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  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/15—Corynebacterium

Definitions

• the invention relates to a process for the preparation of L-serine and to suitable gene sequences, vectors and microorganisms. 5
• the amino acid L-serine is used in human medicine, in the pharmaceutical industry, in the food industry and in animal nutrition. 10
• EP 0931833A2 it is described in EP 0931833A2 that increasing the biosynthetic enzyme phosphoserine phosphatase and phosphoserine transaminase is advantageous for L-serine formation. It is also described that a gene coding for D-3-phosphoglycerate dehydrogenase can be used for L-serine formation (EP 0931833A2, PCT WO 93/12235).
• coryneform bacteria produce L-serine in an improved manner after modification or elimination of the genes coding for folic acid synthesis.
• the bacteria according to the invention and the gene sequences according to the invention for enzymes or regulators which catalyze folic acid synthesis or are involved in the regulation of folic acid synthesis it is now possible to produce L-serine in a yield which is considerably higher than that of the strains not modified according to the invention.
• the L-serine production as well as the production of cysteine, tryptophan and methionine is increased by reducing the folic acid concentration in an amino acid-producing organism.
• the organism already produces L-serine before the modification according to the invention.
• the reduction in folic acid concentration can be achieved by reducing the synthesis of folic acid or its degradation.
• Folic acid can be obtained by targeted or undirected mutation of genes involved in the biosynthesis of folic acid.
• mutations leading to a reduction or elimination of folic acid production are the deletion mutation, insertion mutation, substitution mutation or point mutation of genes involved in the biosynthesis of folic acid.
• genetically modified or unaltered genes of proteins involved in the biosynthesis of folic acid may reduce or eliminate folic acid production by reducing or preventing the expression of genes involved in the biosynthesis of folic acid.
• Reduction or elimination of expression of genes involved in the biosynthetic pathway of folic acid may be achieved by altering, preferably attenuating, more preferably eliminating promoters, such as signal structures, repressor genes, activators, operators, attenuators, ribosome binding sites or start codons, terminators, or further - tion, preferably attenuation, particularly preferably elimination or attenuation of regulators or the stability of the transcripts can be achieved.
• promoters such as signal structures, repressor genes, activators, operators, attenuators, ribosome binding sites or start codons, terminators, or further - tion
• attenuation particularly preferably elimination or attenuation of regulators or the stability of the transcripts can be achieved.
• Farther regulatable promoters in particular attenuated promoters, can be used.
• the activity of the enzymes involved in the biosynthesis of folic acid can be achieved by reducing or eliminating the catalytic activity and the stability of the enzymes. The same effect can be achieved by altering the allosteric center or a feedback inhibition of the enzymes.
• a typical way to reduce or eliminate the activity of the enzymes is by protein modification, for example by phosphorylation or adenylation. It is also possible to increase the proteolytic degradation of enzymes involved in the biosynthesis pathway of folic acid.
• Typical enzymes whose activity can be reduced or eliminated in the manner described are GTP cyclohydrolase, neopterin triphosphate pyrophosphatase, neopterinololase, ⁇ -hydroxymethylpterin pyrophosphokinase, 4-amino-4-deoxy-chorismate synthase, 4-amino-4-deoxybenzoyl chorismate lyase, pteroate synthase, folate synthase and dihydrofolate reductase.
• the invention is also a vector, the one
• the vectors according to the invention contain tools which are suitable for the gene for the synthesis of 4-amino-4- deoxy-chorismate synthase and or 4-amino-4-deoxy-chorismate lyase.
• Seq. No. 1 The sequence for the deletion of the 4-amino-4-deoxy-chorismate synthase gene is shown in Seq. No. 1 shown.
• Seq. No. 2 shows the structure of a vector containing the Seq. No.1 carries.
• Seq. No. 4 shows the structure of a vector containing the Seq. No. 3 carries.
• Seq. No. 5 shows the structure of a vector containing the Seq. No. 5 for the deletion of the 4-amino-4-deoxy- chorismatsynthasegens and the 4-amino-4deoxy-chorismat lyasegens carries.
• Seq. No. 7 is a plasmid which is suitable for additionally increasing L-serine production. It corresponds to the plasmid shown in FIG. 4 with the designation pEC-T18mob2-serA fbr CB-.
• the structures responsible for the deletion can also be incorporated into other vector scaffolds which are suitable for the corresponding organism. Ren as vectorial 'come in addition to the commonly used vectors cyclic and linear vectors or phages in question.
• the figures show vectors which can be used by way of example for the changes according to the invention of the L-serine producing organisms. It shows :
• Fig.2 pK19mobsacB_pabC according to Seq. No. 4.
