatory regulator LuxR, transcriptional regulator LysR1, transcriptional
• Regulator LysR2 malate quinone oxidoreductase, glucose-6-phosphate dehydrogenase,
• Tetrahydrofolate reductase Tetrahydrofolate reductase, phosphoserine aminotransferase, phosphoserine
• threonine synthase threonine export carrier, threonine dehydratase, pyruvate oxidase, lysine exporter, biotin ligase, cysteine synthase, cysteine synthase II coenzyme B12-dependent methionine synthase, coenzyme B12-independent Methionine synthase activity, Sulfatadenyltransferase subunits 1 and 2, phosphoadenosine phosphosulfate reductase, ferredoxin sulfite reductase, ferredoxin NADP reductase, 3-phosphoglycerate dehydrogenase, RXA00655 regulator, RXN2910 regulator, arginyl t-RNA synthetase, phosphoenolpyruvate Carboxylase, threonine efflux protein, serine hydroxymethyltransferase, fruct
• Preferred microorganisms or genetically modified microorganisms are bacteria, algae, fungi or yeasts.
• microorganisms are, in particular, coryneform bacteria.
• Preferred coryneform bacteria are bacteria of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium thermoaminogenes, Corynebacterium molassecola 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
• the abbreviation ATCC means the American strain strain culture collection
• the abbreviation DSM the German Collection of Microorganisms.
• ATCC American Type Culture Collection, Rockville, Md., USA.
• FERM Fermentation Research Institute, Chiba, Japan
• NRRL ARS Culture Collection, Northern Regional Research Laboratory, Peoria, IL, USA
• DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
• nucleic acids with promoter activity according to the invention and the expression units according to the invention make it possible to regulate the metabolic pathways to specific biosynthetic products in the above-described inventive genetically modified microorganisms by means of the above-described inventive methods.
• metabolic pathways which lead to a specific biosynthetic product by causing or increasing the transcription rate or expression rate of genes of this biosynthetic pathway in which the increased protein amount to an increased total activity of these proteins of the desired biosynthetic pathway and thus to an increased metabolic flux to the desired biosynthetic product leads.
• 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 to a reduced total activity of these proteins of the unwanted biosynthetic pathway and thus to increased metabolic flux to the desired biosynthetic product leads.
• 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. men.
• the transcription rate or expression rate of various genes must be increased or reduced.
• At least one altered, ie increased or reduced, transcription rate or expression rate of a gene can be attributed to a nucleic acid according to the invention having promoter activity or expression unit according to the invention.
• Transcription rates or expression rates of other genes in the genetically modified microorganism can, but need not go back to the nucleic acids according to the invention with Promotorassirtör or the expression units of the invention.
• the invention therefore further relates to a process for the preparation of biosynthetic products by culturing genetically modified microorganisms according to the invention.
• Preferred biosynthetic products are fine chemicals.
• fine chemical is well known in the art and includes compounds produced by an organism and used in various industries, such as, but not limited to, the pharmaceutical, agricultural, cosmetics, food and feed industries. These compounds include organic acids such as tartaric acid, itaconic acid and diaminopimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides and nucleotides (as described, for example, in Kuiminaka, A. (1996) Nucleotides and related compounds 6, Rehm et al., Eds.
• VCH Weinheim and the citations contained therein
• lipids saturated and unsaturated fatty acids (for example arachidonic acid), diols (for example propanediol and .alpha., Pp. 561-612 Butanediol), carbohydrates (eg hyaluronic acid and trehalose), aromatic compounds (for example aromatic amines, vanillin and indigo), vitamins and cofactors (as described in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A27, "Vitamins", p. 443-613 (1996) VCH: Weinheim and the citations contained therein, and Ong, AS, Niki, E. and Packer, L.
• amino acids comprise the basic structural units of all proteins and are therefore essential for normal cell function.
• amino acid is known in the art.
• the proteinogenic amino acids of which there are 20 species, serve as structural units for proteins in which they are linked by peptide bonds, whereas the non-proteinogenic amino acids (of which hundreds are known) do not usually occur in proteins (see Ullmann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97 VCH: Weinheim (1985)).
• the amino acids may be in the D or L configuration, although L-amino acids are usually the only type found in naturally occurring proteins. Biosynthesis and degradation pathways of each of the 20 proteinogenic amino acids are well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L.
• the "essential" amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine
• the "essential" amino acids are converted into the remaining 11 by simple biosynthetic pathways converted to "nonessential" amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine).
• Higher animals have the ability to synthesize some of these amino acids, but the essential amino acids must be ingested for normal protein synthesis.
• Lysine is an important amino acid not only for human nutrition, but also for monogastric animals such as poultry and pigs.
• Glutamate is most commonly used as a flavor additive (monosodium glutamate, MSG) as well as widely used in the food industry, as are aspartate, phenylalanine, glycine and cysteine.
• Glycine, L-methionine and tryptophan are all used in the pharmaceutical industry.
• Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are used in the pharmaceutical and cosmetics industries. Threonine, tryptophan and D- / L-methionine are widely used feed additives (Leuchtenberger, W. (1996) Amino acids - technical production and use, pp. 466-502 in Rehm et al., (Ed.) Biotechnology Vol. 6, chapters 14a, VCH: Weinheim).
