 Nucleotide sequences coding for the pgi gene
专利摘要:
The present invention a) a polynucleotide having at least 70% homology with a 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 having at least 70% homology with the amino acid of SEQ ID NO: 2, c) a polynucleotide complementary to the polynucleotides of a) and 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 of the polynucleotide sequence of a), b) or c) above, and the pgi gene To increase the metabolic flux through the pentose phosphate cycle. 公开号:KR20010050449A 申请号:KR1020000054019 申请日:2000-09-14 公开日:2001-06-15 发明作者:맥코맥아쉴링；스테이플턴클리오나；버크케빈；오도나우마이클；막스아힘；뫼켈베티나 申请人:데구사-휠스 악티엔게젤샤프트；내셔널 유니버시티 오브 아일랜드； IPC主号:
专利说明:
Nucleotide sequences coding for the pgi gene} 〈110〉 Degussa-Huls AG National university of Ireland <120> Nucleotide sequences coding for the pgi gene <130> 519990243334; 520000414800 〈150〉 US 09 / 396,478 <151> 1999-09-15 〈160〉 3 〈170〉 KOPATIN 1.5 〈210〉 1 <211> 2811 <212> DNA 213 Corynebacterium glutamicum 〈220〉 <221> CDS 222 (373) .. (2022) <400> 1 aaaacccgag gggcgaaaat tccaccctaa cttttttggg atcccctttt tccggggaat 60 taattggttt gggtttcaat gggaaaacgg gaaacaatgg gccaaaggtt caaaaacccc 120 aaaagggggc cgggttcaaa ttcccaaaaa aaatggcaaa aaaggggggg ccaaaaccaa 180 gttggccccc aaaccaccgg ggcaacggcc cacccacaaa ggggttgggt taaaggaagg 240 acgcccaaag taagcccgga atggcccacg ttcgaaaaag caggccccaa ttaaacgcac 300 cttaaatttg tcgtgtttcc cactttgaac actcttcgat gcgcttggcc acaaaagcaa 360 gctaacctga ag atg tta ttt aac gac aat aaa gga gtt ttc atg 405 Met Leu Phe Asn Asp Asn Lys Gly Val Phe Met 1 5 10 gcg gac att tcg acc acc cag gtt tgg caa gac ctg acc gat cat tac 453 Ala Asp Ile Ser Thr Thr Gln Val Trp Gln Asp Leu Thr Asp His Tyr 15 20 25 tca aac ttc cag gca acc act ctg cgt gaa ctt ttc aag gaa gaa aac 501 Ser Asn Phe Gln Ala Thr Thr Leu Arg Glu Leu Phe Lys Glu Glu Asn 30 35 40 cgc gcc gag aag tac acc ttc tcc gcg gct ggc ctc cac gtc gac ctg 549 Arg Ala Glu Lys Tyr Thr Phe Ser Ala Ala Gly Leu His Val Asp Leu 45 50 55 tcg aag aat ctg ctt gac gac gcc acc ctc acc aag ctc ctt gca ctg 597 Ser Lys Asn Leu Leu Asp Asp Ala Thr Leu Thr Lys Leu Leu Ala Leu 60 65 70 75 acc gaa gaa tct ggc ctt cgc gaa cgc att gac gcg atg ttt gcc ggt 645 Thr Glu Glu Ser Gly Leu Arg Glu Arg Ile Asp Ala Met Phe Ala Gly 80 85 90 gaa cac ctc aac aac acc gaa gac cgc gct gtc ctc cac acc gcg ctg 693 Glu His Leu Asn Asn Thr Glu Asp Arg Ala Val Leu His Thr Ala Leu 95 100 105 cgc ctt cct gcc gaa gct gat ctg tca gta gat ggc caa gat gtt gct 741 Arg Leu Pro Ala Glu Ala Asp Leu Ser Val Asp Gly Gln Asp Val Ala 110 115 120 gct gat gtc cac gaa gtt ttg gga cgc atg cgt gac ttc gct act gcg 789 Ala Asp Val His Glu Val Leu Gly Arg Met Arg Asp Phe Ala Thr Ala 125 130 135 ctg cgc tca ggc aac tgg ttg gga cac acc ggc cac acg atc aag aag 837 Leu Arg Ser Gly Asn Trp Leu Gly His Thr Gly His Thr Ile Lys Lys 140 145 150 155 atc gtc aac att ggt atc ggt ggc tct gac ctc gga cca gcc atg gct 885 Ile Val Asn Ile Gly Ile Gly Gly Ser Asp Leu Gly Pro Ala Met Ala 160 165 170 acg aag gct ctg cgt gca tac gcg acc gct ggt atc tca gca gaa ttc 933 Thr Lys Ala Leu Arg Ala Tyr Ala Thr Ala Gly Ile Ser Ala Glu Phe 175 180 185 gtc tcc aac gtc gac cca gca gac ctc gtt tct gtg ttg gaa gac ctc 981 Val Ser Asn Val Asp Pro Ala Asp Leu Val Ser Val Leu Glu Asp Leu 190 195 200 gat gca gaa tcc aca ttg ttc gtg atc gct tcg aaa act ttc acc acc 1029 Asp Ala Glu Ser Thr Leu Phe Val Ile Ala Ser Lys Thr Phe Thr Thr 205 210 215 cag gag acg ctg tcc aac gct cgt gca gct cgt gct tgg ctg gta gag 1077 Gln Glu Thr Leu Ser Asn Ala Arg Ala Ala Arg Ala Trp Leu Val Glu 220 225 230 235 aag ctc ggt gaa gag gct gtc gcg aag cac ttc gtc gca gtg tcc acc 1125 Lys Leu Gly Glu Glu Ala Val Ala Lys His Phe Val Ala Val Ser Thr 240 245 250 aat gct gaa aag gtc gca gag ttc ggt atc gac acg gac aac atg ttc 1173 Asn Ala Glu Lys Val Ala Glu Phe Gly Ile Asp Thr Asp Asn Met Phe 255 260 265 ggc ttc tgg gac tgg gtc gga ggt cgt tac tcc gtg gac tcc gca gtt 1221 Gly Phe Trp Asp Trp Val Gly Gly Arg Tyr Ser Val Asp Ser Ala Val 270 275 280 ggt ctt tcc ctc atg gca gtg atc ggc cct cgc gac ttc atg cgt ttc 1269 Gly Leu Ser Leu Met Ala Val Ile Gly Pro Arg Asp Phe Met Arg Phe 285 290 295 ctc ggt gga ttc cac gcg atg gat gaa cac ttc cgc acc acc aag ttc 1317 Leu Gly Gly Phe His Ala Met Asp Glu His Phe Arg Thr Thr Lys Phe 300 305 310 315 gaa gag aac gtt cca atc ttg atg gct ctg ctc ggt gtc tgg tac tcc 1365 Glu Glu Asn Val Pro Ile Leu Met Ala Leu Leu Gly Val Trp Tyr Ser 320 325 330 gat ttc tat ggt gca gaa acc cac gct gtc cta cct tat tcc gag gat 1413 Asp Phe Tyr Gly Ala Glu Thr His Ala Val Leu Pro Tyr Ser Glu Asp 335 340 345 ctc agc cgt ttt gct gct tac ctc cag cag ctg acc atg gag acc aat 1461 Leu Ser Arg Phe Ala Ala Tyr Leu Gln Gln Leu Thr Met Glu Thr Asn 350 355 360 ggc aag tca gtc cac cgc gac ggc tcc cct gtt tcc act ggc act ggc 1509 Gly Lys Ser Val His Arg Asp Gly Ser Pro Val Ser Thr Gly Thr Gly 365 370 375 gaa att tac tgg ggt gag cct ggc aca aat ggc cag cac gct ttc ttc 1557 Glu Ile Tyr Trp Gly Glu Pro Gly Thr Asn Gly Gln His Ala Phe Phe 380 385 390 395 cag ctg atc cac cag ggc act cgc ctt gtt cca gct gat ttc att ggt 1605 Gln Leu Ile His Gln Gly Thr Arg Leu Val Pro Ala Asp Phe Ile Gly 400 405 410 ttc gct cgt cca aag cag gat ctt cct gcc ggt gag cgc acc atg cat 1653 Phe Ala Arg Pro Lys Gln Asp Leu Pro Ala Gly Glu Arg Thr Met His 415 420 425 gac ctt ttg atg agc aac ttc ttc gca cag acc aag gtt ttg gct ttc 1701 Asp Leu Leu Met Ser Asn Phe Phe Ala Gln Thr Lys Val Leu Ala Phe 430 435 440 ggt aag aac gct gaa gag atc gct gcg gaa ggt gtc gca cct gag ctg 1749 Gly Lys Asn Ala Glu Glu Ile Ala Ala Glu Gly Val Ala Pro Glu Leu 445 450 455 gtc aac cac aag gtc gtg cca ggt aat cgc cca acc acc acc att ttg 1797 Val Asn His Lys Val Val Pro Gly Asn Arg Pro Thr Thr Thr Ile Leu 460 465 470 475 gcg gag gaa ctt acc cct tct att ctc ggt gcg ttg atc gct ttg tac 1845 Ala Glu Glu Leu Thr Pro Ser Ile Leu Gly Ala Leu Ile Ala Leu Tyr 480 485 490 gaa cac acc gtg atg gtt cag ggc gtg att tgg gac atc aac tcc ttc 1893 Glu His Thr Val Met Val Gln Gly Val Ile Trp Asp Ile Asn Ser Phe 495 500 505 gac caa tgg ggt gtt gaa ctg ggc aaa cag cag gca aat gac ctc gct 1941 Asp Gln Trp Gly Val Glu Leu Gly Lys Gln Gln Ala Asn Asp Leu Ala 510 515 520 ccg gct gtc tct ggt gaa gag gat gtt gac tcg gga gat tct tcc act 1989 Pro Ala Val Ser Gly Glu Glu Asp Val Asp Ser Gly Asp Ser Ser Thr 525 530 535 gat tca ctg att aag tgg tac cgc gca aat agg tagtcgct tgcttatagg 2040 Asp Ser Leu Ile Lys Trp Tyr Arg Ala Asn Arg 540 545 550 gtcaggggcg tgaagaatcc tcgcctcata gcactggccg ctatcatcct gacctcgttc 2100 aatctgcgaa cagctattac tgctttagct ccgctggttt ctgagattcg ggatgattta 2160 ggggttagtg cttctcttat tggtgtgttg ggcatgatcc cgactgctat gttcgcggtt 2220 gctgcgtttg cgcttccgtc gttgaagagg aagttcacta cttcccaact gttgatgttt 2280 gccatgctgt tgactgctgc cggtcagatt attcgtgtcg ctggacctgc ttcgctgttg 2340 atggtcggta ctgtgttcgc gatgtttgcg atcggagtta ccaatgtgtt gcttccgatt 2400 gctgttaggg agtattttcc gcgtcacgtc ggtggaatgt cgacaactta tctggtgtcg 2460 ttccagattg ttcaggcact tgctccgacg cttgccgtgc cgatttctca gtgggctaca 2520 catgtggggt tgaccggttg gagggtgtcg ctcggttcgt gggcgctgct ggggttggtt 2580 gcggcgattt cgtggattcc gctgttgagt ttgcagggtg ccagggttgt tgcggcgccg 2640 tcgaaggttt ctcttcctgt gtggaagtct tcggttggtg tggggctcgg gttgatgttt 2700 gggtttactt cgtttgcgac gtatatcctc atgggtttta tgccgcagat ggtaggtgat 2760 ccaaagaatt caaaaagctt