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    • 专利摘要:
    The present invention a) a polynucleotide at least 70% identical to the polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 2, b) a polynucleotide encoding a polypeptide containing an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO: 2, c) a polynucleotide complementary to the polynucleotide of a) or b) above and d) attenuating an isolated polynucleotide containing a polynucleotide sequence selected from the group consisting of polynucleotides containing at least 15 consecutive bases in the polynucleotide sequences of a), b) or c), and the poxB gene It relates to a fermentative production method of L-amino acid.
    公开号:KR20010051289A
    申请号:KR1020000063500
    申请日:2000-10-27
    公开日:2001-06-25
    发明作者:두쉬니콜레；바테브리기테；칼리노브스키외른；필러알프레트；뫼켈벳티나；바이쎈보른안케；펩페를레발터
    申请人:데구사-휠스 악티엔게젤샤프트；
    IPC主号:
    专利说明:

    Novel nucleotide sequences coding for the poxB gene
    〈110〉 Degussa-Huls AG
    〈120〉 Novel nucleotide sequences coding for the poxB gene
    <130> 5-1999-024333-4
    〈150〉 DE 199 51 975.7
    (151) 1999-10-28
    〈160〉 3
    〈170〉 KOPATIN 1.5
    〈210〉 1
    <211> 2160
    <212> DNA
    213 Corynebacterium glutamicum
    〈220〉
    <221> CDS
    222 (327) .. (2063)
    〈220〉
    〈221〉 -35_signal
    222 (227) .. (232)
    〈220〉
    〈221〉 -10_signal
    222 (256) .. (261)
    <400> 1
    ttagaggcga ttctgtgagg tcactttttg tggggtcggg gtctaaattt ggccagtttt 60
    cgaggcgacc agacaggcgt gcccacgatg tttaaatagg cgatcggtgg gcatctgtgt 120
    ttggtttcga cgggctgaaa ccaaaccaga ctgcccagca acgacggaaa tcccaaaagt 180
    gggcatccct gtttggtacc gagtacccac ccgggcctga aactccctgg caggcgggcg 240
    aagcgtggca acaactggaa tttaagagca caattgaagt cgcaccaagt taggcaacac 300
    aatagccata acgttgagga gttcag atg gca cac agc tac gca gaa caa 350
    Met Ala His Ser Tyr Ala Glu Gln
    1 5
    tta att gac act ttg gaa gct caa ggt gtg aag cga att tat ggt ttg 398
    Leu Ile Asp Thr Leu Glu Ala Gln Gly Val Lys Arg Ile Tyr Gly Leu
    10 15 20
    gtg ggt gac agc ctt aat ccg atc gtg gat gct gtc cgc caa tca gat 446
    Val Gly Asp Ser Leu Asn Pro Ile Val Asp Ala Val Arg Gln Ser Asp
    25 30 35 40
    att gag tgg gtg cac gtt cga aat gag gaa gcg gcg gcg ttt gca gcc 494
    Ile Glu Trp Val His Val Arg Asn Glu Glu Ala Ala Ala Phe Ala Ala
    45 50 55
    ggt gcg gaa tcg ttg atc act ggg gag ctg gca gta tgt gct gct tct 542
    Gly Ala Glu Ser Leu Ile Thr Gly Glu Leu Ala Val Cys Ala Ala Ser
    60 65 70
    tgt ggt cct gga aac aca cac ctg att cag ggt ctt tat gat tcg cat 590
    Cys Gly Pro Gly Asn Thr His Leu Ile Gln Gly Leu Tyr Asp Ser His
    75 80 85
    cga aat ggt gcg aag gtg ttg gcc atc gct agc cat att ccg agt gcc 638
    Arg Asn Gly Ala Lys Val Leu Ala Ile Ala Ser His Ile Pro Ser Ala
    90 95 100
    cag att ggt tcg acg ttc ttc cag gaa acg cat ccg gag att ttg ttt 686
    Gln Ile Gly Ser Thr Phe Phe Gln Glu Thr His Pro Glu Ile Leu Phe
    105 110 115 120
    aag gaa tgc tct ggt tac tgc gag atg gtg aat ggt ggt gag cag ggt 734
    Lys Glu Cys Ser Gly Tyr Cys Glu Met Val Asn Gly Gly Glu Gln Gly
    125 130 135
    gaa cgc att ttg cat cac gcg att cag tcc acc atg gcg ggt aaa ggt 782
    Glu Arg Ile Leu His His Ala Ile Gln Ser Thr Met Ala Gly Lys Gly
    140 145 150
    gtg tcg gtg gta gtg att cct ggt gat atc gct aag gaa gac gca ggt 830
    Val Ser Val Val Val Ile Pro Gly Asp Ile Ala Lys Glu Asp Ala Gly
    155 160 165
    gac ggt act tat tcc aat tcc act att tct tct ggc act cct gtg gtg 878
    Asp Gly Thr Tyr Ser Asn Ser Thr Ile Ser Ser Gly Thr Pro Val Val
    170 175 180
    ttc ccg gat cct act gag gct gca gcg ctg gtg gag gcg att aac aac 926
    Phe Pro Asp Pro Thr Glu Ala Ala Ala Leu Val Glu Ala Ile Asn Asn
    185 190 195 200
    gct aag tct gtc act ttg ttc tgc ggt gcg ggc gtg aag aat gct cgc 974
    Ala Lys Ser Val Thr Leu Phe Cys Gly Ala Gly Val Lys Asn Ala Arg
    205 210 215
    gcg cag gtg ttg gag ttg gcg gag aag att aaa tca ccg atc ggg cat 1022
    Ala Gln Val Leu Glu Leu Ala Glu Lys Ile Lys Ser Pro Ile Gly His
    220 225 230
    gcg ctg ggt ggt aag cag tac atc cag cat gag aat ccg ttt gag gtc 1070
    Ala Leu Gly Gly Lys Gln Tyr Ile Gln His Glu Asn Pro Phe Glu Val
    235 240 245
    ggc atg tct ggc ctg ctt ggt tac ggc gcc tgc gtg gat gcg tcc aat 1118
    Gly Met Ser Gly Leu Leu Gly Tyr Gly Ala Cys Val Asp Ala Ser Asn
    250 255 260
    gag gcg gat ctg ctg att cta ttg ggt acg gat ttc cct tat tct gat 1166
    Glu Ala Asp Leu Leu Ile Leu Leu Gly Thr Asp Phe Pro Tyr Ser Asp
    265 270 275 280
    ttc ctt cct aaa gac aac gtt gcc cag gtg gat atc aac ggt gcg cac 1214
    Phe Leu Pro Lys Asp Asn Val Ala Gln Val Asp Ile Asn Gly Ala His
    285 290 295
    att ggt cga cgt acc acg gtg aag tat ccg gtg acc ggt gat gtt gct 1262
    Ile Gly Arg Arg Thr Thr Val Lys Tyr Pro Val Thr Gly Asp Val Ala
    300 305 310
    gca aca atc gaa aat att ttg cct cat gtg aag gaa aaa aca gat cgt 1310
    Ala Thr Ile Glu Asn Ile Leu Pro His Val Lys Glu Lys Thr Asp Arg
    315 320 325
    tcc ttc ctt gat cgg atg ctc aag gca cac gag cgt aag ttg agc tcg 1358
