CA2593287A1 - Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production - Google Patents
Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production Download PDFInfo
- Publication number
- CA2593287A1 CA2593287A1 CA002593287A CA2593287A CA2593287A1 CA 2593287 A1 CA2593287 A1 CA 2593287A1 CA 002593287 A CA002593287 A CA 002593287A CA 2593287 A CA2593287 A CA 2593287A CA 2593287 A1 CA2593287 A1 CA 2593287A1
- Authority
- CA
- Canada
- Prior art keywords
- nucleic acid
- smp
- protein
- amino acid
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 241000186226 Corynebacterium glutamicum Species 0.000 title claims abstract description 193
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 83
- 108090000623 proteins and genes Proteins 0.000 title description 380
- 102000004169 proteins and genes Human genes 0.000 title description 293
- 230000006860 carbon metabolism Effects 0.000 title description 4
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 243
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 221
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 221
- 238000000034 method Methods 0.000 claims abstract description 140
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 47
- 239000013604 expression vector Substances 0.000 claims abstract description 45
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 43
- 125000003729 nucleotide group Chemical group 0.000 claims description 106
- 239000002773 nucleotide Substances 0.000 claims description 103
- 230000000694 effects Effects 0.000 claims description 74
- 150000001413 amino acids Chemical group 0.000 claims description 73
- 239000013598 vector Substances 0.000 claims description 69
- 235000001014 amino acid Nutrition 0.000 claims description 66
- 239000012847 fine chemical Substances 0.000 claims description 65
- 229940024606 amino acid Drugs 0.000 claims description 62
- 230000014509 gene expression Effects 0.000 claims description 53
- 244000005700 microbiome Species 0.000 claims description 44
- 241000186216 Corynebacterium Species 0.000 claims description 42
- 102000004190 Enzymes Human genes 0.000 claims description 42
- 108090000790 Enzymes Proteins 0.000 claims description 42
- 229920001184 polypeptide Polymers 0.000 claims description 37
- 241000186146 Brevibacterium Species 0.000 claims description 36
- 230000001105 regulatory effect Effects 0.000 claims description 29
- 239000012634 fragment Substances 0.000 claims description 21
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 claims description 20
- 230000000295 complement effect Effects 0.000 claims description 20
- 229940088594 vitamin Drugs 0.000 claims description 19
- 229930003231 vitamin Natural products 0.000 claims description 19
- 235000013343 vitamin Nutrition 0.000 claims description 19
- 239000011782 vitamin Substances 0.000 claims description 19
- 241000186145 Corynebacterium ammoniagenes Species 0.000 claims description 16
- 241000319304 [Brevibacterium] flavum Species 0.000 claims description 16
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 14
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 10
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 claims description 10
- 150000002632 lipids Chemical class 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000002777 nucleoside Substances 0.000 claims description 9
- 241000186227 Corynebacterium diphtheriae Species 0.000 claims description 8
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 8
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 8
- 239000004471 Glycine Substances 0.000 claims description 7
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 7
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 7
- 239000004472 Lysine Substances 0.000 claims description 7
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 7
- 239000004473 Threonine Substances 0.000 claims description 7
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 230000004048 modification Effects 0.000 claims description 7
- 238000012986 modification Methods 0.000 claims description 7
- 150000007524 organic acids Chemical class 0.000 claims description 7
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 7
- 229960002898 threonine Drugs 0.000 claims description 7
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 6
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 6
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 6
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 6
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 6
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 6
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 6
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 6
- 235000018417 cysteine Nutrition 0.000 claims description 6
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 6
- 229960000310 isoleucine Drugs 0.000 claims description 6
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 6
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 6
- 239000004474 valine Substances 0.000 claims description 6
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 6
- 239000004475 Arginine Substances 0.000 claims description 5
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 5
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 5
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 5
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 5
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 5
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 5
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 5
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 5
- 235000004279 alanine Nutrition 0.000 claims description 5
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 5
- 229940009098 aspartate Drugs 0.000 claims description 5
- 206010013023 diphtheria Diseases 0.000 claims description 5
- 229930195712 glutamate Natural products 0.000 claims description 5
- 229940049906 glutamate Drugs 0.000 claims description 5
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 5
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 5
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 4
- 241000187561 Rhodococcus erythropolis Species 0.000 claims description 4
- 150000001491 aromatic compounds Chemical class 0.000 claims description 4
- 150000001720 carbohydrates Chemical class 0.000 claims description 4
- 150000002009 diols Chemical class 0.000 claims description 4
- 229930182817 methionine Natural products 0.000 claims description 4
- 241001517047 Corynebacterium acetoacidophilum Species 0.000 claims description 3
- 230000004071 biological effect Effects 0.000 claims description 3
- 229930001119 polyketide Natural products 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 150000004671 saturated fatty acids Chemical class 0.000 claims description 3
- 235000021122 unsaturated fatty acids Nutrition 0.000 claims description 3
- 150000004670 unsaturated fatty acids Chemical class 0.000 claims description 3
- 150000003881 polyketide derivatives Chemical class 0.000 claims 2
- 229930182852 proteinogenic amino acid Natural products 0.000 claims 2
- 235000012539 Bacterium linens Nutrition 0.000 claims 1
- 241000186310 Brevibacterium linens Species 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract description 68
- 230000000692 anti-sense effect Effects 0.000 abstract description 38
- 102000037865 fusion proteins Human genes 0.000 abstract description 26
- 108020001507 fusion proteins Proteins 0.000 abstract description 26
- 238000003259 recombinant expression Methods 0.000 abstract description 14
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 230000000890 antigenic effect Effects 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 287
- 210000004027 cell Anatomy 0.000 description 170
- 125000003275 alpha amino acid group Chemical group 0.000 description 78
- 235000000346 sugar Nutrition 0.000 description 73
- 108020004414 DNA Proteins 0.000 description 59
- 230000015572 biosynthetic process Effects 0.000 description 44
- 230000004060 metabolic process Effects 0.000 description 44
- 229940088598 enzyme Drugs 0.000 description 41
- 230000037361 pathway Effects 0.000 description 40
- 150000008163 sugars Chemical class 0.000 description 38
- 238000006243 chemical reaction Methods 0.000 description 37
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 36
- 230000008569 process Effects 0.000 description 35
- 101150118911 smp gene Proteins 0.000 description 32
- 108091028043 Nucleic acid sequence Proteins 0.000 description 30
- 230000010627 oxidative phosphorylation Effects 0.000 description 30
- 239000000047 product Substances 0.000 description 29
- 229910052799 carbon Inorganic materials 0.000 description 28
- 239000000543 intermediate Substances 0.000 description 28
- 241000894006 Bacteria Species 0.000 description 26
- 230000006870 function Effects 0.000 description 26
- -1 arachidonic acid) Chemical class 0.000 description 25
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 24
- 108091026890 Coding region Proteins 0.000 description 24
- 238000004458 analytical method Methods 0.000 description 23
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 23
- 239000013612 plasmid Substances 0.000 description 23
- 239000000126 substance Substances 0.000 description 22
- 150000001722 carbon compounds Chemical class 0.000 description 21
- 230000001965 increasing effect Effects 0.000 description 21
- 239000000523 sample Substances 0.000 description 21
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 19
- 108020004999 messenger RNA Proteins 0.000 description 19
- 230000014616 translation Effects 0.000 description 18
- 125000000539 amino acid group Chemical group 0.000 description 17
- 239000002243 precursor Substances 0.000 description 17
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 16
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 15
- 238000003786 synthesis reaction Methods 0.000 description 15
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 14
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 14
- 230000015556 catabolic process Effects 0.000 description 14
- 239000008103 glucose Substances 0.000 description 14
- 230000012010 growth Effects 0.000 description 14
- 239000002609 medium Substances 0.000 description 14
- 238000002703 mutagenesis Methods 0.000 description 14
- 231100000350 mutagenesis Toxicity 0.000 description 14
- 238000013518 transcription Methods 0.000 description 14
- 230000035897 transcription Effects 0.000 description 14
- 230000001413 cellular effect Effects 0.000 description 13
- 230000000875 corresponding effect Effects 0.000 description 13
- 230000004151 fermentation Effects 0.000 description 13
- 238000000855 fermentation Methods 0.000 description 13
- 230000004927 fusion Effects 0.000 description 13
- 238000000746 purification Methods 0.000 description 13
- 238000010561 standard procedure Methods 0.000 description 13
- 238000013519 translation Methods 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 12
- 238000006731 degradation reaction Methods 0.000 description 12
- 238000009396 hybridization Methods 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 241000588724 Escherichia coli Species 0.000 description 11
- 108091034117 Oligonucleotide Proteins 0.000 description 11
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 11
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 11
- 239000007857 degradation product Substances 0.000 description 11
- 230000034659 glycolysis Effects 0.000 description 11
- 238000002744 homologous recombination Methods 0.000 description 11
- 230000006801 homologous recombination Effects 0.000 description 11
- 230000037353 metabolic pathway Effects 0.000 description 11
- 230000035772 mutation Effects 0.000 description 11
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 102000053602 DNA Human genes 0.000 description 10
- 241000196324 Embryophyta Species 0.000 description 10
- 241000233866 Fungi Species 0.000 description 10
- 238000010276 construction Methods 0.000 description 10
- 239000013615 primer Substances 0.000 description 10
- 238000012552 review Methods 0.000 description 10
- 238000012546 transfer Methods 0.000 description 10
- 230000009466 transformation Effects 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 9
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 9
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 9
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 9
- 239000012707 chemical precursor Substances 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 238000012216 screening Methods 0.000 description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 8
- 108091005461 Nucleic proteins Proteins 0.000 description 8
- 238000010367 cloning Methods 0.000 description 8
- 238000012217 deletion Methods 0.000 description 8
- 230000037430 deletion Effects 0.000 description 8
- 108090000994 Catalytic RNA Proteins 0.000 description 7
- 102000053642 Catalytic RNA Human genes 0.000 description 7
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 7
- 229910019142 PO4 Inorganic materials 0.000 description 7
- 229940041514 candida albicans extract Drugs 0.000 description 7
- 230000002255 enzymatic effect Effects 0.000 description 7
- 210000003527 eukaryotic cell Anatomy 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 230000002503 metabolic effect Effects 0.000 description 7
- 235000019161 pantothenic acid Nutrition 0.000 description 7
- 239000011713 pantothenic acid Substances 0.000 description 7
- 239000010452 phosphate Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 210000001236 prokaryotic cell Anatomy 0.000 description 7
- 230000004952 protein activity Effects 0.000 description 7
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 235000019192 riboflavin Nutrition 0.000 description 7
- 229960002477 riboflavin Drugs 0.000 description 7
- 239000002151 riboflavin Substances 0.000 description 7
- 108091092562 ribozyme Proteins 0.000 description 7
- 238000006467 substitution reaction Methods 0.000 description 7
- 239000012138 yeast extract Substances 0.000 description 7
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 6
- KTVPXOYAKDPRHY-SOOFDHNKSA-N D-ribofuranose 5-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O KTVPXOYAKDPRHY-SOOFDHNKSA-N 0.000 description 6
- 239000003155 DNA primer Substances 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 108010021466 Mutant Proteins Proteins 0.000 description 6
- 102000008300 Mutant Proteins Human genes 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 6
- 238000007792 addition Methods 0.000 description 6
- 230000004075 alteration Effects 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000027455 binding Effects 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- 230000007246 mechanism Effects 0.000 description 6
- 230000001404 mediated effect Effects 0.000 description 6
- 229960004452 methionine Drugs 0.000 description 6
- 235000001968 nicotinic acid Nutrition 0.000 description 6
- 239000011664 nicotinic acid Substances 0.000 description 6
- 229940014662 pantothenate Drugs 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 6
- 235000008521 threonine Nutrition 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 108091033380 Coding strand Proteins 0.000 description 5
- 102000018832 Cytochromes Human genes 0.000 description 5
- 108010052832 Cytochromes Proteins 0.000 description 5
- 238000000018 DNA microarray Methods 0.000 description 5
- 108010070675 Glutathione transferase Proteins 0.000 description 5
- 102000005720 Glutathione transferase Human genes 0.000 description 5
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 5
- 241000238631 Hexapoda Species 0.000 description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 108700026244 Open Reading Frames Proteins 0.000 description 5
- 239000007984 Tris EDTA buffer Substances 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 239000002585 base Substances 0.000 description 5
- 230000008238 biochemical pathway Effects 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 239000002537 cosmetic Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- VWWQXMAJTJZDQX-UYBVJOGSSA-N flavin adenine dinucleotide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1CO[P@](O)(=O)O[P@@](O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UYBVJOGSSA-N 0.000 description 5
- 235000019162 flavin adenine dinucleotide Nutrition 0.000 description 5
- 239000011714 flavin adenine dinucleotide Substances 0.000 description 5
- 229940093632 flavin-adenine dinucleotide Drugs 0.000 description 5
- 235000019152 folic acid Nutrition 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 230000010354 integration Effects 0.000 description 5
- 235000018977 lysine Nutrition 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 238000010369 molecular cloning Methods 0.000 description 5
- 229960003512 nicotinic acid Drugs 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 235000016709 nutrition Nutrition 0.000 description 5
- 235000005985 organic acids Nutrition 0.000 description 5
- 230000003647 oxidation Effects 0.000 description 5
- 238000007254 oxidation reaction Methods 0.000 description 5
- 230000004144 purine metabolism Effects 0.000 description 5
- XKMLYUALXHKNFT-UHFFFAOYSA-N rGTP Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O XKMLYUALXHKNFT-UHFFFAOYSA-N 0.000 description 5
- 230000010076 replication Effects 0.000 description 5
- 230000000717 retained effect Effects 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 235000002639 sodium chloride Nutrition 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 230000006820 DNA synthesis Effects 0.000 description 4
- 108091006149 Electron carriers Proteins 0.000 description 4
- 108020004511 Recombinant DNA Proteins 0.000 description 4
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- 239000000370 acceptor Substances 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 4
- 235000009697 arginine Nutrition 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 239000006227 byproduct Substances 0.000 description 4
- 239000011575 calcium Substances 0.000 description 4
- 210000002421 cell wall Anatomy 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 239000003184 complementary RNA Substances 0.000 description 4
- 239000000306 component Substances 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000003623 enhancer Substances 0.000 description 4
- 229940013640 flavin mononucleotide Drugs 0.000 description 4
- FVTCRASFADXXNN-SCRDCRAPSA-N flavin mononucleotide Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-SCRDCRAPSA-N 0.000 description 4
- 239000011768 flavin mononucleotide Substances 0.000 description 4
- FVTCRASFADXXNN-UHFFFAOYSA-N flavin mononucleotide Natural products OP(=O)(O)OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-UHFFFAOYSA-N 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 230000000670 limiting effect Effects 0.000 description 4
- 238000013507 mapping Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 238000002493 microarray Methods 0.000 description 4
- 125000003835 nucleoside group Chemical group 0.000 description 4
- 235000021436 nutraceutical agent Nutrition 0.000 description 4
- 150000002894 organic compounds Chemical class 0.000 description 4
- DTBNBXWJWCWCIK-UHFFFAOYSA-K phosphonatoenolpyruvate Chemical compound [O-]C(=O)C(=C)OP([O-])([O-])=O DTBNBXWJWCWCIK-UHFFFAOYSA-K 0.000 description 4
- 239000002987 primer (paints) Substances 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 235000019231 riboflavin-5'-phosphate Nutrition 0.000 description 4
- 239000013605 shuttle vector Substances 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 230000001131 transforming effect Effects 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 229940011671 vitamin b6 Drugs 0.000 description 4
- OTOIIPJYVQJATP-BYPYZUCNSA-N (R)-pantoic acid Chemical compound OCC(C)(C)[C@@H](O)C(O)=O OTOIIPJYVQJATP-BYPYZUCNSA-N 0.000 description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 3
- RQFCJASXJCIDSX-UHFFFAOYSA-N 14C-Guanosin-5'-monophosphat Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(O)=O)C(O)C1O RQFCJASXJCIDSX-UHFFFAOYSA-N 0.000 description 3
- 108020005544 Antisense RNA Proteins 0.000 description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 3
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical group O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 3
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 3
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 3
- LXJXRIRHZLFYRP-VKHMYHEASA-N D-glyceraldehyde 3-phosphate Chemical compound O=C[C@H](O)COP(O)(O)=O LXJXRIRHZLFYRP-VKHMYHEASA-N 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 3
- 102000005298 Iron-Sulfur Proteins Human genes 0.000 description 3
- 108010081409 Iron-Sulfur Proteins Proteins 0.000 description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 3
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 3
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 3
- 108091092724 Noncoding DNA Proteins 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 3
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 3
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- DJJCXFVJDGTHFX-UHFFFAOYSA-N Uridinemonophosphate Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-UHFFFAOYSA-N 0.000 description 3
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 3
- 229950006790 adenosine phosphate Drugs 0.000 description 3
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 3
- 229960004050 aminobenzoic acid Drugs 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 239000005557 antagonist Substances 0.000 description 3
- 239000000074 antisense oligonucleotide Substances 0.000 description 3
- 238000012230 antisense oligonucleotides Methods 0.000 description 3
- 229960001230 asparagine Drugs 0.000 description 3
- 235000009582 asparagine Nutrition 0.000 description 3
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 3
- 230000001851 biosynthetic effect Effects 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000012539 chromatography resin Substances 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 3
- 239000005515 coenzyme Substances 0.000 description 3
- 239000005516 coenzyme A Substances 0.000 description 3
- 229940093530 coenzyme a Drugs 0.000 description 3
- 238000009833 condensation Methods 0.000 description 3
- 230000005494 condensation Effects 0.000 description 3
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 229960000304 folic acid Drugs 0.000 description 3
- 239000011724 folic acid Substances 0.000 description 3
- 150000002224 folic acids Chemical class 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 230000004110 gluconeogenesis Effects 0.000 description 3
- 229960002989 glutamic acid Drugs 0.000 description 3
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 description 3
- 150000003278 haem Chemical class 0.000 description 3
- 230000001771 impaired effect Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 238000002093 isoelectric focusing polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 238000006241 metabolic reaction Methods 0.000 description 3
- 235000006109 methionine Nutrition 0.000 description 3
- 235000013379 molasses Nutrition 0.000 description 3
- 229950006238 nadide Drugs 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000002417 nutraceutical Substances 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 150000002972 pentoses Chemical class 0.000 description 3
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 3
- 230000026731 phosphorylation Effects 0.000 description 3
- 238000006366 phosphorylation reaction Methods 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 238000001243 protein synthesis Methods 0.000 description 3
- 230000004147 pyrimidine metabolism Effects 0.000 description 3
- 238000006722 reduction reaction Methods 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000004611 spectroscopical analysis Methods 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 235000019157 thiamine Nutrition 0.000 description 3
- 239000011721 thiamine Substances 0.000 description 3
- 229960004072 thrombin Drugs 0.000 description 3
- 241000701447 unidentified baculovirus Species 0.000 description 3
- DJJCXFVJDGTHFX-ZAKLUEHWSA-N uridine-5'-monophosphate Chemical compound O[C@@H]1[C@@H](O)[C@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-ZAKLUEHWSA-N 0.000 description 3
- 229910052720 vanadium Inorganic materials 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- RFLVMTUMFYRZCB-UHFFFAOYSA-N 1-methylguanine Chemical compound O=C1N(C)C(N)=NC2=C1N=CN2 RFLVMTUMFYRZCB-UHFFFAOYSA-N 0.000 description 2
- YSAJFXWTVFGPAX-UHFFFAOYSA-N 2-[(2,4-dioxo-1h-pyrimidin-5-yl)oxy]acetic acid Chemical compound OC(=O)COC1=CNC(=O)NC1=O YSAJFXWTVFGPAX-UHFFFAOYSA-N 0.000 description 2
- FZWGECJQACGGTI-UHFFFAOYSA-N 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one Chemical compound NC1=NC(O)=C2N(C)C=NC2=N1 FZWGECJQACGGTI-UHFFFAOYSA-N 0.000 description 2
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 2
- OIVLITBTBDPEFK-UHFFFAOYSA-N 5,6-dihydrouracil Chemical compound O=C1CCNC(=O)N1 OIVLITBTBDPEFK-UHFFFAOYSA-N 0.000 description 2
- ZLAQATDNGLKIEV-UHFFFAOYSA-N 5-methyl-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CC1=CNC(=S)NC1=O ZLAQATDNGLKIEV-UHFFFAOYSA-N 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 241000195493 Cryptophyta Species 0.000 description 2
- PCDQPRRSZKQHHS-UHFFFAOYSA-N Cytidine 5'-triphosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-UHFFFAOYSA-N 0.000 description 2
- NGHMDNPXVRFFGS-IUYQGCFVSA-N D-erythrose 4-phosphate Chemical compound O=C[C@H](O)[C@H](O)COP(O)(O)=O NGHMDNPXVRFFGS-IUYQGCFVSA-N 0.000 description 2
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 2
- SNPLKNRPJHDVJA-ZETCQYMHSA-N D-panthenol Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCCO SNPLKNRPJHDVJA-ZETCQYMHSA-N 0.000 description 2
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 2
- ZAQJHHRNXZUBTE-NQXXGFSBSA-N D-ribulose Chemical compound OC[C@@H](O)[C@@H](O)C(=O)CO ZAQJHHRNXZUBTE-NQXXGFSBSA-N 0.000 description 2
- FNZLKVNUWIIPSJ-UHNVWZDZSA-N D-ribulose 5-phosphate Chemical compound OCC(=O)[C@H](O)[C@H](O)COP(O)(O)=O FNZLKVNUWIIPSJ-UHNVWZDZSA-N 0.000 description 2
- ZAQJHHRNXZUBTE-UHFFFAOYSA-N D-threo-2-Pentulose Natural products OCC(O)C(O)C(=O)CO ZAQJHHRNXZUBTE-UHFFFAOYSA-N 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 2
- 101710096438 DNA-binding protein Proteins 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- YPZRHBJKEMOYQH-UYBVJOGSSA-L FADH2(2-) Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1COP([O-])(=O)OP([O-])(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C(NC(=O)NC2=O)=C2NC2=C1C=C(C)C(C)=C2 YPZRHBJKEMOYQH-UYBVJOGSSA-L 0.000 description 2
- 108010057573 Flavoproteins Proteins 0.000 description 2
- 102000003983 Flavoproteins Human genes 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 description 2
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 2
- 229930195722 L-methionine Natural products 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- HYVABZIGRDEKCD-UHFFFAOYSA-N N(6)-dimethylallyladenine Chemical compound CC(C)=CCNC1=NC=NC2=C1N=CN2 HYVABZIGRDEKCD-UHFFFAOYSA-N 0.000 description 2
- 102000006746 NADH Dehydrogenase Human genes 0.000 description 2
- 108010086428 NADH Dehydrogenase Proteins 0.000 description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 2
- FNZLKVNUWIIPSJ-UHFFFAOYSA-N Rbl5P Natural products OCC(=O)C(O)C(O)COP(O)(O)=O FNZLKVNUWIIPSJ-UHFFFAOYSA-N 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 108090000190 Thrombin Proteins 0.000 description 2
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 2
- 229910052770 Uranium Inorganic materials 0.000 description 2
- 229930003779 Vitamin B12 Natural products 0.000 description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000000556 agonist Substances 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 2
- 238000005842 biochemical reaction Methods 0.000 description 2
- 230000025938 carbohydrate utilization Effects 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 230000006652 catabolic pathway Effects 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 2
- 238000002742 combinatorial mutagenesis Methods 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- PCDQPRRSZKQHHS-ZAKLUEHWSA-N cytidine-5'-triphosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO[P@](O)(=O)O[P@@](O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-ZAKLUEHWSA-N 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 239000001177 diphosphate Substances 0.000 description 2
- 235000011180 diphosphates Nutrition 0.000 description 2
- 231100000676 disease causative agent Toxicity 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 235000020776 essential amino acid Nutrition 0.000 description 2
- 239000003797 essential amino acid Substances 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 230000005714 functional activity Effects 0.000 description 2
- 229930182830 galactose Natural products 0.000 description 2
- 238000003208 gene overexpression Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 229930195733 hydrocarbon Natural products 0.000 description 2
- 150000002430 hydrocarbons Chemical class 0.000 description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 238000011005 laboratory method Methods 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 238000000464 low-speed centrifugation Methods 0.000 description 2
- 239000012092 media component Substances 0.000 description 2
- 239000012533 medium component Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 2
- 235000013923 monosodium glutamate Nutrition 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 2
- 238000003499 nucleic acid array Methods 0.000 description 2
- 230000037360 nucleotide metabolism Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- 238000002515 oligonucleotide synthesis Methods 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 230000004108 pentose phosphate pathway Effects 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 102000054765 polymorphisms of proteins Human genes 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- FPWMCUPFBRFMLH-UHFFFAOYSA-N prephenic acid Chemical compound OC1C=CC(CC(=O)C(O)=O)(C(O)=O)C=C1 FPWMCUPFBRFMLH-UHFFFAOYSA-N 0.000 description 2
- 210000001938 protoplast Anatomy 0.000 description 2
- 239000002213 purine nucleotide Substances 0.000 description 2
- 150000003212 purines Chemical class 0.000 description 2
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 2
- NHZMQXZHNVQTQA-UHFFFAOYSA-N pyridoxamine Chemical compound CC1=NC=C(CO)C(CN)=C1O NHZMQXZHNVQTQA-UHFFFAOYSA-N 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000035806 respiratory chain Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000007447 staining method Methods 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- 235000019163 vitamin B12 Nutrition 0.000 description 2
- 239000011715 vitamin B12 Substances 0.000 description 2
- 235000019158 vitamin B6 Nutrition 0.000 description 2
- 239000011726 vitamin B6 Substances 0.000 description 2
- GMKMEZVLHJARHF-UHFFFAOYSA-N (2R,6R)-form-2.6-Diaminoheptanedioic acid Natural products OC(=O)C(N)CCCC(N)C(O)=O GMKMEZVLHJARHF-UHFFFAOYSA-N 0.000 description 1
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- LTHDIZOWAIGONP-KODRXGBYSA-N (3r,4s,5r)-3,4,5-trihydroxy-6-phosphonooxyhexanoic acid Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@H](O)CC(O)=O LTHDIZOWAIGONP-KODRXGBYSA-N 0.000 description 1
- MSTNYGQPCMXVAQ-RYUDHWBXSA-N (6S)-5,6,7,8-tetrahydrofolic acid Chemical compound C([C@H]1CNC=2N=C(NC(=O)C=2N1)N)NC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 MSTNYGQPCMXVAQ-RYUDHWBXSA-N 0.000 description 1
- LXJXRIRHZLFYRP-VKHMYHEASA-L (R)-2-Hydroxy-3-(phosphonooxy)-propanal Natural products O=C[C@H](O)COP([O-])([O-])=O LXJXRIRHZLFYRP-VKHMYHEASA-L 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 101150084750 1 gene Proteins 0.000 description 1
- WJNGQIYEQLPJMN-IOSLPCCCSA-N 1-methylinosine Chemical compound C1=NC=2C(=O)N(C)C=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O WJNGQIYEQLPJMN-IOSLPCCCSA-N 0.000 description 1
- HLYBTPMYFWWNJN-UHFFFAOYSA-N 2-(2,4-dioxo-1h-pyrimidin-5-yl)-2-hydroxyacetic acid Chemical compound OC(=O)C(O)C1=CNC(=O)NC1=O HLYBTPMYFWWNJN-UHFFFAOYSA-N 0.000 description 1
- JAHNSTQSQJOJLO-UHFFFAOYSA-N 2-(3-fluorophenyl)-1h-imidazole Chemical compound FC1=CC=CC(C=2NC=CN=2)=C1 JAHNSTQSQJOJLO-UHFFFAOYSA-N 0.000 description 1
- SGAKLDIYNFXTCK-UHFFFAOYSA-N 2-[(2,4-dioxo-1h-pyrimidin-5-yl)methylamino]acetic acid Chemical compound OC(=O)CNCC1=CNC(=O)NC1=O SGAKLDIYNFXTCK-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- VKRFXNXJOJJPAO-UHFFFAOYSA-N 2-amino-4-(2,4-dioxo-1h-pyrimidin-3-yl)butanoic acid Chemical compound OC(=O)C(N)CCN1C(=O)C=CNC1=O VKRFXNXJOJJPAO-UHFFFAOYSA-N 0.000 description 1
- UDOGNMDURIJYQC-UHFFFAOYSA-N 2-amino-6-methyl-1h-pteridin-4-one Chemical compound N1C(N)=NC(=O)C2=NC(C)=CN=C21 UDOGNMDURIJYQC-UHFFFAOYSA-N 0.000 description 1
- XMSMHKMPBNTBOD-UHFFFAOYSA-N 2-dimethylamino-6-hydroxypurine Chemical compound N1C(N(C)C)=NC(=O)C2=C1N=CN2 XMSMHKMPBNTBOD-UHFFFAOYSA-N 0.000 description 1
- VYSRZETUSAOIMP-UHFFFAOYSA-N 2-furanacetic acid Chemical compound OC(=O)CC1=CC=CO1 VYSRZETUSAOIMP-UHFFFAOYSA-N 0.000 description 1
- SMADWRYCYBUIKH-UHFFFAOYSA-N 2-methyl-7h-purin-6-amine Chemical compound CC1=NC(N)=C2NC=NC2=N1 SMADWRYCYBUIKH-UHFFFAOYSA-N 0.000 description 1
- AQSRRZGQRFFFGS-UHFFFAOYSA-N 2-methylpyridin-3-ol Chemical compound CC1=NC=CC=C1O AQSRRZGQRFFFGS-UHFFFAOYSA-N 0.000 description 1
- YQUVCSBJEUQKSH-UHFFFAOYSA-N 3,4-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 description 1
- KOLPWZCZXAMXKS-UHFFFAOYSA-N 3-methylcytosine Chemical compound CN1C(N)=CC=NC1=O KOLPWZCZXAMXKS-UHFFFAOYSA-N 0.000 description 1
- GJAKJCICANKRFD-UHFFFAOYSA-N 4-acetyl-4-amino-1,3-dihydropyrimidin-2-one Chemical compound CC(=O)C1(N)NC(=O)NC=C1 GJAKJCICANKRFD-UHFFFAOYSA-N 0.000 description 1
- OVONXEQGWXGFJD-UHFFFAOYSA-N 4-sulfanylidene-1h-pyrimidin-2-one Chemical compound SC=1C=CNC(=O)N=1 OVONXEQGWXGFJD-UHFFFAOYSA-N 0.000 description 1
- MQJSSLBGAQJNER-UHFFFAOYSA-N 5-(methylaminomethyl)-1h-pyrimidine-2,4-dione Chemical compound CNCC1=CNC(=O)NC1=O MQJSSLBGAQJNER-UHFFFAOYSA-N 0.000 description 1
- WPYRHVXCOQLYLY-UHFFFAOYSA-N 5-[(methoxyamino)methyl]-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CONCC1=CNC(=S)NC1=O WPYRHVXCOQLYLY-UHFFFAOYSA-N 0.000 description 1
- LQLQRFGHAALLLE-UHFFFAOYSA-N 5-bromouracil Chemical compound BrC1=CNC(=O)NC1=O LQLQRFGHAALLLE-UHFFFAOYSA-N 0.000 description 1
- VKLFQTYNHLDMDP-PNHWDRBUSA-N 5-carboxymethylaminomethyl-2-thiouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=S)NC(=O)C(CNCC(O)=O)=C1 VKLFQTYNHLDMDP-PNHWDRBUSA-N 0.000 description 1
- ZFTBZKVVGZNMJR-UHFFFAOYSA-N 5-chlorouracil Chemical compound ClC1=CNC(=O)NC1=O ZFTBZKVVGZNMJR-UHFFFAOYSA-N 0.000 description 1
- LDCYZAJDBXYCGN-VIFPVBQESA-N 5-hydroxy-L-tryptophan Chemical compound C1=C(O)C=C2C(C[C@H](N)C(O)=O)=CNC2=C1 LDCYZAJDBXYCGN-VIFPVBQESA-N 0.000 description 1
- KSNXJLQDQOIRIP-UHFFFAOYSA-N 5-iodouracil Chemical compound IC1=CNC(=O)NC1=O KSNXJLQDQOIRIP-UHFFFAOYSA-N 0.000 description 1
- KELXHQACBIUYSE-UHFFFAOYSA-N 5-methoxy-1h-pyrimidine-2,4-dione Chemical compound COC1=CNC(=O)NC1=O KELXHQACBIUYSE-UHFFFAOYSA-N 0.000 description 1
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 1
- DCPSTSVLRXOYGS-UHFFFAOYSA-N 6-amino-1h-pyrimidine-2-thione Chemical compound NC1=CC=NC(S)=N1 DCPSTSVLRXOYGS-UHFFFAOYSA-N 0.000 description 1
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 1
- 239000007991 ACES buffer Substances 0.000 description 1
- 108010009924 Aconitate hydratase Proteins 0.000 description 1
- ORILYTVJVMAKLC-UHFFFAOYSA-N Adamantane Natural products C1C(C2)CC3CC1CC2C3 ORILYTVJVMAKLC-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 102100027211 Albumin Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 101100014508 Arabidopsis thaliana GDU2 gene Proteins 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 101100161882 Caenorhabditis elegans acr-3 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical class [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 240000001432 Calendula officinalis Species 0.000 description 1
- 235000005881 Calendula officinalis Nutrition 0.000 description 1
- 101150037339 Cavin1 gene Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108020004394 Complementary RNA Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 244000289527 Cordyline terminalis Species 0.000 description 1
- 235000009091 Cordyline terminalis Nutrition 0.000 description 1
- 241000595586 Coryne Species 0.000 description 1
- 241001485655 Corynebacterium glutamicum ATCC 13032 Species 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 102100039868 Cytoplasmic aconitate hydratase Human genes 0.000 description 1
- GSXOAOHZAIYLCY-UHFFFAOYSA-N D-F6P Natural products OCC(=O)C(O)C(O)C(O)COP(O)(O)=O GSXOAOHZAIYLCY-UHFFFAOYSA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 description 1
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 1
- 229930182818 D-methionine Natural products 0.000 description 1
- FFEARJCKVFRZRR-SCSAIBSYSA-N D-methionine Chemical compound CSCC[C@@H](N)C(O)=O FFEARJCKVFRZRR-SCSAIBSYSA-N 0.000 description 1
- 239000011703 D-panthenol Substances 0.000 description 1
- 235000004866 D-panthenol Nutrition 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 230000008265 DNA repair mechanism Effects 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 101100275990 Drosophila melanogaster Naus gene Proteins 0.000 description 1
- 239000004097 EU approved flavor enhancer Substances 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108010013369 Enteropeptidase Proteins 0.000 description 1
- 102100029727 Enteropeptidase Human genes 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 101150092075 FIP1 gene Proteins 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- 206010051998 Febrile infection Diseases 0.000 description 1
- MBMLMWLHJBBADN-UHFFFAOYSA-N Ferrous sulfide Chemical class [Fe]=S MBMLMWLHJBBADN-UHFFFAOYSA-N 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- 102000001390 Fructose-Bisphosphate Aldolase Human genes 0.000 description 1
- 108010068561 Fructose-Bisphosphate Aldolase Proteins 0.000 description 1
- 206010071602 Genetic polymorphism Diseases 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 102000002667 Glycine hydroxymethyltransferase Human genes 0.000 description 1
- 108010043428 Glycine hydroxymethyltransferase Proteins 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 201000005569 Gout Diseases 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 241000288105 Grus Species 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 241001235200 Haemophilus influenzae Rd KW20 Species 0.000 description 1
- 241000701109 Human adenovirus 2 Species 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical class C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 235000000177 Indigofera tinctoria Nutrition 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 235000019766 L-Lysine Nutrition 0.000 description 1
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 101000941450 Lasioglossum laticeps Lasioglossin-1 Proteins 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 101150098959 MON1 gene Proteins 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical class [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 108090000301 Membrane transport proteins Proteins 0.000 description 1
- 102000003939 Membrane transport proteins Human genes 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical class [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- SGSSKEDGVONRGC-UHFFFAOYSA-N N(2)-methylguanine Chemical compound O=C1NC(NC)=NC2=C1N=CN2 SGSSKEDGVONRGC-UHFFFAOYSA-N 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 1
- 229910004619 Na2MoO4 Inorganic materials 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000008763 Neurofilament Proteins Human genes 0.000 description 1
- 108010088373 Neurofilament Proteins Proteins 0.000 description 1
- 101100291875 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) apg-13 gene Proteins 0.000 description 1
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 description 1
- 239000006057 Non-nutritive feed additive Substances 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 101001041669 Oryctolagus cuniculus Corticostatin 1 Proteins 0.000 description 1
- 101710160107 Outer membrane protein A Proteins 0.000 description 1
- ZNXZGRMVNNHPCA-UHFFFAOYSA-N Pantetheine Natural products OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS ZNXZGRMVNNHPCA-UHFFFAOYSA-N 0.000 description 1
- 241000364057 Peoria Species 0.000 description 1
- 108010069013 Phenylalanine Hydroxylase Proteins 0.000 description 1
- 102100038223 Phenylalanine-4-hydroxylase Human genes 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical class [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 101710096655 Probable acetoacetate decarboxylase 1 Proteins 0.000 description 1
- 108010078762 Protein Precursors Proteins 0.000 description 1
- 102000014961 Protein Precursors Human genes 0.000 description 1
- 102000012751 Pyruvate Dehydrogenase Complex Human genes 0.000 description 1
- 108010090051 Pyruvate Dehydrogenase Complex Proteins 0.000 description 1
- 230000006819 RNA synthesis Effects 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 1
- GBFLZEXEOZUWRN-VKHMYHEASA-M S-carboxylatomethyl-L-cysteine(1-) Chemical compound [O-]C(=O)[C@@H]([NH3+])CSCC([O-])=O GBFLZEXEOZUWRN-VKHMYHEASA-M 0.000 description 1
- 229910003798 SPO2 Inorganic materials 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 101100478210 Schizosaccharomyces pombe (strain 972 / ATCC 24843) spo2 gene Proteins 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 241000251131 Sphyrna Species 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 102000019259 Succinate Dehydrogenase Human genes 0.000 description 1
- 108010012901 Succinate Dehydrogenase Proteins 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical group C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical group OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 101100187081 Trichormus variabilis (strain ATCC 29413 / PCC 7937) nifS1 gene Proteins 0.000 description 1
- ZVNYJIZDIRKMBF-UHFFFAOYSA-N Vesnarinone Chemical compound C1=C(OC)C(OC)=CC=C1C(=O)N1CCN(C=2C=C3CCC(=O)NC3=CC=2)CC1 ZVNYJIZDIRKMBF-UHFFFAOYSA-N 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- HXXFSFRBOHSIMQ-UHFFFAOYSA-N [3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] dihydrogen phosphate Chemical compound OCC1OC(OP(O)(O)=O)C(O)C(O)C1O HXXFSFRBOHSIMQ-UHFFFAOYSA-N 0.000 description 1
- JQRLYSGCPHSLJI-UHFFFAOYSA-N [Fe].N1C(C=C2N=C(C=C3NC(=C4)C=C3)C=C2)=CC=C1C=C1C=CC4=N1 Chemical group [Fe].N1C(C=C2N=C(C=C3NC(=C4)C=C3)C=C2)=CC=C1C=C1C=CC4=N1 JQRLYSGCPHSLJI-UHFFFAOYSA-N 0.000 description 1
- 229960004308 acetylcysteine Drugs 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 125000000641 acridinyl group Chemical group C1(=CC=CC2=NC3=CC=CC=C3C=C12)* 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 229960001570 ademetionine Drugs 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- AEMOLEFTQBMNLQ-BKBMJHBISA-N alpha-D-galacturonic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-BKBMJHBISA-N 0.000 description 1
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000037354 amino acid metabolism Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 238000012435 analytical chromatography Methods 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001028 anti-proliverative effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 229940114079 arachidonic acid Drugs 0.000 description 1
- 235000021342 arachidonic acid Nutrition 0.000 description 1
- 150000004982 aromatic amines Chemical class 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- LFYJSSARVMHQJB-QIXNEVBVSA-N bakuchiol Chemical compound CC(C)=CCC[C@@](C)(C=C)\C=C\C1=CC=C(O)C=C1 LFYJSSARVMHQJB-QIXNEVBVSA-N 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Chemical group C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- RNBGYGVWRKECFJ-ARQDHWQXSA-N beta-D-fructofuranose 1,6-bisphosphate Chemical compound O[C@H]1[C@H](O)[C@@](O)(COP(O)(O)=O)O[C@@H]1COP(O)(O)=O RNBGYGVWRKECFJ-ARQDHWQXSA-N 0.000 description 1
- BGWGXPAPYGQALX-ARQDHWQXSA-N beta-D-fructofuranose 6-phosphate Chemical compound OC[C@@]1(O)O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O BGWGXPAPYGQALX-ARQDHWQXSA-N 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000007621 bhi medium Substances 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000001486 biosynthesis of amino acids Effects 0.000 description 1
- GZUXJHMPEANEGY-UHFFFAOYSA-N bromomethane Chemical compound BrC GZUXJHMPEANEGY-UHFFFAOYSA-N 0.000 description 1
- CDQSJQSWAWPGKG-UHFFFAOYSA-N butane-1,1-diol Chemical compound CCCC(O)O CDQSJQSWAWPGKG-UHFFFAOYSA-N 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 238000001818 capillary gel electrophoresis Methods 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000021523 carboxylation Effects 0.000 description 1
- 238000006473 carboxylation reaction Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 108091092328 cellular RNA Proteins 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 238000012824 chemical production Methods 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- ZYWFEOZQIUMEGL-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol;phenol Chemical compound ClC(Cl)Cl.CC(C)CCO.OC1=CC=CC=C1 ZYWFEOZQIUMEGL-UHFFFAOYSA-N 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 125000003346 cobalamin group Chemical group 0.000 description 1
- 239000010941 cobalt Chemical class 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical class [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 235000017471 coenzyme Q10 Nutrition 0.000 description 1
- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000008139 complexing agent Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 244000038559 crop plants Species 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 101150064923 dapD gene Proteins 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000005786 degenerative changes Effects 0.000 description 1
- 230000003413 degradative effect Effects 0.000 description 1
- 238000007357 dehydrogenase reaction Methods 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 101150036185 dnaQ gene Proteins 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000027721 electron transport chain Effects 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000007824 enzymatic assay Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000008713 feedback mechanism Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 150000002211 flavins Chemical class 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 229960002949 fluorouracil Drugs 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000019264 food flavour enhancer Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 235000021550 forms of sugar Nutrition 0.000 description 1
- 235000013611 frozen food Nutrition 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000000417 fungicide Substances 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 229940050410 gluconate Drugs 0.000 description 1
- 229940097042 glucuronate Drugs 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- 230000007407 health benefit Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 229940097275 indigo Drugs 0.000 description 1
- COHYTHOBJLSHDF-UHFFFAOYSA-N indigo powder Natural products N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229960005431 ipriflavone Drugs 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 101150021879 iscS gene Proteins 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- AGBQKNBQESQNJD-UHFFFAOYSA-M lipoate Chemical compound [O-]C(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-M 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 235000019136 lipoic acid Nutrition 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229960003646 lysine Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000011777 magnesium Chemical class 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical class [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- GMKMEZVLHJARHF-SYDPRGILSA-N meso-2,6-diaminopimelic acid Chemical compound [O-]C(=O)[C@@H]([NH3+])CCC[C@@H]([NH3+])C([O-])=O GMKMEZVLHJARHF-SYDPRGILSA-N 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- IZAGSTRIDUNNOY-UHFFFAOYSA-N methyl 2-[(2,4-dioxo-1h-pyrimidin-5-yl)oxy]acetate Chemical compound COC(=O)COC1=CNC(=O)NC1=O IZAGSTRIDUNNOY-UHFFFAOYSA-N 0.000 description 1
- LVHBHZANLOWSRM-UHFFFAOYSA-N methylenebutanedioic acid Natural products OC(=O)CC(=C)C(O)=O LVHBHZANLOWSRM-UHFFFAOYSA-N 0.000 description 1
- 238000013048 microbiological method Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Chemical class 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000004223 monosodium glutamate Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- XJVXMWNLQRTRGH-UHFFFAOYSA-N n-(3-methylbut-3-enyl)-2-methylsulfanyl-7h-purin-6-amine Chemical compound CSC1=NC(NCCC(C)=C)=C2NC=NC2=N1 XJVXMWNLQRTRGH-UHFFFAOYSA-N 0.000 description 1
- 238000001320 near-infrared absorption spectroscopy Methods 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 210000005044 neurofilament Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 101150082753 nifS gene Proteins 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 230000005257 nucleotidylation Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- LDCYZAJDBXYCGN-UHFFFAOYSA-N oxitriptan Natural products C1=C(O)C=C2C(CC(N)C(O)=O)=CNC2=C1 LDCYZAJDBXYCGN-UHFFFAOYSA-N 0.000 description 1
- 239000005022 packaging material Substances 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- ZNXZGRMVNNHPCA-VIFPVBQESA-N pantetheine Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS ZNXZGRMVNNHPCA-VIFPVBQESA-N 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 210000000578 peripheral nerve Anatomy 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- LYCRXMTYUZDUGA-UYRKPTJQSA-N pimeloyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)CCCCCC(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 LYCRXMTYUZDUGA-UYRKPTJQSA-N 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 125000000830 polyketide group Chemical group 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 239000011591 potassium Chemical class 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 235000013324 preserved food Nutrition 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- ULWHHBHJGPPBCO-UHFFFAOYSA-N propane-1,1-diol Chemical compound CCC(O)O ULWHHBHJGPPBCO-UHFFFAOYSA-N 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 229940076376 protein agonist Drugs 0.000 description 1
- 229940076372 protein antagonist Drugs 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 1
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 1
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 1
- 235000008151 pyridoxamine Nutrition 0.000 description 1
- 239000011699 pyridoxamine Substances 0.000 description 1
- 235000008160 pyridoxine Nutrition 0.000 description 1
- 239000011677 pyridoxine Substances 0.000 description 1
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 1
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 description 1
- 239000011764 pyridoxine hydrochloride Substances 0.000 description 1
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical class OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 1
- 150000004053 quinones Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 238000006268 reductive amination reaction Methods 0.000 description 1
- 230000014493 regulation of gene expression Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000003571 reporter gene assay Methods 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 239000010979 ruby Substances 0.000 description 1
- 229910001750 ruby Inorganic materials 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000012409 standard PCR amplification Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 239000005460 tetrahydrofolate Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 229960002663 thioctic acid Drugs 0.000 description 1
- ZEMGGZBWXRYJHK-UHFFFAOYSA-N thiouracil Chemical compound O=C1C=CNC(=S)N1 ZEMGGZBWXRYJHK-UHFFFAOYSA-N 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000005891 transamination reaction Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 125000002264 triphosphate group Chemical group [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 230000004143 urea cycle Effects 0.000 description 1
- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 description 1
- 235000012141 vanillin Nutrition 0.000 description 1
- FGQOOHJZONJGDT-UHFFFAOYSA-N vanillin Natural products COC1=CC(O)=CC(C=O)=C1 FGQOOHJZONJGDT-UHFFFAOYSA-N 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- WCNMEQDMUYVWMJ-JPZHCBQBSA-N wybutoxosine Chemical compound C1=NC=2C(=O)N3C(CC([C@H](NC(=O)OC)C(=O)OC)OO)=C(C)N=C3N(C)C=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O WCNMEQDMUYVWMJ-JPZHCBQBSA-N 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 229910052727 yttrium Inorganic materials 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
- 235000009529 zinc sulphate Nutrition 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Isolated nucleic acid molecules, designated SMP nucleic acid molecules, which encode novel SMP proteins from Corynebacterium glutamicum are described. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing SMP nucleic acid molecules, and host cells into which the expression vectors have been introduced. The invention still further provides isolated SMP proteins, mutated SMP proteins, fusion proteins, antigenic peptides and methods for the improvement of production of a desired compound from C.
glutamicum based on genetic engineering of SMP genes in this organism.
glutamicum based on genetic engineering of SMP genes in this organism.
Description
-, l -CORYNEBACTERIUM GLUTAMICUM GENES ENCODING PROTEINS
INVOLVED IN CARBON METABOLISM AND ENERGY PRODUCTION
This application is a divisional application of Canadian Application Serial No. 2,383,875 filed June 23, 2000.
INVOLVED IN CARBON METABOLISM AND ENERGY PRODUCTION
This application is a divisional application of Canadian Application Serial No. 2,383,875 filed June 23, 2000.
Background of the Invention Certain products and by-products of naturally-occurring metabolic processes in cells have utility in a wide array of industries, including the food, feed, cosmetics, and phanmaceutical industries. These molecules, collectively termed 'fine chemicals', include organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors, and enzymes. Their production is most conveniently performed through the large-scale culture of bacteria developed to produce and secrete large quantities of one or more desired molecules. One particularly useful organism for this purpose is Corynebacterium glutamicum, a gram positive, nonpathogenic bacterium. Through strain selection, a number of mutant strains have been developed which produce an array of desirable compounds. However, selection of strains improved for the production of a particular molecule is a time-consuming and difficult process.
Summary of the Invention The invention provides novel bacterial nucleic acid molecules which have a variety of uses. These uses include the identification of microorganisms which can be used to produce fine chemicals, the modulation of fine chemical production in C.
glutamicum or related bacteria, the typing or identification of C. glutamicum or related bacteria, as reference points for mapping the C. glutamicum genome, and as markers for transformation. These novel nucleic acid molecules encode proteins, referred to herein as sugar metabolism and oxidative phosphorylation (SMP) proteins.
C. glutamicum is a gram positive, aerobic bacterium which is commonly used in industry for the large-scale production of a variety of fine chemicals, and also for the degradation of hydrocarbons (such as in petroleum spills) and for the oxidation of terpenoids. The SMP nucleic acid molecules of the invention, therefore, can be used to identify microorganisms which can be used to produce fine chemicals, e.g., by fermentation processes. Modulation of the expression of the SMP nucleic acids of the invention, or modification of the sequence of the SMP nucleic acid molecules of the invention, can be used to modulate the production of one or more fine chemicals from a microorganism (e.g., to improve the yield or production of one or more fine chemicals from a Corynebacterium or Brevibacterium species).
The SMP nucleic acids of the invention may also be used to identify an organism as being Corynebacterium glutamicum or a close relative thereof, or to identify the presence of C. glutamicum or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of C.
glutamicum genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present. Although Corynebacterium glutamicum itself is nonpathogenic, it is related to species pathogenic in humans, such as Corynebacterium diphtheriae (the causative agent of diphtheria); the detectiori of such organisms is of significant clinical relevance.
The SMP nucleic acid molecules of the invention may also serve as reference points for mapping of the C. glutamicum genome, or of genomes of related organisms.
Similarly, these molecules, or variants or portions thereof, may serve as markers for genetically engineered Corynebacterium or Brevibacterium species.
The SMP ptoteins encoded by the novel nucleic acid molecules of the invention are capable of, for example, performing a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. Given the availability of cloning vectors for use in Corynebacterium glutamicum, such as those disclosed in Sinskey et al., U.S. Patent No. 4,649,119, and techniques for genetic manipulation of C.
glutamicum and the related Brevibacterium species (e.g., lactofermentum) (Yoshihama et al, J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159:
(1984); and Santamaria et al., J. Gen. Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of the invention may be utilized in the genetic engineering of this organism to make it a better or more efficient producer of one or more fine chemicals.
This improved production or efficiency of production of a fine chemical may be due to a direct effect of manipulation of a gene of the invention, or it may be due to an indirect effect of such manipulation.
Summary of the Invention The invention provides novel bacterial nucleic acid molecules which have a variety of uses. These uses include the identification of microorganisms which can be used to produce fine chemicals, the modulation of fine chemical production in C.
glutamicum or related bacteria, the typing or identification of C. glutamicum or related bacteria, as reference points for mapping the C. glutamicum genome, and as markers for transformation. These novel nucleic acid molecules encode proteins, referred to herein as sugar metabolism and oxidative phosphorylation (SMP) proteins.
C. glutamicum is a gram positive, aerobic bacterium which is commonly used in industry for the large-scale production of a variety of fine chemicals, and also for the degradation of hydrocarbons (such as in petroleum spills) and for the oxidation of terpenoids. The SMP nucleic acid molecules of the invention, therefore, can be used to identify microorganisms which can be used to produce fine chemicals, e.g., by fermentation processes. Modulation of the expression of the SMP nucleic acids of the invention, or modification of the sequence of the SMP nucleic acid molecules of the invention, can be used to modulate the production of one or more fine chemicals from a microorganism (e.g., to improve the yield or production of one or more fine chemicals from a Corynebacterium or Brevibacterium species).
The SMP nucleic acids of the invention may also be used to identify an organism as being Corynebacterium glutamicum or a close relative thereof, or to identify the presence of C. glutamicum or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of C.
glutamicum genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present. Although Corynebacterium glutamicum itself is nonpathogenic, it is related to species pathogenic in humans, such as Corynebacterium diphtheriae (the causative agent of diphtheria); the detectiori of such organisms is of significant clinical relevance.
The SMP nucleic acid molecules of the invention may also serve as reference points for mapping of the C. glutamicum genome, or of genomes of related organisms.
Similarly, these molecules, or variants or portions thereof, may serve as markers for genetically engineered Corynebacterium or Brevibacterium species.
The SMP ptoteins encoded by the novel nucleic acid molecules of the invention are capable of, for example, performing a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. Given the availability of cloning vectors for use in Corynebacterium glutamicum, such as those disclosed in Sinskey et al., U.S. Patent No. 4,649,119, and techniques for genetic manipulation of C.
glutamicum and the related Brevibacterium species (e.g., lactofermentum) (Yoshihama et al, J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159:
(1984); and Santamaria et al., J. Gen. Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of the invention may be utilized in the genetic engineering of this organism to make it a better or more efficient producer of one or more fine chemicals.
This improved production or efficiency of production of a fine chemical may be due to a direct effect of manipulation of a gene of the invention, or it may be due to an indirect effect of such manipulation.
There are a number of mechanisms by which the alteration of an SMP protein of the invention may directly affect the yield, production, and/or efficiency of production of a fine chenucal from a C. glutamicum strain incorporating such an altered protein.
The degradation of high-energy carbon molecules such as sugars, and the conversion of compounds such as NADH and FADH2 to compounds containing high energy phosphate bonds via oxidative phosphorylation results in a number of compounds which themselves may be desirable fine chemicals, such as pyruvate, ATP, NADH, and a number of intermediate sugar compounds. Further, the energy molecules (such as ATP) and the reducing equivalents (such as NADH or NADPH) produced by these metabolic pathways are utilized in the cell to drive reactions which would otherwise be energetically unfavorable. Such unfavorable reactions include many biosynthetic pathways for fine chemicals. By improving the ability of the cell to utilize a particular sugar (e.g., by manipulating the genes encoding enzymes involved in the degradation and conversion of that sugar into energy for the cell), one may increase the amount of energy available to permit unfavorable, yet desired metabolic reactions (e.g., the biosynthesis of a desired fine chemical) to occur.
The mutagenesis of one or more SMP genes of the invention may also result in SMP proteins having altered activities which indirectly impact the production of one or more desired fine chemicals from C. glutamicum. For example, by increasing the efficiency of utilization of one or more sugars (such that the conversion of the sugar to useful energy molecules is improved), or by increasing the efficiency of conversion of reducing equivalents to useful energy molecules (e.g., by improving the efficiency of oxidative phosphorylation, or the activity of the ATP synthase), one can increase the amount of these high-energy compounds available to the cell to drive normally unfavorable metabolic processes. These processes include the construction of cell walls, transcription, translation, and the biosynthesis of compounds necessary for growth and division of the cells (e.g., nucleotides, amino acids, vitamins, lipids, etc.) (Lengeler et al.
(1999) Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109; 913-918;
875-899).
By improving the growth and multiplication of these engineered cells, it is possible to increase both the viability of the cells in large-scale culture, and also to improve their rate of division, such that a relatively larger number of cells can survive in fermentor culture. The yield, production, or efficiency of production may be increased, at least due to the presence of a greater number of viable cells, each producing the desired fine chemical. Also, many of the degradation products produced during sugar metabolism are utilized by the cell as precursors or intermediates in the production of other desirable products, such as fine chemicals. So, by increasing the ability of the cell to metabolize sugars, the number of these degradation products available to the cell for other processes should also be increased.
The invention provides novel nucleic acid molecules which encode proteins, referred to herein as SMP proteins, which are capable of, for example, performing a function involved in the metabolism of carbon compounds such as sugars and the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. Nucleic acid molecules encoding an SMP protein are referred to herein as SMP nucleic acid molecules. In a preferred embodiment, the SMP
protein participates in the conversion of carbon molecules and degradation products thereof to energy which is utilized by the cell for metabolic processes.
Examples of such proteins include those encoded by the genes set forth in Table 1.
Accordingly, one aspect of the invention pertains to isolated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotide sequence encoding an SMP protein or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection or amplification of SMP-encoding nucleic acid (e.g., DNA or mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule comprises one of the nucleotide sequences set forth as the odd-numbered SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7....), or the coding region or a complement thereof of one of these nucleotide sequences. In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence set forth as an odd-numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5, SEQ ID NO:7....), or a portion thereof. In other preferred embodiments, the isolated nucleic acid molecule encodes one of the amino acid sequences set forth as an even-numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ
The degradation of high-energy carbon molecules such as sugars, and the conversion of compounds such as NADH and FADH2 to compounds containing high energy phosphate bonds via oxidative phosphorylation results in a number of compounds which themselves may be desirable fine chemicals, such as pyruvate, ATP, NADH, and a number of intermediate sugar compounds. Further, the energy molecules (such as ATP) and the reducing equivalents (such as NADH or NADPH) produced by these metabolic pathways are utilized in the cell to drive reactions which would otherwise be energetically unfavorable. Such unfavorable reactions include many biosynthetic pathways for fine chemicals. By improving the ability of the cell to utilize a particular sugar (e.g., by manipulating the genes encoding enzymes involved in the degradation and conversion of that sugar into energy for the cell), one may increase the amount of energy available to permit unfavorable, yet desired metabolic reactions (e.g., the biosynthesis of a desired fine chemical) to occur.
