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    • 专利摘要:
    The invention relates to a process for the preparation of L-amino acids by fermentation of coryneform bacteria, which comprises carrying out the following steps: a) fermentation of the desired L-amino acid- producing bacteria in which at least the zwf gene is amplified, b) concentration of the L-amino acid in the medium or in the cells of the bacteria andc) isolation of the L-amino acid produced.
    公开号:KR20020012578A
    申请号:KR1020017014792
    申请日:2000-07-05
    公开日:2002-02-16
    发明作者:버크케빈；잠헤르만；에크겔링로타르；모리츠베른트；두니칸엘.카.；므코르맥애쉴링；스테이플톤클리오나；뫼켈벳티나；티어바하게오르크
    申请人:펠드만 마르틴 및 봅 후베르트；데구사 아게；추후제출；포르슝스젠트룸 율리히 게엠베하；내셔널 유니버시티 오브 아일랜드；
    IPC主号:
    专利说明:

    ProcessProcess for the fermentative preparation of L-amino acids with amplification of the zwf gene}
    [1] The present invention relates to a method for preparing fermentation of L-amino acids, in particular L-lysine, L-threonine and L-tryptophan, using coryneform bacteria amplified with at least the zwf gene.
    [2] Prior art
    [3] L-amino acids are used in the nutritional, human drug and pharmaceutical industries of animals.
    [4] Amino acids are known to be produced by fermenting Coryneform bacteria, in particular Corynebacterium glutamicum strains. Because this is very important, research is being carried out to improve the manufacturing process. Improvements to the process can be achieved by means of fermentation means such as agitation and oxygen supply, or nutrient medium composition such as sugar concentration during fermentation, or by post-processing in the form of products by eg ion exchange chromatography, Or inherent productivity of the microorganisms themselves.
    [5] Mutagenesis, selection and mutant selection methods are used to improve the productivity of these microorganisms. Strains that are nutritive and produce L-amino acids, such as threonine, to metabolites that are resistant to or regularly important to antimetabolites, such as threonine analogs α-amino-β-hydroxyvaleric acid (AHV) Is obtained in this way.
    [6] Recombinant DNA technology methods have also been used for many years to improve the Corynebacterium glutamicum strains producing L-amino acids.
    [7] Purpose of the Invention
    [8] We aimed to provide an improved novel method of fermentative preparation of L-amino acids using coryneform bacteria.
    [9] Description of the invention
    [10] L-amino acids are used in the human drug, pharmaceutical industry, food industry, and in particular animal nutrition. Therefore, it is a general concern to provide improved novel methods of preparing amino acids.
    [11] The present invention provides a method for the production of L-amino acids, in particular L-lysine, L-threonine and L-tryptophan, by fermentation using coryneform bacteria which have been amplified, in particular overexpressed, a nucleotide sequence encoding the zwf protein (zwf gene). To provide.
    [12] Preferably the strains used already produce L-amino acids prior to amplification of the zwf gene.
    [13] Preferred embodiments are described in the claims.
    [14] The term "amplification" in this context refers to, for example, using a strong promoter or using a gene encoding a correspondingly active enzyme (protein) and optionally combining these means to increase the number of copies of the gene or genes. By increasing the intracellular activity of one or more enzymes (proteins) of the microorganism encoded by the corresponding DNA.
    [15] Microorganisms provided by the present invention can prepare L-amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. These are especially representative of coryneform bacteria of the genus Corynebacterium. Among the genera Corynebacterium, mention may be made of the species Corynebacterium glutamicum, which is known to those skilled in the art for the production of L-amino acids.
    [16] Suitable strains of the genus Corynebacterium, in particular Corynebacterium glutamicum species, are for example known wild type strains.
    [17] Corynebacterium glutamicum ATCC13032,
    [18] Corynebacterium acetoglutamicum ATCC15806,
    [19] Corynebacterium acetoacidophilum ATCC13870,
    [20] Corynebacterium thermoaminogenes FERM BP-1539,
    [21] Brevibacterium flavum ATCC14067,
    [22] Brevibacterium lactofermentum ATCC13869 and
    [23] Brevibacterium divaricatum ATCC14020, and
    [24] Mutants that produce L-amino acids prepared therefrom, for example, strains that produce L-threonine
    [25] Corynebacterium glutamicum ATCC21649,
    [26] Brevibacterium plaboom BB69,
    [27] Brevibacterium plaboom DSM5399,
    [28] Brevibacterium lactofermentum FERM-BP 269 and
    [29] Brevibacterium lactopfermentum TBB-10, and
    [30] For example, a strain that produces L-isoleucine
    [31] Corynebacterium glutamicum ATCC14309,
    [32] Corynebacterium glutamicum ATCC14310,
    [33] Corynebacterium glutamicum ATCC14311,
    [34] Corynebacterium glutamicum ATCC15168 and
    [35] Corynebacterium ammoniagenes ATCC6871, and
    [36] For example, a strain that produces L-tryptophan
    [37] Corynebacterium glutamicum ATCC21850 and
    [38] Corynebacterium glutamicum KY9218 (pKW9901), and
    [39] For example, a strain that produces L-lysine
    [40] Corynebacterium glutamicum FERM-P 1709,
    [41] Brevibacterium plaboom FERM-P 1708,
    [42] Brevibacterium lactofermentum FERM-P 1712,
    [43] Corynebacterium glutamicum FERM-P 6463,
    [44] Corynebacterium glutamicum FERM-P 6464,
    [45] Corynebacterium glutamicum ATCC13032,
    [46] Corynebacterium glutamicum DM58-1 and
    [47] Corynebacterium glutamicum DSM12866.
    [48] Coryneform bacteria have been found to produce L-amino acids, in particular L-lysine, L-threonine and L-tryptophan, in an improved way after overexpression of the zwf gene encoding zwf protein.
    [49] In addition, alleles of the zwf gene due to degeneracy of the genetic code or sensory mutations of neutral function can also be used.
    [50] Japanese Patent Application Laid-Open No. 9-224661 describes the nucleotide sequence of the glucose 6-phosphate dehydrogenase gene called zwf of Brevibacterium flavum MJ-223 (FERM BP-1497). . Japanese Laid-Open Patent Publication No. 9-224661 describes the N-terminal amino acid sequence of a zwf polypeptide as Met Val Ile Phe Gly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu.
    [51] However, this could not be confirmed. Instead, the following N-terminal amino acid sequence was found: Val Ser Thr Asn Thr Thr Pro Ser Ser Trp Thr Asn Pro Leu Arg Asp. Valyl radicals in the N-position can be separated off in the context of post-translational modification, and then Ser Thr Asn Thr Thr Pro Ser Ser Trp Thr Asn Pro Leu Arg Asp is obtained as the N-terminal amino acid sequence. do.
    [52] To amplify (eg, overexpress), the copy number of the corresponding gene is increased or the ribosome binding site of the promoter and regulatory region or upstream of the structural gene is mutated. Expression cassettes incorporated into the upstream stream of structural genes work in the same way. By inducible promoter, it is also possible to increase expression in the course of fermentative production of L-amino acids. Expression is likewise enhanced by means of extending the life of m-RNA. In addition, enzyme activity is also increased by preventing degradation of enzyme proteins. Genes or gene constructs are present in plasmids with varying copy numbers or integrated and amplified in chromosomes. In addition, the gene composition may be further overexpressed by changing the medium composition and culture process.