    Ser Phe Leu Asp Arg Met Leu Lys Ala His Glu Arg Lys Leu Ser Ser
    330 335 340
    gtg gta gag acg tac aca cat aac gtc gag aag cat gtg cct att cac 1406
    Val Val Glu Thr Tyr Thr His Asn Val Glu Lys His Val Pro Ile His
    345 350 355 360
    cct gaa tac gtt gcc tct att ttg aac gag ctg gcg gat aag gat gcg 1454
    Pro Glu Tyr Val Ala Ser Ile Leu Asn Glu Leu Ala Asp Lys Asp Ala
    365 370 375
    gtg ttt act gtg gat acc ggc atg tgc aat gtg tgg cat gcg agg tac 1502
    Val Phe Thr Val Asp Thr Gly Met Cys Asn Val Trp His Ala Arg Tyr
    380 385 390
    atc gag aat ccg gag gga acg cgc gac ttt gtg ggt tca ttc cgc cac 1550
    Ile Glu Asn Pro Glu Gly Thr Arg Asp Phe Val Gly Ser Phe Arg His
    395 400 405
    ggc acg atg gct aat gcg ttg cct cat gcg att ggt gcg caa agt gtt 1598
    Gly Thr Met Ala Asn Ala Leu Pro His Ala Ile Gly Ala Gln Ser Val
    410 415 420
    gat cga aac cgc cag gtg atc gcg atg tgt ggc gat ggt ggt ttg ggc 1646
    Asp Arg Asn Arg Gln Val Ile Ala Met Cys Gly Asp Gly Gly Leu Gly
    425 430 435 440
    atg ctg ctg ggt gag ctt ctg acc gtt aag ctg cac caa ctt ccg ctg 1694
    Met Leu Leu Gly Glu Leu Leu Thr Val Lys Leu His Gln Leu Pro Leu
    445 450 455
    aag gct gtg gtg ttt aac aac agt tct ttg ggc atg gtg aag ttg gag 1742
    Lys Ala Val Val Phe Asn Asn Ser Ser Leu Gly Met Val Lys Leu Glu
    460 465 470
    atg ctc gtg gag gga cag cca gaa ttt ggt act gac cat gag gaa gtg 1790
    Met Leu Val Glu Gly Gln Pro Glu Phe Gly Thr Asp His Glu Glu Val
    475 480 485
    aat ttc gca gag att gcg gcg gct gcg ggt atc aaa tcg gta cgc atc 1838
    Asn Phe Ala Glu Ile Ala Ala Ala Ala Gly Ile Lys Ser Val Arg Ile
    490 495 500
    acc gat ccg aag aaa gtt cgc gag cag cta gct gag gca ttg gca tat 1886
    Thr Asp Pro Lys Lys Val Arg Glu Gln Leu Ala Glu Ala Leu Ala Tyr
    505 510 515 520
    cct gga cct gta ctg atc gat atc gtc acg gat cct aat gcg ctg tcg 1934
    Pro Gly Pro Val Leu Ile Asp Ile Val Thr Asp Pro Asn Ala Leu Ser
    525 530 535
    atc cca cca acc atc acg tgg gaa cag gtc atg gga ttc agc aag gcg 1982
    Ile Pro Pro Thr Ile Thr Trp Glu Gln Val Met Gly Phe Ser Lys Ala
    540 545 550
    gcc acc cga acc gtc ttt ggt gga gga gta gga gcg atg atc gat ctg 2030
    Ala Thr Arg Thr Val Phe Gly Gly Gly Val Gly Ala Met Ile Asp Leu
    555 560 565
    gcc cgt tcg aac ata agg aat att cct act cca tgatgattga tacacctgct 2083
    Ala Arg Ser Asn Ile Arg Asn Ile Pro Thr Pro
    570 575
    gttctcattg accgcgagcg cttaactgcc aacatttcca ggatggcagc tcacgccggt 2143
    gcccatgaga ttgccct 2160
    〈210〉 2
    <211> 579
    <212> PRT
    213 Corynebacterium glutamicum
    <400> 2
    Met Ala His Ser Tyr Ala Glu Gln Leu Ile Asp Thr Leu Glu Ala Gln
    1 5 10 15
    Gly Val Lys Arg Ile Tyr Gly Leu Val Gly Asp Ser Leu Asn Pro Ile
    20 25 30
    Val Asp Ala Val Arg Gln Ser Asp Ile Glu Trp Val His Val Arg Asn
    35 40 45
    Glu Glu Ala Ala Ala Phe Ala Ala Gly Ala Glu Ser Leu Ile Thr Gly
    50 55 60
    Glu Leu Ala Val Cys Ala Ala Ser Cys Gly Pro Gly Asn Thr His Leu
    65 70 75 80
    Ile Gln Gly Leu Tyr Asp Ser His Arg Asn Gly Ala Lys Val Leu Ala
    85 90 95
    Ile Ala Ser His Ile Pro Ser Ala Gln Ile Gly Ser Thr Phe Phe Gln
    100 105 110
    Glu Thr His Pro Glu Ile Leu Phe Lys Glu Cys Ser Gly Tyr Cys Glu
    115 120 125
    Met Val Asn Gly Gly Glu Gln Gly Glu Arg Ile Leu His His Ala Ile
    130 135 140
    Gln Ser Thr Met Ala Gly Lys Gly Val Ser Val Val Val Ile Pro Gly
    145 150 155 160
    Asp Ile Ala Lys Glu Asp Ala Gly Asp Gly Thr Tyr Ser Asn Ser Thr
    165 170 175
    Ile Ser Ser Gly Thr Pro Val Val Phe Pro Asp Pro Thr Glu Ala Ala
    180 185 190
    Ala Leu Val Glu Ala Ile Asn Asn Ala Lys Ser Val Thr Leu Phe Cys
    195 200 205
    Gly Ala Gly Val Lys Asn Ala Arg Ala Gln Val Leu Glu Leu Ala Glu
    210 215 220
    Lys Ile Lys Ser Pro Ile Gly His Ala Leu Gly Gly Lys Gln Tyr Ile
    225 230 235 240
    Gln His Glu Asn Pro Phe Glu Val Gly Met Ser Gly Leu Leu Gly Tyr
    245 250 255
    Gly Ala Cys Val Asp Ala Ser Asn Glu Ala Asp Leu Leu Ile Leu Leu
    260 265 270
    Gly Thr Asp Phe Pro Tyr Ser Asp Phe Leu Pro Lys Asp Asn Val Ala
    275 280 285
    Gln Val Asp Ile Asn Gly Ala His Ile Gly Arg Arg Thr Thr Val Lys
    290 295 300
    Tyr Pro Val Thr Gly Asp Val Ala Ala Thr Ile Glu Asn Ile Leu Pro
    305 310 315 320
    His Val Lys Glu Lys Thr Asp Arg Ser Phe Leu Asp Arg Met Leu Lys
    325 330 335
    Ala His Glu Arg Lys Leu Ser Ser Val Val Glu Thr Tyr Thr His Asn
    340 345 350
    Val Glu Lys His Val Pro Ile His Pro Glu Tyr Val Ala Ser Ile Leu
    355 360 365
    Asn Glu Leu Ala Asp Lys Asp Ala Val Phe Thr Val Asp Thr Gly Met
    370 375 380
    Cys Asn Val Trp His Ala Arg Tyr Ile Glu Asn Pro Glu Gly Thr Arg
    385 390 395 400
    Asp Phe Val Gly Ser Phe Arg His Gly Thr Met Ala Asn Ala Leu Pro
    405 410 415
    His Ala Ile Gly Ala Gln Ser Val Asp Arg Asn Arg Gln Val Ile Ala
    420 425 430
    Met Cys Gly Asp Gly Gly Leu Gly Met Leu Leu Gly Glu Leu Leu Thr
    435 440 445
    Val Lys Leu His Gln Leu Pro Leu Lys Ala Val Val Phe Asn Asn Ser
    450 455 460