ctcgagagta cttctagagc ggccgcgggc c 2811 〈210〉 2 <211> 550 <212> PRT 213 Corynebacterium glutamicum <400> 2 Met Leu Phe Asn Asp Asn Lys Gly Val Phe Met Ala Asp Ile Ser Thr 1 5 10 15 Thr Gln Val Trp Gln Asp Leu Thr Asp His Tyr Ser Asn Phe Gln Ala 20 25 30 Thr Thr Leu Arg Glu Leu Phe Lys Glu Glu Asn Arg Ala Glu Lys Tyr 35 40 45 Thr Phe Ser Ala Ala Gly Leu His Val Asp Leu Ser Lys Asn Leu Leu 50 55 60 Asp Asp Ala Thr Leu Thr Lys Leu Leu Ala Leu Thr Glu Glu Ser Gly 65 70 75 80 Leu Arg Glu Arg Ile Asp Ala Met Phe Ala Gly Glu His Leu Asn Asn 85 90 95 Thr Glu Asp Arg Ala Val Leu His Thr Ala Leu Arg Leu Pro Ala Glu 100 105 110 Ala Asp Leu Ser Val Asp Gly Gln Asp Val Ala Ala Asp Val His Glu 115 120 125 Val Leu Gly Arg Met Arg Asp Phe Ala Thr Ala Leu Arg Ser Gly Asn 130 135 140 Trp Leu Gly His Thr Gly His Thr Ile Lys Lys Ile Val Asn Ile Gly 145 150 155 160 Ile Gly Gly Ser Asp Leu Gly Pro Ala Met Ala Thr Lys Ala Leu Arg 165 170 175 Ala Tyr Ala Thr Ala Gly Ile Ser Ala Glu Phe Val Ser Asn Val Asp 180 185 190 Pro Ala Asp Leu Val Ser Val Leu Glu Asp Leu Asp Ala Glu Ser Thr 195 200 205 Leu Phe Val Ile Ala Ser Lys Thr Phe Thr Thr Gln Glu Thr Leu Ser 210 215 220 Asn Ala Arg Ala Ala Arg Ala Trp Leu Val Glu Lys Leu Gly Glu Glu 225 230 235 240 Ala Val Ala Lys His Phe Val Ala Val Ser Thr Asn Ala Glu Lys Val 245 250 255 Ala Glu Phe Gly Ile Asp Thr Asp Asn Met Phe Gly Phe Trp Asp Trp 260 265 270 Val Gly Gly Arg Tyr Ser Val Asp Ser Ala Val Gly Leu Ser Leu Met 275 280 285 Ala Val Ile Gly Pro Arg Asp Phe Met Arg Phe Leu Gly Gly Phe His 290 295 300 Ala Met Asp Glu His Phe Arg Thr Thr Lys Phe Glu Glu Asn Val Pro 305 310 315 320 Ile Leu Met Ala Leu Leu Gly Val Trp Tyr Ser Asp Phe Tyr Gly Ala 325 330 335 Glu Thr His Ala Val Leu Pro Tyr Ser Glu Asp Leu Ser Arg Phe Ala 340 345 350 Ala Tyr Leu Gln Gln Leu Thr Met Glu Thr Asn Gly Lys Ser Val His 355 360 365 Arg Asp Gly Ser Pro Val Ser Thr Gly Thr Gly Glu Ile Tyr Trp Gly 370 375 380 Glu Pro Gly Thr Asn Gly Gln His Ala Phe Phe Gln Leu Ile His Gln 385 390 395 400 Gly Thr Arg Leu Val Pro Ala Asp Phe Ile Gly Phe Ala Arg Pro Lys 405 410 415 Gln Asp Leu Pro Ala Gly Glu Arg Thr Met His Asp Leu Leu Met Ser 420 425 430 Asn Phe Phe Ala Gln Thr Lys Val Leu Ala Phe Gly Lys Asn Ala Glu 435 440 445 Glu Ile Ala Ala Glu Gly Val Ala Pro Glu Leu Val Asn His Lys Val 450 455 460 Val Pro Gly Asn Arg Pro Thr Thr Thr Ile Leu Ala Glu Glu Leu Thr 465 470 475 480 Pro Ser Ile Leu Gly Ala Leu Ile Ala Leu Tyr Glu His Thr Val Met 485 490 495 Val Gln Gly Val Ile Trp Asp Ile Asn Ser Phe Asp Gln Trp Gly Val 500 505 510 Glu Leu Gly Lys Gln Gln Ala Asn Asp Leu Ala Pro Ala Val Ser Gly 515 520 525 Glu Glu Asp Val Asp Ser Gly Asp Ser Ser Thr Asp Ser Leu Ile Lys 530 535 540 Trp Tyr Arg Ala Asn Arg 545 550 〈210〉 3 <211> 462 <212> DNA 213 Corynebacterium glutamicum <400> 3 atggagacca atggcaagtc agtccaccgc gacggctccc ctgtttccac tggcactggc 60 gaaatttact ggggtgagcc tggcacaaat ggccagcacg ctttcttcca gctgatccac 120 cagggcactc gccttgttcc agctgatttc attggtttcg ctcgtccaaa gcaggatctt 180 cctgccggtg agcgcaccat gcatgacctt ttgatgagca acttcttcgc acagaccaag 240 gttttggctt tcggtaagaa cgctgaagag atcgctgcgg aaggtgtcgc acctgagctg 300 gtcaaccaca aggtcgtgcc aggtaatcgc ccaaccacca ccattttggc ggaggaactt 360 accccttcta ttctcggtgc gttgatcgct ttgtacgaac acaccgtgat ggttcagggc 420 gtgatttggg acatcaactc cttcgaccaa tggggcgtgg aa 462 The present invention provides a nucleotide sequence from a Coryneform bacterium encoding a pgi gene, and a method of attenuating the pgi gene to increase metabolic rate through the pentose phosphate cycle. Nucleotides, vitamins and especially L-amino acids, more particularly lysine and tryptophan, are used in the foodstuff industry, animal feed, human pharmaceutical and pharmaceutical industries. As is known, these materials are produced by fermentation using Corynebacterium strains, in particular Corynebacterium glutamicum. Because of their enormous importance, attempts have been made to improve the manufacturing method. Improvements to the method include fermentation techniques (e.g., agitation and oxygenation), composition of the nutrient medium (e.g. sugar concentrations during fermentation), post-treatment of the product (e.g. ion exchange chromatography), or the inherent in the microorganism itself. To means associated with performance characteristics. Performance characteristics of these microorganisms are improved using mutagenesis, screening and mutant selection. In this way, strains are obtained that are resistant to anti-metabolites or that are nutritional components for important regulatory intermediates and that produce nucleotides, vitamins or amino acids. For many years, methods of recombinant DNA technology have also been used to improve Corynebacterium strains that produce nucleotides, vitamins and L-amino acids. Typical raw materials for producing these compounds are glucose commonly used in the form of starch hydrolysates. Sucrose is also used as a raw material. By cell uptake, glucose was phosphorylated with the consumption of phosphoroenolpyruvate (phosphotransferase system) (Malin & Bourd, Journal of Applied Bacteriology 71, 517-523 (1993)) and then glucose Cells are available as -6-phosphate. Sucrose is a phosphotransferase system [Shio et al., Agricultural and Biological Chemistry 54, 1513-1519 (1990)] and a conversion enzyme reaction [Yamamoto et al., Journal of Fermentation Technology 64, 285-291]. (1986)] to fructose and glucose-6-phosphate. In glucose catabolism, the enzymes glucose-6-phosphate dehydrogenase (EC 1.1.14.9) and glucose-6-phosphate isomerase (EC 5.3.1.9) compete for the substrate glucose-6-phosphate. The enzyme glucose-6-phosphate isomerase catalyzes the first reaction step of the Embden-Meyerhof-Parnas pathway or the corresponding pathway, ie the conversion to fructose-6-phosphate. The enzyme glucose-6-phosphate dehydrogenase catalyzes the first reaction step of the oxidized portion of the pentose phosphate cycle, ie the conversion to 6-phosphoglucolacnolactone. In the oxidized portion of the pentose phosphate cycle, glucose-6-phosphate is converted to ribulose-5-phosphate to produce a reducing equivalent in the form of NADPH. As the pentose phosphate cycle further proceeds, the pentose phosphate, hexose phosphate and triose phosphate are interconverted. Pentose phosphate (eg 5-phosphoribosyl-1-pyrophosphate) is required, for example, in nucleotide biosynthesis. 5-phosphoribosyl-1-pyrophosphate is also a precursor of aromatic amino acids and amino acid L-histidine. NADPH acts as a reducing equivalent in many assimilation biosynthesis. Therefore, four molecules of NADPH are consumed to biosynthesize one molecule of L-lysine from oxal acetic acid. The importance of the pentose phosphate cycle in the biosynthesis and production of amino acids, in particular L-lysine, by coryneform bacteria is known and has been the focus of attention of many experts. Therefore, Oishi & Aida (Agricultural and Biological Chemistry 29, 83-89 (1965)) reported "hexose monophosphate avoidance" by Brevibacterium ammoniagenes. Studies using the 13 C isotope method of Ishino et al., Journal of General and Applied Microbiology 37, 157-165 (1991) on glucose metabolism during fermentation have been conducted through the pentose phosphate cycle. Correlation between lysine production and metabolic rate is shown. It is an object of the present inventors to provide a method for increasing the metabolic rate through the pentose phosphate cycle. 