The mutagenesis of one or more SMP genes of the invention may also result in SMP proteins having altered activities which indirectly impact the production of one or more desired fine chemicals from C. glutamicum. For example, by increasing the efficiency of utilization of one or more sugars (such that the conversion of the sugar to useful energy molecules is improved), or by increasing the efficiency of conversion of reducing equivalents to useful energy molecules (e.g., by improving the efficiency of oxidative phosphorylation, or the activity of the ATP synthase), one can increase the amount of these high-energy compounds available to the cell to drive normally unfavorable metabolic processes. These processes include the construction of cell walls, transcription, translation, and the biosynthesis of compounds necessary for growth and division of the cells (e.g., nucleotides, amino acids, vitamins, lipids, etc.) (Lengeler et al.
(1999) Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109; 913-918;
875-899).
By improving the growth and multiplication of these engineered cells, it is possible to increase both the viability of the cells in large-scale culture, and also to improve their rate of division, such that a relatively larger number of cells can survive in fermentor culture. The yield, production, or efficiency of production may be increased, at least due to the presence of a greater number of viable cells, each producing the desired fine chemical. Also, many of the degradation products produced during sugar metabolism are utilized by the cell as precursors or intermediates in the production of other desirable products, such as fine chemicals. So, by increasing the ability of the cell to metabolize sugars, the number of these degradation products available to the cell for other processes should also be increased.
The invention provides novel nucleic acid molecules which encode proteins, referred to herein as SMP proteins, which are capable of, for example, performing a function involved in the metabolism of carbon compounds such as sugars and the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. Nucleic acid molecules encoding an SMP protein are referred to herein as SMP nucleic acid molecules. In a preferred embodiment, the SMP
protein participates in the conversion of carbon molecules and degradation products thereof to energy which is utilized by the cell for metabolic processes.
Examples of such proteins include those encoded by the genes set forth in Table 1.
Accordingly, one aspect of the invention pertains to isolated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotide sequence encoding an SMP protein or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection or amplification of SMP-encoding nucleic acid (e.g., DNA or mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule comprises one of the nucleotide sequences set forth as the odd-numbered SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7....), or the coding region or a complement thereof of one of these nucleotide sequences. In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence set forth as an odd-numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5, SEQ ID NO:7....), or a portion thereof. In other preferred embodiments, the isolated nucleic acid molecule encodes one of the amino acid sequences set forth as an even-numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:6, SEQ ID NO:8....).. The preferred SMP proteins of the present invention also preferably possess at least one of the SMP activities described herein.
In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence having an even-numbered SEQ ID NO: in the Sequence Listing), e.g., sufficiently homologous to an amino acid sequence of the invention such that the protein or portion thereof maintains an SMP activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to perform a function involved in the metabolism of carbon compounds such as sugars or the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. In one embodiment, the protein encoded by the nucleic acid molecule is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80%, or 90% and most preferably at least about 95%, 96%, 97%, 98%, or 99% or more homologous to an amino acid sequence of the invention (e.g., an entire amino acid sequence selected those having an even-numbered SEQ ID NO in the Sequence Listing). In another preferred embodiment, the protein is a full length C.
glutamicum protein which is substantially homologous to an entire amino acid sequence of the invention (encoded by an open reading frame shown in the corresponding odd-numbered SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7....).
In another preferred embodiment, the isolated nucleic acid molecule is derived from C. glutamicum and encodes a protein (e.g., an SMP fusion protein) which includes a biologically active domain which is at least about 50% or more homologous to one of the amino acid sequences of the invention (e.g., a sequence of one of the even-numbered SEQ ID NOs in the Sequence Listing) and is able to perform a function involved in the metabolism of carbon compounds such as sugars or the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum, or has one or more of the activities set forth in Table 1, and which also includes heterologous nucleic acid sequences encoding a heterologous polypeptide or regulatory regions.
In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence having an even-numbered SEQ ID NO: in the Sequence Listing), e.g., sufficiently homologous to an amino acid sequence of the invention such that the protein or portion thereof maintains an SMP activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to perform a function involved in the metabolism of carbon compounds such as sugars or the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. In one embodiment, the protein encoded by the nucleic acid molecule is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80%, or 90% and most preferably at least about 95%, 96%, 97%, 98%, or 99% or more homologous to an amino acid sequence of the invention (e.g., an entire amino acid sequence selected those having an even-numbered SEQ ID NO in the Sequence Listing). In another preferred embodiment, the protein is a full length C.
glutamicum protein which is substantially homologous to an entire amino acid sequence of the invention (encoded by an open reading frame shown in the corresponding odd-numbered SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7....).
In another preferred embodiment, the isolated nucleic acid molecule is derived from C. glutamicum and encodes a protein (e.g., an SMP fusion protein) which includes a biologically active domain which is at least about 50% or more homologous to one of the amino acid sequences of the invention (e.g., a sequence of one of the even-numbered SEQ ID NOs in the Sequence Listing) and is able to perform a function involved in the metabolism of carbon compounds such as sugars or the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum, or has one or more of the activities set forth in Table 1, and which also includes heterologous nucleic acid sequences encoding a heterologous polypeptide or regulatory regions.
In another embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence of the invention (e.g., a sequence of an odd-numbered SEQ ID NO in the Sequence Listing) A. Preferably, the isolated nucleic acid molecule corresponds to a naturally-occurring nucleic acid molecule. More preferably, the isolated nucleic acid encodes a naturally-occurring C. glutamicum SMP
protein, or a biologically active portion thereof.
Another aspect of the invention pertains to vectors, e.g., recombinant expression vectors, containing the nucleic acid molecules of the invention, and host cells into which such vectors have been introduced. In one embodiment, such a host cell is used to produce an SMP protein by culturing the host cell in a suitable medium. The SMP
protein can be then isolated from the medium or the host cell.
Yet another aspect of the invention pertains to a genetically altered microorganism in which an SMP gene has been introduced or altered. In one embodiment, the genome of the microorganism has been altered by introduction of a nucleic acid molecule of the invention encoding wild-type or mutated SMP
sequence as a transgene. In another embodiment, an endogenous SMP gene within the genome of the microorganism has been altered, e.g., functionally disrupted, by homologous recombination with an altered SMP gene. In another embodiment, an endogenous or introduced SMP gene in a microorganism has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional SMP
protein. In still another embodiment, one or more of the regulatory regions (e.g., a promoter, repressor, or inducer) of an SMP gene in a microorganism has been altered (e.g., by deletion, truncation, inversion, or point mutation) such that the expression of the SMP
gene is modulated. In a preferred embodiment, the microorganism belongs to the genus Corynebacterium or Brevibacterium, with Corynebacterium glutamicum being particularly preferred. In a preferred embodiment, the microorganism is also utilized for the production of a desired compound, such as an amino acid, with lysine being particularly preferred.
In another aspect, the invention provides a method of identifying the presence or activity of Cornyebacterium diphtheriae in a subject. This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention (e.g., the sequences set forth in the Sequence Listing as SEQ ID NOs I through 782) in a subject, thereby detecting the presence or activity of Corynebacterium diphtheriae in the subject.
Still another aspect of the invention pertains to an isolated SMP protein or a portion, e.g., a biologically active portion, thereof. In a preferred embodiment, the isolated SMP protein or portion thereof is capable of performing a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. In another preferred embodiment, the isolated SMP
protein or portion thereof is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: in the Sequence Listing) such that the protein or portion thereof maintains the ability to perfonm a function involved in the metabolism of.carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum.
The invention also provides an isolated preparation of an SMP protein. In preferred embodiments, the SMP protein comprises an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing).
In another preferred embodiment, the invention pertains to an isolated full length protein which is substantially homologous to an entire amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) (encoded by an open reading frame set forth in a corresponding odd-numbered SEQ ID NO:
of the Sequence Listing). In yet another embodiment, the protein is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80%, or 90%, and most preferably at least about 95%, 96%, 97%, 98%, or 99% or more homologous to an entire amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing). In other embodiments, the isolated SMP
protein comprises an amino acid sequence which is at least about 50% or more homologous to one of the amino acid sequences of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) and is able to perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum, or has one or more of the activities set forth in Table 1.
protein, or a biologically active portion thereof.
Another aspect of the invention pertains to vectors, e.g., recombinant expression vectors, containing the nucleic acid molecules of the invention, and host cells into which such vectors have been introduced. In one embodiment, such a host cell is used to produce an SMP protein by culturing the host cell in a suitable medium. The SMP
protein can be then isolated from the medium or the host cell.
Yet another aspect of the invention pertains to a genetically altered microorganism in which an SMP gene has been introduced or altered. In one embodiment, the genome of the microorganism has been altered by introduction of a nucleic acid molecule of the invention encoding wild-type or mutated SMP
sequence as a transgene. In another embodiment, an endogenous SMP gene within the genome of the microorganism has been altered, e.g., functionally disrupted, by homologous recombination with an altered SMP gene. In another embodiment, an endogenous or introduced SMP gene in a microorganism has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional SMP
protein. In still another embodiment, one or more of the regulatory regions (e.g., a promoter, repressor, or inducer) of an SMP gene in a microorganism has been altered (e.g., by deletion, truncation, inversion, or point mutation) such that the expression of the SMP
gene is modulated. In a preferred embodiment, the microorganism belongs to the genus Corynebacterium or Brevibacterium, with Corynebacterium glutamicum being particularly preferred. In a preferred embodiment, the microorganism is also utilized for the production of a desired compound, such as an amino acid, with lysine being particularly preferred.
In another aspect, the invention provides a method of identifying the presence or activity of Cornyebacterium diphtheriae in a subject. This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention (e.g., the sequences set forth in the Sequence Listing as SEQ ID NOs I through 782) in a subject, thereby detecting the presence or activity of Corynebacterium diphtheriae in the subject.
Still another aspect of the invention pertains to an isolated SMP protein or a portion, e.g., a biologically active portion, thereof. In a preferred embodiment, the isolated SMP protein or portion thereof is capable of performing a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. In another preferred embodiment, the isolated SMP
protein or portion thereof is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: in the Sequence Listing) such that the protein or portion thereof maintains the ability to perfonm a function involved in the metabolism of.carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum.
The invention also provides an isolated preparation of an SMP protein. In preferred embodiments, the SMP protein comprises an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing).
In another preferred embodiment, the invention pertains to an isolated full length protein which is substantially homologous to an entire amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) (encoded by an open reading frame set forth in a corresponding odd-numbered SEQ ID NO:
of the Sequence Listing). In yet another embodiment, the protein is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80%, or 90%, and most preferably at least about 95%, 96%, 97%, 98%, or 99% or more homologous to an entire amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing). In other embodiments, the isolated SMP
protein comprises an amino acid sequence which is at least about 50% or more homologous to one of the amino acid sequences of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) and is able to perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum, or has one or more of the activities set forth in Table 1.
Altematively, the isolated SMP protein can comprise an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80%, or 90%, and even more preferably at least about 95%, 96%, 97%, 98,%, or 99% or more homologous to a nucleotide sequence of one of the even-numbered SEQ ID NOs set forth in the Sequence Listing. It is also preferred that the preferred fonns of SMP proteins also have one or more of the SMP
bioactivities described herein.
The SMP polypeptide, or a biologically active portion thereof, can be operatively linked to a non-SMP polypeptide to form a fusion protein. In preferred embodiments, this fusion protein has an activity which differs from that of the SMP protein alone. In other preferred embodiments, this fusion protein performs a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. In particularly preferred embodiments, integration of this fusion protein into a host cell modulates production of a desired compound from the cell.
In another aspect, the invention provides methods for screening molecules which modulate the activity of an SMP protein, either by interacting with the protein itself or a substrate or binding partner of the SMP protein, or by modulating the transcription or translation of an SMP nucleic acid molecule of the invention.
Another aspect of the invention pertains to a method for producing a fine chemical. This method involves the culturing of a cell containing a vector directing the expression of an SMP nucleic acid molecule of the invention, such that a fine chemical is produced. In a preferred embodiment, this method further includes the step of obtaining a cell containing such a vector, in which a cell is transfected with a vector directing the expression of an SMP nucleic acid. In another preferred embodiment, this method further includes the step of recovering the fine chemical from the culture. In a particularly preferred embodiment, the cell is from the genus Corynebacterium or Brevibacterium, or is selected from those strains set forth in Table 3.
Another aspect of the invention pertains to methods for modulating production of a molecule from a microorganism. Such methods include contacting the cell with an agent which modulates SMP protein activity or SMP nucleic acid expression such that a celi associated activity is altered relative to this same activity in the absence of the agent. In a preferred embodiment, the cell is modulated for one or more C.
glutamicum carbon metabolism pathways or for the production of energy through processes such as oxidative phosphorylation, such that the yields or rate of production of a desired fine chemical by this microorganism is improved. The agent which modulates SMP
protein activity can be an agent which stimulates SMP protein activity or SMP nucleic acid expression. Examples of agents which stimulate SMP protein activity or SMP
nucleic acid expression include small molecules, active SMP proteins, and nucleic acids encoding SMP proteins that have been introduced into the cell. Examples of agents which inhibit SMP activity or expression include small molecules and antisense SMP
nucleic acid molecules.
Another aspect of the invention pertains to methods for modulating yields of a desired compound from a cell, involving the introduction of a wild-type or mutant SMP
gene into a cell, either maintained on a separate plasmid or integrated into the genome of the host cell. If integrated into the genome, such integration can be random, or it can take place by homologous recombination such that the native gene is replaced by the introduced copy, causing the production of the desired compound from the cell to be modulated. In a preferred embodiment, said yields are increased. In another preferred embodiment, said chemical is a fine chemical. In a particularly preferred embodiment, said fine chemical is an amino acid. In especially preferred embodiments, said amino acid is L-lysine.
Detailed Description of the Invention The present invention provides SMP nucleic acid and protein molecules which are involved in the metabolism of carbon compounds such as sugars and the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. The molecules of the invention may be utilized in the modulation of production of fine chemicals from microorganisms, such as C.
glutamicum, either directly (e.g., where overexpression or optimization of a glycolytic pathway protein has a direct impact on the yield, production, and/or efficiency of production of, e.g., pyruvate from modified C. glutamicum), or may have an indirect = ' .
-II-impact which nonetheless results in an increase of yield, production, and/or efficiency of production of the desired compound (e.g., where modulation of proteins involved in oxidative phosphorylation results in alterations in the amount of energy available to perform necessary metabolic processes and other cellular functions, such as nucleic acid and protein biosynthesis and transcription/translation). Aspects of the invention are further explicated below.
1. Fine Chemicals The term 'fine chemical' is art-recognized and includes molecules produced by an organism which have applications in various industries, such as, but not limited to, the pharmaceutical, agriculture, and cosmetics industries. Such compounds include organic acids, such as tartaric acid, itaconic acid, and diaminopimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides, and nucleotides (as described e.g. in Kuninaka, A. (1996) Nucleotides and related compounds, p. 561-612, in Biotechnology vol. 6, Rehm et al., eds. VCH:
Weinheim, and references contained therein), lipids, both saturated and unsaturated fatty acids (e.g., arachidonic acid), diols (e.g., propane diol, and butane diol), carbohydrates (e.g., hyaluronic acid and trehalose), aromatic compounds (e.g., aromatic amines, vanillin, and indigo), vitamins and cofactors (as described in Ullmann's Encyclopedia of Industrial Chemistry, vol. A27, "Vitamins", p. 443-613 (1996) VCH: Weinheim and references therein; and Ong, A.S., Niki, E. & Packer, L. (1995) "Nutrition, Lipids, Health, and Disease" Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research -Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes, polyketides (Cane el al. (1998) Science 282: 63-68), and all other chemicals described in Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN:
0818805086 and references therein. The metabolism and uses of certain of these fine chemicals are further explicated below.
A. Amino Acid Metabolism and Uses Amino acids comprise the basic structural units of all proteins, and as such are essential for normal cellular functioning in all organisms. The term "amino acid" is art-recognized. The proteinogenic amino acids, of which there are 20 species, serve as structural units for proteins, in which they are linked by peptide bonds, while the nonproteinogenic amino acids (hundreds of which are known) are not normally found in proteins (see Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH:
Weinheim (1985)). Amino acids may be in the D- or L- optical configuration, though L-amino acids are generally the only type found in naturally-occurring proteins.
Biosynthetic and degradative pathways of each of the 20 proteinogenic amino acids have been well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3d edition, pages 578-590 (1988)). The 'essential' amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine), so named because they are generally a nutritional requirement due to the complexity of their biosyntheses, are readily converted by simple biosynthetic pathways to the remaining 11 'nonessential' amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, and tyrosine).
Higher animals do retain the ability to synthesize some of these amino acids, but the essential amino acids must be supplied from the diet in order for nonnal protein synthesis to occur.
Aside from their function in protein biosynthesis, these amino acids are interesting chemicals in their own right, and many have been found to have various applications in the food, feed, chemical, cosmetics, agriculture, and pharmaceutical industries. Lysine is an important amino acid in the nutrition not only of humans, but also of monogastric animals such as poultry and swine. Glutamate is most conunonly used as a flavor additive (mono-sodium glutamate, MSG) and is widely used throughout the food industry, as are aspartate, phenylalanine, glycine, and cysteine.
Glycine, L-methionine and tryptophan are all utilized in the pharmaceutical industry.
Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are of use in both the pharmaceutical and cosmetics industries. Threonine, tryptophan, and D/ L-methionine are common feed additives. (Leuchtenberger, W. (1996) Amino aids -technical production and use, p. 466-502 in Rehm et al. (eds.) Biotechnology vol. 6, chapter 14a, VCH: Weinheim). Additionally, these amino acids have been found to be useful as precursors for the synthesis of synthetic amino acids and proteins, such as N-acetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan, and others described in Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97, VCH:
Weinheim, 1985.
The biosynthesis of these natural amino acids in organisms capable of producing them, such as bacteria, has been well characterized (for review of bacterial amino acid biosynthesis and regulation thereof, see Umbarger, H.E.(1978) Ann.
Rev.
Biochem. 47: 533-606). Glutamate is synthesized by the reductive amination of a-ketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline, and arginine are each subsequently produced from glutamate. The biosynthesis of serine is a three-step process beginning with 3-phosphoglycerate (an intermediate in glycolysis), and resulting in this amino acid after oxidation, transamination, and hydrolysis steps. Both cysteine and glycine are produced from serine; the former by the condensation of homocysteine with serine, and the latter by the transferal of the side-chain 0-carbon atom to tetrahydrofolate, in a reaction catalyzed by serine transhydroxymethylase.
Phenylalanine, and tyrosine are synthesized from the glycolytic and pentose phosphate pathway precursors erythrose 4-phosphate and phosphoenolpyruvate in a 9-step biosynthetic pathway that differ only at the final two steps after synthesis of prephenate.
Tryptophan is also produced from these two initial molecules, but its synthesis is an 11-step pathway. Tyrosine may also be synthesized from phenylalanine, in a reaction catalyzed by phenylalanine hydroxylase. Alanine, valine, and leucine are all biosynthetic products of pyruvate, the final product of glycolysis. Aspartate is formed from oxaloacetate, an intermediate of the citric acid cycle. Asparagine, methionine, threonine, and lysine are each produced by the conversion of aspartate.
Isoleucine is formed from threonine. A complex 9-step pathway results in the production of histidine from 5-phosphoribosyl-l-pyrophosphate, an activated sugar.
Amino acids in excess of the protein synthesis needs of the cell cannot be stored, and are instead degraded to provide intermediates for the major metabolic pathways of the cell (for review see Stryer, L. Biochemistry 3'd ed. Ch. 21 "Amino Acid Degradation and the Urea Cycle" p. 495-516 (1988)). Although the cell is able to convert unwanted amino acids into useful metabolic intermediates, amino acid production is costly in tenms of energy, precursor molecules, and the enzymes necessary to synthesize them.
Thus it is not surprising that amino acid biosynthesis is regulated by feedback inhibition, in which the presence of a particular amino acid serves to slow or entirely stop its own production (for overview of feedback mechanisms in amino acid biosynthetic pathways, see Stryer, L. Biochemistry, 3rd ed. Ch. 24: "Biosynthesis of Amino Acids and Heme" p.
575-600 (1988)). Thus, the output of any particular amino acid is limited by the amount of that amino acid present in the cell.
B. Vitamin, Cofactor, and Nutraceutical Metabolism and Uses Vitamins, cofactors, and nutraceuticals comprise another group of molecules which the higher animals have lost the ability to synthesize and so must ingest, although they are readily synthesized by other organisms such as bacteria. These molecules are either bioactive substances themselves, or are precursors of biologically active substances which may serve as electron carriers or intermediates in a variety of metabolic pathways. Aside from their nutritive value, these compounds also have significant industrial value as coloring agents, antioxidants, and catalysts or other processing aids. (For an overview of the structure, activity, and industrial applications of these compounds, see, for example, Ullman's Encyclopedia of Industrial Chemistry, "Vitamins" vol. A27, p. 443-613, VCH: Weinheim, 1996.) The term "vitamin" is art-recognized, and includes nutrients which are required by an organism for normal functioning, but which that organism cannot synthesize by itself. The group of vitamins may encompass cofactors and nutraceutical compounds. The language "cofactor"
includes nonproteinaceous compounds required for a normal enzymatic activity to occur. Such compounds may be organic or inorganic; the cofactor molecules of the invention are preferably organic. The term "nutraceutical" includes dietary supplements having health benefits in plants and animals, particularly humans. Examples of such molecules are vitamins, antioxidants, and also certain lipids (e.g., polyunsaturated fatty acids).
The biosynthesis of these molecules in organisms capable of producing them, such as bacteria, has been largely characterized (Uliman's Encyclopedia of Industrial Chemistry, "Vitamins" vol. A27, p. 443-613, VCH: Weinheim, 1996; Michal, G.
(1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley & Sons; Ong, A.S., Niki, E. & Packer, L. (1995) "Nutrition, Lipids, Health, and Disease" Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research - Asia, held Sept.
1-3, 1994 at Penang, Malaysia, AOCS Press: Champaign, IL X, 374 S).
Thiamin (vitamin B i) is produced by the chemical coupling of pyrimidine and thiazole moieties. Riboflavin (vitamin BZ) is synthesized from guanosine-5'-triphosphate (GTP) and ribose-5'-phosphate. Riboflavin, in turn, is utilized for the synthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The family of compounds collectively termed 'vitamin B6' (e.g., pyridoxine, pyridoxamine, pyridoxa-5'-phosphate, and the commercially used pyridoxin hydrochloride) are all derivatives of the common structural unit, 5-hydroxy-6-methylpyridine. Pantothenate (pantothenic acid, (R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-l-oxobutyl)-p-alanine) can be produced either by chemical synthesis or by fermentation. The final steps in pantothenate biosynthesis consist of the ATP-driven condensation of 0-alanine and pantoic acid. The enzymes responsible for the biosynthesis steps for the conversion to pantoic acid, to 0-alanine and for the condensation to panthotenic acid are known. The metabolically active form of pantothenate is Coenzyme A, for which the biosynthesis proceeds in 5 enzymatic steps. Pantothenate, pyridoxal-5'-phosphate, cysteine and ATP are the precursors of Coenzyme A. These enzymes not only catalyze the formation of panthothante, but also the production of (R)-pantoic acid, (R)-pantolacton, (R)-panthenol (provitamin B5), pantetheine (and its derivatives) and coenzyme A.
Biotin biosynthesis from the precursor molecule pimeloyl-CoA in microorganisms has been studied in detail and several of the genes involved have been identified. Many of the corresponding proteins have been found to also be involved in Fe-cluster synthesis and are members of the nifS class of proteins. Lipoic acid is derived from octanoic acid, and serves as a coenzyme in energy metabolism, where it becomes part of the pyruvate dehydrogenase complex and the a-ketoglutarate dehydrogenase complex. The folates are a group of substances which are all derivatives of folic acid, which is turn is derived from L-glutamic acid, p-amino-benzoic acid and 6-methylpterin. The biosynthesis of folic acid and its derivatives, starting from the metabolism intermediates guanosine-5'-triphosphate (GTP), L-glutamic acid and p-amino-benzoic acid has been studied in detail in certain microorganisms.
Corrinoids (such as the cobalamines and particularly vitamin B12) and porphyrines belong to a group of chemicals characterized by a tetrapyrole ring system.
t . - ' The biosynthesis of vitamin B12 is sufficiently complex that it has not yet been completely characterized, but many of the enzymes and substrates involved are now known. Nicotinic acid (nicotinate), and nicotinamide are pyridine derivatives which are also tenmed 'niacin'. Niacin is the precursor of the important coenzymes NAD
5(nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms.
The large-scale production of these compounds has largely relied on cell-free chemical syntheses, though some of these chemicals have also been produced by large-scale culture of microorganisms, such as riboflavin, Vitamin B6, pantothenate, and biotin. Only Vitamin BiZ is produced solely by fermentation, due to the complexity of its synthesis. In vitro methodologies require significant inputs of materials and time, often at great cost.
C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses Purine and pyrimidine metabolism genes and their corresponding proteins are important targets for the therapy of tumor diseases and viral infections. The language "purine" or "pyrimidine" includes the nitrogenous bases which are constituents of nucleic acids, co-enzymes, and nucleotides. The term "nucleotide" includes the basic structural units of nucleic acid molecules, which are comprised of a nitrogenous base, a pentose sugar (in the case of RNA, the sugar is ribose; in the case of DNA, the sugar is D-deoxyribose), and phosphoric acid. The language "nucleoside" includes molecules which serve as precursors to nucleotides, but which are lacking the phosphoric acid moiety that nucleotides possess. By inhibiting the biosynthesis of these molecules, or their mobilization to form nucleic acid molecules, it is possible to inhibit RNA and DNA
synthesis; by inhibiting this activity in a fashion targeted to cancerous cells, the ability of tumor cells to divide and replicate may be inhibited. Additionally, there are nucleotides which do not form nucleic acid molecules, but rather serve as energy stores (i.e., AMP) or as coenzymes (i.e., FAD and NAD).
Several publications have described the use of these chemicals for these medical indications, by influencing purine and/or pyrimidine metabolism (e.g.
Christopherson, R.I. and Lyons, S.D. (1990) "Potent inhibitors of de novo pyrimidine and purine biosynthesis as chemotherapeutic agents." Med Res. Reviews 10: 505-548).
Studies of enzymes involved in purine and pyrimidine metabolism have been focused on the development of new drugs which can be used, for example, as immunosuppressants or anti-proliferants (Smith, J.L., (1995) "Enzymes in nucleotide synthesis."
Curr. Opin.
Struct. Biol. 5: 752-757; (1995) Biochem Soc. Transact. 23: 877-902). However, purine and pyrimidine bases, nucleosides and nucleotides have other utilities: as intermediates in the biosynthesis of several fine chemicals (e.g., thiamine, S-adenosyl-methionine, folates, or riboflavin), as energy carriers for the cell (e.g., ATP or GTP), and for chemicals themselves, commonly used as flavor enhancers (e.g., IMP or GMP) or for several medicinal applications (see, for example, Kuninaka, A. (1996) Nucleotides and Related Compounds in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, p.
612). Also, enzymes involved in purine, pyrimidine, nucleoside, or nucleotide metabolism are increasingly serving as targets against which chemicals for crop protection, including fungicides, herbicides and insecticides, are developed.
The metabolism of these compounds in bacteria has been characterized (for reviews see, for example, Zalkin, H. and Dixon, J.E. (1992) "de novo purine nucleotide biosynthesis", in: Progress in Nucleic Acid Research and Molecular Biology, vol. 42, Academic Press:, p. 259-287; and Michal, G. (1999) "Nucleotides and Nucleosides", Chapter 8 in: Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York). Purine metabolism has been the subject of intensive research, and is essential to the normal functioning of the cell. Impaired purine metabolism in higher animals can cause severe disease, such as gout. Purine nucleotides are synthesized from ribose-5-phosphate, in a series of steps through the intermediate compound inosine-5'-phosphate (IMP), resulting in the production of guanosine-5'-monophosphate (GMP) or adenosine-5'-monophosphate (AMP), from which the triphosphate forms utilized as nucleotides are readily formed. These compounds are also utilized as energy stores, so their degradation provides energy for many different biochemical processes in the cell.
Pyrimidine biosynthesis proceeds by the formation of uridine-5'-monophosphate (UMP) from ribose-5-phosphate. UMP, in turn, is converted to cytidine-5'-triphosphate (CTP).
The deoxy- forms of all of these nucleotides are produced in a one step reduction reaction from the diphosphate ribose form of the nucleotide to the diphosphate deoxyribose form of the nucleotide. Upon phosphorylation, these molecules are able to participate in DNA synthesis.
D. Trehalose Metabolism and Uses Trehalose consists of two glucose molecules, bound in a, a-1,1 linkage. It is commonly used in the food industry as a sweetener, an additive for dried or frozen foods, and in beverages. However, it also has applications in the pharmaceutical, cosmetics and biotechnology industries (see, for example, Nishimoto et al., (1998) U.S.
Patent No. 5,759,610; Singer, M.A. and Lindquist, S. (1998) Trends Biotech.
16: 460-467; Paiva, C.L.A. and Panek, A.D. (1996) Biotech. Ann. Rev. 2: 293-314; and Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose is produced by enzymes from many microorganisms and is naturally released into the surrounding medium, from which it can be collected using methods known in the art.
II. Sugar and Carbon Molecule Utilization and Oxidative Phosphorylation Carbon is a critically important element for the formation of all organic compounds, and thus is a nutritional requirement not only for the growth and division of C. glutamicum, but also for the overproduction of fine chemicals from this microorganism. Sugars, such as mono-, di-, or polysaccharides, are particularly good carbon sources, and thus standard growth media typically contain one or more of:
glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, or cellulose (Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes", VCH: Weinheim). Alternatively, more complex forms of sugar may be utilized in the media, such as molasses, or other by-products of sugar refinement. Other compounds aside from the sugars may be used as alternate carbon sources, including alcohols (e.g., ethanol or methanol), alkanes, sugar alcohols, fatty acids, and organic acids (e.g., acetic acid or lactic acid). For a review of carbon sources and their utilization by microorganisms in culture, see: Ullman's Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes", VCH: Weinheim; Stoppok, E. and Buchholz, K. (1996) "Sugar-based raw materials for fennentation applications"
in Biotechnology (Rehm, H.J. et al., eds.) vol. 6, VCH: Weinheim, p. 5-29; Rehm, H.J.
(1980) Industrielle Mikrobiologie, Springer: Berlin; Bartholomew, W.H., and Reiman, H.B. (1979). Economics of Fermentation Processes, in: Peppler, H.J. and Perlman, D., eds. Microbial Technology 2"d ed., vol. 2, chapter 18, Academic Press: New York; and Kockova-Kratachvilova, A. (1981) Characteristics of Industrial Microorganisms, in:
Rehm, H.J. and Reed, G., eds. Handbook of Biotechnology, vol. 1, chapter 1, Verlag Chemie: Weinheim.
After uptake, these energy-rich carbon molecules must be processed such that they are able to be degraded by one of the major sugar metabolic pathways.
Such pathways lead directly to useful degradation products, such as ribose-5-phosphate and phosphoenolpyruvate, which may be subsequently converted to pyruvate. Three of the most important pathways in bacteria for sugar metabolism include the Embden-Meyerhoff-Pamas (EMP) pathway (also known as the glycolytic or fructose bisphosphate pathway), the hexosemonophosphate (HMP) pathway (also known as the pentose shunt or pentose phosphate pathway), and the Entner-Doudoroff (ED) pathway (for review, see Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York, and Stryer, L. (1988) Biochemistry, Chapters 13-19, Freeman: New York, and references therein).
The EMP pathway converts hexose molecules to pyruvate, and in the process produces 2 molecules of ATP and 2 molecules of NADH. Starting with glucose-l-phosphate (which may be either directly taken up from the medium, or altematively may be generated from glycogen, starch, or cellulose), the glucose molecule is isomerized to fructose-6-phosphate, is phosphorylated, and split into two 3-carbon molecules of glyceraldehyde-3-phosphate. After dehydrogenation, phosphorylation, and successive rearrangements, pyruvate results.
The HMP pathway converts glucose to reducing equivalents, such as NADPH, and produces pentose and tetrose compounds which are necessary as intenmediates and precursors in a number of other metabolic pathways. In the HMP pathway, glucose-6-phosphate is converted to ribulose-5-phosphate by two successive dehydrogenase reactions (which also release two NADPH molecules), and a carboxylation step.
Ribulose-5-phosphate may also be converted to xyulose-5-phosphate and ribose-5-phosphate; the former can undergo a series of biochemical steps to glucose-6-phosphate, which may enter the EMP pathway, while the latter is commonly utilized as an intermediate in other biosynthetic pathways within the cell.
The ED pathway begins with the compound glucose or gluconate, which is subsequently phosphorylated and dehydrated to form 2-dehydro-3-deoxy-6-P-gluconate.
Glucuronate and galacturonate may also be converted to 2-dehydro-3-deoxy-6-P-gluconate through more complex biochemical pathways. This product molecule is subsequently cleaved into glyceraldehyde-3-P and pyruvate; glyceraldehyde-3-P
may itself also be converted to pyruvate.
The EMP and HMP pathways share many features, including intermediates and enzymes. The EMP pathway provides the greatest amount of ATP, but it does not produce ribose-5-phosphate, an important precursor for, e.g., nucleic acid biosynthesis, nor does it produce erythrose-4-phosphate, which is important for amino acid biosynthesis. Microorganisms that are capable of using only the EMP pathway for glucose utilization are thus not able to grow on simple media with glucose as the sole carbon source. They are referred to as fastidious organisms, and their growth requires inputs of complex organic compounds, such as those found in yeast extract.
In contrast, the HMP pathway produces all of the precursors necessary for both nucleic acid and amino acid biosynthesis, yet yields only half the amount of ATP energy that the EMP pathway does. The HMP pathway also produces NADPH, which may be used for redox reactions in biosynthetic pathways. The HMP pathway does not directly produce pyruvate, however, and thus these microorganisms must also possess this portion of the EMP pathway. It is therefore not surprising that a number of microorganisms, particularly the facultative anerobes, have evolved such that they possess both of these pathways.
The ED pathway has thus far has only been found in bacteria. Although this pathway is linked partly to the HMP pathway in the reverse direction for precursor formation, the ED pathway directly forms pyruvate by the aldolase cleavage of ketodeoxy-6-phosphogluconate. The ED pathway can exist on its own and is utilized by the majority of strictly aerobic microorganisms. The net result is similar to that of the HMP pathway, although one mole of ATP can be formed only if the carbon atoms are converted into pyruvate, instead of into precursor molecules.
The pyruvate molecules produced through any of these pathways can be readily converted into energy via the Krebs cycle (also known as the citric acid cycle, the citrate cycle, or the tricarboxylic acid cycle (TCA cycle)). In this process, pyruvate is first decarboxylated, resulting in the production of one molecule of NADH, I
molecule of acetyl-CoA, and I molecule of CO2. The acetyl group of acetyl CoA then reacts with the 4 carbon unit, oxaolacetate, leading to the formation of citric acid, a 6 carbon organic acid. Dehydration and two additional COZ molecules are released.
Ultimately, oxaloacetate is regenerated and can serve again as an acetyl acceptor, thus completing the cycle. The electrons released during the oxidation of intermediates in the TCA cycle are transferred to NAD+ to yield NADH.
During respiration, the electrons from NADH are transferred to molecular oxygen or other terminal electron acceptors. This process is catalyzed by the respiratory chain, an electron transport system containing both integral membrane proteins and membrane associated proteins. This system serves two basic functions: first, to accept electrons from an electron donor and to transfer them to an electron acceptor, and second, to conserve some of the energy released during electron transfer by the synthesis of ATP. Several types of oxidation-reduction enzymes and electron transport proteins are known to be involved in such processes, including the NADH dehydrogenases, flavin-containing electron carriers, iron sulfur proteins, and cytochromes.
The NADH
dehydrogenases are located at the cytoplasmic surface of the plasma membrane, and transfer hydrogen atoms from NADH to flavoproteins, in turn accepting electrons from NADH. The flavoproteins are a group of electron carriers possessing a flavin prosthetic group which is alternately reduced and oxidized as it accepts and transfers electrons.
Three flavins are known to participate in these reactions: riboflavin, flavin-adenine dinucleotide (FAD) and flavin-mononucleotide (FMN). Iron sulfur proteins contain a cluster of iron and sulfur atoms which are not bonded to a heme group, but which still are able to participate in dehydration and rehydration reactions. Succinate dehydrogenase and aconitase are exemplary iron-sulfur proteins; their iron-sulfur complexes serve to accept and transfer electrons as part of the overall electron-transport chain. The cytochromes are proteins containing an iron porphyrin ring (heme).
There are a number of different classes of cytochromes, differing in their reduction potentials.
Functionally, these cytochromes form pathways in which electrons may be transferred to other cytochromes having increasingly more positive reduction potentials. A
further class of non-protein electron carriers is known: the lipid-soluble quinones (e.g., coenzyme Q). These molecules also serve as hydrogen atom acceptors and electron donors.
The action of the respiratory chain generates a proton gradient across the cell membrane, resulting in proton motive force. This force is utilized by the cell to synthesize ATP, via the membrane-spanning enzyme, ATP synthase. This enzyme is a multiprotein complex in which the transport of H+ molecules through the membrane results in the physicaI rotation of the intracellular subunits and concomitant phosphorylation of ADP to form ATP (for review, see Fillingame, R.H. and Divall, S.
(1999) Novartis Found. Symp. 221: 218-229, 229-234).
Non-hexose carbon substrates may also serve as carbon and energy sources for cells. Such substrates may first be converted to hexose sugars in the gluconeogenesis pathway, where glucose is first synthesized by the cell and then is degraded to produce energy. The starting material for this reaction is phosphoenolpyruvate (PEP), which is one of the key intermediates in the glycolytic pathway. PEP may be formed from substrates other than sugars, such as acetic acid, or by decarboxylation of oxaloacetate (itself an intermediate in the TCA cycle). By reversing the glycolytic pathway (utilizing a cascade of enzymes different than those of the original glycolysis pathway), glucose-6-phosphate may be formed. The conversion of pyruvate to glucose requires the utilization of 6 high energy phosphate bonds, whereas glycolysis only produces in the conversion of glucose to pyruvate. However, the complete oxidation of glucose (glycolysis, conversion of pyruvate into acetyl CoA, citric acid cycle, and oxidative phosphorylation) yields between 36-38 ATP, so the net loss of high energy phosphate bonds experienced during gluconeogenesis is offset by the overall greater gain in such high-energy molecules produced by the oxidation of glucose.
III. Elements and Methods of the Invention The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as SMP nucleic acid and protein molecules, which participate in the conversion of sugars to useful degradation products and energy (e.g., ATP) in C. glutamicum or which may participate in the production of useful energy-rich molecules (e.g., ATP) by other processes, such as oxidative phosphorylation.
In one embodiment, the SMP molecules participate in the metabolism of carbon compounds such as sugars or the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. In a preferred embodiment, the activity of the SMP molecules of the present invention to contribute to carbon metabolism or energy production in C. glutamicum has an impact on the production of a desired fine chemical by this organism. In a particularly prefen-ed embodiment, the SMP
molecules of the invention are modulated in activity, such that the C.
glutamicum metabolic and energetic pathways in which the SMP proteins of the invention participate are modulated in yield, production, and/or efficiency of production, which either directly or indirectly modulates the yield, production, and/or efficiency of production of a desired fine chemical by C. glutamicum.
The language, "SMP protein" or "SMP polypeptide" includes proteins which are capable of performing a function involved in the metabolism of carbon compounds such as sugars and the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. Examples of SMP proteins include those encoded by the SMP genes set forth in Table 1 and by the odd-numbered SEQ ID
NOs. The tenns "SMP gene" or "SMP nucleic acid sequence" include nucleic acid sequences encoding an SMP protein, which consist of a coding region and also corresponding untranslated 5' and 3' sequence regions. Examples of SMP genes include those set forth in Table 1. The tenns "production" or "productivity" are art-recognized and include the concentration of the fermentation product (for example, the desired fine chemical) formed within a given time and a given fermentation volume (e.g., kg product per hour per liter). The term "efficiency of production" includes the time required for a.
particular level of production to be achieved (for example, how long it takes for the cell to attain a particular rate of output of a fine chemical). The term "yield" or "product/carbon yield" is art-recognized and includes the efficiency of the conversion of the carbon source into the product (f.e., fine chemical). This is generally written as, for example, kg product per kg carbon source. By increasing the yield or production of the compound, the quantity of recovered molecules, or of useful recovered molecules of that compound in a given amount of culture over a given amount of time is increased. The terms "biosynthesis" or a "biosynthetic pathway" are art-recognized and include the synthesis of a compound, preferably an organic compound, by a cell from intenmediate compounds in what may be a multistep and highly regulated process. The terms "degradation" or a "degradation pathway" are art-recognized and include the breakdown of a compound, preferably an organic compound, by a cell to degradation products (generally speaking, smaller or less complex molecules) in what may be a multistep and highly regulated process. The term "degradation product" is art-recognized and includes breakdown products of a compound. Such products may themselves have utility as precursor (starting point) or intermediate molecules necessary for the biosynthesis of other compounds by the cell. The language "metabolism"
is art-recognized and includes the totality of the biochemical reactions that take place in an organism. The metabolism of a particular compound, then, (e.g., the metabolism of an amino acid such as glycine) comprises the overall biosynthetic, modification, and degradation pathways in the cell related to this compound.
In another embodiment, the SMP molecules of the invention are capable of modulating the production of a desired molecule, such as a fine chemical, in a microorganism such as C. glutamicum. There are a number of mechanisms by which the alteration of an SMP protein of the invention may directly affect the yield, production, and/or efficiency of production of a fine chemical from a C.
glutamicum strain incorporating such an altered protein. The degradation of high-energy carbon molecules such as sugars, and the conversion of compounds such as NADH and to more useful forms via oxidative phosphorylation results in a number of compounds which themselves may be desirable fine chemicals, such as pyruvate, ATP, NADH, and a number of intermediate sugar compounds. Further, the energy molecules (such as ATP) and the reducing equivalents (such as NADH or NADPH) produced by these metabolic pathways are utilized in the cell to drive reactions which would otherwise be energetically unfavorable. Such unfavorable reactions include many biosynthetic pathways for fine chemicals. By improving the ability of the cell to utilize a particular sugar (e.g., by manipulating the genes encoding enzymes involved in the degradation and conversion of that sugar into energy for the cell), one may increase the amount of energy available to pemiit unfavorable, yet desired metabolic reactions (e.g., the biosynthesis of a desired fine chemical) to occur.
The mutagenesis of one or more SMP genes of the invention may also result in SMP proteins having altered activities which indirectly impact the production of one or more desired fine chemicals from C. glutamicum. For example, by increasing the efficiency of utilization of one or more sugars (such that the conversion of the sugar to useful energy molecules is improved), or by increasing the efficiency of conversion of reducing equivalents to useful energy molecules (e.g., by improving the efficiency of oxidative phosphorylation, or the activity of the ATP synthase), one can increase the amount of these high-energy compounds available to the cell to drive normally unfavorable metabolic processes. These processes include the construction of cell walls, transcription, translation, and the biosynthesis of compounds necessary for growth and division of the cells (e.g., nucleotides, amino acids, vitamins, lipids, etc.) (Lengeler et al.
(1999) Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109; 913-918;
875-899).
By improving the growth and multiplication of these engineered cells, it is possible to increase both the viability of the cells in large-scale culture, and also to improve their rate of division, such that a relatively larger number of cells can survive in fennentor culture. The yield, production, or efficiency of production may be increased, at least due to the presence of a greater number of viable cells, each producing the desired fine chemical. Further, a number of the degradation and intermediate compounds produced during sugar metabolism are necessary precursors and intermediates for other biosynthetic pathways throughout the cell. For example, many amino acids are synthesized directly from compounds normally resulting from glycolysis or the TCA
cycle (e.g_, serine is synthesized from 3-phosphoglycerate, an intermediate in glycolysis). Thus, by increasing the efficiency of conversion of sugars to useful energy molecules, it is also possible to increase the amount of useful degradation products as well.
The isolated nucleic acid sequences of the invention are contained within the genome of a Corynebacterium glutamicum strain available through the American Type Culture Collection, given designation ATCC 13032. The nucleotide sequence of the isolated C. glutamicum SMP DNAs and the predicted amino acid sequences of the C.
glutamicum SMP proteins are shown in the Sequence Listing as odd-numbered SEQ
ID
NOs and even-numbered SEQ ID NOs, respectively. Computational analyses were performed which classified and/or identified these nucleotide sequences as sequences which encode proteins having a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum.
The present invention also pertains to proteins which have an amino acid sequence which is substantially homologous to an amino acid sequence of the invention (e.g., the sequence of an even-numbered SEQ ID NO of the Sequence Listing). As used herein, a protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence is least about 50% homologous to the selected amino acid sequence, e.g., the entire selected amino acid sequence. A protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence can also be least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%, 80-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to the selected amino acid sequence.
An SMP protein or a biologically active portion or fragment thereof of the invention can participate in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium gtutamrcum, or can have one or more of the activities set forth in Table 1.
Various aspects of the invention are described in further detail in the following subsections:
A. Isolated Nucleic Acid Molecules One aspect of the invention pertains to isolated nucleic acid molecules that encode SMP polypeptides or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes or primers for the identification or amplification of SMP-encoding nucleic acid (e.g., SMP DNA). As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. This term also encompasses untranslated sequence located at both the 3' and 5' ends of the coding region of the gene: at least about 100 nucleotides of sequence upstream from the 5' end of the coding region and at least about nucleotides of sequence downstream from the 3'end of the coding region of the gene.
The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated SMP nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g, a C. glutamicum cell). Moreover, an "isolated"
nucleic acid molecule, such as a DNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having a nucleotide sequence of an odd-numbered SEQ ID NO of the Sequence Listing, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a C glutamicum SMP DNA can be isolated from a C. glutamicum library using all or portion of one of the odd-numbered SEQ ID NO sequences of the Sequence Listing as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
Moreover, a nucleic acid molecule encompassing all or a portion of one of the nucleic acid sequences of the invention (e.g., an odd-numbered SEQ ID NO:) can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence (e.g., a nucleic acid molecule encompassing all or a portion of one of the nucleic acid sequences of the invention (e.g., an odd-numbered SEQ ID NO of the Sequence Listing) can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this same sequence). For example, mRNA
can be isolated from normal endothelial cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and DNA
can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from GibcoBRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in the Sequence Listing. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to an SMP nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises one of the nucleotide sequences shown in the Sequence Listing. The nucleic acid sequences of the invention, as set forth in the Sequence Listing , correspond to the Corynebacterium gtutamicum SMP DNAs of the invention. This DNA comprises sequences encoding SMP proteins (i.e., the "coding region", indicated in each odd-numbered SEQ ID NO: sequence in the Sequence Listing), as well as 5' untranslated sequences and 3' untranslated sequences, also indicated in each odd-numbered SEQ ID
NO: in the Sequence Listing.. Alternatively, the nucleic acid molecule can comprise only the coding region of any of the sequences in nucleic acid sequences of the Sequence Listing.
For the purposes of this application, it will be understood that each of the nucleic acid and amino acid sequences set forth in the Sequence Listing has an identifying RXA, RXN, or RXS number having the designation "RXA," "RXN," or "RXS" followed by 5 digits (i.e., RXA01626, RXN00043, or RXS0735). Each of the nucleic acid sequences comprises up to three parts: a 5' upstream region, a coding region, and a downstream region. Each of these three regions is identified by the same RXA, RXN, or RXS
designation to eliminate confusion. The recitation "one of the odd-numbered sequences of the Sequence Listing", then, refers to any of the nucleic acid sequences in the Sequence Listing, which may also be distinguished by their differing RXA, RXN, or RXS designations. The coding region of each of these sequences is translated into a corresponding amino acid sequence, which is also set forth in the Sequence Listing, as an even-numbered SEQ ID NO: immediately following the corresponding nucleic acid sequence. For example, the coding region for RXA02735 is set forth in SEQ ID
NO:1, while the amino acid sequence which it encodes is set forth as SEQ ID NO:2.
The sequences of the nucleic acid molecules of the invention are identified by the same RXA, RXN, or RXS designations as the amino acid molecules which they encode, such that they can be readily correlated. For example, the amino acid sequence designated RXA00042 is a translation of the coding region of the nucleotide sequence of nucleic acid molecule RXA00042, and the amino acid sequence designated RXN00043 is a translation of the coding region of the nucleotide sequence of nucleic acid molecule RXN00043. The correspondence between the RXA, RXN and RXS nucleotide and amino acid sequences of the invention and their assigned SEQ ID NOs is set forth in Table 1.
Several of the genes of the invention are "F-designated genes". An F-designated gene includes those genes set forth in Table 1 which have an 'F' in front of the RXAdesignation. For example, SEQ ID NO:11, designated, as indicated on Table 1, as "F RXA01312", is an F-designated gene, as are SEQ ID NOs: 29, 33, and 39 (designated on Table I as "F RXA02803", "F RXA02854", and "F RXA01365", respectively).
In one embodiment, the nucleic acid molecules of the present invention are not intended to include those compiled in Table 2. In the case of the dapD gene, a sequence for this gene was published in Wehrmann, A., et al. (1998) J. Bacteriol.
180(12): 3159-3165. However, the sequence obtained by the inventors of the present application is significantly longer than the published version. It is believed that the published version relied on an incorrect start codon, and thus represents only a fragment of the actual coding region.
In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of one of the nucleotide sequences of the invention (e.g., a sequence of an odd-numbered SEQ
ID
NO: of the Sequence Listing), or a portion thereof. A nucleic acid molecule which is complementary to one of the nucleotide sequences of the invention is one which is sufficiently complementary to one of the nucleotide sequences shown in the Sequence Listing (e.g., the sequence of an odd-numbered SEQ ID NO:) such that it can hybridize to one of the nucleotide sequences of the invention, thereby forming a stable duplex.
In still another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which is at least about 50'/0, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62010, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing), or a portion thereof. Ranges and identity values intermediate to the above-recited ranges, (e.g., 70-90% identical or 80-95% identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended, to be included. In an additional preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one of the nucleotide sequences of the invention, or a portion thereof.
Moreover, the nucleic acid molecule of the invention can comprise only a portion of the coding region of the sequence of one of the odd-numbered SEQ ID
NOs of the Sequence Listing, for example a fragment which can be used as a probe or primer - or a fragment encoding a biologically active portion of an SMP protein. The nucleotide sequences determined from the cloning of the SMP genes from C. glutamicum allows for the generation of probes and primers designed for use in identifying and/or cloning SMP homologues in other cell types and organisms, as well as SMP homologues from other Corynebacteria or related species. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand of one of the nucleotide sequences of the invention (e.g., a sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing), an anti-sense sequence of one of these sequences, or naturally occurring mutants thereof. Primers based on a nucleotide sequence of the invention can be used in PCR reactions to clone SMP
homologues. Probes based on the SMP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g.
the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells which misexpress an SMP protein, such as by measuring a level of an SMP-encoding nucleic acid in a sample of cells, e.g., detecting SMP mRNA levels or determining whether a genomic SMP gene has been mutated or deleted.
In one embodiment, the nucleic acid molecule of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO of the Sequence Listing) such that the protein or portion thereof maintains the ability to perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. As used herein, the language "sufficiently homologous" refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain as an amino acid residue in a sequence of one of the even-numbered SEQ ID NOs of the Sequence Listing) amino acid residues to an amino acid sequence of the invention such that the protein or portion thereof is able to perfonn a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum.
Protein members of such sugar metabolic pathways or energy producing systems, as described herein, may play a role in the production and secretion of one or more fine chemicals. Examples of such activities are also described herein. Thus, "the function of an SMP protein" contributes either directly or indirectly to the yield, production, and/or efficiency of production of one or more fine chemicals. Examples of SMP
protein activities are set forth in Table 1.
In another embodiment, the protein is at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%, 80-90%, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to an entire amino acid sequence of the invention(e.g., a sequence of an even-numbered SEQ ID NO:
of the Sequence Listing).
Portions of proteins encoded by the SMP nucleic acid molecules of the invention are preferably biologically active portions of one of the SMP proteins. As used herein, the term "biologically active portion of an SMP protein" is intended to include a portion, e.g., a domain/motif, of an SMP protein that participates in the metabolism of carbon compounds such as sugars, or in energy-generating pathways in C glutamicum, or has an activity as set forth in Table 1. To determine whether an SMP protein or a biologically active portion thereof can participate in the metabolism of carbon compounds or in the production of energy-rich molecules in C. glutamicum, an assay of enzymatic activity may be performed. Such assay methods are well known to those of ordinary slcili in the art, as detailed in Example 8 of the Exemplification.
Additional nucleic acid fragments encoding biologically active portions of an SMP protein can be prepared by isolating a portion of one of the amino acid sequences of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing), expressing the encoded portion of the SMP protein or peptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the SMP protein or peptide.
The invention further encompasses nucleic acid molecules that differ from one of the nucleotide sequences of the invention (e.g., a sequence of an odd-numbered SEQ ID
NO: of the Sequence Listing) (and portions thereof) due to degeneracy of the genetic code and thus encode the same SMP protein as that encoded by the nucleotide sequences of the invention. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in the Sequence Listing (e.g., an even-numbered SEQ ID NO:). In a still further embodiment, the nucleic acid molecule of the invention encodes a full length C.
glutamicum protein which is substantially homologous to an amino acid of the invention (encoded by an open reading frame shown in an odd-numbered SEQ ID NO: of the Sequence Listing).
It will be understood by one of ordinary skill in the art that in one embodiment the sequences of the invention are not meant to include the sequences of the prior art, such as those Genbank sequences set forth in Tables 2 or 4 which were available prior to the present invention. In one embodiment, the invention includes nucleotide and amino acid sequences having a percent identity to a nucleotide or amino acid sequence of the invention which is greater than that of a sequence of the prior art (e.g., a Genbank sequence (or the protein encoded by such a sequence) set forth in Tables 2 or 4). For example, the invention includes a nucleotide sequence which is greater than and/or at least 58% identical to the nucleotide sequence designated RXA00014 (SEQ ID
NO:41), a nucleotide sequence which is greater than and/or at least % identical to the nucleotide sequence designated RXA00195 (SEQ ID NO:399), and a nucleotide sequence which is greater than and/or at least 42% identical to the nucleotide sequence designated RXA00196 (SEQ ID NO:401). One of ordinary skill in the art would be able to calculate the lower threshold of percent identity for any given sequence of the invention by examining the GAP-calculated percent identity scores set forth in Table 4 for each of the three top hits for the given sequence, and by subtracting the highest GAP-calculated percent identity from 100 percent. One of ordinary skill in the art will also appreciate that nucleic acid and amino acid sequences having percent identities greater than the lower threshold so calculated (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more identical) are also encompassed by the invention.
In addition to the C. glutamicum SMP nucleotide sequences set forth in the Sequence Listing as odd-numbered SEQ ID NOs, it will be appreciated by those of ordinary skill in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of SMP proteins may exist within a population (e.g., the C.
glutamicum population). Such genetic polymorphism in the SMP gene may exist among individuals within a population due to natural variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding an SMP protein, preferably a C. glutamicum SMP protein.
Such natural variations can typically result in 1-5% variance in the nucleotide sequence of the SMP gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in SMP that are the result of natural variation and that do not alter the functional activity of SMP proteins are intended to be within the scope of the invention.
Nucleic acid molecules corresponding to natural variants and non-C. glutamicum homologues of the C. glutamicum SMP DNA of the invention can be isolated based on their homology to the C. glutamicum SMP nucleic acid disclosed herein using the C.
glutamicum DNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of of an odd-numbered SEQ ID NO: of the Sequence Listing. In other embodiments, the nucleic acid is at least 30, 50, 100, 250 or more nucleotides in length. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those of ordinary skill in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65 C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a nucleotide sequence of the invention corresponds to a naturally-occurring nucleic acid molecule. As used herein, a"naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). In one embodiment, the nucleic acid encodes a natural C.
glutamicum SMP protein.
In addition to naturally-occurring variants of the SMP sequence that may exist in the population, one of ordinary skill in the art will further appreciate that changes can be introduced by mutation into a nucleotide sequence of the invention, thereby leading to changes in the amino acid sequence of the encoded SMP protein, without altering the functional ability of the SMP protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in a nucleotide sequence of the invention. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of one of the SMP proteins (e.g., an even-numbered SEQ ID NO: of the Sequence Listing) without altering the activity of said SMP protein, whereas an "essential" amino acid residue is required for SMP protein activity. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved in the domain having SMP activity) may not be essential for activity and thus are likely to be amenable to alteration without altering SMP activity.
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding SMP proteins that contain changes in amino acid residues that are not essential for SMP activity. Such SMP proteins differ in amino acid sequence from a sequence of an even-numbered SEQ ID NO: of the Sequence Listing yet retain at least one of the SMP activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50% homologous to an amino acid sequence of the invention and is capable of participate in the metabolism of carbon compounds such as sugars, or in the biosynthesis of high-energy compounds in C. glutamicum, or has one or more activities set forth in Table 1. Preferably, the protein encoded by the nucleic acid molecule is at least about 50-60% homologous to the amino acid sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing, more preferably at least about 60-70% homologous to one of these sequences, even more preferably at least about 80%, 80-90%, 90-95% homologous to one of these sequences, and most preferably at least about 96%, 97%, 98%, or 99% homologous to one of the amino acid sequences of the invention.