    Ser Leu Gly Met Val Lys Leu Glu Met Leu Val Glu Gly Gln Pro Glu
    465 470 475 480
    Phe Gly Thr Asp His Glu Glu Val Asn Phe Ala Glu Ile Ala Ala Ala
    485 490 495
    Ala Gly Ile Lys Ser Val Arg Ile Thr Asp Pro Lys Lys Val Arg Glu
    500 505 510
    Gln Leu Ala Glu Ala Leu Ala Tyr Pro Gly Pro Val Leu Ile Asp Ile
    515 520 525
    Val Thr Asp Pro Asn Ala Leu Ser Ile Pro Pro Thr Ile Thr Trp Glu
    530 535 540
    Gln Val Met Gly Phe Ser Lys Ala Ala Thr Arg Thr Val Phe Gly Gly
    545 550 555 560
    Gly Val Gly Ala Met Ile Asp Leu Ala Arg Ser Asn Ile Arg Asn Ile
    565 570 575
    Pro Thr Pro
    〈210〉 3
    <211> 875
    <212> DNA
    213 Corynebacterium glutamicum
    <400> 3
    tgcgagatgg tgaatggtgg tgagcagggt gaacgcattt tgcatcacgc gattcagtcc 60
    accatggcgg gtaaaggtgt gtcggtggta gtgattcctg gtgatatcgc taaggaagac 120
    gcaggtgacg gtacttattc caattccact atttcttctg gcactcctgt ggtgttcccg 180
    gatcctactg aggctgcagc gctggtggag gcgattaaca acgctaagtc tgtcactttg 240
    ttctgcggtg cgggcgtgaa gaatgctcgc gcgcaggtgt tggagttggc ggagaagatt 300
    aaatcaccga tcgggcatgc gctgggtggt aagcagtaca tccagcatga gaatccgttt 360
    gaggtcggca tgtctggcct gcttggttac ggcgcctgcg tggatgcgtc caatgaggcg 420
    gatctgctga ttctattggg tacggatttc ccttattctg atttccttcc taaagacaac 480
    gttgcccagg tggatatcaa cggtgcgcac attggtcgac gtaccacggt gaagtatccg 540
    gtgaccggtg atgttgctgc aacaatcgaa aatattttgc ctcatgtgaa ggaaaaaaca 600
    gatcgttcct tccttgatcg gatgctcaag gcacacgagc gtaagttgag ctcggtggta 660
    gagacgtaca cacataacgt cgagaagcat gtgcctattc accctgaata cgttgcctct 720
    attttgaacg agctggcgga taaggatgcg gtgtttactg tggataccgg catgtgcaat 780
    gtgtggcatg cgaggtacat cgagaatccg gagggaacgc gcgactttgt gggttcattc 840
    cgccacggca cgatggctaa tgcgttgcct catgc 875
    The present invention provides nucleotide sequences from coryneform bacteria encoding the poxB gene and methods of fermentative production of amino acids, in particular L-lysine, by attenuating said poxB gene.
    L-amino acids, in particular lysine, are used in human medicine, the pharmaceutical industry, the food industry, in particular animal nutrition.
    Amino acids are known to be produced by fermentation of coryneform bacteria, in particular Corynebacterium glutamicum strains. Due to their great importance, efforts have been made to improve the production method. Improvements in this method can be achieved by means of fermentation techniques, eg, agitation and oxygenation, or the composition of the nutrient medium, eg, sugar concentration during fermentation, or after the product, for example by ion exchange chromatography. Or inherent performance characteristics of the microorganisms themselves.
    The performance characteristics of these microorganisms improve the performance characteristics of these microorganisms using mutagenesis, selection and mutant selection methods. In this way, a strain is obtained that is aminotrophic and produces amino acids for metabolites that are resistant to or metabolically resistant to antimetabolites.
    For several years, recombinant DNA technology has also been used to ameliorate Corynebacterium strains producing L-amino acids.
    We aim to provide new means for improved fermentative production of amino acids, in particular L-lysine.
    1 is a map of the plasmid pCR2.1poxBint.
    Abbreviations and names are defined as follows:
    ColE1 ori: origin of replication of plasmid ColE1
    lacZ: 5'-terminus of β-galactosidase gene
    f1 ori: origin of f1 phage replication
    KmR: Kanamycin Resistance
    ApR: Ampicillin Resistance
    BamHI: restriction site of the restriction enzyme BamHI
    EcoRI: restriction site of the restriction enzyme EcoRI
    poxBint: the internal fragment of the poxB gene
    L-amino acids, in particular lysine, are used in human medicine, the pharmaceutical industry, the food industry, in particular animal nutrition. Therefore, there is a general interest in providing new and improved methods for the production of amino acids, especially L-lysine.
    The present invention
    a) a polynucleotide at least 70% identical to the polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 2,
    b) a polynucleotide encoding a polypeptide containing an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO: 2,
    c) a polynucleotide complementary to the polynucleotide of a) or b) above and
    d) providing an isolated polynucleotide containing a polynucleotide sequence selected from the group consisting of polynucleotides containing at least 15 consecutive bases in the polynucleotide sequence of a), b) or c).
    The invention also
    (i) the nucleotide sequence set forth in SEQ ID NO: 1,
    (ii) one or more sequences that match the sequence of (i) within the degenerate range of the genetic code, or
    (iii) one or more sequences that hybridize with sequences complementary to the sequences of (i) or (ii), and optionally
    (iv) A polynucleotide according to claim 1, which preferably comprises a replicable DNA containing a functionally neutral sense mutation in (i).