• amino acids are also useful as precursors for the synthesis of synthetic amino acids and proteins, such as N-acetylcysteine, S-carboxymethyl-L-cysteine, (S) -5-hydroxytryptophan, and others, in Ullmann's Encyclopedia of Industrial Chemistry. Bd. A2, pp. 57-97, VCH, Weinheim, 1985.
• Ketoglutarate an intermediate in the citric acid cycle.
• Glutamine, proline and arginine are each produced sequentially from glutamate.
• the biosynthesis of serine is carried out in a three-step process and starts with 3-phosphoglycerate (an intermediate in glycolysis), and this oxidation results after oxidation, transamination and hydrolysis steps.
• Cysteine and glycine are each produced from serine, the former by condensation of homocysteine with serine, and the latter by transfer of the side chain ⁇ -carbon atom to tetrahydrofolate, in a serine transhydroxymethylase catalyzed reaction.
• Phenylalanine and tyrosine are synthesized from the precursors of the glycolysis and pentose phosphate pathway, erythrose 4-phosphate and phosphoenolpyruvate in a 9-step biosynthetic pathway that differs only in the last two steps after the synthesis of prephenate. Tryptophan is also produced from these two starting molecules, but its synthesis takes place in an 11-step pathway.
• Tyrosine can also be prepared from phenylalanine in a reaction catalyzed by phenylalanine hydroxylase.
• Alanine, valine and leucine are biosynthetic products of pyruvate, the end product of glycolysis. Aspartate is formed from oxalacetate, an intermediate of the citrate cycle.
• Asparagine, methionine, threonine and lysine are each produced by conversion of aspartate.
• Isoleucine is formed from threonine.
• histidine is formed from 5-phosphoribosyl-1-pyrophosphate, an activated sugar.
• Amino acids the amount of which exceeds the protein biosynthetic demand of the cell, can not be stored, and instead are degraded to provide intermediates for the major metabolic pathways of the cell (for review, see Stryer, L., Biochemistry, 3rd Ed. 21 "Amino Acid Degradation and the Urea Cycle, 495-516 (1988)).
• Vitamins, cofactors and nutraceuticals comprise another group of molecules. Higher animals have lost the ability to synthesize them and thus need to ingest them, although they are readily synthesized by other organisms, such as bacteria. These molecules are either biologically active molecules per se or precursors of biologically active substances that serve as electron carriers or intermediates in a variety of metabolic pathways. In addition to their nutritional value, these compounds also have significant industrial value as dyes, antioxidants and catalysts or other processing aids. (For an overview of the structure, activity and industrial applications of these compounds see, for example, Ullmann's Encyclopedia of Industrial Chemistry, "Vitamins", Vol. A27, pp. 443-613, VCH: Weinheim, 1996).
• vitamin is known in the art and includes nutrients that are needed by an organism for normal function, but can not be synthesized by that organism itself.
• the group of vitamins may include cofactors and nutraceutical compounds.
• cofactor includes non-proteinaceous compounds that are necessary for the occurrence of normal enzyme activity. These compounds may be organic or inorganic; the cofactor molecules of the invention are preferably organic.
• nutraceutical includes food additives that are beneficial to the health of plants and animals, especially humans. Examples of such molecules are vitamins, antioxidants and also certain lipids (e.g., polyunsaturated fatty acids).
• Thiamine (Vitamin B ⁇ is produced by chemical coupling of pyrimidine and thiazole
• Riboflavin (vitamin B 2 ) is synthesized from guanosine 5'-triphosphate (GTP) and ribose 5'-phosphate. In turn, riboflavin is used to synthesize flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).
• the family of compounds collectively referred to as "vitamin B6" eg, pyridoxine, pyridoxamine, pyridoxal-5'-phosphate and the commercially used pyridoxine hydrochloride) are all derivatives of the common structural unit 5-hydroxy-6-methylpyridine.
• Panthothenate (pantothenic acid, R - (+) - N- (2,4-dihydroxy-3,3-dimethyl-1-oxobutyl) - ⁇ -alanine) can be prepared either by chemical synthesis or by fermentation.
• the last steps in pantothenate biosynthesis consist of the ATP-driven condensation of ⁇ -alanine and pantoic acid.
• the enzymes responsible for the biosynthetic steps for the conversion to pantoic acid, to ⁇ -alanine and to the condensation in pantothenic acid are known.
• the metabolically active form of pantothenate is coenzyme A, whose biosynthesis proceeds through 5 enzymatic steps.
• Pantothenate pyridoxal-5'-phosphate, cysteine and ATP are the precursors of coenzyme A. These enzymes not only catalyze the formation of pantothenate but also the production of (R) -pantoic acid, (R) -pantolactone, (R) - Panthenol (provitamin B 5 ), pantethein (and its derivatives) and coenzyme A.
• the biosynthesis of biotin from the precursor molecule pimeloyl-CoA in microorganisms has been extensively studied, and several of the genes involved have been identified. It has been found that many of the corresponding proteins are involved in Fe cluster synthesis and belong to the class of nifS proteins.
• the lipoic acid is derived from octanoic acid and serves as a coenzyme in energy metabolism, where it becomes part of the pyruvate dehydrogenase complex and the ⁇ -ketoglutarate dehydrogenase complex.