• the tools of sequences 1, 3 and 5 are introduced into vectors in L-serine production organisms which already produce L-serine prior to alteration.
• Suitable organisms are, for example, Corynebacteria, such as Corynebacterium glutamicum or Brevibacterium. Enterobacteria, bacillaceae or hay species, which have a reduced folic acid concentration, can also be used as production organisms.
• Corynebacteria such as Corynebacterium glutamicum or Brevibacterium.
• Enterobacteria, bacillaceae or hay species, which have a reduced folic acid concentration can also be used as production organisms.
• the invention will be described in more detail:
• the invention relates to a process for the production of L-serine by fermentation using coryneform bacteria, in which the genes coding for folic acid synthesis are modified, switched off or altered in their expression or in Folic acid bacteria naturally a folic acid deficiency is induced or the regulation of folic acid synthesis is influenced in such a way that folic acid deficiency arises. Since the folic acid synthesis starts from an intermediate of the nucleotide synthesis as well as an intermediate of the synthesis of aromatic amino acids, these corresponding reactions can in principle also be modified or eliminated, provided that nucleotides and aromatic amino acids themselves are still sufficiently available, as for example by external supplementation the case is. In addition, folic acid deficiency may also be induced by deletion or modification of regulators that control the expression of genes of folic acid or its associated metabolic pathways.
• the strains used preferably produce L-serine before the folic acid synthesis is modified.
• the term modification includes the attenuation of folic acid synthesis genes and the complete deletion of polysynthetic genes. These include non-directed mutagenesis and directed recombinant DNA techniques. With the help of these methods, for example, genes of folic acid synthesis in the chromosome can be deleted. Suitable methods are described in Shufer et al. (Gene (1994) 145: 69-73) or also Link et al. (J. Bacteriology (1998) 179: 6228-6237). Also, only parts of the gene can be deleted or mutated fragments of genes can be exchanged.
• An advantageous embodiment of the method according to the invention is, for example, the inventively modified C. glutamicum strain ATCC13032DpykDsdaADpabABpserABC, which, inter alia, carries a deletion in the pabAB gene.
• mutagenesis methods include undirected methods involving chemical reagents such as. B. N-methyl-N-nitro-N-nitrosoguanidine or UV irradiation for mutagenesis use, with subsequent search of the desired microorganisms for reduction or loss of folic acid synthesis activity.
• genes of folic acid synthesis can be reduced by altering the signal structures for gene expression.
• Signal structures are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators. Information on this is the expert z. In Patent Application WO 96/15246, Boyd and Murphy (J. Bacteriol 1988: 170: 5949), Voskuil and Chambliss (Nucleic Acids Res. 1998. 26: 3548, Jensen and Hammer (Biotechnol. 1998 58: 191), in Patek et al.
• a regulation of the translation is possible by, for example, the stability of the m-RNA is reduced. This can be achieved by weakening the stability by additional and / or altered sequences at the 5 'end or 3' end of the gene. Examples are for genes from Bacillus subtilis (Microbiology (2001) 147: 1331-41) or yeast (Trends Biotechnol., 1994, 12: 444-9). It is also possible to influence enzyme activity by intrinsic proteolytic activity as described (Mol Microbiol. (2005) 57: 576-91).
• genes coding for the corresponding enzyme of low activity folic acid synthesis can be used. Mutations that lead to a change or reduction of the catalytic activity of enzyme proteins are known. Examples can be found in the work of Qiu and Goodman (J Biological Chemistry (1997) 272: 8611-8617), Sugimoto et al. (Bioscience Biotechnology and Biochemistry (1997) 61: 1760-1762) and Möckel ("The threonine dehydratase from Corynebacterium glutamicum: abolition of the allosteric regulation and structure of the enzyme", Reports from the Jülich Research Center, Jül-2906, ISSN09442952, Jülich , Germany, 1994).
• Patent 4,601,893, Schwarzer and Pühler Bio / Technology 9, 84-87 (1991), Reinscheid et al (Applied and Environmental Microbiology 60, 126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993) ) and in patent application WO 96/15246.
• genes of the folic acid synthesis of C. glutamicum can be reduced expressed or deleted or the enzyme activities can be reduced.
• L-serine in addition to the induced deficiency of folic acid, one or more of the genes selected from the group, • the serA gene coding for the 3-phosphoglycerate dehydrogenase,
• the serB gene coding for the phosphoserine phosphatase in particular to overexpress or alleles of these genes, in particular
• metC gene coding for cystathionin lyase The metC gene coding for cystathionin lyase, the sdaA gene coding for serine dehydratase,
• the microorganisms according to the invention comprise bacteria of the genus Corynebacterium or Brevibacterium, which are modified by classical and / or molecular genetic methods such that their metabolic flux increasingly proceeds in the direction of the biosynthesis of amino acids or their derivatives.