1 is a map of plasmid pAMC1. 2 is a map of plasmid pMC1. Abbreviations and names used in FIGS. 1 and 2 are defined as follows: Neo r: neomycin / kanamycin resistance ColE1 ori: origin of replication of plasmid ColE1 CMV: cytomegalovirus promoter lacP: lactose promoter pgi: phosphoglucose isomerase gene lacZ: 5'-terminus of β-galactosidase gene SV40 3 'Splice: 3' splicing site of Simian virus 40 SV40 polyA: polyadenylation site of Simian virus 40 f1 (-) ori: origin of replication of filamentous grip f1 SV40 ori: origin of replication of simian virus 40 kan r: kanamycin resistant pgi insert: the inner fragment of the gene pgi ori: origin of replication of plasmid pBGS8 AccI: cleavage site of restriction enzyme AccI ApaI: cleavage site of restriction enzyme ApaI BamHI: cleavage site of the restriction enzyme BamHI ClaI: cleavage site of restriction enzyme ClaI DraI: cleavage site of the restriction enzyme DraI EcoRI: cleavage site of restriction enzyme EcoRI HindIII: cleavage site of the restriction enzyme HindIII MluI: cleavage site of the restriction enzyme MluI MstII: cleavage site of restriction enzyme MstII NheI: cleavage site of the restriction enzyme NheI NsiI: cleavage site of the restriction enzyme NsiI PstI: cleavage site of restriction enzyme PstI PvuII: cleavage site of the restriction enzyme PvuII SacI: cleavage site of restriction enzyme SacI SalI: cleavage site of restriction enzyme SalI SmaI: cleavage site of the restriction enzyme SmaI SpeI: cleavage site of restriction enzyme SpeI SspI: cleavage site of restriction enzyme SspI Figure 3 [6- 13 C] is the nuclear magnetic resonance spectra for strain DSM5715 :: pMC1 cultured on dextrose. Coupled to the echo data, (b) carbon atom position C-2 to C-6 of lysine from - L-2 to L-6, 13 C- dissociation exists under measurement (a) and 13 C- dissociation member under Spin: Acronym Nuclear magnetic resonance of protons. 4 is nuclear magnetic resonance spectra for strain DSM5715 :: pMC1 cultured on [ 1-13 C] dextrose. Coupled to the echo data, (b) carbon atom position C-2 to C-6 of lysine from - L-2 to L-6, 13 C- dissociation exists under measurement (a) and 13 C- dissociation member under Spin: Acronym Nuclear magnetic resonance of protons. 5 is a nuclear magnetic resonance spectrum for strain DSM5715 cultured on [6- 13 C] dextrose. Coupled to the echo data, (b) carbon atom position C-2 to C-6 of lysine from - L-2 to L-6, 13 C- dissociation exists under measurement (a) and 13 C- dissociation member under Spin: Acronym Nuclear magnetic resonance of protons. 6 is nuclear magnetic resonance spectra for strain DSM5715 cultured on [ 1-13 C] dextrose. Coupled to the echo data, (b) carbon atom position C-2 to C-6 of lysine from - L-2 to L-6, 13 C- dissociation exists under measurement (a) and 13 C- dissociation member under Spin: Acronym Nuclear magnetic resonance of protons. FIG. 7 is the correlation of pentose phosphate pathway velocity with enhancement ratio [(BA) / B]. By computer simulation and in Marx et al., 1996 (Biotechnology and Bioengineering 49: 111-129), Marx et al., 1997 (Biotechnology and Bioengineering 56, 168-180), Marx et al., 1999 ( Metabolic Engineering 1: 35-48), Sonntag et al., 1993 (European Journal of Biochemistry 213: 1325-1331) and Sonntag et al., 1995 (Applied Microbiology and Biotechnology 44: 489-495). By using the hyperbolic and linear functions for the correlation of PPP rate and reinforcement ratio [(BA) / B], the phosphoglucoisomerase (Pgi) is present to balance (@) or absent overall (-), respectively. If found in the metabolic network. Speed through the PPP is derived from the ratio (BA) / B (wherein, A represents the entire 13 C strengthening of the lysine produced from the reinforced A, B indicates the total 13 C strengthening of the lysine prepared from experiment B) ( Table 2). Experimental and corresponding PPP rates for (BA) / B are shown for strains DSM5715 (□) and DSM5715 :: pMC1 (□). The PPP rate represents the dextrose absorption ratio as mol per 100 mol and the strengthening ratio [(BA) / B] is expressed as%. Nucleotides, vitamins and especially L-amino acids, more particularly L-lysine and L-tryptophan, are used in the foodstuff industry, animal feed, human pharmaceutical and pharmaceutical industries. Therefore, it is a general concern to provide improved methods for the production of these products. The present invention a) a polynucleotide having at least 70% homology with a 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 having at least 70% homology with the amino acid of SEQ ID NO: 2, c) a polynucleotide complementary to the polynucleotides 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 of the polynucleotide sequence of a), b) or c) above. In addition, the present invention preferably comprises (i) the nucleotide sequence set forth in SEQ ID NO: 1 or (ii) one or more sequences that match sequence (i) above within the degeneracy range of the genetic code, or (iii) one or more sequences that hybridize with the complementary sequence to sequence (i) or (ii) above and optionally (iv) providing a polynucleotide according to claim 1 comprising a reproducible DNA containing the functional neutral sense mutation in (i) above. The present invention also provides a polynucleotide according to claim 2 containing the nucleotide sequence set forth in SEQ ID NO: 1, A polynucleotide according to claim 2 encoding a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 2, The polynucleotide of claim 1, indent d, in particular E. Vector containing pMCl deposited with E. coli DSM12969 and There is provided a Coryneform bacterium which acts as a host cell containing the vector according to claim 6. "Isolated" means separated from its natural environment. "Polynucleotides" generally refer to polyribonucleotides and polydeoxyribonucleotides in which RNA or DNA may or may not be modified. "Polypeptide" means a peptide or protein containing two or more amino acids linked by peptide bonds. Polypeptides according to the invention are at least 70% homologous, preferably with a polypeptide having the biological activity of the polypeptide according to SEQ ID NO: 2, in particular glucose-6-phosphate isomerase and also a polypeptide according to SEQ ID NO: 2 Polypeptides having at least 80% and especially at least 95% homology with the polypeptides according to SEQ ID NO: 2 and having the activities described above. The present invention also particularly relates to nucleotides, vitamins and especially L-amino acids, more particularly lysine and tryptophan, using coryneform bacteria which already produce the above-mentioned substances and attenuate, especially at low levels, nucleotide sequences encoding the pgi gene. It provides a method of producing fermentation. In this regard, the term “attenuation” refers to the use of enzymes or genes or alleles that encode, for example, weak promoters or low activity of corresponding enzymes or inactivate the corresponding enzymes (proteins), or by optionally combining these means. It describes the reduction or blocking of the intracellular activity of one or more enzymes (proteins) in a microorganism encoded by the corresponding DNA. Microorganisms