• the folates are a group of substances that are all derived from folic acid, which in turn is derived from L-glutamic acid, p-aminobenzoic acid and 6-methylpterin.
• the biosynthesis of folic acid and its derivatives, starting from the metabolic intermediates guanosine 5'-triphosphate (GTP), L-glutamic acid and p-aminobenzoic acid has been extensively studied in certain microorgan
• Corrinoids such as the cobalamins and especially vitamin B 12
• the porphyrins belong to a group of chemicals that are characterized by a tetrapyrrole ring system.
• the biosynthesis of vitamin B 12 is sufficiently complex that it has not yet been fully characterized, but now much of the enzymes and substrates known.
• Nicotinic acid (nicotinate) and nicotinamide are pyridine derivatives, also referred to as "niacin”.
• Niacin is the precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adrenine dinucleotide phosphate) and their reduced forms.
• purine and pyrimidine metabolism and their corresponding proteins are important targets for the treatment of tumors and viral infections.
• purine or pyrimidine includes nitrogenous bases which are part of the nucleic acids, coenzymes and nucleotides.
• nucleotide includes the basic structural units of the nucleic acid molecules that contain a nitrogen-containing base, a pentose sugar (in the case of RNA, the sugar is ribose;
• nucleoside includes molecules which serve as precursors of nucleotides, but unlike the nucleotides have no phosphoric acid moiety.
• nucleotides that do not form nucleic acid molecules but serve as energy stores (i.e., AMPs) or coenzymes (i.e., FAD and NAD).
• the purine and pyrimidine bases, nucleosides and nucleotides also have other uses: as intermediates in the biosynthesis of various fine chemicals (eg thiamine, S-adenosyl-methionine, folates or riboflavin), as energy sources for the cell (eg ATP or GTP) and for chemicals themselves are commonly used as flavor enhancers (eg, IMP or GMP) or for many medical applications (see, for example, Kuninaka, A., (1996) Nucleotides and Related Compounds in Biotechnology Vol. 6, Rehm et al., Ed. VCH: Weinheim, pp. 561-612.) Enzymes involved in purine, pyrimidine, nucleoside or nucleotide metabolism are also increasingly serving as targets against which chemicals for plant protection, including fungicides, herbicides and insecticides, are being developed ,
• the purine nucleotides are synthesized via a series of steps via the lnosine 5'-phosphate (IMP) intermediate from ribose-5-phosphate, resulting in the production of guanosine 5'-monophosphate (GMP) or adenosine 5'-monophosphate (AMP ), from which the triphosphate forms used as nucleotides can be easily prepared. These compounds are also used as energy stores, so that their degradation provides energy for many different biochemical processes in the cell.
• the Pyrimidinbiosynthe- se via the formation of uridine 5'-monophosphate (UMP) from ribose-5-phosphate. In turn, UMP is converted to cytidine 5'-triphosphate (CTP).
• the deoxy forms of all nucleotides are prepared in a one-step reduction reaction from the diphosphate ribose form of the nucleotide to the diphosphate-deoxyribose form of the nucleotide. After phosphorylation, these molecules can participate in DNA synthesis.
• Trehalose consists of two molecules of glucose linked together by ⁇ , ⁇ -1, 1 bonding. It is commonly used in the food industry as a sweetener, as an additive for dried or frozen foods, and in beverages. However, it is also used in the pharmaceutical, cosmetics and biotechnology industries (see, for example, Nishimoto et al., (1998) U.S. Patent No. 5,759,610; Singer, MA and Lindquist, S. Trends Biotech. 16 (1998) 460-467; Paiva, CLA and Panek, AD Biotech Ann. Rev. 2 (1996) 293-314; and Shiosaka, MJ Japan 172 (1997) 97-102). Trehalose is produced by enzymes from many microorganisms and naturally released into the surrounding medium from which it can be recovered by methods known in the art.
• biosynthetic products are selected from the group of organic acids, proteins, nucleotides and nucleosides, both proteinogenic and non-proteinogenic amino acids, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors, enzymes and proteins.
• Preferred organic acids are tartaric acid, itaconic acid and diaminopimelic acid
• nucleosides and nucleotides are described, for example, in Kuninaka, A. (1996) Nucleotides and related compounds, pp. 561-612, Biotechnology Vol. 6, Rehm et al., Ed. VCH: Weinheim and the citations contained therein.
• Preferred biosynthetic products are also lipids, saturated and unsaturated fatty acids such as arachidonic acid, diols such as propanediol and butanediol, carbohydrates such as hyaluronic acid and trehalose, aromatic compounds such as aromatic amines, vanillin and indigo, vitamins and cofactors such for example, described in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A27, "Vitamins", pp. 443-613 (1996) VCH: Weinheim and the citations contained therein; and Ong, AS, Niki, E. and Packer, L.