    [53] The guidelines herein refer to experts, in particular Martin et al. (Bio / Technology 5, 137-146 (1987)), Guerrero et al. (Gen 138, 35-41 (1994)), Tsuchiya ( Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), Eikmanns et al. (Gen 102, 93-98 (1991)), European Patent Specification 0 472 869, US Pat. No. 4,601,893, Schwarzer and Puhler (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)], WO 96/15246, Malumbres et al. Reference: Gene 134, 15-24 (1993)], Japanese Patent Application Laid-Open No. 10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)). And is also described in known genetics and molecular biology textbooks.
    [54] For example, zwf proteins are overexpressed using plasmids. For this purpose the E. coli-C. glutamicum shuttle vector pEC-T18mob2 shown in FIG. 1 is used. After incorporating the zwf gene into the KpnI / SalI cleavage region of pEC-T18mob2, the plasmid pEC-T18mob2zwf shown in FIG. 2 is formed.
    [55] Other plasmid vectors capable of replicating in C. glutamicum, for example pEKEx1 (Gen 102: 93-98 (1991), Eikmanns et al.) Or pZ8-1 [European Patent Publication 0 375 889] can be used.
    [56] In addition, for the preparation of L-amino acids, it is advantageous not only to amplify the zwf gene, but also to amplify one or more enzymes of a specific biosynthetic pathway, glycolysis, anaplerosis, pentose phosphate pathway or amino acid transport.
    [57] Thus, for example, especially for the production of L-threonine,
    [58] Hom gene encoding homoserine dehydrogenase (Molecular Microbiology 2, 63-72 (1988), Peoples et al.) Or hom dr allele encoding "feedback resistant" homoserine dehydrogenase [ See Gene 107, 53-59 (1991), Archer et al.,
    [59] Gap gene encoding glyceraldehyde 3-phosphate dehydrogenase (Journal of Bacteriology 174: 6076-6068 (1992), Eikmanns et al.),
    [60] Pyc gene encoding pyruvate carboxylase (Microbiology 144: 915-927 (1998), Peters-Wendisch et al.),
    [61] Maleate: mqo gene encoding quinone oxidoreductase (European Journal of Biochemistry 254, 395-403 (1998), Molenaar et al.),
    [62] Tkt gene encoding transketolase (Accession No. AB023377 of the European Molecular Biologicals Laboratories Databank (EMBL, Heidelberg, Germany)),
    [63] Gnd gene encoding 6-phosphogluconate dehydrogenase (Japanese Patent Laid-Open No. 9-224662);
    [64] ThrE gene, encoding threonine transport, see, eg, German Patent Application No. 199 41 478.5; DSM 12840],
    [65] Zwal gene [see: German Patent Application No. 199 59 328.0; DSM 13115] and
    [66] One or more genes selected from the group consisting of eno genes encoding enolase (see German Patent Application No. 199 41 478.5) can be amplified, in particular overexpressed, simultaneously.
    [67] Thus, for example, for the production of L-lysine,
    [68] DapA gene encoding dihydrodipicolinate synthase (European Patent Publication No. 0 197 335),
    [69] LysC gene encoding feedback resistant aspartate kinase (Molecular and General Genetics 224: 317-324 (1990), Kalinowski et al.),
    [70] Gap gene encoding glycerolaldehyde 3-phosphate dehydrogenase (Journal of Bacteriology 174: 6076-6068 (1992), Eikmanns),
    [71] A pyc gene encoding pyruvate carboxylase (German Patent Application No. 198 31 609),
    [72] Tkt gene encoding transketolase (Accession No. AB023377 of the European Molecular Biologicals Laboratories Databank (EMBL, Heidelberg, Germany)),
    [73] Gnd gene encoding 6-phosphogluconate dehydrogenase (Japanese Patent Laid-Open No. 9-224662);
    [74] LysE gene encoding lysine transport (German Patent Application No. 195 48 222),
    [75] Zwal gene [see: German Patent Application No. 199 59 328.0; DSM 13115] and
    [76] One or more genes selected from the group consisting of eno genes encoding enolase (see German Patent Application No. 199 47 791.4) can be amplified, in particular overexpressed, simultaneously.
    [77] In addition to the amplification of the zwf gene,
    [78] Pck gene encoding phosphoenol pyruvate carboxykinase (see German Patent Application No. 199 50 409.1; DSM 13047],
    [79] Pgi gene encoding glucose 6-phosphate isomerase (US Patent Application No. 09 / 396,478, DSM 12969),
    [80] PoxB gene, which encodes pyruvate oxidase, see German Patent Application No. 1995 197 975.7; DSM 13114] and
    [81] Zwa2 gene [see: German Patent Application No. 199 59 327.2; Simultaneously attenuating one gene selected from the group consisting of DSM 13113 may be more advantageous for the production of L-amino acids.
    [82] In addition to overexpression of the zwf protein, eliminating undesirable side reactions may be more advantageous for the production of L-amino acids. Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta , Vanek (eds.), Academic Press, London, UK, 1982.
    [83] The microorganisms prepared according to the present invention can be cultured continuously or discontinuously in a batch process (batch culture) or feed batch (feed process) or repeat feed batch process (repeated feed process) for the production of L-amino acids. Known culture methods are described in Chmiel's textbook (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Bioprocess Technology 1. Introduction to Biopress Technology (Gustav Fisher Verlag, Stuttgart, 1991)) or Storhas textbook). See Bioreaktoren und periphere Einrichtungen (Bioreactions and Peripheral Equipment) (Vieweg Verlag, Braunschweig / Wiesbaden, 1994).
    [84] The culture medium to be used must meet the requirements of the particular microorganism in a suitable way. Information on the culture medium of various microorganisms is described in the Handbook [Manual of Methods for General Bacteriology] of the American Society for Bacteriology (Washington DC, USA, 1981.) Sugars and carbohydrates such as glucose, water Cross, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as soybean oil, sunflower oil, peanut oil and coconut fat, fatty acids such as palmitic acid, stearic acid and linoleic acid, Alcohols such as glycerol and ethanol and organic acids such as acetic acid can be used as carbon sources These materials can be used alone or as a mixture Organic nitrogen-containing compounds such as pentose, Yeast extract, meat extract, malt extract, corn steep liquor, soy flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, carbonate rock Nitrogen and ammonium nitrate can be used as the nitrogen source Nitrogen source can be used alone or as a mixture Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium containing salt can be used as the phosphorus source. The medium should contain metal salts such as magnesium sulphate or iron sulphate necessary for growth, and finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used, and further suitable precursors can be added to the culture medium. The starting materials can be added to the culture in a single batch or fed during the culturing in a suitable manner.
    [85] Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH. Antifoams such as fatty acid polyglycol esters can be used to control bubble generation. Suitable substances with selective action, such as antibiotics, can be added to the medium to maintain the stability of the plasmid. An oxygen or oxygen containing gas mixture, such as air, is introduced into the culture to maintain anaerobic conditions. The temperature of the culture is generally 20 to 45 ° C, preferably 25 to 40 ° C. Continue incubation until the maximum amount of L-amino acid is formed. This goal is generally achieved within 10 to 160 hours.
    [86] Analysis of L-amino acids can be performed by anion exchange chromatography followed by ninhydrin derivatization as described by Spackman et al., Analytical Chemistry, 30, (1958), 1190, or It can be performed by reverse phase HPLC as described by Lindroth et al., Analytical Chemistry (1979) 51: 1167-1174.