To determine the percent homology of two amino acid sequences (e.g., one of the amino acid sequences of the invention and a mutant form thereof) or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence (e.g., one of the amino acid sequences the invention) is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence (e.g., a mutant fon=n of the amino acid sequence), then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions/total # of positions x 100).
An isolated nucleic acid molecule encoding an SMP protein homologous to a protein sequence of the invention (e.g., a sequence of an even-numbered SEQ ID
NO: of the Sequence Listing) can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of the invention such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into one of the nucleotide sequences of the invention by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., al.anine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an SMP protein is preferably replaced with another amino acid residue from the same side chain family. Altematively, in another embodiment, mutations can be introduced randomly along all or part of an SMP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an SMP activity described herein to identify mutants that retain SMP activity. Following mutagenesis of the nucleotide sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing, the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, assays described herein (see Example 8 of the Exemplification).
In addition to the nucleic acid molecules encoding SMP proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded DNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire SMP coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an SMP protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the entire coding region of NO.
3(RXA01626) comprises nucleotides I to 345). In another embodiment, the antisense nucleic acid molecule is antisense to a"noncoding region" of the coding strand of a nucleotide sequence encoding SMP. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not transtated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding SMP disclosed herein (e.g., the sequences set forth as odd-numbered SEQ ID NOs in the Sequence Listing), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of SMP mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of SMP mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of SMP mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA
and/or genomic DNA encoding an SMP protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to fonn a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucteic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ~-units, the strands run parallel to each other (Gaultier e1 al. (1987) Nucleic Acids.
Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et aL (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave SMP mRNA transcripts to thereby inhibit translation of SMP
mRNA. A ribozyme having specificity for an SMP-encoding nucleic acid can be designed based upon the nucleotide sequence of an SMP cDNA disclosed herein (i.e., SEQ ID NO. 3(RXA01626)). For example, a derivative of a Teirahymena L-19 IVS
RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an SMP-encoding mRNA.
See, e.g., Cech et al. U.S. Patent No. 4,987,071 and Cech et al. U.S. Patent No.
5,116,742. Alternatively, SMP mRNA can be used to select a catalytic RNA
having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.
Altematively, SMP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of an SMP nucleotide sequence (e.g., an SMP promoter and/or enhancers) to form triple helical structures that prevent transcription of an SMP gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N. Y. Acad.
Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14(12):807-15.
B. Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an SMP protein (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA
techniques are often in the fonn.n of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.
However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells.
Preferred regulatory sequences are, for example, promoters such as cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, Ipp-lac-, IacIq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, amy, SPO2, X-PR-or X PL, which are used preferably in bacteria. Additional regulatory sequences are, for example, promoters from yeasts and fungi, such as ADC 1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4, usp, STLS 1, B33, nos or ubiquitin- or phaseolin-promoters. It is also possible to use artificial promoters. It will be appreciated by those of ordinary skill in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., SMP proteins, mutant forms of SMP proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of SMP proteins in prokaryotic or eukaryotic cells. For example, SMP genes can be expressed in bacterial cells such as C. glutamicum, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos, M.A. et al.
(1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488; van den Hondel, C.A.M.J.J. et al. (1991) "Heterologous gene expression in filamentous fungi"
in: More Gene Manipulations in Fungi, J.W. Bennet & L.L. Lasure, eds., p. 396-428:
Academic Press: San Diego; and van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds., p. 1-28, Cambridge University Press:
Cambridge), algae and multicellular plant cells (see Schmidt, R. and Willmitzer, L. (1988) High efficiency Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana leaf and cotyledon explants" Plant Cell Rep: 583-586), or mammalian cells.
Suitable host cells are discussed further in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein but also to the C-terminus or fused within suitable regions in the proteins. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase.
Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence of the SMP protein is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X protein. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin.
Recombinant SMP protein unfused to GST can be recovered by cleavage of the fusion protein with thrombin.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315), pLG338, pACYC184, pBR322, pUC18, pUC 19, pKC30, pRep4, pHS 1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-I1I113-B1, kgt11, pBdCl, and pET I id (Studier et al., Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Califonva (1990) 60-89;
and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).
Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET I ld vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident k prophage harboring a gn 1 gene under the transcriptional control of the lacUV 5 promoter. For transformation of other varieties of bacteria, appropriate vectors may be selected. For example, the plasmids pIJ101, pIJ364, pIJ702 and pIJ361 are known to be useful in transforming Streptomyces, while plasmids pUB 110, pC 194, or pBD214 are suited for transformation of Bacillus species. Several plasmids of use in the transfer of genetic information into Corynebacterium include pHM1519, pBL1, pSA77, or pAJ667 (Pouwels et al., eds.
(1985) Cloning Vectors. Elsevier: New York IBSN 0 444 90401 8)_ One strategy to maximize recombinant protein expression is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Califomia (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression, such as C.
glutamicum (Wada et aL (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the SMP protein expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari, et al., (1987) EmboJ. 6:229-234), 2 , pAG-1, Yep6, Yep13, pEMBLYe23, pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, include those detailed in: van den Hondel, C.A.M.J.J. &
Punt, P.J.
(1991) "Gene transfer systems and vector development for filamentous fungi, in:
Applied Molecular Genetics of Fungi, J.F. Peberdy, et al., eds., p. 1-28, Cambridge University Press: Cambridge, and Pouwels et al., eds. (1985) Cloning Vectors.
Elsevier:
New York (IBSN 0 444 904018).
Alternatively, the SMP proteins of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.
(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
In another embodiment, the SMP proteins of the invention may be expressed in unicellular plant cells (such as algae) or in plant cells from higher plants (e.g., the spermatophytes, such as crop plants). Examples of plant expression vectors include those detailed in: Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acid. Res. 12: 8711-8721, and include pLGV23, pGHlac+, pBIN19, pAK2004, and pDH51 (Pouwels et al., eds. (1985) Cloning Vectors.
Elsevier:
New York IBSN 0 444 904018). .
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC
(Kaufman et al. (1987) EMBOJ. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific;
Pinkert et al.
(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBOJ. 8:729-733) and immunoglobulins (Banerji et al, (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to SMP mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be deterrnined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. l(1) 1986.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. It is understood that such tenns refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, an SMP
protein can be expressed in bacterial cells such as C. glutamicum, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
Other suitable host cells are known to one of ordinary skill in the art.
Microorganisms related to Corynebacterium glutamicum which may be conveniently used as host cells for the nucleic acid and protein molecules of the invention are set forth in Table 3.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection", "conjugation" and "transduction" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., linear DNA or RNA (e.g., a linearized vector or a gene construct alone without a vector) or nucleic acid in the form of a vector (e.g., a plasmid, phage, phasmid, phagemid, transposon or other DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al.
(Molecular Cloning: A Laboratory ManuaL 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
Preferred selectable markers include those which confer resistance to drugs, such as G41 8, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an SMP protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by, for example, drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
To create a homologous recombinant microorganism, a vector is prepared which contains at least a portion of an SMP gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the SMP
gene.
Preferably, this SMP gene is a Corynebacterium glutamicum SMP gene, but it can be a homologue from a related bacterium or even from a mammalian, yeast, or insect source.
In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous SMP gene is functionally disrupted (I.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
Altematively, the vector can be designed such that, upon homologous recombination, the endogenous SMP gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous SMP protein). In the homologous recombination vector, the altered portion of the SMP gene is flanked at its 5' and 3' ends by additional nucleic acid of the SMP
gene to allow for homologous recombination to occur between the exogenous SMP
gene carried by the vector and an endogenous SMP gene in a microorganism. The additional flanking SMP nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA
(both at the 5' and 3' ends) are included in the vector (see e.g., Thomas, K.R., and Capecchi, M.R. (1987) Cell 51: 503 for a description of homologous recombination vectors). The vector is introduced into a microorganism (e.g., by electroporation) and cells in which the introduced SMP gene has homologously recombined with the endogenous SMP gene are selected, using art-known techniques.
In another embodiment, recombinant microorganisms can be produced which contain selected systems which allow for regulated expression of the introduced gene.
For example, inclusion of an SMP gene on a vector placing it under control of the lac operon permits expression of the SMP gene only in the presence of IPTG. Such regulatory systems are well known in the art.
In another embodiment, an endogenous SMP gene in a host cell is disrupted (e.g., by homologous recombination or other genetic means known in the art) such that expression of its protein product does not occur. In another embodiment, an endogenous or introduced SMP gene in a host cell has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional SMP protein. In still another embodiment, one or more of the regulatory regions (e.g., a promoter, repressor, or inducer) of an SMP gene in a microorganism has been altered (e.g., by deletion, truncation, inversion, or point mutation) such that the expression of the SMP
gene is modulated. One of ordinary skill in the art will appreciate that host cells containing more than one of the described SMP gene and protein modifications may be readily produced using the methods of the invention, and are meant to be included in the present invention.
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) an SMP protein. Accordingly, the invention further provides methods for producing SMP proteins using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding an SMP protein has been introduced, or into which genome has been introduced a gene encoding a wild-type or altered SMP protein) in a suitable medium until SMP protein is produced. In another embodiment, the method further comprises isolating SMP proteins from the medium or the host cell.
C. Isolated SMP Proteins Another aspect of the invention pertains to isolated SMP proteins, and biologically active portions thereof. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material"
includes preparations of SMP protein in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of SMP protein having less than about 30% (by dry weight) of non-SMP protein (also referred to herein as a"contaminating protein"), more preferably less than about 20% of non-SMP
protein, still more preferably less than about 10% of non-SMP protein, and most preferably less than about 5% non-SMP protein. When the SMP protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The language "substantially free of chemical precursors or other chemicals" includes preparations of SMP protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of SMP protein having less than about 30% (by dry weight) of chemical precursors or non-SMP chemicals, more preferably less than about 20% chemical precursors or non-SMP chemicals, still more preferably less than about 10% chemical precursors or non-SMP chemicals, and most preferably less than about 5% chemical precursors or non-SMP chemicals. In preferred embodiments, isolated proteins or biologically active portions thereof lack contaminating proteins from the same organism from which the SMP protein is derived. Typically, such proteins are produced by recombinant expression of, for example, a C. glutamicum SMP
protein in a microorganism such as C. glutamicum.
An isolated SMP protein or a portion thereof of the invention can participate in the metabolism of carbon compounds such as sugars, or in the production of energy compounds (e.g., by oxidative phosphorylation) utilized to drive unfavorable metabolic pathways, or has one or more of the activities set forth in Table 1. In preferred embodiments, the protein or portion thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) such that the protein or portion thereof maintains the ability to perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. The portion of the protein is preferably a biologically active portion as described herein. In another preferred embodiment, an SMP protein of the invention has an amino acid sequence set forth as an even-numbered SEQ ID NO: of the Sequence Listing. In yet another preferred embodiment, the SMP protein has an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of the invention (e.g., a sequence of an odd-numbered SEQ
ID NO:
of the Sequence Listing). In still another preferred embodiment, the SMP
protein has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to one of the nucleic acid sequences of the invention, or a portion thereof. Ranges and identity values intermediate to the above-recited values, (e.g., 70-90% identical or 80-95%
identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. The preferred SMP proteins of the present invention also preferably possess at least one of the SMP activities described herein. For example, a preferred SMP protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of the invention, and which can perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum, or which has one or more of the activities set forth in Table 1.
In other embodiments, the SMP protein is substantially homologous to an amino acid sequence of of the invention (e.g., a sequence of an even-numbered SEQ ID
NO: of the Sequence Listing)and retains the functional activity of the protein of one of the amino acid sequences of the invention yet differs in amino acid sequence due to natural variation or mutagenesis, as described in detail in subsection I above.
Accordingly, in another embodiment, the SMP protein is a protein which comprises an amino acid sequence which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to an entire amino acid sequence of the invention and which has at least one of the SMP activities described herein. Ranges and identity values intermediate to the above-recited values, (e.g., 70-90% identical or 80-95%
identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. In another embodiment, the invention pertains to a full length C. glutamrcum protein which is substantially homologous to an entire amino acid sequenoe of the invention.
Biologically active portions of an SMP protein include peptides comprising amino acid sequences derived from the amino acid sequence of an SMP protein, e.g., an amino acid sequence of an even-numbered SEQ ID NO: of the Sequence Listing or the amino acid sequence of a protein homologous to an SMP protein, which include fewer amino acids than a full length SMP protein or the full length protein which is homologous to an SMP protein, and exhibit at least one activity of an SMP
protein.
Typically, biologically active portions (peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif with at least one activity of an SMP protein. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein. Preferably, the biologically active portions of an SMP
protein include one or more selected domains/motifs or portions thereof having biological activity.
SMP proteins are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the SMP protein is expressed in the host cell. The SMP
protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Altemative to recombinant expression, an SMP
protein, polypeptide, or peptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, native SMP protein can be isolated from cells (e.g., endothelial cells), for example using an anti-SMP antibody, which can be produced by standard techniques utilizing an SMP protein or fragment thereof of this invention.
The invention also provides SMP chimeric or fusion proteins. As used herein, an SMP "chimeric protein" or "fusion protein" comprises an SMP polypeptide operatively linked to a non-SMP polypeptide. An "SMP polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an SMP protein, whereas a "non-SMP
polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the SMP protein, e.g., a protein which is different from the SMP protein and which is derived from the same or a different organism. Within the fusion protein, the term "operatively linked" is intended to indicate that the SMP polypeptide and the non-SMP polypeptide are fused in-frame to each other. The non-SMP polypeptide can be fused to the N-terminus or C-terminus of the SMP polypeptide. For example, in one embodiment the fusion protein is a GST-SMP fusion protein in which the SMP sequences are fused to the C-terminus of the GST
sequences. Such fusion proteins can facilitate the purification of recombinant SMP
proteins. In another embodiment, the fusion protein is an SMP protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of an SMP protein can be increased through use of a heterologous signal sequence.
Preferably, an SMP chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA
synthesizers.
Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST
polypeptide).
An SMP-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the SMP protein.
Homologues of the SMP protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the SMP protein. As used herein, the term "homologue"
refers to a variant form of the SMP protein which acts as an agonist or antagonist of the activity of the SMP protein. An agonist of the SMP protein can retain substantially the same, or a subset, of the biological activities of the SMP protein. An antagonist of the SMP protein can inhibit one or more of the activities of the naturally occurring form of the SMP protein, by, for example, competitively binding to a downstream or upstream member of the sugar molecule metabolic cascade or the energy-producing pathway which includes the SMP protein.
In an alternative embodiment, homologues of the SMP protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the SMP
protein for SMP protein agonist or antagonist activity. In one embodiment, a variegated library of SMP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of SMP
variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential SMP
sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of SMP sequences therein.
There are a variety of methods which can be used to produce libraries of potential SMP
homologues from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential SMP sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3;
Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
In addition, libraries of fragments of the SMP protein coding can be used to generate a variegated population of SMP fragments for screening and subsequent selection of homologues of an SMP protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an SMP coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the SMP protein.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA
libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of SMP
homologues. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify SMP homologues (Arkin and Yourvan (1992) PNAS
89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
In another embodiment, cell based assays can be exploited to analyze a variegated SMP library, using methods well known in the art.
D. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologues, fusion proteins, primers, vectors, and host cells described herein can be used in one or more of the following methods: identification of C. glutamicum and related organisms;
mapping of genomes of organisms related to C. glutamicum; identification and localization of C.
glutamicum sequences of interest; evolutionary studies; determination of SMP
protein regions required for function; modulation of an SMP protein activity;
modulation of the metabolism of one or more sugars; modulation of high-energy molecule production in a cell (i.e., ATP, NADPH); and modulation of cellular production of a desired compound, such as a fine chemical.
The SMP nucleic acid molecules of the invention have a variety of uses. First, they may be used to identify an organism as being Corynebacterium glutamicum or a close relative thereof. Also, they may be used to identify the presence of C.
glutamicum or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of C. glutamicum genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present.
Although Corynebacterium glutamicum itself is nonpathogenic, it is related to pathogenic species, such as Corynebacterium diphtheriae. Corynebacterium diphtheriae is the causative agent of diphtheria, a rapidly developing, acute, febrile infection which involves both local and systemic pathology. In this disease, a local lesion develops in the upper respiratory tract and involves necrotic injury to epithelial cells;
the bacilli secrete toxin which is disseminated through this lesion to distal susceptible tissues of the body. Degenerative changes brought about by the inhibition of protein synthesis in these tissues, which include heart, muscle, peripheral nerves, adrenals, kidneys, liver and spleen, result in the systemic pathology of the disease. Diphtheria continues to have high incidence in many parts of the world, including Africa, Asia, Eastern Europe and the independent states of the former Soviet Union. An ongoing epidemic of diphtheria in the latter two regions has resulted in at least 5,000 deaths since 1990.
In one embodiment, the invention provides a method of identifying the presence or activity of Cornyebacterium diphtheriae in a subject. This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention (e.g., the sequences set forth as odd-numbered or even-numbered SEQ ID NOs, respectively, in the Sequence Listing) in a subject, thereby detecting the presence or activity of Corynebacterium diphiheriae in the subject. C. glutamicum and C. diphtheriae are related bacteria, and many of the nucleic acid and protein molecules in C.
glutamicum are homologous to C. diphtheriae nucleic acid and protein molecules, and can therefore be used to detect C. diphtheriae in a subject.
The nucleic acid and protein molecules of the invention may also serve as markers for specific regions of the genome. This has utility not only in the mapping of the genome, but also for functional studies of C. glutamicum proteins. For example, to identify the region of the genome to which a particular C. glutamicum DNA-binding protein binds, the C. glutarnicum genome could be digested, and the fragments incubated with the DNA-binding protein. Those which bind the protein may be additionally probed with the nucleic acid molecules of the invention, preferably with readily detectable labels; binding of such a nucleic acid molecule to the genome fragment enables the localization of the fragment to the genome map of C. glutamicum, and, when performed multiple times with different enzymes, facilitates a rapid determination of the nucleic acid sequence to which the protein binds. Further, the nucleic acid molecules of the invention may be sufficiently homologous to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related bacteria, such as Brevibacterium lactofermentum.
The SMP nucleic acid molecules of the invention are also useful for evolutionary and protein structural studies. The metabolic and energy-releasing processes in which the molecules of the invention participate are utilized by a wide variety of prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the evolutionary relatedness of the organisms can be assessed. Similarly, such a comparison permits an assessment of which regions of the sequence are conserved and which are not, which may aid in determining those regions of the protein which are essential for the functioning of the enzyme. This type of determination is of value for protein engineering studies and may give an indication of what the protein can tolerate in terms of mutagenesis without losing function.
Manipulation of the SMP nucleic acid molecules of the invention may result in the production of SMP proteins having functional differences from the wild-type SMP
proteins. These proteins may be improved in efficiency or activity, may be present in greater numbers in the cell than is usual, or may be decreased in efficiency or activity.
The invention provides methods for screening molecules which modulate the activity of an SMP protein, either by interacting with the protein itself or a substrate or binding partner of the SMP protein, or by modulating the transcription or translation of an SMP nucleic acid molecule of the invention. In such methods, a microorganism expressing one or more SMP proteins of the invention is contacted with one or more test compounds, and the effect of each test compound on the activity or level of expression of the SMP protein is assessed.
There are a number of mechanisms by which the alteration of an SMP protein of the invention may directly affect the yield, production, and/or efficiency of production of a fine chemical from a C. glutamicum strain incorporating such an altered protein.
The degradation of high-energy carbon molecules such as sugars, and the conversion of compounds such as NADH and FADH2 to more useful fon;ns via oxidative phosphorylation results in a number of compounds which themselves may be desirable fine chemicals, such as pyruvate, ATP, NADH, and a number of intermediate sugar compounds. Further, the energy molecules (such as ATP) and the reducing equivalents (such as NADH or NADPH) produced by these metabolic pathways are utilized in the cell to drive reactions which would otherwise be energetically unfavorable.
Such unfavorable reactions include many biosynthetic pathways for fine chemicals.
By improving the ability of the cell to utilize a particular sugar (e,g., by manipulating the genes encoding enzymes involved in the degradation and conversion of that sugar into energy for the cell), one may increase the amount of energy available to permit unfavorable, yet desired metabolic reactions (e.g., the biosynthesis of a desired fine chemical) to occur.
Further, modulation of one or more pathways involved in sugar utilization permits optimization of the conversion of the energy contained within the sugar molecule to the production of one or more desired fine chemicals. For example, by reducing the activity of enzymes involved in, for example, gluconeogenesis, more ATP
is available to drive desired biochemical reactions (such as fine chemical biosyntheses) in the cell. Also, the overall production of energy molecules from sugars may be modulated to ensure that the cell maximizes its energy production from each sugar molecule. Inefficient sugar utilization can lead to excess COz production and excess energy, which may result in futile metabolic cycles. By improving the metabolism of sugar molecules, the cell should be able to function more efficiently, with a need for fewer carbon molecules. This should result in an improved fine chemical product: sugar molecule ratio (improved carbon yield), and permits a decrease in the amount of sugars that must be added to the medium in large-scale fermentor culture of such engineered C.
glutamicum.
The mutagenesis of one or more SMP genes of the invention may also result in SMP proteins having altered activities which indirectly impact the production of one or more desired fine chemicals from C. glutamicum. For example, by increasing the efficiency of utilization of one or more sugars (such that the conversion of the sugar to useful energy molecules is improved), or by increasing the efficiency of conversion of reducing equivalents to useful energy molecules (e.g., by improving the efficiency of oxidative phosphorylation, or the activity of the ATP synthase), one can increase the amount of these high-energy compounds available to the cell to drive normally unfavorable metabolic processes. These processes include the construction of cell walls, transcription, translation, and the biosynthesis of compounds necessary for growth and division of the cells (e.g., nucleotides, amino acids, vitamins, lipids, etc.) (Lengeler et al.
(1999) Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109; 913-918;
875-899).
By improving the growth and multiplication of these engineered cells, it is possible to increase both the viability of the cells in large-scale culture, and also to improve their rate of division, such that a relatively larger number of cells can survive in fermentor culture. The yield, production, or efficiency of production may be increased, at least -5g-due to the presence of a greater number of viable cells, each producing the desired fine chemical.
Further, many of the degradation products produced during sugar metabolism are themselves utilized by the cell as precursors or intermediates for the production of a number of other useful compounds, some of which are fine chemicals. For example, pyruvate is converted into the amino acid alanine, and ribose-5-phosphate is an integral part of, for example, nucleotide molecules. The amount and efficiency of sugar metabolism, then, has a profound effect on the availability of these degradation products in the cell. By increasing the ability of the cell to process sugars, either in terms of efficiency of existing pathways (e.g., by engineering enzymes involved in these pathways such that they are optimized in activity), or by increasing the availability of the enzymes involved in such pathways (e.g., by increasing the number of these enzymes present in the cell), it is possible to also increase the availability of these degradation products in the cell, which should in turn increase the production of many different other desirable compounds in the cell (e.g., fine chemicals).
The aforementioned mutagenesis strategies for SMP proteins to result in increased yields of a fine chemical from C. glutamicum are not meant to be limiting;
variations on these strategies will be readily apparent to one of ordinary skill in the art.
Using such strategies, and incorporating the mechanisms disclosed herein, the nucleic acid and protein molecules of the invention may be utilized to generate C.
glutamicum or related strains of bacteria expressing mutated SMP nucleic acid and protein molecules such that the yield, production, and/or efficiency of production of a desired compound is improved. This 'desired compound may be any product produced by C. glutamicum, which includes the final products of biosynthesis pathways and intermediates of naturally-occurring metabolic pathways, as well as molecules which do not naturally occur in the metabolism of C. glutamicum, but which are produced by a C.
glutamicum strain of the invention.
This invention is further illustrated by the following examples which should not be construed as limiting.
uj a w w a w M
z j a 0 Wz _ i~~ z m m QF- N ~ z >
N N Zo W z 1u tu ~'N' ~
~ ri F- F- Z~ a U g h ~ N
~u O O O
z ww p p a a a g O N W >~] > O~ O_~ N N N N N N
~ LL I~L S~~.- ~ N fV N N fV N
R = Y V ~-W W W W W~ U U ui vi n tj ~j V ~ a U) < y W W W W
J W W W W W
a v a N W W Ill W W W
a o~~~+ ad' R ~F a a a a a U a 8l, W ~ =ya y S~~aW~ ~
v0 0 J U 0 0 w w ww~0~ W J
t~i .,L Q W W W ~ W == J ~NJ J J
_ ~Ng 8 8 ~a ~ ~N
I y ~ J N I U I U T U U Q~ OI 0 a Y d= d f a V a~ a V
Z ll m J~~ U. N~(n.-fnlALLil tL vQ Wa Wa Wa w W
~,wD N < p O n n LLJ Z
~; W~~ ~ W f0 V(~pn~ONO F~ ~~ ~ g M
MJ NNN
r fp =
Q C
.gl 1D > N O p ~
V ~p ep J"_ y C /~0 y~ t0 t~h! ~ ~I N N CO
Z ~ ~ ~I n ~ N r, N f0 Y1 ' = N N n = h ~ f 0 h.-N
N ~ M~ N M pi t,j N Of ac" ce ~~c~~ c ~
O N _ C M
alll~~~ N 10 1n U_1 N ptf~ p ,}i ~ N~ = O M M M M~ N l7 Ys ~ O M ry (np N
b 0000 ~
zig Z ~
gW
N N= 10 ID Q VI ~ ~ .=- ~~ N N ~ C O ~p p ~ fA N N N l'7 M ~y th Z fn ~- M1An ~"" z vv~~N LLI 9 NN N p~ ~y N M tf ~
y r'= U~U rv~i, ~ ~ ~QN ~~=~-~=~-.~-~
~ W=W NNN4~Y
N [V N fV N N N fV NN
O YMf ~ eM- UUUUC~ U UC~U p.C
x ' !~
V < ~ 1yw 4 a.
y y W iTJlrl~~~~~ Z
00 nn O 0 ~a.. ~ ..v (i~~~ W~=-~W
a 4 NNfVN=-=W~C~Qc%1 ~
~ O~ V a ~ tn 1f/ W y1 S YYI ~S1 N W
N N N g ~ ~ .~r.
v N
t7_y~ 3OQQg~'- =-~-fV N
VVU U
.' WW~jnj wFdFW U UUUQUUUiIgo WW
a- ~n yf/~V~ ~~g~~ WWWWWWW~7~y m ~G~O
y~ S S g}} W W WW W W W W W W U U VW
99a<GQWW WW~WU
yhr~555WWhF- y~ ~~.-yyyyyNyyyyyy W W_ W W W I~~y 222 WWWWW,~j q F~q<WWW ~igy4 ~Y NNNNrqFqpWW WW(~(WWW(~(W~ ~ yyyyV~
~ d aaMS-1-ts-[~~,~~~yW~c3~a~LF~_WW_W_W~~O~~OOOQOOO~y~~~ye~jq~gS~~S}~
U U 0O_ww-t)Q ~~y}OOd9~<VjyWWy}Y~0~0~~}GG~O_O>pmW
}}W~! <Qa }
u=i 202 d ' ~~}>=}}OOCya u~}ra22z J J 6565 Zt~
W W W W W W q q v q Q
-~ J J J LL ~ y UY-Y-Y-Y-O Qd d ~~ ~tlW2ZdOdO0000 2 UU~ yU~U1L~}
p Wk66 ~!SN!yO t~S=TUSS~~~~~ oW
.~ a v a v, Q a a a a y y y ~ W W W a 0.
y t>>> a a q>>>>>>> a q~ 1~ q q a; W
~ viyunUUy 0y00~ >yy~> 7~~ ~ ~~~ V
~ ~ ~S0JJg~~ga~"'*ttt 'o }ioo rcaaoo~rc~~acx~~~
C LL aWaWt9c9aaaa~6~rLL~~~waa}aa~~aaaaaaaao}aaa(L9 O
Z ~ = aD ~ N N aaD 1f1 1[f (~j N fQ N~ m N N O~~ tyj ~ t~f YMi N 1[i ONi Nry a0 N- ~ ~ O MI=~-h . '~f ~ 1ff N ~~Qj 1ri ~Oa l7 NN WfV V 1n~.~~A {p.-m 1Q yl ~D tf, Cf .- N ~fJ h b N N~ I~fl ~~~ 01 ~ O~ _ ~ n 1~ N O ~ff ~ f~ O tD Oi sp Co .-tp Y M N t~l A
p~yf ~p ~ r4 m=-=-N.-fOQO N~ N 4DYINNN~ fN.NNYNf ~NiDM.-~'f ~N Y ~+-dDaMDN N
M M~ggg~ 1~ sgpgogo-l y Q~ O1 f~ ~h 1~ h O!
U
a ~ ~ ~ m N O~y N i7 A~ Nc~ N ~Ny N~ N h~ M h ~ff ~~ aQ ~~ M O~! N~~~ ~ " O
U.
~ Z ~ 01+0f xOe,x~7'{-N?~Zx_ ~RLL
~~~~~ LL LL
9 U.
LL K LL LL
~LLKLL~~~LL~LL LL~
3C~(J~p eV ~p ' y M O ~P~~V~iui' NI~iYitOt6~Dlab~f:nn~aOeDaDa~aDOiQN1~i0iOloO~00~.r-~~~DNNN
~ h N~i Ff ~~ V ~~Yf~(OM~tOtOl~ M1P~~aD~~ o~ ~ 00~00~ ~~ w~~~j ~f~~-~t ~ff a~DaoO~OMi~iO~~~ ~.-.-.-...' v W d dr '".-~
-oR W õr, =cr3ocZZ=io c>vv ~i_ag,rn o~=~i ~~.rZW<WX WW~
NtV ~ s fKb~
fV ' Ym ~ a a W
WWyUj vWWWW y WW W0~~ ~~ 00 WW~j WwwWw a ~ wW t~ U WW
~ N N N N N,~ WwW w W ~ m 3o ~Kw gm eiry~e ~ ry rn= w w _~ ~
g s '~'~w~
w '~ aW a ~
2 Q~f w ~~ N~ ~7$0~~~ EEWSS
W~ v~ W W W W~ W WI I WQ ~S y V W W9WEW9 S=~ C7 CJ
W W W W W W W ~ {y~ W
yNqt<nptlfAN<h~(0W0y~0w0~y0W w~ = 00Ny~N< ~'QOW8~KW
OG_WO ~iFl-~HF
Mc~f,iW~ll1W~WWFFFhF 8W w FF~ W
YOI~w~ F OZZ*Z~~WpOZO~C~ FFF.FrF-viNaNN0OoC ONVai8F~FW-OOGn= OV
oC~K-~a ww<aa<ooo=OOw ~C~~~a~~ = Q=-QQ
g ~ cI
JJ~Sg~55~M~ClIf~J~~=3J~aa~~~~~~s ~ 2 c LL ~~JddJod dddddooa44~~~dda =aa JiCU=J(.U U. U' oC7WOO
o f/1 1AO 1~ n~U p~ a~o (~01~ dOOO)Y ~ W tp l7 u) ~" 01 V7 10a0aYo N
V 1- oo.N1p ~n~4D~p l'!e~ ap NNO~1p~~pp~N NO)~ ip~ t7 NONe~ of~
Z Nttf~ aD~~ NAUS~ {y N ~f~N'VNN~~~ aDNN aD~pN~
N N ~Ntp.-~f1 t0'-Nl7 CD N ~N ~N 17 Z N !-m ti r C7 OI 4D '- f=n p~ 1~ Y Mp~ pU f0 ~ f~ M V) Pl Ifl ~ t~/a pl~ltl~~.-~NMNM1~1+1~ N~n w1f~lOlt"f O
~*p N 1n o l'7 ~- p~ pp O~ p 4'!
gO1~~ONLLK LL ~~y"pK~
~ Q'~2 ~<Z Z ~ Z ~ Z ~t91 W ~ i ~!( X ~X LL LL LL LLK x 9199 ~ LL LL~~ 9 E x K LLIII
LLLL ~( QLL
~
.11 ~ gc ~ N~3~i~g~a~~~~A~ I~-3 8 2 P -~~P 8~ X
~ 9 68-5l-9 ~ ~ ~~~~~ ~
W 1~01~ h ~AYf1r01~ 1~ ~h b ~ h ii~,~~~~~~~~~ ~~m~m- - - - - - - c~ Z~
~ o ti (i Ci Ci .~ V$ ~ W~IWW iD~
N 7~ $ ~tt t h:~n zzzz mm r W W 3 .=r r NNNVj VUr' WWw WW~
Z Z ~ W W W W 000000 ~~
W
V W W W WN Wy ~~~ N W
= z a m m ~ ~ N~N<<NNW Z
~a~zzzoo~~5~25~~rz~ aO
I~y~~~~
N U 3 v wU~~~~~SSS2xNNNN QF~
4 zw=w=oooo'~'~'<a~< ~il4l~
o'8 'o~'www 'b ~ ~
v N
-p 4 UVV~~~ ~~{{~OOOOwwwww J d' --~ ~ ~' 7 U C) U J U J q ~~ U U U U IL y~v c u~{ c~ac~a~oc~ aa~a~aa~a~aaaaaa ~
o m Ny~ aND c=1 n ~rnf ~ ~n n N $ 1~0 p '~ ~ ppp~~1(~ en e7i N -- 4 p! v o co ~y(~ ~" l~!
N N z ~~~~N~ V~~e~f~t9a0~~ Z ~~ONO
~ O~ N h- " Y7 p~pW{V r{p- n~p N ppp~ (ap~y I e0 tD ~
-W ~ ao ~ l~f Vi n ~ O ~ ~ ~[l 1if N rn ~ ~ C N N N ~
t N A
~ ~1. t"9t~f170i~~1N 1nNh ~ V~ "O
E ~
_ ~ _ ~tc (~J 8 epV ~ ~E, ~{~7J ~pp~p oNa{OWO~yN~yfry~Np aND ~y ~p F~ d{y ~pp e N~ N N ~ C Q N NNNNNNNNNNNN~NNN Q NNa ~Z c Z
N N N N~~ N N~C4 N W N~ N N
Z Z N N
W W W jijO1& e 1a~.
(~ N~'.~ NNNp ~~' r'. N IA Ih ~ V
Z Z W 1+~ njN tip:nr~ t~
W N V V ~ N N N ti yVjw-W NUo()N
X X W W c~i U.W.=IL IY
W W
W W
W Q ~ oro~~m Z It1yN~W~ ao ~ =~ ~
W UUUU W ipJuliUW
~ ~ ~ JJ~W W
OC 40COJ 446 k ~~CQCQF
~ O O
y~ W Z W
~UO 0 ~ev~0 O O~y1 s'~~y~~j Z~V~j11tll~y~ ~,U
Ft~ie~i~ h F' W ~ }
~ ~ :4~oyo= ooW n ONtWAj ~ ~ pOOy~ ~yWfsnZ~a~~rN O
ZWUIUlluui ~
~xa s = cnrnrnv,v~
'~~y$ a a a a a ~g~uc ~m~ Z
O_ = O g.. a ~ US -~ 6 'O J~ ti ~
~ 'I lo ~0.~ ~ a70r 7~~(09~CJ9CJ
= LL rb~b~b w {Q N ~p m 1{~y1~P 'Ma O
~ ~ Na p~p~ r ,~ ~ V~tf f0 ~ ~~ Qj ~f1 N~p N aD OI N h N
Nf N9 h~V01~Nff.N-~.l.- Z Pf ~ Z
W N
t" N N~ M A N '='yjN N N n~M N~ N N ~
z ~~10.N- N l~ I ~Olhm~l~f~~ Y IrN Z V
K
0000 c W N ~O 1~
~p ppJ~ 1D~~Nhq tO~O 1~ ~~A N $
C C+) II b ~ A 9U.U.11i i u.
U.
e ~
0 3 0 _ p~y ~p m ~ N~NNN N ~ m~ N NNNtVN A~~NNNNNNAN
~ 1 R z ~ y NN'NN ~~NNNNNNtVN C N
.T. N N
"%
N
07 ~
OI
~ W W W W W
w-pj pj a 0 W W
UU r a V ~ ~ U U U ~~~
~W W soab~aDa0~i0V W O ~ fVNN
w rW w ___ _ ::W.=~ w w w9wW
W W ""
WN
~'~-~~UUUC~UU Ua~ W,~
WW WWWw~WU' ~ v .r~ O d 99 xi oojiiw~~~lww~W WN'~N~
Z
~8888881p~ 8Sl ~~,~,; ~ ~ X x X
mddsd}ddXO O~ U rpiJC~~i80~~W W W ~M-F
NNNO hh VYy~~SSi2q gdWWW~UUjNf.ci9 ~ O O O a O O W W W W {~~y ry1~y Q p r~~4R44~~~ i7> - WW<wOWIOW~WWW~j > > S~4 NNNNNNr'N y a -j' JJJJ ,,r J~= ~2ZZ qU'V~~'~~ LL
O~ 0000$O~OOCU! 44-i~~-m~Z r "N ~ ~ ~~tD
FF t~FFFFtt~-Q N N-~W_W..W..U11...WWW
iQ/}fQ~ fQnVJ(~ tQ~ NNpUIc'v WWO~Z~WY~ ~0~ OyaoMmMaotlfl~M
N V
ZZ~ZZ2~2Z~ZOp O OOZZZOOF- c~i fy F~ OV
J
~~~
~ ~ dazaaaaaaaa ~~ ~~~(d~LLcoLL aWa~~~~090 OYY}Y J~a C u X?ZZ 2YYY ~~~~
o N N P1 W f0 ~ (((yyy ~ p O
1p O Pf a Z ~~ N~.-~ h t~1 ~ i0 ~
~ ~ I~ dD ~C!
~ ~== = ~'f ' i. N N
'õ2 = <~f Nf = 'f N f- N
~ ~~ tP tf) tWtf ~~~ f~ f~- e' 0~1 ry tV ~~ 1f1 W N~ vf ~ r ~~~ 1+/ f0 ~ ~~ OD
"=-'-8~O~O~
~ N =~ ~
r$'O~~Wm~Wb~NN ~ ~PCNn ~ ~ NaO~O O ~~O
LL ~~~LL R: LL
aLLLL XLLX LL
K
xLL
Ir xKLL ~tL
lm _ ~ p w~~~~~~~~~~~~~
l _ 2 1 W ~R~NYv~Ne~~~e~i~i-~n =~~! ~ N afnf~Me~r~~~t~i t~+~ ~t~~~ ~ ~3 e~e~M
N
W
M (~1 ~
r __ W W U.
~.j IV g O y ovwiai __ r =- ~~7iu~
aDW~W ~ 'rUU R9;;.g Q I[i uiW
U UNlclt'>NNNNN WwhNN VUWWW~ Ny WV aa UU~
v~viitf WWVVr U ~ ~~....WW
Z
IV N Z_ W
VUU"'~~ C y,1 wWWWWUUV. N ~UWy~y~WW~~lin Wa J
.~ .~~OC
~~K~WW~JUUUUU Uy~ M9J~ZWWW
~~ Z~NN
FFF-< LLLLLLJJJ ~}Y~ NO>NN~n~nWU~ 4WTrJ~O~W_GWC~~Uwa_0.
Z a~~ ~=-= ~. .hp KJVVg 00 OOCpjZ<VUVrrOC J~~~~~lf~~ ZZZZyUW~wW ~~W
agi mm Z=SS W 7d~WW<~=jih ~~'z paata~a~ '~
=ja~ ~
aQUU ~~~i 4 00 O~u ~W W W W Z
~C3 ~ ~ Op pNvCJ OC
O== ~ Wa SS=~WytN2ZWW w s~j7~'~~~7~~Ww" GO
R4a3,woO~aaaa~g ~gu~ YYWWEmi3 JJ~JJJJJJ=?~~~~~O~m~
C7cJc9oo ~ -v zz6zzZZ >> Fwwyv~ ~ $x.
WWWQ WWW(~QW{~~71 IDCL~ W <Q d x ~T Wu1WW p.~1 W W
888 (9C900UOOO~~N'~ZZypjy~T~YY}~~~~~~j~~K!-FFFOOVO
=C o 2-+C010 UO p~J ~
J~[ OX ~0000~ a QQQy7U ~~UmmaD
ZZZ !
4YJJ}JJJJ Jj=}m~Oq-~ Z ~ ~~ < 3C LL ~~oaa~--~r ~~~i_c~c7C7c~c9(7c~~UC~xxzKZ~bo?. izTZZzOC9~ ~7C7g~
:.:.
O
V tA~ tD Nt~H)O~ff NiD ~ h~+ YNIM ~OD N Nfpp hOi1'~ aD ~ T Vni hPf aD
O M M 1+ N ~pp a0 OD O ha h M ~hp M M CD Cal.Y1 Yf f~ M aD CI h O c~ O cn N;- .~ Y! O
NI~~reN-~ ef~~ MNNtM~fYiM~N tDa ~r~r NiDN <
m Y
~ M t+1 m~ r ht{pp fM~pV O~i0Mt~0 O{~ M1~ V~~ nh~p+fN y~~y Nrp~p (Oh yp~~ ~p~ 01 ~Y ~~(f OP1 O cn~Oh ~~Yf~~N f+-~.N~N ~r.Y-N~ 1~ t~h tONN~
~ N M~ff r ~.- M r.- M N M N r.- f H M/~ r~- N N O~ a0 M N t0 m 0 r h r ~p l+~ M {~ 01l~ ~y W In o ~pp N h 01 M V' N N
Q~ Rf N h~(O h e~ '. /n 0 = q r.
AnO~M N
M~ N h h O p~ Nr N fh~lr hM h U p ~ ~~p l+1 OM h NYf ~i t+f OI ~ ~
pfp~ Mf'l'r' ~ r haD~~pp h Sj {0~~ h N~mN~Gh0/ M N~OIN NCp O~y~$~pNp1 Npp=
~'f 'Q~Np ~y ~ ~~ ~~yN N~haDNrQffry n~V h{~ p~ p'o' h< AQa E~D O hM N pN V
C ~~aqTr5OO~Q~rQ~Q~SOaO'CSj~~}Q4 O O~GrqJs4~~~{G ~Z~O pQ O~r~PN O
LL ~ ~ZLL ~X Z~(LLBLL~~~ ~8LL~~ LL~ LLILLLLL~~S LL~
- ~ LL ~ Q LL K
~ Q
~
N ~ iV N
W ~ ~ V Y E
W uj': U U U ~ $ E
a:
~
px uiW v y ' w w~ y 8 _ N UN W v ~ 40) a~ ~ ~~
r 17 i - W Q W ' -' Na ~ 7 K Vi' U~. W N N u W O 'wS oC
~~NN N~O lcm + -a- 4 ~ ~1~ U E
W W
N ~c wWWniyUjW a=-WXa~ LL W FW-Ua 'L 4 c ~~~UUvmv JE 2 z U~ 0 sN vW) UUWW 1~ w~W = nj ~_ ~
"
N {- W U 0 =- 9 1 $~ $Vi O~O yW W ~VWIc U7 etiW ~yK ~ c= S, ~
N !%J N y~ 91 =7~ p] N ~'! ~- g'~ ~. c~9 K4Q a~ 1Sa gFerWNW YU w t9 hWwi1LC~W~ W ~ ,"=.:QOOLLN t"~vU.lISF o . aa GOLLy ~
o ~ p x w o iowa~ ~~ ~~c< a a ~'g' Z~~~I r~ aw o ~.g. ~.sg.ssc y~ q~bQ~b FF SWv~(?.:>W NK W N NW W
~ I a 0 WvaO~ ~t~p WyZO O wr~' t 0 ~>_YyWNOO ~r80mw~ S~ZyW-7 O -~ ';~ c OOZZj N _==d] O ~p~~ O a WU c.~ I W{j/ W~~j ~<<2Z 7~~~Cpj ~ ~~~YNpLL ~. o~p~
QjQj p,,jJJ y~SZ_NqNpaod~wWa~p '.Y N ~a rfW ~~ z NN W N~ G C c C ~ ~ ~~~.~ ~.~!
~.Q 1 1 1 T T~~UJ ~~~ F-'JlV $
~ - -l ~ LCC' _" r-~ Ua;O p .p s~
oaaaaQaaQooo a Oa6"~WOWawO~Qc~ Qg- m- 9~ 9 E~ ) vtJi r~ ap~ F o c ~ U. rv o~~ p~Q pFaa~t~U~a3c9~~~c}c} i n u'~a < 0U
:z O o W~ ~ cp 1 N W W N O Yf ~p - W M dp ~ = fDa~on F ~p e~ W1p Y'I
O 't W O
NNO~ O p ~p p~ ~y~NN~ SZ
r V f OD.-nmNOWi~-~N ~'1 ~ ~NNt90 3 iG
~ ' NN Ot~O1N ~~ ~~~ n W O~OlO~ n +j (~ 171O~N~YWp~~ y ~
'fNA~V bn~~~~NNIOYI f{~y~17~~.-Nh ~
~tly ~=y 1~~ ~ ~ W
~ IffN N w n n 17 ~t p~ ~4Q ~AQN~O $ O_ Q.- ~~ W N Uf U~ C~C7C7ClS~~~ S~~NNNQ$~
S
W ~ n rr N
zzz u.LL4'C~~~~a~ LL KLLKKKKKK K4'~a"~a~~K
~FZ
G
V<-W :v vvV:! VV
W OWi ~ On! W' n ~ lW fffffffV~FO~f~f M~rY~'f~f Vf~ = fV~1Yf8f~Qf17i ' iffu~'~=V- N
IpNNN~~f NO t W~.WiD
~ e~l .. tO b i0 0 NRNi E
W N=~u~i,~_~ NW
OvVCliUWX
~ZWWW WW j~
O~W
rr..., ~ ~av A WW ~ q00 SU~WWW yv ~iNCCW, pp E E ~~W3"G~VC~U fgfg!!~~
< w~
$ ~ZG UUUU~~~yWWyWNW" Z ;C
WWW ~YWWZZ~M~~Nqf,VYi o E t UU~~WN = WW=-.=-~>_~+
Wmoca'r~'aroi~~~~~+L~~~~~~-"~
x~yy=WWWW~~
2 20 ~4$$~~r Z~WW=WS~!==W ~<O ~Z~~~~~~"
~~$ S~ C2 E 2 Y}F-1-FF~F~-~~YOOG O' _ k-~ bI
~~~oQ~~~~aQ'oo 0 c U.
w V N I ~t~ogb~~~a~
Z Pf ~ N N N~o N7 01 V' r tn r ZI in N.- C'! ~ c7 N P'f 1f! r l'1 N "
~ h N
~ ~~' ~00 0)Yf NN~~~~1~ Np N
~y~y C ~by 'N' Or II UU~UUt~U~ U~( ~y Z~ZX~ Z Z
z ~ ~~LL a $ ~~~LL~LLRW~~ ~~2LLLL R'LLILK K
~~ Z
0 ~
~~~~~h~~~~ m ~~0 W) ~ Q a u,~~~~~~~x -9 NNq sag W W U
mm > > m tu zz uwwLLl '='~NfVww Y11N Yi~~_NN~V9~_ rivi~?~r~r00:
v4fea WW .r.... ~ fJ
C)VW 4 <
WW W UW e rT iracccm'.w0y pft s~nn~iiill-JdJ~z wõ sszzz Z~~
t=WWXX O
~R
~ LLI H1I ~l ~~~~~~c (O f 1+1 f~. ~ N O~1 f!1 Qpp1~~
o ~p Q 46 _ p QQQ
V y 1~ 1 1. V~ Ih F- ~ ~ ~o p ~ m Of Of ~f Z V - NtD TOI
m C C
Z ~<N~fVHft+l Z t+10~~~Off~~lf~NYfO~O/NOl ~~~o~~~ =c~ N~~pO~~OC~~~'~~ .C' ~~~
UI ~O~~bt7 vl ~C9t9t~t~~~i~C7t9~00 UUU3I ~n(~
p p a N cn a &
co $j~g"~P 1L~~ L ~4N4N~~K~LL6 fQ+~
~3Zs~Qqo~[ Z
LL ~(K~LL~~~K~~oz~(NX~pxa ~ ~3a ~K K KLL~
~ ~o L ~e ~ 111 O a IMM ~ ~ Z ~~~~s~mmmmm~~ Z ~! ~~~
y P9 Nf l+l C' O G O
VUU y(,~ '-vi~ < Z ~
W~W4~Wei ai~ vi u. 9, J ~
j p1 W OaDa~ __ _~ _ ~ OjOi ?
NiVi~ai ~~tU~~'=- Ny _ _ _ 222Z2ZUtlUUWU eqr> ~+fMZ_p 0'O12 R aWOi1YW-W wz ro,viO.mb~
UUUU 1y t~~u <~ .=4m.~ ; ~-"'- .-U W
> O
Wow WWi~Wp~gF W~ UZZUUUVUUWW
W~WW W
y W--W_WWWW_W_yV1 !q-FFa--a- QGOSJN ~_O.ZO.WW0. p U Wg JJi iJJV
x G
y~ ZZZZZZ
xici}m~ ~ ~ ,}z_ic W
z r< ss aiat~hai OOO((~~ vai~~UO j A_U_~ t _~i~_IK
iijm Oj~O~OGQ~~~ ~~~Wy<Wy~( Wy<l Q~o'f W WW O_dap-~~__wKU + W2~ZZO0~
Q
F' 'mm} y~XXXXX LLLL O1 >>F F~ ((~~ W y O O O O O O U~ Oa yFF 8 (~ W W W((~~ W((~~ W
VUUU GGOC}ISGUUUUUU~~OW }~~~~~~~Z~V
} y}} W W W W W W W W W W W W __ JJJJ ~~ ~Te U ZZzsa OO v C9C9 ~~~~~~00~~00p~0 ~pXx XZZ W QQ_~
c 8=2 225===2KTW SW~20~~~Q~~000007y~ti ol Io~ a PfKKaWC~
a US== =2 ~il aia= ~iUcic.~t.~u 0 c.>Ut.~cici~ac~.>LLIWi.IWi.WW~~ZZZ~~zZ~' o p _ pp tA t~0 ~'g<NO~iT N1~ ~~i O~~ 1~0 eA0 V c"I YN1 y NaDN ~ N V N ~t")00011 ~p N {~
Oi p/+/ O~ N~~1")Of f a0 p l"!1p ~ N~O ~ OD.-aD1D~tON V ~D=-~ ~=- N~ NN~.-h e!N V V
C {p 1f+~1 ~pp Nppp ~V~ ~+i.~-O~N01~lp+~N~1+~y Vl~11~f~.N=-N~=pt~ttnVf ~ =
yF-Ii ~p~pp v1 N Vl ~t~ n ~ N ~f ~
~ ZI N~ OOi~ .a- N.- N N 1~ cD Pf N n~ ~~ Nf .~- 'f N N N N ~~ 00 N c7 N
1~-.01C/ f~~N W O/ O W 1~N
~~~~~~8888go~8880 9> VI OU (9nn(.'1~~(9oS~7C7(~9(~?oC7?(7>CJ~C7C9~
sZ ~vO
I ~$g~
~~~~x( QX~~~~~~{Oq OQ~' ~[ a5g N
~LL~K CLLLL~ ~~4.Y{L Ip*IIIK~G~~~~~ IL~CLLI~~~
m Q o 2 W
w W V c~, W W oi aa -- ? N D a U
q,Z
Z pW ?~
~~ " o Q
W siiUU~W Z a c~i0 W_N rp~-~~
,oaor..rZ'~ U < <orom F W- "WW~~ Z e'l ~ e7 IJO W Q W~ W WV=?z ~ 4W) W Ww~ Hp ZU~UWgOWz~W ?~a IhI
yro zTX X} c W a0 ~~ O O F O <<~ m m~
2U88 ~ZZW~~ZZW._Z_ ~a a~~ ZwZ~~c4~~c WWW
M
WZ 5 Ug WW~ oa aWW W~~vO9 NWW
de~~oo sgws'~u7go~0.0~~~ W~~ Ox~~
~ ''4 i "~"r~o oa~~~~Te a ~xpxF~,,~~~ o 111111 rrrr2~~ZI Ux~Sp~~000cU=-V=S L tAt!)V1V1tAa) lL~ 2WZC1C~~t Z~20~LLLLan.~s(9V UZ21~= I QQQQQ<
n'~ie ~pr ~~~npa~po$'i ~m O mmp~~p fOnticX o 8 ~+ma vt' 1p ,'~i"' ~epOO~1 N N~f. t7 N ~~d0~ naDt7r biO~ 1710 .n NInO lMP) MN~D Y L Y Y' al r M~ O N t9 ~~ b ~ C~ ~p 01 = n Np ~ N l+f M N 1m p ~D M yl 1~ uf m F T1~/'bN ~ U1~Np ~p aD(p ONO Op ~[f~p {- ~ nNh ZN GN~OI~D=~~P70NNi0e(y011A ~ If/ NNN f 1~7P'1 O Z ~f0~='~
q ~ ~~~0~~ ~~Q C~o aDUf Np n ly I nnN O N~~ ~
V~ C7 C9 ~ ~ ~
pp~p pp ~p ~y ~pp ~- W{p~
l'70D O ~ N ~ N~OM' N
N aOD ~
ZigIZ
~~LL9~~~ti,L~ LLa ~ ~
r IS~
0 f}~~
~, ~ O~ 00000.'-~~~~'(~{{y~ NNNe+f ~+f~ ln+fe+f Y Yf af~fM
Q Z~ n n 1~ 1~ 1~ F
b n n n H n n f- A/~ f~ A f~ n n n f~ h n n n n n n P h. 1-"i Q
~, a '~WU '=h rotCWZW
UuZ~
wwl(~ Wm ZZ~p~= apu C) L) a W~~
UUOwQ orc wUJ U) wwa a ~Z ~v m o ~
z zz o }}}}}? o Ut ~ . hNV~hym ~
a aa~a c C LL QQQQaQ U. uS
~
p ~ o ~ ~~ ' V N
t7tOrtA n N t'~Y Z f~V
.G ~9I N
z lh fV M ~MON1 F~) ~~
NlzN
N CD ~ ~ ~ Q N
>8SS~
S g U V
s c g~sro~ E
u.8~aa~
.0 ~z m 0o E ao yw nr;~~~~.
.mc w ~ ~ o c Z gtz N
u o {s :o ~
to cc C
E
' h o m c v7 o ~ a ~
C a~ N C C~ w~ 4~
ooS gn~ ~.,._yv c ~y O 7 m~ v v O
0000 Ei 4l E 00 y N
E
~~a~.3'o Rwoo a~~~'a1 ~
z W
CF
mN =S~ ~
h Y ~. T,~ = -=fi ' x SE
,~oG m~~H~Ern ~[mN3oa:G
u W w N o >
~
~ ..aa o Ly~p3 y E==1 ~ v1 N y T V y C C t7 ~ 4 ~ ~~i~ ~ ~ g =; ~' 4) 6 15111 d QG ~ N M M N~p r O Nvnvi v1 v1 O 00 N CO
~=i h ~ N~ V1 O0 00 N N N V~'~ g M M ~
~~ mSS 9 9 w~~~
aa aaaa a a aaaa a<a a aaa<
d ~~ v u y 0 0'~ E v ' ep = o 00 '' ~ =C o N y o00 c~ a~U ~-w U o = 4 ~
u p V x y0- C/
p =~y o y v a u vVi A N V N W~= C
= n C
E
u a r,:~ u 1 u a.
C3a ~c~ X
o, u U 0 u o N c ~
~ u t 79 2 V y~~ N
o a ' or = ~
F~v to uZ 'c_ O Ci y . w E12 $ y[ , ' ~ Y
,o = ~ a r~ " C .~ '~ y ?~ Q= y E ' = W l-~'- '-~ E y C N p L O=U x-, O y C==~ =O N N V ~V ~ T.17 ...- a ~ V/~
~$s o _~ v~ u~ u u ti c$A u E~~ ~_ ~' A 3~ y e uE C C ~~ ~ ~'C ,a u ~i &._ =c~
p c o u ns=e c e o o o o u ~=- o o~u ~'9 ~ao~ E
n y A a o T~ QL o~ a ~ E ~
o~'mo eow E pn~o o o 0 a aQ 5 a Z o a Loa Z a V
ts 0 Q ~p $'O vy h h h rl 00 ~ .~ ~ N a a wa aaQa~ ~~4 a a~a U. a Q a aa o = ~ - ICU
i+ Y ~p $~~~~~õni ei ~o o i3v=~=~ o o~ ' ~7 ~o ~=~ 0 5~'u o eo~ ~ E ~ ~~.E $h u u ~~
en _, F uC o_ Y p oo a S~ y u u N~ .~
~~p '~" Fi1 ~y . ~ ~ .. ~ pld d= 'pp 3 Y i'Q y tpT~ L' ~~ V r'j M~1 0~ 'p =!j r l0 bp~C h Y p='s p C t0 C~i O~ i0 Yu M a V~~ u C ~2 Y QO O~
=~ vYi~p= y C ~ ~=~p Mt...~ O~ti 5Ge C v~ ~
a O~'~.~l0 =~ O Y O N~~ R. E~' C'~% p~~p =O ' ~, T. p E t-' =O O Vi7~ y C O~ O 7 O O O Y CGL
Y - v'~= ~~M '~=~ '~ pa W O. Opo t~ ~~ ~N C E y ON=.C" õj õj ~~N
e~ ~' $ o'Ca E ca: o o ~m r E= E DOO 1.2 o u.yu.uc~g,'~; ~'=5 ~d ' ~= vu~~a.uu u L oho 7dL C Y a GT7~ ~OT. ,T ~=C O~W,= Q071"~
{>i ~eE$ ~ Q r~
E.