• 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 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 genes in the microorganism is regulated by expression units according to the invention or by expression units according to the invention with increased specific expression activity according to embodiment ch), the genes being heterologous with respect to the expression units,
• genes are selected from the group of nucleic acids encoding an aspartate kinase, nucleic acids encoding an aspartate semialdehyde dehydrogenase, nucleic acids encoding a 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 Reg
• one or more expression units according to the invention into the genome of the microorganism brings, so that the expression of one or more of these endogenous genes under the control of the introduced expression units according to the invention, optionally with increased specific expression activity occurs, or
• dh2 introduces one or more of these genes into the genome of the microorganism such that the expression of one or more of the introduced genes is under the control of the endogenous expression units of the invention, optionally with increased specific expression activity, or
• nucleic acid constructs containing an expression unit according to the invention, optionally with increased specific expression activity, and functionally linked to one or more nucleic acids to be expressed, into which microorganism introduces.
• a further preferred embodiment of the process for the production 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 the group aspartate kinase activity, aspartate-semialdehyde dehydrogenase activity , Diaminoperilate dehydrogenase activity, diaminopimelate decarboxylase activity, dihydrodipicolinate synthetase activity, dihydridipicolinate reductase activity, glyceraldehyde-3-phosphate dehydrogenase activity, 3-phosphoglycerate kinase activity, pyruvate carboxylase activity , Triosephosphate isomerase activity, transcriptional regulator activity LuxR, activity of transcriptional regulator LysR1, activity of transcriptional regulator LysR2, malate-quinone-oxo-reductase activity, glucose-6-phosphate de-dehydrogenase activity, 6-phosphoglucon
• 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 Have decarboxylase activity and threonine synthase activity.
• 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 genes in the microorganism is regulated by expression units according to the invention or by expression units according to the invention with increased specific expression activity according to embodiment ch), wherein the genes are heterologous with respect to the expression units,
• genes are selected from the group of nucleic acids encoding an aspartate kinase, nucleic acids encoding an aspartate semialdehyde dehydrogenase, nucleic acids encoding a homoserine dehydrogenase, nucleic acids encoding a glyceraldehyde-3-phosphate dehydrogenase, nucleic acids encoding a 3-phosphoglycerate kinase, encoding nucleic acids Pyruvate carboxylase, nucleic acids encoding a triosephosphate isomerase, nucleic acids encoding a 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 aspartate
• dh1 introduces one or more expression units according to the invention, optionally with increased specific expression activity, into the genome of the microorganism so that the expression of one or more of these endogenous genes takes place under the control of the introduced expression units according to the invention, optionally with increased specific expression activity, or
• dh2 introduces one or more of these genes into the genome of the microorganism, so that the expression of one or more of the genes introduced takes place under the control of the endogenous expression units according to the invention, optionally with increased specific expression activity, or
• nucleic acid constructs containing an expression unit according to the invention, optionally with increased specific expression activity, and functionally linked to one or more nucleic acids to be expressed, into which microorganism introduces.
• 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 Hor ⁇ oserine 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 expression of genes in the microorganism is regulated by expression units according to the invention or by expression units according to the invention with increased specific expression activity according to embodiment ch), the genes being heterologous with respect to the expression units,
• genes are selected from the group of nucleic acids encoding an aspartate kinase, nucleic acids encoding an aspartate semialdehyde dehydrogenase, nucleic acids encoding a glyceraldehyde-3-phosphate dehydrogenase, nucleic acids encoding a 3-phosphoglycerate kinase, nucleic acids encoding a pyruvate carboxylase, encoding nucleic acids 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 quin
• dh1 introduces one or more expression units according to the invention, optionally with increased specific expression activity, into the genome of the microorganism so that the expression of one or more of these endogenous genes takes place under the control of the introduced expression units according to the invention, optionally with increased specific expression activity, or
• dh2 introduces one or more of these genes into the genome of the microorganism such that the expression of one or more of the introduced genes is under the control of the endogenous expression units of the invention, optionally with increased specific expression activity, or
• nucleic acid constructs containing an expression unit according to the invention, optionally with increased specific expression activity, and functionally linked to one or more nucleic acids to be expressed, into which microorganism introduces.
• a further preferred embodiment of the method for the production of threonine described above is characterized in that the genetically modified microorganisms in addition to the wild type additionally an increased activity, at least one of the activities selected from the group aspartate kinase activity, aspartate semialdehyde dehydrogenase activity , Glycerol-dehyd-3-phosphate dehydrogenase activity, 3-phosphoglycerate kinase activity, pyruvate carboxylase activity, triosephosphate isomerase activity, threonine synthase activity, threonine export carrier activity, transaldolase activity, transketolase se activity, glucose-6-phosphate dehydrogenase activity, malate-quinone
• Oxidoreductase activity Oxidoreductase activity, homoserine kinase activity, biotin ligase activity,
• OpcA activity 1-phosphofructokinase activity, 6-phosphofructokinase activity, fructose 1, 6 bisphosphatase activity, 6-phosphogluconate dehydrogenase and homoserine
• a further particularly 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 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, dihydrodipicolinate reductase activity, asparaginase Activity, aspartate decarboxylase activity, lysine exporter activity, acetolactate synthase activity, ketol-A reductoisomerase activity, branched chain aminotransferase activity,
• nucleic acid according to the invention having promoter activity and / or an expression unit according to the invention.
• activity of a protein in enzymes means the enzyme activity of the corresponding protein, in other proteins, for example structure or transport proteins, the physiological activity of the proteins.