    [87] Under the Budapest Treaty, the following microorganisms have been deposited in Deutsche Zaumlong Pureorgorgizmen und Celkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany):
    [88] Escherichia coli K-12 DH5α / pEC-T18mob2 (Accession No. DSM 13244)
    [89] The following figures are attached:
    [90] Figure 1: Map of plasmid pEC-T18mob2
    [91] Figure 2: Map of the plasmid pEC-T18mob2zwf
    [92] Figure 3: Map of plasmid pAMC1
    [93] Figure 4: Map of plasmid pMC1
    [94] Figure 5: Map of plasmid pCR2.1poxBint
    [95] Base pair numbers mentioned are approximate values obtained in the sense of reproducibility.
    [96] With respect to FIGS. 1 and 2:
    [97] The abbreviations used have the following meanings:
    [98] Tet: resistance gene to tetracycline
    [99] oriV: flami-encoding replication initiation of E. coli
    [100] RP4mob: mob region for moving the plasmid
    [101] rep: plasmid-encoded replication initiation point from C. glutakum plasmid pGA1
    [102] per: gene controlling the number of copies from pGA1
    [103] lacZ-alpha: lacZα gene fragment (N-terminus) of β-galactosidase gene
    [104] lacZalpha ': 5'-terminus of the lacZα gene fragment
    [105] 'lacZalpha: the 3'-terminus of the lacZα gene fragment
    [106] With respect to FIGS. 3 and 4:
    [107] The abbreviations used have the following meanings:
    [108] Neo r: neomycin / kanamycin resistance
    [109] ColE1 ori: Initiation point for replication of plasmid ColE1
    [110] CMV: cytomegalovirus promoter
    [111] lacP: lactose promoter
    [112] pgi: phosphoglucose isomerase gene
    [113] lacZ: part of β-galactosidase gene
    [114] SV40 3 'splice: 3' splice region of simian virus 40
    [115] SV40 polyA: polyadenylation domain of monkey virus 40
    [116] f1 (-) ori: origin of replication of filamentous phage f1
    [117] SV40 ori: Initiation point for replication of monkey virus 40
    [118] kan r: kanamycin resistant
    [119] pgi insert: an internal fragment of the pgi gene
    [120] ori: initiation of replication of plasmid pBGS8
    [121] Regarding Figure 5:
    [122] The abbreviations used have the following meanings:
    [123] ColE1 ori: Initiation point for replication of plasmid ColE1
    [124] lacZ: cloning residue of lacZα gene fragment
    [125] f1 ori: Initiation point for phage f1
    [126] KmR: Kanamycin Resistance
    [127] ApR: Ampicillin Resistance
    [128] poxBint: the internal fragment of the poxB gene
    [129] The meanings of the abbreviations for various restriction enzymes (eg BamHI, EcoRI, etc.) are known in the prior art and are described, for example, in Kessler and Holtke (Gen 47, 1-153 (1986)). Or Roberts et al., Nucleic Acids Research 27, 312-313 (1999).
    [130] Example
    [131] The following examples further illustrate the invention. The molecular biology techniques used, such as plasmid DNA isolation, restriction enzyme processing, ligation, standard transformation of E. coli (if not stated otherwise), are described in Sambrook et al. Molecular Cloning. A Laboratory Manual (1989) Cold Spring Harbor Laboratories, USA.
    [132] Example 1
    [133] Expression of the zwf gene
    [134] 1.1 Preparation of Plasmid pEC-T18mob2
    [135] E. coli -C. Glutamicum shuttle vector pEC-T18mob2 is constructed according to the prior art. Vectors are described in US Pat. No. 5,175,108; Replication region rep of plasmid pGA1 containing the replication effector per according to Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997); US Pat. No. 5,175,108; Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997), the tetracycline resistance-conferring tetA (Z) gene of plasmid pAG1 (see US Pat. No. 5,158,891; gene library entry at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA), accession number AF121000], replication region oriV of plasmid pMB1 [Sutcliffe, Cold Spring Harbor Symposium on Quantitative Biology 43, 77-90 (1979) )], lacZα gene fragment comprising lac promoter and multiple cloning region (mcs) [Norrander et al. Gene 26, 101-106 (1983)] and the mob region of plasmid RP4 (Simon et al, (1983) Bio / Technology 1: 784-791). The constructed vector is transformed in E. coli strain DH5α (Brown (ed.) Molecular Biology Labfax, BIOS Scientific Publishers, Oxford, UK, 1991). The selection of plasmid-carrying cells was determined by LB agar supplemented with 5 mg / l tetracycline (Sambrook et al., Molecular Cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep Kit (Qiagen) and tested by restriction enzymes EcoRI and HindIII followed by agarose gel electrophoresis (0.8%).
    [136] This plasmid is called pEC-T18mob2 and is shown in FIG. 1. This was deposited as DSM 13244 in the form of strain E. coli K-12 strain DH5αpEC-T18mob2 in Deutsche Zamlong pur microorganisms und Chellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany).
    [137] 1.2 Preparation of Plasmid pEC-T18mob2zwf
    [138] First the gene from Corynebacterium glutamicum ATCC13032 is amplified by polymerase chain reaction (PCR) using the following oligonucleotide primers:
    [139] zwf-forward:
    [140] 5'-TCG ACG CGG TTC TGG AGC AG-3 '
    [141] zwf-reverse:
    [142] 5'-CTA AAT TAT GGC CTG CGC CAG-3 '
    [143] PCR reactions were carried out in a thermocycler (PTC-100, MJ Research, Inc., Watertown, USA) under 200 μM of deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), in each case the corresponding oligonucleotide 1 μM, 100 ng of chromosomal DNA from Corynebacterium glutamicum ATCC13032, 1/10 volume reaction buffer 1/10 volume and heat resistant Taq- / Pwo-DNA polymerase mixture with Expand High Fidelity PCR System (manufactured by Roche Diagnotics, Mannheim, Germany) )) Perform 30 cycles in the presence of 2.6 units: 30 seconds at 94 ° C, 1 minute at 64 ° C and 3 minutes at 68 ° C.
    [144] The amplified fragment of about 1.8 kb in size is then linked into the SmaI cleavage region of the vector pUC18 using the SureClone Ligation Kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. E. coli strain DH5αmcr (Grant et al., Proceedings of the National Academy of Sciences of the United States of America USA (1990) 87: 4645-4649) is transformed in a full ligation batch. Transformants are identified using carbenicillin resistance on LB-agar plates containing 50 μg / ml carbenicillin. Flamismids are prepared from seven of the transformants and examined for the presence of 1.8 kb PCR fragments as inserts by restriction analysis. The recombinant plasmid thus formed is hereinafter referred to as pUC18zwf.
    [145] For the construction of pEC-T18mob2zwf, pUC18zwf was digested with KpnI and SalI, and the product was isolated using the NucleoSpin Extraction Kit (manufacturer: Macherey-Nagel (Duren, Germany)) according to the manufacturer's instructions, followed by KpnI and SalI. The vector pEC-T18mob2 was cleaved and dephosphorylated. E. coli strain DH5αmcr (Grant et al., Proceedings of the National Academy of Sciences of the United States of America USA (1990) 87: 4645-4649) is transformed in a full ligation batch. Transformants are identified using tetracycline resistance on LB-agar plates containing 5 μg / ml tetracycline. Flamismids are prepared from 12 of the transformants and examined for the presence of 1.8 kb PCR fragments as inserts by restriction analysis. The recombinant plasmid thus separated is referred to as pEC-T18mob2zwf (FIG. 2).