    The invention also
    A polynucleotide according to claim 2 containing a nucleotide sequence set forth in SEQ ID NO: 1,
    A polynucleotide according to claim 2 encoding a polypeptide containing an amino acid sequence set forth in SEQ ID NO: 2,
    Accession number A vector containing a polynucleotide sequence according to claims 1, d), in particular pCR.1poxBint, deposited as E. coli No. DSM 13114 and
    Provided are coryneform bacteria that act as host cells, containing insertions or deletions in the pox gene.
    In addition, the present invention provides a hybridization of a suitable gene library containing a complete gene having a polynucleotide sequence according to SEQ ID NO: 1 with a probe or fragment thereof containing the polynucleotide sequence described above according to SEQ ID NO: 1, and the DNA sequence mentioned above. Provided are polynucleotides consisting essentially of certain polynucleotide sequences obtainable by screening by separation, and methods of isolation of such DNA sequences.
    The polynucleotide sequence according to the present invention isolates a full-length cDNA encoding pyruvate oxidase and isolates such cDNA or gene, wherein the sequence exhibits a high degree of similarity with the sequence of the pyruvate oxidase gene. Are suitable as hybridization probes for RNA, cDNA and DNA.
    In addition, the polynucleotide sequences according to the present invention are suitable as primers for DNA production of genes encoding pyruvate oxidase by polymerase chain reaction (PCR).
    Such oligonucleotides which act as probes or primers contain at least 30, preferably at least 20, particularly preferably at least 15 consecutive nucleotides. Also suitable are oligonucleotides having a length of 40 to 50 or more base pairs.
    "Isolated" means to be separated from its natural environment.
    In general, "polynucleotide" refers to polyribonucleotides and polydeoxyribonucleotides where RNA or DNA may be unmodified or modified.
    A "polypeptide" is understood to mean a peptide or protein containing two or more amino acids joined via peptide bonds.
    Polypeptides according to the invention comprise at least 70%, preferably at least 80%, in particular at least 90, of the polypeptide according to SEQ ID NO: 2, in particular the polypeptide having the biological activity of pyruvate oxidase, and also the polypeptide according to SEQ ID NO: 2. Polypeptides that are 95% identical and have the aforementioned activities.
    The present invention also provides a method for the fermentative production of amino acids, in particular using coryneform bacteria which pre-produce amino acids, in particular L-lysine, wherein the nucleotide sequence encoding the poxB gene is attenuated, especially at low levels. .
    In this regard, the term “attenuation” uses, for example, a gene or an allele that encodes a weak promoter or a corresponding enzyme with low activity or inactivates a corresponding gene or enzyme (protein), and optionally these means. By means of combined use it is meant to lower or inhibit the intracellular activity of one or more enzymes (proteins) encoded by the corresponding DNA in the microorganism.
    Microorganisms provided by the present invention can produce amino acids, in particular lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose, or glycerol and ethanol. Such microorganisms may in particular include representative coryneform bacteria of the genus Corynebacterium. Among the genus Corynebacterium, mention may be made specifically of Corynebacterium glutamicum which is known to those skilled in the art for its ability to produce L-amino acids.
    Suitable strains of the genus Corynebacterium, in particular Corynebacterium glutamicum species, are, for example, known wild type strains:
    Corynebacterium glutamicum ATCC13032,
    Corynebacterium acetoglutamicum ATCC15806,
    Corynebacterium acetoacidophilum ATCC13870,
    Corynebacterium melassecola ACTT17965,
    Corynebacterium thermoaminogenes FERM-BP 1539,
    Brevibacterium flavum ATCC14067,
    Brevibacterium lactofermentum ATCC13869 and
    Brevibacterium divaricatum ATCC14020, and L-amino acid producing mutants or strains prepared therefrom,
    For example, L-lysine producing strains:
    Corynebacterium glutamicum FERM-P 1709
    Brevibacterium plaboom FERM-P 1708,
    Brevibacterium lactofermentum FERM-P 1712,
    Corynebacterium glutamicum FERM-P 6463,
    Corynebacterium glutamicum FERM-P 6464 and
    Corynebacterium glutamicum DSM 5714.
    The inventors have found a seed encoding the enzyme pyruvate oxidase (EC 1.2.2.2). The poxB gene of glutamicum was successfully isolated.
    Seed. Glutamicum's genetic library first. By constructing in E. coli, the poxB gene or other genes are isolated from the microorganism. Construction of gene libraries is generally described in well-known textbooks and manuals. Examples that may be mentioned include textbooks [Winnacker, Gene und Klone, Eine Einfuhrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or handbook [Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring) Harbor Laboratory Press, 1989) One of the well known gene libraries is E. coli constructed in λ vectors by Kohara et al., Cell 50, 495-508 (1987). A library of K-12 strain W3110. Bathe et al., Molecular and General Genetics, 252: 255-265, 1996, described in the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of). Seeds constructed in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575) using the National Academy of Sciences USA 84: 2160-2164. A genetic library of glutamicum ATCC13032 has been described, see also Bormann et al., Mo lecular Microbiology 6 (3), 317-326, 1992 describes a gene library of C. glutamicum ATCC13032 using cosmid pHC79 (Ref .: Hohn and Collins, Gene 11, 291-298 (1980)). O'Donohue, The Cloning and Molecular Analysis of Four Common Aromatic Amino Acid Biosynthetic Genes from Corynebacterium glutamicum. Ph.D. Thesis, National University of Ireland, Galway, 1997. Seeds using the λ Zap expression system described in Short et al., Nucleic Acids Research, 16: 7583. Cloning of the glutamicum gene is described.
    Also, this. Mr. Coli. The gene library of glutamicum is either plasmid such as pBR322 (Bolivar, Life Science, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268). It can be prepared using. E. with restriction defects and recombinant defects. E. coli strains are particularly suitable hosts, examples of which include Jeffrey H. Miller, "A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria", Cold Spring Harbor Laboratory Press, 1992. There is the DH5α strain described.
    The long chain DNA fragment cloned with the aid of cosmid or other λ-vector can then be sub-cloned in a conventional vector suitable for DNA sequencing.
    DNA sequencing methods are described in particular in Sanger et al., Proceedings of the National Academy of Sciences of the United States of America USA, 74: 5463-5467, 1997.
    The DNA sequences obtained are known algorithms or sequencing programs, for example, Staden's program (Nucleic Acids Research 14, 217-232 (1986)), Butler's GCG program [ References: Methods of Biochemical Analysis 39, 74-97 (1998), Pearson & Lipman's FASTA algorithm (Proceedings of the National Academy of Sciences USA 85, 2444-2448 (1988)) or The BLAST algorithm of Altschul (Nature Genetics 6, 119-129 (1994)) can be examined and compared with the list of sequences available in publicly accessible databases. The publicly accessible nucleotide sequence database can be, for example, the European Molecular Biology Laboratory (EMBL), Heidelberg, Germany, or the National Center for Biotechnology Information database (NCBI; Bethesda, Midland, USA). Material) database.