G' r~ _ ~i a E o CL Ta e -=u m" U~ o a ' a ae ~ou.
c 'u E S
'~$~ =- Coo ax r+E= =$.
u~ e~ >'=? e I1t!!_!...H c u V ~> Q~~ o o~ o~i?~ b o o Q ae o ~=a ~ .~ _ Q~ V y N ~ r' n' $ C=C Y vYi o E
~=C y ~,C ~ Y O.~ E ~ 7~ E O
.G .~ t0 N ~ vWi Q E L' .Y. 4u t~ O L C
t,l_C, g ~ T p 6, Y~ t3a ~~~'j ~ u t wp vTi r C V x'Y~' ~ põ ~= Y~ E~ o~ C ~ O N
't, ~ ~ a7 X ~ T= ~C ,y d 'C y ~ 0 ~ 'g =t~.
c E > E ~ E R u u E
~ s u Y 6 u O E T v q OGQ ~ y=7 T ~ yN V V~,,, Y 'C
O ~~ G Y5 pC y E ~ V' t{~ ~ '~'7 C O{~ r~
~ b ~~ O f! ~~. N Y 'G G ~ OD
~$ o G~~ ~ o~ s E Q''o EL-~ a za F N
$ ~
eo w ' A 8='~ ~a ~ ~ 8. ~ = ~' ~, ~ a~i ~o ~E o s M ~ M ~ O~ ~ vpi O
8 ~ S C ~ N N M ~ O ~ M ~h'1 M
a a a a aa a< A A ~~~~
~
tr ~y u -' y w .5 r ~ oao o'~o e ~
~
V uo~+' y~'= $;; ~~, a, 2i E E~
O = ONO u O . ~D Z 7 G C V p~t~
p~ p O p O 'J a~'0 l0 =.~. tyi ;O ,='~3 =w E ~ O~
{8 .'a .O C N = MN 4 a Y =C N ty G. C1.
O~ O. F =~ M --.
0 Q T ttl 'G. ~ =fi JR 4== K ~ ~y OVON V~ v== E. n= ~ P .D O a O.
c_oM
c~ = o',! 0 8 e c ~~'= u g a, u ai 10~ w h .v o ;c~ ~o=e =~ o u u S o r oDo ~X.c$ g oo a c e u~n e_ eo-~ e c a~~ ~3 =.e.~ = =z~p s s s Q = ==.~, dG ~- _ C V Q=~ N =E N Ofl a a$ a C ~+M '+M C N_ O O
E
~;
Q~zd e~_ N oQ
~ v 'U xoM EM E=oM~M ~N+~~.~._.~~p~
oo.Z ca g= G~ u~ u~ ~$ v~ uo+ ao~~c.v~ ~i~ vs ~< ~a a~ a .~~ = ~p v 5 710 O
Z' io c t7 u 7 v v_ M M- M(~ n y . ~y .
M M iV N~ a. {i .~. id lV PM'1 ~ c'1 a'V'Jj . r ~L
V~ E V C C 37 O~ N iY+ N N~1 ~p N~ : O. ~7 p~ V p~ N"~
V~' ~~: Y a.+ 0 00 OO IZ V1 ~O C
c o o e~ "~ E-- 0. F. a v v o Y Q o0e cd ~+ af cv o ea ~o R o a~c v~ o~ ~d~
au o E.2 ~ E E'~ oeo Ei;; c ~a o x E ~ o~ oo o ~ a A ~ c y t . E oo E oo _ a e $ oo e v r ~e ~e : = . . .~ o .~ o S ocno ~
C
V
N u u '~ pEp C
~ A a0 O
C C V O :7 ~ o ~ s'a =~ .~ .c e a, T d a ~ = v o~ ~ u y s S Ta saT.r . =3 a X ~p, v G G. ~ y 'v V .V+ E a ~ppQ,=V w O. N
~U Vl C W V C =~ C ~ Y=
r E O e E 7i L' a O ~ tnl= 3 O S:4 'ro- ((-7%
.. a a e oo =o ~y~vt wo V E M ~Np GV.
~ ~ ~'"' ~~ ~ O. G G Z. T1 ~ O. 0 1'- =G C ,C L
[o~
n.< oa v~ " G~ u == a -, -, a, ~b $.
[DIII! V C o0 .~ c G x ~ ~ ~ C C ~ C C
C C C V .N .S A 7 7 3 =7 7 OA r ~ ~y y jd C~ ~..0"y.C' DP ~iftY1 G~=~p,~...p, V V V V V
c oz oz 41>a o~ o~~ v v e ~t ao. u e"_ c =~ Ep a ~ m~ a Ea 0 0 ~a ~y n~ P3 C~ o~~ c~ M E~ ~~'~ o 0 0 0 0.~ td'~ ~ O O V V
., a'~av ia E õ g f g= _ D Q
z z a.~ ~g m~' U=v u'o z~zMS - - Q Q 4='~s noo aoo aoo oao ~yoo E'... E:. E A G r = E- E:Z ~ Z= Z-" ~ E,=i E; E E E
- p e c~ o a~ ' ppyp p do QoQoQoQo aQ ~~ o O c ul~ !r- 0i'~ o Gs.~ .-CC ZS '.a a .as.as.a ..D C7 = C7 =~ C7 N~ N ~ N~ tC r ~
- v~ vY ~f ea Y iar Te w, w_ af~.
'a E ps -e s vcr, N iSN ~iN Y+N
z r r r r r r oo Y: 73~ o ~o~oo.~o~ooo.
E oo o o .. o e ~ $ T g+ T , aqo e o~ o v -cu 'g Ce vlyv ao C c C C C
v c.inLO-W inw QatO.= ~-d~~ 00 ;
o v N V
V C
=V
er o E
= C n.
o v v ~ '- o > u u re g e x S~ E P Z x H u a =a 'gq UN C C m ~- E L O, A aVi O '~
~, ~ ~ =O ~, :G =~ y5 b0 V =
Q" c c y 5 a~8 q a Y W
N O N ~ r 00 0_0 ONO tNry M ~ R ~ O' O' 01 O . h~ W ~ h r ~p N N ~D ~p v1 r r r r r r P-R7 f~7 ~tl 41 I~ Lf] I~ It~ 47 W [Ot7 W LOi~ Ii7 W W W W W W Lt] W
N
~ O0 ~ C 7%'~ ~P~= aC N N a! ~v~1 V V
a y v1 '~ 000 3 ~~ $ev T C
C~p a! p~=F~ C.: CO ~ ~õO~ O y ~o 7 '~ =- O a.l =~ y1 ..i .~ O yC v~ 'G y N g~o .~
=C ~~ i~d a.... ~=h o Q ~ u~ ~m V C C C
~ ob~ ~' ' u o ~X =y o ~$~ ao o0 _icr,~ o_y U au; o=ot- u ~S+ u ao a ~ - == ~.a e ~
.~ p~ fn U O 'a =~ .C C ai !t m- t~ 0~ O~ ~'=. ~. 0' p, S~
.C =~ iC0 "~ 7 'R' ' C" ~ =~j pb i.~~". l~ ~" < u v t> co u v p, 's Op, 'p A1~ ~ o Q Z~ C ~v" ~= di' ~7~ C 4~ S7=~ ~7 t~
+% ~=p~ ~p~ U O, =~, W ,~ 'C1 ryi ~y C t ~ .~ Q C ~ ~ ~ ~ V
aNR ~j=V OO~.fn(~ ypr~ C~ ~TP ty app0 C 'n~4õ W"
p Z V~_. IyV =~ w pU=a N.C ~'in ~ C 7 C 7 t pl-~= H ~ ~O = u =y < 21 af SJ bD G~ N Z ~~j ~. O aC0 y=~=~ C=r o,~p_, ~ C~ C af 2 -'nt g W E eL E
E '- Ee~i~~u u o e2~ ~ o~ ~~ u mp v~ u I E v oV 7 m y E
u u o en o ~v N o a E W ~ i'i =~ ._ F't u aa . ~e .a :.
Z
. E v '~-~-s S'v ucN!y~ ~~'~' v~'y ~~a~i S5'-~S
?'. C -l V ~ Q'. o U 0 ,? V =~ ~O G . Y N .D } fV fV
o~o,o=~o >=~ 6 i'i~~n~vFpV, <
0 N yõ y =~ W=N C~O O t . .~ V~ d' LS C O~ 4o O l~C A~ N
? Z o~ c~. U eo4 lL Ev, u, aa -, eo~'~ ~'v -v.U.:. O c=i r,.U n~v~mv, v, c~j C 'e o a N ~y a H
EO Op C y T o = ~ o u ~u N N a0 C rJC
Q ~ t'3 ~. X X X 0 Cp~ .CC _~ '~ .O =vp ~p p~' ~?
a a N z z - - ~
g g v~i O $ M n fMV N $~ r~+~ ~c ~b ~
~o ~n oo n C4 oo n 3 ~ ~ ~
tn A 5e~~ YC on ~ - 5~ .
N FTi ~y v ~ 4 ,=t~c~
C=r0 O G O
$0 ~>.=aCY t~
F~ F en eD u~ v Y ~= O ~- p p~ a C 5 W 5~
Y~ C7zQ C7zd a ~~ E u ~
0 Zo~ Zt~ pt~~_ a z a a ~ p!n Q(A N ~,, ~ q( Vv A G g S. I~ -=~vl ~((V~~~~.CN N V 00 00 i 00 00 0 0_ sOD~ ~QO= avuQ u~ L; t54'' upci o~=_'~n =+5~a 7~ <
v 'N ~ae = ~~n~oo -a g~$'~'~%~t1'~
c r (j e .. u o o u o v.'~'3 .~ 3 5>o ao'M.~ gN~~ 8 ~~ u pCai ~
o~ ~~' ~ '.2~~U C)~
C n CN u c..E-v u~ ~ "F-C7 u n t~
e u v' c~ g =
u d eov..=_r~
E~ 5d 5~ c~' N 7 i==.
o~M r~ E~ E_~.Y-0.c o$ EV C ~... o ,o <~ a urCi ~(~~ O u V ~o~ EO~Y~~~OV e~ t0 ~~ t~ ~0 f.0 v~ CLi 7- a0 W N
>VC7 w~c7 ae~ ~?' eo=. .~~ p eu V~n,y : oos 30_~ 0 30+ 3v E
w ~ ~V" = ~ = ~S O 'O e0 u d ' 7 Y=' - Y=' ti~ V~~' 'C =C =-a', ~N V Q y b ~ a0 W u.~ V V~ V.~ ~
m s u o ~'~({~~ o' - 3 Y v p u u'; _u a~ vi c v_~ c vi ~ a N ~ ~ \ 1.. O Np~ Q ~ L=~+ ~ ~ W N f' 1 F= =~ Y ~~l' =_ V~1i ~'=~ x N ~
~ rC+t 'p vV C O r C ~ C ~
~ z] U
tC 7 p Z='u' p: ,LW ' O ?'mi u E
~ Qa euoG a u~oG a2r ~. U~i ~
u _ w 1 ~ u N $ y, a K 0 w u u a 'u Z' N O
u u a i u c o " > Y o ='e E+ k '~r 'v '~+ =n. ~ ~ u E ~ -e", > ~ ~ 8 n y'~~~ + x=
=Y =Y ~ ~ ~
$ z vJ eq = u o c ~" ~
E a N N < E~~ .5 ~"a' oc be T x o a' 8 Q a v1 [V N eF Vl rO~
In eh0 0~0 ONO O' _~ M ~~ ~ N
v ~ . c ~ w od E~ opq o~ =~ " e _ 0 7 00 c t3 ~ r ~'i3 a 3~ ~=~~'-'~3 ~~
F. -;3 Y a E
eEd? ~Y w c~ ~~-~ W ~~~lo-~
vN o~t~~ o u o Eo~ooEi~v u ~u~' aci _ oo w< u_~ t o E E V ~ ~ c Y C~ V.. q ~ .C =~~
~~._ o ae Y ro~_ o~ eo _ pC
Ns 00 Y~ ~, O Y
qE vouE~b veo t- Arn xeo u oE
v v y .e ~p T [y~ E =rJ ~'p~p Y N~o =.. Y Y
=E-_ Q . ~i E
Y Tv Y
E~ ~=~.Y+ X N =wY.. V G V~ O~ y L N Y '~ V~=O 7(.J ~ O'=~
E'-E ~
L~ 1'~ U aY= ~ V ~ ~. . O 7 V ~ 7 .~ H 'O
e N
u~ OD ~ Y y'.' pp i ,YC= (7 p~,' ' U=Gi U Q Y g ~p eo a ~._v ~ 00~ rn~-...= _ YZ V ~'Z{=3 +' ,O_ on C N a~ v=~ F=C C~~ z ~_ E3 U e C _i eoV ~ d X oQ-4s - V E ~~=~ aS ep~ eu~t~$..z Y ., ' 0 3 N u Ea Y E: _:s ~j y'~ E E~NS d v o ' , u o u a o 3, yO
e Yr u~~ Y N m=u ~ ~c u > Z'u eo''3 y'E i a+= ~-. A E ~o a Eo~ Y o 5'~ v Y e _ I~-=~ ae o e~
~. Y .Y
~vY~'E~.: -~ 3~'~n' C7cY'~c'Eoy~=id~p '~ o-y"V..
E~t~ ~ u m c C3 ~o -W o KrO 0 0~ ~~ ? ~~w o ~ ri~ S~V-~ a~=, ~'-- 5-~- ooC~ii~V Y: > h v o a$UVo ~
O ~ =n i V Y
Y
Y V .5 0 e~u O
o c_ Y y e Ec D.V.~ bN 'C ai ~'a m.E c Ee w s u_ >
~ 3 K t ui;lIi 00 n ~ m 7~ =Mtg~~~ a~ a u~ .N.Saa <
00 Gp fj r! ~
U ~= g ly M ,.,;
~. m fi ~~ g=~i w ~ R
00 N C~~1 M0~0~ b fM ~ M M ~
M M M V ~/1 ~ O ~ ~ ~ ~
> x x x x x x w E
E
o o E id~ '~ u A & e~'~ v Eri al ~7 O O
O 5~,, u =u- ~ ~ u 3~;
7 ~~ õ Y 0 W~ O~~ O J~pTp~ V N p~ V i+ V N' O V
M
~ p Y
~ =;. ~~y=- (p Vi o_ Uy QQD ~ Vl u VI' = =F. v Y-0 7 c ~C~'O m ~ o G=
q~ >' G r+ p,~' yp R
Y ~ x ~{n . ~1 y -C v t"1 ~ u ~ U .~ Y ~ C u t~,7 F }j ~~==~ ~b Y OYwO =~+o.C~N N~bD~~ ~< $a w S~ Rc v 'Y' E ' a YO . =pc 3' p ,,,~ o~'gvo a o ~_ Ã,c o- u A 7Ce;o u O~ u~_ Es l0 .~,, o O~D V y~ O y s 3 N +' ~=F~. ~ U
ot-~'~' ~- y~ _v .. TW o Q K y ~~~oE~ u ''=~~~ ~E
i-~ 2D yQ. Y u! C7 py ? F T p C 'rJY Q N C~~ 4. =O y T
O N u CL u u~ .S 7 V EO A=OG 7 i~ S= 0' b Y 3 ONO C=Y= yõ d aE
~~~~dUo .Y~us ouo$ Ao oa ~~-c~~E~ ~'~ o~
E E E
a ~ a u a c ? ~ .~ n >
un.-~ u ~Yq~ a u 3 .~ 7 s . oo Y a E' '~ e'a c a e v ~~g o~:.a~:ao~vm_v'a ~en Y$ - o~+ 1dr~ s :;~~ -$
T ~= id m E~ o Si w E'_ E L 78 tt0 Z
_ .
~, Z n. _o, v _~ v tG ~u >, w .. ~ rs v o~c. ar o0 o Y u O N y~ q ~J a pp Y 2~ pU O u u d; s ..] O V y~
G c~ c~p ~.Y~p 3 0 3~~ ~~= D~~ Ih!fl-i ~+ ai ~.U VoV'eovi=~ C~ aTi ~ 2 s~z ~G a7~G ~.~ v~ aoN, a qam~-O Y Y
Q
. v~ = y ~ u yR u r ~ o c c ~ a u a~ ~ s }q > Y D0 ~'m o e c Y '~ ~ ~ Y e'C
n gj~
~ V C Y Y = u ~ Y 'u ~ =a ~ 0~7 a1 v c~ y~ a 4~ S; C~ a~ C7 P N
'/~ ..T+ _ =p W =~
t 24 n o x x x x x x x x xx x - gl -N y >
Y o o W ~Cy~ ~9~. 00 9 fi! x O, t~,i bp =' Y o~ o~ t v~i ~!1 '~t~. =~
E v S a $~
Y ~.r a mY
Yo.~ . o a,W .a aDO ' Y 'n"v Y! u -0 Y~w ~{L
a_ ~
O ~ Ly i. " y M '=' ~' t~f pp p 7 ~Ci =v ~ O
'C S~ W sC.gp~0 aY0y(,,,, O~i aJ0 p~, a a~ V J~ fip1, ~.C t+1 .~' ~ C ld Cl N'~ Y lC0 W' v w E" v~j O p0 O_ A J ad .~, N Y o r' ,n ~i11 = o a v1 o a v~ U a=p ''"b 3 3 Q'e 0 E x v~r J, o o U ~$~u ~~vum"$r ?E"NCE"ry~~,Y~ SO~v Y g E v~i ~e ~p~ al O ~G 0 ~O eC C ~~ ia o $ 'D o o e~
.N p p ~~+' 7 'Lj ~ = C V1~ ,q O Y 4R7., d' ~ euE ti E'fl CH o y u~ H~~
a~- a E :: Eoe w._ c Y._ c=;, ., .. E N
oo uU E
~ u ~M auoe'OO ea0o~' ' ~e$r .4 o oc oo t Q'_ U~.'4 v V er ~ ~b M
S) Eo~- $- B Q~u ~.t ,rj~ J ~=..Eotc~ '=3y~~Q~ ' 7 O~ r=-~p '0 ~ !C ~(y. = 00 p,~~C's 0. V' !y W L' 3 u w o cv V~l ~". N..1a '.. oo V Gei ~ .;~ , vYl . w eo .l E m,~ o) aa -: - i Y v E,oa '.~~ p OL~~4'~ W ~ v~'~ L] oj~ 3~ 4C
N N G W~' Y Z7 J ..3 tC0 ~L=" 3 ~1' Y J~C Cy+~. a W G T~'r1 C'~ =..
:9 ~ ~ C Q 0V' n Q A ~ Y C ~ U 0 Y ~.' Y ~C Y O .O t++ '~=,~,' Y ,D ~
Y' oo~r onu_ Y ~aec' '3,. E i ~ u o x>sEY~~ 'r~oo~p : u 3~:~
~ v o Y E~+ _ u E E~+
pa yi,~ ovh- Uo~oYCVoo3 m3 Jg, o~'omU a z~"'~-eu~3'S~
N
~ Q p u e .o d+ Y
~ ' ~ =~ F a y E
Y =C V
l0 ~ .C W N ~Y Q =
u Y o y N~ 'O
Q ~~ ie id eYO ~ E
Q U J, a~ < x a ob m ~
v~i 60 0 e~o eo~0 O r e~+i o0 C~ N~p O~ 9 k-I
~ N O ~1 vl ko r n ~ p ~
l~ h f+ h l- I- 00 Oo x x xxx x x x x x x x x x ~
J, =~
a U ~ =~ ~j =~ r ~p. =~V~.r yC..~ C 'M~~v =O ~ ,O
=~ YC1 4 ~ ~ =T'~ ~\ C 1p y o C c7 O, O ,O C~ ~O
00.C QD.E O~.C N~. ~O+ C y d~ 00w=" 004+" 00 OD4;
= o'~ E~o ES Eo ~o ca tn E
u~ ,N~ =n 7 m O= N wrT E~ ~~tL~ ~~~ Vc~ d~i U np u v~i C> y _G a G O .n y O h O ~.CC ip ~ " ,O O ~ y z N~ N a =N O fJ ~' .p .~ C~ C C C C
m V
J~ 'n t~- fn .C ~~~ a~0 G t C~ e7~ V c S Vo Vo y !tl w. ~, E Ew E E
oa o a o a~ o Si ~~ a~~>; L es E c t o~.2 o~' u e G~' m o v'~i v'~i 4 oE .. E
~
uh~ o ~~
E~
s V a7 Qv c6 Qv, V-, 5 y w V.e 6 ' i,' :[ a=T.p.~:
> .C a ~E VcZN5 y p~
~ v o " o'- .~ C4.
iO VI E~3 a C O 1 .Y ~~
~ N V N
~' .G ~' =~ pQ, "yN~ ~ ~y =7 =Li .C N Y J. ~1 0-~ F~ C Vt ~ V 6J . d 'd ~. ,C 67 C~ tC e}' tV e{ tV d N a-Q aG .vvv m~nva eo~rn~ y aG o na E- a E -a E -n. E
~ ~e e E r;f c el E
,a_ aci eo o on ~. -'a~~ 61 v Z y ~ u u u E -a E coyy~Ie E a E
E y - E ~ r? wuyi y v~ w ,Q~ o~ u E
E $ _ "
E a Ep u E E
E
<
a < -en~ av a c~ a ~O id Qa ~D w 2 8 ~a a h vOOi va\i N 00 OPNO o0 ao '00 00 9 X iC >C iC >C iC iC x x giC
w ' =
.s ~ 9 _ _ .~ .9 ~v ~
=C ~~ O ~ =O O O ,p1p~ ,~ p N~,~=~~~_~
0 3 ~ g o 6 $ 7 o 0 o fi u x~ oo-o E...ppp o~ Ey~ o E Q ~
o o ~ o E~ oc ~w aN~ E~ o:CC
+ i ~~ E P X ~
aow: e~~: o e eow: ao~.: mw eu~= m eow E~r E o u~
Eo = Eo .Eo E8 Eo Eo o Eo t~ X~" ~~wm E E ~=~ E E .2 y .2 y E E 9 g o~ F fi E
~ ~ ~ ~ eu w u u N v H -cl u V1 (71 u~ u N ~ Vl ~~ '1j yj E,~ ~'=
C C C V C C C C C C C 'V fQAd u u u ~ aun~ .~i 0 U1 U: ao a U~e Ua ae ~m ~p uyx 1R
L Y L F. L L L C .-= V t3 m O.~
Ea w w Ew Ew . E~ . v~S
g.~ ~gt t gr ~s gt ~ afU ~~.5 c G~ G~v~.
S~ ~~ ~S~ }! L = g~~~
N N +r N w N V r ~ N N N V C= C U r,. N
0 0 o e,,o e ov v..~v..ov oE
B~'- 4-E o b; E ~o E o -' o e o~ p ~ g'~ p ~ a tc ~ m o ~e u ~ y eou$ Ea vao Tvp~ Y. ~vp~ s E
~. TvQ~ Y ~pv~ Y ~~.p/~. fn vp~. = Tvp~ Y, vp~ lY C"~ Y p C v lt1 R O N ed O W id /0 O ad RI O ad RI O i0 lOT O W~ O N O L! ~=C. O o~~=T N
m E v ~ o ua e ~'.S
ov! csc a o N =C ~ ~C u =~ .~' p '$ ;q U
Q aC
a~
E ~~ L'_Ov ~>~2~
N r0 ~ W OE r E -a ~Y tQ ... E h= N o tO t0 E E E E "' E-v~ ~a N a U a C
~ d E e F y 'N
p~ y N r, N ~d (~ v~ e! vf O O C
.. ri c~i. Q~
u A. A. A.
Eto E E t E EEF.~ ~ ..
Lu 111111111 ~ ~ a E =~ ~~
ct E
<
pM pM oM QtM ~p+'1 Mp fp~l Mp Mp M1 Yl O~ O. O~ ST O+ O~ P O~ O~ 0~ O~ Q\
x x x x x x x x x x x x tz 8 V w {~ /1 = =~1 U~ u y N M U e E q, N N F ~ V .C fff~~7~!!! ~
.E 'a E l'~G' =--= C
h~=fi y ot"v~9 Qo~o E 5~ eox~ o~c E
E an u. ry u a~ u=v - o v o a'~ 3 ~ a~
~.. ao'c c C
O O g E a~ N ts E.g U r '!1 ~,. N
~'.=.~' c.~ ~i~ a oZ ~ c-~g ~'f vi gur c~
oa ~ 3 +
. ug U S u _, ~'00V v F- ~ o o.! F=5 =p~op'~vE o~ ~ ~'~ o1 NTRx ~ooE_o~
vi c~ EO G~ Ci 'a U U V u O~p l6 C~ ~' =~ ='wX p v W C_ ~3 G C
d ,au3 u ~> o ooV v:: u eu im v~ e oe 0 i 4~ uuVo'I '~ o# co>~', u~ ~' E? L,~
e ~ p~+ E uppoo~ ~'.~ W o_ti uc~
U O~ L V y Y p~ O Z,~ W= N N o~'~ o'~ c3 .
o cv c u v ca E 1_5 U E ~~:. U v v~~'~n u n Vi Y'i ='Fs~ a'~u > 3~ _.~uo~~ =~.nT.~
la ~ Z e c ~ $ ~ ~~
c V o ? E o, ~ c .c a ~C N U a ~ uu7 Ae . a~ a;o;_~ .~~ ~~'" ~C wWS Eu' ~7 U.~ ~+ w ~~.O ~d v~ C~ p,; =~ i0 d voi 'C ao L C~ V C p um u u r v~ u ~ o e o~.y ,a e o'd ~, o v u o 2 " v E ~&' V_ dv-8a.w~uVe "O EO ~~rETO~~ac.ldÃ+aoeoo V ~~> ' =~' uoa'o. d V cp u v a Q~44 ~n o a,V o ...-ac~..S o0o mva oo~
a a u o~
u '~ c o o e_ ~, ~a 0 0E
a E.~ C r -O c v i m E
n l0 ,V~ =ld yn C Q Q S u 5i '~ r ~ I w w hG= G ~
~~ c 0 0 C ~! =E O~ ~ O H
~ U O L y GO A'~ a t '' .~U
~"~ ~,a u =v ~ co c ~ ~ ~ a T~~ ~ o C ~~ =Y ~C ~ .U., Tg "
a o ~ ~ e~ vs o u z c e~ > 0. E e E~~ vVi O U S O~ 4>
Mr A ~w x x x x a'v' ax r, Q
x n>
cgo 0~0 N~~cc 00 ef V1 r ecct O v r h ~~I
~n ~ N ~ =- ~n ~n N N
x ~C K g g i- g Y ~ $ $ ~- ~--g5-..
E d o T6-V pq ?, o eEiV C~y=~
b Y~ ~N Y Y p a. . =~ y u~ U_ EM ~
w E
A~
R Y1 >>~ C U
p ON y E=~ _d0 ~'jf~ ~=x G. G. ~~' rU ~itf oO
z 95 'Bo'~T v~~ oo uv Y~ ~iy EQ
~ac ~ u~ E~
~:-->.? uy~ .~o 00 v aw oo Y a.~ a eDco J! ~ u O
C~ e g.5 x~ s e v v fi ~ p H E
vV, ~e~uu~oE~' _~" Eo e ~ w C~~ u y~ =yo'~ OQ 3~=~=r =~ Y
e Y y o u Q M v, u o ~ w Y=~ W c. y.~~ E U C y O" ~. M U>~ U 1 O C C Q L ld Y 1~ d y qp L C
ocnpor;_ p i ai = ~i ~r V=~~ u v', IN t o e~ u E
u a $ Y'op~ Y~~ l ; ~~- ~ E
viE~'~ ~~ Q~ d m uo~
eo Y-, o~ ?. =~ u -,~{ ~a %0 Q E~~
p y.]L V O~Võ Qp y Y Ct h L~ ~ Q~ tO Y S 7> Y'~ p v Y 00.5r .c Q~-Ys aCi ~.c0 7Q:~ GLm.t...~~.-~.. 0 Rv10C0 00~ Av~U ~O=p v u y n 0 c N Q U v ~.c ~ ~ YO- Y C~ dr al 'X O C
E a~ z u u c u u Y p G ' u_ ~ y N y Q~ ,p, ~ N wA ? Q ~ ~, o a =- y ~o w b E
e Q E u E ,u y ~j $
co (hgX v h C7 5 < Q v L] v ~ C7 in U v~a c~' E~ u s 7?
>
T =p Q w lm 3 a 'yY
o ~y v y b h N M I,. R ~
a~0 v1 h 00 ONO N
?= i= >- N N 1~ N l~~V ~ v ~ g u N N Q ~
TABLE 3: Corynebacterium and Brevibacterium Strains Which May be Used in the Practice of the Invention Brevibacterium ammoniagenes 2i054 Brevibacterium ammoniagenes 19350 Brevibacterium ammoniagenes 19351 Brevibacterium ammoniagenes 19352 Brevibacterium ammoniagenes 19353 Brevibacterium ammoniagenes 19354 Brevibacterium ammoniagenes 19355 Brevibacterium ammoniagenes 19356 Brevibacterium ammoniagenes 21055 Brevibacterium ammoniagenes 21077 Brevibacterium ammoniagenes 21553 Brevibacterium ammoniagenes 21580 Brevibacterium ammoniagenes 39101 Brevibacterium butanicum 21196 Brevibacterium divaricatum 21792 P928 Brevibacterium flavum 21474 Brevibacterium flavum 21129 Brevibacterium flavum 21518 Brevibacterium flavum B 11474 Brevibacterium flavum B 11472 Brevibacterium flavum 21127 Brevibacterium flavum 21128 Brevibacterium flavum 21427 Brevibacterium flavum 21475 Brevibacterium flavum 21517 Brevibacterium flavum 21528 Brevibacterium flavum 21529 Brevibacterium flavum B11477 Brevibacterium flavum B 11478 Brevi acterium flavum 21127 Brevibacterium flavum B 11474 Brevibacterium heatii 15527 Brevibacterium ketoglutamicum 21004 Brevibacterium ketoglutamicum 21089 Brevibacterium ketosoreductum 21914 Brevtbacterium actofermentum 70 Brevibacterium actofermentum 74 Brevibacterium actofermentum 77 Brevibacterium actofertnentum 21798 Brevibacterium actoferrnentum 21799 Brevibacterium actofermentum 21800 Brevt cterium acto ermentum 21801 Brevibacterium lactofennentum B11470 Brevibacterium lactofermentum B 11471 (~u3 FERM ' NR1ti:. CEGT = C11MB CW;: NCTG DSm Brevibacterium lactofetmentum 21086 Brevibacterium lactofermentum 21420 Brevibacterium actofermentum 21086 Brevibacterium iactofermentum 312 9 Brevibacterium nens 9174 Bravibacterium nens 19391 Brevibacterium nens 8377 Brevibacterium paraffinolyticum 11160 Brevibacterium spec. 717.73 Brevibacterium spec. 717.73 Brevibacterium spec. 14604 Brevibacterium spec. 21860 Brevibacterium spec. 21864 Brevibacterium spec. 21865 Brevibacterium spec. 21866 Bnevibacterium spec. 19240 Corynebacterium acetoacidophilum 21476 Corynebacterium acetoacidophilum 13870 Corynebacterium acetoglutamicum BI1473 Corynebacterium acetoglutamicum B11475 Corynebacterium acetoglutamicum 15806 Corynebacterium acetoglutamicum 21491 Corynebacterium acetoglutamicum 31270 Corynebacterium acetophilum B3671 Corynebacterium ammontagenes 6872 2399 Corynebacterium ammoniagenes 15511 Corynebacterium fi-jiokense 21496 Corynebacterium glutamicum 14067 Corynebacterium glutamicum 39137 Corynebacterium glutamicum 21254 Corynebacterium glutamicum 21255 Corynebacterium glutamicum 31830 Corynebacterium glutamicum 13032 Corynebacterium glutamicum 14305 Corynebacterium glutamicum 15455 Corynebactertum glutamicum 13058 Corynebacterium glutamicum 13059 Corynebacterium glutamicum 13060 Corynebacterium glutamicum 21492 Corynebacterium glutamicum 21513 Corynebacterium g utamicum 21526 Corynebacterium glutamicum 21543 Corynebacterium glutamicum 13287 Corynebacterium glutamicum 21851 Corynebacterium glutamicum 21253 Corynebacterium glutamicum 21514 Corynebacterium glutamicum 21516 Corynebacterium glutamicum 21299 .. : _. .. __.
:.; . , .. .:., usss.-, s es.:: _;;:~; x!.=. .'. ,=.' _ 4TN
' Corynebacterium glutamicum 21300 Corynebacterium glutamicum 39684 Corynebacterium glutamicum 21488 Corynebactertum glutamicum 21649 Corynebacterium glutamicum 21650 Corynebacterium glutamicum 19223 Corynebacterium glutamieum 13869 Corynebacterium glutamicum 21157 Corynebacterium glutamicum 21158 Corynebacterium glutamicum 21159 Corynebacterium glutamicum 21355 Corynebacterium glutamicum 31808 Corynebacterium glutamicum 21674 Corynebacterium glutamicum 21562 Corynebacterium glutamicum 21563 Corynebacterium glutamicum 21564 Corynebacterium glutamicum 21565 Corynebacterium glutamicum 21566 Corynebacterium glutamicum 21567 Corynebacterium glutamicum 21568 Corynebacterium glutamicum 21569 Corynebacterium glutamicum 21570 Corynebacterium glutamicum 21571 Corynebacterium glutamicum 21572 Corynebacterium glutamicum 21573 Corynebacterium glutamicum 21579 Corynebacterium glutamicum 19049 Corynebacterium glutamicum 19050 Corynebacterium glutamicum 19051 Corynebacterium glutam icum 19052 Corynebacterium glutamicum 19053 Corynebacterium glutamicum 19054 Corynebacterium glutamicum 19055 Corynebacterium glutamicum 19056 Corynebacterium glutamicum 19057 Corynebacterium glutamicum 19058 Corynebacterium glutamicum 19059 Corynebacterium glutamicum 19060 Corynebacterium glutamicum 19185 Coryne acterium glutamicum 13286 Corynebacterium glutamicum 21515 Corynebacterium glutamicum 21527 Corynebacterium glutamicum 21544 Corynebacterium glutamicum 21492 Corynebacterium glutamicum B8183 Corynebacterium glutamicum B8182 Corynebacterium glutamicum B124I6 Corynebacterium glutamicum B12417 . .......... ._, ._. .
' P2~
Corynebacterium glutamicum B12418 Corynebacterium glutamicum B11476 Corynebacterium glutamicum 21608 Corynebacterium lilium P973 Corynebacterium nitrilophilus 21419 11594 Corynebacterium spec. P4445 Corynebacterium spec. P4446 Corynebacterium spec. 31088 Corynebacterlum spec. 31089 Corynebacterium spec. 31090 Corynebacterium spec. 31090 Corynebacterium spec. 31090 Corynebacterium spec. 15954 20145 Corynebacterium spec. 21857 Corynebacterium spec. 21862 Corynebacterium spec. 21863 ATCC: American Type Culture Collection, Rockville, MD, USA
FERM: Fermentation Research Institute, Chiba, Japan NRRL: ARS Culture Collection, Northern Regional Research Laboratory, Peoria, IL, USA
CECT: Coleccion Espanola de Cultivos Tipo, Valencia, Spain NCIMB: National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, UK
CBS: Centraalbureau voor Schimmelcultures, Baam, NL
NCTC: National Collection of Type Cultures, London, UK
DSMZ: Deutsche Sammiung von Mikroorganismen und Zellkulturen, Braunschweig, Gennany For reference see Sugawara, H. et al. (1993) World directory of collections of cultures of microorganisms: Bacteria, fungi and yeasts (41 edn), World federation for culture collections world data center on microorganisms, Saimata, Japen.
&' ~' 0t+ o~,~~ $-' r F= y F~ HH F~~-_ ~~ ~ N CQ N N N N N O U O N ~ G.) O
cn Q
Y ~ E .2 C, V
p E E E
Y~1 = G o bU O OYYY Y U S~ J
E
N
~ a~ a $ .~, o~gc $ %7 a. a g c~'i ~ ~ E ~ o gn !
~p ~p $
o~ O z O Z in E E 5 E
z_ J y~ W ~ W 'Z
E ~ ~ ~ ' 1=0 mrn Ij v ~
a o 8 0,2 Od e ~ ~ c r A
C _ Y ~ 0 _~~~ oa ~ a=r~W W LL N .g a uE~ p~9 ~N c ~
r y Ot a 8 a ~~ 5$cm~+ E'~m L' d vi c'S ~ cq ~ it~~ v~ nK E c E v w v~
1~0 x C)so I S ~o c ' u E E3$ 9 E 4~u ~~ Cy m 6 Us $~ JjIH1JI ~ ~ w~$~ W E vi g cl~ i~
A? o Fo V y, a~Q e e.~~n z m ~
m D 2 w g i+ a g =~
~ =~ N a g ~ ~
i Q~a $ n"i N~X~ Q U Q~ 4 W ~G W ~.
~ 1n .=. ~ c., '~f as col ~ 0 1D !7 tO ~D
N
~= N N
Pf ti n ~+ M t~~O O~ f~ Q oMp atoG
w_ w w w ~ ~ o~ a a~ a a ~ i i -Q1iq~i i fQi ~ i C~ (9 C9 U' [7 C7 t7 C9 (7 ~ C9 C9 W C7 C7 W C7 In LC LC
W C $'j o~ 0, ' ~~ W f~ t~ ~ OO O 1'7 O f- d1 dl dl ~~j N N ~ 7 C N C < ~y N 80 tD Y
E E E E E E E E E
~~ tofo ~ otfo o s'~~d h a E E
a T II T a a T~ D T L ~ T~ $ T~ 7 7 U~U v, _~ .232352~2 .32 a 2 3:'2ao: 2 ptf1 {A ~
m (O m m INO m INO ~~ t0 (np m N N N N ~ (a E E ~ c u d ~ g ?W
4 O O T ~ Y' O O O Q V/ pFpF
.7 8 c c 0 N CI C C C ~ O~ V C~ n rn rn uo ao a O o, ~ o=r V 8 N'~ E E E w ~ E E
~p 8 8 8 8 8 8 8 8 8 8 o a a Z 8 ~
lx-~ N~~ ao a '2= i , = s = x = 2 UmJ ~ 2 Y~I
My_ h N Ny OI N 0 y~ N q y rl V) ~_ y y q*
~ h' "' O O O O O O O O 8 O OO O W 7 O O
o~ Eo '"
E s ,~$ a E~ E E E E E E ~ E WNc8v1S . EE
c c 7 7 _3 7 _7 7 5 ~ o~ Q~ 7 ~ 8~ a acr u m ~ m m v o m o m ~~ ~ s~ i. ~i y't t0 ~o tS i5 tf ~ 25 t5 ti ti 2f tf tS 2i ~ ri YõA o Q
f C~ M 0 N /0 N /C W m w ~= u 7~.
ac v) E
~ o h_ _o 0 ppp ~ N (O < p p ~ ~ ~
~ p N ~~py N~p Np N ~ < ~ ~ ~ N
Of ~ O~ O~i V~' a0 V V V
W $ N 8 $ 8 N
=- =- ~ ~ g o o g o g ~ bi ~ ~i ~ r~ ~ ~. aNO a )o m M r ~
a o 1~ ~ o N ~i ~ ~i n N ~ w r~r. ~v v O~t ~ ~ ~
O r$ N ~ vf N N N ~V ~ ~~iS Q ~ ~ a g~ ~ U U (~ ~ F U C~ (~,? TU U ~~ ~+~f g J
lt~+W W
~ (a 0) I ' z~ di af i W ~ ~ 0 1008 8 n ~ ~ s CR n a ~
~ ~ ~ W 01 g ~ ~ ~ ~
St ~ N N N r =- =~ pi N N 1~ ~- r c I Q Y t~+) c1 =~ M l~+f ~ Y'f 1~O ~ N kn f =P '~f ~ tq f s' ~ 8 "~
8 ti'cV q q E
p N
W
L=a taQF_~ ,$ 7 p 7 W C .~
6 N ~
WI c c cn~m a a w~~z wZ 8 a o m > > a o ~ a ~+ ~ m 'c W rn c N
n ~$ o, fn m 1 2.
~ f f a g r ce n 2 < Q
x R Q.
W
g >? b y ~ ~ m m~$a 8 t W ~ ~ "
> E $ a ~ V o, o, w ~n ~ $
C 2 n a a E
~~ o a c y a6 3 v!~ ~c t~r o a W u~o N--m c ~ 8' m ~ ~ ~ r F q=
T i a = g IQ p a8o o~e rvgEtf"'~Q'~' Vm r- n a~'n E N V E y $~C O V ! _C c7 ~ O O s f~0 O C C
M W p _ =~ c :o ~i~
~ nt O = ~ 9 9 owc~aE W n 8.~ 2- ym $ .8 A .8 1.- c C X
n = ic~a'~~E
~ d~ ~ ?c ~iw E a'rE ~n ~ ~~8 7 8~~~C~x W=pc E ~ ~~~rgQ
O~ 'd U O f( CQ C~:~ l~Y C~~y tl L V V V O } p N E
y 8 ? 8= ~ W 'C ~ g f p n }~ F W p Q C W y. a t~ - n a~y iz V ~ o C
m !~a ~ ox(~ 2 tCZS W z :N W W c.. wa o~ ai ~ ~ ~ a lll~~~S!! aQw ao tn cn >
A tti aD
~, t0 N"
w a n < N
-+ < a a d~ s o N a~ d m a ~ w N ~ aD m ~ N ~ t~~~ (O1D m ~ Nf O m c~Nf (~
O N awp wQ Q 9 !R LL' Um ad a ~ '~~ 4 a ui Q ~a a ~ ~mi N
m m m m m m m m m m m m m m m m m m C7 t7 U' L9 C9 C9 C9 t9 (9 (9 t7 t? C9 ~ C7 U' (~ C~ C7 C7 l'M M O
a N N N ~V N
st o, :
O lIIVVV O N~-= ~ N O W
/") < M f P9 l~+f {'7 l+) {''1 l"1 M t~~7 l'pH V 17 l'7 l7 {~ t9 l'7 I
_u ~'0 2 2 u~p g Ll N y N ~ C C 7 ~
fft~ r t EEx~ sf ~1 E g~s~ 2 2 1 2 2 0 p i f Z~ J V1 N v1 I~ ~.r~ a V
iW ~ Z Z g N 2 2 c $ v ~ a Z_ Z
E 2 ~i i cT 0 E az c9 Z O
V) 8$ W N f.~ 1 a W W 1 Ea ~O ttN dl J = Z 2 v) g ~ n o E =UW
-zo C W W E ~
N
G_~ 27' 0 a ~ 0 X y o N ~1 ~1 r a n ~ a E ~a ~ E~ O
R 8 ~8 = o ~ ~ ~a i1hiI ililfi o~~- {- N " 0 rnG %
~ C ~
E ~' o E o~~ c 3 c a o ~r~~'rSE
p -f G o ya p W c ~ c ~ a Q n g n ~ c c 1-F- c v~ U cU SIc 9"~'N - aa a a a~o em ~
~N~" n = '~ o~ y 1 ~ c a a ; o q E o.4 ~ zs~t~i~W
~'no5i '- 3 m 4 ' qw ~'a~>=~X'- Ee ag=cWo ~n $t P oN ntp o E .~a oc '3 = W
Z
~ ~={ ~$ O ~W 10 = N~~ O~~-= i= CE G ~ S >~! > S a S J J f/) fQ (n 31 U 4. G
OD N O p h ~ OI ~ Sf qpj C~0 p N _ p n a0 N N op O1 ~ N~ N ~~Nyy N
p U V p~1NV O(p'q1~ U i0 f0 !o' t+01 O U V V v 1L ~ O
Q Q r J X Q Q X X J J J Q Q Q Q Q Q Q
OD to7 f yp f~f N 1D oD 10 ~~ a~Q qf~~ Ipwp ~+
N N V r~ ~- 1 c0 CD ID ~ ON~! M N O~f N N
OD
$ U! g~~
auNi a I
~ t7 ~u m'U ~U' C~9 C9 ~U' ~ c~ Um' C~ tm7 tm7 n t7 ~ c~ cm~ Cm7 c~9 c~9 C~9 ~
go cn ' W
N 'C~ t~1 O N O N N N ~
n a~0 q ~
h ~p N ~ V N
M
O = O f~l ~(f N lh 10 f0 N t7 ~ lr1 M
n n Q y ; co ;
t c c E E
c E ~ ~ y m a y a E
E -'~1 8 ~ Sn m ~ o ~' ~ ~ n $ a =~ ~ 'g 8~8g~ g 0 ~
x aa N~, N 14 ~~ Z ~m+ g W 10 y _~ E _a U t (~ g N rLL~' .- ~ (o ~ 'J Ep 9' , Z y Z tO mC 'j v Q al Yuj E q2 o fnQ ~ $ y p w ? ~ E
L Cm y~, ~ a rn M U x a N N c n oO U'oO ~ y~ g OC r ~
~ =E ~
c =a T g U ~ WZ
4 ~
_~ c v c Z m'~S o -_ ' W ~ rn p ~ m c C3 ~i ~ V g o E E ~-u c V v) N aQ~{ "' E N ,QE 'yt m o~ n} t S = c_v i, a E
? m Z' S~ Z' ~~(7 n~~ " v m~~ V q~ yj V ~ O W W
~ E E o E ?3 >
q ~ ~ E (r 0 n p S ~ fV y y = t0 l6 ~ p 1=~) E w E o QH n n m y Q~, V Cl ~4 h= E U t~ a N n ~ n 'n r m m ~ O E E l ~ E '~'n 0 R W o p o n oy E k o I y m m W ~ p, A Y ~ ~ _C ~ O n C C ~
Q c m EY .Y ~ i c Z Q~ =~ ~ E
. c ~ N c ~ n ~ tn ~ fn ~
p F.. <~ =_ 0 mrn ~ 'c) ~~ !~MV~a$ y m v aU m m .p 2 ~
Z?i ZS 's W ~ m c h Z(n L~ ~
E NI 2 C? ~ Q G~ C7 cry+ m a t i~S n6 A
LL N 8~5 ~O8 8 mC'S ~b Co~' ~~a 7 4)O ~ o 0 m <xx x xai~ cia= ~~ E ~Ca 8 ~ ~ M ~ ~ ~ N O U 1n x 8 S p~
09 O ~ y) $ M Y1 1~
CL
Q Q N N Q } Q N N N
i ~_p _ ~ in oMi ~ p ~ Y N ~ Y'1 n ~ Y Y ~ !~+! 1y N ~ ~ " C7 ~ N
N N
N p~ ~ p O yMj O xN N U U1 1A ~ ~ ~ CO
N. a _ ~ N N N q = ~ Vl q U ~ U Vj U ~ N U) ' ~ 2 ~ ~ ~ _ ~ N = = i22~=
Lr ,W, 0 a On (D 0 ~ 04 ' N N
g 8 09 s m' N ~
C&
~p m a0 W tp pM fD M th C~O O) O Np Otp M n nCp T iT 'a ri cr) v v Ili a o a vMi u~i l~! cTi {'~7 c~i e~ r) {! r~i r~i E ~ y E
a ~o c E S N m a c c e ~ L
E
a n &
c co ~ O V N w L
s s $ $ o 0 0 w U U o~ - ~ a 0 E ~ E ~ ~ oE 0 w v) w O x O a~o a~u < a < x < < = x =
= 2 $ a$, 8 $ z z w ~ L nc E B.
~ o t ni O~ N
8 8 z z O w w a a ~ W' x >
E E g g y~
z z z a ad Q "' m E m E
Z 2 c E 8mt d a~t .E v 8 o ,n 9 vi 'a 0 n A ,a t o > ~ n Z N a a o j E U a c O O 8 8 aC 8 - O E ~
(n N y ~ a v N u lt U. O Wt Wt N
y ap = O ~
V t o < z O O Da U < E o ro m m 8 c U c6 U U a M O ~ m n lT ' P "' "
~ ~ c - -p 1 g 9 E ~ ) 8 E 4 yyyc ~ 0 ~ o a n a m 7~
E~ C ~ 7 E O~~ t0 N y~ N O O Q ~1 r' ~ 10 3 2S S t t q E a E ~ E E m c ~ c c ~ x~ O Q O Dy y L ~ Q 8 Y m o E x N 16 OI W Vi ~~C ~ N ~ O = 7 O 8 0 ~ p OI OE; z c 2~ Y c fS E c~S .~ Nc 8 - 8 E E m 8 c' 8 L o ~
~?_._ 2 !3 0L 0~ ?
~ ' 1A 4 1! n M t n Q= :. w ~+ = $ N 8 W N . lO a N a a W ~1 U a '~ ~.'~ =' t~o E $0 L$~~5 Z~ a-68 $,.~i o oF r ~ ~ c a c~+y ~~ Y
~ E~0 O O 4 Q 2 3 u_O N O N YWl O
~ ~ uWi K v~i N o a= ~ p Q
~
E c c c c~~p ~ E E m n ~~~o ~~ E a a E~ E~ E
wc'0i~c~r'~ wcnr nwQ wa Q. a Q < Q i Q a~iax ai 1~ 2 m N M N A
~pO $$ o p fD Q ~'~QY( (p y M f~0 O O ~pp ~C < Q >> _n m m m N O l'Mf M O Cp O m O O p aa ~a x x a x a Q, ic Q Q ~ Q
N a _ _ m g _ o a ~ ~ ~ m f fO a ~ ~ v rmi ~ ~ 1Ip .~
~ aMD h I.
O/ W cD
M M 0D m aD OD
cr) Z z Yl P. p~ N~p ~p up 9 w 10 N
-~ h O O N on0 gY tD ~p ~ f~ ~ t= s m c~ ~ U c) ~ >> b~ ~ C~ o o Y cS a Y co w fV N 4' v~ q 4 a a ~ ao 0 o t~ t~
S cF~ a S
W W W W I 1 I 1 I 1 1 i I 1 ~g a~ 1 I I I I
(7 ~ O C 7 ( S O I 1 CJ 0 (7 (7 (9 0 C'J C7 (~ (~ (~ (9 (9 C~ C9 D G(p o) N M OD ~ N
M N
LC
~ ~ ~
r- co 7Tp-QR 5 >
U~
O N
N OI ~ N N N N CE00 N
N Y'1 M r- C! N OI h~p P N OOD N O Q! U, ~O M
'~Qp {Yp f~ O~p ~ -p~ 1~ l~1 O t0 Ol M M p CCC000 M
t~1 < lh ~ ~ Y f+1 U) 1 M ~+) ~ tMo (NO ~ l+~) lN V~1 Y
W y O
G~O W~ C C C _7 ~ W~yy~ E W C C ~ Fj ~W 4Il mC q 9 E 7 .a .~ .~ 7 v d . L~ t ~ . .2 !A _/
A m Q.9 Wa4 g .3t'imEbQ $ ~ n N N W 7 2i W W
~co ce g E oE o 0 o E E ap o NQ o h W " ' " a E ~
N O E LL >O, ~ C EO 0 EO CO
o~ x x x Uv~~23x x x x 222 a = x x o Ci Q r ? Z
~ ~ V J _~ N W H
--. " g ' o ~ u a u a w W
~ E 0 c rq~ 3~ u o c c~~ _ _m c E a c o E g E
o tci ui 1 - 9 '8 4 x $' 'm E E
a~ 9 ~ $ O õ c o vi ~ a H E y E Hf 'C a E W v .9- v~ ir E rn o o ~ x E nE az~ Om 0L ~ a m? E a ~ s cNl C04 I
n W Y Y ~e 1 w m C7 I I x o .. ~U.1's LLI~
a D) Q N N 01 O ~ r'E~E ~ Eo Eo v N ~ YE~
0 0 o Nro ~,~o c a W o '" n 0I8 mva a c E a E _ M
a ~ o~
2 o p o aci p o py~l m ~ L t E
L r,m QV~m _W a Wl N V m V v ,~ ~ V O m ~ O ~ M H
p- Q Qpp c sc= s: g EN E1~~ dn ~ v Q'o Q~ cr c, Z>
~ Ut=/~ V a M Ca p v: V t 70 .2 1 1 m~ 1O W W GDU2 0 ~g WI
~ Z= ~ 7 t.- y.- y N x ~== W ~~ ~ E 8 m 8 c y m H W 1- ~i c y ? w~ a ~
o o c7~ U m~o o W U t Q $ E'o E a W ~w o ~ 0 a E~ 9 ca'o m m Z W ih - H o ~~ E~ o s m $ ? E a 'n F E "a j -~ 4 ~ 0 E a L
c a E.r c Ecv ZL ZU ging cing W m y W n~ ENa n2y~ o 2 L Wp~~ Z~y o H Q ( 7 U My W~ 1~.. S~ Wm T ? ~?~ .~..
ZS NI y Z C Z G~~ N E tn O C ~ C 2 _I O 0 O N w 7 j A y UC Z C Z C 7 ao O tQ o O N 7 Z nZ Z
= = x' wD ~ m m ml m t0 ~ m Cm c VnU U n{~ c ~ V! ~
W N
W ~Cy O M O O~ Q + O W N W
1~. U L O C O o~ om o~
m i> o W ~
8x a'xW vw v x i ix l'7 ~ N l~ _ h OD w N M l\ 89 2 e~ 8 Qfg V ~ O O a a~ b O O g (D M N
D ~ Q Q N
W J J ~ LV a J ~ 10 I OM O) c~=c a< a a xa Am z A~d a a a~
M {+~
A Np NpN h Q~~j CO ~+j N ~
O! n ~ O
O N" Or M ~ CO S ~ V N ~~ W CD ~n+l n 1~') N t~
p O ~ ]yff~y[I Q~,~I I~ ~p M ~ =' ~ ~ N 1' ~ ~ h_ 1_q ui m _ 4 4 aV >- '' $
U ~J Q ~ U U ~ U S
aN ~ O N N N ~ O (J fn ~ Re.'~ O ~ ~'Zj ~ ~~ N N 4 I' f6 Z W W W LU ~ m m m U' W W m ai ~ _ ~ fD
1 1 1 1 1 t I 1 I 1 I I I 1 I !
~
cp n 8 8 n M ~
~ 8 oE 52 g E $ d ~6 E
OD m p~f .- ~+i1 ~ll N ~p ~f! N ~py ' f ~p 10 ptD 1~~p pa P
~~lYf ~? ~~p0 ~~p p M N~p N~p 1f1 f~ P ~p! h~p ~p M~p tD a t0 tn~l l0 l+f m +f '7 M V ~ ~ t~ t'9 t~f H V d 91 0 o ~ a E "~
> c~ E
~ E.n H o $ ~ '0 $
x a ~
~ 10 ~ o ~a õ a a ,~ I
~ QM~ E a ~ C~ o ~ E .~ .~ ~ =- o 1wlO1- N
c qoe~~'~ a~Eg$ 2. 5- Yo ci W tq ~o~Cc~cH S 2~ ~~mo~ o~p C~ ~yao W n g~yfW a o G~ x~ O LL d C
~ T ~C p. .. ~ ~ > ,~ 5 10 l'=
- LL~~.
G L
c_o m a 3tvtnvme~ c (n O 01 aov) !0 vUN~ gwW c =v_l Z'C ww= cg V ~ ~w F- o a VU E~ ~=q vi~ o ~ m o E~
$ H V " < g .
a w z n zS 14 N ELL~
F3a Ac~c_Z~
qc Z t i a~ ri a~ o ~ V v' To i3 $~ V
D2O -~S W ~'r. t3 2S o~ E ~Z W
ct ~ Q 2 > 4 E F E
~ 8 W~ W ~~ ~ a o W S W~ g~ t N~ i W
N H StA H (D N x~ USN=t~ UU~ U S t!) M V) N~p m O~ N N
<Z x c'4 a f N O O mm ~ R ti m Y~ N aNND !~l f ~ ~ 00 40 ~ m m ~ ~O iD C~0 cq a-~ ih -' ~f 000 fz tPSN
S ax ~CtJ U ~c v N
(D 0 N
C! O) Q! , v ap ap W (,~ '~l+ ~7Tj l 7 ? V Fa 1 R N N r N tV N ci N ??~., o N $
aF a ci s 2 ig N' ~ N
9t ~9t~ N
E E
gY ~ ~ a tf E
õ
W a w o 9 9 ~ g 0 a z z õ
z z N o =
z F d a c c c Z 2 r N
N 1; ~
E
d 1 1 5 "~ N j N tn 1 ~ Nt _ ~ C~~ f Tr rr R a&i o d $ o 0 0 .5 ~fD c V ~ao~e' .~ .~ , E W
$
8r ~ U U o ~ ~ ~ ~ r0 ~a wa ab n~ ~i'?c' E
m 0 'aF ~ ~=~~=~~ ~ ~~ a ~~~ g d ~ ~ .~ g ~ ~ ~ ~ n mf/~t v a U ai S ,n.~ mn y@ o E ~T i aZf a 0 m w t~0 w q x ~~~ s _ ~ x E a~ c x x xI
I m y a Z vo a o ~g a a Fn ~~ EIE _ ji87S8~Z h a N
n~= rn pC7. N u~> ~F E c_ E E E E E F~ o E qF~D8~~
C Z ~ p (A f/) ~ .L 7 7 _7 y C C C d (7 .Ytn tn o ~ o arc ~ u c ~ N ~~ =~ Q o a'~ o a N>ZU. _N a di~d pv ~o~ z iS iS $ 2'~
Waa2 rn L'iiq~=
n y~
A ~ey ~~ a n aro m eo r~ c~
p~ Cj ~ ~Q 01 ' N_ h ~ ~p 1~ny 1~0V ~Np n ~ p (Oj N ~ ONi O Ool~ ~ V V V q N N~
Q T.j ui ~ ~ Q Q ~ J N Q N Q Q 4 ~ Q
(~1 ~ l~ AQ1~J p) t0 N{7 ~ ~
~N ~ l'f ~ f h dD ~ MN ((~y ((~y n dD N iV ~ l7 f n m ~O ~ F~
.~=-m r tn n g c'3 c'~ t~.> >- ao Q n c~
~ U n Ur =
t9 ~ C7 G7 ~ ~ ~ ~ ~ 4U) _~ ~~ m ~ " A~j Z W W d4o a 1 q~I 1 mt t i~p 1 cp I q11ap1~p1 Ipp I I 1 q~1 ~J1 1 1 f~ (~ (9 G7 O C7 C~ (~ C7 (9 (7 C~ p t7 (7 (9 C7 (7 O C7 C7 C7 Cb ~ ~ $
.