    [146] Example 2
    [147] Preparation of Amino Acid Producing Strains Using Amplified zwf Gene
    [148] L-lysine producing strain Corynebacterium glutamicum DSM 5715 is described in European Patent Publication No. 0 435 132, and L-threonine producing strain Brevibacterium flavomb DSM5399 to European Patent Publication No. 0 385 940 It is described. Both strains were deposited in the German Collection of Microorganisms and Cell Cultures, Deutsche Zamlong Pureorgorganizmen, Braunschweig, Germany, under the Budapest Treaty.
    [149] 2.1 Preparation of strains DSM5715 / pEC-T18mob2zwf and DSM5399 / pEC-T18mob2zwf
    [150] Strains DSM5715 and DSM5399 are transformed with plasmid pEC-T18mob2zwf using electroporation described in Lieb et al., FEMS Microbiology Letters, 53: 299-303 (1989). Selection of the transformants was 18.5 g / l Brain-Hert broth medium, 0.5 M sorbitol, 5 g / l Bakto-trypton, 2.5 g / l Bakto-east extract, supplemented with 5 mg / l tetracycline , LBHIS agar containing 5 g / l NaCl and 18 g / l bacto-agar. Incubate at 33 ° C. for 2 days.
    [151] In each case, the plasmid DNA was isolated from the transformation in a conventional manner (Peters-Wendisch et al., 1998, Microbiology 144, 915-927), digested with restriction endonucleases XbaI and KpnI, and then plasmid Is examined by agarose gel electrophoresis. The strains thus obtained are referred to as DSM5715 / pEC-T18mob2zwf and DSM5399 / pEC-T18mob2zwf.
    [152] 2.2 Preparation of L-Threonine
    [153] C. glutamicum strain DSM5399 / pEC-T18mob2zwf obtained in Example 2.1 is incubated in a nutrient medium suitable for threonine preparation and the threonine content in the culture supernatant is measured.
    [154] To this end, the strains are first incubated for 24 hours at 33 ° C. on agar plates containing the corresponding antibiotic (brain-heart agar containing 5 mg / l tetracycline). Starting from this agar plate culture, the preculture is seeded (10 ml of medium in a 100 ml Erlenmeyer flask). Complete medium CgIII is used as the medium for preculture.
    [155] Badge Cg III
    [156] NaCl 2.5g / ℓ
    [157] Bakto-Peptone 10g / ℓ
    [158] Bakto-Yeast Extract 10g / ℓ
    [159] Glucose (separately autoclaved) 2% (w / v)
    [160] pH is 7.4.
    [161] Tetracycline (5 mg / L) was added thereto. The precultures are incubated for 16 hours at 33 ° C. at 240 rpm on a shaker. The main culture is seeded from this preculture so that the initial OD (660 nm) of the main culture is 0.1. Medium MM is used as the main culture.
    [162] Badge MM
    [163] CSL (corn immersion liquid) 5g / ℓ
    [164] MOPS (morpholinopropanesulfonic acid) 20 g / ℓ
    [165] Glucose (separately autoclaved) 50 g / l
    [166] (NH 4 ) 2 SO 4 25g / ℓ
    [167] KH 2 PO 4 0.1 g / ℓ
    [168] MgSO 4 7H 2 O 1.0 g / ℓ
    [169] CaCl 2 · 2H 2 O 10mg / l
    [170] FeSO 4 7H 2 O 10mg / ℓ
    [171] MnSO 4 · H 2 O 5.0mg / ℓ
    [172] Biotin (sterile-filtered) 0.3 mg / l
    [173] Thiamine / HCl (sterile-filtered) 0.2 mg / l
    [174] L-Leucine (sterile-filtered) 0.1 g / l
    [175] CaCO 3 25g / ℓ
    [176] Aqueous ammonia is used to adjust the pH of CLS, MOPS and salt solution to 7 and autoclave. Subsequently, sterile substrate and vitamin solution are added as well as anhydrous autoclaved CaCO 3 .
    [177] Incubate in a 10 ml volume in a 100 ml Erlenmeyer flask with baffle. Tetracycline (5 mg / L) is added. Incubate at 33 ° C. and 80% atmospheric humidity.
    [178] After 72 hours, the OD value is measured at a wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of threonine produced is measured using an amino acid analyzer (Eppendorf-BioTronik (Hamburg, Germany)) by post-column derivatization using ion exchange chromatography and ninhydrin detection.
    [179] The experimental results are shown in Table 1.
    [180] StrainODL-threonine (g / ℓ) DSM539912.30.74 DSM5399 / pEC-T18mob2zwf10.21.0
    [181] 2.3 Preparation of L-Lysine
    [182] C. glutamicum strain DSM5715 / pEC-T18mob2zwf obtained in Example 2.1 is incubated in a nutrient medium suitable for lysine preparation and the lysine content in the culture supernatant is measured.
    [183] To this end, the strain is first incubated for 24 hours at 33 ° C. on an agar plate containing the corresponding antibiotic (brain-hert agar containing tetracycline (5 mg / L)). Starting from this agar plate culture, the preculture is seeded (10 ml of medium in a 100 ml Erlenmeyer flask). Complete medium CgIII is used as the medium for preculture.
    [184] Badge Cg III
    [185] NaCl 2.5g / ℓ
    [186] Bakto-Peptone 10g / ℓ
    [187] Bakto-Yeast Extract 10g / ℓ
    [188] Glucose (separately autoclaved) 2% (w / v)
    [189] pH is 7.4.
    [190] Tetracycline (5 mg / L) was added thereto. The precultures are incubated for 16 hours at 33 ° C. at 240 rpm on a shaker. The main culture is seeded from this preculture so that the initial OD (660 nm) of the main culture is 0.1. Medium MM is used for main culture.
    [191] Badge MM
    [192] CSL (corn immersion liquid) 5g / ℓ
    [193] MOPS (morpholinopropanesulfonic acid) 20 g / ℓ
    [194] Glucose (separately autoclaved) 58 g / l
    [195] (NH 4 ) 2 SO 4 25g / ℓ
    [196] KH 2 PO 4 0.1 g / ℓ
    [197] MgSO 4 7H 2 O 1.0 g / ℓ
    [198] CaCl 2 · 2H 2 O 10mg / l
    [199] FeSO 4 7H 2 O 10mg / ℓ
    [200] MnSO 4 · H 2 O 5.0mg / ℓ
    [201] Biotin (sterile-filtered) 0.3 mg / l
    [202] Thiamine / HCl (sterile-filtered) 0.2 mg / l
    [203] L-Leucine (sterile-filtered) 0.1 g / l
    [204] CaCO 3 25g / ℓ
    [205] Aqueous ammonia is used to adjust the pH of CLS, MOPS and salt solution to 7 and autoclave. Subsequently, sterilized substrate and vitamin solution are added as well as anhydrous autoclaved CaCO 3 .
    [206] Incubate in a 10 ml volume in a 100 ml Erlenmeyer flask with baffle. Tetracycline (5 mg / L) is added. Incubate at 33 ° C. and 80% atmospheric humidity.