• the present invention encompasses all known amino acid production strains.
• those production strains are included, which the skilled person can produce by analogy with findings from other microorganisms, for example enterobacteria, bacillaceae or yeast species, by the conventional method.
• such amino acid production strains are also included in which the degradation of L-serine is altered or attenuated.
• the present invention also relates to a pabAB and pabC gene sequence Nos. 1, 3 and 5, which is characterized in that a portion of the sequence was cut out by means of a defined deletion, so that only inactivated or attenuated Aminodeoxy- chorismatsynthase or Aminodeoxychorismatlyase activity can result ,
• the pabAB and pabC gene sequences are preferably isolated from microorganisms of the genus Corynebacterium or Brevibacterium. By way of example, a few more specific microorganisms are listed here:
• the pabAB gene of C. glutamicum is replaced by known methods in the chromosome by a pABAB gene shortened by 1734 bp (J. Bacteriol. (1997) 179: 6228-37, Gene (1994) 145: 69-73).
• the following primers were synthesized derived from the publicly available genome sequence (NCBI Accession Number YP_225287; NC_006958):
• the primer pabAB-del-A starts 522 bp before the start of translation and pabAB-del-D 436 bp behind the translation stop of the pabAB gene.
• the primer pabAB-del-B is 21 bp behind the translation start
• the primer pabAB-del-C is 66 bp before the translation stop and both have complementary linker regions, as in Link et al. (J. Bacteriol. (1997) 179: 6228-37).
• PCR amplifications were carried out with the primer combination pabAB-del-A and pabAB-del-B and the primer combination pABAB-del-B and pabAB-del-C with chromosomal DNA of C.
• the PCR reaction was carried out in 30 cycles in the presence of 200 ⁇ M deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP), each 600 nM of the corresponding oligonucleotides,
• the amplification was carried out in 35 cycles in the presence of 200 ⁇ M deoxynucleotide triphosphates, 600 nM each of the corresponding oligonucleotide, 20 ng each of the isolated template DNA from the first PCR, 1/10 volume 10-fold Reaction buffer and 2.6 units of the Taq / Pwo ⁇ DNA polymerase mixture under the following conditions:
• the vector pK19mobsacBDpabC suitable for gene replacement was constructed for the deletion of the pabC gene of C. glutamicum.
• the primers required for PCR amplification were in turn derived from the publicly available genome sequence (NCBI accession number YP_225288.1, NC_006958). They are listed below:
• pabC-del-A 5'-GAGGATCCAATCATTGCTGAGCTGCGCAG-3 ' pabC-del-B:
• pabC-del-C 5 '-TGTTTAAGTTTAGTGGATGGGTCGGTGAAGCCCTGGAATGAA-3'
• the primer pabC-del-A starts 500 bp before the start of translation and pabC-del-D 500 bp behind the translation stop of the pabC gene.
• the primer pabC-del-B is 51 bp behind the translation start and pabC-del-C 48 bp before the translation stop.
• the last two primers each have complementary linker regions.
• the primer combination pabC-del-A and pabC-del-B became a 602 bp 5 'flanking region and the primer combination pabC-del-C and pabC-del-D a 597 bp 3' flanking region of deletierenden fragment amplified.
• the PCR reaction was carried out according to the standard methods, such as in example 1 with chromosomal DNA of C. glutamicum ATCC13032. After the PCR reaction, the DNA fragments obtained were isolated with the QIAExII gel extraction kit (Qiagen) and both fragments were isolated as
• the vector pK19mobsacBDpabABC suitable for gene replacement was constructed for the deletion of the pabABC gene of C. glutamicum.
• the PCR amplification primers required for this derived from the publicly available genome sequence (NCBI accession number YP_225287; YP_225288.1; NC_006958), are indicated below:
• the primer pabABC-del-A starts 500 bp before the start of translation and pabABC-del-D 500 bp behind the translation stop of the pabABC gene.
• Primer pabABC-del-B is 51 bp beyond the translation start of pabAB and pabABC-del-C 48 bp before the translation stop of pabC.
• the latter two primers each have complementary linker regions.