provided by the present invention can produce nucleotides, vitamins and in particular L-amino acids, more particularly lysine and tryptophan, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or ethanol . These microorganisms may typically include Corynebacterium bacteria and in particular Corynebacterium genus. Among the Corynebacterium genus, Corynebacterium glutamicum can be mentioned in particular, which is well known in the art for the production capacity of L-amino acids. Suitable strains of the genus Corynebacterium, especially suitable strains of Corynebacterium glutamicum species, are known wild type strains that produce nucleotides, vitamins or L-amino acids, for example Corynebacterium glutamicum ATCC13032; Corynebacterium acetoglutamicum ATCC15806; Corynebacterium acetoacidophilum ATCC13870; Corynebacterium termoaminogenes (ferm bp-1539) Brevibacterium flavum ATCC14067; Brevibacterium lactofermentum ATCC13869; And Brevibacterium divaricatum ATCC14020; And mutants thereof or strains generated therefrom, For example, as a 5'-inosinic acid producing strain Corynebacterium ammonia genes ATCC15190; Corynebacterium ammonia genes ATCC15454 and Corynebacterium glutamicum ATCC14998; or For example, as a 5'-guanylic acid producing strain Corynebacterium glutamicum ATCC21171 and Corynebacterium ammonia genes ATCC19216; or For example, as a D-pantothenic acid producing strain Corynebacterium glutamicum ATCC13032 / pECM3ilvBNCD, pEKEX2panBC and Corynebacterium glutamicum ATCC13032 / pND-D2; or For example, as an L-lysine producing strain 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; or For example, as L-tryptophan producing strain Corynebacterium glutamicum ATCC21850 and Corynebacterium glutamicum KY9218 (pKW9901). We have successfully isolated a new pgi gene encoding the enzyme glucose-6-phosphate isomerase (EC 5.3.1.9) from Corynebacterium glutamicum. In order to isolate pgi genes or other genes from Corynebacterium glutamicum, the genetic library of the microorganisms can be extracted from E. coli. It is first constructed in E. coli. The construction of gene libraries is generally described in well-known textbooks and manuals. Examples that may be mentioned include textbooks [Winnacker, Genes und Klones, Eine Einfuhrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or manuals [Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring) Harbor Laboratory Press, 1989) A very well known gene library is E. coli K-12 strain constructed in a λ vector by Kohara et al., Cell 50, 495-508 (1987). A library of W3110, Bathe et al., Molecular and General Genetics, 252, 255-265 (1996), described in the cosmid vector SuperCos I (WahI et al., 1987, Proceedings of the National Academy of Science USA 84, 2160-2164], Corynebacterium glutamicum ATCC constructed in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16, 1563-1575). A gene library of 13032 is described. See also Bormann et al. (Molecular Micro) biology 6 (3), 317-326, describes a gene library of Corynebacterium glutamicum ATCC 13032 using cosmid pHC79 (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] clones of the Corynebacterium glutamicum gene using the λZap expression system described by Short et al., Nucleic Acids Research, 16: 7583. Describe. Also, this. The gene library of Corynebacterium glutamicum in E. coli is either pBR322 [Bolivar, Life Science, 25, 807-818 (1979)] or pUC9 [Vieira et al., Gene 19, 259-268 (1982). Can be constructed using a plasmid such as). Limitation- 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 gene library was then directed by transformation (Hanahan, Journal of Molecular Biology 166, 557-580, 1983) or electroporation (Tauch et al., 1994, FEMS Microbiological Letters, 123: 343-347). Insert into strain. The characteristic of the indicator strain is that it has a mutation in the gene of interest that results in a detectable phenotype. The E. coli mutant DF1311 described in Kupor & Fraenkel (Journal of Bacteriology 100: 1296-1301 (1969)) is important for the purposes of the present invention, which includes mutations in the pgi and pgl genes, Growth on glucose is significantly inhibited by transformation with a vector containing the pgi gene, followed by re-establishment of growth on glucose One example of such a vector containing the pgi gene is pAMC1 (FIG. 1). The long chain DNA fragment cloned with the aid of cosmid or other λ-vector can then be subcloned in turn into a conventional vector suitable for DNA sequencing. DNA cloning 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 described, for example, in the program of Staden (Nucleic Acids Research 14, 217-232 (1986)), GCG of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)). The program, FASTA algorithm of Pearson and Lipman (Proceedings of the National Academy of Sciences USA 85, 2444-2448 (1988)) or Altschul et al. (Nature Genetics 6, 119-129 (1994) A known algorithm such as the BLAST algorithm or a sequence analysis program can be tested to compare with a list of sequences present in a database accessible to the public, for example, a nucleotide sequence database accessible to the public. EMBL, Heidelberg, Germany), or the National Center for Biotechnology Information database (NCBI, Bethesda, MD, USA). These are the methods used to obtain novel DNA sequences encoding the pgi gene from Corynebacterium glutamicum, provided by the present invention as SEQ ID NO: 1. 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 pgi gene product. Coding DNA sequences generated from SEQ ID NO: 1 by degeneracy of the genetic code are also provided by the present invention. Similarly, DNA sequences that hybridize with SEQ ID NO: 1 or a portion of SEQ ID NO: 1 are provided by the present invention. Finally, a DNA sequence generated by polymerase chain reaction (PCR) using a primer obtained from SEQ ID NO: 1 is provided by the present invention. One skilled in the art will be able to understand in particular, the "The DIG System Users Guide for Filter Hybridization" from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and Liebl et al. In the International Journal of Systematic Bacteriology (1991) 41: 255-260, you will find instructions for identifying DNA by hybridization. One skilled in the art will specifically refer to Gait: Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1994); Newton & Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994) can find instructions for amplifying DNA sequences using polymerase chain reaction (PCR). We have shown that after attenuating the pgi gene, coryneform bacteria exhibit an improved metabolic rate through the pentose phosphate cycle and in an improved way nucleotides, vitamins and in particular L-amino acids, particularly preferably L-lysine and L -Found to produce tryptophan. Attenuation can be achieved by reducing or blocking the expression of the pgi gene or the catalytic properties of the enzyme protein. The two means may be arbitrarily combined. Reduction of expression levels 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. A person skilled in the art can obtain information related to this, see 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); Patek et al., Microbiology 142: 1297 (1996) or known textbooks on gene and molecular biology (Knippers, "Molekulare