    [207] After 72 hours, the OD value is measured at a wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of lysine produced is measured using an amino acid analyzer (Eppendorf-BioTronik (Hamburg, Germany)) by post-column derivatization using ion exchange chromatography and ninhydrin detection.
    [208] The experimental results are shown in Table 2.
    [209] StrainODL-lysine HCl (g / L) DSM571510.816.0 DSM5715 / pEC-T18mob2zwf7.217.1
    [210] Example 3
    [211] Construction of Gene Library of Corynebacterium glutamicum strain AS019
    [212] DNA library of Corynebacterium glutamicum strain AS019 (Yoshihama et al., Journal of Bacteriology 162, 591-597 (1985)) by O'Donohue, O'Donohue, M (1997). The Cloning and Molecular Analysis of Four Common Aromatic Amono Acid Biosynthetic Genes form Corynebacterium glutamicum. Ph.D. It is constructed using the λ Zap Express ™ system (Short et al., (1988) Nucleic Acids Research, 16: 7583-7600) as described in Thesis, National University of Ireland, Galway. λ Zap Express ™ kit is purchased from Stratagene (Stratagene, 11011 North Torrey Pines Rd., La Jolla, California 92037) and used according to the manufacturer's instructions. AS019-DNA is digested with restriction enzyme Sau3A and linked to BamHI treated and dephosphorylated λ Zap Express ™ arms.
    [213] Example 4
    [214] Cloning and Sequencing of the pgi Gene
    [215] 1. Cloning
    [216] Escherichia coli strain DF1311 with mutagenicity in the pgi and pgl genes, as described by Kupor and Fraenkel (Journal of Bacteriology 100: 1296-1301 (1969)) Transformation is performed using about 500 ng of AS019 λ Zap Express ™ plasmid library described in Example 3. The selection of the transformants was determined by M9 minimal medium containing kanamycin at a concentration of 50 mg / L (Sambrook et al., (1989). Molecular Cloning. A Laboratory Manual Cold Spring Harbor Laboratories, USA] is carried out by incubation at 37 ℃ for 48 hours. Plasmid DNA was isolated from one transformant according to Birnboim and Doly (Nucleic Acids Reserach 7: 1513-1523 (1979)) and represented as pAMC1 (FIG. 3).
    [217] 2. Sequencing
    [218] For sequencing of cloned inserts of pAMC1, Sanger et al., using primers differentially labeled with colored fluorescent labels (Proceedings of the National Academy of Sciences USA 74, 5463-5467 (1977). )] Applies. This is done using the ABI prism 310 gene analyzer (Perkin Elmer Corporation, Norwalk, Connecticut, USA) purchased from Perkin Elmer Applied Biosystems and the ABI prism Big Dye ™ Terminator Cycle Sequencing Ready Reaction kit, also purchased from Perkin Elmer.
    [219] Initial sequence analysis is performed using conventional forward and M13 reverse primers purchased from Pharmacia Biotech (St. Albans, Herts, AL1 3AW, UK):
    [220] Typical Forward Primer: GTA ATA CGA CTC ACT ATA GGG C
    [221] M13 reverse primer: GGA AAC AGC TAT GAC CAT G
    [222] Internal primers are then designed from the obtained sequences to estimate the total pgi gene. The sequence of the inner primer is as follows:
    [223] Internal primer 1: GGA AAC AGG GGA GCC GTC
    [224] Internal primer 2: TGC TGA GAT ACC AGC GGT
    [225] The resulting sequence was then run on an Apple Macintosh computer with the DNA Strider program (Marck, (1988). Nucleic Acids Research 16: 1829-1836], version 1.0. The program allows you to perform analyzes such as constrained area usage, open reading frame analysis, and codon usage measurements. The investigation between the obtained DNA sequence and the DNA sequences in the EMBL and Genbank databases is described in the BLAST program (Altschul et al., (1997). Nucleic Acids Reserach, 25: 3389-3402. DNA and protein sequences are arranged using the Clustal V and Clustal W programs (Higgins and Sharp, 1988 Gene 73: 238-244).
    [226] The sequence thus obtained is shown in SEQ ID NO: 1. Analysis of the obtained nucleotide sequence shows an open reading frame of 1650 base pairs, which is expressed as the pgi gene. This encodes a protein of 550 amino acids set forth in SEQ ID NO: 2.
    [227] Example 5
    [228] Preparation of Integration Vectors for Integrative Mutagenesis of the pgi Gene
    [229] As a template, the genomic DNA isolated from Corynebacterium glutamicum AS019 (Heery and Dunican, (1993) Applied and Environmental Microbiology 59: 791-799) was used to determine the pgi gene by polymerase chain reaction (PCR). Amplify the internal fragments. The pgi primers used were as follows:
    [230] Forward Primer: ATG GAR WCC AAY GGH AA
    [231] Reverse primer: YTC CAC GCC CCA YTG RTC
    [232] Where R = A + G; Y = C + T; W = A + T; H = A + T + C.
    [233] PCR parameters are as follows: 35 cycles
    [234] 1 minute at 94 ℃
    [235] 1 minute at 47 ℃
    [236] 30 seconds at 72 ° C
    [237] MgCl 2 1.5 mM
    [238] DNA template about 150-200 ng.
    [239] Commercially available pGEM-T vector supplied from Promega Corp. (Promega UK, Southampton.) Using strain E. coli JM109 (Yanisch-Perron et al., 1985. Gene, 33: 103-119). The PCR product obtained is cloned. The sequence of the PCR product is shown as SEQ ID NO: 3. The cloned insert is then cleaved as an EcoRI fragment and linked to plasmid pBGS8 (Spart et al., Gene 41: 337-342 (1986)) pretreated with EcoRI. Restriction enzymes used were purchased from Boehringer Mannheim UK Ltd. (Bell Lane, Lewes East Sussex BN7 1LG, UK.) And used according to the manufacturer's instructions. E. coli JM109 was then transformed using the ligation mixture and electrotransformants were subjected to IPTG (isopropyl-β-D-thiogalactopyrano) at concentrations of 1 mM, 0.02% and 50 mg / l, respectively. Seed), XGAL (5-bromo-4-chloro-3-indolyl-D-galactopyranoside), and Luria agar supplemented with kanamycin. Agar plates are incubated at 37 ° C. for 12 hours. Plasmid DNA was isolated from the transformants specified by restriction enzyme analysis using EcoRI, BamHI and SalI and represented as pMC1 (FIG. 4).
    [240] Plasmid pMC1 was deposited in the form of E. coli strain DH5α / pMC1 in Deutsche Zaulung Microorgangansmen Unt Chelculturene (DSMZ, Braunschweig, Germany) as DSM 12969 according to the Budapest Treaty.