    A seed encoding the poxB gene provided by the present invention as SEQ ID NO: 1. New DNA sequences from glutamicum are obtained in this manner. The amino acid sequence of the corresponding protein is also inferred from the DNA sequence using the method described above. SEQ ID NO: 2 shows the resulting amino acid sequence of the poxB gene product.
    Also provided by the present invention is a coding DNA sequence resulting from SEQ ID NO: 1 due to the degeneracy of the genetic code. DNA sequences that hybridize with SEQ ID NO: 1 or a portion of SEQ ID NO: 1 are provided similarly by the present invention. Finally, DNA sequences prepared by polymerase chain reaction (PCR) using primers obtained from SEQ ID NO: 1 are also provided by the present invention.
    Those skilled in the art are particularly familiar with the handbook ["The DIG System Users Guide for Filter Hybridization", Boehringer Mannheim GmbH (Mannheim, Germany, 1993)) and Liebl et al., International Journal of Systematic Bacteriology (1991) 41: 255. Guidance on identifying DNA by hybridization can be found in [260]. Those skilled in the art are particularly concerned with polymerase chain reaction (PCR) in the handbook (Gait, Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and Newton & Graham, PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994)). Can be used to find instructions for amplifying a DNA sequence.
    We found that attenuating the poxB gene produces coryneform bacteria in an improved manner, L-amino acids, in particular L-lysine.
    Attenuation can be achieved by lowering or inhibiting the expression of the poxB gene or the catalytic properties of the enzyme protein. Two means can also be used together.
    Degraded gene expression can be achieved by appropriate regulation of the culture or by genetic modification (mutation) of the signal structure for gene expression. Signal structures for gene expression are, for example, inhibitor genes, activator genes, operators, promoters, attenuators, ribosomal binding sites, start codons and terminators. Those skilled in the art can find information in this regard, for example, in patent application WO 96/15246; Boyd & Murphy, Journal of Bacteriology 170: 5949 (1988); Voskuil & Chambliss, Nucleic Acids Research 26: 3548 (1998); Jensen & Hammer, Biotechnology and Bioengineering 58: 191 (1998); And Patek et al., Microbiology 142: 1297 (1996) and, for example, known textbooks of gene and molecular biology (Knippers, "Molekulare Genetik", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995 Or Winnacker, "Gene und Klone", VCH Verlagsgesellschaft, Weinheim, Germany, 1990.
    Mutations that modify or degrade the catalytic properties of enzyme proteins are known in the art, and examples that may be mentioned are Qiu and Goodman, Journal of Biological Chemistry 272: 8611-8617 (1997); Sugimoto et al., Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997); Mockel, "Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der allosterischen Regulation und Struktur des Enzyms", Berichte des Forschungszentrums Julichs, Jul-2906, ISSN09442952, Julich, Germany, 1994. A summary presentation can be found, for example, in known textbooks of gene and molecular biology (see Hagemann, "Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986).
    Mutations that can be considered are metastasis, conversion, insertion and deletion. Depending on the exchange effect of amino acids on enzyme activity, mutations are known as missense mutations or nonsense mutations. Insertion or deletion of one or more base pairs in a gene results in a frame shift mutation, whereby an inappropriate amino acid is inserted or prematurely terminates translation. Deletion of two or more codons typically disrupts enzymatic activity completely. Instructions for generating such mutations are included in the prior art and include, for example, known textbooks of genetics and molecular biology, for example, Knippers, "Molekulare Genetik", 6th edition, Georg Thieme Verlag , Stuttgart, Germany, 1995; Winnacker, "Gene und Klone", VCH Verlagsgesellschaft, Weinheim, Germany, 1990; And Hagemann, "Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986.
    One example of a plasmid capable of insert mutagenesis of the poxB gene is pCR2.1poxBint (FIG. 1).
    Plasmid pCR2.1poxBint is a plasmid pCR2.1-TOPO into which an internal fragment of the poxB gene set forth in SEQ ID NO: 3 is described, described in Mead et al., Bio / Technology 9: 657-663 (1991). Consists of. After transformation and homologous recombination into the chromosome poxB gene, this plasmid completely loses enzyme function. By way of example, strain DSM 5715 :: pCR2.1poxBint with pyruvate oxidase inhibition is prepared in this manner. Guidance and explanations for insertion mutagenesis are described, for example, in Schwarzer and Puhler, Bio / Technology 9, 84-87 (1991) or Fitzpatrick et al., Applied Microbiology and Biotechnology 42, 575-580 (1994). )].
    In addition, in the production of L-amino acids, especially L-lysine, in addition to attenuating the poxB gene, it would be advantageous to amplify, in particular overexpress, one or more enzymes of specific biosynthetic pathways, glycolysis, complement metabolism, citric acid cycles or amino acid excretion. Can be.
    As such, for example, to produce L-lysine, the following genes can be overexpressed simultaneously:
    DapA gene encoding dehydropicolinate synthase (reference: EP-B 0 197 335),
    DapD encoding tetradihydrodipicolinate succinylase (Wehrmann et al., Journal of Bacteriology 180, 3159-3165 (1998)),
    DapE gene encoding succinyldiaminopimelate desuccinylase (Wehrmann et al., Journal of Bacteriology 177: 5991-5993 (1995)),
    Gap gene encoding glyceraldehyde 3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
    Pyc gene encoding pyruvate carboxylase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
    Malate: mqo gene encoding quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)), or
    LysE gene encoding lysine excretion (reference: DE-A-195 48 222).
    In addition, in order to produce amino acids, especially L-lysine, it may be advantageous to inhibit unwanted secondary reactions in addition to attenuating the poxB gene. See also Nakayama: "Breeding of Amino Acid Producing Micro-organisms." ", Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982].
    The microorganism according to claim 1 is provided by the present invention and can be cultured continuously or discontinuously in batch or fed-batch or repeated fed-batch for the purpose of producing L-amino acids, in particular L-lysine. . A summary of known culture methods is provided in the textbook [Chmiel, Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991) and Storhas, Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994). It is.
    The culture medium used must suitably meet the requirements of the particular strain. Culture media for various microorganisms are described in "Manual of Methods for General Bacteriology", American Society for Bacteriology (Washington D.C., USA, 1981). As carbon sources are sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids For example, palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid can be used. These materials can be used alone or as a mixture. Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, soy flour and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate And ammonium nitrate can be used. The nitrogen source can be used alone or as a mixture. As the phosphorus source, phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or the corresponding sodium containing salts can be used. The culture medium should also contain metal salts such as magnesium sulfate or iron sulfide which are essential for propagation. Finally, in addition to the substances mentioned above, it is possible to use essential growth-promoting substances such as amino acids and vitamins. In addition, suitable precursors may also be added to the culture medium. The feed materials mentioned can be added to the culture as a single batch or can be fed as appropriate during the culture.
    Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds such as phosphoric acid or sulfuric acid can be used to adjust the pH of the culture. Defoamers such as fatty acid polyglycol esters can be used to control foam formation. In order to maintain the stability of the plasmid, a suitable substance which optionally acts, for example antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or an oxygen containing gas mixture, such as air, can be introduced into the culture. The culture temperature is generally 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. Incubation is continued until the maximum amount of side chain amino acid is produced. This object is generally achieved within 10 to 160 hours.
    Methods for measuring L-amino acids are known in the art. Analyzes by anion exchange chromatography followed by ninhydrin derivatization as described in Spackman et al., Analytical Chemistry, 30, (1958), 1990, or by Lindroth et al. , Analytical Chemistry (1979) 51: 1167-1174, can be analyzed by reverse phase HPLC.
    The following microorganisms were entrusted to the Deutsche Sammlung fur Mikrorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany:
    Escherichia coli strain DH5αpCR2.1poxBint as DSM 13114.
    Example
    The invention will be described in more detail with reference to the following examples.
    Example 1
    Preparation of Genomic Cosmid Gene Library from Corynebacterium glutamicum ATCC13032
    Chromosome DNA from Corynebacterium glutamicum ATCC13032 was isolated as described in Tauch et al., 1995, Plasmid 33: 168-179, and the restriction enzyme Sau3AI (Amersham Pharmacia; Freiburg, Germany) Material, product description Sau3AI, code no. 27-0913-02), partially cut. DNA fragments are dephosphorylated using Shrimp alkaline phosphatase (Roche Molecular Biochemicals; Mannheim, Germany, product description SAP, code no. 1758250). Cosmid Vector SuperCos1 (purchased from Stratagene; La Jolla, USA, product description SuperCos1 Cosmid Vektor Kit, Code No. 251301) .Ref. Wahl et al., 1987, Proceedings of the National Academy of Sciences USA 84: 2160. -2164] is cleaved with restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, product description XbaI, code no. 27-0948-02) and also dephosphorylation with Schrim alkaline phosphatase. The cosmid DNA is then cut with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI, code no. 27-0868-04). The cosmid DNA treated in this manner was mixed with the treated ATCC13032 DNA, and then the batch was T4 DNA ligase (manufactured by Amersham Pharmacia, Freiburg, Germany, product description T4-DNA-Ligase, code no. 27-0870). -04). The ligation mixture is then packed into phage using Gigapack II XL Packing Extracts (Stratagene; La Jolla, USA product description Gigapack II XL Packing Extract, code no. 200217). this. To infect E. coli strain NM554 (Raleigh et al., 1988, Nucleic Acid Research 16: 1563-1575), cells are suspended in 10 mM MgSO 4 and mixed with aliquots of phage suspension. Infection and titer analysis of the cosmid library was performed as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, where cells were LB agar containing 100 μg / ml of ampicillin. [Ref .: Lennox, 1955, Virology, 1: 190]. After incubation overnight at 37 ° C., individual recombinant clones are selected.
    Example 2
    Isolation and Sequencing of the poxB Gene
    Cosmid DNA of individual colonies was isolated using a Qiaprep Spin Miniprep Kit (Product No. 27106, manufactured by Qiagen, Hilden, Germany) according to the manufacturer's instructions, and the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany). cut partly with product description Sau3AI, part number 27-0913-02). The DNA fragments are dephosphorylated with Shrimp alkaline phosphatase (Roche Molecular Biochemicals; Mannheim, Germany, product description SAP, product no. 1758250). After separation by gel electrophoresis, cosmid fragments of 1500 to 2000 bp in size are separated using a QiaExII Gel Extraction Kit (Product No. 20021, manufactured by Qiagen, Hilden, Germany). DNA of the sequencing vector pZero-1 (Invitrogen; Groningen, The Netherlands, product description Zero Background Cloning Kit, product no.K2500-01) was restricted to the enzyme BamHI (Amersham Pharmacia, Freiburg, Germany). product description BamHI, product number 27-0868-04). Binding of cosmid fragments in the sequencing vector pZero-1 was performed as described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, and the DNA mixture was T4 ligase. Incubate overnight using Pharmacia Biotech (Frybuck, Germany). Thereafter, the binding mixture Electroporation into E. coli strain DH5αMCR [Grant, 1990, Proceedings of the National Academy of Sciences USA, 87: 4645-4649] [Tauch et al., 1994, FEMS Microbiol Letters, 123: 343-7] And then plated on LB agar (Lennox, 1955, Virology, 1: 190) containing 50 μg / ml zeocin. Plasmid preparation of the recombinant clones is performed using Biorobot 9600 (product no. 900200, Qiagen, Hilden, Germany). Sequencing is described by Zimmermann et al., 1990, Nucleic Acids Research, 18: 1067, Sanger et al., 1977, Proceedings of the National Academy of Sciences USA, 74: 5463-. 5467, using the dideoxy chain-termination method. "RR dRhodamin Terminator Cycle Sequencing Kit" (manufactured by PE Applied Biosystems, product no. 403044; Weiterstadt, Germany). Analysis of isolation and sequencing reactions by gel electrophoresis was carried out using an "ABI Prism 377" sequencer (PE Applied Biosystems, Weiterstadt, Germany) using a "Rotiphoresis NF acrylamide / bisacrylamide" gel (29 : 1) (Product No. A124.1, manufactured by Roth; Karlsruhe, Germany).
    The raw sequence data obtained is processed using the Staden software package (Ref. 1986, Nucleic Acids Research, 14: 217-231) 97-0. Individual sequences of pZero1 derivatives are assembled into sticky contigs. Computer-assisted cryptographic domain analysis is performed using XNIP software (Staden, 1986, Nucleic Acids Research, 14: 217-231). Further analysis is provided by the "BLAST search program" for non-redundant databanks of the National Center for Biotechnology Information (NCBI; Bethesda, Midland, USA) [Altschul et al., 1997, Nucleic Acids Research, 25: 3389-3402.
    The obtained nucleotide sequence is shown in SEQ ID NO: 1. Analysis of this nucleotide sequence revealed that the open reading frame was 1737 base pairs, which is termed the poxB gene. The poxB gene encodes a polypeptide of 579 amino acids.
    Example 3
    Preparation of Insertion Vector for Mutagenization of poxB Gene
    Chromosome DNA is isolated from strain ATCC13032 as described in Eikmanns et al., Microbiology 140: 1817-1828 (1994). Seeds known from Example 2. Based on the sequence of the poxB gene for glutamicum, the following oligonucleotides are selected for polymerase chain reaction:
    poxBint1:
    5 'TGC GAG ATG GTG AAT GGT GG 3'
    poxBint2:
    5 'GCA TGA GGC AAC GCA TTA GC 3'
    The primers presented were synthesized by MWG Biotech (Eversberg, Germany) and PCR reactions were performed using Pwo polymerase (Boehringer), Innis et al., PCR-Protocols. A guide to methods and applications, 1990, Academic Press]. DNA fragments containing an internal fragment of the poxB gene and having a size of about 0.9 kb as set forth in SEQ ID NO: 3 are isolated by polymerase chain reaction.