Genetik", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995; Winnacker, "Gene und Klone", VCH Verlagsgesellschaft, Weinheim, Germany, 1990. Mutations that modify or reduce the catalytic properties of enzyme proteins are known in the art. Examples that may be mentioned are: Qiu & 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", Forschungszentrum Julich reports, Jul-2906, ISSN09442952, Julich, Germany, 1994. Representative summaries are known textbooks of genetic and molecular biology. , "Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986. Mutations that can be considered are substitutions, base pair substitutions, insertions, deletions and translocations. Depending on the exchange effect of amino acids on enzymatic activity, the 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 incorrect amino acid is inserted or premature termination of translation. Deletion of two or more codons usually completely destroys enzyme activity. Instructions for generating such mutations are included in prior art and are known textbooks in 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; Hagemann, "Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986. One example of insertional mutagenesis is the plasmid pMC1 (FIG. 2) capable of mutating the pgi gene. Plasmid pMC1 consists of plasmid pBGS8 described in Spratt et al., Gene 41: 337 (1986), into which an internal fragment of the pgi gene shown in SEQ ID NO: 3 is inserted. After transformation and homologous recombination (insertion) into the pgi gene, this plasmid completely loses its enzyme function. Guidance and explanations regarding insertional mutagenesis are described, for example, in Schwarzer & Puhler, Bio / Technology 9, 84-87 (1991); Fitzpatrick et al., Applied Microbiology and Biotechnology 42, 575-580 (1994). In addition to attenuation of the pgi gene, it may be advantageous to amplify, in particular overexpress, one or more enzymes of the biosynthetic pathway, in particular to produce nucleotides, vitamins and especially L-amino acids, more particularly L-lysine and L-tryptophan. For example, when generating nucleotides, one can simultaneously overexpress the purF gene encoding glutamine-PRPP amidotransferase and / or the b) carAB gene encoding carbamoyl synthase. For example, when producing L-lysine, simultaneously overexpresses the dapA gene encoding dihydrodipicolinate synthase (EP-B 0 197 335), and / or Simultaneously overexpress and / or express gdh gene encoding glutamate dehydrogenase (Bormann et al., Molecular Microbiology 6, 317-326 (1992)), DNA fragments that provide S- (2-aminoethyl) cysteine resistance (EP-A 0 088 166) can be amplified simultaneously. For example, when producing L-tryptophan, concurrently overexpresses the tkt gene encoding transketolase, and / or The prs gene encoding phosphoribosylpyrophosphate synthase can be simultaneously overexpressed. Apart from attenuating the pgi gene, it may be desirable to block unwanted secondary reactions in order to produce nucleotides, vitamins and especially L-amino acids, more particularly L-lysine and L-tryptophan. Nakayama: " Breeding of Amino Acid Producing Micro-organisms ", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982]. Microorganisms containing the polynucleotides according to claim 1 are also provided by the present invention and are batchwise or fed-batch or repeatable to produce nucleotides, vitamins and especially L-amino acids, more particularly L-lysine and L-tryptophan. It is fed on a fed-batch, continuous or discontinuously. A summary of known culture methods can be found in the textbook [Chmiel, Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991); Storhas, Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994). The culture medium used must meet the requirements of the particular strain in a suitable manner. Culture media for various microorganisms are described in Manual of Methods for General Bacteriology from American Society for Bacteriology (Washington DC, USA, 1981). 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 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 may be used as carbon sources These materials may be used alone or as a mixture Organic nitrogen-containing compounds such as peptone, yeast extract, meat extract, malt extract, corn Dipping solutions, soybean wheat and urea) or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate may be used as the nitrogen source. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium containing salts may be used as the phosphorus source The culture medium should also contain metal salts such as magnesium sulfate or iron sulfide required for propagation. Finally, in addition to the substances mentioned above, essential growth-promoting substances such as amino acids and vitamins can be used In addition, suitable precursors can also be added to the culture medium. Can be added or appropriately supplied 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 (eg 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. Oxygen or oxygen containing gas mixtures such as air may be introduced into the culture to maintain aerobic conditions. The culture temperature is generally 20 ° C to 45 ° C and preferably 25 ° C to 40 ° C. Incubation is continued until the maximum amount of desired product is produced. This object is generally achieved within 10 to 160 hours. The metabolic rate through the pentose phosphate cycle is measured using a culture containing C-1 labeled 13C glucose, for example as a carbon source. This method of analysis is based on the known fact that when glucose is metabolized by the pentose phosphate cycle, the C-1 position is converted to carbon dioxide, whereas when catalyzed by that 13C-1 label is passed for the C-3 position of pyruvate. Based. The 13C content of the C-3 position of pyruvate is measured at an appropriate time by examining extracellular metabolites such as lactate and especially lysine using nuclear magnetic resonance or mass spectroscopy methods. Alternatively, amino acids can be obtained by acid hydrolysis from biomass and then determine the 13C content in the individual carbon atoms of a particular amino acid. Those skilled in the art will find comprehensive guidelines, in particular relating to the evaluation of computer-assisted data of 13 C content in various carbon atoms of investigated metabolites, see Sonntag et al., European Journal of Biochemistry 213, 1325-1331 (1993); Sonntag et al., Applied Microbiology and Biotechnology 44, 489-495 (1995); Marx et al., Biotechnology and Bioengineering 49, 111-129 (1996); Marx et al., Biotechnology and Bioengineering 56, 168-180 (1997). Methods of measuring nucleotides, vitamins and L-amino acids are known from the prior art. L-amino acids are analyzed using anion exchange chromatography followed by ninhydrin derivatization as described, for example, in Spackman et al., Analytical Chemistry, 30, (1958), 1990, or Analysis may be by reverse phase HPLC as described in Lindroth et al., Analytical Chemistry (1979) 51: 1167-1174. The following microorganisms have been deposited in Deutsche Zaumlong von Microorganismen Unt Chelculturene (DSMZ, Brunswick, Germany) under the Budapest Treaty: E. coli strain DH5α / pMC1 as DSM 12969 Example The following examples will further illustrate the present invention. The molecular biology