    [241] Example 6
    [242] Integrated Mutation of pgi Gene in Lysine-producing Strain DSM 5715
    [243] The vector pMC1 mentioned in Example 5 is electroporated by Tauch et al. Electroporation (FEMS Microbiology Letters, 123: 343-347 (1994)) in Corynebacterium glutamicum DSM 5715. Strain DSM 5715 is an AEC-resistant lysine producing strain. Vector pMC1 cannot be replicated independently in DSM5715 and remains in the cell once it is integrated into the chromosome of DSM 5715. Selection of clones with pMC1 integrated into the chromosome is performed by plating electroporation batches on LB agar supplemented with kanamycin 15 mg / l. Sambrook et al., Molecular Cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. To detect integration, the internal pgi fragment (Example 5) was labeled with a Dig hybridization kit (Boehringer Mannheim) according to the "The Dig System Users Guide for Filter Hybridization" method of Boehringer Mannheim GmbH (Mannheim, Germany, 1993). Let's do it. The chromosomal DNA of the transformants is isolated by Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) and in each case digested with restriction enzymes SalI, SacI and HindIII. The formed fragments are separated by agarose gel electrophoresis and hybridized at 68 ° C. using a Dig hybridization kit (Boehringer Mannheim). In this way, the plasmid pMC1 is inserted into the chromosome pgi gene of strain DSM5715. This strain is called DSM5715 :: pMC1.
    [244] Example 7
    [245] Effect on Lysine Production by Overexpressing the zwf Gene While Removing the pgi Gene
    [246] Preparation of 7.1 strain DSM5715 :: pMC1 / pEC-T18mob2zwf
    [247] Corynebacterium glutamicum DSM5715 :: pMC1 is electroporated with the vector pEC-T18mob2zwf mentioned in Example 1.2 by electroporation such as Touch et al. (FEMS Microbiology Letters, 123: 343-347 (1994)). . The selection of plasmid carrier cells is performed by plating electroporation batches on LB agar supplemented with 15 mg / l kanamycin and 5 mg / l tetracycline. See Sambrook et al., Molecular Cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Chromosomal DNA is isolated from the transformants by conventional methods (Peters-Wendisch et al., 1998, Microbiology 144, 915-927), treated with restriction enzymes KpnI and SalI and examined by agarose gel electrophoresis. . This strain is called DSM5715 :: pMC1 / pEC-T18mob2zwf.
    [248] 7.2 Preparation of Lysine
    [249] C. glutamicum strain DSM5715 :: pMC1 / pEC-T18mob2zwf obtained in Example 7.1 is incubated in a nutrient medium suitable for lysine preparation and the lysine content in the culture supernatant is measured.
    [250] To this end, the strains are first incubated for 24 hours at 33 ° C. on agar plates containing the corresponding antibiotics (brain-heart agar containing tetracycline (5 mg / l) and kanamycin (25 mg / l)). The cultures of the comparative strains are supplemented according to their resistance to antibiotics. Starting from this agar plate culture, the preculture is seeded (10 ml of medium in a 100 ml Erlenmeyer flask). Complete medium CgIII is used as the medium for preculture.
    [251] Badge Cg III
    [252] NaCl 2.5g / ℓ
    [253] Bakto-Peptone 10g / ℓ
    [254] Bakto-Yeast Extract 10g / ℓ
    [255] Glucose (separately autoclaved) 2% (w / v)
    [256] pH is 7.4.
    [257] To this was added tetracycline (5 mg / l) and kanamycin (5 mg / l). The precultures are incubated for 16 hours at 33 ° C. at 240 rpm on a shaker. The main culture is seeded from this preculture so that the initial OD (660 nm) of the main culture is 0.1. Medium MM is used for main culture.
    [258] Badge MM
    [259] CSL (corn immersion liquid) 5g / ℓ
    [260] MOPS (morpholinopropanesulfonic acid) 20 g / ℓ
    [261] Glucose (separately autoclaved) 50 g / l
    [262] (NH 4 ) 2 SO 4 25g / ℓ
    [263] KH 2 PO 4 0.1 g / ℓ
    [264] MgSO 4 7H 2 O 1.0 g / ℓ
    [265] CaCl 2 · 2H 2 O 10mg / l
    [266] FeSO 4 7H 2 O 10mg / ℓ
    [267] MnSO 4 · H 2 O 5.0mg / ℓ
    [268] Biotin (sterile-filtered) 0.3 mg / l
    [269] Thiamine / HCl (sterile-filtered) 0.2 mg / l
    [270] L-Leucine (sterile-filtered) 0.1 g / l
    [271] CaCO 3 25g / ℓ
    [272] Aqueous ammonia is used to adjust the pH of CLS, MOPS and salt solution to 7 and autoclave. Subsequently, sterile substrate and vitamin solution are added as well as anhydrous autoclaved CaCO 3 .
    [273] Incubate in a 10 ml volume in a 100 ml Erlenmeyer flask with baffle. Tetracycline (5 mg / l) and kanamycin (25 mg / l) are added. Incubate at 33 ° C. and 80% atmospheric humidity.
    [274] After 72 hours, the OD value is measured at a wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of lysine produced is measured using an amino acid analyzer of Eppendorf-BioTronik (Hamburg, Germany) by post-column derivatization using ion exchange chromatography and ninhydrin detection.
    [275] The experimental results are shown in Table 3.
    [276] StrainODL-lysine HCl (g / L) DSM57157.314.3 DSM5715 / pEC-T18mob2zwf7.114.6 DSM5715 :: pMC1 / pEC-T18mob2zwf10.415.2
    [277] Example 8
    [278] Preparation of Genomic Cosmid Gene Library from Corynebacterium glutamicum ATCC 13032
    [279] Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolated as described by Touch et al., Plasmid 33: 168-179, 1995, and the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description). Partial cut using Sau3AI, Code no. 27-0913-02). Dephosphorylation of DNA fragments using shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Code no. 1758250) Stratagene (La Jolla, USA, Product Description SuperCos1 Cosmid Vektor Kit, Code no. 251301) Cosmid vector SuperCos1 obtained from Wahl et al. (1987) Prodeedings of the National Academy of Sciences USA 84: 2160-2164] were digested with restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product Description XbaI, Code no. 27-0948-02) and similarly alkaline in shrimp. Dephosphorylation with phosphatase. Cosmid DNA is then digested with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04). The cosmid DNA thus treated is mixed with the treated ATCC13032 DNA and the batch is treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-Ligase, Code no. 27-0870-04). The ligation mixture is then charged into phage using Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, Product Descripton Gigapack II XL Packing Extract, Code no. 200217). E. coli strain NM554 [Raleigh et al. 1988, Nucleic Acid Res. 16: 1563-1575], cells are lysed in 10 mM MgSO 4 and mixed with the phage suspension fractions. Infection and titration of the cosmid library was performed as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (1989) Cold Spring Harbor, and cells were treated with LB agar with 100 μg / ml of ampicillin. (See Lennox, Virology, 1: 190 (1995)). After overnight incubation at 37 ° C., recombinant individual clones are selected.