N N N
N O N O~ V O G U N
Cf ~8 1~ O ~pO~ ~S Nt C~D pfp o< Oi Np~
C~9 r m m l r /'A7 M M A
!+1 ~
$ ~ E E
=
E
EL arn m~ E~ ~ ~n a ~ is E E ~ E tj o ~ s = a ' s~ ~ ~ o t ~ ~ t~ ~ =
g QQ QQ o $~
VV
A eo 8 =- -C
m < <
N N V
GGX C - M fA
cLL, W
Q bv ' z O ~ .~ r m o 0 8 a rn ~ n n a~ U Z ? a a n ~ n ~ ao BE ~ w c m ao ~_ p Z Z a C y p a (~ g E .ic E c ia N w W W an E E $ ~N i c 0D m ~ N dW W ~ ; r t 's p ar' > g ~ C c I
0 = ~
a o e ~o W = ~ E E
~-'~g-=gs " u 3$ ~ Ã "'~ "'t c> ~ c~ ~ ? E E g ~ o O a ~ Q c ~ 8 dF g ~ E o, z n~ ao o a ~ Z rn or g c c c,~ O E. a > O 0 ~=. E ~ ~ ~ o 0 0 g ~ o tn E
a0o aVo oC E E n y%
p C ~ pppp C C. C. C. ~ E ~ E 7 OI
V O ro~~ C V =~ n=~ n=~ 40 W O O ~ U
aN a u a 8 ~. c a ~ o Ew ~ o o~ p EO~ a m $ o' o, a U~U u~ t~ U n ~i ss ~x g= x ~~(j m ~ t6 Ci % An W) I " i I
o < 5 No I i X x pp ~ n n ~ ~~ tM0 i0 {~ N IN N N{p N N
~qy e+f W
- i i ~ i i i i ~ i ~~0 CD (D 0 c ~, cma on 0 CD~ otD w n u~ n a 4m +~ 1Qj!1L~l! cw ik A r, ~ m (a ~ 4" V) U) U) n 10 $~ !
E E ~ .2 ~ y p o c c a c a n u3 O y~p p ~ ~ C =~ =~ n~ ~ ~ "' t"a L ~ ~ ~ ~ =~Q 'N W
{7 ~ H tnl O
~~~~ ai r%1 Uw~ _~ :EarUt7~ O Ni~~ ~ SS x y ~p N
w t= t ~G~r,'~ ~Q. N N
N i N m X a~ N f =
' V a' V
W '' o m a m d w ui So a z s a Z ~z Z a ~~ z z m Z Q8 ~ W U W y a~2 Z
_ z m rn rn g = qz z Y W W
W QQ== o =4~ H a o ~ C7 O~
tp.i ao Vrn õ '~ V! fA CC o L.Y N~ N tWq tWn ~N W C O > L ~" ~ U
Wt Q~ C a (p x ~~ (Jy o ~ 2 8 a= ~ LL
C G ~ C C O~c ~ C ~ y ~ ~ p O ~ ? ~ y~= CO y.
sQ E ,C ttYyll ONS $ GGG ~ ~ (-T ~f G 7 a N a C~ y~~ a N Ã n~ ~ Z' 7 7 C C~
v0._ C.~COI
ffi a c c _ _ m E M c ~~~SSS pG. c c pn N N N ~~=O to N C~ a A 7~~ 7 O rl coW
E n w a o a n P mm H $ ~o vi 2 ai ai a i'. x x 2 a ci 'n ~ o c ~ o g~ m E~ x xS x~
N aND t~~f p~y S ep Np W~p Nti~~ N
ph t0 a0 O! = In l!'~~ '"' Of O 1f1 p O
A ~f -~ '~ a ~ a a a aa <
I go on - - - N
M (ey7 ~A p N e~
NN
A J
J g LO ow 8 ~
a rn rn q i1, ai a m~mmmmm~m~mmm~m~m~m~m~ m~ co CD to N N N N N N
4~~~~~~~~~
N N ~ .- r- N N ~=- N N N p C4 N ~p O
M ~ M Nf p ~ff M Nf 1'7 t~7 M Y f M t9 M ~ cn _a = a .f~ ~,~
S
x W 2.. x = _ O
v p~ fa W v t v a Q ~_ u) 'i' vNi I
C ~ a ~ _= p m N N N w R
V) V) w tl ) q~ - a w w o '~ ,;~~~_~"~ d d O ct_7 0 87~V c~+ E o C9 C9 ~ z W
~ Z Z ~?
- ~ 8' s "' Do ~~ LLõ ~Y U a z z C'9 w]
W Wt ~ Z' ~j g E~ c 8 c B E W Z Z
C a E W $' ~
C =g d ~ ~ ~ S g O - ~, ~ a p ~ Y ~ d -' ~ W W W
~=, 7 M R o ~_ 3!0 f' 'Q -~ ~ p i a C W U. pnto cl) s i i O WfQ W y~
u a g c t ~ o y 5 . y m MYYL q~ h O=~~P~~} -~..C [A
A m~E E E ~ tQ ~ rn 9 g a g .. c'3 '4 X Y o M t à ~ c L a O~ (n _p p tn = ~ t~ !~ h Q T ~.
V IL
g ~a..:j I~~
S
m O r a g a ~ o qpyZ_ Q Y $ z z Z w C _ " N
Z QW1 . " ~ .
~ Z. pp~Z X cY CS I/ Y_ ~ ~ L $tll t OI t Oi C ~~~ ~
EZZZ cg~ c c.~ CU..89 c., o $$ W az~W -~ o o m g .n,7, nSnm~ y g'ao a=~ n Q~ g ~~{~ a t E O crf ~~ ~ p t np ~ a ~ C
~ad~"" ~ ' ~orn y1=~' rEio E
,0 g y'~ 0~~~~0~5~ ~ ~~~o~~ og~zN
w~
hU
UU 0 ~j S ivui x > >w lco S a~ ~x ~oar O
~
On N
~ S~ N O N
OOODDD ~ n ad N f~ ~< a 5~ a~~~~ ~ a a~ a a0 p p P, v "
Y N N N Y! N N
~
O M N Y7Y h ~ ~
a n ~ a $
o LL a aa~33 a0 w ~ C~Q7 i~- ~~~ g c~ C~ c~ (~, 4 co co 81 co~
O 0'b N -~ ~- A M
OD '~ p a ~f1 ~ N ~ N Yf O O ~ r- M 1~) l''=
~ I $ I
si ~a$N
!~f Yco9 M ~ 1+001 O t~) M lIn~) l7 l~~l A p/
o o Z E E _ s c e e c 8 $ c n E w a n Y
o 32 = QU K S ~3< ~s3 S Vl U oi= oaW lt~=
_ ~ (A fA ~ V r a O
N W
~~Xr1 CC C O g Y Ol < UZ W O _~(7 o d' N c rn r W ul a g q a o CE~M U ~
z z m fA a ~ Z Z Z 2 a tS c ri ri ~~i 8 E r u~ O~
W W O O n ~ = N L Eo +a xai 0 ~ ~ a Z Z ~p ~~Q d wpom~
p W c: n e o c n ~ a QK ~g x~ El~
Cl ai e~i E'n o 5 3 E '~
eleQ ~o ~'I8~' ~ ~10 y~JE
O
~ N g N _ O qp N N
~ ~ ~~ ~ ~o' 93C C m m m O a O r r EOL L
E X~ U~Q Q a ~ Z
0 c a 9 w M V c Y '~~=,LD toi o ~'" Y ~~ ~~~F~ ~i~ 8=.4d 8 ~ u 'Scc+'d Z ~'a . n M .!! = E E C C 1- c E a S~ at 8N . c_ 0 ~ c.~ E E v a~~~~~d ~ ~}W
~ t ~ o E IL hi ~riq ~ o r3U =~U YJ 0~ ~~?
_ ui v ii g aS
O O N N n Ch n ~Q N A
O
x wR
~ ~a a N a N CD 0) 0 N Of ~ 17 m t~f .- N N N
~V n n r ~ r h O) ~ g yj r ''34 4 ~4 4 ~ b m ~ g y~ u~ q, O c7 o~o V' U U FW- ~ri~
~ ~ ~S1 ~ ~ a ~l w ~ o r r lol r O O O
t ~ ~
~' N Jj N 4 la ~ N YN) ~ O h O~ Of ~ O O ~ A N In 1[) CO M~p N~p N t0 f(~p ~{O O h t0 N 0 1~( 1n ~ p~ m-8 ~$~ _~ m O '~s l õ ~ 8 z ~ E E EÃ a õ~~ E > E
tsE~'o ='r ~=g ~'~ E I I ~ s ~mQoc Ep ti 8~~ x in~in c4s~rn2 ~no a in into x Ny N
-5 m m 3 e E
E
W po ~ v p $ $ $ ~pp ~
m c c - p W
E
~ z v E
c CD ~ ~ Ã W Ã
z o c n C7 C9 ~ T QSp p C Ol 0 p1 ~ ~ N W Wg Q O
c E
w õ ~ o 0 0 O.2C7 nF a o n t E .0 E
n a2 a 'p~s ~i8 ~ N
- P
R~ E E E
E E B = m x E 8 Y 4 E
3 gy y ~y W~o ~ ~ a , aNOB
a E~ o S E ~ p ~~ o~ u d O A A o .0 ' m ~ v, i U m m c ys - ~~
m u $' U p~ p~ y 1V _T
8 p p C ~ p T p J
~ ~ O U ~ U a L7 ~E 2 d b C C ~ Cl Cl E 10 z J~Wõ y õ N 7 E 7 >> E 7 ;
= v C .C W Yl E 7 T Z
c C ~1 i0 C r LL
e" T T ~$ a y , o o m oF v W W_ ~~$ u n O
o~~a o M 10~
Qm ~ 0~~8 8 ~8 ~ 8 c~
kn ) Qf a0 N p ~t7 ~ pa paf lh f7 OCp Q ~ J ~ J O p~j J O n O OLL, ~ {OL LL 01 1~L, a a a ~ a~ n, a a ~~ Q o a a N c yo~
pf A N ~ O_ O~p~ inNN pp M 4m N
N Y th N ~ to ~ ~
N ~
W) n M N
~ N
W
N ~i= q_Ui v ~='J~i= 3 ,~r, ~ ~ a doS ~y ~ .~ ~ ~ o SLL' Q QQ _ a mi , ZI
cc m m mm mm m mm m m mm m A In N 4m ti cc O
O C
ic ~ ~ /D~ pCD~ Wp~
~p tn ~1~ CD LLa'1 ptrf ( N Nt{ Na CD {a~0 ~ N O
r f M cn') pOj pOj O
7yy 7 ~ 7 q Ny W
ry~~1 uq >
C C p So a c n C C C V ~ N H y~ W
~, .n ,~ 2 r5 CL a acr 3 b b o- a10i n p a O g Q O Q O O
~ L' L u E E E E E 0 E 0 V
Z V) (q (n vJ U c 0 x = T T O 0 = m v rn p p W ~ W nQ~ E E $ d p Y T ~ 0 c n W O 0 O O U N U~ O O 0.' 0 0 ~ W W
$ a n n n ~ E o Z~i t~ o Q4 K ~ ' ao m a o E (7 'p8 a _ ~c> O M o 0 o 00:
O e VaE N" a1s o p v, E S ? o ? rn a u Q Q 'Q Q ~ n N E 0 E ar $z Z z z U
Q y o y o e~ - F co oi W F- c c o E !? - U v U
41 o O O O O 2~ co O0 n A 0- U W Ud yZj E t k ~~
~gr ' v v v o so c 4ao ma à 4~ ~ ~ g N N w O O O O o~ ~ Cc7 ~ tc ~ o O c Fo W ei a6 W o' w fn 3 o<n orn Qo E~ ~=
O e c ai ~ ~ ~t ~ ~ o r> > c~ ~ ~ f ~ c u c 0 0 ~~ Ti~ T u c~ hc~ ~N a ~ a y 4 ~ N un, C) i U ? -c 1 m a o ~i ar ~ ~
m ' 0 ~ 0 E e N ~ & ~ ~ ~ = 7 , n nrNi n ~ a~ E c~l a ~ E ~
L m 'n Q~ c 10 LL i; v) c c E
.~ W~' c ~ c ~ t ia t m c c E~ ~ ar ar ~~ w rn v ~ a ~ g a m L ~ 8 d m ~ y ~ a. N O a) O O I I C ~~ O T W W~~C W ~' 7 n 0. ~
g Q u ~ c c c E 8 _~~
E f a y~ rn or rn 2 E2 c W
Q=y x Y X y a m W n O n ~ p IE-t=a y N N ~ Yl õ N ~ L q Ip ~ t1py C O N q ~ 01 L
m o E~ n~ a N ~ ~ E Y C1 E m q ~O+~=8 ~ 88 8 m~ 1 'a N
W .Ø ~Ø. E..V. ..d. O yY+ O' c h' Ol A L N W 10 ? f iY ~ fV N N l0 l0 0 n O 9t O O g1 O~ .YI p N p tll N d N m t O E n' N N L E
o ~ L E S a g 'r'I a "~.~ N ~ z Z ' $_~
za~n v~ v~ v~8 tt rnx,n =,n x>xBxm m , ~
e~ c~ ao o v N i(i tp ~
p) Q ~ m N ~ O ~ ~NCI h~ p N C-4 04 fpNp C cG4 ~ N N y, o p~ Op~~ O
J Q Q D I < Q Q Q m N N Q
O O O a0 p N~ h e~i O
co a aD
N N Y N Ci < N N ~
a ~
y 'n p p Q O ~ _N a VRQ
z _Q _< Q Q V
U. ~ eqD Q ~ ~~ 4 m N <
m m m ~
~m~ ~m~ m ~ y ~ m m ~ 5 m m~ I ~I !, A
m m m i i C7 cD a~
Cm7 ~
P ~
~ O N ~
V
g ~ ~
it W
t a t ~a a ~, ~ a b >
r r r N) ~ e~-P ~
pO t~0p A~p OpI~ h N~ N/ m (~Dp O O N O V') ~N~ p h Nf M l'~ Of 1~+1 !9 c~+l l~~f lh 1~0 1(1 tN0 ~O ~Nff 10+f a 9 (O 1'~07 V c+! 1~f d y ' .1Q .i~
a = E
X
E
CL o ~ Jjj:jj S h O S
põ c ~ a o C'i W fN0 ~ Ci S C S cC
E q z a O D ~i/ C
Q
G ON crU = m V .Y
o c ro g ~-~' g 16 E rn ~ ~ o~~ m ~ y r o E m a d C ~o g ! o $ Q s 8 m 1 C 1O _ v o C n N Ci C
V
N E ~
r= E r:'~ ~$ M y y E y p~ a~ r~ ~S 2 m t F N
~ E o E a -~ o S o U ~
~~~~ m~~y~' L L E
rn ~o1 o~
W E S o E .2 y_G a c _c Y e rn E ~
m,p 7 7 ~ E 7 X E
o am eo coI Z L E F' m o ~ n . Q ~ y ~ c0 O~ ~ Q
Q NI ~ = ED a O
Yirlf egl~ E1 o N
U ~8v~ UX..= ~r '2 2 ~ w=wN V) 4~ 00= vi N M ~ry~rypp n ~ ~ ~
1~ Y M NY N tln+~ _ O = ~ 10 ~ h N Q $$~
m !~ ~y r4 1p h~ M G Y ~ O $Uf~ ayX, N f Nf ~~ ~ a V lJ
N ~ X ~ Q Q N Q N Nr G N U ~ N N Q < Q
O 01 O O a~O 1~ ~ t~ ~ 01 T N
~~yp _ ~a~app 1n U1 N N N 0 C9 ~~f/ V) V N P1 tn h O ~ ~~p N 0~y GN h f ~f ~'~' I~O NO G ~ CO mNQ I 1 m J N N V ~ r tW ~
~ U ~ ~ r U w ~ ~ C1 8b~ C U
(m ?a~ =m 'w ad~
~ 1 I 1 I I I 1 I ~g 1 I 1 1c~1 I 1 I I I 1 1 mI ~1 (9 C9~ (m7 C7(7 ~ C~ ~ mU' ~t7~ m G ~ 0 ~
(7 t7 r $ ~
~ $
' c5 c5 o IL~
O O N
SIR Isim R v E c c H ~m'~.lL'~.lQ rin n 72 .a W 'n w E E
43 5=S 3N fnT T Q C
m ='~n W . - s cp v~
a~ Z E ~.j p t7ia ~ iW
m a w ~o E W aj y pvi 2~ m ~" G W W y ~
U ~1 q ~ tl~ d , c~E c..E EV1~0 O -.0 oz a a ~ N(?
7i a c z = N =~ ~ -~ Z Z N j n d~'> E z (23 j U U m v~ H 9.0 12 ++ D E- 125 w W~r' r C ma E
~W W G rf ri 8 Goc H
crr aa~E,~,Ca~~Eo 31n E
e ~ E Erot 4 o v~U~' a ~~ ' g g4 ~ A
o~~~~N D~ v~,~~V o V~~W~~ ~ ~ v$m ~y U
~ a' A H ~ ~' 3 r ui p e~ 3a ~ i - m oe~4~N 7E
WL E ~~~ -8 =~$N~~Q ~
~ ~ oc~iqggo c ~ 31QZ
~ ~~cE
LZU~ E.
n 4Y v m 7 rm O ~ .JQL mdQQ~~ c~ m ~E. ~j' ~i 7 1f~ =g ya a ya (L A
~
E N ~ a ~ E N 11 m W C Q ~ 2 E ~ N H
t~z..En~ ~ c~~ W WG'Je ~~v, 2 SS=$~ a v ~nota,~t~ o ~ N C ~ O1 ~
W
~ ~
M ~ t+1 ~ ~ ~ I Yl h ~ CD
~O (0 1+1 ~.:
~ {+1 (Qp f~ A ~
C Q CD ~~ ~ N ~ N A V ~ ~ ~
IL
U) ~ ~ i~~ ~ rn ~4 ~0 4 4 d(D
ml m m 1 1 tn ml~p1 fnl ml m t~ 1 ml mlpp1 1 ml ~1 ~1 t~ (9 C~ ~ ~ C~ U' (~ C7 (~ C~ t? (! (9 (7 ~ (9 ~
c, ~ ~ ~
CP ~ ~ ~ b?w Cf { {R N h N~Vp N~p ~ t~+p~ O! p~ N N fi0~ 1~ N f0 <
l~! V O M M - b 0 N t~0 ~f t0 ~f t~~! g Y M ~ M tp m{~
w p c E 3 "~ a g E p E
3 w~ w C at.Q
$~ a~ ~=~ ~ ~ a.~ g E E
c N x' ~a"$3~~ ~Zz~n i~-m~ ~
V ui ~ $ E =o dcr ~ c~i Y; 0 2 A $ c Aa N ~'~ ~ $
o O1 $
4m cl) E
E E $ (9 0, U S C ~ Q U ~ $ N N ~
R' K a F o W aLLO aci & m c 3 tZ+~ a o E~ EI
S' ~ t2 w O w E a Q c v E y~ o W q p~ Q c J 1 c r EO w V m QO1 cn ~s p n i3 $~ 2 df b U $ ~õi o = 'fi ~'o ~. La ~ p o~ $
w x w rn ~1 c rnI~5p~ EQ ~o ~ oa cli ~' G .~ g~ L~ m a J a ~ G C N~ C N=
~ tl L~ ai ? '3 ~ o w$ U p g VI $ Q Wll w a$ m c E 10 Q wc = c 8~ ~'c 9 c o c~
a w E p I!1 ww$ E a à ~9~ ~ Z o gg s iS S a ~j q -E a 2 a. ~ 3 e o c' c i5 E N .Y ~ c~~~~ c ~
a a n g rn u ~ a cv -~ 'n w ~~UsN
J ~ ~ Z W w oo= E= Q U ~~~tn xc1 Q xr~ S~S ~mat~~ ov~m am h Yj ~ N ~ ~ ~ W p N
I ? z~
a a ~ Y Q~ Q N -j< K x a Q a Q K ~N ~ aiOc N~ X~
~y Q O ~pp N i_A h_ O
N/ N ~ ' O O ~ N
N p~ ~ ~pp }j~~ pp ~y Of M m t' ~ ~ ~ 17 l'~i 1.) a~ ~ ~b0 ~ N ~~p W ' ~ ' N O~
1 1'N ~ bf N O~f N
O O ) ~ (~V Uy OI
' w~~ ~ W ab ~ '~ O1 N ~ ~ ~ G
~X" LU InUZIN
_~
m wg w ma, m' m~ m~ m m~m~ m~ m~ m m m~ m m' m~ tp~ ~p~ m m mop~
o c7 (9 Ca C~ c7 C~ C~ C~ c7 c7 (~ 0 C7 c7 C~ c7 C7 c7 (7 c7 C~ C~ c7 OD ~ se ~ ~ ~
cri a W
N ly .- =- N C) ~p ~my {p m y~ ~y pp ~pp~ fp ap Nf c7 -~ ID i w N f f~ f~ 10 ntp N1~ ~ N a0 Pf ~'9 ~fpD N
''7 ~1 t0 N t~ 0~1 p~i In I~+f Y1 t7 a 1() ~ Cd { j ~.~j IA ~f! l+!
g E W e $ u a a > ;
E ~
-~ n N N ~2~Q~~0~~~~ ~.~>.~~ ~ ~ ~ ~~Er$$
N ~+aUa3~~r31~ NO = ~y0 W = _ 2 n ~
E
~ 6 ~ m N Z tV
!0 L H a ~ ~ N i ~ 10 C C c ui O g in $ a ~ a ~ n ~ i~ c ~ ~- 1' ~ c 2 ~
h a E
E
= E ~ .E3 m I ~ o E o ~+ ~ ~ a ~ t g+ ~ m p J u~ ~ a~3 O1 s~ 'n g N, $ x~~o C
V t n E a' aE~oV
a o U p' } V
=d ~ J ~ ~ '0 a Q ~ a N ud dl 0 o m~ Qa rn z 0S
(0 c~ o a ,ae ~ E _ =- H $ ~' V o ' ~ = a ~ N 3 n m m ~~ pp C~ H O O 8~ v O ~ CiI Od U~ y Y U~ ~ N Q W Y ~~~~ .Y!
e oC
E> E m ~c~j ~'t3'o ~ ~~
à Z ~a 3 >'~ ' r ~ ' U~' ~oc~z~rc c c'-~i c c_Z or; 2{L > > N ~ EEnE
E Z3 tS t5 a m ga U =' __=- 3 c c $ E .- tn =~ =~ 3 tE~a ~ ~ '~wr o~_$~goo ; a t 'o x1,1uit~~i1t~
V. 4 p $ 0 ~a ~S$-r'-_ ~ a a ~ ~ 8 H~ $
~~~ a W
E 2 r2m =~ ~ _ t~ Qf N_ ~
q ~ Q ~ N 7 W a~D O
~< Q Q xQ << f3 ~ S~~ ~~ ~g J J Q ry ~ ss ~ ~ $ ~~ em WN g (y,~
W Q ~ V J !} Q q.- =- f=Q- ~
~ i VUJ
= = ~ OQ
a ~"p~ ~~ l ~ g U' c9 c~ (7 c9 U' C~ (7 C~ (~ C7 c~7 ~ c7 ~ ~ a s o ~ ~
'3 N 1!f r ~- r r .- r r r N N r r e- M M O ~ l7 q~p ~f f0 CD n ~ l0 Y n ~ 7 N
M
~ V f {+f If1 A l+~f ~ l+f 1+f V ~~f f0 m C9 ~7 Ol Y
VO
E '~ y E
8 c~ca~E E
, Y c~.8'c~ c c ZI. n E 1 2 E
IIIIIH x s s s s z oC~ o a 3~~ aC s c~'oa~
~~~ ~j w 9 a C _ Q ~Q$~~ ;_ ~tapp ~ ~p ~a.C
C C V7 (/} C7 t~ N = gj p ~ W C G
. o' gi Qc ElA+
m CL n Cj E ~ E 3r ~ i6 $ N
~~~=_ ~~~~
g ~ A
c~~o O
C
0 Q ~"=a rn ~~N~ a " ES 3 p~~8 . ,. ,. ~
mmM v~ para~ vx ~ ~~ c3 F m im l0a M a.2 M~ b o__Jj E~ C), E cc~"
a~ al wEmEB ~EEEY1 oi~iy ~N
y= g (~, C ;o C 9 U O. V 3 H J. B~ c rn ~ rn=~N- 7~ E ?~~E
=~N$ ~.~ .~ ~o,Y $~~' d' ~ nc c E~c c c V! NZ ?0"iA g i -IL'~
~9 ~~~~r n w_ $ ~r ~
r 2 o~E H~ i1ti Id V < ~+f N
~ M ~ N
N ~p to M
i r0 aI0 aD 04 N N N
r N ON ir., cr~ Y1 _h r m A
pp~ o1~ (.~ a Q ~
Q Q N J J N Q a 4 Q a Q K N
r r W p M ~ Np O~ ~ ~pQ~ l f Ih IL! {'~ y ~~ O l n9 M 10 M g !~~'f l~f N A N N ~
~ U ~I g n~~pp ~~npp ~ u~
G n C~ G ul O~ r n o M }- M ~ N N M
W ~_ Rf W ~ W ~
~p1 1 1 mI 1 I I 1 ~p I ~p I (~ I 1 1 1 ~p 1 1Q I 1~p1 1 t~ I 1 1 (9 t7 C9 O t~ (7 C7 C7 t9 U 17 C7 C7 (~ C7 C7 L7 (? (~
O t~l r p ~
r W ==-s dCNDO r r r W W C~ ~ F N Op 8p Op $
n~ ~p dtq0 N W O O O
01 l~f df f P'f 1 Lh f0 1 1D 10 W r '" s E
E $ ? F
~=~ ~ ~ ~ ~~ ~ fliJhiIllfl ap a 8 ~ Z' S
s~ 8~~ ~ ~~}8 ~TB~ 0 w g o- o o 0 5F~f w N ~ ~ = G .~ C ~ ~ U L ~(~ ~ YI jY~I G
~ ~ ~~ ~E = m m ' ~ p O ap E
~m $
La U. a 'fl C l0 ~ L' C~ 0 1nf ~ V $ LL~ D~ y GY N
La 0, i -o a 8~ a .r ~ 1 1 2 C C~ b C~ O~ d yv~~r: -- 8 t~ ~ $ a w n I -i I Cq~. $ w a x q C
~ ~ E
o S
s'Q ~ o~$~~aa~'~ ~o= ~~ ~ n n a 8yn t ~~= g"~ 0 n W ' L t 122 O1 =(7~~a2c Y~NSv ag= ~Bg~ n~ n E
~m~'i~ W~~ga.p $81! g Q E
bioactivities described herein.
The SMP polypeptide, or a biologically active portion thereof, can be operatively linked to a non-SMP polypeptide to form a fusion protein. In preferred embodiments, this fusion protein has an activity which differs from that of the SMP protein alone. In other preferred embodiments, this fusion protein performs a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. In particularly preferred embodiments, integration of this fusion protein into a host cell modulates production of a desired compound from the cell.
In another aspect, the invention provides methods for screening molecules which modulate the activity of an SMP protein, either by interacting with the protein itself or a substrate or binding partner of the SMP protein, or by modulating the transcription or translation of an SMP nucleic acid molecule of the invention.
Another aspect of the invention pertains to a method for producing a fine chemical. This method involves the culturing of a cell containing a vector directing the expression of an SMP nucleic acid molecule of the invention, such that a fine chemical is produced. In a preferred embodiment, this method further includes the step of obtaining a cell containing such a vector, in which a cell is transfected with a vector directing the expression of an SMP nucleic acid. In another preferred embodiment, this method further includes the step of recovering the fine chemical from the culture. In a particularly preferred embodiment, the cell is from the genus Corynebacterium or Brevibacterium, or is selected from those strains set forth in Table 3.
Another aspect of the invention pertains to methods for modulating production of a molecule from a microorganism. Such methods include contacting the cell with an agent which modulates SMP protein activity or SMP nucleic acid expression such that a celi associated activity is altered relative to this same activity in the absence of the agent. In a preferred embodiment, the cell is modulated for one or more C.
glutamicum carbon metabolism pathways or for the production of energy through processes such as oxidative phosphorylation, such that the yields or rate of production of a desired fine chemical by this microorganism is improved. The agent which modulates SMP
protein activity can be an agent which stimulates SMP protein activity or SMP nucleic acid expression. Examples of agents which stimulate SMP protein activity or SMP
nucleic acid expression include small molecules, active SMP proteins, and nucleic acids encoding SMP proteins that have been introduced into the cell. Examples of agents which inhibit SMP activity or expression include small molecules and antisense SMP
nucleic acid molecules.
Another aspect of the invention pertains to methods for modulating yields of a desired compound from a cell, involving the introduction of a wild-type or mutant SMP
gene into a cell, either maintained on a separate plasmid or integrated into the genome of the host cell. If integrated into the genome, such integration can be random, or it can take place by homologous recombination such that the native gene is replaced by the introduced copy, causing the production of the desired compound from the cell to be modulated. In a preferred embodiment, said yields are increased. In another preferred embodiment, said chemical is a fine chemical. In a particularly preferred embodiment, said fine chemical is an amino acid. In especially preferred embodiments, said amino acid is L-lysine.
Detailed Description of the Invention The present invention provides SMP nucleic acid and protein molecules which are involved in the metabolism of carbon compounds such as sugars and the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. The molecules of the invention may be utilized in the modulation of production of fine chemicals from microorganisms, such as C.
glutamicum, either directly (e.g., where overexpression or optimization of a glycolytic pathway protein has a direct impact on the yield, production, and/or efficiency of production of, e.g., pyruvate from modified C. glutamicum), or may have an indirect = ' .
-II-impact which nonetheless results in an increase of yield, production, and/or efficiency of production of the desired compound (e.g., where modulation of proteins involved in oxidative phosphorylation results in alterations in the amount of energy available to perform necessary metabolic processes and other cellular functions, such as nucleic acid and protein biosynthesis and transcription/translation). Aspects of the invention are further explicated below.
1. Fine Chemicals The term 'fine chemical' is art-recognized and includes molecules produced by an organism which have applications in various industries, such as, but not limited to, the pharmaceutical, agriculture, and cosmetics industries. Such compounds include organic acids, such as tartaric acid, itaconic acid, and diaminopimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides, and nucleotides (as described e.g. in Kuninaka, A. (1996) Nucleotides and related compounds, p. 561-612, in Biotechnology vol. 6, Rehm et al., eds. VCH:
Weinheim, and references contained therein), lipids, both saturated and unsaturated fatty acids (e.g., arachidonic acid), diols (e.g., propane diol, and butane diol), carbohydrates (e.g., hyaluronic acid and trehalose), aromatic compounds (e.g., aromatic amines, vanillin, and indigo), vitamins and cofactors (as described in Ullmann's Encyclopedia of Industrial Chemistry, vol. A27, "Vitamins", p. 443-613 (1996) VCH: Weinheim and references therein; and Ong, A.S., Niki, E. & Packer, L. (1995) "Nutrition, Lipids, Health, and Disease" Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research -Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes, polyketides (Cane el al. (1998) Science 282: 63-68), and all other chemicals described in Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN:
0818805086 and references therein. The metabolism and uses of certain of these fine chemicals are further explicated below.
A. Amino Acid Metabolism and Uses Amino acids comprise the basic structural units of all proteins, and as such are essential for normal cellular functioning in all organisms. The term "amino acid" is art-recognized. The proteinogenic amino acids, of which there are 20 species, serve as structural units for proteins, in which they are linked by peptide bonds, while the nonproteinogenic amino acids (hundreds of which are known) are not normally found in proteins (see Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH:
Weinheim (1985)). Amino acids may be in the D- or L- optical configuration, though L-amino acids are generally the only type found in naturally-occurring proteins.
Biosynthetic and degradative pathways of each of the 20 proteinogenic amino acids have been well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3d edition, pages 578-590 (1988)). The 'essential' amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine), so named because they are generally a nutritional requirement due to the complexity of their biosyntheses, are readily converted by simple biosynthetic pathways to the remaining 11 'nonessential' amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, and tyrosine).
Higher animals do retain the ability to synthesize some of these amino acids, but the essential amino acids must be supplied from the diet in order for nonnal protein synthesis to occur.
Aside from their function in protein biosynthesis, these amino acids are interesting chemicals in their own right, and many have been found to have various applications in the food, feed, chemical, cosmetics, agriculture, and pharmaceutical industries. Lysine is an important amino acid in the nutrition not only of humans, but also of monogastric animals such as poultry and swine. Glutamate is most conunonly used as a flavor additive (mono-sodium glutamate, MSG) and is widely used throughout the food industry, as are aspartate, phenylalanine, glycine, and cysteine.
Glycine, L-methionine and tryptophan are all utilized in the pharmaceutical industry.
Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are of use in both the pharmaceutical and cosmetics industries. Threonine, tryptophan, and D/ L-methionine are common feed additives. (Leuchtenberger, W. (1996) Amino aids -technical production and use, p. 466-502 in Rehm et al. (eds.) Biotechnology vol. 6, chapter 14a, VCH: Weinheim). Additionally, these amino acids have been found to be useful as precursors for the synthesis of synthetic amino acids and proteins, such as N-acetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan, and others described in Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97, VCH:
Weinheim, 1985.
The biosynthesis of these natural amino acids in organisms capable of producing them, such as bacteria, has been well characterized (for review of bacterial amino acid biosynthesis and regulation thereof, see Umbarger, H.E.(1978) Ann.
Rev.
Biochem. 47: 533-606). Glutamate is synthesized by the reductive amination of a-ketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline, and arginine are each subsequently produced from glutamate. The biosynthesis of serine is a three-step process beginning with 3-phosphoglycerate (an intermediate in glycolysis), and resulting in this amino acid after oxidation, transamination, and hydrolysis steps. Both cysteine and glycine are produced from serine; the former by the condensation of homocysteine with serine, and the latter by the transferal of the side-chain 0-carbon atom to tetrahydrofolate, in a reaction catalyzed by serine transhydroxymethylase.
Phenylalanine, and tyrosine are synthesized from the glycolytic and pentose phosphate pathway precursors erythrose 4-phosphate and phosphoenolpyruvate in a 9-step biosynthetic pathway that differ only at the final two steps after synthesis of prephenate.
Tryptophan is also produced from these two initial molecules, but its synthesis is an 11-step pathway. Tyrosine may also be synthesized from phenylalanine, in a reaction catalyzed by phenylalanine hydroxylase. Alanine, valine, and leucine are all biosynthetic products of pyruvate, the final product of glycolysis. Aspartate is formed from oxaloacetate, an intermediate of the citric acid cycle. Asparagine, methionine, threonine, and lysine are each produced by the conversion of aspartate.
Isoleucine is formed from threonine. A complex 9-step pathway results in the production of histidine from 5-phosphoribosyl-l-pyrophosphate, an activated sugar.
Amino acids in excess of the protein synthesis needs of the cell cannot be stored, and are instead degraded to provide intermediates for the major metabolic pathways of the cell (for review see Stryer, L. Biochemistry 3'd ed. Ch. 21 "Amino Acid Degradation and the Urea Cycle" p. 495-516 (1988)). Although the cell is able to convert unwanted amino acids into useful metabolic intermediates, amino acid production is costly in tenms of energy, precursor molecules, and the enzymes necessary to synthesize them.
Thus it is not surprising that amino acid biosynthesis is regulated by feedback inhibition, in which the presence of a particular amino acid serves to slow or entirely stop its own production (for overview of feedback mechanisms in amino acid biosynthetic pathways, see Stryer, L. Biochemistry, 3rd ed. Ch. 24: "Biosynthesis of Amino Acids and Heme" p.
575-600 (1988)). Thus, the output of any particular amino acid is limited by the amount of that amino acid present in the cell.
B. Vitamin, Cofactor, and Nutraceutical Metabolism and Uses Vitamins, cofactors, and nutraceuticals comprise another group of molecules which the higher animals have lost the ability to synthesize and so must ingest, although they are readily synthesized by other organisms such as bacteria. These molecules are either bioactive substances themselves, or are precursors of biologically active substances which may serve as electron carriers or intermediates in a variety of metabolic pathways. Aside from their nutritive value, these compounds also have significant industrial value as coloring agents, antioxidants, and catalysts or other processing aids. (For an overview of the structure, activity, and industrial applications of these compounds, see, for example, Ullman's Encyclopedia of Industrial Chemistry, "Vitamins" vol. A27, p. 443-613, VCH: Weinheim, 1996.) The term "vitamin" is art-recognized, and includes nutrients which are required by an organism for normal functioning, but which that organism cannot synthesize by itself. The group of vitamins may encompass cofactors and nutraceutical compounds. The language "cofactor"
includes nonproteinaceous compounds required for a normal enzymatic activity to occur. Such compounds may be organic or inorganic; the cofactor molecules of the invention are preferably organic. The term "nutraceutical" includes dietary supplements having health benefits in plants and animals, particularly humans. Examples of such molecules are vitamins, antioxidants, and also certain lipids (e.g., polyunsaturated fatty acids).
The biosynthesis of these molecules in organisms capable of producing them, such as bacteria, has been largely characterized (Uliman's Encyclopedia of Industrial Chemistry, "Vitamins" vol. A27, p. 443-613, VCH: Weinheim, 1996; Michal, G.
(1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley & Sons; Ong, A.S., Niki, E. & Packer, L. (1995) "Nutrition, Lipids, Health, and Disease" Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research - Asia, held Sept.
1-3, 1994 at Penang, Malaysia, AOCS Press: Champaign, IL X, 374 S).
Thiamin (vitamin B i) is produced by the chemical coupling of pyrimidine and thiazole moieties. Riboflavin (vitamin BZ) is synthesized from guanosine-5'-triphosphate (GTP) and ribose-5'-phosphate. Riboflavin, in turn, is utilized for the synthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The family of compounds collectively termed 'vitamin B6' (e.g., pyridoxine, pyridoxamine, pyridoxa-5'-phosphate, and the commercially used pyridoxin hydrochloride) are all derivatives of the common structural unit, 5-hydroxy-6-methylpyridine. Pantothenate (pantothenic acid, (R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-l-oxobutyl)-p-alanine) can be produced either by chemical synthesis or by fermentation. The final steps in pantothenate biosynthesis consist of the ATP-driven condensation of 0-alanine and pantoic acid. The enzymes responsible for the biosynthesis steps for the conversion to pantoic acid, to 0-alanine and for the condensation to panthotenic acid are known. The metabolically active form of pantothenate is Coenzyme A, for which the biosynthesis proceeds in 5 enzymatic steps. Pantothenate, pyridoxal-5'-phosphate, cysteine and ATP are the precursors of Coenzyme A. These enzymes not only catalyze the formation of panthothante, but also the production of (R)-pantoic acid, (R)-pantolacton, (R)-panthenol (provitamin B5), pantetheine (and its derivatives) and coenzyme A.
Biotin biosynthesis from the precursor molecule pimeloyl-CoA in microorganisms has been studied in detail and several of the genes involved have been identified. Many of the corresponding proteins have been found to also be involved in Fe-cluster synthesis and are members of the nifS class of proteins. Lipoic acid is derived from octanoic acid, and serves as a coenzyme in energy metabolism, where it becomes part of the pyruvate dehydrogenase complex and the a-ketoglutarate dehydrogenase complex. The folates are a group of substances which are all derivatives of folic acid, which is turn is derived from L-glutamic acid, p-amino-benzoic acid and 6-methylpterin. The biosynthesis of folic acid and its derivatives, starting from the metabolism intermediates guanosine-5'-triphosphate (GTP), L-glutamic acid and p-amino-benzoic acid has been studied in detail in certain microorganisms.
Corrinoids (such as the cobalamines and particularly vitamin B12) and porphyrines belong to a group of chemicals characterized by a tetrapyrole ring system.
t . - ' The biosynthesis of vitamin B12 is sufficiently complex that it has not yet been completely characterized, but many of the enzymes and substrates involved are now known. Nicotinic acid (nicotinate), and nicotinamide are pyridine derivatives which are also tenmed 'niacin'. Niacin is the precursor of the important coenzymes NAD
5(nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms.
The large-scale production of these compounds has largely relied on cell-free chemical syntheses, though some of these chemicals have also been produced by large-scale culture of microorganisms, such as riboflavin, Vitamin B6, pantothenate, and biotin. Only Vitamin BiZ is produced solely by fermentation, due to the complexity of its synthesis. In vitro methodologies require significant inputs of materials and time, often at great cost.
C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses Purine and pyrimidine metabolism genes and their corresponding proteins are important targets for the therapy of tumor diseases and viral infections. The language "purine" or "pyrimidine" includes the nitrogenous bases which are constituents of nucleic acids, co-enzymes, and nucleotides. The term "nucleotide" includes the basic structural units of nucleic acid molecules, which are comprised of a nitrogenous base, a pentose sugar (in the case of RNA, the sugar is ribose; in the case of DNA, the sugar is D-deoxyribose), and phosphoric acid. The language "nucleoside" includes molecules which serve as precursors to nucleotides, but which are lacking the phosphoric acid moiety that nucleotides possess. By inhibiting the biosynthesis of these molecules, or their mobilization to form nucleic acid molecules, it is possible to inhibit RNA and DNA
synthesis; by inhibiting this activity in a fashion targeted to cancerous cells, the ability of tumor cells to divide and replicate may be inhibited. Additionally, there are nucleotides which do not form nucleic acid molecules, but rather serve as energy stores (i.e., AMP) or as coenzymes (i.e., FAD and NAD).
Several publications have described the use of these chemicals for these medical indications, by influencing purine and/or pyrimidine metabolism (e.g.
Christopherson, R.I. and Lyons, S.D. (1990) "Potent inhibitors of de novo pyrimidine and purine biosynthesis as chemotherapeutic agents." Med Res. Reviews 10: 505-548).
Studies of enzymes involved in purine and pyrimidine metabolism have been focused on the development of new drugs which can be used, for example, as immunosuppressants or anti-proliferants (Smith, J.L., (1995) "Enzymes in nucleotide synthesis."
Curr. Opin.
Struct. Biol. 5: 752-757; (1995) Biochem Soc. Transact. 23: 877-902). However, purine and pyrimidine bases, nucleosides and nucleotides have other utilities: as intermediates in the biosynthesis of several fine chemicals (e.g., thiamine, S-adenosyl-methionine, folates, or riboflavin), as energy carriers for the cell (e.g., ATP or GTP), and for chemicals themselves, commonly used as flavor enhancers (e.g., IMP or GMP) or for several medicinal applications (see, for example, Kuninaka, A. (1996) Nucleotides and Related Compounds in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, p.
612). Also, enzymes involved in purine, pyrimidine, nucleoside, or nucleotide metabolism are increasingly serving as targets against which chemicals for crop protection, including fungicides, herbicides and insecticides, are developed.
The metabolism of these compounds in bacteria has been characterized (for reviews see, for example, Zalkin, H. and Dixon, J.E. (1992) "de novo purine nucleotide biosynthesis", in: Progress in Nucleic Acid Research and Molecular Biology, vol. 42, Academic Press:, p. 259-287; and Michal, G. (1999) "Nucleotides and Nucleosides", Chapter 8 in: Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York). Purine metabolism has been the subject of intensive research, and is essential to the normal functioning of the cell. Impaired purine metabolism in higher animals can cause severe disease, such as gout. Purine nucleotides are synthesized from ribose-5-phosphate, in a series of steps through the intermediate compound inosine-5'-phosphate (IMP), resulting in the production of guanosine-5'-monophosphate (GMP) or adenosine-5'-monophosphate (AMP), from which the triphosphate forms utilized as nucleotides are readily formed. These compounds are also utilized as energy stores, so their degradation provides energy for many different biochemical processes in the cell.
Pyrimidine biosynthesis proceeds by the formation of uridine-5'-monophosphate (UMP) from ribose-5-phosphate. UMP, in turn, is converted to cytidine-5'-triphosphate (CTP).
The deoxy- forms of all of these nucleotides are produced in a one step reduction reaction from the diphosphate ribose form of the nucleotide to the diphosphate deoxyribose form of the nucleotide. Upon phosphorylation, these molecules are able to participate in DNA synthesis.
D. Trehalose Metabolism and Uses Trehalose consists of two glucose molecules, bound in a, a-1,1 linkage. It is commonly used in the food industry as a sweetener, an additive for dried or frozen foods, and in beverages. However, it also has applications in the pharmaceutical, cosmetics and biotechnology industries (see, for example, Nishimoto et al., (1998) U.S.
Patent No. 5,759,610; Singer, M.A. and Lindquist, S. (1998) Trends Biotech.
16: 460-467; Paiva, C.L.A. and Panek, A.D. (1996) Biotech. Ann. Rev. 2: 293-314; and Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose is produced by enzymes from many microorganisms and is naturally released into the surrounding medium, from which it can be collected using methods known in the art.
II. Sugar and Carbon Molecule Utilization and Oxidative Phosphorylation Carbon is a critically important element for the formation of all organic compounds, and thus is a nutritional requirement not only for the growth and division of C. glutamicum, but also for the overproduction of fine chemicals from this microorganism. Sugars, such as mono-, di-, or polysaccharides, are particularly good carbon sources, and thus standard growth media typically contain one or more of:
glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, or cellulose (Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes", VCH: Weinheim). Alternatively, more complex forms of sugar may be utilized in the media, such as molasses, or other by-products of sugar refinement. Other compounds aside from the sugars may be used as alternate carbon sources, including alcohols (e.g., ethanol or methanol), alkanes, sugar alcohols, fatty acids, and organic acids (e.g., acetic acid or lactic acid). For a review of carbon sources and their utilization by microorganisms in culture, see: Ullman's Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes", VCH: Weinheim; Stoppok, E. and Buchholz, K. (1996) "Sugar-based raw materials for fennentation applications"
in Biotechnology (Rehm, H.J. et al., eds.) vol. 6, VCH: Weinheim, p. 5-29; Rehm, H.J.
(1980) Industrielle Mikrobiologie, Springer: Berlin; Bartholomew, W.H., and Reiman, H.B. (1979). Economics of Fermentation Processes, in: Peppler, H.J. and Perlman, D., eds. Microbial Technology 2"d ed., vol. 2, chapter 18, Academic Press: New York; and Kockova-Kratachvilova, A. (1981) Characteristics of Industrial Microorganisms, in:
Rehm, H.J. and Reed, G., eds. Handbook of Biotechnology, vol. 1, chapter 1, Verlag Chemie: Weinheim.
After uptake, these energy-rich carbon molecules must be processed such that they are able to be degraded by one of the major sugar metabolic pathways.
Such pathways lead directly to useful degradation products, such as ribose-5-phosphate and phosphoenolpyruvate, which may be subsequently converted to pyruvate. Three of the most important pathways in bacteria for sugar metabolism include the Embden-Meyerhoff-Pamas (EMP) pathway (also known as the glycolytic or fructose bisphosphate pathway), the hexosemonophosphate (HMP) pathway (also known as the pentose shunt or pentose phosphate pathway), and the Entner-Doudoroff (ED) pathway (for review, see Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York, and Stryer, L. (1988) Biochemistry, Chapters 13-19, Freeman: New York, and references therein).
The EMP pathway converts hexose molecules to pyruvate, and in the process produces 2 molecules of ATP and 2 molecules of NADH. Starting with glucose-l-phosphate (which may be either directly taken up from the medium, or altematively may be generated from glycogen, starch, or cellulose), the glucose molecule is isomerized to fructose-6-phosphate, is phosphorylated, and split into two 3-carbon molecules of glyceraldehyde-3-phosphate. After dehydrogenation, phosphorylation, and successive rearrangements, pyruvate results.
The HMP pathway converts glucose to reducing equivalents, such as NADPH, and produces pentose and tetrose compounds which are necessary as intenmediates and precursors in a number of other metabolic pathways. In the HMP pathway, glucose-6-phosphate is converted to ribulose-5-phosphate by two successive dehydrogenase reactions (which also release two NADPH molecules), and a carboxylation step.
Ribulose-5-phosphate may also be converted to xyulose-5-phosphate and ribose-5-phosphate; the former can undergo a series of biochemical steps to glucose-6-phosphate, which may enter the EMP pathway, while the latter is commonly utilized as an intermediate in other biosynthetic pathways within the cell.
The ED pathway begins with the compound glucose or gluconate, which is subsequently phosphorylated and dehydrated to form 2-dehydro-3-deoxy-6-P-gluconate.
Glucuronate and galacturonate may also be converted to 2-dehydro-3-deoxy-6-P-gluconate through more complex biochemical pathways. This product molecule is subsequently cleaved into glyceraldehyde-3-P and pyruvate; glyceraldehyde-3-P
may itself also be converted to pyruvate.
The EMP and HMP pathways share many features, including intermediates and enzymes. The EMP pathway provides the greatest amount of ATP, but it does not produce ribose-5-phosphate, an important precursor for, e.g., nucleic acid biosynthesis, nor does it produce erythrose-4-phosphate, which is important for amino acid biosynthesis. Microorganisms that are capable of using only the EMP pathway for glucose utilization are thus not able to grow on simple media with glucose as the sole carbon source. They are referred to as fastidious organisms, and their growth requires inputs of complex organic compounds, such as those found in yeast extract.
In contrast, the HMP pathway produces all of the precursors necessary for both nucleic acid and amino acid biosynthesis, yet yields only half the amount of ATP energy that the EMP pathway does. The HMP pathway also produces NADPH, which may be used for redox reactions in biosynthetic pathways. The HMP pathway does not directly produce pyruvate, however, and thus these microorganisms must also possess this portion of the EMP pathway. It is therefore not surprising that a number of microorganisms, particularly the facultative anerobes, have evolved such that they possess both of these pathways.
The ED pathway has thus far has only been found in bacteria. Although this pathway is linked partly to the HMP pathway in the reverse direction for precursor formation, the ED pathway directly forms pyruvate by the aldolase cleavage of ketodeoxy-6-phosphogluconate. The ED pathway can exist on its own and is utilized by the majority of strictly aerobic microorganisms. The net result is similar to that of the HMP pathway, although one mole of ATP can be formed only if the carbon atoms are converted into pyruvate, instead of into precursor molecules.
The pyruvate molecules produced through any of these pathways can be readily converted into energy via the Krebs cycle (also known as the citric acid cycle, the citrate cycle, or the tricarboxylic acid cycle (TCA cycle)). In this process, pyruvate is first decarboxylated, resulting in the production of one molecule of NADH, I
molecule of acetyl-CoA, and I molecule of CO2. The acetyl group of acetyl CoA then reacts with the 4 carbon unit, oxaolacetate, leading to the formation of citric acid, a 6 carbon organic acid. Dehydration and two additional COZ molecules are released.
Ultimately, oxaloacetate is regenerated and can serve again as an acetyl acceptor, thus completing the cycle. The electrons released during the oxidation of intermediates in the TCA cycle are transferred to NAD+ to yield NADH.
During respiration, the electrons from NADH are transferred to molecular oxygen or other terminal electron acceptors. This process is catalyzed by the respiratory chain, an electron transport system containing both integral membrane proteins and membrane associated proteins. This system serves two basic functions: first, to accept electrons from an electron donor and to transfer them to an electron acceptor, and second, to conserve some of the energy released during electron transfer by the synthesis of ATP. Several types of oxidation-reduction enzymes and electron transport proteins are known to be involved in such processes, including the NADH dehydrogenases, flavin-containing electron carriers, iron sulfur proteins, and cytochromes.
The NADH
dehydrogenases are located at the cytoplasmic surface of the plasma membrane, and transfer hydrogen atoms from NADH to flavoproteins, in turn accepting electrons from NADH. The flavoproteins are a group of electron carriers possessing a flavin prosthetic group which is alternately reduced and oxidized as it accepts and transfers electrons.
Three flavins are known to participate in these reactions: riboflavin, flavin-adenine dinucleotide (FAD) and flavin-mononucleotide (FMN). Iron sulfur proteins contain a cluster of iron and sulfur atoms which are not bonded to a heme group, but which still are able to participate in dehydration and rehydration reactions. Succinate dehydrogenase and aconitase are exemplary iron-sulfur proteins; their iron-sulfur complexes serve to accept and transfer electrons as part of the overall electron-transport chain. The cytochromes are proteins containing an iron porphyrin ring (heme).
There are a number of different classes of cytochromes, differing in their reduction potentials.
Functionally, these cytochromes form pathways in which electrons may be transferred to other cytochromes having increasingly more positive reduction potentials. A
further class of non-protein electron carriers is known: the lipid-soluble quinones (e.g., coenzyme Q). These molecules also serve as hydrogen atom acceptors and electron donors.
The action of the respiratory chain generates a proton gradient across the cell membrane, resulting in proton motive force. This force is utilized by the cell to synthesize ATP, via the membrane-spanning enzyme, ATP synthase. This enzyme is a multiprotein complex in which the transport of H+ molecules through the membrane results in the physicaI rotation of the intracellular subunits and concomitant phosphorylation of ADP to form ATP (for review, see Fillingame, R.H. and Divall, S.
(1999) Novartis Found. Symp. 221: 218-229, 229-234).
Non-hexose carbon substrates may also serve as carbon and energy sources for cells. Such substrates may first be converted to hexose sugars in the gluconeogenesis pathway, where glucose is first synthesized by the cell and then is degraded to produce energy. The starting material for this reaction is phosphoenolpyruvate (PEP), which is one of the key intermediates in the glycolytic pathway. PEP may be formed from substrates other than sugars, such as acetic acid, or by decarboxylation of oxaloacetate (itself an intermediate in the TCA cycle). By reversing the glycolytic pathway (utilizing a cascade of enzymes different than those of the original glycolysis pathway), glucose-6-phosphate may be formed. The conversion of pyruvate to glucose requires the utilization of 6 high energy phosphate bonds, whereas glycolysis only produces in the conversion of glucose to pyruvate. However, the complete oxidation of glucose (glycolysis, conversion of pyruvate into acetyl CoA, citric acid cycle, and oxidative phosphorylation) yields between 36-38 ATP, so the net loss of high energy phosphate bonds experienced during gluconeogenesis is offset by the overall greater gain in such high-energy molecules produced by the oxidation of glucose.
III. Elements and Methods of the Invention The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as SMP nucleic acid and protein molecules, which participate in the conversion of sugars to useful degradation products and energy (e.g., ATP) in C. glutamicum or which may participate in the production of useful energy-rich molecules (e.g., ATP) by other processes, such as oxidative phosphorylation.
In one embodiment, the SMP molecules participate in the metabolism of carbon compounds such as sugars or the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. In a preferred embodiment, the activity of the SMP molecules of the present invention to contribute to carbon metabolism or energy production in C. glutamicum has an impact on the production of a desired fine chemical by this organism. In a particularly prefen-ed embodiment, the SMP
molecules of the invention are modulated in activity, such that the C.
glutamicum metabolic and energetic pathways in which the SMP proteins of the invention participate are modulated in yield, production, and/or efficiency of production, which either directly or indirectly modulates the yield, production, and/or efficiency of production of a desired fine chemical by C. glutamicum.
The language, "SMP protein" or "SMP polypeptide" includes proteins which are capable of performing a function involved in the metabolism of carbon compounds such as sugars and the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. Examples of SMP proteins include those encoded by the SMP genes set forth in Table 1 and by the odd-numbered SEQ ID
NOs. The tenns "SMP gene" or "SMP nucleic acid sequence" include nucleic acid sequences encoding an SMP protein, which consist of a coding region and also corresponding untranslated 5' and 3' sequence regions. Examples of SMP genes include those set forth in Table 1. The tenns "production" or "productivity" are art-recognized and include the concentration of the fermentation product (for example, the desired fine chemical) formed within a given time and a given fermentation volume (e.g., kg product per hour per liter). The term "efficiency of production" includes the time required for a.
particular level of production to be achieved (for example, how long it takes for the cell to attain a particular rate of output of a fine chemical). The term "yield" or "product/carbon yield" is art-recognized and includes the efficiency of the conversion of the carbon source into the product (f.e., fine chemical). This is generally written as, for example, kg product per kg carbon source. By increasing the yield or production of the compound, the quantity of recovered molecules, or of useful recovered molecules of that compound in a given amount of culture over a given amount of time is increased. The terms "biosynthesis" or a "biosynthetic pathway" are art-recognized and include the synthesis of a compound, preferably an organic compound, by a cell from intenmediate compounds in what may be a multistep and highly regulated process. The terms "degradation" or a "degradation pathway" are art-recognized and include the breakdown of a compound, preferably an organic compound, by a cell to degradation products (generally speaking, smaller or less complex molecules) in what may be a multistep and highly regulated process. The term "degradation product" is art-recognized and includes breakdown products of a compound. Such products may themselves have utility as precursor (starting point) or intermediate molecules necessary for the biosynthesis of other compounds by the cell. The language "metabolism"
is art-recognized and includes the totality of the biochemical reactions that take place in an organism. The metabolism of a particular compound, then, (e.g., the metabolism of an amino acid such as glycine) comprises the overall biosynthetic, modification, and degradation pathways in the cell related to this compound.