techniques used, such as plasmid DNA isolation, restriction enzyme processing, ligation, standard transformation of Escherichia coli, are described in Sambrook et al., Molecular Cloning. A Laboratory Manual (1989) Cold Spring Harbor Laboratories, USA (unless otherwise stated). Example 1 Construction of Gene Library of Corynebacterium glutamicum strain AS019 DNA library of Corynebacterium glutamicum strain ASO19 (Yoshihama et al., Journal of Bacteriology 162, 591-597 (1985)) is described in O'Donohue (O'Donohue, M. (1997)). . the Cloning and Molecular Analysis of Four Common Aromatic Amino Acid biosynthetic Genes from Corynebacterium glutamicum. Ph. D. Thesis, National University of Ireland, Galway] λ Zap Express TM system (Short et al as described in., (1988) Nucleic Acids Research, 16: 5783-7600) The 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 ™ cancer. Example 2 Cloning and Sequencing of the pgi Gene 1. Cloning Escherichia coli strain DF1311 comprising mutations in the gpi and pgl genes, as described in Kupor & Fraenkel, (Journal of Bacteriology 100: 1296-1301), was described in AS019 λ Zap Express ™ described in Example 1. Transform with approximately 500ng of plasmid library. Screening for transformants included M9 minimal medium containing kanamycin at a concentration of 50 mg / L and incubated at 37 ° C. for 48 hours (Sambrook et al., (1989). Molecular Cloning. A Laboratory Manual Cold Spring Harbor Laboratories, USA. Plasmid DNA is isolated from specific transformants according to Birnboim & Doly (Nucleic Acids Research 7: 1513-1523 (1979)) and named pAMC1 (FIG. 1). 2. Sequencing To sequence the cloned insert of pAMC1, the method of Sanger et al., Proceedings of the National Academy of Sciences USA 74, 5463-5467 (1997) is differentially stained with a fluorescent tag. Apply using labeled primers. It is an ABI Prism 310 gene analyzer from Perkin Elmer Applied Biosystems, Perkin Elmer Corporation, Norwalk, Connecticut, USA and also ABI Prism Big Dye ™ Terminator Cycle Sequencing Ready from Perkin Elmer. Reaction kit is performed. Initial sequencing is performed using pluripotent omni and M13 reverse primers from Pharmacia Biotech (St. Albans, Herts, AL1 3AW, UK): Universal Omnidirectional Primer: GTA ATA CGA CTC ACT ATA GGG C M13 reverse primer: GGA AAC AGC TAT GAC CAT G Internal primers are subsequently designed from the sequences obtained so that the entire PGI gene is estimated. The sequence of the inner primer is as follows: Internal primer 1: GGA AAC AGG GGA GCC GTC Internal primer 2: TGC TGA GAT ACC AGC GGT Subsequently, the obtained sequences were prepared using the DNA Strider program (Marck, (1988); Nucleic Acids Research 16: 1829-1836, version 1.0 on an Apple Macintosh computer. This program enables analysis such as restriction site use, open reading frame analysis, and codon usage determination. The search between the DNA sequences obtained and the sequences in the EMBL and Genbank databases is described in the BLAST program (Altschul et al., (1997). Nucleic Acids Research, 25: 3389-3402. DNA and protein sequences are aligned using the Clustal V and Clustal W programs (Higgins and Sharp, 1988 Gene 73: 237-244). The sequence thus obtained is shown in SEQ ID NO: 1. Analysis of the obtained nucleotide sequence showed an open reading frame of 1650 base pairs designated as the pgi gene. It encodes a protein of 550 amino acids set forth in SEQ ID NO: 2. Example 3 mutagenesis of the pgi gene 1. Construction of pgi crushing vector The internal fragment of the pgi gene is a polymerase chain reaction using genomic DNA isolated from Corynebacterium glutamicum AS019 (Heery & Dunican, (1993), Applied and Environmental Microbiology 59: 791-799) as a template. Amplification by PCR). The pgi primers used were as follows: Omni-directional primer: ATG GAR WCC AAY GGH AA Reverse primer: YTC CAC GCC CCA YTG RTC Where R = A + G; Y = C + T; W = A + T; H = A + T + C. PCR parameters were as follows: 35 cycles; 1 minute at 94 ° C., 1 minute at 47 ° C., 30 seconds at 72 ° C .; 1.5 mM MgCl 2 ; Approximately 150-200 ng DNA template. The obtained PCR product was strained as a host. Cloned into a commercially available pGEM-T vector obtained from Promega Corporation (Promega UK, Southampton) using E. coli JM109 (Yanisch-Perron et al., 1985. Gene, 33: 103-119). . The sequence of the PCR product is shown in SEQ ID NO: 3. The cloned insert is then digested with EcoRI fragments and linked to plasmid pBGS8 pretreated with EcoRI (Spratt et al., Gene 41: 337-342 (1986)). Restriction enzymes used are obtained from Boehringer Mannheim UK Ltd., Bell Lane, Lewes, East Sussex BN 7 1LG, UK and used according to the manufacturer's instructions. Then, this. E. coli JM109 was transformed with the ligation mixture and the electrotransformers were IPTG (isopropyl-β-D-thiogalactopyranoside), XGAL (5-bromo-4-chloro-3-indolyl-D Galapyranoside) and kanamycin are selected on Luria agar supplemented at concentrations of 1 mM, 0.02% and 50 mg / L, respectively. Agar plates are incubated at 37 ° C. for 12 hours. Plasmid DNA is isolated from one transformant and characterized by restriction enzyme analysis using EcoRI, BamHI and SalI and named pMC1 (FIG. 2). 2. Insertion Mutation of pgi Gene in Strain DSM5715 Strain DSM 5715 is then transformed into plasmid pMC1 using the electroporation method described in Liebl et al., FEMS Microbiology Letters, 53: 299-303 (1989). Transformant selection was performed with 18.5 g / L brain-heart injection bouillon, 0.5 M sorbitol, 5 g / L bacto tryptone, 2.5 g / L bacto yeast extract, supplemented with 15 mg / L kanamycin and 1% fructose , Proceed on LBHIS agar consisting of 5 g / L NaCl and 18 g / L bacto agar. Incubation is performed at 33 ° C. for 2 days. Transformants 1, 2 and 3 are obtained. The transformants obtained are tested using polymerase chain reaction (PCR). To this end, chromosomal DNA is isolated from the resulting transformants and strain DSM5715 described in Eikmanns et al., Microbiology 140: 1817-1828 (1994). The following primer oligonucleotides are selected for PCR based on the DNA sequence of the pgi gene, which is shown in SEQ ID NO: 1: pgi-1: 5 'ACC CAC GCT GTC CTA CCT TA 3' pgi-2: 5 'TGT CCC AAA TCA CGC CCT AG 3' pgi-3: 5 'gat gat agc ggc cag tgc at 3' The primers presented were synthesized by MWG Biotech (Ebersberg, Germany) and PCR reactions were described in Innis et al., PCR-Protocols. A guide to methods and applications, 1990, Academic Press]. The chromosomal DNA of the transformant is used as a template, and the chromosomal DNA of DSM5715 is used as a control. Each template is used for two PCR reactions, one using primer pair pgi-1 / pgi-2 and the other using primer pair pgi-1 / pgi-3. PCR batches are separated on 0.8% agarose gel by puncture. Using primer pairs pgi-1 / pgi-2, each of the four PCR reactions produced a 0.5 kb long DNA fragment. Using primer pair pgi-1 / pgi-3, the control with DSM5715 DNA showed only 0.7 kb length of amplification product. No PCR product was detected in the batch using chromosomal DNA from the transformants. Transformant 3 characterized in this way is named strain DSM5715 :: pMC1: Example 4 Formation of lysine Corynebacterium glutamicum strain DSM5715 :: pMC1 obtained in Example 3 is cultured in a suitable nutrient medium to produce