    DNA fragments amplified using the TOPO TA Cloning Kit (Invitrogen Corporation; Carlsbad, Calif .; Cat. No. K4500-01) were subjected to the vector pCR2.1-TOPO [Mead et al. (1991) Bio / Technology 9: 657-663. Next, this. Linked batch of E. coli strain DH5α [Ref. Hanahan, DNA Cloning. A practical approach. Vol.I. IRL-Press, Oxford, Washington DC, USA, 1985]. LB agar supplemented with 25 mg / l kanamycin for transforming batches of plasmid-bearing cells [Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989]. Plasmid DNA is isolated from the transformants using the QIAprep Spin Miniprep Kit (Qiagen), limited to the restriction enzyme EcoRI, and demonstrated by agarose gel electrophoresis (0.8%). This plasmid is named pCR2.1poxBint.
    Example 4
    Induced Mutation of the poxB Gene into the Lysine Producer DSM 5715
    A vector designated pCR2.1poxBint in Example 2 was corynebacterium glutamicum DSM using electroporation (Tauch et al., 1994, FEMS Microbiological Letters, 123: 343-347 (1994)). Electroporate into 5715. Strain DSM 5715 is an AEC-resistant lysine producer. The vector pCR2.1poxBint cannot independently replicate in DSM 5715 and is retained in cells only when inserted into the chromosome of DSM 5715. Clones containing pCR2.1poxBint inserted into the chromosome were LB agar supplemented with kanamycin 15 mg / L electroporation batch [Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Insertion was performed by labeling poxBint fragments using a Dig hybridization kit (Boehringer) according to the method according to "The DIG System Users Guide for Filter Hybridization", Boehringer Mannheim GmbH (Mannheim, Germany, 1993). Detect. Chromosome DNA of latent inserts is isolated using the method according to Eikmanns et al., Microbiology 140: 1817-1828 (1994), and in each case are cleaved with restriction enzymes SalI, SacI and HinDIII. The resulting fragments are separated by agarose gel electrophoresis and hybridized at 68 ° C. using a Dig hydration kit (Boehringer). A plasmid named pCR2.1poxBint in Example 3 is inserted into the chromosome poxB gene in the chromosome of DSM 5715. This strain is named DSM 5715 :: pCR2.1poxBint.
    Example 5
    Preparation of Lysine
    Seed obtained in Example 3. Glutamicum strain DSM 5715 :: pCR2.1poxBint is incubated in a nutrient medium suitable for lysine production and the lysine content of the culture supernatant is measured.
    To this end, the strains are first incubated at 33 ° C. for 24 hours on agar plates containing suitable antibiotics (brain / heart agar containing kanamycin (25 mg / L)). Starting from this agar plate culture, precultures are inoculated (10 ml of medium in a 100 ml Erlenmeyer flask). Complete medium CgIII is used as this preculture medium. Kanamycin (25 mg / L) is added to the medium. The preculture is incubated for 48 hours at 33 ° C. on a 240 rpm shaker. The culture is inoculated from this preculture so that the initial optical density (OD, 660 nm) of the culture is 0.1 OD. Medium MM is used for this culture.
    Badge MM:
    CSL (corn immersion liquid) 5 g / ℓ
    MOPS 20g / ℓ
    50 g / l glucose (autoclaved separately)
    salt:
    (NH 4 ) 2 SO 4 25g / ℓ
    KH 2 PO 4 0.1 g / ℓ
    MgSO 4 * 7H 2 O 1.0 g / ℓ
    CaCl 2 * 2H 2 O 10mg / L
    FeSO 4 * 7H 2 O 10 mg / l
    MnSO 4 * H 2 O 5.0 mg / l
    Biotin (Sterile-filtration) 0.3 mg / ℓ
    Thiamine * HCl (sterile-filtration) 0.2 mg / l
    Leucine (sterile-filtration) 0.1 g / ℓ
    CaCO 3 25g / ℓ
    CSL, MOPS and salt solutions are adjusted to pH 7 using ammonia solution and autoclaved. Sterile substrate and vitamin solution are then added with autoclaved dried CaCO 3 .
    Incubation is carried out in a 10 ml volume in a 100 ml Erlenmeyer flask equipped with an outlet spoiler. Kanamycin (25 mg / L) is added. Incubation is performed at 33 ° C. and 80% atmospheric humidity.
    After 48 hours, the OD is measured at a measurement wavelength of 660 nm using a Biomek 1000 from Beckmann Instruments GmbH; The amount of lysine formed is determined by post-column derivatization using ion exchange chromatography and ninhydrin detection using an amino acid analyzer (Eppendorf-BioTronik, Hamburg, Germany).
    The experimental results are shown in Table 1.
    StrainOD (660)Lysine HCl (g / L) DSM 571513.19.5 DSM 5715 :: pCR2.1poxBint12.512.9
    Example 6
    Induced Mutation of the poxB Gene into Valine Producer FERM-BP 1763
    The vector named pCR2.1poxBint in Example 2 was used to electroporation (Tauch et al., 1994, FEMS Microbiological Letters, 123: 343-347 (1994)) Brevibacterium lactofermentum FERM Electroporate into BP 1763. Strain FERM-BP 1763 is a mycophenolic acid-resistant valine producer (US Pat. No. US-A-5,188,948). The vector pCR2.1poxBint cannot independently replicate in FERM-BP 1763 and is retained in cells only when inserted into the chromosome of FERM-BP 1763. Clones containing pCR2.1poxBint inserted into the chromosome were LB agar supplemented with kanamycin 15 mg / L electroporation batch [Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Insertion was performed by labeling poxBint fragments using a Dig hybridization kit (Boehringer) according to the method according to "The DIG System Users Guide for Filter Hybridization", Boehringer Mannheim GmbH (Mannheim, Germany, 1993). Detect. Chromosome DNA of latent inserts is isolated using the method according to Eikmanns et al., Microbiology 140: 1817-1828 (1994), and in each case are cleaved with restriction enzymes SalI, SacI and HinDIII. The resulting fragments are separated by agarose gel electrophoresis and hybridized at 68 ° C. using a Dig hydration kit (Boehringer). The plasmid named pCR2.1poxBint in Example 3 is inserted into the chromosome poxB gene of the chromosome of FERM-BP 1763. This strain is named FERM-BP 1763 :: pCR2.1poxBint.
    Example 7
    Manufacture of valine
    Ratio obtained in Example 6. The lactofermentum strain FERM-BP 1763 :: pCR2.1poxBint is incubated in a nutrient medium suitable for valine production and the valine content of the culture supernatant is measured.