    [280] Example 9
    [281] Isolation and Sequencing of the poxB Gene
    [282] The individual colonies of cosmid DNA (Example 7) were isolated using the QIAprep Spin Miniprep Kit (product no. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions and the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description). Part is cut using Sau3AI, Article No. 27-0913-02). Shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Part No. 1758250) is used to dephosphorylate DNA fragments. After separation by gel electrophoresis, cosmid fragments ranging in size from 1500 to 2000 bp are separated using a QiaExII Gel Extraction Kit (product number 20021, Qiagen, Hilden, Germany). DNA of the sequencing vector pZero-1 obtained from Invitrogen (Groningen, Holland, Product Description Zero Background Cloning Kit, Article No. K2500-01) was transferred to the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Article No. 27-). 0868-04). Linkage of cosmid fragments in the sequencing vector pZero-1 was performed as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (1989) Cold Spring Harbor, and the DNA mixture was subjected to T4 ligase (Pharmacia Biotech, Freiburg). , Germany) overnight. The ligation mixture was then electroporated to E. coli strain DH5αMCR (Grant, Proceedings of the National Academy of Sciences USA, 87: 4645-4649) (Tauch et al., FEMS Microbiology Letters, 123: 343-). 7 (1994)] and plated on LB agar containing 50 μg / ml zeocin (Lennox, Virology, 1: 190 (1995)). Plasmid preparation of recombinant clones is performed using Biorobot 9600 (product number 900200, Qiagen, Hilden, Germany). Dideoxy chain-stopping method of Sanger et al., Modified according to Zimmermann et al., Nucleic Acids Research, 18: 1067, 1990 [Proceedings of the National Academies of Sciences USA, 74: 5463] -5467 (1977). Use the "RP dRhodamin Terminator Cycle Sequencing Kit" (product no. 403044, Weiterstadt, Germany) purchased from PE Applied Biosystems. Gel electrophoresis on "Rotiphoresis NF Acrylamide / Bisacrylamide" gel (29: 1) (product no. A124.1, Roth, Karlsruhe, Germany) using an "ABI Prism 377" sequencer purchased from PE Applied Biosystems (Weiterstadt, Germany) Analysis of the separation and sequencing reactions is performed.
    [283] The original sequence data obtained is then processed using the Staden program package (Nucleic Acids Research, 14: 217-231 (1986)) version 97-0. Individual sequences of the pZero1 derivatives are assembled into continuous contig. Computer-assisted cryptographic domain analysis is prepared by the XNIP program (Staden, Nucleic Acids Research, 14: 217-231 (1986)). For insufficient databanks of the National Center for Biotechnology Information (NCBI, Bethesda, MD. USA), see the BLAST search program (Altschul et al., Nucleic Acids Reserach, 25: 3389-3402 (1997)). For further analysis.
    [284] The obtained nucleotide sequence is shown by SEQ ID NO: 4. Nucleotide sequence analysis revealed an open reading frame of 1737 base pairs, which is referred to as the poxB gene. The poxB gene encodes a polypeptide consisting of 579 amino acids (SEQ ID NO: 5).
    [285] Example 10
    [286] Preparation of Integration Vectors for Integrative Mutation of the poxB Gene
    [287] Chromosomal DNA is isolated from strain ATCC 13032 by Ichmans et al. (Microbiology 140: 1817-1828 (1994)). Based on the sequence of poxB gene known for C. glutamicum from Example 8, the following oligonucleotides are selected for polymerase chain reaction:
    [288] poxBint1:
    [289] 5 'TGC GAG ATG GTG AAT GGT GG 3'
    [290] poxBint2:
    [291] 5 'GCA TGA GGC AAC GCA TTA GC 3'
    [292] The primers presented are synthesized by MWG Biotech (Ebersberg, Germany), and standard PCR methods such as Innis et al. Using Pwo-Polymerase (manufactured by Boehringer) [PCR protocols. A guide to methods and applications, 1990, Academic Press]. The polymerase chain reaction is used to isolate DNA fragments of about 0.9 kb in size, which carry the internal fragment of the poxB gene and are represented by SEQ ID NO: 6.
    [293] Amplified DNA fragments were prepared using vector TPOP TA Cloning Kit purchased from Invitrogen Corporation (Carlsbad, CA, USA; Listing No. K4500-01), vector pCR2.1-TOPO [Mead et al. (1991) Bio / Techology 9: 657-663. The E. coli strain DH5α is then electroporated using a linkage batch. Hanahan, In: DNA Cloning, A practical approach. Vol. I. IRL-Press, Oxford, Washington DC, USA, 1985] The selection of plasmid carrier cells was determined by LB agar supplemented with kanamycin 25 mg / L [Sambrook et al., Molecular Cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989]. Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep Kit (purchased from Qiagen) and examined by restriction by the restriction enzyme EcoRI followed by agarose gel electrophoresis (0.8%). This plasmid is called pCR2.1poxBint (FIG. 5).
    [294] The plasmid pCR2.1poxBint is in the form of strain E. coli DH5α / pCR2.1poxBint as DSM 13114 in Deutsche Zamlong pur microorganisms und Chellkulturen (DSMZ = German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) according to the Budapest Treaty. Deposited.
    [295] Example 11
    [296] Integration of the poxB Gene in Lysine-producing Strain DSM 5715
    [297] The vector pCR2.1poxBint mentioned in Example 10 is electroporated in Corynebacterium glutamicum DSM 5715 by electroporation such as Touch et al. (FEMS Microbiology Letters, 123: 343-347 (1994)). Strain DSM 5715 is an AEC-resistant lysine producing strain. The vector pCR2.1poxBint cannot be replicated independently in DSM5715 and remains in the cell once it is integrated into the chromosome of DSM 5715. Selection of clones with pCR2.1poxBint incorporated into the chromosome is performed by plating electroporation batches on LB agar supplemented with kanamycin 15 mg / l. Sambrook et al., Molecular Cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. To detect integration, poxBint fragments are labeled with a Dig hybridization kit (Boehringer Mannheim) according to the "The DIG System Users Guide for Filter Hybridization" method of Boehringer Mannheim GmbH (Mannheim, Germany, 1993). Potential integrant chromosomal DNA is isolated by Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) and in each case digested with restriction enzymes SalI, SacI and HindIII. The formed fragments are separated by agarose gel electrophoresis and hybridized at 68 ° C. using a Dig hybridization kit (manufactured by Boehringer Mannheim). In this way, the plasmid pCR2.1poxBint mentioned in Example 9 is inserted into the chromosome poxB gene of DSM5715. This strain is called DSM5715 :: pCR2.1poxBint.
    [298] Example 12
    [299] Effect on Lysine Production by Simultaneous Removal of the poxB Gene and Overexpression of the zwf Gene
    [300] 12.1 Preparation of strain DSM5715 :: pCR2.1poxBint / pEC-T18mob2zwf
    [301] Strain DSM5715 :: pCR2.1poxBint is transformed with plasmid pEC-T18mob2zwf using an electroporation method such as Liebhl et al. (FEMS Microbiology Letters, 53: 299-303 (1989)). Selection of the transformants was 18.5 g / l Brain-Hert broth medium, 0.5 M sorbitol, 5 g / l bacto-tryptone, 2.5 g / l, supplemented with 25 mg / l kanamycin and 5 mg / l tetracycline It is performed on LB agar containing bacto-yeast extract, 5 g / l NaCl and 18 g / l bacto-agar. Incubate at 33 ° C. for 2 hours.
    [302] The chromosomal DNA was isolated from the transformants in each case by conventional methods (Peters-Wendisch et al., 1998, Microbiology 144, 915-927) and cleaved with restriction endonucleases XbaI and KpnI, followed by plasmid Examination by agarose gel electrophoresis. The strain thus obtained is referred to as DSM5715 :: pCR2.1poxBint / pEC-T18mob2zwf.
    [303] 12.2 Preparation of L-Lysine
    [304] C. glutamicum strain DSM5715 :: pCR2.1poxBint / pEC-T18mob2zwf obtained in Example 12.1 is incubated in a nutrient medium suitable for lysine preparation and the lysine content in the culture supernatant is measured.