lysine and the lysine content in the culture supernatant is measured. To this end, the strains are first incubated at 33 ° C. for 24 hours on agar plates with the corresponding antibiotics (brain-heart agar containing kanamycin (25 mg / L)). The preculture is inoculated using the agar plate culture (10 ml medium in a 100 ml Ellenmeyer flask). Complete medium CgIII (Kase & Nakayama, Agricultural and Biological Chemistry 36 (9) 1611-1621 (1972)) is used as the medium for preculture. Kanamycin (25 mg / L) is added to the preculture. The precultures are incubated at 33 ° C. at 240 rpm on a shaker for 24 hours. The main culture is inoculated from the preculture so that the initial OD (660 nm) of the main culture is 0.1. Medium CGCs are used for the main culture. Badge CGC(NH 4 ) 2 SO 4 5g / L Urea5g / L Corn Dipping Liquid (CSL)5g / L Glucose (Separate Autoclaving)36 g / L KH 2 PO 4 / K 2 HPO 4 0.5g / L each MgSO 4 * 7H 2 O0.25g / L CaCl 2 * 2H 2 O10mg / L Biotin (Sterile Filtration)0.2mg / L FeSO 4 * 7H 2 O10mg / L MnSO 4 * H 2 O10.0mg / L CuSO 4 0.2mg / L ZnSO 4 * 7H 2 O1mg / L NiCl 2 * 6H 2 O0.02mg / L Leucine0.15 g / L The CSL and salt solutions are adjusted to pH 7 with ammonia water and autoclaved. Then, sterile substrate vitamin solution is added. Incubation is carried out in a 10 ml volume batch in a 100 ml Elenmeyer flask with a septum. Add kanamycin (25 mg / L). Cultivation 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 (Beckmann Instruments GmbH, Munich). The concentration of lysine formed is performed by an amino acid analyzer from Eppendorf-Biotronik (Hamburg, Germany) by postcolumn derivatization using ion exchange chromatography and ninhydrin detection. The experimental results are shown in Table 1. StrainOD (660)Lysine HCl (g / L) DSM571514.84.8 DSM5715 :: pMC111.57.2 Example 5 Amplification of metabolic rate via pentose phosphate pathway (PPP) Cells are preincubated in 10 mL CGIII (Menkel et al., 1989. Applied and Environmental Microbiology 55: 684-688). A 100 mL- shake flask with a septum is used and the incubation is carried out on a shaker with a rotating diameter of 50 mm at 33 ° C. and 250 rpm at an initial pH 7.0 for 24 hours. Cells are washed with 9 g / L NaCl and used to inoculate the main culture with an optical density of 0.1 at 600 nm (Biochrom Novaspec 4049, LKB Instrument GmbH, Grafelfing, Germany, Cubec Navi 10 mm). Main culture is carried out in 10 mL CGC (Schrumpf et al., 1991. Journal of Bacteriology 173: 4510-4516) modified by addition of 5 g / L corn steep liquor, in addition, 30 g / L ( 1-13 C) dex. the Desert rose (experiment a) or the addition of 15g / L unlabeled dextrose + 15g / L [6- 13 C ] dextrose (experiment B) to the medium. [1- 13 C] dextrose (99 % Enrichment) and [6- 13 C] dextrose (99% enrichment) are purchased from Cambridge Isotope Laboratories, Cambridge, Mass., USA, using a 100 mL- shake flask with a septum , Cultivation is carried out on a shaker with a rotation diameter of 50 mm at an initial pH of 7.0 at 250 ° C. at 250 ° C. At the end of fermentation, the optical density at 660 nm and the concentration of lysine HCl (amino acid analyzer, Eppendorf-Biotronik, Hamburg, Germany) The biomass is also removed by centrifugation at 15000 g and the cell-free supernatant is freeze-dried. The dried powder is redissolved in 1 mL D 2 O (99.98%, Deutero GmbH, Kastellaun, Germany) and 3-trimethylsilyl-propionate-2,2,3,3, d 4 (MSD Isotopes, Montreal, Canada ) Is added as a standard. Nuclear magnetic resonance spectroscopy experiments and differential spectral analysis, including spin echo techniques, are described in Marx et al., 1996, Biotechnology and Bioengineering 49: 111-129; Marx et al., 1999, Metabolic Engineering 1: 35-48; and Wendisch et al., 1997, analytical Biochemistry 245: 196-202, on an AMX 400-WB spectrometer (Bruker analytik GmbH, Karlsruhe, Germany). 13 C enrichment within the lysine carbon atom position is measured and described in Marx et al., 1996, Biotechnology and Bioengineering 49: 111-129; Marx et al., 1997, Biotechnology and Bioengineering 56: 168-180; Marx et al., 1999, Metabolic Engineering 1: 35-48; Sonntag et al., 1993, European Journal of Biochemistry 213: 1325-1331; and the principles described in Sonntag et al., 1995, Applied Microbiology and Biotechnology 44: 489-495, to assess the metabolic rate through the pentose phosphate pathway (PPP) as described below. The results of nuclear magnetic resonance spectroscopy are shown in Figures 3-6. Panel a shows the integration over 13 C-separated spectra. The differential integration shown in panel b is obtained when the integral for 13 C-enhancing for a particular carbon atom position is obtained by dividing the integral from panel b by the integral from panel a. For lysine carbon atom positions C-4 (indicated as L-4 in FIGS. 3A and 3B), the differential spectral integration of 38.286 is divided by the integral for the 13 C-separated spectra of 198.867 and the result is divided by 1.95. 9.9% strengthening is obtained (Table 2). The efficacy of spin echo experiments varies the partition coefficient from 1.80 to 1.99, varying for each single carbon atom position of lysine as described in Wendisch et al., 1997, analytical Biochemistry 245: 196-202. The results described in Figures 3 and 5 [6- 13 C] dextrose is one (experiment B) is obtained for the culture added to the medium. The results described in FIGS. 4 and 6 were obtained for cultures in which [ 1-13 C] dextrose was added to the medium (Experiment A). 13 C enrichment at the carbon atom position of lysine against the parent strain DSM5715 and pgi mutant DSM5715 :: pMC1. The carbon atom positions of lysine are represented by L-2 to L-6 (see FIGS. 3-6). The last column shows the metabolic rate through the pentose phosphate pathway normalized by the dextrose uptake rate. The eighth column shows the enrichment ratio [(BA) / B]. StrainDex 1 L-2 (%)L-3 (%)L-4 (%)L-5 (%)L-6 (%)er 4 (%)speed(%) 1 2 [1- 13 C]11.628.017.028.45.6 1 2 [6- 13 C]11.923.68.424.51.443 ± 460 ± 6 2 3 [1- 13 C]1.71.514.61.71.4 2 3 [6- 13 C]5.027.79.928.14.398 ± 198 ± 2 1 Enhanced Dextrose 2 strain 1 is DSM5715. 3 strain 2 is DSM5715 :: pMC1. 4 er is the reinforcement ratio [(BA) / B]. Different 13 C enhancers within the carbon atom position of lysine are obtained for cells grown in Experiment A and grown in Experiment B (Table 2). In particular, for strain DSM5715 :: pMC1, the loss of enrichment in lysine positions C3 and C5 in Experiment A indicates high PPP rates. Speed through the PPP is the ratio [(BA) / B] (wherein, A represents the entire 13 C enhanced in the lysine prepared from experiment A, B indicates the total 13 C enhanced in the lysine prepared from experiment B) derived from (Equation 1; Equation 2; Equation 3; Table 2). For example, LYS_2_A is enhanced in experiment A 13 C carbon atom position 2 of lysine in, GLC_6_B is 13 C in Experiment A reinforced index root carbon atom position 6 in the Rhodes substrate. Using Equation 3, the enrichment is normalized by dividing the enrichment of 99% dextrose position C1 and 49% C6 for Experiment A and Experiment B, respectively. For strain DSM5715, the 13 C enrichment in trehalose is important for the measurement of rate via PPP as derived from the ratio [(BA) / B], cytoplasmic pool of glucose 6-phosphate and