• the enzymes are usually able to convert a substrate into a product or to catalyze this conversion step.
• the "activity" of an enzyme is understood to mean the amount of substrate or amount of product converted by the enzyme in a certain time.
• the amount of substrate converted by the enzyme is compared to the wild type or the amount of product formed increases.
• this increase in "activity" in all the activities described above and below is at least 5%, more preferably at least 20%, more preferably at least 50%, even more preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, especially at least 600% of the "wild-type activity".
• the amount of substrate or the amount of product formed is thus reduced by the enzyme compared to the wild type in a certain time.
• a reduced activity is preferably understood to mean the partial or substantially complete interruption or blocking of the functionality of this enzyme in a microorganism, which is 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 activity)
• the activity in the microorganism is reduced by at least 5%, more preferably by at least 5%, more preferably by at least 50%, more preferably by 100% compared to the wild type.
• “reduction” also means the complete absence of the corresponding activity.
• the activity of certain enzymes in genetically modified microorganisms and in the wild type and thus the increase or reduction of the enzyme activity can be determined by known methods, such as enzyme assays.
• a pyruvate carboxylase is understood as meaning a protein which has the enzymatic activity of converting pyruvate into oxaloacetate.
• a pyruvate carboxylase activity is understood as meaning the amount of pyruvate reacted or the amount of oxaloacetate formed in a certain time by the protein pyruvate carboxylase.
• the amount of pyruvate reacted or the amount of oxaloacetate formed is thus increased by the protein pyruvate carboxylase compared to the wild type in a specific time.
• this increase in pyruvate carboxylase activity is at least 5%, more preferably at least 20%, more preferably at least 50%, even more preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, especially at least 600% of the pyruvate carboxylase activity of wild type.
• a phosphoenolpyruvate carboxykinase activity as the enzyme activity of a phosphoenolpyruvate carboxykinase.
• a phosphoenolpyruvate carboxykinase is meant a protein having the enzymatic activity of converting oxaloacetate to phosphoenolpyruvate.
• phosphoenolpyruvate carboxykinase activity is understood as meaning the amount of oxaloacetate or the amount of phosphoenolpyruvate reacted in a certain time by the protein phosphoenolpyruvate.
• the amount of oxaloacetate or the amount of phosphoenolpyruvate formed is thus reduced in a certain time by the protein phosphoenolpyruvate carboxykinase in comparison to the wild type.
• a reduction in phosphoenolpyruvate carboxykinase activity includes a reduction in the amount of phosphoenolpyruvate carboxykinase to a substantially complete absence of phosphoenolpyruvate carboxykinase (i.e., lack of detectability of phosphoenolpyruvate carboxykinase activity or lack of immunological detectability of the phosphoenolpyruvate carboxykinase).
• the phosphoenolpyruvate carboxykinase activity is reduced by at least 5%, more preferably by at least 20%, more preferably by at least 50%, more preferably by 100%, compared to the wild type.
• “reduction” also means the complete absence of phosphoenolpyruvate carboxykinase activity.
• the additional increase of activities can be effected by different ways, for example by switching off inhibitory regulation mechanisms on expression and protein level or by increasing the gene expression of nucleic acids coding the proteins described above against the wild type.
• the increase in the gene expression of the nucleic acids encoding the proteins described above with respect to the wild type can also be effected by different routes, for example by induction of the gene by activators or as described above by increasing the promoter activity or increasing the expression activity or by introducing one or more gene copies into the microorganism.
• 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
• biosynthetic products in particular L-lysine, L-methionine and L-threonine
• L-lysine in addition to the expression or amplification of a gene to eliminate unwanted side reactions
• 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.
• Introduction of the nucleic acid can be chromosomally or extrachromosomally, ie by increasing the number of copies on the chromosome and / or a copy of the gene on a replicating plasmid in the host microorganism.
• the introduction of the nucleic acid preferably takes place chromosomally, in particular by the SacB method described above.
• any gene encoding one of the proteins described above can be used for this purpose.
• genomic nucleic acid sequences from eukaryotic sources containing introns in the event that the host microorganism is unable or unable to express the corresponding proteins, preferably already processed nucleic acid sequences, such as the corresponding cDNAs to use.
• the reduction of the above-described activities in microorganisms is carried out by at least one of the following processes:
• Introducing at least one sense ribonucleic acid sequence for inducing a cosuppression or an expression cassette ensuring its expression Introducing at least one DNA- or protein-binding factor against the corresponding gene, RNA or protein or an expression cassette ensuring its expression; introducing at least one RNA-degrading viral nucleic acid sequence or an expression cassette ensuring its expression
• 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.
• 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 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 discontinuously in the batch process (batch cultivation) or in the fed batch (feed batch). method) or repeated fed batch method (repetitive feed method) for the production of biosynthetic products, in particular L-lysine, L-methionine and L-threonine, are cultured.
• method or repeated fed batch method (repetitive feed method) for the production of biosynthetic products, in particular L-lysine, L-methionine and L-threonine, are cultured.
• a summary of known cultivation methods can be found in the textbook by Chmiel (Bioreatechnik 1. Introduction to bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994) ) to 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.O, 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
• sulfur source for the production of fine chemicals in particular of methionine
• inorganic compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, but also organic sulfur compounds, such as mercaptans and thiols.
• Phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.
• Chelating agents can be added to the medium to keep the metal ions in solution.
• Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
• the fermentation media used according to the invention usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine.
• growth factors and salts are often derived from complex 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 about 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 can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like.
• All media components are sterilized either by heat (20 min at 1, 5 bar and 121 ° C) or by sterile filtration.
• the components can 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 batchwise, as desired.
• the temperature of the culture is normally between 15 ° C and 45 C C, preferably at 25 ° C to 40 ° C and can be kept constant during the experiment or altered.
• 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.
• antifoams such as As fatty acid polyglycol, are used.
• the medium can be selected selectively acting substances such. As antibiotics, are added.
• oxygen or oxygen-containing gas mixtures such. B. ambient air, registered in the culture.
• the temperature of the culture is usually 20 ° 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 Fermentatio ⁇ sbrühe can then be further processed, for example.
• the biomass may be wholly or partly by Separationsmetho- 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, thickened or concentrated.
• This concentrated fermentation broth can then be worked up by freeze drying, spray drying, spray granulation or by other methods.
• biosynthetic products in particular L-lysine, L-
• Methionine and L-threonine continue to purify.
• 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, with the same or different re chromatography resins are used.
• the person skilled in the art is familiar with the choice of suitable chromatography resins and their most effective use.
• the purified product may be concentrated by filtration or ultrafiltration and stored at a temperature at which the stability of the product is maximized.
• biosynthetic products can be obtained in different forms, for example in the form of their salts or esters.
• the identity and purity of the isolated compound (s) can be determined by techniques of the prior art. These include high performance
• SEQ. ID. NO. 6 grol 1: 5'- agtcgacacgatgaatccctccatgagaaaa-3 '
• the primers were used in a PCR reaction with chromosomal DNA of C. glutamicum ATCC 13032. With this approach, a DNA fragment corresponding to the expected size of 427 bp could be amplified.
• the following oligonucleotides have been defined.
• SEQ. ID. NO. 7 SEQ. ID. NO. 7: pyc6: 5'-tttttctcatggagggattcatcgtgtcgactcacatcttcaacgcttccag-3 '
• SEQ. ID. NO. 8 pyc3: 5'-cccgcagcaacgcacgcaagaaa-3 '
• the primers were used in a PCR reaction with chromosomal DNA of C. glutamicum ATCC13032. With this approach, a DNA fragment corresponding to the expected size of 1344 bp could be amplified.
• the primers grol 1 and pyc6 contain an overlapping sequence and are homologous to each other at their 5 'ends.
• PCR products obtained above were used as template for another PCR in which the following primers were used.
• SEQ. ID. NO. 12 pyd 4: 5'- ccggcgaagtgtctgctcgcgtga-3 '
• the primers were used in a PCR reaction with chromosomal DNA of C. glutamicum ATCC13032. With this approach, a DNA fragment corresponding to the expected size of 487 bp could be amplified. This DNA fragment was cloned into the vector pCR2.1 (Invertrogen GmbH, Düsseldorf, Germany). A 593 bp SpeTi XbaI fragment was then subsequently cloned into the vector pK19 mob sacB Psod ask, which had previously been digested with the restriction enzyme Nhel. The resulting plasmid was named pK19 mob sacB Pgro pycA + US (SEQ ID NO: 14). Until this step, all cloning was performed in Escherichia coli XL-1 Blue (Stratagene, Amsterdam, Nierderouche).
• CM plates (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 A-gar (Difco)) with kanamycin (25 ⁇ g / ml), several transconjugants were obtained.
• sucrose-resistant clones were checked for their kanamycin sensitivity. For 15 of the tested clones, in addition to the resistance to sucrose, a sensitivity to kanamycin was also detected. Whether the desired exchange of the natural expression unit by the Pgro expression unit had also occurred was checked by polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• chromosomal DNA was isolated from the parent strain and the 15 clones. For this purpose, the respective clones were removed from the agar plate using a toothpick and suspended in 100 ⁇ l H 2 O and boiled at 95 ° C. for 10 min. 10 ⁇ l each The resulting solution was used as a template in the PCR.
• the primers used were oligonucleotides which are homologous to the Pgro expression unit and the pycA gene.
• PCR conditions were chosen as follows: pre-denaturation: 5 min at 95 ° C; Denaturation for 30 sec at 95 ° C; Hybridization for 30 sec at 56 ° C; Amplification for 1 min at 72 ° C; 30 cycles ,; End extension 5 min at 72 ° C.
• pre-denaturation 5 min at 95 ° C
• Denaturation for 30 sec at 95 ° C
• Hybridization for 30 sec at 56 ° C
• Amplification for 1 min at 72 ° C
• 30 cycles 30 cycles
• End extension 5 min at 72 ° C End extension 5 min at 72 ° C.
• the digestion of the cells was carried out with the aid of a Ribolyzers (3 x 30 sec at level 6, Fa.
• FIG. 1 shows a 10% SDS gel of Pgro pycA clones.
• ampicillin resistance and replication origin of the vector pBR322 with the oligonucleotide primers SEQ ID NO: 15 and SEQ ID NO: 16 were amplified by means of the polymerase chain reaction (PCR).