• the pabABC-del-A and pabABC-del-B pri- mary combination resulted in a 593 bp 5 'flanking region and, with the primer combination pabABC-del-C and pabABC-del-D, a 597 bp 3' flanking area of the to be deleted
• the PCR reaction was carried out according to standard methods such as in Example 1 with chromosomal DNA of C. glutamicum ATCC13032. After the PCR reaction, the DNA fragments obtained were isolated with the QIAExII gel extraction kit (Qiagen), and both fragments were used as template in another PCR. The primers pabABC-del-A and pabABC-del-D were used as primers.
• the PCR reaction was carried out by the standard methods such as, for example, in Example 1 with chromosomal DNA of C. glutamicum ATCC13032.
• the resulting 1169 bp DNA fragment containing the inactivated pabABC gene with a 2475 bp central deletion was isolated from a 0.8% agarose gel and ligated with the vector pK19mobsacB (Schäfer et Gene 145: 69-73 (1994). Escherichiae was used with the ligation mixture coli strain DH5 ⁇ mcr (Grant et al., Proceedings of the National Academy of Sciences of the United States of America (1990) 87: 4645-4649). The resulting plasmid pK19mobsacBDpabC (FIG. 3) was checked for its accuracy by restriction digestion and sequencing.
• EXAMPLE 4 Construction of folic acid auxotrophic strains C. glutamicum DsdaADpabAB, C. glutamicum DsdaADpabC, C. glutamicum DsdaADpabABC.
• pabAB-del-pr-u2 5'-TGGCTCACTTCGCTGGTCTTGTTG-S 'pabAB-del-pr-12 5'-GAATGGTTGCGGCGAGTGTCA-S'
• pabC-del-pr-u2 5 '-GTTGGGGGAGCAGGACGAGTGGT-3' pabC-del-pr-12 5'-TACGCGCATCTGGAAGCCTGGTTA-B '
• pabABC-del -pr-u2 5 '-CGTTCCGGCATCATTCTGGCTAAG-3'
• pabABC-del-pr-12 5 '-GCGACTCCGGGTTGTTCCTGATAA-3'
• the successful deletion gave a band of 1426 bp for the pabAB locus, a band of 1663 bp for the pabC genort, and a band of pabABC for the gene locus
• strains were designated as C. glutamicum DsdaADpabAB, C. glutamicum DsdaADpabC, and C. glutamicum DsdaADpabABC, respectively.
• the plasmid (FIG. 4) is composed of the vector pEC-T18mob2 (Curr. Microbiol. (2002) 45, 362-367), the corynebacterial genes serAfbr (Appl Microbiol Biotechnol. (2002) 60: 437-41) and serC and serB (German Patent Application 100 44 831.3).
• Tetracycline-resistant clones were tested by known standard methods for the presence and integrity of the plasmid pEC-T18mob2-serAfbrserCserB (Sambrook et al., (1989) Molecular Cloning, A Laboratory Manual, ColD Spring Harbor Laboratory Press) and a clone designated C. glutamicum DsdaADpabAB pserABC ,
• the strain C. glutamicum DsdaADpabAB pserABC was grown in complex medium (CgIII with 2% glucose, 5 ⁇ g / l tetracycline) and the fermentation cationsraedium CGXII (J Bacteriol (1993) 175: 5595-5603).
• the fermentation medium CGXII contained 0.1 or 1 mM folic acid.
• the strain C. glutamicum DsdaA pserABC was cultivated in the same way. At least two independent fermentations were ever carried out. After culturing for 30 hours at 30 0 C on a rotary shaker at 120 rpm accumulated in the medium L-serine quantity was determined.
• the analysis of the amino acid concentration was carried out by means of high pressure liquid chromatography (J Chromat)
• the plasmid pK19mobsacBDpyk (Arch Microbiol. (2004) 182: 354-63) was introduced into the strain C. glutamicum 13032DsdaADpabABC and selected for kanamycin resistance. Only such
• Clones in which the plasmid was integrated into the chromosomal pyk gene locus via homologous recombination were kanamycin resistant. A clone was excluded. which, when checked, showed the plasmid-mediated sucrose sensitivity and cultured in 50 ml of BHI medium (Brain Heart Infusion Medium, Difco Laboratories, Detroit, USA) without kanamycin and sucrose. Subsequently, 100 ⁇ l each of a 10-2,
• the parent strain C. glutamicum DpykDsdaADpabAB could be obtained from the starting strain.
• This strain was transformed with pEC-T18mob2-serAfbrserCserB as described in Example 5, thereby obtaining the strain C. glutamicum DpykDsdaADpabAB pserABC.
• This strain was used for L-serine formation as described in Example 5 in comparison with C. glutamicum DsdaADpabAB pserABC. The result is shown in Table 3.

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