    [305] To this end, the strains are first incubated for 24 hours at 33 ° C. on agar plates containing the corresponding antibiotics (brain-heart agar containing tetracycline (5 mg / l) and kanamycin (25 mg / l)). The comparative strains are supplemented according to their resistance to antibiotics. Starting from this agar plate culture, the preculture is seeded (10 ml of medium in a 100 ml Erlenmeyer flask). Complete medium CgIII is used as the medium for preculture.
    [306] Badge Cg III
    [307] NaCl 2.5g / ℓ
    [308] Bakto-Peptone 10g / ℓ
    [309] Bakto-Yeast Extract 10g / ℓ
    [310] Glucose (separately autoclaved) 2% (w / v)
    [311] pH is 7.4.
    [312] To this was added tetracycline (5 mg / L) and kanamycin (25 mg / L). The precultures are incubated for 16 hours at 33 ° C. at 240 rpm on a shaker. The main culture is seeded from this preculture so that the initial OD (660 nm) of the main culture is 0.1. Medium MM is used for main culture.
    [313] Badge MM
    [314] CSL (corn immersion liquid) 5g / ℓ
    [315] MOPS (morpholinopropanesulfonic acid) 20 g / ℓ
    [316] Glucose (separately autoclaved) 58 g / l
    [317] (NH 4 ) 2 SO 4 25g / ℓ
    [318] KH 2 PO 4 0.1 g / ℓ
    [319] MgSO 4 7H 2 O 1.0 g / ℓ
    [320] CaCl 2 · 2H 2 O 10mg / l
    [321] FeSO 4 7H 2 O 10mg / ℓ
    [322] MnSO 4 · H 2 O 5.0mg / ℓ
    [323] Biotin (sterile-filtered) 0.3 mg / l
    [324] Thiamine / HCl (sterile-filtered) 0.2 mg / l
    [325] L-Leucine (sterile-filtered) 0.1 g / l
    [326] CaCO 3 25g / ℓ
    [327] Aqueous ammonia is used to adjust the pH of CLS, MOPS and salt solution to 7 and autoclave. Subsequently, sterile substrate and vitamin solution are added as well as anhydrous autoclaved CaCO 3 .
    [328] Incubate in a 10 ml volume in a 100 ml Erlenmeyer flask with baffle. Tetracycline (5 mg / l) and kanamycin (25 mg / l) are added. Incubate at 33 ° C. and 80% atmospheric humidity.
    [329] After 72 hours, OD values are measured at a wavelength of 660 nm with Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount of threonine produced is measured using an amino acid analyzer (Eppendorf-BioTronik (Hamburg, Germany)) by post-column derivatization using ion exchange chromatography and ninhydrin detection.
    [330] The experimental results are shown in Table 4.
    [331] StrainODL-lysine HCl (g / L) DSM571510.816.0 DSM5715 / pEC-T18mob2zwf8.317.1 DSM5715 :: pCR2.1poxBint7.116.7 DSM5715 :: pCR2.1poxBint / pEC-T18mob2zwf7.817.7
    权利要求:
    Claims (10)
    [1" claim-type="Currently amended] Fermenting a bacterium that produces the desired L-amino acid, at least amplified by the zwf gene (a),
    (B) concentrating the L-amino acid in the medium or bacterial cell and
    A method for producing L-amino acid by fermentation of coryneform bacteria, comprising the step (c) of separating the resulting L-amino acid.
    [2" claim-type="Currently amended] The method according to claim 1, wherein a bacterium in which an additional gene of the biosynthetic pathway of the desired L-amino acid is further amplified, in particular overexpressed, is used.
    [3" claim-type="Currently amended] The method of claim 1, wherein coryneform bacteria are used to prepare L-threonine, L-lysine or L-tryptophan.
    [4" claim-type="Currently amended] 4. The method of claim 3, wherein coryneform bacteria for producing L-lysine are used.
    [5" claim-type="Currently amended] The method according to claim 2, in particular in coryneform microorganisms already producing L-lysine,
    DapA gene encoding 5.1 dihydrodipicolinate synthase,
    5.2 lysC gene, which encodes a feedback resistant aspartate kinase,
    5.3 gap gene encoding glycerolaldehyde 3-phosphate dehydrogenase,
    5.4 pyc gene encoding pyruvate carboxylase,
    5.5 tkt gene encoding transketolase,
    5.6 gnd gene encoding glucose 6-phosphate dehydrogenase,
    5.7 lysE gene encoding lysine export,
    5.8 zwal gene and
    5.9 A method for fermenting L-lysine, wherein at least one gene selected from the group consisting of eno genes encoding enolase is simultaneously amplified or overexpressed.
    [6" claim-type="Currently amended] The method according to claim 2, in particular in coryneform microorganisms already producing L-threonine,
    6.1 hom gene encoding homoserine dehydrogenase or hom dr allele encoding “feedback resistant” homoserine dehydrogenase,
    6.2 gap gene encoding glycerolaldehyde 3-phosphate dehydrogenase,
    6.3 pyc gene encoding pyruvate carboxylase,
    6.4 malate: mqo gene encoding quinone oxidoreductase,
    6.5 the tkt gene encoding transketolase,
    6.6 zwf gene encoding glucose 6-phosphate dehydrogenase,
    6.7 thrE gene, encoding threonine transport,
    6.8 zwal gene and
    6.9 A method for fermenting production of L-threonine, wherein at least one gene selected from the group consisting of eno genes encoding enolase is simultaneously amplified, in particular overexpressed.
    [7" claim-type="Currently amended] The method of claim 2,
    7.1 pck gene encoding phosphoenol pyruvate carboxykinase,
    7.2 pgi gene encoding glucose 6-phosphate isomerase,
    7.3 the poxB gene encoding pyruvate oxidase and
    7.4 A method for producing L-amino acids, in particular L-lysine or L-threonine, wherein at least one gene selected from the group consisting of zwa2 genes is fermented simultaneously.
    [8" claim-type="Currently amended] The method according to any one of claims 2 to 6, wherein in order to perform amplification, the copy number of the gene or nucleotide sequence is increased by transforming the microorganism using a plasmid vector comprising these gene or nucleotide sequences.
    [9" claim-type="Currently amended] Plasmid vector pEC-T18mob2 deposited with accession number DSM13244 in E. coli K-12 DH5α shown in FIG. 2.
    [10" claim-type="Currently amended] A genus of coryneform microorganisms, in particular Corynebacterium, transformed by introducing a plasmid vector as claimed in claim 9, which further contains the zwf gene.
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    AU6821900A|2001-10-03|
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2000-03-20|Priority to US53126900A
    2000-03-20|Priority to US09/531,269
    2000-07-05|Application filed by 펠드만 마르틴 및 봅 후베르트, 데구사 아게, 추후제출, 포르슝스젠트룸 율리히 게엠베하, 내셔널 유니버시티 오브 아일랜드
    2000-07-05|Priority to PCT/EP2000/006303
    2002-02-16|Publication of KR20020012578A
    2007-05-08|Application granted
    2007-05-08|Publication of KR100715641B1
    优先权:
    申请号 | 申请日 | 专利标题
    US53126900A| true| 2000-03-20|2000-03-20|
    US09/531,269|2000-03-20|
    PCT/EP2000/006303|WO2001070995A1|2000-03-20|2000-07-05|Process for the fermentative preparation of l-amino acids with amplification of the zwf gene|
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