• the oligonucleotide primer SEQ ID NO: 15 in 5'-3 'direction contains the restriction endonuclease sites Smal, BamHI, Nhel and Ascl and the oligonucleotide primer SEQ ID NO: 16 in 5'-3 'direction, the restriction endonuclease sites Xbal, Xhol, Notl and Dral.
• 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 Jolla, 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 blunt ends of the DNA fragment were ligated together with the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation mixture according to standard methods as in Sambrook et al. (Molecular Clooning, A Laboratory Manual, Cold Spring Harbor, described (1989)), transformed into competent E. coli XL-1Blue (Stratagene, La Jolla, 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: 17 and SEQ ID NO: 18.
• SEQ ID NO: 17 5'-GAGATCTAGACCCGGGGATCCGCTAGCGGGCTGCTAAAGGAAGCGGA-3 '
• SEQ ID NO: 18 5'-GAGAGGCGCGCCGCTAGCGTGGGCGAAGAACTCCAGCA-3 '
• the oligonucleotide primer SEQ ID NO: 17 in the 5'-3 'direction contains the restriction endonuclease Xbal, SmaI, BamHI, NheI and oligonucleotide primers SEQ ID NO: 18 in the 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 Jolla, 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 subsequently re-gated with GFX TM PCR, DNA and Gel Band Purification. fication kit (Amersham Pharmacia, Freiburg) according to the manufacturer.
• 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 (Röche 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-1Blue (Stratagene, La Jolla, USA).
• Selection for plasmid-carrying cells was achieved by plating on ampicillin (50 ⁇ g / ml) and kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virolgy, 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 (Röche 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-1Blue (Stratagene, La Jolla, 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: 19 and SEQ ID NO: 20.
• the oligonucleotide primers SEQ ID NO: 19 and SEQ ID NO: 20 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 Jolla, 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 (Röche 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, (1989)), into competent E. coli XL-1Blue (Stratagene, La Jolla,
• 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 pCUK5.
• SEQ ID NO: 21 For the extension of pCLiK ⁇ by a "multiple cloning site" (MCS), the two synthetic, largely complementary oligonucleotides SEQ ID NO: 21 and SEQ ID NO: 22, the restriction endonuclease sites Swal, Xhol, Aatl, Apal, Asp718, Mlul, Ndel, Spei, EcoRV, Sall, Clal, BamHI, XbaI and Smal, by heating together to 95 ° C and slowly cooling to a double-stranded DNA fragment.
• the vector pCLiK ⁇ 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 aid of the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer with the synthetic double-stranded DNA fragment 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 Jolla, 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 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: 23.
• 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 SEQ ID NO 24 and SEQ ID NO 25, 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.
• 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. This was followed by restriction enzymes
• the vector pClik ⁇ MCS SEQ ID NO: 23 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 (Röche 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 Jolla, 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 PmetA metA 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 method was modified in such a way that the oligonucleotidepri SEQ ID NO: 27 and SEQ ID NO: 30 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: 26 was cleaved with the restriction enzymes Ncol and Spei (Röche 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: 23 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 used together with the PCR fragment and the metA-
• 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 pCLiK5MCS PGroESmetA is listed as SEQ ID NO: 31.
• Example 10 MetA Activities The strain Corynebacterium glutamicum ATCC13032 was in each case transformed with the plasmids pClik ⁇ MCS, pClik MCS PmetA metA and pCLiK5MCS PGroESmetA according to the method described (Liebl, et al. (1989) FEMS Microbiology Letters 53: 299-303). The transformation mixture was plated on CM plates, which additionally contained 20mg / l kanamycin, to achieve selection for plasmid-containing cells. Obtained Kan-resistant clones were picked and separated.
• C. glutamicum strains containing one of these plasmid constructs were dissolved in MMA medium (40 g / l sucrose, 20 g / l (NH 4 ) 2 SO 4 , 1 g / l KH 2 PO 4 , 1 g / l K 2 HPO 4 , 0.25 g / l MgSO 4 x 7H 2 O, 54 g aces, 1 ml CaCl 2 (10 g / l), 1 ml protocatechoate (300 mg / 10 ml), 1 ml of trace element solution (10 g / l FeSO 4) x / H 2 O, 10 g / l MnSO 4 ⁇ H 2 O, 2 g / l ZnSO 4 ⁇ 7 H 2 O, 0.2 g / l CuSO 4 , 0.02 g / l NiCl 2 ⁇ 6 H 2 O), 100 ⁇ g / l vitamin B 12 , 0.3 mg /
• 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. A kinetics was then recorded at 412 nm for 10 min.
• the activity of MetA could be significantly increased by using the heterologous expression unit.
• 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 32 to SEQ ID NO 33, the chromosomal DNA as template and Pfu Turbo Polymerase (Stratagene) were amplified 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, amplifies the terminus region of the groEL gene.
• the obtained DNA fragments of about 60 bp size were 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 XbaI and BcnI (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: 23 was cut with the restriction enzyme XbaI 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 60 bp fragment using the Rapid DNA Ligation Kit (Röche 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 Jolla, 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.