cytoplasm of fructose 6-phosphate The overall equilibrium exists between pools. LYS_A = LYS_2_A + LYS_3_A + LYS_5_A + LYS_6_A-4.4 LYS_B = LYS_2_B + LYS_3_B + LYS_5_B + LYS_6_B -4.4 (B-A) / B = [LYS_B * 99 / (GLC_6_B-1.1)-LYS_ A * 99 / (GLC_1_A-1.1)] / [LYS_B * 99 / (GLC_6_B-1.1)] By computer simulation and in Marx et al., 1996, Biotechnology and Bioengineering 49: 111-129; Marx et al., 1997, Biotechnology and Bioengineering 56: 168-180; Marx et al., 1999, Metabolic Engineering 1: 35-48; Sonntag et al., 1993, European Journal of Biochemstry 213: 1325-1331; By using the metabolic model described in and and Sonntag et al., 1995, Applied Microbiology and Biotechnology 44: 489-495, hyperbolic or linear functions for the correlation between PPP rate and enrichment rate [(BA) / B] Each was found in the metabolic network where phosphoglucoisomerase was present to balance or absent overall (FIG. 7). Comparing the experimental data for the ratio [(BA) / B] shown in Table 2 with the data calculated by the computer simulation, the molar PPP rate was about 60 mol per 100 mol dextrose for the parent strain DSM5715 ( 5). This compares well with the metabolic rate data in the literature for strain DSM5715. Marx et al., 1996. Biotechnology and Bioengineering 49: 111-129; Marx et al., 1997. Biotechnology and Bioengineering 56: 168-180; Marx et al., 1998. Preprints of the 7 th International Conference on Computer Applications in Biotechnology, Osaka, Japan, May 31 to June 4, 1998. PP. 387-392; Marx et al., 1999. Metabolic Engineering 1: 35-48; Sonntag et al., 1995. Applied Microbiology and Biotechnology 44: 489-495]. The molar PPP rate for strain DSM5715 :: pMC1 was found to be 98 mol per 100 mol dextrose. The results clearly indicate that the overall metabolic rate of dextrose uptake has been reinduced to PPP due to the knock out mutation of the pgi gene. Example 6 Reduction of By-Product Formation Strains DSM5715 and DSM5715 :: pMC1 are cultured and analyzed as described in Example 5. Nuclear magnetic resonance spectroscopy (FIGS. 3-6) of the culture supernatants showed by-products (eg trehalose, isopropyl malate, lactate, oxoglutarate and valine) to strain DSM5715 :: pMC1. The concentration of was found to be significantly reduced compared to the parent strain DSM5715. This data is summarized in Table 3. After fermentation for 72 hours in shake flask, the concentration of various extracellular compounds (mM) measured by proton nuclear magnetic resonance spectroscopy. See FIGS. 5 and 6 for the assignment of chemical shifts in the nuclear magnetic resonance spectrum. compoundDSM5715DSM5715 :: pMC1 Trehalose2.9<0.2 Oxoglutarate10.70.7 Lactate3.50.3 Valine3.3〈0.1 Isopropyl Maleate5.51.0 Lee Sin32.254.4 According to the method of the present invention, a method of increasing the metabolic rate through the pentose phosphate cycle is provided.
权利要求:
Claims (17) [1" claim-type="Currently amended] a) a polynucleotide having at least 70% homology with a 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 having at least 70% homology with the amino acid of SEQ ID NO: 2, c) a polynucleotide complementary to the polynucleotides 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 sequence of a), b) or c) above. [2" claim-type="Currently amended] The polynucleotide of claim 1, wherein the polynucleotide is a renewable, preferably recombinant DNA. [3" claim-type="Currently amended] The polynucleotide of claim 1, wherein the polynucleotide is RNA. [4" claim-type="Currently amended] 3. The polynucleotide of claim 2 containing 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 polynucleotide of claim 1, indent d, in particular E. Vector containing pMCl deposited with E. coli DSM12969. [7" claim-type="Currently amended] Coryneform bacteria that act as host cells containing the vector of claim 6. [8" claim-type="Currently amended] A method for increasing metabolic rate via the pentose phosphate cycle in coryneform bacteria, comprising attenuating, in particular expressing or blocking, low levels of the polynucleotides of claim 1. [9" claim-type="Currently amended] 9. A method according to claim 8, wherein the Coryneform bacteria producing nucleotides, vitamins and especially L-amino acids are fermented. [10" claim-type="Currently amended] The method of claim 8, wherein the expression of the polynucleotide of claim 1 is reduced. [11" claim-type="Currently amended] The method of claim 8, wherein the catalytic properties of the polypeptide (enzyme protein) encoding the polynucleotide of claim 1 are reduced. [12" claim-type="Currently amended] The method of claim 8, which is attenuated using the insertion mutation method with plasmid pMC1 as shown in FIG. 2 and deposited as DSM12969. [13" claim-type="Currently amended] 10. The method of claim 9, wherein the nucleotide is produced by fermenting a bacterium which simultaneously overexpresses a) a purF gene encoding glutamine-PRPP amidotransferase and / or b) a carAB gene encoding carbamoyl phosphate synthase. [14" claim-type="Currently amended] 10. The method of claim 9, wherein the dapA gene encoding the dihydropicolinate synthase is simultaneously overexpressed and / or Simultaneously overexpress the gdh gene encoding glutamate dehydrogenase, and / or A method of producing L-lysine by fermenting a bacterium that simultaneously amplifies a DNA fragment that provides S- (2-aminoethyl) cysteine resistance. [15" claim-type="Currently amended] The method of claim 9, wherein simultaneously overexpresses the tkt gene encoding transketolase, and / or A method for producing L-tryptophan by fermenting a bacterium that simultaneously overexpresses the prs gene encoding phosphoribosylpyrophosphate synthase. [16" claim-type="Currently amended] a) fermenting the microorganism according to any one of claims 1 to 15, wherein at least the pgi gene is attenuated or blocked, optionally amplifying the further gene, b) accumulating the desired product in medium or microbial cells; and c) separating the product, the method of producing a nucleotide, vitamin or L-amino acid. [17" claim-type="Currently amended] The method according to any one of claims 8 to 16, wherein a microorganism of the genus Corynebacterium glutamicum is used.
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同族专利:
公开号 | 公开日 JP2001136988A|2001-05-22| CA2318507A1|2001-03-15| HU0003651A3|2005-07-28| BR0004208A|2001-04-10| ZA200004911B|2002-03-13| EP1087015A3|2003-07-09| MXPA00008965A|2002-12-16| DE60045180D1|2010-12-16| SK13362000A3|2003-01-09| AT486942T|2010-11-15| HU0003651D0|2000-09-14| CN1288058A|2001-03-21| RU2261912C2|2005-10-10| US6586214B1|2003-07-01| EP1087015A2|2001-03-28| EP1087015B1|2010-11-03| ID27238A|2001-03-15| AU5505500A|2001-03-22| HU0003651A2|2003-04-28|
引用文献:
公开号 | 申请日 | 公开日 | 申请人 | 专利标题
法律状态:
1999-09-15|Priority to US09/396,478 1999-09-15|Priority to US9/396,478 2000-09-14|Application filed by 데구사-휠스 악티엔게젤샤프트, 내셔널 유니버시티 오브 아일랜드 2001-06-15|Publication of KR20010050449A
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申请号 | 申请日 | 专利标题 US09/396,478|US6586214B1|1999-09-15|1999-09-15|Method for increasing the metabolic flux through the pentose phosphate cycle in coryneform bacteria by regulation of the phosphoglucose isomerase | US9/396,478|1999-09-15| 相关专利
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