In another embodiment, the SMP molecules of the invention are capable of modulating the production of a desired molecule, such as a fine chemical, in a microorganism such as C. glutamicum. There are a number of mechanisms by which the alteration of an SMP protein of the invention may directly affect the yield, production, and/or efficiency of production of a fine chemical from a C.
glutamicum strain incorporating such an altered protein. The degradation of high-energy carbon molecules such as sugars, and the conversion of compounds such as NADH and to more useful forms via oxidative phosphorylation results in a number of compounds which themselves may be desirable fine chemicals, such as pyruvate, ATP, NADH, and a number of intermediate sugar compounds. Further, the energy molecules (such as ATP) and the reducing equivalents (such as NADH or NADPH) produced by these metabolic pathways are utilized in the cell to drive reactions which would otherwise be energetically unfavorable. Such unfavorable reactions include many biosynthetic pathways for fine chemicals. By improving the ability of the cell to utilize a particular sugar (e.g., by manipulating the genes encoding enzymes involved in the degradation and conversion of that sugar into energy for the cell), one may increase the amount of energy available to pemiit unfavorable, yet desired metabolic reactions (e.g., the biosynthesis of a desired fine chemical) to occur.
The mutagenesis of one or more SMP genes of the invention may also result in SMP proteins having altered activities which indirectly impact the production of one or more desired fine chemicals from C. glutamicum. For example, by increasing the efficiency of utilization of one or more sugars (such that the conversion of the sugar to useful energy molecules is improved), or by increasing the efficiency of conversion of reducing equivalents to useful energy molecules (e.g., by improving the efficiency of oxidative phosphorylation, or the activity of the ATP synthase), one can increase the amount of these high-energy compounds available to the cell to drive normally unfavorable metabolic processes. These processes include the construction of cell walls, transcription, translation, and the biosynthesis of compounds necessary for growth and division of the cells (e.g., nucleotides, amino acids, vitamins, lipids, etc.) (Lengeler et al.
(1999) Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109; 913-918;
875-899).
By improving the growth and multiplication of these engineered cells, it is possible to increase both the viability of the cells in large-scale culture, and also to improve their rate of division, such that a relatively larger number of cells can survive in fennentor culture. The yield, production, or efficiency of production may be increased, at least due to the presence of a greater number of viable cells, each producing the desired fine chemical. Further, a number of the degradation and intermediate compounds produced during sugar metabolism are necessary precursors and intermediates for other biosynthetic pathways throughout the cell. For example, many amino acids are synthesized directly from compounds normally resulting from glycolysis or the TCA
cycle (e.g_, serine is synthesized from 3-phosphoglycerate, an intermediate in glycolysis). Thus, by increasing the efficiency of conversion of sugars to useful energy molecules, it is also possible to increase the amount of useful degradation products as well.
The isolated nucleic acid sequences of the invention are contained within the genome of a Corynebacterium glutamicum strain available through the American Type Culture Collection, given designation ATCC 13032. The nucleotide sequence of the isolated C. glutamicum SMP DNAs and the predicted amino acid sequences of the C.
glutamicum SMP proteins are shown in the Sequence Listing as odd-numbered SEQ
ID
NOs and even-numbered SEQ ID NOs, respectively. Computational analyses were performed which classified and/or identified these nucleotide sequences as sequences which encode proteins having a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum.
The present invention also pertains to proteins which have an amino acid sequence which is substantially homologous to an amino acid sequence of the invention (e.g., the sequence of an even-numbered SEQ ID NO of the Sequence Listing). As used herein, a protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence is least about 50% homologous to the selected amino acid sequence, e.g., the entire selected amino acid sequence. A protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence can also be least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%, 80-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to the selected amino acid sequence.
An SMP protein or a biologically active portion or fragment thereof of the invention can participate in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium gtutamrcum, or can have one or more of the activities set forth in Table 1.
Various aspects of the invention are described in further detail in the following subsections:
A. Isolated Nucleic Acid Molecules One aspect of the invention pertains to isolated nucleic acid molecules that encode SMP polypeptides or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes or primers for the identification or amplification of SMP-encoding nucleic acid (e.g., SMP DNA). As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. This term also encompasses untranslated sequence located at both the 3' and 5' ends of the coding region of the gene: at least about 100 nucleotides of sequence upstream from the 5' end of the coding region and at least about nucleotides of sequence downstream from the 3'end of the coding region of the gene.
The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated SMP nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g, a C. glutamicum cell). Moreover, an "isolated"
nucleic acid molecule, such as a DNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having a nucleotide sequence of an odd-numbered SEQ ID NO of the Sequence Listing, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a C glutamicum SMP DNA can be isolated from a C. glutamicum library using all or portion of one of the odd-numbered SEQ ID NO sequences of the Sequence Listing as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
Moreover, a nucleic acid molecule encompassing all or a portion of one of the nucleic acid sequences of the invention (e.g., an odd-numbered SEQ ID NO:) can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence (e.g., a nucleic acid molecule encompassing all or a portion of one of the nucleic acid sequences of the invention (e.g., an odd-numbered SEQ ID NO of the Sequence Listing) can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this same sequence). For example, mRNA
can be isolated from normal endothelial cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and DNA
can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from GibcoBRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in the Sequence Listing. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to an SMP nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises one of the nucleotide sequences shown in the Sequence Listing. The nucleic acid sequences of the invention, as set forth in the Sequence Listing , correspond to the Corynebacterium gtutamicum SMP DNAs of the invention. This DNA comprises sequences encoding SMP proteins (i.e., the "coding region", indicated in each odd-numbered SEQ ID NO: sequence in the Sequence Listing), as well as 5' untranslated sequences and 3' untranslated sequences, also indicated in each odd-numbered SEQ ID
NO: in the Sequence Listing.. Alternatively, the nucleic acid molecule can comprise only the coding region of any of the sequences in nucleic acid sequences of the Sequence Listing.
For the purposes of this application, it will be understood that each of the nucleic acid and amino acid sequences set forth in the Sequence Listing has an identifying RXA, RXN, or RXS number having the designation "RXA," "RXN," or "RXS" followed by 5 digits (i.e., RXA01626, RXN00043, or RXS0735). Each of the nucleic acid sequences comprises up to three parts: a 5' upstream region, a coding region, and a downstream region. Each of these three regions is identified by the same RXA, RXN, or RXS
designation to eliminate confusion. The recitation "one of the odd-numbered sequences of the Sequence Listing", then, refers to any of the nucleic acid sequences in the Sequence Listing, which may also be distinguished by their differing RXA, RXN, or RXS designations. The coding region of each of these sequences is translated into a corresponding amino acid sequence, which is also set forth in the Sequence Listing, as an even-numbered SEQ ID NO: immediately following the corresponding nucleic acid sequence. For example, the coding region for RXA02735 is set forth in SEQ ID
NO:1, while the amino acid sequence which it encodes is set forth as SEQ ID NO:2.
The sequences of the nucleic acid molecules of the invention are identified by the same RXA, RXN, or RXS designations as the amino acid molecules which they encode, such that they can be readily correlated. For example, the amino acid sequence designated RXA00042 is a translation of the coding region of the nucleotide sequence of nucleic acid molecule RXA00042, and the amino acid sequence designated RXN00043 is a translation of the coding region of the nucleotide sequence of nucleic acid molecule RXN00043. The correspondence between the RXA, RXN and RXS nucleotide and amino acid sequences of the invention and their assigned SEQ ID NOs is set forth in Table 1.
Several of the genes of the invention are "F-designated genes". An F-designated gene includes those genes set forth in Table 1 which have an 'F' in front of the RXAdesignation. For example, SEQ ID NO:11, designated, as indicated on Table 1, as "F RXA01312", is an F-designated gene, as are SEQ ID NOs: 29, 33, and 39 (designated on Table I as "F RXA02803", "F RXA02854", and "F RXA01365", respectively).
In one embodiment, the nucleic acid molecules of the present invention are not intended to include those compiled in Table 2. In the case of the dapD gene, a sequence for this gene was published in Wehrmann, A., et al. (1998) J. Bacteriol.
180(12): 3159-3165. However, the sequence obtained by the inventors of the present application is significantly longer than the published version. It is believed that the published version relied on an incorrect start codon, and thus represents only a fragment of the actual coding region.
In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of one of the nucleotide sequences of the invention (e.g., a sequence of an odd-numbered SEQ
ID
NO: of the Sequence Listing), or a portion thereof. A nucleic acid molecule which is complementary to one of the nucleotide sequences of the invention is one which is sufficiently complementary to one of the nucleotide sequences shown in the Sequence Listing (e.g., the sequence of an odd-numbered SEQ ID NO:) such that it can hybridize to one of the nucleotide sequences of the invention, thereby forming a stable duplex.
In still another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which is at least about 50'/0, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62010, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing), or a portion thereof. Ranges and identity values intermediate to the above-recited ranges, (e.g., 70-90% identical or 80-95% identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended, to be included. In an additional preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one of the nucleotide sequences of the invention, or a portion thereof.
Moreover, the nucleic acid molecule of the invention can comprise only a portion of the coding region of the sequence of one of the odd-numbered SEQ ID
NOs of the Sequence Listing, for example a fragment which can be used as a probe or primer - or a fragment encoding a biologically active portion of an SMP protein. The nucleotide sequences determined from the cloning of the SMP genes from C. glutamicum allows for the generation of probes and primers designed for use in identifying and/or cloning SMP homologues in other cell types and organisms, as well as SMP homologues from other Corynebacteria or related species. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand of one of the nucleotide sequences of the invention (e.g., a sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing), an anti-sense sequence of one of these sequences, or naturally occurring mutants thereof. Primers based on a nucleotide sequence of the invention can be used in PCR reactions to clone SMP
homologues. Probes based on the SMP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g.
the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells which misexpress an SMP protein, such as by measuring a level of an SMP-encoding nucleic acid in a sample of cells, e.g., detecting SMP mRNA levels or determining whether a genomic SMP gene has been mutated or deleted.
In one embodiment, the nucleic acid molecule of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO of the Sequence Listing) such that the protein or portion thereof maintains the ability to perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum. As used herein, the language "sufficiently homologous" refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain as an amino acid residue in a sequence of one of the even-numbered SEQ ID NOs of the Sequence Listing) amino acid residues to an amino acid sequence of the invention such that the protein or portion thereof is able to perfonn a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum.
Protein members of such sugar metabolic pathways or energy producing systems, as described herein, may play a role in the production and secretion of one or more fine chemicals. Examples of such activities are also described herein. Thus, "the function of an SMP protein" contributes either directly or indirectly to the yield, production, and/or efficiency of production of one or more fine chemicals. Examples of SMP
protein activities are set forth in Table 1.
In another embodiment, the protein is at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%, 80-90%, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to an entire amino acid sequence of the invention(e.g., a sequence of an even-numbered SEQ ID NO:
of the Sequence Listing).
Portions of proteins encoded by the SMP nucleic acid molecules of the invention are preferably biologically active portions of one of the SMP proteins. As used herein, the term "biologically active portion of an SMP protein" is intended to include a portion, e.g., a domain/motif, of an SMP protein that participates in the metabolism of carbon compounds such as sugars, or in energy-generating pathways in C glutamicum, or has an activity as set forth in Table 1. To determine whether an SMP protein or a biologically active portion thereof can participate in the metabolism of carbon compounds or in the production of energy-rich molecules in C. glutamicum, an assay of enzymatic activity may be performed. Such assay methods are well known to those of ordinary slcili in the art, as detailed in Example 8 of the Exemplification.
Additional nucleic acid fragments encoding biologically active portions of an SMP protein can be prepared by isolating a portion of one of the amino acid sequences of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing), expressing the encoded portion of the SMP protein or peptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the SMP protein or peptide.
The invention further encompasses nucleic acid molecules that differ from one of the nucleotide sequences of the invention (e.g., a sequence of an odd-numbered SEQ ID
NO: of the Sequence Listing) (and portions thereof) due to degeneracy of the genetic code and thus encode the same SMP protein as that encoded by the nucleotide sequences of the invention. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in the Sequence Listing (e.g., an even-numbered SEQ ID NO:). In a still further embodiment, the nucleic acid molecule of the invention encodes a full length C.
glutamicum protein which is substantially homologous to an amino acid of the invention (encoded by an open reading frame shown in an odd-numbered SEQ ID NO: of the Sequence Listing).
It will be understood by one of ordinary skill in the art that in one embodiment the sequences of the invention are not meant to include the sequences of the prior art, such as those Genbank sequences set forth in Tables 2 or 4 which were available prior to the present invention. In one embodiment, the invention includes nucleotide and amino acid sequences having a percent identity to a nucleotide or amino acid sequence of the invention which is greater than that of a sequence of the prior art (e.g., a Genbank sequence (or the protein encoded by such a sequence) set forth in Tables 2 or 4). For example, the invention includes a nucleotide sequence which is greater than and/or at least 58% identical to the nucleotide sequence designated RXA00014 (SEQ ID
NO:41), a nucleotide sequence which is greater than and/or at least % identical to the nucleotide sequence designated RXA00195 (SEQ ID NO:399), and a nucleotide sequence which is greater than and/or at least 42% identical to the nucleotide sequence designated RXA00196 (SEQ ID NO:401). One of ordinary skill in the art would be able to calculate the lower threshold of percent identity for any given sequence of the invention by examining the GAP-calculated percent identity scores set forth in Table 4 for each of the three top hits for the given sequence, and by subtracting the highest GAP-calculated percent identity from 100 percent. One of ordinary skill in the art will also appreciate that nucleic acid and amino acid sequences having percent identities greater than the lower threshold so calculated (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more identical) are also encompassed by the invention.
In addition to the C. glutamicum SMP nucleotide sequences set forth in the Sequence Listing as odd-numbered SEQ ID NOs, it will be appreciated by those of ordinary skill in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of SMP proteins may exist within a population (e.g., the C.
glutamicum population). Such genetic polymorphism in the SMP gene may exist among individuals within a population due to natural variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding an SMP protein, preferably a C. glutamicum SMP protein.
Such natural variations can typically result in 1-5% variance in the nucleotide sequence of the SMP gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in SMP that are the result of natural variation and that do not alter the functional activity of SMP proteins are intended to be within the scope of the invention.
Nucleic acid molecules corresponding to natural variants and non-C. glutamicum homologues of the C. glutamicum SMP DNA of the invention can be isolated based on their homology to the C. glutamicum SMP nucleic acid disclosed herein using the C.
glutamicum DNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of of an odd-numbered SEQ ID NO: of the Sequence Listing. In other embodiments, the nucleic acid is at least 30, 50, 100, 250 or more nucleotides in length. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those of ordinary skill in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65 C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a nucleotide sequence of the invention corresponds to a naturally-occurring nucleic acid molecule. As used herein, a"naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). In one embodiment, the nucleic acid encodes a natural C.
glutamicum SMP protein.
In addition to naturally-occurring variants of the SMP sequence that may exist in the population, one of ordinary skill in the art will further appreciate that changes can be introduced by mutation into a nucleotide sequence of the invention, thereby leading to changes in the amino acid sequence of the encoded SMP protein, without altering the functional ability of the SMP protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in a nucleotide sequence of the invention. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of one of the SMP proteins (e.g., an even-numbered SEQ ID NO: of the Sequence Listing) without altering the activity of said SMP protein, whereas an "essential" amino acid residue is required for SMP protein activity. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved in the domain having SMP activity) may not be essential for activity and thus are likely to be amenable to alteration without altering SMP activity.
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding SMP proteins that contain changes in amino acid residues that are not essential for SMP activity. Such SMP proteins differ in amino acid sequence from a sequence of an even-numbered SEQ ID NO: of the Sequence Listing yet retain at least one of the SMP activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50% homologous to an amino acid sequence of the invention and is capable of participate in the metabolism of carbon compounds such as sugars, or in the biosynthesis of high-energy compounds in C. glutamicum, or has one or more activities set forth in Table 1. Preferably, the protein encoded by the nucleic acid molecule is at least about 50-60% homologous to the amino acid sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing, more preferably at least about 60-70% homologous to one of these sequences, even more preferably at least about 80%, 80-90%, 90-95% homologous to one of these sequences, and most preferably at least about 96%, 97%, 98%, or 99% homologous to one of the amino acid sequences of the invention.
To determine the percent homology of two amino acid sequences (e.g., one of the amino acid sequences of the invention and a mutant form thereof) or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence (e.g., one of the amino acid sequences the invention) is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence (e.g., a mutant fon=n of the amino acid sequence), then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions/total # of positions x 100).
An isolated nucleic acid molecule encoding an SMP protein homologous to a protein sequence of the invention (e.g., a sequence of an even-numbered SEQ ID
NO: of the Sequence Listing) can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of the invention such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into one of the nucleotide sequences of the invention by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., al.anine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an SMP protein is preferably replaced with another amino acid residue from the same side chain family. Altematively, in another embodiment, mutations can be introduced randomly along all or part of an SMP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an SMP activity described herein to identify mutants that retain SMP activity. Following mutagenesis of the nucleotide sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing, the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, assays described herein (see Example 8 of the Exemplification).
In addition to the nucleic acid molecules encoding SMP proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded DNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire SMP coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an SMP protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the entire coding region of NO.
3(RXA01626) comprises nucleotides I to 345). In another embodiment, the antisense nucleic acid molecule is antisense to a"noncoding region" of the coding strand of a nucleotide sequence encoding SMP. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not transtated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding SMP disclosed herein (e.g., the sequences set forth as odd-numbered SEQ ID NOs in the Sequence Listing), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of SMP mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of SMP mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of SMP mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA
and/or genomic DNA encoding an SMP protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to fonn a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucteic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ~-units, the strands run parallel to each other (Gaultier e1 al. (1987) Nucleic Acids.
Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et aL (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave SMP mRNA transcripts to thereby inhibit translation of SMP
mRNA. A ribozyme having specificity for an SMP-encoding nucleic acid can be designed based upon the nucleotide sequence of an SMP cDNA disclosed herein (i.e., SEQ ID NO. 3(RXA01626)). For example, a derivative of a Teirahymena L-19 IVS
RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an SMP-encoding mRNA.
See, e.g., Cech et al. U.S. Patent No. 4,987,071 and Cech et al. U.S. Patent No.
5,116,742. Alternatively, SMP mRNA can be used to select a catalytic RNA
having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.
Altematively, SMP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of an SMP nucleotide sequence (e.g., an SMP promoter and/or enhancers) to form triple helical structures that prevent transcription of an SMP gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N. Y. Acad.
Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14(12):807-15.
B. Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an SMP protein (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA
techniques are often in the fonn.n of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.
However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells.
Preferred regulatory sequences are, for example, promoters such as cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, Ipp-lac-, IacIq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, amy, SPO2, X-PR-or X PL, which are used preferably in bacteria. Additional regulatory sequences are, for example, promoters from yeasts and fungi, such as ADC 1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4, usp, STLS 1, B33, nos or ubiquitin- or phaseolin-promoters. It is also possible to use artificial promoters. It will be appreciated by those of ordinary skill in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., SMP proteins, mutant forms of SMP proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of SMP proteins in prokaryotic or eukaryotic cells. For example, SMP genes can be expressed in bacterial cells such as C. glutamicum, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos, M.A. et al.
(1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488; van den Hondel, C.A.M.J.J. et al. (1991) "Heterologous gene expression in filamentous fungi"
in: More Gene Manipulations in Fungi, J.W. Bennet & L.L. Lasure, eds., p. 396-428:
Academic Press: San Diego; and van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds., p. 1-28, Cambridge University Press:
Cambridge), algae and multicellular plant cells (see Schmidt, R. and Willmitzer, L. (1988) High efficiency Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana leaf and cotyledon explants" Plant Cell Rep: 583-586), or mammalian cells.
Suitable host cells are discussed further in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein but also to the C-terminus or fused within suitable regions in the proteins. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase.
Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence of the SMP protein is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X protein. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin.
Recombinant SMP protein unfused to GST can be recovered by cleavage of the fusion protein with thrombin.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315), pLG338, pACYC184, pBR322, pUC18, pUC 19, pKC30, pRep4, pHS 1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-I1I113-B1, kgt11, pBdCl, and pET I id (Studier et al., Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Califonva (1990) 60-89;
and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).
Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET I ld vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident k prophage harboring a gn 1 gene under the transcriptional control of the lacUV 5 promoter. For transformation of other varieties of bacteria, appropriate vectors may be selected. For example, the plasmids pIJ101, pIJ364, pIJ702 and pIJ361 are known to be useful in transforming Streptomyces, while plasmids pUB 110, pC 194, or pBD214 are suited for transformation of Bacillus species. Several plasmids of use in the transfer of genetic information into Corynebacterium include pHM1519, pBL1, pSA77, or pAJ667 (Pouwels et al., eds.
(1985) Cloning Vectors. Elsevier: New York IBSN 0 444 90401 8)_ One strategy to maximize recombinant protein expression is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Califomia (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression, such as C.
glutamicum (Wada et aL (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the SMP protein expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari, et al., (1987) EmboJ. 6:229-234), 2 , pAG-1, Yep6, Yep13, pEMBLYe23, pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, include those detailed in: van den Hondel, C.A.M.J.J. &
Punt, P.J.
(1991) "Gene transfer systems and vector development for filamentous fungi, in:
Applied Molecular Genetics of Fungi, J.F. Peberdy, et al., eds., p. 1-28, Cambridge University Press: Cambridge, and Pouwels et al., eds. (1985) Cloning Vectors.
Elsevier:
New York (IBSN 0 444 904018).
Alternatively, the SMP proteins of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.
(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
In another embodiment, the SMP proteins of the invention may be expressed in unicellular plant cells (such as algae) or in plant cells from higher plants (e.g., the spermatophytes, such as crop plants). Examples of plant expression vectors include those detailed in: Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acid. Res. 12: 8711-8721, and include pLGV23, pGHlac+, pBIN19, pAK2004, and pDH51 (Pouwels et al., eds. (1985) Cloning Vectors.
Elsevier:
New York IBSN 0 444 904018). .
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC
(Kaufman et al. (1987) EMBOJ. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific;
Pinkert et al.
(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBOJ. 8:729-733) and immunoglobulins (Banerji et al, (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to SMP mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be deterrnined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. l(1) 1986.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. It is understood that such tenns refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, an SMP
protein can be expressed in bacterial cells such as C. glutamicum, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
Other suitable host cells are known to one of ordinary skill in the art.
Microorganisms related to Corynebacterium glutamicum which may be conveniently used as host cells for the nucleic acid and protein molecules of the invention are set forth in Table 3.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection", "conjugation" and "transduction" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., linear DNA or RNA (e.g., a linearized vector or a gene construct alone without a vector) or nucleic acid in the form of a vector (e.g., a plasmid, phage, phasmid, phagemid, transposon or other DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al.
(Molecular Cloning: A Laboratory ManuaL 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
Preferred selectable markers include those which confer resistance to drugs, such as G41 8, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an SMP protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by, for example, drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
To create a homologous recombinant microorganism, a vector is prepared which contains at least a portion of an SMP gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the SMP
gene.
Preferably, this SMP gene is a Corynebacterium glutamicum SMP gene, but it can be a homologue from a related bacterium or even from a mammalian, yeast, or insect source.
In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous SMP gene is functionally disrupted (I.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
Altematively, the vector can be designed such that, upon homologous recombination, the endogenous SMP gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous SMP protein). In the homologous recombination vector, the altered portion of the SMP gene is flanked at its 5' and 3' ends by additional nucleic acid of the SMP
gene to allow for homologous recombination to occur between the exogenous SMP
gene carried by the vector and an endogenous SMP gene in a microorganism. The additional flanking SMP nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA
(both at the 5' and 3' ends) are included in the vector (see e.g., Thomas, K.R., and Capecchi, M.R. (1987) Cell 51: 503 for a description of homologous recombination vectors). The vector is introduced into a microorganism (e.g., by electroporation) and cells in which the introduced SMP gene has homologously recombined with the endogenous SMP gene are selected, using art-known techniques.
In another embodiment, recombinant microorganisms can be produced which contain selected systems which allow for regulated expression of the introduced gene.
For example, inclusion of an SMP gene on a vector placing it under control of the lac operon permits expression of the SMP gene only in the presence of IPTG. Such regulatory systems are well known in the art.
In another embodiment, an endogenous SMP gene in a host cell is disrupted (e.g., by homologous recombination or other genetic means known in the art) such that expression of its protein product does not occur. In another embodiment, an endogenous or introduced SMP gene in a host cell has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional SMP protein. In still another embodiment, one or more of the regulatory regions (e.g., a promoter, repressor, or inducer) of an SMP gene in a microorganism has been altered (e.g., by deletion, truncation, inversion, or point mutation) such that the expression of the SMP
gene is modulated. One of ordinary skill in the art will appreciate that host cells containing more than one of the described SMP gene and protein modifications may be readily produced using the methods of the invention, and are meant to be included in the present invention.
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) an SMP protein. Accordingly, the invention further provides methods for producing SMP proteins using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding an SMP protein has been introduced, or into which genome has been introduced a gene encoding a wild-type or altered SMP protein) in a suitable medium until SMP protein is produced. In another embodiment, the method further comprises isolating SMP proteins from the medium or the host cell.
C. Isolated SMP Proteins Another aspect of the invention pertains to isolated SMP proteins, and biologically active portions thereof. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material"
includes preparations of SMP protein in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of SMP protein having less than about 30% (by dry weight) of non-SMP protein (also referred to herein as a"contaminating protein"), more preferably less than about 20% of non-SMP
protein, still more preferably less than about 10% of non-SMP protein, and most preferably less than about 5% non-SMP protein. When the SMP protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The language "substantially free of chemical precursors or other chemicals" includes preparations of SMP protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of SMP protein having less than about 30% (by dry weight) of chemical precursors or non-SMP chemicals, more preferably less than about 20% chemical precursors or non-SMP chemicals, still more preferably less than about 10% chemical precursors or non-SMP chemicals, and most preferably less than about 5% chemical precursors or non-SMP chemicals. In preferred embodiments, isolated proteins or biologically active portions thereof lack contaminating proteins from the same organism from which the SMP protein is derived. Typically, such proteins are produced by recombinant expression of, for example, a C. glutamicum SMP
protein in a microorganism such as C. glutamicum.
An isolated SMP protein or a portion thereof of the invention can participate in the metabolism of carbon compounds such as sugars, or in the production of energy compounds (e.g., by oxidative phosphorylation) utilized to drive unfavorable metabolic pathways, or has one or more of the activities set forth in Table 1. In preferred embodiments, the protein or portion thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) such that the protein or portion thereof maintains the ability to perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules by processes such as oxidative phosphorylation in Corynebacterium glutamicum. The portion of the protein is preferably a biologically active portion as described herein. In another preferred embodiment, an SMP protein of the invention has an amino acid sequence set forth as an even-numbered SEQ ID NO: of the Sequence Listing. In yet another preferred embodiment, the SMP protein has an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of the invention (e.g., a sequence of an odd-numbered SEQ
ID NO:
of the Sequence Listing). In still another preferred embodiment, the SMP
protein has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to one of the nucleic acid sequences of the invention, or a portion thereof. Ranges and identity values intermediate to the above-recited values, (e.g., 70-90% identical or 80-95%
identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. The preferred SMP proteins of the present invention also preferably possess at least one of the SMP activities described herein. For example, a preferred SMP protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of the invention, and which can perform a function involved in the metabolism of carbon compounds such as sugars or in the generation of energy molecules (e.g., ATP) by processes such as oxidative phosphorylation in Corynebacterium glutamicum, or which has one or more of the activities set forth in Table 1.
In other embodiments, the SMP protein is substantially homologous to an amino acid sequence of of the invention (e.g., a sequence of an even-numbered SEQ ID
NO: of the Sequence Listing)and retains the functional activity of the protein of one of the amino acid sequences of the invention yet differs in amino acid sequence due to natural variation or mutagenesis, as described in detail in subsection I above.
Accordingly, in another embodiment, the SMP protein is a protein which comprises an amino acid sequence which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to an entire amino acid sequence of the invention and which has at least one of the SMP activities described herein. Ranges and identity values intermediate to the above-recited values, (e.g., 70-90% identical or 80-95%
identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. In another embodiment, the invention pertains to a full length C. glutamrcum protein which is substantially homologous to an entire amino acid sequenoe of the invention.
Biologically active portions of an SMP protein include peptides comprising amino acid sequences derived from the amino acid sequence of an SMP protein, e.g., an amino acid sequence of an even-numbered SEQ ID NO: of the Sequence Listing or the amino acid sequence of a protein homologous to an SMP protein, which include fewer amino acids than a full length SMP protein or the full length protein which is homologous to an SMP protein, and exhibit at least one activity of an SMP
protein.
Typically, biologically active portions (peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif with at least one activity of an SMP protein. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein. Preferably, the biologically active portions of an SMP
protein include one or more selected domains/motifs or portions thereof having biological activity.
SMP proteins are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the SMP protein is expressed in the host cell. The SMP
protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Altemative to recombinant expression, an SMP
protein, polypeptide, or peptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, native SMP protein can be isolated from cells (e.g., endothelial cells), for example using an anti-SMP antibody, which can be produced by standard techniques utilizing an SMP protein or fragment thereof of this invention.
The invention also provides SMP chimeric or fusion proteins. As used herein, an SMP "chimeric protein" or "fusion protein" comprises an SMP polypeptide operatively linked to a non-SMP polypeptide. An "SMP polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an SMP protein, whereas a "non-SMP
polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the SMP protein, e.g., a protein which is different from the SMP protein and which is derived from the same or a different organism. Within the fusion protein, the term "operatively linked" is intended to indicate that the SMP polypeptide and the non-SMP polypeptide are fused in-frame to each other. The non-SMP polypeptide can be fused to the N-terminus or C-terminus of the SMP polypeptide. For example, in one embodiment the fusion protein is a GST-SMP fusion protein in which the SMP sequences are fused to the C-terminus of the GST
sequences. Such fusion proteins can facilitate the purification of recombinant SMP
proteins. In another embodiment, the fusion protein is an SMP protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of an SMP protein can be increased through use of a heterologous signal sequence.
Preferably, an SMP chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA
synthesizers.
Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST
polypeptide).
An SMP-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the SMP protein.
Homologues of the SMP protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the SMP protein. As used herein, the term "homologue"
refers to a variant form of the SMP protein which acts as an agonist or antagonist of the activity of the SMP protein. An agonist of the SMP protein can retain substantially the same, or a subset, of the biological activities of the SMP protein. An antagonist of the SMP protein can inhibit one or more of the activities of the naturally occurring form of the SMP protein, by, for example, competitively binding to a downstream or upstream member of the sugar molecule metabolic cascade or the energy-producing pathway which includes the SMP protein.
In an alternative embodiment, homologues of the SMP protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the SMP
protein for SMP protein agonist or antagonist activity. In one embodiment, a variegated library of SMP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of SMP
variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential SMP
sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of SMP sequences therein.
There are a variety of methods which can be used to produce libraries of potential SMP
homologues from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential SMP sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3;
Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
In addition, libraries of fragments of the SMP protein coding can be used to generate a variegated population of SMP fragments for screening and subsequent selection of homologues of an SMP protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an SMP coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the SMP protein.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA
libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of SMP
homologues. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify SMP homologues (Arkin and Yourvan (1992) PNAS
89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
In another embodiment, cell based assays can be exploited to analyze a variegated SMP library, using methods well known in the art.
D. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologues, fusion proteins, primers, vectors, and host cells described herein can be used in one or more of the following methods: identification of C. glutamicum and related organisms;
mapping of genomes of organisms related to C. glutamicum; identification and localization of C.
glutamicum sequences of interest; evolutionary studies; determination of SMP
protein regions required for function; modulation of an SMP protein activity;
modulation of the metabolism of one or more sugars; modulation of high-energy molecule production in a cell (i.e., ATP, NADPH); and modulation of cellular production of a desired compound, such as a fine chemical.
The SMP nucleic acid molecules of the invention have a variety of uses. First, they may be used to identify an organism as being Corynebacterium glutamicum or a close relative thereof. Also, they may be used to identify the presence of C.
glutamicum or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of C. glutamicum genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present.
Although Corynebacterium glutamicum itself is nonpathogenic, it is related to pathogenic species, such as Corynebacterium diphtheriae. Corynebacterium diphtheriae is the causative agent of diphtheria, a rapidly developing, acute, febrile infection which involves both local and systemic pathology. In this disease, a local lesion develops in the upper respiratory tract and involves necrotic injury to epithelial cells;
the bacilli secrete toxin which is disseminated through this lesion to distal susceptible tissues of the body. Degenerative changes brought about by the inhibition of protein synthesis in these tissues, which include heart, muscle, peripheral nerves, adrenals, kidneys, liver and spleen, result in the systemic pathology of the disease. Diphtheria continues to have high incidence in many parts of the world, including Africa, Asia, Eastern Europe and the independent states of the former Soviet Union. An ongoing epidemic of diphtheria in the latter two regions has resulted in at least 5,000 deaths since 1990.
In one embodiment, the invention provides a method of identifying the presence or activity of Cornyebacterium diphtheriae in a subject. This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention (e.g., the sequences set forth as odd-numbered or even-numbered SEQ ID NOs, respectively, in the Sequence Listing) in a subject, thereby detecting the presence or activity of Corynebacterium diphiheriae in the subject. C. glutamicum and C. diphtheriae are related bacteria, and many of the nucleic acid and protein molecules in C.
glutamicum are homologous to C. diphtheriae nucleic acid and protein molecules, and can therefore be used to detect C. diphtheriae in a subject.
The nucleic acid and protein molecules of the invention may also serve as markers for specific regions of the genome. This has utility not only in the mapping of the genome, but also for functional studies of C. glutamicum proteins. For example, to identify the region of the genome to which a particular C. glutamicum DNA-binding protein binds, the C. glutarnicum genome could be digested, and the fragments incubated with the DNA-binding protein. Those which bind the protein may be additionally probed with the nucleic acid molecules of the invention, preferably with readily detectable labels; binding of such a nucleic acid molecule to the genome fragment enables the localization of the fragment to the genome map of C. glutamicum, and, when performed multiple times with different enzymes, facilitates a rapid determination of the nucleic acid sequence to which the protein binds. Further, the nucleic acid molecules of the invention may be sufficiently homologous to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related bacteria, such as Brevibacterium lactofermentum.
The SMP nucleic acid molecules of the invention are also useful for evolutionary and protein structural studies. The metabolic and energy-releasing processes in which the molecules of the invention participate are utilized by a wide variety of prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the evolutionary relatedness of the organisms can be assessed. Similarly, such a comparison permits an assessment of which regions of the sequence are conserved and which are not, which may aid in determining those regions of the protein which are essential for the functioning of the enzyme. This type of determination is of value for protein engineering studies and may give an indication of what the protein can tolerate in terms of mutagenesis without losing function.
Manipulation of the SMP nucleic acid molecules of the invention may result in the production of SMP proteins having functional differences from the wild-type SMP
proteins. These proteins may be improved in efficiency or activity, may be present in greater numbers in the cell than is usual, or may be decreased in efficiency or activity.
The invention provides methods for screening molecules which modulate the activity of an SMP protein, either by interacting with the protein itself or a substrate or binding partner of the SMP protein, or by modulating the transcription or translation of an SMP nucleic acid molecule of the invention. In such methods, a microorganism expressing one or more SMP proteins of the invention is contacted with one or more test compounds, and the effect of each test compound on the activity or level of expression of the SMP protein is assessed.
There are a number of mechanisms by which the alteration of an SMP protein of the invention may directly affect the yield, production, and/or efficiency of production of a fine chemical from a C. glutamicum strain incorporating such an altered protein.
The degradation of high-energy carbon molecules such as sugars, and the conversion of compounds such as NADH and FADH2 to more useful fon;ns via oxidative phosphorylation results in a number of compounds which themselves may be desirable fine chemicals, such as pyruvate, ATP, NADH, and a number of intermediate sugar compounds. Further, the energy molecules (such as ATP) and the reducing equivalents (such as NADH or NADPH) produced by these metabolic pathways are utilized in the cell to drive reactions which would otherwise be energetically unfavorable.
Such unfavorable reactions include many biosynthetic pathways for fine chemicals.
By improving the ability of the cell to utilize a particular sugar (e,g., by manipulating the genes encoding enzymes involved in the degradation and conversion of that sugar into energy for the cell), one may increase the amount of energy available to permit unfavorable, yet desired metabolic reactions (e.g., the biosynthesis of a desired fine chemical) to occur.
Further, modulation of one or more pathways involved in sugar utilization permits optimization of the conversion of the energy contained within the sugar molecule to the production of one or more desired fine chemicals. For example, by reducing the activity of enzymes involved in, for example, gluconeogenesis, more ATP
is available to drive desired biochemical reactions (such as fine chemical biosyntheses) in the cell. Also, the overall production of energy molecules from sugars may be modulated to ensure that the cell maximizes its energy production from each sugar molecule. Inefficient sugar utilization can lead to excess COz production and excess energy, which may result in futile metabolic cycles. By improving the metabolism of sugar molecules, the cell should be able to function more efficiently, with a need for fewer carbon molecules. This should result in an improved fine chemical product: sugar molecule ratio (improved carbon yield), and permits a decrease in the amount of sugars that must be added to the medium in large-scale fermentor culture of such engineered C.
glutamicum.
The mutagenesis of one or more SMP genes of the invention may also result in SMP proteins having altered activities which indirectly impact the production of one or more desired fine chemicals from C. glutamicum. For example, by increasing the efficiency of utilization of one or more sugars (such that the conversion of the sugar to useful energy molecules is improved), or by increasing the efficiency of conversion of reducing equivalents to useful energy molecules (e.g., by improving the efficiency of oxidative phosphorylation, or the activity of the ATP synthase), one can increase the amount of these high-energy compounds available to the cell to drive normally unfavorable metabolic processes. These processes include the construction of cell walls, transcription, translation, and the biosynthesis of compounds necessary for growth and division of the cells (e.g., nucleotides, amino acids, vitamins, lipids, etc.) (Lengeler et al.
(1999) Biology of Prokaryotes, Thieme Verlag: Stuttgart, p. 88-109; 913-918;
875-899).
By improving the growth and multiplication of these engineered cells, it is possible to increase both the viability of the cells in large-scale culture, and also to improve their rate of division, such that a relatively larger number of cells can survive in fermentor culture. The yield, production, or efficiency of production may be increased, at least -5g-due to the presence of a greater number of viable cells, each producing the desired fine chemical.
Further, many of the degradation products produced during sugar metabolism are themselves utilized by the cell as precursors or intermediates for the production of a number of other useful compounds, some of which are fine chemicals. For example, pyruvate is converted into the amino acid alanine, and ribose-5-phosphate is an integral part of, for example, nucleotide molecules. The amount and efficiency of sugar metabolism, then, has a profound effect on the availability of these degradation products in the cell. By increasing the ability of the cell to process sugars, either in terms of efficiency of existing pathways (e.g., by engineering enzymes involved in these pathways such that they are optimized in activity), or by increasing the availability of the enzymes involved in such pathways (e.g., by increasing the number of these enzymes present in the cell), it is possible to also increase the availability of these degradation products in the cell, which should in turn increase the production of many different other desirable compounds in the cell (e.g., fine chemicals).
The aforementioned mutagenesis strategies for SMP proteins to result in increased yields of a fine chemical from C. glutamicum are not meant to be limiting;
variations on these strategies will be readily apparent to one of ordinary skill in the art.
Using such strategies, and incorporating the mechanisms disclosed herein, the nucleic acid and protein molecules of the invention may be utilized to generate C.
glutamicum or related strains of bacteria expressing mutated SMP nucleic acid and protein molecules such that the yield, production, and/or efficiency of production of a desired compound is improved. This 'desired compound may be any product produced by C. glutamicum, which includes the final products of biosynthesis pathways and intermediates of naturally-occurring metabolic pathways, as well as molecules which do not naturally occur in the metabolism of C. glutamicum, but which are produced by a C.
glutamicum strain of the invention.
This invention is further illustrated by the following examples which should not be construed as limiting.
uj a w w a w M
z j a 0 Wz _ i~~ z m m QF- N ~ z >
N N Zo W z 1u tu ~'N' ~
~ ri F- F- Z~ a U g h ~ N
~u O O O
z ww p p a a a g O N W >~] > O~ O_~ N N N N N N
~ LL I~L S~~.- ~ N fV N N fV N
R = Y V ~-W W W W W~ U U ui vi n tj ~j V ~ a U) < y W W W W
J W W W W W
a v a N W W Ill W W W
a o~~~+ ad' R ~F a a a a a U a 8l, W ~ =ya y S~~aW~ ~
v0 0 J U 0 0 w w ww~0~ W J
t~i .,L Q W W W ~ W == J ~NJ J J
_ ~Ng 8 8 ~a ~ ~N
I y ~ J N I U I U T U U Q~ OI 0 a Y d= d f a V a~ a V
Z ll m J~~ U. N~(n.-fnlALLil tL vQ Wa Wa Wa w W
~,wD N < p O n n LLJ Z
~; W~~ ~ W f0 V(~pn~ONO F~ ~~ ~ g M
MJ NNN
r fp =
Q C
.gl 1D > N O p ~
V ~p ep J"_ y C /~0 y~ t0 t~h! ~ ~I N N CO
Z ~ ~ ~I n ~ N r, N f0 Y1 ' = N N n = h ~ f 0 h.-N
N ~ M~ N M pi t,j N Of ac" ce ~~c~~ c ~
O N _ C M
alll~~~ N 10 1n U_1 N ptf~ p ,}i ~ N~ = O M M M M~ N l7 Ys ~ O M ry (np N
b 0000 ~
zig Z ~
gW
N N= 10 ID Q VI ~ ~ .=- ~~ N N ~ C O ~p p ~ fA N N N l'7 M ~y th Z fn ~- M1An ~"" z vv~~N LLI 9 NN N p~ ~y N M tf ~
y r'= U~U rv~i, ~ ~ ~QN ~~=~-~=~-.~-~
~ W=W NNN4~Y
N [V N fV N N N fV NN
O YMf ~ eM- UUUUC~ U UC~U p.C
x ' !~
V < ~ 1yw 4 a.
y y W iTJlrl~~~~~ Z
00 nn O 0 ~a.. ~ ..v (i~~~ W~=-~W
a 4 NNfVN=-=W~C~Qc%1 ~
~ O~ V a ~ tn 1f/ W y1 S YYI ~S1 N W
N N N g ~ ~ .~r.
v N
t7_y~ 3OQQg~'- =-~-fV N
VVU U
.' WW~jnj wFdFW U UUUQUUUiIgo WW
a- ~n yf/~V~ ~~g~~ WWWWWWW~7~y m ~G~O
y~ S S g}} W W WW W W W W W W U U VW
99a<GQWW WW~WU
yhr~555WWhF- y~ ~~.-yyyyyNyyyyyy W W_ W W W I~~y 222 WWWWW,~j q F~q<WWW ~igy4 ~Y NNNNrqFqpWW WW(~(WWW(~(W~ ~ yyyyV~
~ d aaMS-1-ts-[~~,~~~yW~c3~a~LF~_WW_W_W~~O~~OOOQOOO~y~~~ye~jq~gS~~S}~
U U 0O_ww-t)Q ~~y}OOd9~<VjyWWy}Y~0~0~~}GG~O_O>pmW
}}W~! <Qa }
u=i 202 d ' ~~}>=}}OOCya u~}ra22z J J 6565 Zt~
W W W W W W q q v q Q
-~ J J J LL ~ y UY-Y-Y-Y-O Qd d ~~ ~tlW2ZdOdO0000 2 UU~ yU~U1L~}
p Wk66 ~!SN!yO t~S=TUSS~~~~~ oW
.~ a v a v, Q a a a a y y y ~ W W W a 0.
y t>>> a a q>>>>>>> a q~ 1~ q q a; W
~ viyunUUy 0y00~ >yy~> 7~~ ~ ~~~ V
~ ~ ~S0JJg~~ga~"'*ttt 'o }ioo rcaaoo~rc~~acx~~~
C LL aWaWt9c9aaaa~6~rLL~~~waa}aa~~aaaaaaaao}aaa(L9 O
Z ~ = aD ~ N N aaD 1f1 1[f (~j N fQ N~ m N N O~~ tyj ~ t~f YMi N 1[i ONi Nry a0 N- ~ ~ O MI=~-h . '~f ~ 1ff N ~~Qj 1ri ~Oa l7 NN WfV V 1n~.~~A {p.-m 1Q yl ~D tf, Cf .- N ~fJ h b N N~ I~fl ~~~ 01 ~ O~ _ ~ n 1~ N O ~ff ~ f~ O tD Oi sp Co .-tp Y M N t~l A
p~yf ~p ~ r4 m=-=-N.-fOQO N~ N 4DYINNN~ fN.NNYNf ~NiDM.-~'f ~N Y ~+-dDaMDN N
M M~ggg~ 1~ sgpgogo-l y Q~ O1 f~ ~h 1~ h O!
U
a ~ ~ ~ m N O~y N i7 A~ Nc~ N ~Ny N~ N h~ M h ~ff ~~ aQ ~~ M O~! N~~~ ~ " O
U.
~ Z ~ 01+0f xOe,x~7'{-N?~Zx_ ~RLL
~~~~~ LL LL
9 U.
LL K LL LL
~LLKLL~~~LL~LL LL~
3C~(J~p eV ~p ' y M O ~P~~V~iui' NI~iYitOt6~Dlab~f:nn~aOeDaDa~aDOiQN1~i0iOloO~00~.r-~~~DNNN
~ h N~i Ff ~~ V ~~Yf~(OM~tOtOl~ M1P~~aD~~ o~ ~ 00~00~ ~~ w~~~j ~f~~-~t ~ff a~DaoO~OMi~iO~~~ ~.-.-.-...' v W d dr '".-~
-oR W õr, =cr3ocZZ=io c>vv ~i_ag,rn o~=~i ~~.rZW<WX WW~
NtV ~ s fKb~
fV ' Ym ~ a a W
WWyUj vWWWW y WW W0~~ ~~ 00 WW~j WwwWw a ~ wW t~ U WW
~ N N N N N,~ WwW w W ~ m 3o ~Kw gm eiry~e ~ ry rn= w w _~ ~
g s '~'~w~
w '~ aW a ~
2 Q~f w ~~ N~ ~7$0~~~ EEWSS
W~ v~ W W W W~ W WI I WQ ~S y V W W9WEW9 S=~ C7 CJ
W W W W W W W ~ {y~ W
yNqt<nptlfAN<h~(0W0y~0w0~y0W w~ = 00Ny~N< ~'QOW8~KW
OG_WO ~iFl-~HF
Mc~f,iW~ll1W~WWFFFhF 8W w FF~ W
YOI~w~ F OZZ*Z~~WpOZO~C~ FFF.FrF-viNaNN0OoC ONVai8F~FW-OOGn= OV
oC~K-~a ww<aa<ooo=OOw ~C~~~a~~ = Q=-QQ
g ~ cI
JJ~Sg~55~M~ClIf~J~~=3J~aa~~~~~~s ~ 2 c LL ~~JddJod dddddooa44~~~dda =aa JiCU=J(.U U. U' oC7WOO
o f/1 1AO 1~ n~U p~ a~o (~01~ dOOO)Y ~ W tp l7 u) ~" 01 V7 10a0aYo N
V 1- oo.N1p ~n~4D~p l'!e~ ap NNO~1p~~pp~N NO)~ ip~ t7 NONe~ of~
Z Nttf~ aD~~ NAUS~ {y N ~f~N'VNN~~~ aDNN aD~pN~
N N ~Ntp.-~f1 t0'-Nl7 CD N ~N ~N 17 Z N !-m ti r C7 OI 4D '- f=n p~ 1~ Y Mp~ pU f0 ~ f~ M V) Pl Ifl ~ t~/a pl~ltl~~.-~NMNM1~1+1~ N~n w1f~lOlt"f O
~*p N 1n o l'7 ~- p~ pp O~ p 4'!
gO1~~ONLLK LL ~~y"pK~
~ Q'~2 ~<Z Z ~ Z ~ Z ~t91 W ~ i ~!( X ~X LL LL LL LLK x 9199 ~ LL LL~~ 9 E x K LLIII
LLLL ~( QLL
~
.11 ~ gc ~ N~3~i~g~a~~~~A~ I~-3 8 2 P -~~P 8~ X
~ 9 68-5l-9 ~ ~ ~~~~~ ~
W 1~01~ h ~AYf1r01~ 1~ ~h b ~ h ii~,~~~~~~~~~ ~~m~m- - - - - - - c~ Z~
~ o ti (i Ci Ci .~ V$ ~ W~IWW iD~
N 7~ $ ~tt t h:~n zzzz mm r W W 3 .=r r NNNVj VUr' WWw WW~
Z Z ~ W W W W 000000 ~~
W
V W W W WN Wy ~~~ N W
= z a m m ~ ~ N~N<<NNW Z
~a~zzzoo~~5~25~~rz~ aO
I~y~~~~
N U 3 v wU~~~~~SSS2xNNNN QF~
4 zw=w=oooo'~'~'<a~< ~il4l~
o'8 'o~'www 'b ~ ~
v N
-p 4 UVV~~~ ~~{{~OOOOwwwww J d' --~ ~ ~' 7 U C) U J U J q ~~ U U U U IL y~v c u~{ c~ac~a~oc~ aa~a~aa~a~aaaaaa ~
o m Ny~ aND c=1 n ~rnf ~ ~n n N $ 1~0 p '~ ~ ppp~~1(~ en e7i N -- 4 p! v o co ~y(~ ~" l~!
N N z ~~~~N~ V~~e~f~t9a0~~ Z ~~ONO
~ O~ N h- " Y7 p~pW{V r{p- n~p N ppp~ (ap~y I e0 tD ~
-W ~ ao ~ l~f Vi n ~ O ~ ~ ~[l 1if N rn ~ ~ C N N N ~
t N A
~ ~1. t"9t~f170i~~1N 1nNh ~ V~ "O
E ~
_ ~ _ ~tc (~J 8 epV ~ ~E, ~{~7J ~pp~p oNa{OWO~yN~yfry~Np aND ~y ~p F~ d{y ~pp e N~ N N ~ C Q N NNNNNNNNNNNN~NNN Q NNa ~Z c Z
N N N N~~ N N~C4 N W N~ N N
Z Z N N
W W W jijO1& e 1a~.
(~ N~'.~ NNNp ~~' r'. N IA Ih ~ V
Z Z W 1+~ njN tip:nr~ t~
W N V V ~ N N N ti yVjw-W NUo()N
X X W W c~i U.W.=IL IY
W W
W W
W Q ~ oro~~m Z It1yN~W~ ao ~ =~ ~
W UUUU W ipJuliUW
~ ~ ~ JJ~W W
OC 40COJ 446 k ~~CQCQF
~ O O
y~ W Z W
~UO 0 ~ev~0 O O~y1 s'~~y~~j Z~V~j11tll~y~ ~,U
Ft~ie~i~ h F' W ~ }
~ ~ :4~oyo= ooW n ONtWAj ~ ~ pOOy~ ~yWfsnZ~a~~rN O
ZWUIUlluui ~
~xa s = cnrnrnv,v~
'~~y$ a a a a a ~g~uc ~m~ Z
O_ = O g.. a ~ US -~ 6 'O J~ ti ~
~ 'I lo ~0.~ ~ a70r 7~~(09~CJ9CJ
= LL rb~b~b w {Q N ~p m 1{~y1~P 'Ma O
~ ~ Na p~p~ r ,~ ~ V~tf f0 ~ ~~ Qj ~f1 N~p N aD OI N h N
Nf N9 h~V01~Nff.N-~.l.- Z Pf ~ Z
W N
t" N N~ M A N '='yjN N N n~M N~ N N ~
z ~~10.N- N l~ I ~Olhm~l~f~~ Y IrN Z V
K
0000 c W N ~O 1~
~p ppJ~ 1D~~Nhq tO~O 1~ ~~A N $
C C+) II b ~ A 9U.U.11i i u.
U.
e ~
0 3 0 _ p~y ~p m ~ N~NNN N ~ m~ N NNNtVN A~~NNNNNNAN
~ 1 R z ~ y NN'NN ~~NNNNNNtVN C N
.T. N N
"%
N
07 ~
OI
~ W W W W W
w-pj pj a 0 W W
UU r a V ~ ~ U U U ~~~
~W W soab~aDa0~i0V W O ~ fVNN
w rW w ___ _ ::W.=~ w w w9wW
W W ""
WN
~'~-~~UUUC~UU Ua~ W,~
WW WWWw~WU' ~ v .r~ O d 99 xi oojiiw~~~lww~W WN'~N~
Z
~8888881p~ 8Sl ~~,~,; ~ ~ X x X
mddsd}ddXO O~ U rpiJC~~i80~~W W W ~M-F
NNNO hh VYy~~SSi2q gdWWW~UUjNf.ci9 ~ O O O a O O W W W W {~~y ry1~y Q p r~~4R44~~~ i7> - WW<wOWIOW~WWW~j > > S~4 NNNNNNr'N y a -j' JJJJ ,,r J~= ~2ZZ qU'V~~'~~ LL
O~ 0000$O~OOCU! 44-i~~-m~Z r "N ~ ~ ~~tD
FF t~FFFFtt~-Q N N-~W_W..W..U11...WWW
iQ/}fQ~ fQnVJ(~ tQ~ NNpUIc'v WWO~Z~WY~ ~0~ OyaoMmMaotlfl~M
N V
ZZ~ZZ2~2Z~ZOp O OOZZZOOF- c~i fy F~ OV
J
~~~
~ ~ dazaaaaaaaa ~~ ~~~(d~LLcoLL aWa~~~~090 OYY}Y J~a C u X?ZZ 2YYY ~~~~
o N N P1 W f0 ~ (((yyy ~ p O
1p O Pf a Z ~~ N~.-~ h t~1 ~ i0 ~
~ ~ I~ dD ~C!
~ ~== = ~'f ' i. N N
'õ2 = <~f Nf = 'f N f- N
~ ~~ tP tf) tWtf ~~~ f~ f~- e' 0~1 ry tV ~~ 1f1 W N~ vf ~ r ~~~ 1+/ f0 ~ ~~ OD
"=-'-8~O~O~
~ N =~ ~
r$'O~~Wm~Wb~NN ~ ~PCNn ~ ~ NaO~O O ~~O
LL ~~~LL R: LL
aLLLL XLLX LL
K
xLL
Ir xKLL ~tL
lm _ ~ p w~~~~~~~~~~~~~
l _ 2 1 W ~R~NYv~Ne~~~e~i~i-~n =~~! ~ N afnf~Me~r~~~t~i t~+~ ~t~~~ ~ ~3 e~e~M
N
W
M (~1 ~
r __ W W U.
~.j IV g O y ovwiai __ r =- ~~7iu~
aDW~W ~ 'rUU R9;;.g Q I[i uiW
U UNlclt'>NNNNN WwhNN VUWWW~ Ny WV aa UU~
v~viitf WWVVr U ~ ~~....WW
Z
IV N Z_ W
VUU"'~~ C y,1 wWWWWUUV. N ~UWy~y~WW~~lin Wa J
.~ .~~OC
~~K~WW~JUUUUU Uy~ M9J~ZWWW
~~ Z~NN
FFF-< LLLLLLJJJ ~}Y~ NO>NN~n~nWU~ 4WTrJ~O~W_GWC~~Uwa_0.
Z a~~ ~=-= ~. .hp KJVVg 00 OOCpjZ<VUVrrOC J~~~~~lf~~ ZZZZyUW~wW ~~W
agi mm Z=SS W 7d~WW<~=jih ~~'z paata~a~ '~
=ja~ ~
aQUU ~~~i 4 00 O~u ~W W W W Z
~C3 ~ ~ Op pNvCJ OC
O== ~ Wa SS=~WytN2ZWW w s~j7~'~~~7~~Ww" GO
R4a3,woO~aaaa~g ~gu~ YYWWEmi3 JJ~JJJJJJ=?~~~~~O~m~
C7cJc9oo ~ -v zz6zzZZ >> Fwwyv~ ~ $x.
WWWQ WWW(~QW{~~71 IDCL~ W <Q d x ~T Wu1WW p.~1 W W
888 (9C900UOOO~~N'~ZZypjy~T~YY}~~~~~~j~~K!-FFFOOVO
=C o 2-+C010 UO p~J ~
J~[ OX ~0000~ a QQQy7U ~~UmmaD
ZZZ !
4YJJ}JJJJ Jj=}m~Oq-~ Z ~ ~~ < 3C LL ~~oaa~--~r ~~~i_c~c7C7c~c9(7c~~UC~xxzKZ~bo?. izTZZzOC9~ ~7C7g~
:.:.
O
V tA~ tD Nt~H)O~ff NiD ~ h~+ YNIM ~OD N Nfpp hOi1'~ aD ~ T Vni hPf aD
O M M 1+ N ~pp a0 OD O ha h M ~hp M M CD Cal.Y1 Yf f~ M aD CI h O c~ O cn N;- .~ Y! O
NI~~reN-~ ef~~ MNNtM~fYiM~N tDa ~r~r NiDN <
m Y
~ M t+1 m~ r ht{pp fM~pV O~i0Mt~0 O{~ M1~ V~~ nh~p+fN y~~y Nrp~p (Oh yp~~ ~p~ 01 ~Y ~~(f OP1 O cn~Oh ~~Yf~~N f+-~.N~N ~r.Y-N~ 1~ t~h tONN~
~ N M~ff r ~.- M r.- M N M N r.- f H M/~ r~- N N O~ a0 M N t0 m 0 r h r ~p l+~ M {~ 01l~ ~y W In o ~pp N h 01 M V' N N
Q~ Rf N h~(O h e~ '. /n 0 = q r.