• the oligonucleotide primers SEQ ID NO 35 and SEQ ID NO 36, the chromosomal DNA as template, and Pfu Turbo Polymerase (Stratagene) were amplified 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 the metA gene without sartcodone.
• the resulting DNA fragment of approximately 1.2 kb in 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 Ndel and Bcnl (Roche Diagnostics, Mannheim) and the DNA fragment was purified with GFX TM PCR, DNA and Gel Band Purification Kit.
• the vector pClik ⁇ MCS groEL term SEQ ID NO: 34 was cut with the restriction enzymes Ndel and Bcnl 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 (Röche 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 Jolla, 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 metA without start codon is listed as SEQ ID NO: 37.
• oligonucleotide primers SEQ ID NO 38 to SEQ ID NO 43, the chromosomal DNA as template and Pfu Turbo Polymerase (Stratagene) were amplified 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 the various expression units.
• Oligonucleotide primer 1701 (SEQ ID NO 38) served as the sense primer and was combined with the other oligonucleotide primers.
• Oligonucleotide primer 1828 5'-ctctcatatgcAATCCCTCCATGAGAAAATT-3 '
• oligonucleotide primer 1832 5'-ctctcatatgcAAtctctcATGAGAAAATTTTGTGTG-3 '
• SEQ ID NO 42 oligonucleotide primer 1833 ⁇ '-ctctcatatgcAActcctcATGAGAAAATTTTGTGTG-3 ' ⁇
• SEQ ID NO 43 oligonucleotide primer 1834 5'-cttfcatatgcAAtcccttcATGAGAAAATTTTGTGTG-3'
• the obtained DNA fragments of about 200 bp size were purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the vector pBS KS + (SEQ ID NO: 44) was cut with the restriction enzyme EcoRV ge-5 and a 2.9 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 fragments using the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer's instructions0 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 Jolla, USA).
• plasmid-carrying cells were achieved by plating on kanagycin (20 ⁇ g / ml) -containing LB agar (Lennox, 1955, Virology, 1: 190) .5
• the preparation of the plasmid DNA was carried out by methods and with materials from Quiagen , Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-6467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Weiherstadt).
• the resulting plasmids were designated pKS Pgro 1701/1828, pKS Pgro 1701/1831, pKS Pgro 1701/1832, pKS Pgro 1701/1833 and pKS Pgro 1701/1834.
• These plasmids were then cut with the restriction enzymes Ndel and Xhol.
• the obtained DNA fragments of about 200 bp size were isolated and purified with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
• the vector pCLiK ⁇ MCS metA without sartcodone SEQ ID NO: 37 was cut with the restriction enzymes Ndel and Xhol and a 5 kb fragment was isolated after electrophoretic separation with GFX TM PCR, DNA and Gel Band Purification Kit, ⁇
• the vector fragment was used together with the 200 bp fragment using the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer ligated and the ligation mixture according to standard methods as described in Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, described (1989)), into competent E. coli XL-1 Blue (Stratagene, La Jolla, USA). Selection for plasmid-bearing cells was achieved by plating on kanamycin (20 ⁇ g / ml) -containing LB agar (Lennox, 19 ⁇ 5, Virology, 1: 190).
• the preparation of the plasmid DNA was carried out according to methods and materials of the Fa. Quiagen. Sequencing reactions were performed according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-6467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt). 0 The resulting plasmids pCLiK5MCS Pgro 1701/1828 metA, pCLiK ⁇ MCS Pgro 1701/1831 metA, pCLiK5MCS Pgro 1701/1832 metA, pCLiK5MCS Pgro 1701/1833 metA and pCLiK5MCS Pgro 1701/1834 metA are listed as SEQ ID NO: 45 to 49. 5 The expression unit Pgro was changed by the choice of Olugonukleotide as described in Figure 2.
• the strain Corynebacterium glutamicum ATCC 13032 was in each case with the plasmids pClik ⁇ MCS, pClik MCS Pgro metA, pCLiK ⁇ MCS Pgro 1701/1828 metA,, 0 pCLiK ⁇ MCS Pgro 1701/1831 metA, pCLiK ⁇ MCS Pgro 1701/1832 metA, pCLiK ⁇ MCS Pgro 1701/1833 metA and pCLiK ⁇ MCS Pgro 1701/1834 according to the method described (Liebl, et al. (1989) FEMS Microbiology Letters 63: 299-303) transformed.
• the transformation mixture was plated on CM plates containing an additional 20 mg / L kanamycin to achieve selection for plasmid-containing cells. Obtained ⁇ Kan-resistant clones were picked and separated.
• the cells were spun down at 4 ° C and washed twice with cold Tris-HCl buffer (0.1%, pH 8.0). After re-centrifugation, the cells were taken up in cold Tris-HCl buffer (0.1%, pH 8.0) and an OD 600 of 160 was set.
• 1 ml of this cell suspension was transferred to 2 ml of ribolyser tubes from Hybaid and lysed in a Ribolyser from Hybaid at a rotation setting of 6.0 three times for 30 sec each.
• the lysate was clarified by centrifugation at 16,000 rpm at 4 ° C. for 30 minutes in an Eppendorf centrifuge, and the supernatant was transferred to a new Eppendorf cup.
• 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, ⁇ 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. The results are shown in Table 2a.

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