    To this end, the strains are first incubated for 24 hours at 33 ° C. on agar plates containing a suitable antibiotic (brain / heart agar containing kanamycin (25 mg / L)). Starting from this agar plate culture, precultures are inoculated (10 ml of medium in a 100 ml Erlenmeyer flask). Complete medium CgIII is used as this preculture medium. Kanamycin (25 mg / L) is added to the medium. The preculture is incubated for 24 hours at 33 ° C. on a 240 rpm shaker. The culture is inoculated from this preculture so that the initial optical density (OD, 660 nm) of the culture is 0.1 OD. Medium MM is used for this culture.
    Badge MM:
    CSL 5g / ℓ
    MOPS 20g / ℓ
    50 g / l glucose (autoclaved separately)
    salt:
    (NH 4 ) 2 SO 4 25g / ℓ
    KH 2 PO 4 0.1 g / ℓ
    MgSO 4 * 7H 2 O 1.0 g / ℓ
    CaCl 2 * 2H 2 O 10mg / L
    FeSO 4 * 7H 2 O 10 mg / l
    MnSO 4 * H 2 O 5.0 mg / l
    Isoleucine (sterile-filtration) 0.1 g / ℓ
    Methionine (sterile-filtration) 0.1 g / ℓ
    Thiamine * HCl (sterile-filtration) 0.2 mg / l
    Leucine (sterile-filtration) 0.1 g / ℓ
    CaCO 3 25g / ℓ
    CSL (corn immersion), MOPS (morpholinopropanesulfonic acid) and salt solution are adjusted to pH 7 using ammonia solution and autoclaved. Sterile substrate and vitamin solution are then added with autoclaved dried CaCO 3 .
    Incubation is carried out in a 10 ml volume in a 100 ml Erlenmeyer flask equipped with an outlet spoiler. Kanamycin (25 mg / L) is added. Incubation is performed at 33 ° C. and 80% atmospheric humidity.
    After 48 hours, the OD is measured at a measurement wavelength of 660 nm using a Biomek 1000 from Beckmann Instruments GmbH; The amount of valine formed is determined by post-column derivatization using ion exchange chromatography and ninhydrin detection using an amino acid analyzer (Eppendorf-BioTronik, Hamburg, Germany).
    The experimental results are shown in Table 2.
    StrainOD (660)Valine HCl (g / L) FERM-BP 17638.612.1 FERM-BP 1763 :: pCR2.1poxBint9.513.0
    The present invention enables the production of L-amino acids in an improved fermentative method by attenuating the poxB gene.
    权利要求:
    Claims (16)
    [1" claim-type="Currently amended] a) a polynucleotide at least 70% identical to the polynucleotide encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 2,
    b) a polynucleotide encoding a polypeptide containing an amino acid sequence at least 70% identical to the amino acid sequence of SEQ ID NO: 2,
    c) a polynucleotide complementary to the polynucleotide of a) or b) above and
    d) an isolated polynucleotide containing a polynucleotide sequence selected from the group consisting of polynucleotides containing at least 15 contiguous bases of the polynucleotide sequences of a), b) or c).
    [2" claim-type="Currently amended] The polynucleotide of claim 1, wherein the polynucleotide is replicable, preferably recombinant DNA.
    [3" claim-type="Currently amended] The polynucleotide of claim 1, which is RNA.
    [4" claim-type="Currently amended] The polynucleotide of claim 2 comprising the nucleotide sequence set forth in SEQ ID NO: 1.
    [5" claim-type="Currently amended] The polynucleotide of claim 2 encoding a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 2.
    [6" claim-type="Currently amended] The method of claim 2,
    (i) the nucleotide sequence set forth in SEQ ID NO: 1,
    (ii) one or more sequences that match the sequence of (i) within the degenerate range of the genetic code, or
    (iii) one or more sequences that hybridize with sequences complementary to the sequences of (i) or (ii), and optionally
    (iv) a polynucleotide which is a replicable DNA containing a functionally neutral sense mutation in (i).
    [7" claim-type="Currently amended] this. A vector containing a polynucleotide as claimed in claim 1, in particular d), deposited with E. coli DSM 13114.
    [8" claim-type="Currently amended] Coryneform bacteria that act as host cells containing deletions or insertions in the poxB gene.
    [9" claim-type="Currently amended] a) fermenting a bacterium producing the desired L-amino acid, which at least attenuates the poxB gene,
    b) accumulating the desired L-amino acid in the medium or cells of the bacterium, and
    c) a process for producing L-amino acids, in particular L-lysine, characterized in that the step of separating the L-amino acids is carried out.
    [10" claim-type="Currently amended] 10. The method of claim 9, wherein a bacterium further amplified with an additional gene in the biosynthetic pathway of the desired L-amino acid is used.
    [11" claim-type="Currently amended] 10. The method of claim 9, wherein a bacterium is used wherein at least partially inhibited metabolic pathways that reduce the production of the desired L-amino acids.
    [12" claim-type="Currently amended] 10. The method according to claim 9, characterized in that the expression of polynucleotides as claimed in claims 1, in particular 1a) to 1c) is reduced.
    [13" claim-type="Currently amended] 10. The method according to claim 9, characterized in that the catalytic properties of the polypeptide (enzyme protein) encoded by the polynucleotide as claimed in claims 1, in particular 1a) to 1c) are reduced.
    [14" claim-type="Currently amended] 10. The method of claim 9, wherein a bacterium attenuated using insertion mutagenesis by the plasmid pCR2.1poxBint or one of its components shown in FIG. 1 and deposited as DSM 13114 is used.
    [15" claim-type="Currently amended] The method of claim 9,
    DapA gene, which encodes a dihydropicolinate synthase,
    DNA fragments providing S- (2-aminoethyl) cysteine resistance,
    A pyc gene encoding pyruvate carboxylase,
    DapE gene encoding succinyldiaminopimelate desuccinase,
    Gap gene encoding glyceraldehyde 3-phosphate dehydrogenase,
    Maleate: mqo gene encoding quinone oxidoreductase or
    L-lysine is produced by fermenting bacteria in which at least one gene selected from the group consisting of lysE genes encoding lysine excretion is simultaneously overexpressed.
    [16" claim-type="Currently amended] The method according to any one of claims 9 to 15, characterized in that a microorganism of the genus Corynebacterium glutamicum is used.
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    CA2322553A1|2001-04-28|
    EP1096013A2|2001-05-02|
    BR0005091A|2001-06-19|
    AU6807500A|2001-05-03|
    EP1096013A3|2005-06-01|
    ZA200006039B|2001-05-03|
    DE19951975A1|2001-05-03|
    HU0004188D0|2001-01-29|
    SK15732000A3|2001-11-06|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1999-10-28|Priority to DE19951975.7
    1999-10-28|Priority to DE1999151975
    2000-10-27|Application filed by 데구사-휠스 악티엔게젤샤프트
    2001-06-25|Publication of KR20010051289A
    优先权:
    申请号 | 申请日 | 专利标题
    DE19951975.7|1999-10-28|
    DE1999151975|DE19951975A1|1999-10-28|1999-10-28|New Nuleotide sequences coding for the poxB gene|
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