AnO~M N
M~ N h h O p~ Nr N fh~lr hM h U p ~ ~~p l+1 OM h NYf ~i t+f OI ~ ~
pfp~ Mf'l'r' ~ r haD~~pp h Sj {0~~ h N~mN~Gh0/ M N~OIN NCp O~y~$~pNp1 Npp=
~'f 'Q~Np ~y ~ ~~ ~~yN N~haDNrQffry n~V h{~ p~ p'o' h< AQa E~D O hM N pN V
C ~~aqTr5OO~Q~rQ~Q~SOaO'CSj~~}Q4 O O~GrqJs4~~~{G ~Z~O pQ O~r~PN O
LL ~ ~ZLL ~X Z~(LLBLL~~~ ~8LL~~ LL~ LLILLLLL~~S LL~
- ~ LL ~ Q LL K
~ Q
~
N ~ iV N
W ~ ~ V Y E
W uj': U U U ~ $ E
a:
~
px uiW v y ' w w~ y 8 _ N UN W v ~ 40) a~ ~ ~~
r 17 i - W Q W ' -' Na ~ 7 K Vi' U~. W N N u W O 'wS oC
~~NN N~O lcm + -a- 4 ~ ~1~ U E
W W
N ~c wWWniyUjW a=-WXa~ LL W FW-Ua 'L 4 c ~~~UUvmv JE 2 z U~ 0 sN vW) UUWW 1~ w~W = nj ~_ ~
"
N {- W U 0 =- 9 1 $~ $Vi O~O yW W ~VWIc U7 etiW ~yK ~ c= S, ~
N !%J N y~ 91 =7~ p] N ~'! ~- g'~ ~. c~9 K4Q a~ 1Sa gFerWNW YU w t9 hWwi1LC~W~ W ~ ,"=.:QOOLLN t"~vU.lISF o . aa GOLLy ~
o ~ p x w o iowa~ ~~ ~~c< a a ~'g' Z~~~I r~ aw o ~.g. ~.sg.ssc y~ q~bQ~b FF SWv~(?.:>W NK W N NW W
~ I a 0 WvaO~ ~t~p WyZO O wr~' t 0 ~>_YyWNOO ~r80mw~ S~ZyW-7 O -~ ';~ c OOZZj N _==d] O ~p~~ O a WU c.~ I W{j/ W~~j ~<<2Z 7~~~Cpj ~ ~~~YNpLL ~. o~p~
QjQj p,,jJJ y~SZ_NqNpaod~wWa~p '.Y N ~a rfW ~~ z NN W N~ G C c C ~ ~ ~~~.~ ~.~!
~.Q 1 1 1 T T~~UJ ~~~ F-'JlV $
~ - -l ~ LCC' _" r-~ Ua;O p .p s~
oaaaaQaaQooo a Oa6"~WOWawO~Qc~ Qg- m- 9~ 9 E~ ) vtJi r~ ap~ F o c ~ U. rv o~~ p~Q pFaa~t~U~a3c9~~~c}c} i n u'~a < 0U
:z O o W~ ~ cp 1 N W W N O Yf ~p - W M dp ~ = fDa~on F ~p e~ W1p Y'I
O 't W O
NNO~ O p ~p p~ ~y~NN~ SZ
r V f OD.-nmNOWi~-~N ~'1 ~ ~NNt90 3 iG
~ ' NN Ot~O1N ~~ ~~~ n W O~OlO~ n +j (~ 171O~N~YWp~~ y ~
'fNA~V bn~~~~NNIOYI f{~y~17~~.-Nh ~
~tly ~=y 1~~ ~ ~ W
~ IffN N w n n 17 ~t p~ ~4Q ~AQN~O $ O_ Q.- ~~ W N Uf U~ C~C7C7ClS~~~ S~~NNNQ$~
S
W ~ n rr N
zzz u.LL4'C~~~~a~ LL KLLKKKKKK K4'~a"~a~~K
~FZ
G
V<-W :v vvV:! VV
W OWi ~ On! W' n ~ lW fffffffV~FO~f~f M~rY~'f~f Vf~ = fV~1Yf8f~Qf17i ' iffu~'~=V- N
IpNNN~~f NO t W~.WiD
~ e~l .. tO b i0 0 NRNi E
W N=~u~i,~_~ NW
OvVCliUWX
~ZWWW WW j~
O~W
rr..., ~ ~av A WW ~ q00 SU~WWW yv ~iNCCW, pp E E ~~W3"G~VC~U fgfg!!~~
< w~
$ ~ZG UUUU~~~yWWyWNW" Z ;C
WWW ~YWWZZ~M~~Nqf,VYi o E t UU~~WN = WW=-.=-~>_~+
Wmoca'r~'aroi~~~~~+L~~~~~~-"~
x~yy=WWWW~~
2 20 ~4$$~~r Z~WW=WS~!==W ~<O ~Z~~~~~~"
~~$ S~ C2 E 2 Y}F-1-FF~F~-~~YOOG O' _ k-~ bI
~~~oQ~~~~aQ'oo 0 c U.
w V N I ~t~ogb~~~a~
Z Pf ~ N N N~o N7 01 V' r tn r ZI in N.- C'! ~ c7 N P'f 1f! r l'1 N "
~ h N
~ ~~' ~00 0)Yf NN~~~~1~ Np N
~y~y C ~by 'N' Or II UU~UUt~U~ U~( ~y Z~ZX~ Z Z
z ~ ~~LL a $ ~~~LL~LLRW~~ ~~2LLLL R'LLILK K
~~ Z
0 ~
~~~~~h~~~~ m ~~0 W) ~ Q a u,~~~~~~~x -9 NNq sag W W U
mm > > m tu zz uwwLLl '='~NfVww Y11N Yi~~_NN~V9~_ rivi~?~r~r00:
v4fea WW .r.... ~ fJ
C)VW 4 <
WW W UW e rT iracccm'.w0y pft s~nn~iiill-JdJ~z wõ sszzz Z~~
t=WWXX O
~R
~ LLI H1I ~l ~~~~~~c (O f 1+1 f~. ~ N O~1 f!1 Qpp1~~
o ~p Q 46 _ p QQQ
V y 1~ 1 1. V~ Ih F- ~ ~ ~o p ~ m Of Of ~f Z V - NtD TOI
m C C
Z ~<N~fVHft+l Z t+10~~~Off~~lf~NYfO~O/NOl ~~~o~~~ =c~ N~~pO~~OC~~~'~~ .C' ~~~
UI ~O~~bt7 vl ~C9t9t~t~~~i~C7t9~00 UUU3I ~n(~
p p a N cn a &
co $j~g"~P 1L~~ L ~4N4N~~K~LL6 fQ+~
~3Zs~Qqo~[ Z
LL ~(K~LL~~~K~~oz~(NX~pxa ~ ~3a ~K K KLL~
~ ~o L ~e ~ 111 O a IMM ~ ~ Z ~~~~s~mmmmm~~ Z ~! ~~~
y P9 Nf l+l C' O G O
VUU y(,~ '-vi~ < Z ~
W~W4~Wei ai~ vi u. 9, J ~
j p1 W OaDa~ __ _~ _ ~ OjOi ?
NiVi~ai ~~tU~~'=- Ny _ _ _ 222Z2ZUtlUUWU eqr> ~+fMZ_p 0'O12 R aWOi1YW-W wz ro,viO.mb~
UUUU 1y t~~u <~ .=4m.~ ; ~-"'- .-U W
> O
Wow WWi~Wp~gF W~ UZZUUUVUUWW
W~WW W
y W--W_WWWW_W_yV1 !q-FFa--a- QGOSJN ~_O.ZO.WW0. p U Wg JJi iJJV
x G
y~ ZZZZZZ
xici}m~ ~ ~ ,}z_ic W
z r< ss aiat~hai OOO((~~ vai~~UO j A_U_~ t _~i~_IK
iijm Oj~O~OGQ~~~ ~~~Wy<Wy~( Wy<l Q~o'f W WW O_dap-~~__wKU + W2~ZZO0~
Q
F' 'mm} y~XXXXX LLLL O1 >>F F~ ((~~ W y O O O O O O U~ Oa yFF 8 (~ W W W((~~ W((~~ W
VUUU GGOC}ISGUUUUUU~~OW }~~~~~~~Z~V
} y}} W W W W W W W W W W W W __ JJJJ ~~ ~Te U ZZzsa OO v C9C9 ~~~~~~00~~00p~0 ~pXx XZZ W QQ_~
c 8=2 225===2KTW SW~20~~~Q~~000007y~ti ol Io~ a PfKKaWC~
a US== =2 ~il aia= ~iUcic.~t.~u 0 c.>Ut.~cici~ac~.>LLIWi.IWi.WW~~ZZZ~~zZ~' o p _ pp tA t~0 ~'g<NO~iT N1~ ~~i O~~ 1~0 eA0 V c"I YN1 y NaDN ~ N V N ~t")00011 ~p N {~
Oi p/+/ O~ N~~1")Of f a0 p l"!1p ~ N~O ~ OD.-aD1D~tON V ~D=-~ ~=- N~ NN~.-h e!N V V
C {p 1f+~1 ~pp Nppp ~V~ ~+i.~-O~N01~lp+~N~1+~y Vl~11~f~.N=-N~=pt~ttnVf ~ =
yF-Ii ~p~pp v1 N Vl ~t~ n ~ N ~f ~
~ ZI N~ OOi~ .a- N.- N N 1~ cD Pf N n~ ~~ Nf .~- 'f N N N N ~~ 00 N c7 N
1~-.01C/ f~~N W O/ O W 1~N
~~~~~~8888go~8880 9> VI OU (9nn(.'1~~(9oS~7C7(~9(~?oC7?(7>CJ~C7C9~
sZ ~vO
I ~$g~
~~~~x( QX~~~~~~{Oq OQ~' ~[ a5g N
~LL~K CLLLL~ ~~4.Y{L Ip*IIIK~G~~~~~ IL~CLLI~~~
m Q o 2 W
w W V c~, W W oi aa -- ? N D a U
q,Z
Z pW ?~
~~ " o Q
W siiUU~W Z a c~i0 W_N rp~-~~
,oaor..rZ'~ U < <orom F W- "WW~~ Z e'l ~ e7 IJO W Q W~ W WV=?z ~ 4W) W Ww~ Hp ZU~UWgOWz~W ?~a IhI
yro zTX X} c W a0 ~~ O O F O <<~ m m~
2U88 ~ZZW~~ZZW._Z_ ~a a~~ ZwZ~~c4~~c WWW
M
WZ 5 Ug WW~ oa aWW W~~vO9 NWW
de~~oo sgws'~u7go~0.0~~~ W~~ Ox~~
~ ''4 i "~"r~o oa~~~~Te a ~xpxF~,,~~~ o 111111 rrrr2~~ZI Ux~Sp~~000cU=-V=S L tAt!)V1V1tAa) lL~ 2WZC1C~~t Z~20~LLLLan.~s(9V UZ21~= I QQQQQ<
n'~ie ~pr ~~~npa~po$'i ~m O mmp~~p fOnticX o 8 ~+ma vt' 1p ,'~i"' ~epOO~1 N N~f. t7 N ~~d0~ naDt7r biO~ 1710 .n NInO lMP) MN~D Y L Y Y' al r M~ O N t9 ~~ b ~ C~ ~p 01 = n Np ~ N l+f M N 1m p ~D M yl 1~ uf m F T1~/'bN ~ U1~Np ~p aD(p ONO Op ~[f~p {- ~ nNh ZN GN~OI~D=~~P70NNi0e(y011A ~ If/ NNN f 1~7P'1 O Z ~f0~='~
q ~ ~~~0~~ ~~Q C~o aDUf Np n ly I nnN O N~~ ~
V~ C7 C9 ~ ~ ~
pp~p pp ~p ~y ~pp ~- W{p~
l'70D O ~ N ~ N~OM' N
N aOD ~
ZigIZ
~~LL9~~~ti,L~ LLa ~ ~
r IS~
0 f}~~
~, ~ O~ 00000.'-~~~~'(~{{y~ NNNe+f ~+f~ ln+fe+f Y Yf af~fM
Q Z~ n n 1~ 1~ 1~ F
b n n n H n n f- A/~ f~ A f~ n n n f~ h n n n n n n P h. 1-"i Q
~, a '~WU '=h rotCWZW
UuZ~
wwl(~ Wm ZZ~p~= apu C) L) a W~~
UUOwQ orc wUJ U) wwa a ~Z ~v m o ~
z zz o }}}}}? o Ut ~ . hNV~hym ~
a aa~a c C LL QQQQaQ U. uS
~
p ~ o ~ ~~ ' V N
t7tOrtA n N t'~Y Z f~V
.G ~9I N
z lh fV M ~MON1 F~) ~~
NlzN
N CD ~ ~ ~ Q N
>8SS~
S g U V
s c g~sro~ E
u.8~aa~
.0 ~z m 0o E ao yw nr;~~~~.
.mc w ~ ~ o c Z gtz N
u o {s :o ~
to cc C
E
' h o m c v7 o ~ a ~
C a~ N C C~ w~ 4~
ooS gn~ ~.,._yv c ~y O 7 m~ v v O
0000 Ei 4l E 00 y N
E
~~a~.3'o Rwoo a~~~'a1 ~
z W
CF
mN =S~ ~
h Y ~. T,~ = -=fi ' x SE
,~oG m~~H~Ern ~[mN3oa:G
u W w N o >
~
~ ..aa o Ly~p3 y E==1 ~ v1 N y T V y C C t7 ~ 4 ~ ~~i~ ~ ~ g =; ~' 4) 6 15111 d QG ~ N M M N~p r O Nvnvi v1 v1 O 00 N CO
~=i h ~ N~ V1 O0 00 N N N V~'~ g M M ~
~~ mSS 9 9 w~~~
aa aaaa a a aaaa a<a a aaa<
d ~~ v u y 0 0'~ E v ' ep = o 00 '' ~ =C o N y o00 c~ a~U ~-w U o = 4 ~
u p V x y0- C/
p =~y o y v a u vVi A N V N W~= C
= n C
E
u a r,:~ u 1 u a.
C3a ~c~ X
o, u U 0 u o N c ~
~ u t 79 2 V y~~ N
o a ' or = ~
F~v to uZ 'c_ O Ci y . w E12 $ y[ , ' ~ Y
,o = ~ a r~ " C .~ '~ y ?~ Q= y E ' = W l-~'- '-~ E y C N p L O=U x-, O y C==~ =O N N V ~V ~ T.17 ...- a ~ V/~
~$s o _~ v~ u~ u u ti c$A u E~~ ~_ ~' A 3~ y e uE C C ~~ ~ ~'C ,a u ~i &._ =c~
p c o u ns=e c e o o o o u ~=- o o~u ~'9 ~ao~ E
n y A a o T~ QL o~ a ~ E ~
o~'mo eow E pn~o o o 0 a aQ 5 a Z o a Loa Z a V
ts 0 Q ~p $'O vy h h h rl 00 ~ .~ ~ N a a wa aaQa~ ~~4 a a~a U. a Q a aa o = ~ - ICU
i+ Y ~p $~~~~~õni ei ~o o i3v=~=~ o o~ ' ~7 ~o ~=~ 0 5~'u o eo~ ~ E ~ ~~.E $h u u ~~
en _, F uC o_ Y p oo a S~ y u u N~ .~
~~p '~" Fi1 ~y . ~ ~ .. ~ pld d= 'pp 3 Y i'Q y tpT~ L' ~~ V r'j M~1 0~ 'p =!j r l0 bp~C h Y p='s p C t0 C~i O~ i0 Yu M a V~~ u C ~2 Y QO O~
=~ vYi~p= y C ~ ~=~p Mt...~ O~ti 5Ge C v~ ~
a O~'~.~l0 =~ O Y O N~~ R. E~' C'~% p~~p =O ' ~, T. p E t-' =O O Vi7~ y C O~ O 7 O O O Y CGL
Y - v'~= ~~M '~=~ '~ pa W O. Opo t~ ~~ ~N C E y ON=.C" õj õj ~~N
e~ ~' $ o'Ca E ca: o o ~m r E= E DOO 1.2 o u.yu.uc~g,'~; ~'=5 ~d ' ~= vu~~a.uu u L oho 7dL C Y a GT7~ ~OT. ,T ~=C O~W,= Q071"~
{>i ~eE$ ~ Q r~
E.
G' r~ _ ~i a E o CL Ta e -=u m" U~ o a ' a ae ~ou.
c 'u E S
'~$~ =- Coo ax r+E= =$.
u~ e~ >'=? e I1t!!_!...H c u V ~> Q~~ o o~ o~i?~ b o o Q ae o ~=a ~ .~ _ Q~ V y N ~ r' n' $ C=C Y vYi o E
~=C y ~,C ~ Y O.~ E ~ 7~ E O
.G .~ t0 N ~ vWi Q E L' .Y. 4u t~ O L C
t,l_C, g ~ T p 6, Y~ t3a ~~~'j ~ u t wp vTi r C V x'Y~' ~ põ ~= Y~ E~ o~ C ~ O N
't, ~ ~ a7 X ~ T= ~C ,y d 'C y ~ 0 ~ 'g =t~.
c E > E ~ E R u u E
~ s u Y 6 u O E T v q OGQ ~ y=7 T ~ yN V V~,,, Y 'C
O ~~ G Y5 pC y E ~ V' t{~ ~ '~'7 C O{~ r~
~ b ~~ O f! ~~. N Y 'G G ~ OD
~$ o G~~ ~ o~ s E Q''o EL-~ a za F N
$ ~
eo w ' A 8='~ ~a ~ ~ 8. ~ = ~' ~, ~ a~i ~o ~E o s M ~ M ~ O~ ~ vpi O
8 ~ S C ~ N N M ~ O ~ M ~h'1 M
a a a a aa a< A A ~~~~
~
tr ~y u -' y w .5 r ~ oao o'~o e ~
~
V uo~+' y~'= $;; ~~, a, 2i E E~
O = ONO u O . ~D Z 7 G C V p~t~
p~ p O p O 'J a~'0 l0 =.~. tyi ;O ,='~3 =w E ~ O~
{8 .'a .O C N = MN 4 a Y =C N ty G. C1.
O~ O. F =~ M --.
0 Q T ttl 'G. ~ =fi JR 4== K ~ ~y OVON V~ v== E. n= ~ P .D O a O.
c_oM
c~ = o',! 0 8 e c ~~'= u g a, u ai 10~ w h .v o ;c~ ~o=e =~ o u u S o r oDo ~X.c$ g oo a c e u~n e_ eo-~ e c a~~ ~3 =.e.~ = =z~p s s s Q = ==.~, dG ~- _ C V Q=~ N =E N Ofl a a$ a C ~+M '+M C N_ O O
E
~;
Q~zd e~_ N oQ
~ v 'U xoM EM E=oM~M ~N+~~.~._.~~p~
oo.Z ca g= G~ u~ u~ ~$ v~ uo+ ao~~c.v~ ~i~ vs ~< ~a a~ a .~~ = ~p v 5 710 O
Z' io c t7 u 7 v v_ M M- M(~ n y . ~y .
M M iV N~ a. {i .~. id lV PM'1 ~ c'1 a'V'Jj . r ~L
V~ E V C C 37 O~ N iY+ N N~1 ~p N~ : O. ~7 p~ V p~ N"~
V~' ~~: Y a.+ 0 00 OO IZ V1 ~O C
c o o e~ "~ E-- 0. F. a v v o Y Q o0e cd ~+ af cv o ea ~o R o a~c v~ o~ ~d~
au o E.2 ~ E E'~ oeo Ei;; c ~a o x E ~ o~ oo o ~ a A ~ c y t . E oo E oo _ a e $ oo e v r ~e ~e : = . . .~ o .~ o S ocno ~
C
V
N u u '~ pEp C
~ A a0 O
C C V O :7 ~ o ~ s'a =~ .~ .c e a, T d a ~ = v o~ ~ u y s S Ta saT.r . =3 a X ~p, v G G. ~ y 'v V .V+ E a ~ppQ,=V w O. N
~U Vl C W V C =~ C ~ Y=
r E O e E 7i L' a O ~ tnl= 3 O S:4 'ro- ((-7%
.. a a e oo =o ~y~vt wo V E M ~Np GV.
~ ~ ~'"' ~~ ~ O. G G Z. T1 ~ O. 0 1'- =G C ,C L
[o~
n.< oa v~ " G~ u == a -, -, a, ~b $.
[DIII! V C o0 .~ c G x ~ ~ ~ C C ~ C C
C C C V .N .S A 7 7 3 =7 7 OA r ~ ~y y jd C~ ~..0"y.C' DP ~iftY1 G~=~p,~...p, V V V V V
c oz oz 41>a o~ o~~ v v e ~t ao. u e"_ c =~ Ep a ~ m~ a Ea 0 0 ~a ~y n~ P3 C~ o~~ c~ M E~ ~~'~ o 0 0 0 0.~ td'~ ~ O O V V
., a'~av ia E õ g f g= _ D Q
z z a.~ ~g m~' U=v u'o z~zMS - - Q Q 4='~s noo aoo aoo oao ~yoo E'... E:. E A G r = E- E:Z ~ Z= Z-" ~ E,=i E; E E E
- p e c~ o a~ ' ppyp p do QoQoQoQo aQ ~~ o O c ul~ !r- 0i'~ o Gs.~ .-CC ZS '.a a .as.as.a ..D C7 = C7 =~ C7 N~ N ~ N~ tC r ~
- v~ vY ~f ea Y iar Te w, w_ af~.
'a E ps -e s vcr, N iSN ~iN Y+N
z r r r r r r oo Y: 73~ o ~o~oo.~o~ooo.
E oo o o .. o e ~ $ T g+ T , aqo e o~ o v -cu 'g Ce vlyv ao C c C C C
v c.inLO-W inw QatO.= ~-d~~ 00 ;
o v N V
V C
=V
er o E
= C n.
o v v ~ '- o > u u re g e x S~ E P Z x H u a =a 'gq UN C C m ~- E L O, A aVi O '~
~, ~ ~ =O ~, :G =~ y5 b0 V =
Q" c c y 5 a~8 q a Y W
N O N ~ r 00 0_0 ONO tNry M ~ R ~ O' O' 01 O . h~ W ~ h r ~p N N ~D ~p v1 r r r r r r P-R7 f~7 ~tl 41 I~ Lf] I~ It~ 47 W [Ot7 W LOi~ Ii7 W W W W W W Lt] W
N
~ O0 ~ C 7%'~ ~P~= aC N N a! ~v~1 V V
a y v1 '~ 000 3 ~~ $ev T C
C~p a! p~=F~ C.: CO ~ ~õO~ O y ~o 7 '~ =- O a.l =~ y1 ..i .~ O yC v~ 'G y N g~o .~
=C ~~ i~d a.... ~=h o Q ~ u~ ~m V C C C
~ ob~ ~' ' u o ~X =y o ~$~ ao o0 _icr,~ o_y U au; o=ot- u ~S+ u ao a ~ - == ~.a e ~
.~ p~ fn U O 'a =~ .C C ai !t m- t~ 0~ O~ ~'=. ~. 0' p, S~
.C =~ iC0 "~ 7 'R' ' C" ~ =~j pb i.~~". l~ ~" < u v t> co u v p, 's Op, 'p A1~ ~ o Q Z~ C ~v" ~= di' ~7~ C 4~ S7=~ ~7 t~
+% ~=p~ ~p~ U O, =~, W ,~ 'C1 ryi ~y C t ~ .~ Q C ~ ~ ~ ~ V
aNR ~j=V OO~.fn(~ ypr~ C~ ~TP ty app0 C 'n~4õ W"
p Z V~_. IyV =~ w pU=a N.C ~'in ~ C 7 C 7 t pl-~= H ~ ~O = u =y < 21 af SJ bD G~ N Z ~~j ~. O aC0 y=~=~ C=r o,~p_, ~ C~ C af 2 -'nt g W E eL E
E '- Ee~i~~u u o e2~ ~ o~ ~~ u mp v~ u I E v oV 7 m y E
u u o en o ~v N o a E W ~ i'i =~ ._ F't u aa . ~e .a :.
Z
. E v '~-~-s S'v ucN!y~ ~~'~' v~'y ~~a~i S5'-~S
?'. C -l V ~ Q'. o U 0 ,? V =~ ~O G . Y N .D } fV fV
o~o,o=~o >=~ 6 i'i~~n~vFpV, <
0 N yõ y =~ W=N C~O O t . .~ V~ d' LS C O~ 4o O l~C A~ N
? Z o~ c~. U eo4 lL Ev, u, aa -, eo~'~ ~'v -v.U.:. O c=i r,.U n~v~mv, v, c~j C 'e o a N ~y a H
EO Op C y T o = ~ o u ~u N N a0 C rJC
Q ~ t'3 ~. X X X 0 Cp~ .CC _~ '~ .O =vp ~p p~' ~?
a a N z z - - ~
g g v~i O $ M n fMV N $~ r~+~ ~c ~b ~
~o ~n oo n C4 oo n 3 ~ ~ ~
tn A 5e~~ YC on ~ - 5~ .
N FTi ~y v ~ 4 ,=t~c~
C=r0 O G O
$0 ~>.=aCY t~
F~ F en eD u~ v Y ~= O ~- p p~ a C 5 W 5~
Y~ C7zQ C7zd a ~~ E u ~
0 Zo~ Zt~ pt~~_ a z a a ~ p!n Q(A N ~,, ~ q( Vv A G g S. I~ -=~vl ~((V~~~~.CN N V 00 00 i 00 00 0 0_ sOD~ ~QO= avuQ u~ L; t54'' upci o~=_'~n =+5~a 7~ <
v 'N ~ae = ~~n~oo -a g~$'~'~%~t1'~
c r (j e .. u o o u o v.'~'3 .~ 3 5>o ao'M.~ gN~~ 8 ~~ u pCai ~
o~ ~~' ~ '.2~~U C)~
C n CN u c..E-v u~ ~ "F-C7 u n t~
e u v' c~ g =
u d eov..=_r~
E~ 5d 5~ c~' N 7 i==.
o~M r~ E~ E_~.Y-0.c o$ EV C ~... o ,o <~ a urCi ~(~~ O u V ~o~ EO~Y~~~OV e~ t0 ~~ t~ ~0 f.0 v~ CLi 7- a0 W N
>VC7 w~c7 ae~ ~?' eo=. .~~ p eu V~n,y : oos 30_~ 0 30+ 3v E
w ~ ~V" = ~ = ~S O 'O e0 u d ' 7 Y=' - Y=' ti~ V~~' 'C =C =-a', ~N V Q y b ~ a0 W u.~ V V~ V.~ ~
m s u o ~'~({~~ o' - 3 Y v p u u'; _u a~ vi c v_~ c vi ~ a N ~ ~ \ 1.. O Np~ Q ~ L=~+ ~ ~ W N f' 1 F= =~ Y ~~l' =_ V~1i ~'=~ x N ~
~ rC+t 'p vV C O r C ~ C ~
~ z] U
tC 7 p Z='u' p: ,LW ' O ?'mi u E
~ Qa euoG a u~oG a2r ~. U~i ~
u _ w 1 ~ u N $ y, a K 0 w u u a 'u Z' N O
u u a i u c o " > Y o ='e E+ k '~r 'v '~+ =n. ~ ~ u E ~ -e", > ~ ~ 8 n y'~~~ + x=
=Y =Y ~ ~ ~
$ z vJ eq = u o c ~" ~
E a N N < E~~ .5 ~"a' oc be T x o a' 8 Q a v1 [V N eF Vl rO~
In eh0 0~0 ONO O' _~ M ~~ ~ N
v ~ . c ~ w od E~ opq o~ =~ " e _ 0 7 00 c t3 ~ r ~'i3 a 3~ ~=~~'-'~3 ~~
F. -;3 Y a E
eEd? ~Y w c~ ~~-~ W ~~~lo-~
vN o~t~~ o u o Eo~ooEi~v u ~u~' aci _ oo w< u_~ t o E E V ~ ~ c Y C~ V.. q ~ .C =~~
~~._ o ae Y ro~_ o~ eo _ pC
Ns 00 Y~ ~, O Y
qE vouE~b veo t- Arn xeo u oE
v v y .e ~p T [y~ E =rJ ~'p~p Y N~o =.. Y Y
=E-_ Q . ~i E
Y Tv Y
E~ ~=~.Y+ X N =wY.. V G V~ O~ y L N Y '~ V~=O 7(.J ~ O'=~
E'-E ~
L~ 1'~ U aY= ~ V ~ ~. . O 7 V ~ 7 .~ H 'O
e N
u~ OD ~ Y y'.' pp i ,YC= (7 p~,' ' U=Gi U Q Y g ~p eo a ~._v ~ 00~ rn~-...= _ YZ V ~'Z{=3 +' ,O_ on C N a~ v=~ F=C C~~ z ~_ E3 U e C _i eoV ~ d X oQ-4s - V E ~~=~ aS ep~ eu~t~$..z Y ., ' 0 3 N u Ea Y E: _:s ~j y'~ E E~NS d v o ' , u o u a o 3, yO
e Yr u~~ Y N m=u ~ ~c u > Z'u eo''3 y'E i a+= ~-. A E ~o a Eo~ Y o 5'~ v Y e _ I~-=~ ae o e~
~. Y .Y
~vY~'E~.: -~ 3~'~n' C7cY'~c'Eoy~=id~p '~ o-y"V..
E~t~ ~ u m c C3 ~o -W o KrO 0 0~ ~~ ? ~~w o ~ ri~ S~V-~ a~=, ~'-- 5-~- ooC~ii~V Y: > h v o a$UVo ~
O ~ =n i V Y
Y
Y V .5 0 e~u O
o c_ Y y e Ec D.V.~ bN 'C ai ~'a m.E c Ee w s u_ >
~ 3 K t ui;lIi 00 n ~ m 7~ =Mtg~~~ a~ a u~ .N.Saa <
00 Gp fj r! ~
U ~= g ly M ,.,;
~. m fi ~~ g=~i w ~ R
00 N C~~1 M0~0~ b fM ~ M M ~
M M M V ~/1 ~ O ~ ~ ~ ~
> x x x x x x w E
E
o o E id~ '~ u A & e~'~ v Eri al ~7 O O
O 5~,, u =u- ~ ~ u 3~;
7 ~~ õ Y 0 W~ O~~ O J~pTp~ V N p~ V i+ V N' O V
M
~ p Y
~ =;. ~~y=- (p Vi o_ Uy QQD ~ Vl u VI' = =F. v Y-0 7 c ~C~'O m ~ o G=
q~ >' G r+ p,~' yp R
Y ~ x ~{n . ~1 y -C v t"1 ~ u ~ U .~ Y ~ C u t~,7 F }j ~~==~ ~b Y OYwO =~+o.C~N N~bD~~ ~< $a w S~ Rc v 'Y' E ' a YO . =pc 3' p ,,,~ o~'gvo a o ~_ Ã,c o- u A 7Ce;o u O~ u~_ Es l0 .~,, o O~D V y~ O y s 3 N +' ~=F~. ~ U
ot-~'~' ~- y~ _v .. TW o Q K y ~~~oE~ u ''=~~~ ~E
i-~ 2D yQ. Y u! C7 py ? F T p C 'rJY Q N C~~ 4. =O y T
O N u CL u u~ .S 7 V EO A=OG 7 i~ S= 0' b Y 3 ONO C=Y= yõ d aE
~~~~dUo .Y~us ouo$ Ao oa ~~-c~~E~ ~'~ o~
E E E
a ~ a u a c ? ~ .~ n >
un.-~ u ~Yq~ a u 3 .~ 7 s . oo Y a E' '~ e'a c a e v ~~g o~:.a~:ao~vm_v'a ~en Y$ - o~+ 1dr~ s :;~~ -$
T ~= id m E~ o Si w E'_ E L 78 tt0 Z
_ .
~, Z n. _o, v _~ v tG ~u >, w .. ~ rs v o~c. ar o0 o Y u O N y~ q ~J a pp Y 2~ pU O u u d; s ..] O V y~
G c~ c~p ~.Y~p 3 0 3~~ ~~= D~~ Ih!fl-i ~+ ai ~.U VoV'eovi=~ C~ aTi ~ 2 s~z ~G a7~G ~.~ v~ aoN, a qam~-O Y Y
Q
. v~ = y ~ u yR u r ~ o c c ~ a u a~ ~ s }q > Y D0 ~'m o e c Y '~ ~ ~ Y e'C
n gj~
~ V C Y Y = u ~ Y 'u ~ =a ~ 0~7 a1 v c~ y~ a 4~ S; C~ a~ C7 P N
'/~ ..T+ _ =p W =~
t 24 n o x x x x x x x x xx x - gl -N y >
Y o o W ~Cy~ ~9~. 00 9 fi! x O, t~,i bp =' Y o~ o~ t v~i ~!1 '~t~. =~
E v S a $~
Y ~.r a mY
Yo.~ . o a,W .a aDO ' Y 'n"v Y! u -0 Y~w ~{L
a_ ~
O ~ Ly i. " y M '=' ~' t~f pp p 7 ~Ci =v ~ O
'C S~ W sC.gp~0 aY0y(,,,, O~i aJ0 p~, a a~ V J~ fip1, ~.C t+1 .~' ~ C ld Cl N'~ Y lC0 W' v w E" v~j O p0 O_ A J ad .~, N Y o r' ,n ~i11 = o a v1 o a v~ U a=p ''"b 3 3 Q'e 0 E x v~r J, o o U ~$~u ~~vum"$r ?E"NCE"ry~~,Y~ SO~v Y g E v~i ~e ~p~ al O ~G 0 ~O eC C ~~ ia o $ 'D o o e~
.N p p ~~+' 7 'Lj ~ = C V1~ ,q O Y 4R7., d' ~ euE ti E'fl CH o y u~ H~~
a~- a E :: Eoe w._ c Y._ c=;, ., .. E N
oo uU E
~ u ~M auoe'OO ea0o~' ' ~e$r .4 o oc oo t Q'_ U~.'4 v V er ~ ~b M
S) Eo~- $- B Q~u ~.t ,rj~ J ~=..Eotc~ '=3y~~Q~ ' 7 O~ r=-~p '0 ~ !C ~(y. = 00 p,~~C's 0. V' !y W L' 3 u w o cv V~l ~". N..1a '.. oo V Gei ~ .;~ , vYl . w eo .l E m,~ o) aa -: - i Y v E,oa '.~~ p OL~~4'~ W ~ v~'~ L] oj~ 3~ 4C
N N G W~' Y Z7 J ..3 tC0 ~L=" 3 ~1' Y J~C Cy+~. a W G T~'r1 C'~ =..
:9 ~ ~ C Q 0V' n Q A ~ Y C ~ U 0 Y ~.' Y ~C Y O .O t++ '~=,~,' Y ,D ~
Y' oo~r onu_ Y ~aec' '3,. E i ~ u o x>sEY~~ 'r~oo~p : u 3~:~
~ v o Y E~+ _ u E E~+
pa yi,~ ovh- Uo~oYCVoo3 m3 Jg, o~'omU a z~"'~-eu~3'S~
N
~ Q p u e .o d+ Y
~ ' ~ =~ F a y E
Y =C V
l0 ~ .C W N ~Y Q =
u Y o y N~ 'O
Q ~~ ie id eYO ~ E
Q U J, a~ < x a ob m ~
v~i 60 0 e~o eo~0 O r e~+i o0 C~ N~p O~ 9 k-I
~ N O ~1 vl ko r n ~ p ~
l~ h f+ h l- I- 00 Oo x x xxx x x x x x x x x x ~
J, =~
a U ~ =~ ~j =~ r ~p. =~V~.r yC..~ C 'M~~v =O ~ ,O
=~ YC1 4 ~ ~ =T'~ ~\ C 1p y o C c7 O, O ,O C~ ~O
00.C QD.E O~.C N~. ~O+ C y d~ 00w=" 004+" 00 OD4;
= o'~ E~o ES Eo ~o ca tn E
u~ ,N~ =n 7 m O= N wrT E~ ~~tL~ ~~~ Vc~ d~i U np u v~i C> y _G a G O .n y O h O ~.CC ip ~ " ,O O ~ y z N~ N a =N O fJ ~' .p .~ C~ C C C C
m V
J~ 'n t~- fn .C ~~~ a~0 G t C~ e7~ V c S Vo Vo y !tl w. ~, E Ew E E
oa o a o a~ o Si ~~ a~~>; L es E c t o~.2 o~' u e G~' m o v'~i v'~i 4 oE .. E
~
uh~ o ~~
E~
s V a7 Qv c6 Qv, V-, 5 y w V.e 6 ' i,' :[ a=T.p.~:
> .C a ~E VcZN5 y p~
~ v o " o'- .~ C4.
iO VI E~3 a C O 1 .Y ~~
~ N V N
~' .G ~' =~ pQ, "yN~ ~ ~y =7 =Li .C N Y J. ~1 0-~ F~ C Vt ~ V 6J . d 'd ~. ,C 67 C~ tC e}' tV e{ tV d N a-Q aG .vvv m~nva eo~rn~ y aG o na E- a E -a E -n. E
~ ~e e E r;f c el E
,a_ aci eo o on ~. -'a~~ 61 v Z y ~ u u u E -a E coyy~Ie E a E
E y - E ~ r? wuyi y v~ w ,Q~ o~ u E
E $ _ "
E a Ep u E E
E
<
a < -en~ av a c~ a ~O id Qa ~D w 2 8 ~a a h vOOi va\i N 00 OPNO o0 ao '00 00 9 X iC >C iC >C iC iC x x giC
w ' =
.s ~ 9 _ _ .~ .9 ~v ~
=C ~~ O ~ =O O O ,p1p~ ,~ p N~,~=~~~_~
0 3 ~ g o 6 $ 7 o 0 o fi u x~ oo-o E...ppp o~ Ey~ o E Q ~
o o ~ o E~ oc ~w aN~ E~ o:CC
+ i ~~ E P X ~
aow: e~~: o e eow: ao~.: mw eu~= m eow E~r E o u~
Eo = Eo .Eo E8 Eo Eo o Eo t~ X~" ~~wm E E ~=~ E E .2 y .2 y E E 9 g o~ F fi E
~ ~ ~ ~ eu w u u N v H -cl u V1 (71 u~ u N ~ Vl ~~ '1j yj E,~ ~'=
C C C V C C C C C C C 'V fQAd u u u ~ aun~ .~i 0 U1 U: ao a U~e Ua ae ~m ~p uyx 1R
L Y L F. L L L C .-= V t3 m O.~
Ea w w Ew Ew . E~ . v~S
g.~ ~gt t gr ~s gt ~ afU ~~.5 c G~ G~v~.
S~ ~~ ~S~ }! L = g~~~
N N +r N w N V r ~ N N N V C= C U r,. N
0 0 o e,,o e ov v..~v..ov oE
B~'- 4-E o b; E ~o E o -' o e o~ p ~ g'~ p ~ a tc ~ m o ~e u ~ y eou$ Ea vao Tvp~ Y. ~vp~ s E
~. TvQ~ Y ~pv~ Y ~~.p/~. fn vp~. = Tvp~ Y, vp~ lY C"~ Y p C v lt1 R O N ed O W id /0 O ad RI O ad RI O i0 lOT O W~ O N O L! ~=C. O o~~=T N
m E v ~ o ua e ~'.S
ov! csc a o N =C ~ ~C u =~ .~' p '$ ;q U
Q aC
a~
E ~~ L'_Ov ~>~2~
N r0 ~ W OE r E -a ~Y tQ ... E h= N o tO t0 E E E E "' E-v~ ~a N a U a C
~ d E e F y 'N
p~ y N r, N ~d (~ v~ e! vf O O C
.. ri c~i. Q~
u A. A. A.
Eto E E t E EEF.~ ~ ..
Lu 111111111 ~ ~ a E =~ ~~
ct E
<
pM pM oM QtM ~p+'1 Mp fp~l Mp Mp M1 Yl O~ O. O~ ST O+ O~ P O~ O~ 0~ O~ Q\
x x x x x x x x x x x x tz 8 V w {~ /1 = =~1 U~ u y N M U e E q, N N F ~ V .C fff~~7~!!! ~
.E 'a E l'~G' =--= C
h~=fi y ot"v~9 Qo~o E 5~ eox~ o~c E
E an u. ry u a~ u=v - o v o a'~ 3 ~ a~
~.. ao'c c C
O O g E a~ N ts E.g U r '!1 ~,. N
~'.=.~' c.~ ~i~ a oZ ~ c-~g ~'f vi gur c~
oa ~ 3 +
. ug U S u _, ~'00V v F- ~ o o.! F=5 =p~op'~vE o~ ~ ~'~ o1 NTRx ~ooE_o~
vi c~ EO G~ Ci 'a U U V u O~p l6 C~ ~' =~ ='wX p v W C_ ~3 G C
d ,au3 u ~> o ooV v:: u eu im v~ e oe 0 i 4~ uuVo'I '~ o# co>~', u~ ~' E? L,~
e ~ p~+ E uppoo~ ~'.~ W o_ti uc~
U O~ L V y Y p~ O Z,~ W= N N o~'~ o'~ c3 .
o cv c u v ca E 1_5 U E ~~:. U v v~~'~n u n Vi Y'i ='Fs~ a'~u > 3~ _.~uo~~ =~.nT.~
la ~ Z e c ~ $ ~ ~~
c V o ? E o, ~ c .c a ~C N U a ~ uu7 Ae . a~ a;o;_~ .~~ ~~'" ~C wWS Eu' ~7 U.~ ~+ w ~~.O ~d v~ C~ p,; =~ i0 d voi 'C ao L C~ V C p um u u r v~ u ~ o e o~.y ,a e o'd ~, o v u o 2 " v E ~&' V_ dv-8a.w~uVe "O EO ~~rETO~~ac.ldÃ+aoeoo V ~~> ' =~' uoa'o. d V cp u v a Q~44 ~n o a,V o ...-ac~..S o0o mva oo~
a a u o~
u '~ c o o e_ ~, ~a 0 0E
a E.~ C r -O c v i m E
n l0 ,V~ =ld yn C Q Q S u 5i '~ r ~ I w w hG= G ~
~~ c 0 0 C ~! =E O~ ~ O H
~ U O L y GO A'~ a t '' .~U
~"~ ~,a u =v ~ co c ~ ~ ~ a T~~ ~ o C ~~ =Y ~C ~ .U., Tg "
a o ~ ~ e~ vs o u z c e~ > 0. E e E~~ vVi O U S O~ 4>
Mr A ~w x x x x a'v' ax r, Q
x n>
cgo 0~0 N~~cc 00 ef V1 r ecct O v r h ~~I
~n ~ N ~ =- ~n ~n N N
x ~C K g g i- g Y ~ $ $ ~- ~--g5-..
E d o T6-V pq ?, o eEiV C~y=~
b Y~ ~N Y Y p a. . =~ y u~ U_ EM ~
w E
A~
R Y1 >>~ C U
p ON y E=~ _d0 ~'jf~ ~=x G. G. ~~' rU ~itf oO
z 95 'Bo'~T v~~ oo uv Y~ ~iy EQ
~ac ~ u~ E~
~:-->.? uy~ .~o 00 v aw oo Y a.~ a eDco J! ~ u O
C~ e g.5 x~ s e v v fi ~ p H E
vV, ~e~uu~oE~' _~" Eo e ~ w C~~ u y~ =yo'~ OQ 3~=~=r =~ Y
e Y y o u Q M v, u o ~ w Y=~ W c. y.~~ E U C y O" ~. M U>~ U 1 O C C Q L ld Y 1~ d y qp L C
ocnpor;_ p i ai = ~i ~r V=~~ u v', IN t o e~ u E
u a $ Y'op~ Y~~ l ; ~~- ~ E
viE~'~ ~~ Q~ d m uo~
eo Y-, o~ ?. =~ u -,~{ ~a %0 Q E~~
p y.]L V O~Võ Qp y Y Ct h L~ ~ Q~ tO Y S 7> Y'~ p v Y 00.5r .c Q~-Ys aCi ~.c0 7Q:~ GLm.t...~~.-~.. 0 Rv10C0 00~ Av~U ~O=p v u y n 0 c N Q U v ~.c ~ ~ YO- Y C~ dr al 'X O C
E a~ z u u c u u Y p G ' u_ ~ y N y Q~ ,p, ~ N wA ? Q ~ ~, o a =- y ~o w b E
e Q E u E ,u y ~j $
co (hgX v h C7 5 < Q v L] v ~ C7 in U v~a c~' E~ u s 7?
>
T =p Q w lm 3 a 'yY
o ~y v y b h N M I,. R ~
a~0 v1 h 00 ONO N
?= i= >- N N 1~ N l~~V ~ v ~ g u N N Q ~
TABLE 3: Corynebacterium and Brevibacterium Strains Which May be Used in the Practice of the Invention Brevibacterium ammoniagenes 2i054 Brevibacterium ammoniagenes 19350 Brevibacterium ammoniagenes 19351 Brevibacterium ammoniagenes 19352 Brevibacterium ammoniagenes 19353 Brevibacterium ammoniagenes 19354 Brevibacterium ammoniagenes 19355 Brevibacterium ammoniagenes 19356 Brevibacterium ammoniagenes 21055 Brevibacterium ammoniagenes 21077 Brevibacterium ammoniagenes 21553 Brevibacterium ammoniagenes 21580 Brevibacterium ammoniagenes 39101 Brevibacterium butanicum 21196 Brevibacterium divaricatum 21792 P928 Brevibacterium flavum 21474 Brevibacterium flavum 21129 Brevibacterium flavum 21518 Brevibacterium flavum B 11474 Brevibacterium flavum B 11472 Brevibacterium flavum 21127 Brevibacterium flavum 21128 Brevibacterium flavum 21427 Brevibacterium flavum 21475 Brevibacterium flavum 21517 Brevibacterium flavum 21528 Brevibacterium flavum 21529 Brevibacterium flavum B11477 Brevibacterium flavum B 11478 Brevi acterium flavum 21127 Brevibacterium flavum B 11474 Brevibacterium heatii 15527 Brevibacterium ketoglutamicum 21004 Brevibacterium ketoglutamicum 21089 Brevibacterium ketosoreductum 21914 Brevtbacterium actofermentum 70 Brevibacterium actofermentum 74 Brevibacterium actofermentum 77 Brevibacterium actofertnentum 21798 Brevibacterium actoferrnentum 21799 Brevibacterium actofermentum 21800 Brevt cterium acto ermentum 21801 Brevibacterium lactofennentum B11470 Brevibacterium lactofermentum B 11471 (~u3 FERM ' NR1ti:. CEGT = C11MB CW;: NCTG DSm Brevibacterium lactofetmentum 21086 Brevibacterium lactofermentum 21420 Brevibacterium actofermentum 21086 Brevibacterium iactofermentum 312 9 Brevibacterium nens 9174 Bravibacterium nens 19391 Brevibacterium nens 8377 Brevibacterium paraffinolyticum 11160 Brevibacterium spec. 717.73 Brevibacterium spec. 717.73 Brevibacterium spec. 14604 Brevibacterium spec. 21860 Brevibacterium spec. 21864 Brevibacterium spec. 21865 Brevibacterium spec. 21866 Bnevibacterium spec. 19240 Corynebacterium acetoacidophilum 21476 Corynebacterium acetoacidophilum 13870 Corynebacterium acetoglutamicum BI1473 Corynebacterium acetoglutamicum B11475 Corynebacterium acetoglutamicum 15806 Corynebacterium acetoglutamicum 21491 Corynebacterium acetoglutamicum 31270 Corynebacterium acetophilum B3671 Corynebacterium ammontagenes 6872 2399 Corynebacterium ammoniagenes 15511 Corynebacterium fi-jiokense 21496 Corynebacterium glutamicum 14067 Corynebacterium glutamicum 39137 Corynebacterium glutamicum 21254 Corynebacterium glutamicum 21255 Corynebacterium glutamicum 31830 Corynebacterium glutamicum 13032 Corynebacterium glutamicum 14305 Corynebacterium glutamicum 15455 Corynebactertum glutamicum 13058 Corynebacterium glutamicum 13059 Corynebacterium glutamicum 13060 Corynebacterium glutamicum 21492 Corynebacterium glutamicum 21513 Corynebacterium g utamicum 21526 Corynebacterium glutamicum 21543 Corynebacterium glutamicum 13287 Corynebacterium glutamicum 21851 Corynebacterium glutamicum 21253 Corynebacterium glutamicum 21514 Corynebacterium glutamicum 21516 Corynebacterium glutamicum 21299 .. : _. .. __.
:.; . , .. .:., usss.-, s es.:: _;;:~; x!.=. .'. ,=.' _ 4TN
' Corynebacterium glutamicum 21300 Corynebacterium glutamicum 39684 Corynebacterium glutamicum 21488 Corynebactertum glutamicum 21649 Corynebacterium glutamicum 21650 Corynebacterium glutamicum 19223 Corynebacterium glutamieum 13869 Corynebacterium glutamicum 21157 Corynebacterium glutamicum 21158 Corynebacterium glutamicum 21159 Corynebacterium glutamicum 21355 Corynebacterium glutamicum 31808 Corynebacterium glutamicum 21674 Corynebacterium glutamicum 21562 Corynebacterium glutamicum 21563 Corynebacterium glutamicum 21564 Corynebacterium glutamicum 21565 Corynebacterium glutamicum 21566 Corynebacterium glutamicum 21567 Corynebacterium glutamicum 21568 Corynebacterium glutamicum 21569 Corynebacterium glutamicum 21570 Corynebacterium glutamicum 21571 Corynebacterium glutamicum 21572 Corynebacterium glutamicum 21573 Corynebacterium glutamicum 21579 Corynebacterium glutamicum 19049 Corynebacterium glutamicum 19050 Corynebacterium glutamicum 19051 Corynebacterium glutam icum 19052 Corynebacterium glutamicum 19053 Corynebacterium glutamicum 19054 Corynebacterium glutamicum 19055 Corynebacterium glutamicum 19056 Corynebacterium glutamicum 19057 Corynebacterium glutamicum 19058 Corynebacterium glutamicum 19059 Corynebacterium glutamicum 19060 Corynebacterium glutamicum 19185 Coryne acterium glutamicum 13286 Corynebacterium glutamicum 21515 Corynebacterium glutamicum 21527 Corynebacterium glutamicum 21544 Corynebacterium glutamicum 21492 Corynebacterium glutamicum B8183 Corynebacterium glutamicum B8182 Corynebacterium glutamicum B124I6 Corynebacterium glutamicum B12417 . .......... ._, ._. .
' P2~
Corynebacterium glutamicum B12418 Corynebacterium glutamicum B11476 Corynebacterium glutamicum 21608 Corynebacterium lilium P973 Corynebacterium nitrilophilus 21419 11594 Corynebacterium spec. P4445 Corynebacterium spec. P4446 Corynebacterium spec. 31088 Corynebacterlum spec. 31089 Corynebacterium spec. 31090 Corynebacterium spec. 31090 Corynebacterium spec. 31090 Corynebacterium spec. 15954 20145 Corynebacterium spec. 21857 Corynebacterium spec. 21862 Corynebacterium spec. 21863 ATCC: American Type Culture Collection, Rockville, MD, USA
FERM: Fermentation Research Institute, Chiba, Japan NRRL: ARS Culture Collection, Northern Regional Research Laboratory, Peoria, IL, USA
CECT: Coleccion Espanola de Cultivos Tipo, Valencia, Spain NCIMB: National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, UK
CBS: Centraalbureau voor Schimmelcultures, Baam, NL
NCTC: National Collection of Type Cultures, London, UK
DSMZ: Deutsche Sammiung von Mikroorganismen und Zellkulturen, Braunschweig, Gennany For reference see Sugawara, H. et al. (1993) World directory of collections of cultures of microorganisms: Bacteria, fungi and yeasts (41 edn), World federation for culture collections world data center on microorganisms, Saimata, Japen.
&' ~' 0t+ o~,~~ $-' r F= y F~ HH F~~-_ ~~ ~ N CQ N N N N N O U O N ~ G.) O
cn Q
Y ~ E .2 C, V
p E E E
Y~1 = G o bU O OYYY Y U S~ J
E
N
~ a~ a $ .~, o~gc $ %7 a. a g c~'i ~ ~ E ~ o gn !
~p ~p $
o~ O z O Z in E E 5 E
z_ J y~ W ~ W 'Z
E ~ ~ ~ ' 1=0 mrn Ij v ~
a o 8 0,2 Od e ~ ~ c r A
C _ Y ~ 0 _~~~ oa ~ a=r~W W LL N .g a uE~ p~9 ~N c ~
r y Ot a 8 a ~~ 5$cm~+ E'~m L' d vi c'S ~ cq ~ it~~ v~ nK E c E v w v~
1~0 x C)so I S ~o c ' u E E3$ 9 E 4~u ~~ Cy m 6 Us $~ JjIH1JI ~ ~ w~$~ W E vi g cl~ i~
A? o Fo V y, a~Q e e.~~n z m ~
m D 2 w g i+ a g =~
~ =~ N a g ~ ~
i Q~a $ n"i N~X~ Q U Q~ 4 W ~G W ~.
~ 1n .=. ~ c., '~f as col ~ 0 1D !7 tO ~D
N
~= N N
Pf ti n ~+ M t~~O O~ f~ Q oMp atoG
w_ w w w ~ ~ o~ a a~ a a ~ i i -Q1iq~i i fQi ~ i C~ (9 C9 U' [7 C7 t7 C9 (7 ~ C9 C9 W C7 C7 W C7 In LC LC
W C $'j o~ 0, ' ~~ W f~ t~ ~ OO O 1'7 O f- d1 dl dl ~~j N N ~ 7 C N C < ~y N 80 tD Y
E E E E E E E E E
~~ tofo ~ otfo o s'~~d h a E E
a T II T a a T~ D T L ~ T~ $ T~ 7 7 U~U v, _~ .232352~2 .32 a 2 3:'2ao: 2 ptf1 {A ~
m (O m m INO m INO ~~ t0 (np m N N N N ~ (a E E ~ c u d ~ g ?W
4 O O T ~ Y' O O O Q V/ pFpF
.7 8 c c 0 N CI C C C ~ O~ V C~ n rn rn uo ao a O o, ~ o=r V 8 N'~ E E E w ~ E E
~p 8 8 8 8 8 8 8 8 8 8 o a a Z 8 ~
lx-~ N~~ ao a '2= i , = s = x = 2 UmJ ~ 2 Y~I
My_ h N Ny OI N 0 y~ N q y rl V) ~_ y y q*
~ h' "' O O O O O O O O 8 O OO O W 7 O O
o~ Eo '"
E s ,~$ a E~ E E E E E E ~ E WNc8v1S . EE
c c 7 7 _3 7 _7 7 5 ~ o~ Q~ 7 ~ 8~ a acr u m ~ m m v o m o m ~~ ~ s~ i. ~i y't t0 ~o tS i5 tf ~ 25 t5 ti ti 2f tf tS 2i ~ ri YõA o Q
f C~ M 0 N /0 N /C W m w ~= u 7~.
ac v) E
~ o h_ _o 0 ppp ~ N (O < p p ~ ~ ~
~ p N ~~py N~p Np N ~ < ~ ~ ~ N
Of ~ O~ O~i V~' a0 V V V
W $ N 8 $ 8 N
=- =- ~ ~ g o o g o g ~ bi ~ ~i ~ r~ ~ ~. aNO a )o m M r ~
a o 1~ ~ o N ~i ~ ~i n N ~ w r~r. ~v v O~t ~ ~ ~
O r$ N ~ vf N N N ~V ~ ~~iS Q ~ ~ a g~ ~ U U (~ ~ F U C~ (~,? TU U ~~ ~+~f g J
lt~+W W
~ (a 0) I ' z~ di af i W ~ ~ 0 1008 8 n ~ ~ s CR n a ~
~ ~ ~ W 01 g ~ ~ ~ ~
St ~ N N N r =- =~ pi N N 1~ ~- r c I Q Y t~+) c1 =~ M l~+f ~ Y'f 1~O ~ N kn f =P '~f ~ tq f s' ~ 8 "~
8 ti'cV q q E
p N
W
L=a taQF_~ ,$ 7 p 7 W C .~
6 N ~
WI c c cn~m a a w~~z wZ 8 a o m > > a o ~ a ~+ ~ m 'c W rn c N
n ~$ o, fn m 1 2.
~ f f a g r ce n 2 < Q
x R Q.
W
g >? b y ~ ~ m m~$a 8 t W ~ ~ "
> E $ a ~ V o, o, w ~n ~ $
C 2 n a a E
~~ o a c y a6 3 v!~ ~c t~r o a W u~o N--m c ~ 8' m ~ ~ ~ r F q=
T i a = g IQ p a8o o~e rvgEtf"'~Q'~' Vm r- n a~'n E N V E y $~C O V ! _C c7 ~ O O s f~0 O C C
M W p _ =~ c :o ~i~
~ nt O = ~ 9 9 owc~aE W n 8.~ 2- ym $ .8 A .8 1.- c C X
n = ic~a'~~E
~ d~ ~ ?c ~iw E a'rE ~n ~ ~~8 7 8~~~C~x W=pc E ~ ~~~rgQ
O~ 'd U O f( CQ C~:~ l~Y C~~y tl L V V V O } p N E
y 8 ? 8= ~ W 'C ~ g f p n }~ F W p Q C W y. a t~ - n a~y iz V ~ o C
m !~a ~ ox(~ 2 tCZS W z :N W W c.. wa o~ ai ~ ~ ~ a lll~~~S!! aQw ao tn cn >
A tti aD
~, t0 N"
w a n < N
-+ < a a d~ s o N a~ d m a ~ w N ~ aD m ~ N ~ t~~~ (O1D m ~ Nf O m c~Nf (~
O N awp wQ Q 9 !R LL' Um ad a ~ '~~ 4 a ui Q ~a a ~ ~mi N
m m m m m m m m m m m m m m m m m m C7 t7 U' L9 C9 C9 C9 t9 (9 (9 t7 t? C9 ~ C7 U' (~ C~ C7 C7 l'M M O
a N N N ~V N
st o, :
O lIIVVV O N~-= ~ N O W
/") < M f P9 l~+f {'7 l+) {''1 l"1 M t~~7 l'pH V 17 l'7 l7 {~ t9 l'7 I
_u ~'0 2 2 u~p g Ll N y N ~ C C 7 ~
fft~ r t EEx~ sf ~1 E g~s~ 2 2 1 2 2 0 p i f Z~ J V1 N v1 I~ ~.r~ a V
iW ~ Z Z g N 2 2 c $ v ~ a Z_ Z
E 2 ~i i cT 0 E az c9 Z O
V) 8$ W N f.~ 1 a W W 1 Ea ~O ttN dl J = Z 2 v) g ~ n o E =UW
-zo C W W E ~
N
G_~ 27' 0 a ~ 0 X y o N ~1 ~1 r a n ~ a E ~a ~ E~ O
R 8 ~8 = o ~ ~ ~a i1hiI ililfi o~~- {- N " 0 rnG %
~ C ~
E ~' o E o~~ c 3 c a o ~r~~'rSE
p -f G o ya p W c ~ c ~ a Q n g n ~ c c 1-F- c v~ U cU SIc 9"~'N - aa a a a~o em ~
~N~" n = '~ o~ y 1 ~ c a a ; o q E o.4 ~ zs~t~i~W
~'no5i '- 3 m 4 ' qw ~'a~>=~X'- Ee ag=cWo ~n $t P oN ntp o E .~a oc '3 = W
Z
~ ~={ ~$ O ~W 10 = N~~ O~~-= i= CE G ~ S >~! > S a S J J f/) fQ (n 31 U 4. G
OD N O p h ~ OI ~ Sf qpj C~0 p N _ p n a0 N N op O1 ~ N~ N ~~Nyy N
p U V p~1NV O(p'q1~ U i0 f0 !o' t+01 O U V V v 1L ~ O
Q Q r J X Q Q X X J J J Q Q Q Q Q Q Q
OD to7 f yp f~f N 1D oD 10 ~~ a~Q qf~~ Ipwp ~+
N N V r~ ~- 1 c0 CD ID ~ ON~! M N O~f N N
OD
$ U! g~~
auNi a I
~ t7 ~u m'U ~U' C~9 C9 ~U' ~ c~ Um' C~ tm7 tm7 n t7 ~ c~ cm~ Cm7 c~9 c~9 C~9 ~
go cn ' W
N 'C~ t~1 O N O N N N ~
n a~0 q ~
h ~p N ~ V N
M
O = O f~l ~(f N lh 10 f0 N t7 ~ lr1 M
n n Q y ; co ;
t c c E E
c E ~ ~ y m a y a E
E -'~1 8 ~ Sn m ~ o ~' ~ ~ n $ a =~ ~ 'g 8~8g~ g 0 ~
x aa N~, N 14 ~~ Z ~m+ g W 10 y _~ E _a U t (~ g N rLL~' .- ~ (o ~ 'J Ep 9' , Z y Z tO mC 'j v Q al Yuj E q2 o fnQ ~ $ y p w ? ~ E
L Cm y~, ~ a rn M U x a N N c n oO U'oO ~ y~ g OC r ~
~ =E ~
c =a T g U ~ WZ
4 ~
_~ c v c Z m'~S o -_ ' W ~ rn p ~ m c C3 ~i ~ V g o E E ~-u c V v) N aQ~{ "' E N ,QE 'yt m o~ n} t S = c_v i, a E
? m Z' S~ Z' ~~(7 n~~ " v m~~ V q~ yj V ~ O W W
~ E E o E ?3 >
q ~ ~ E (r 0 n p S ~ fV y y = t0 l6 ~ p 1=~) E w E o QH n n m y Q~, V Cl ~4 h= E U t~ a N n ~ n 'n r m m ~ O E E l ~ E '~'n 0 R W o p o n oy E k o I y m m W ~ p, A Y ~ ~ _C ~ O n C C ~
Q c m EY .Y ~ i c Z Q~ =~ ~ E
. c ~ N c ~ n ~ tn ~ fn ~
p F.. <~ =_ 0 mrn ~ 'c) ~~ !~MV~a$ y m v aU m m .p 2 ~
Z?i ZS 's W ~ m c h Z(n L~ ~
E NI 2 C? ~ Q G~ C7 cry+ m a t i~S n6 A
LL N 8~5 ~O8 8 mC'S ~b Co~' ~~a 7 4)O ~ o 0 m <xx x xai~ cia= ~~ E ~Ca 8 ~ ~ M ~ ~ ~ N O U 1n x 8 S p~
09 O ~ y) $ M Y1 1~
CL
Q Q N N Q } Q N N N
i ~_p _ ~ in oMi ~ p ~ Y N ~ Y'1 n ~ Y Y ~ !~+! 1y N ~ ~ " C7 ~ N
N N
N p~ ~ p O yMj O xN N U U1 1A ~ ~ ~ CO
N. a _ ~ N N N q = ~ Vl q U ~ U Vj U ~ N U) ' ~ 2 ~ ~ ~ _ ~ N = = i22~=
Lr ,W, 0 a On (D 0 ~ 04 ' N N
g 8 09 s m' N ~
C&
~p m a0 W tp pM fD M th C~O O) O Np Otp M n nCp T iT 'a ri cr) v v Ili a o a vMi u~i l~! cTi {'~7 c~i e~ r) {! r~i r~i E ~ y E
a ~o c E S N m a c c e ~ L
E
a n &
c co ~ O V N w L
s s $ $ o 0 0 w U U o~ - ~ a 0 E ~ E ~ ~ oE 0 w v) w O x O a~o a~u < a < x < < = x =
= 2 $ a$, 8 $ z z w ~ L nc E B.
~ o t ni O~ N
8 8 z z O w w a a ~ W' x >
E E g g y~
z z z a ad Q "' m E m E
Z 2 c E 8mt d a~t .E v 8 o ,n 9 vi 'a 0 n A ,a t o > ~ n Z N a a o j E U a c O O 8 8 aC 8 - O E ~
(n N y ~ a v N u lt U. O Wt Wt N
y ap = O ~
V t o < z O O Da U < E o ro m m 8 c U c6 U U a M O ~ m n lT ' P "' "
~ ~ c - -p 1 g 9 E ~ ) 8 E 4 yyyc ~ 0 ~ o a n a m 7~
E~ C ~ 7 E O~~ t0 N y~ N O O Q ~1 r' ~ 10 3 2S S t t q E a E ~ E E m c ~ c c ~ x~ O Q O Dy y L ~ Q 8 Y m o E x N 16 OI W Vi ~~C ~ N ~ O = 7 O 8 0 ~ p OI OE; z c 2~ Y c fS E c~S .~ Nc 8 - 8 E E m 8 c' 8 L o ~
~?_._ 2 !3 0L 0~ ?
~ ' 1A 4 1! n M t n Q= :. w ~+ = $ N 8 W N . lO a N a a W ~1 U a '~ ~.'~ =' t~o E $0 L$~~5 Z~ a-68 $,.~i o oF r ~ ~ c a c~+y ~~ Y
~ E~0 O O 4 Q 2 3 u_O N O N YWl O
~ ~ uWi K v~i N o a= ~ p Q
~
E c c c c~~p ~ E E m n ~~~o ~~ E a a E~ E~ E
wc'0i~c~r'~ wcnr nwQ wa Q. a Q < Q i Q a~iax ai 1~ 2 m N M N A
~pO $$ o p fD Q ~'~QY( (p y M f~0 O O ~pp ~C < Q >> _n m m m N O l'Mf M O Cp O m O O p aa ~a x x a x a Q, ic Q Q ~ Q
N a _ _ m g _ o a ~ ~ ~ m f fO a ~ ~ v rmi ~ ~ 1Ip .~
~ aMD h I.
O/ W cD
M M 0D m aD OD
cr) Z z Yl P. p~ N~p ~p up 9 w 10 N
-~ h O O N on0 gY tD ~p ~ f~ ~ t= s m c~ ~ U c) ~ >> b~ ~ C~ o o Y cS a Y co w fV N 4' v~ q 4 a a ~ ao 0 o t~ t~
S cF~ a S
W W W W I 1 I 1 I 1 1 i I 1 ~g a~ 1 I I I I
(7 ~ O C 7 ( S O I 1 CJ 0 (7 (7 (9 0 C'J C7 (~ (~ (~ (9 (9 C~ C9 D G(p o) N M OD ~ N
M N
LC
~ ~ ~
r- co 7Tp-QR 5 >
U~
O N
N OI ~ N N N N CE00 N
N Y'1 M r- C! N OI h~p P N OOD N O Q! U, ~O M
'~Qp {Yp f~ O~p ~ -p~ 1~ l~1 O t0 Ol M M p CCC000 M
t~1 < lh ~ ~ Y f+1 U) 1 M ~+) ~ tMo (NO ~ l+~) lN V~1 Y
W y O
G~O W~ C C C _7 ~ W~yy~ E W C C ~ Fj ~W 4Il mC q 9 E 7 .a .~ .~ 7 v d . L~ t ~ . .2 !A _/
A m Q.9 Wa4 g .3t'imEbQ $ ~ n N N W 7 2i W W
~co ce g E oE o 0 o E E ap o NQ o h W " ' " a E ~
N O E LL >O, ~ C EO 0 EO CO
o~ x x x Uv~~23x x x x 222 a = x x o Ci Q r ? Z
~ ~ V J _~ N W H
--. " g ' o ~ u a u a w W
~ E 0 c rq~ 3~ u o c c~~ _ _m c E a c o E g E
o tci ui 1 - 9 '8 4 x $' 'm E E
a~ 9 ~ $ O õ c o vi ~ a H E y E Hf 'C a E W v .9- v~ ir E rn o o ~ x E nE az~ Om 0L ~ a m? E a ~ s cNl C04 I
n W Y Y ~e 1 w m C7 I I x o .. ~U.1's LLI~
a D) Q N N 01 O ~ r'E~E ~ Eo Eo v N ~ YE~
0 0 o Nro ~,~o c a W o '" n 0I8 mva a c E a E _ M
a ~ o~
2 o p o aci p o py~l m ~ L t E
L r,m QV~m _W a Wl N V m V v ,~ ~ V O m ~ O ~ M H
p- Q Qpp c sc= s: g EN E1~~ dn ~ v Q'o Q~ cr c, Z>
~ Ut=/~ V a M Ca p v: V t 70 .2 1 1 m~ 1O W W GDU2 0 ~g WI
~ Z= ~ 7 t.- y.- y N x ~== W ~~ ~ E 8 m 8 c y m H W 1- ~i c y ? w~ a ~
o o c7~ U m~o o W U t Q $ E'o E a W ~w o ~ 0 a E~ 9 ca'o m m Z W ih - H o ~~ E~ o s m $ ? E a 'n F E "a j -~ 4 ~ 0 E a L
c a E.r c Ecv ZL ZU ging cing W m y W n~ ENa n2y~ o 2 L Wp~~ Z~y o H Q ( 7 U My W~ 1~.. S~ Wm T ? ~?~ .~..
ZS NI y Z C Z G~~ N E tn O C ~ C 2 _I O 0 O N w 7 j A y UC Z C Z C 7 ao O tQ o O N 7 Z nZ Z
= = x' wD ~ m m ml m t0 ~ m Cm c VnU U n{~ c ~ V! ~
W N
W ~Cy O M O O~ Q + O W N W
1~. U L O C O o~ om o~
m i> o W ~
8x a'xW vw v x i ix l'7 ~ N l~ _ h OD w N M l\ 89 2 e~ 8 Qfg V ~ O O a a~ b O O g (D M N
D ~ Q Q N
W J J ~ LV a J ~ 10 I OM O) c~=c a< a a xa Am z A~d a a a~
M {+~
A Np NpN h Q~~j CO ~+j N ~
O! n ~ O
O N" Or M ~ CO S ~ V N ~~ W CD ~n+l n 1~') N t~
p O ~ ]yff~y[I Q~,~I I~ ~p M ~ =' ~ ~ N 1' ~ ~ h_ 1_q ui m _ 4 4 aV >- '' $
U ~J Q ~ U U ~ U S
aN ~ O N N N ~ O (J fn ~ Re.'~ O ~ ~'Zj ~ ~~ N N 4 I' f6 Z W W W LU ~ m m m U' W W m ai ~ _ ~ fD
1 1 1 1 1 t I 1 I 1 I I I 1 I !
~
cp n 8 8 n M ~
~ 8 oE 52 g E $ d ~6 E
OD m p~f .- ~+i1 ~ll N ~p ~f! N ~py ' f ~p 10 ptD 1~~p pa P
~~lYf ~? ~~p0 ~~p p M N~p N~p 1f1 f~ P ~p! h~p ~p M~p tD a t0 tn~l l0 l+f m +f '7 M V ~ ~ t~ t'9 t~f H V d 91 0 o ~ a E "~
> c~ E
~ E.n H o $ ~ '0 $
x a ~
~ 10 ~ o ~a õ a a ,~ I
~ QM~ E a ~ C~ o ~ E .~ .~ ~ =- o 1wlO1- N
c qoe~~'~ a~Eg$ 2. 5- Yo ci W tq ~o~Cc~cH S 2~ ~~mo~ o~p C~ ~yao W n g~yfW a o G~ x~ O LL d C
~ T ~C p. .. ~ ~ > ,~ 5 10 l'=
- LL~~.
G L
c_o m a 3tvtnvme~ c (n O 01 aov) !0 vUN~ gwW c =v_l Z'C ww= cg V ~ ~w F- o a VU E~ ~=q vi~ o ~ m o E~
$ H V " < g .
a w z n zS 14 N ELL~
F3a Ac~c_Z~
qc Z t i a~ ri a~ o ~ V v' To i3 $~ V
D2O -~S W ~'r. t3 2S o~ E ~Z W
ct ~ Q 2 > 4 E F E
~ 8 W~ W ~~ ~ a o W S W~ g~ t N~ i W
N H StA H (D N x~ USN=t~ UU~ U S t!) M V) N~p m O~ N N
<Z x c'4 a f N O O mm ~ R ti m Y~ N aNND !~l f ~ ~ 00 40 ~ m m ~ ~O iD C~0 cq a-~ ih -' ~f 000 fz tPSN
S ax ~CtJ U ~c v N
(D 0 N
C! O) Q! , v ap ap W (,~ '~l+ ~7Tj l 7 ? V Fa 1 R N N r N tV N ci N ??~., o N $
aF a ci s 2 ig N' ~ N
9t ~9t~ N
E E
gY ~ ~ a tf E
õ
W a w o 9 9 ~ g 0 a z z õ
z z N o =
z F d a c c c Z 2 r N
N 1; ~
E
d 1 1 5 "~ N j N tn 1 ~ Nt _ ~ C~~ f Tr rr R a&i o d $ o 0 0 .5 ~fD c V ~ao~e' .~ .~ , E W
$
8r ~ U U o ~ ~ ~ ~ r0 ~a wa ab n~ ~i'?c' E
m 0 'aF ~ ~=~~=~~ ~ ~~ a ~~~ g d ~ ~ .~ g ~ ~ ~ ~ n mf/~t v a U ai S ,n.~ mn y@ o E ~T i aZf a 0 m w t~0 w q x ~~~ s _ ~ x E a~ c x x xI
I m y a Z vo a o ~g a a Fn ~~ EIE _ ji87S8~Z h a N
n~= rn pC7. N u~> ~F E c_ E E E E E F~ o E qF~D8~~
C Z ~ p (A f/) ~ .L 7 7 _7 y C C C d (7 .Ytn tn o ~ o arc ~ u c ~ N ~~ =~ Q o a'~ o a N>ZU. _N a di~d pv ~o~ z iS iS $ 2'~
Waa2 rn L'iiq~=
n y~
A ~ey ~~ a n aro m eo r~ c~
p~ Cj ~ ~Q 01 ' N_ h ~ ~p 1~ny 1~0V ~Np n ~ p (Oj N ~ ONi O Ool~ ~ V V V q N N~
Q T.j ui ~ ~ Q Q ~ J N Q N Q Q 4 ~ Q
(~1 ~ l~ AQ1~J p) t0 N{7 ~ ~
~N ~ l'f ~ f h dD ~ MN ((~y ((~y n dD N iV ~ l7 f n m ~O ~ F~
.~=-m r tn n g c'3 c'~ t~.> >- ao Q n c~
~ U n Ur =
t9 ~ C7 G7 ~ ~ ~ ~ ~ 4U) _~ ~~ m ~ " A~j Z W W d4o a 1 q~I 1 mt t i~p 1 cp I q11ap1~p1 Ipp I I 1 q~1 ~J1 1 1 f~ (~ (9 G7 O C7 C~ (~ C7 (9 (7 C~ p t7 (7 (9 C7 (7 O C7 C7 C7 Cb ~ ~ $
.
N N N
N O N O~ V O G U N
Cf ~8 1~ O ~pO~ ~S Nt C~D pfp o< Oi Np~
C~9 r m m l r /'A7 M M A
!+1 ~
$ ~ E E
=
E
EL arn m~ E~ ~ ~n a ~ is E E ~ E tj o ~ s = a ' s~ ~ ~ o t ~ ~ t~ ~ =
g QQ QQ o $~
VV
A eo 8 =- -C
m < <
N N V
GGX C - M fA
cLL, W
Q bv ' z O ~ .~ r m o 0 8 a rn ~ n n a~ U Z ? a a n ~ n ~ ao BE ~ w c m ao ~_ p Z Z a C y p a (~ g E .ic E c ia N w W W an E E $ ~N i c 0D m ~ N dW W ~ ; r t 's p ar' > g ~ C c I
0 = ~
a o e ~o W = ~ E E
~-'~g-=gs " u 3$ ~ Ã "'~ "'t c> ~ c~ ~ ? E E g ~ o O a ~ Q c ~ 8 dF g ~ E o, z n~ ao o a ~ Z rn or g c c c,~ O E. a > O 0 ~=. E ~ ~ ~ o 0 0 g ~ o tn E
a0o aVo oC E E n y%
p C ~ pppp C C. C. C. ~ E ~ E 7 OI
V O ro~~ C V =~ n=~ n=~ 40 W O O ~ U
aN a u a 8 ~. c a ~ o Ew ~ o o~ p EO~ a m $ o' o, a U~U u~ t~ U n ~i ss ~x g= x ~~(j m ~ t6 Ci % An W) I " i I
o < 5 No I i X x pp ~ n n ~ ~~ tM0 i0 {~ N IN N N{p N N
~qy e+f W
- i i ~ i i i i ~ i ~~0 CD (D 0 c ~, cma on 0 CD~ otD w n u~ n a 4m +~ 1Qj!1L~l! cw ik A r, ~ m (a ~ 4" V) U) U) n 10 $~ !
E E ~ .2 ~ y p o c c a c a n u3 O y~p p ~ ~ C =~ =~ n~ ~ ~ "' t"a L ~ ~ ~ ~ =~Q 'N W
{7 ~ H tnl O
~~~~ ai r%1 Uw~ _~ :EarUt7~ O Ni~~ ~ SS x y ~p N
w t= t ~G~r,'~ ~Q. N N
N i N m X a~ N f =
' V a' V
W '' o m a m d w ui So a z s a Z ~z Z a ~~ z z m Z Q8 ~ W U W y a~2 Z
_ z m rn rn g = qz z Y W W
W QQ== o =4~ H a o ~ C7 O~
tp.i ao Vrn õ '~ V! fA CC o L.Y N~ N tWq tWn ~N W C O > L ~" ~ U
Wt Q~ C a (p x ~~ (Jy o ~ 2 8 a= ~ LL
C G ~ C C O~c ~ C ~ y ~ ~ p O ~ ? ~ y~= CO y.
sQ E ,C ttYyll ONS $ GGG ~ ~ (-T ~f G 7 a N a C~ y~~ a N Ã n~ ~ Z' 7 7 C C~
v0._ C.~COI
ffi a c c _ _ m E M c ~~~SSS pG. c c pn N N N ~~=O to N C~ a A 7~~ 7 O rl coW
E n w a o a n P mm H $ ~o vi 2 ai ai a i'. x x 2 a ci 'n ~ o c ~ o g~ m E~ x xS x~
N aND t~~f p~y S ep Np W~p Nti~~ N
ph t0 a0 O! = In l!'~~ '"' Of O 1f1 p O
A ~f -~ '~ a ~ a a a aa <
I go on - - - N
M (ey7 ~A p N e~
NN
A J
J g LO ow 8 ~
a rn rn q i1, ai a m~mmmmm~m~mmm~m~m~m~m~ m~ co CD to N N N N N N
4~~~~~~~~~
N N ~ .- r- N N ~=- N N N p C4 N ~p O
M ~ M Nf p ~ff M Nf 1'7 t~7 M Y f M t9 M ~ cn _a = a .f~ ~,~
S
x W 2.. x = _ O
v p~ fa W v t v a Q ~_ u) 'i' vNi I
C ~ a ~ _= p m N N N w R
V) V) w tl ) q~ - a w w o '~ ,;~~~_~"~ d d O ct_7 0 87~V c~+ E o C9 C9 ~ z W
~ Z Z ~?
- ~ 8' s "' Do ~~ LLõ ~Y U a z z C'9 w]
W Wt ~ Z' ~j g E~ c 8 c B E W Z Z
C a E W $' ~
C =g d ~ ~ ~ S g O - ~, ~ a p ~ Y ~ d -' ~ W W W
~=, 7 M R o ~_ 3!0 f' 'Q -~ ~ p i a C W U. pnto cl) s i i O WfQ W y~
u a g c t ~ o y 5 . y m MYYL q~ h O=~~P~~} -~..C [A
A m~E E E ~ tQ ~ rn 9 g a g .. c'3 '4 X Y o M t à ~ c L a O~ (n _p p tn = ~ t~ !~ h Q T ~.
V IL
g ~a..:j I~~
S
m O r a g a ~ o qpyZ_ Q Y $ z z Z w C _ " N
Z QW1 . " ~ .
~ Z. pp~Z X cY CS I/ Y_ ~ ~ L $tll t OI t Oi C ~~~ ~
EZZZ cg~ c c.~ CU..89 c., o $$ W az~W -~ o o m g .n,7, nSnm~ y g'ao a=~ n Q~ g ~~{~ a t E O crf ~~ ~ p t np ~ a ~ C
~ad~"" ~ ' ~orn y1=~' rEio E
,0 g y'~ 0~~~~0~5~ ~ ~~~o~~ og~zN
w~
hU
UU 0 ~j S ivui x > >w lco S a~ ~x ~oar O
~
On N
~ S~ N O N
OOODDD ~ n ad N f~ ~< a 5~ a~~~~ ~ a a~ a a0 p p P, v "
Y N N N Y! N N
~
O M N Y7Y h ~ ~
a n ~ a $
o LL a aa~33 a0 w ~ C~Q7 i~- ~~~ g c~ C~ c~ (~, 4 co co 81 co~
O 0'b N -~ ~- A M
OD '~ p a ~f1 ~ N ~ N Yf O O ~ r- M 1~) l''=
~ I $ I
si ~a$N
!~f Yco9 M ~ 1+001 O t~) M lIn~) l7 l~~l A p/
o o Z E E _ s c e e c 8 $ c n E w a n Y
o 32 = QU K S ~3< ~s3 S Vl U oi= oaW lt~=
_ ~ (A fA ~ V r a O
N W
~~Xr1 CC C O g Y Ol < UZ W O _~(7 o d' N c rn r W ul a g q a o CE~M U ~
z z m fA a ~ Z Z Z 2 a tS c ri ri ~~i 8 E r u~ O~
W W O O n ~ = N L Eo +a xai 0 ~ ~ a Z Z ~p ~~Q d wpom~
p W c: n e o c n ~ a QK ~g x~ El~
Cl ai e~i E'n o 5 3 E '~
eleQ ~o ~'I8~' ~ ~10 y~JE
O
~ N g N _ O qp N N
~ ~ ~~ ~ ~o' 93C C m m m O a O r r EOL L
E X~ U~Q Q a ~ Z
0 c a 9 w M V c Y '~~=,LD toi o ~'" Y ~~ ~~~F~ ~i~ 8=.4d 8 ~ u 'Scc+'d Z ~'a . n M .!! = E E C C 1- c E a S~ at 8N . c_ 0 ~ c.~ E E v a~~~~~d ~ ~}W
~ t ~ o E IL hi ~riq ~ o r3U =~U YJ 0~ ~~?
_ ui v ii g aS
O O N N n Ch n ~Q N A
O
x wR
~ ~a a N a N CD 0) 0 N Of ~ 17 m t~f .- N N N
~V n n r ~ r h O) ~ g yj r ''34 4 ~4 4 ~ b m ~ g y~ u~ q, O c7 o~o V' U U FW- ~ri~
~ ~ ~S1 ~ ~ a ~l w ~ o r r lol r O O O
t ~ ~
~' N Jj N 4 la ~ N YN) ~ O h O~ Of ~ O O ~ A N In 1[) CO M~p N~p N t0 f(~p ~{O O h t0 N 0 1~( 1n ~ p~ m-8 ~$~ _~ m O '~s l õ ~ 8 z ~ E E EÃ a õ~~ E > E
tsE~'o ='r ~=g ~'~ E I I ~ s ~mQoc Ep ti 8~~ x in~in c4s~rn2 ~no a in into x Ny N
-5 m m 3 e E
E
W po ~ v p $ $ $ ~pp ~
m c c - p W
E
~ z v E
c CD ~ ~ Ã W Ã
z o c n C7 C9 ~ T QSp p C Ol 0 p1 ~ ~ N W Wg Q O
c E
w õ ~ o 0 0 O.2C7 nF a o n t E .0 E
n a2 a 'p~s ~i8 ~ N
- P
R~ E E E
E E B = m x E 8 Y 4 E
3 gy y ~y W~o ~ ~ a , aNOB
a E~ o S E ~ p ~~ o~ u d O A A o .0 ' m ~ v, i U m m c ys - ~~
m u $' U p~ p~ y 1V _T
8 p p C ~ p T p J
~ ~ O U ~ U a L7 ~E 2 d b C C ~ Cl Cl E 10 z J~Wõ y õ N 7 E 7 >> E 7 ;
= v C .C W Yl E 7 T Z
c C ~1 i0 C r LL
e" T T ~$ a y , o o m oF v W W_ ~~$ u n O
o~~a o M 10~
Qm ~ 0~~8 8 ~8 ~ 8 c~
kn ) Qf a0 N p ~t7 ~ pa paf lh f7 OCp Q ~ J ~ J O p~j J O n O OLL, ~ {OL LL 01 1~L, a a a ~ a~ n, a a ~~ Q o a a N c yo~
pf A N ~ O_ O~p~ inNN pp M 4m N
N Y th N ~ to ~ ~
N ~
W) n M N
~ N
W
N ~i= q_Ui v ~='J~i= 3 ,~r, ~ ~ a doS ~y ~ .~ ~ ~ o SLL' Q QQ _ a mi , ZI
cc m m mm mm m mm m m mm m A In N 4m ti cc O
O C
ic ~ ~ /D~ pCD~ Wp~
~p tn ~1~ CD LLa'1 ptrf ( N Nt{ Na CD {a~0 ~ N O
r f M cn') pOj pOj O
7yy 7 ~ 7 q Ny W
ry~~1 uq >
C C p So a c n C C C V ~ N H y~ W
~, .n ,~ 2 r5 CL a acr 3 b b o- a10i n p a O g Q O Q O O
~ L' L u E E E E E 0 E 0 V
Z V) (q (n vJ U c 0 x = T T O 0 = m v rn p p W ~ W nQ~ E E $ d p Y T ~ 0 c n W O 0 O O U N U~ O O 0.' 0 0 ~ W W
$ a n n n ~ E o Z~i t~ o Q4 K ~ ' ao m a o E (7 'p8 a _ ~c> O M o 0 o 00:
O e VaE N" a1s o p v, E S ? o ? rn a u Q Q 'Q Q ~ n N E 0 E ar $z Z z z U
Q y o y o e~ - F co oi W F- c c o E !? - U v U
41 o O O O O 2~ co O0 n A 0- U W Ud yZj E t k ~~
~gr ' v v v o so c 4ao ma à 4~ ~ ~ g N N w O O O O o~ ~ Cc7 ~ tc ~ o O c Fo W ei a6 W o' w fn 3 o<n orn Qo E~ ~=
O e c ai ~ ~ ~t ~ ~ o r> > c~ ~ ~ f ~ c u c 0 0 ~~ Ti~ T u c~ hc~ ~N a ~ a y 4 ~ N un, C) i U ? -c 1 m a o ~i ar ~ ~
m ' 0 ~ 0 E e N ~ & ~ ~ ~ = 7 , n nrNi n ~ a~ E c~l a ~ E ~
L m 'n Q~ c 10 LL i; v) c c E
.~ W~' c ~ c ~ t ia t m c c E~ ~ ar ar ~~ w rn v ~ a ~ g a m L ~ 8 d m ~ y ~ a. N O a) O O I I C ~~ O T W W~~C W ~' 7 n 0. ~
g Q u ~ c c c E 8 _~~
E f a y~ rn or rn 2 E2 c W
Q=y x Y X y a m W n O n ~ p IE-t=a y N N ~ Yl õ N ~ L q Ip ~ t1py C O N q ~ 01 L
m o E~ n~ a N ~ ~ E Y C1 E m q ~O+~=8 ~ 88 8 m~ 1 'a N
W .Ø ~Ø. E..V. ..d. O yY+ O' c h' Ol A L N W 10 ? f iY ~ fV N N l0 l0 0 n O 9t O O g1 O~ .YI p N p tll N d N m t O E n' N N L E
o ~ L E S a g 'r'I a "~.~ N ~ z Z ' $_~
za~n v~ v~ v~8 tt rnx,n =,n x>xBxm m , ~
e~ c~ ao o v N i(i tp ~
p) Q ~ m N ~ O ~ ~NCI h~ p N C-4 04 fpNp C cG4 ~ N N y, o p~ Op~~ O
J Q Q D I < Q Q Q m N N Q
O O O a0 p N~ h e~i O
co a aD
N N Y N Ci < N N ~
a ~
y 'n p p Q O ~ _N a VRQ
z _Q _< Q Q V
U. ~ eqD Q ~ ~~ 4 m N <
m m m ~
~m~ ~m~ m ~ y ~ m m ~ 5 m m~ I ~I !, A
m m m i i C7 cD a~
Cm7 ~
P ~
~ O N ~
V
g ~ ~
it W
t a t ~a a ~, ~ a b >
r r r N) ~ e~-P ~
pO t~0p A~p OpI~ h N~ N/ m (~Dp O O N O V') ~N~ p h Nf M l'~ Of 1~+1 !9 c~+l l~~f lh 1~0 1(1 tN0 ~O ~Nff 10+f a 9 (O 1'~07 V c+! 1~f d y ' .1Q .i~
a = E
X
E
CL o ~ Jjj:jj S h O S
põ c ~ a o C'i W fN0 ~ Ci S C S cC
E q z a O D ~i/ C
Q
G ON crU = m V .Y
o c ro g ~-~' g 16 E rn ~ ~ o~~ m ~ y r o E m a d C ~o g ! o $ Q s 8 m 1 C 1O _ v o C n N Ci C
V
N E ~
r= E r:'~ ~$ M y y E y p~ a~ r~ ~S 2 m t F N
~ E o E a -~ o S o U ~
~~~~ m~~y~' L L E
rn ~o1 o~
W E S o E .2 y_G a c _c Y e rn E ~
m,p 7 7 ~ E 7 X E
o am eo coI Z L E F' m o ~ n . Q ~ y ~ c0 O~ ~ Q
Q NI ~ = ED a O
Yirlf egl~ E1 o N
U ~8v~ UX..= ~r '2 2 ~ w=wN V) 4~ 00= vi N M ~ry~rypp n ~ ~ ~
1~ Y M NY N tln+~ _ O = ~ 10 ~ h N Q $$~
m !~ ~y r4 1p h~ M G Y ~ O $Uf~ ayX, N f Nf ~~ ~ a V lJ
N ~ X ~ Q Q N Q N Nr G N U ~ N N Q < Q
O 01 O O a~O 1~ ~ t~ ~ 01 T N
~~yp _ ~a~app 1n U1 N N N 0 C9 ~~f/ V) V N P1 tn h O ~ ~~p N 0~y GN h f ~f ~'~' I~O NO G ~ CO mNQ I 1 m J N N V ~ r tW ~
~ U ~ ~ r U w ~ ~ C1 8b~ C U
(m ?a~ =m 'w ad~
~ 1 I 1 I I I 1 I ~g 1 I 1 1c~1 I 1 I I I 1 1 mI ~1 (9 C9~ (m7 C7(7 ~ C~ ~ mU' ~t7~ m G ~ 0 ~
(7 t7 r $ ~
~ $
' c5 c5 o IL~
O O N
SIR Isim R v E c c H ~m'~.lL'~.lQ rin n 72 .a W 'n w E E
43 5=S 3N fnT T Q C
m ='~n W . - s cp v~
a~ Z E ~.j p t7ia ~ iW
m a w ~o E W aj y pvi 2~ m ~" G W W y ~
U ~1 q ~ tl~ d , c~E c..E EV1~0 O -.0 oz a a ~ N(?
7i a c z = N =~ ~ -~ Z Z N j n d~'> E z (23 j U U m v~ H 9.0 12 ++ D E- 125 w W~r' r C ma E
~W W G rf ri 8 Goc H
crr aa~E,~,Ca~~Eo 31n E
e ~ E Erot 4 o v~U~' a ~~ ' g g4 ~ A
o~~~~N D~ v~,~~V o V~~W~~ ~ ~ v$m ~y U
~ a' A H ~ ~' 3 r ui p e~ 3a ~ i - m oe~4~N 7E
WL E ~~~ -8 =~$N~~Q ~
~ ~ oc~iqggo c ~ 31QZ
~ ~~cE
LZU~ E.
n 4Y v m 7 rm O ~ .JQL mdQQ~~ c~ m ~E. ~j' ~i 7 1f~ =g ya a ya (L A
~
E N ~ a ~ E N 11 m W C Q ~ 2 E ~ N H
t~z..En~ ~ c~~ W WG'Je ~~v, 2 SS=$~ a v ~nota,~t~ o ~ N C ~ O1 ~
W
~ ~
M ~ t+1 ~ ~ ~ I Yl h ~ CD
~O (0 1+1 ~.:
~ {+1 (Qp f~ A ~
C Q CD ~~ ~ N ~ N A V ~ ~ ~
IL
U) ~ ~ i~~ ~ rn ~4 ~0 4 4 d(D
ml m m 1 1 tn ml~p1 fnl ml m t~ 1 ml mlpp1 1 ml ~1 ~1 t~ (9 C~ ~ ~ C~ U' (~ C7 (~ C~ t? (! (9 (7 ~ (9 ~
c, ~ ~ ~
CP ~ ~ ~ b?w Cf { {R N h N~Vp N~p ~ t~+p~ O! p~ N N fi0~ 1~ N f0 <
l~! V O M M - b 0 N t~0 ~f t0 ~f t~~! g Y M ~ M tp m{~
w p c E 3 "~ a g E p E
3 w~ w C at.Q
$~ a~ ~=~ ~ ~ a.~ g E E
c N x' ~a"$3~~ ~Zz~n i~-m~ ~
V ui ~ $ E =o dcr ~ c~i Y; 0 2 A $ c Aa N ~'~ ~ $
o O1 $
4m cl) E
E E $ (9 0, U S C ~ Q U ~ $ N N ~
R' K a F o W aLLO aci & m c 3 tZ+~ a o E~ EI
S' ~ t2 w O w E a Q c v E y~ o W q p~ Q c J 1 c r EO w V m QO1 cn ~s p n i3 $~ 2 df b U $ ~õi o = 'fi ~'o ~. La ~ p o~ $
w x w rn ~1 c rnI~5p~ EQ ~o ~ oa cli ~' G .~ g~ L~ m a J a ~ G C N~ C N=
~ tl L~ ai ? '3 ~ o w$ U p g VI $ Q Wll w a$ m c E 10 Q wc = c 8~ ~'c 9 c o c~
a w E p I!1 ww$ E a à ~9~ ~ Z o gg s iS S a ~j q -E a 2 a. ~ 3 e o c' c i5 E N .Y ~ c~~~~ c ~
a a n g rn u ~ a cv -~ 'n w ~~UsN
J ~ ~ Z W w oo= E= Q U ~~~tn xc1 Q xr~ S~S ~mat~~ ov~m am h Yj ~ N ~ ~ ~ W p N
I ? z~
a a ~ Y Q~ Q N -j< K x a Q a Q K ~N ~ aiOc N~ X~
~y Q O ~pp N i_A h_ O
N/ N ~ ' O O ~ N
N p~ ~ ~pp }j~~ pp ~y Of M m t' ~ ~ ~ 17 l'~i 1.) a~ ~ ~b0 ~ N ~~p W ' ~ ' N O~
1 1'N ~ bf N O~f N
O O ) ~ (~V Uy OI
' w~~ ~ W ab ~ '~ O1 N ~ ~ ~ G
~X" LU InUZIN
_~
m wg w ma, m' m~ m~ m m~m~ m~ m~ m m m~ m m' m~ tp~ ~p~ m m mop~
o c7 (9 Ca C~ c7 C~ C~ C~ c7 c7 (~ 0 C7 c7 C~ c7 C7 c7 (7 c7 C~ C~ c7 OD ~ se ~ ~ ~
cri a W
N ly .- =- N C) ~p ~my {p m y~ ~y pp ~pp~ fp ap Nf c7 -~ ID i w N f f~ f~ 10 ntp N1~ ~ N a0 Pf ~'9 ~fpD N
''7 ~1 t0 N t~ 0~1 p~i In I~+f Y1 t7 a 1() ~ Cd { j ~.~j IA ~f! l+!
g E W e $ u a a > ;
E ~
-~ n N N ~2~Q~~0~~~~ ~.~>.~~ ~ ~ ~ ~~Er$$
N ~+aUa3~~r31~ NO = ~y0 W = _ 2 n ~
E
~ 6 ~ m N Z tV
!0 L H a ~ ~ N i ~ 10 C C c ui O g in $ a ~ a ~ n ~ i~ c ~ ~- 1' ~ c 2 ~
h a E
E
= E ~ .E3 m I ~ o E o ~+ ~ ~ a ~ t g+ ~ m p J u~ ~ a~3 O1 s~ 'n g N, $ x~~o C
V t n E a' aE~oV
a o U p' } V
=d ~ J ~ ~ '0 a Q ~ a N ud dl 0 o m~ Qa rn z 0S
(0 c~ o a ,ae ~ E _ =- H $ ~' V o ' ~ = a ~ N 3 n m m ~~ pp C~ H O O 8~ v O ~ CiI Od U~ y Y U~ ~ N Q W Y ~~~~ .Y!
e oC
E> E m ~c~j ~'t3'o ~ ~~
à Z ~a 3 >'~ ' r ~ ' U~' ~oc~z~rc c c'-~i c c_Z or; 2{L > > N ~ EEnE
E Z3 tS t5 a m ga U =' __=- 3 c c $ E .- tn =~ =~ 3 tE~a ~ ~ '~wr o~_$~goo ; a t 'o x1,1uit~~i1t~
V. 4 p $ 0 ~a ~S$-r'-_ ~ a a ~ ~ 8 H~ $
~~~ a W
E 2 r2m =~ ~ _ t~ Qf N_ ~
q ~ Q ~ N 7 W a~D O
~< Q Q xQ << f3 ~ S~~ ~~ ~g J J Q ry ~ ss ~ ~ $ ~~ em WN g (y,~
W Q ~ V J !} Q q.- =- f=Q- ~
~ i VUJ
= = ~ OQ
a ~"p~ ~~ l ~ g U' c9 c~ (7 c9 U' C~ (7 C~ (~ C7 c~7 ~ c7 ~ ~ a s o ~ ~
'3 N 1!f r ~- r r .- r r r N N r r e- M M O ~ l7 q~p ~f f0 CD n ~ l0 Y n ~ 7 N
M
~ V f {+f If1 A l+~f ~ l+f 1+f V ~~f f0 m C9 ~7 Ol Y
VO
E '~ y E
8 c~ca~E E
, Y c~.8'c~ c c ZI. n E 1 2 E
IIIIIH x s s s s z oC~ o a 3~~ aC s c~'oa~
~~~ ~j w 9 a C _ Q ~Q$~~ ;_ ~tapp ~ ~p ~a.C
C C V7 (/} C7 t~ N = gj p ~ W C G
. o' gi Qc ElA+
m CL n Cj E ~ E 3r ~ i6 $ N
~~~=_ ~~~~
g ~ A
c~~o O
C
0 Q ~"=a rn ~~N~ a " ES 3 p~~8 . ,. ,. ~
mmM v~ para~ vx ~ ~~ c3 F m im l0a M a.2 M~ b o__Jj E~ C), E cc~"
a~ al wEmEB ~EEEY1 oi~iy ~N
y= g (~, C ;o C 9 U O. V 3 H J. B~ c rn ~ rn=~N- 7~ E ?~~E
=~N$ ~.~ .~ ~o,Y $~~' d' ~ nc c E~c c c V! NZ ?0"iA g i -IL'~
~9 ~~~~r n w_ $ ~r ~
r 2 o~E H~ i1ti Id V < ~+f N
~ M ~ N
N ~p to M
i r0 aI0 aD 04 N N N
r N ON ir., cr~ Y1 _h r m A
pp~ o1~ (.~ a Q ~
Q Q N J J N Q a 4 Q a Q K N
r r W p M ~ Np O~ ~ ~pQ~ l f Ih IL! {'~ y ~~ O l n9 M 10 M g !~~'f l~f N A N N ~
~ U ~I g n~~pp ~~npp ~ u~
G n C~ G ul O~ r n o M }- M ~ N N M
W ~_ Rf W ~ W ~
~p1 1 1 mI 1 I I 1 ~p I ~p I (~ I 1 1 1 ~p 1 1Q I 1~p1 1 t~ I 1 1 (9 t7 C9 O t~ (7 C7 C7 t9 U 17 C7 C7 (~ C7 C7 L7 (? (~
O t~l r p ~
r W ==-s dCNDO r r r W W C~ ~ F N Op 8p Op $
n~ ~p dtq0 N W O O O
01 l~f df f P'f 1 Lh f0 1 1D 10 W r '" s E
E $ ? F
~=~ ~ ~ ~ ~~ ~ fliJhiIllfl ap a 8 ~ Z' S
s~ 8~~ ~ ~~}8 ~TB~ 0 w g o- o o 0 5F~f w N ~ ~ = G .~ C ~ ~ U L ~(~ ~ YI jY~I G
~ ~ ~~ ~E = m m ' ~ p O ap E
~m $
La U. a 'fl C l0 ~ L' C~ 0 1nf ~ V $ LL~ D~ y GY N
La 0, i -o a 8~ a .r ~ 1 1 2 C C~ b C~ O~ d yv~~r: -- 8 t~ ~ $ a w n I -i I Cq~. $ w a x q C
~ ~ E
o S
s'Q ~ o~$~~aa~'~ ~o= ~~ ~ n n a 8yn t ~~= g"~ 0 n W ' L t 122 O1 =(7~~a2c Y~NSv ag= ~Bg~ n~ n E
~m~'i~ W~~ga.p $81! g Q E
11 W ~ ~~8 c~~'~ E a PL
aa,~ ~..~ a E E aE~$ ~ a ~
? E E$xvt~?.~310Lg $ac9O1E~E $> > E E E E
c e ' ' ~ y ~ '"$ S ~o ~ v E $ g =~ ~ ~, v o ~_i ~ ~i ~
RZ t~LE Em 9 .
= t/l W a JD a 7 -Yj :3 t, r ~a 1 S ~ Jilhili 90 U U U U a a W=N aiq~ ~ i N
W- " im ~' W ~ N
~ s ~ ~j ~ Q X 4 N X N f~G 2 !R ~ Q )C
a ~ v)o N Y Y U
~' CD a U
~ a V
~ ~ ~ C=~), U
CIL
o7 ol t9 L~ t7 (9 ~7 C~ t9 (7 t7 ~7 th ~ t9 C7 ~ ~ ~ ~
.- .- n t- ~ ti ~ i3 ~
g g C C C C ~ 'EJ ~ C C E ,~ (S 7 y ~ ~i ~ ~ ~ O O o o ~ E m ~ S~~
E ~2o~ 5~ oC
i x = ~ _~ ~ ~nU o '52 3 U rnU 4m~~ U SiU o~~ dr M~~
B $
a v ~ $ = N o N
N = y y ~~y 8 vp~ 'o n p p Q
g W pN? a Q p L g 11 Q~ R' o pi~ Y Cj Epj E
E ~' = a E
p CV) a a >
Q ? ? w!' " n a o Z_ Z_ m OC
_Vo U U N m~ O a a ax v~ O1 Ã' Y! > > am ~~E~~$ $ = a s = ' ' $ ~
s ~1~=~ >
Vl' : 2 >. W f0 0 1 ~ 11O t=. ~ T T M, i R's r s E E = E E = E E
F ~ o d ~ ~ ~ ~ g o Y Q E
g z z F -- ~ ~ ~ ~ ~ ~ ~=m o N ~ $
tn > > 5 0 Q
rn o~ B rn o~ ~ a a an ~ E
~
~a 'ri.~8ea.. E E F E E Es c Yt U 8 13 U, C C y cm C
aa,~ ~..~ a E E aE~$ ~ a ~
? E E$xvt~?.~310Lg $ac9O1E~E $> > E E E E
c e ' ' ~ y ~ '"$ S ~o ~ v E $ g =~ ~ ~, v o ~_i ~ ~i ~
RZ t~LE Em 9 .
= t/l W a JD a 7 -Yj :3 t, r ~a 1 S ~ Jilhili 90 U U U U a a W=N aiq~ ~ i N
W- " im ~' W ~ N
~ s ~ ~j ~ Q X 4 N X N f~G 2 !R ~ Q )C
a ~ v)o N Y Y U
~' CD a U
~ a V
~ ~ ~ C=~), U
CIL
o7 ol t9 L~ t7 (9 ~7 C~ t9 (7 t7 ~7 th ~ t9 C7 ~ ~ ~ ~
.- .- n t- ~ ti ~ i3 ~
g g C C C C ~ 'EJ ~ C C E ,~ (S 7 y ~ ~i ~ ~ ~ O O o o ~ E m ~ S~~
E ~2o~ 5~ oC
i x = ~ _~ ~ ~nU o '52 3 U rnU 4m~~ U SiU o~~ dr M~~
B $
a v ~ $ = N o N
N = y y ~~y 8 vp~ 'o n p p Q
g W pN? a Q p L g 11 Q~ R' o pi~ Y Cj Epj E
E ~' = a E
p CV) a a >
Q ? ? w!' " n a o Z_ Z_ m OC
_Vo U U N m~ O a a ax v~ O1 Ã' Y! > > am ~~E~~$ $ = a s = ' ' $ ~
s ~1~=~ >
Vl' : 2 >. W f0 0 1 ~ 11O t=. ~ T T M, i R's r s E E = E E = E E
F ~ o d ~ ~ ~ ~ g o Y Q E
g z z F -- ~ ~ ~ ~ ~ ~ ~=m o N ~ $
tn > > 5 0 Q
rn o~ B rn o~ ~ a a an ~ E
~
~a 'ri.~8ea.. E E F E E Es c Yt U 8 13 U, C C y cm C
'G
c a EE~~~~~e~
_$_ ~= 7 m~ S en W~ ~ U ~ U CJ ~ U ~ a~ 4 2 y b 1n uD
01 O1 f0 p~Qg ON_) Nqp 1A m QO a0 M c7 O p ~p ' a ~ Mo!
N
p m QQ
to Q~1 M.ap h p~ N t f N N ~ ~ N /~ (~O {p ~ 10 N ~[1 ~f (D N~ O N ~ cq tD ~ cAn ~ a M V) ~ ~ ~ fff VVV~ C~~ lIIVVV~ c~l O, p p co O Q t~ N q ~U U a.
W
~~~
p O C
C', !0 ~
,~
co Ln ~
P, orn rna, ~i~i ~+
a~ d p p~j t~ t0 e7 N N . - N
o 25 rn o m c~ y~ rn r oi n rn aop dpp ~ ~ 2(pf', ~~ ~
p m t~ v~ g ~ ao ao r o ~ Of O~f T f0 l~+) ) 1') ~ C~f M ~+f M ~~'1 M l ~f1 Y {~'1 E
o _3 p M Y1 pp V
'c ='c 'c y y y~ m n E y y E E W E
~ p p p W c c c W c O c O c O
r E , c w W i o.5 v'' $ F ~ 2 w y y 0 y ~~ y y W b W J
E E v o o a ~c W E o~ o Z t o o $+~ o ~ o L~ y o $~
o c~ o, U c, ~ 0 o, c ~ ' a x x x c.~ 00 x x 2 x in x (a n a8 x ao N ~c or il) ''' q,y E N
_ 9 ~ a N - h y p ~ V tN0 O E p ~ C = . ~ lu $ a c M
~{pa ~a W c9Ci E ~ Q
E
OZ c JJ 2pSZ~ O E $
8 E= p=o ~ 2 = s $
Z o! m N W W x Z
W t w a U UZ a F 0 E O
a LL m ~
U~ Z y W p a y O
E w w a~ n ~Z c c rn U cp C p uwi r '~ m 'r d E w E A y K p c ~ ~ = y N (w/1 c~i .~ u E~ =~ m o ~ a a u c a ~ E ~ OZ a o y y$
V''~ n y d U.
g~ c y U a a~ y 4 4 d E~ ~S m OE o m N S 0. 4 p a m E~ O~~ cL rn ~~
m E~ m E E a _ = x ..W-E
/0 c c E N
c~i v i '0 vpi ayp~ y pN p~ Z p !3 0 V C L 4 " G O C
W o 'r O
a; y c E c~ E U.
~+ I I.- Y a a m al 9 od~
n a oa U c Qy UI c $
w 1O E c E ~ Q E
W LL
c y n t~ U W Z V E 'a c c y E $ c Qy E
~~ ~n y Z c E Z CQ 2 v> j g V c' y p M Z c W~ E E p cy c ~ rl U O O. a d E O O y~ _ y ~1 p W a p~ y p d' p rNE E 8 -E a _ a.~3 C" "~ ~ pQI_~ vi$~W_o2pEw W ~
~ 3rn w c E b' N E v' E ~~~
v~ci U ~nU c 3 a i xx0 a D 0 8 ~ N o N g ZEU; im ms C~D O Q~ h N Na c7 ~p ~ ~p ~
~ 17 N A tp N N
O) OI t 1 N
O O ~y N p y) ap n g x g aa~ ~ 2 a < e v pp I~ N 4~ V' ED N a a 1~ m U) Nf f'! 111ppp OD
O l~l l+1 N Ny lh 04 4lf N ~ h ~ Of M N ~ 2 ~ aa00 f~f V eh M N t~~l E ~o N
N N r, go N2 N N go c+f mll ( U (4 N_G N
p W V
0 4gq v .- v m a m f6 4. m I O~ W W m W W
m m m m m m m m m m m m m 1 m m m m 0 0 0 0 0 (D 0 0 0 0 c3 c~ C7 o a oo a cl) 04 ao ~O Oi a~o ~'o O NN O O O N
7~ m O N N N .~
- A ? N
Y~'7~ O m1(~ V~ O ~p ~{p ~~p f~ io0 t0 O O O~D_ ~~m A
t~ ~ ~O (D M M li1 f~l {D ~ m ~f Y~1 lN'f l+f l~+!
~
W ICO
Y ~_ O O y E
F3 y ~ Y A ~q ~q ~ E 'C 0 C CCC õ .y1 W F'J C C .C"
D
g~8 a. 3~ a. B h a a ~
2.2 1 t ~ a m mm W ~
m QQQ (7'5 Q
N a 7 N xY c ~ ~ y 1 X !l' m Q
m ~o S ? k o w i~'J ~'J I G Y $ Y Y f/1V)LL= Y~=-C o~ ~ W W 3~
K~~
~ o o pgN gN ~'o 9 m a a E
I A~' V 4 ~ E ~=xY='~ e 3 r ' So o ts $ 3 g " ' g aa ' g~=-a Ã
m < E 'u " ~ o $ P d ~ Q Z Z'yffi~
E p = ~ a Q> a. _ ~~ n C O1 a d+ V S ~ c Y g E E -U n o ~ e Z Z e 0 0 ~ 7-5 j j ~ $ U a g m Y ~ a n16v~ 8 L' 2 'rnI
2 Vm Y uic tWi~y E
n~'~a~~
E E 2 ~ 5 & r I~ E~
0 0 ce in 4Y in ~ ~ ~'w$'r': wN m~ a'~v rn~m 'a r mm'v~~~C7 Uma ~ N y N N O~ Uf gj N OD ~ V ~ ~p pppppp yIIVV'~ ~
1~ O LL ~ ~ ~
6 < 5 Q O O N
In oIi~i n ~ ~ p2G ~ r'X e~ n c$ '~ ~n g v ~+i~cn~i V) W ~ ~ ~ '' N N ~ (V
W .. ~
a~
~ c7 ~ (m7 G~ C7 (7 cm7 c7 (m7 ~ n Lm'~tm7 ptm.~~ n~ n M ~
LC
LC
~ L~
si =' N N N
r r N O
m h OW~i- 1N0 V h in Y O tt> ~ EWD- 10 N M~f t0 1~ f ~
t0 M W p h O ~p p h m m A ~p 1~ O O! Qi W Oi t+f ~[) r M W h lh tD ~() N1 ~l) 1!f pf M Uf t~9 M Y) Pf l'~'1 t"f ~ q C C
~' o= s E ~ E E E F~ E E E
~It~~~ 0 ~9E~ y ~ ~$ a a E o L i ~ o ~~~~a :r3~~s3~B aU oo~a'~ a a< < x n L Q g c c c c C d $~ 8 E
O QQ p f tl) =W~= FF h_ h_ - ~S C
n 0 O'~ 0 . eb t~ C C
s " W
~
s g h L m 0 n Z~ a a ~r E ~ W
c E ~ ~
v 8 g a ~
L
8g x E 2oS 2S F~ ~ 8 = 3 '4 A. m. u 8 oi $ o g w, s, i a a~ m'E
~ c a 4~ > c o a g Q~ r Q1 aa ao P
~! a h a a m g M -~ x x Ng u) x 7 >= 4 _ ~C Q u p y = Y~
~ o ~
75 5~ o o c E $ ~1S -~ Q Wa o 0 '~ ._<
n ~ 'bd 2g a~ cE
Z 3 3 .4~ ~ p~ ~c E E $(.E E E E E E E E 10 ~ ~~~ E ~0 /0 y~6 y $
~ Z m o X~ u ~g d o g G $ N E'e N 41 l'11 ~ 9 OI$ ~0 Gg 'o4lueo a ao ia ~~
1~ $i $11~ fflfltit ~ w$UUON m O tD W_ p~ N r Y i0 {h ~ O l+~f N t0 N p W N NO b r- O~ M M h N O
0 (O a N ~ ~f ~
< <
pp p ~
w O~ Oi h A O Yf r h Q~ A N- ~ r. O1 r r) g Mp ~ T N W } Y ~p} } ~ M_ O NQ Y1 n M
m~ ~ ~~ ~i a ~ ~
~ tm7 r N
~ ~ ~ ~ ~
W
O~ OMl ~ M ~ ~ st W
~ N N N N N N
N N N
W M M -. n h 1~ 1~ ~{ ~ W W iyA~ M N OW! ~a h 'Q 'C M N N f~ ~p O ~ f0 ~ l+w N
) {Np M Y
V M !+f 4Y in t~[l N e~ f'~~) ~ M 0) pW) ~ OI fW0 PW) ~
N
C
H E
d C E
YI nR n C C E o E
o E 'B~ >>~~5' LP
o 0.a.a ~.2 4 0 x Cg u 0- co Q ia S o~ Qp a0 O x O x U a~ tAU rntAU)U m a M
e 2a - Z E o N 1v~6 W ~
z_$ g ~ E E E ~ E~ ~ a 8 ~ .: N .:
0 g Q C 8 fWA &n Z ~n Z (04 In Z C Y
'J al W ~ Z a IE LL~ ~ E a w E c 8~z~ E ciz - 0 E
E~_aN~w~~. u 80~~ z~i ~z cz ei O~ EoO a ffio J ~= pz =a D
0 V a~$ E 8 E~~ E ~"g, E Y 8 Z W q Q Q E o c Z~sg~ pg v 2~5 Q tn o t/~ = W _ $ a ~ ~
E00?~ a ~Q~ c ~t ht x ~ ~ x ~p V h ea . ~ Z a Q in x i ~ n N a L~~~} a vEi n E E x ~ ~ o aLi x M x M y U.
M 'n = W Q' $
E y ~ ~~? E xu~,~ - ~ n~ ~ n $~ $~ y U~ S E
.~ 8r a8 mx a Nw w~ ~ "~ E ~
q a w E ~r ~ 75 o o c~.Q c u p a 8 o m m ~'r c8$~_ 8C c8g~ n'o Ea a~mm8m~' . ~~~ t~8 W ~$c?~~o 8 9 $'1 W (q tq 7[ ~ cq ~ N~ anO q ~ ~ fD N M U1 N ~ N cl, V O I~. Sj CI < qOq~ q ~j IA M
Q Q J fhV J~~ Q Q Q Q ~ W N w ~~MQ
a r ~p N h o ~ O I~
p~ N N N ~
a ~ a N N ~ M ~ ~ ~ N~ G n="~ M N a ~~ N a U. N
~
n V t~/ ~~ ~ ~ ~ tp OMi h m w U UU ~ Q ~ o~ V ~fT~+f ~vSN
O F- d q U
W (A W N tf~
W W W
~ ~~~ 0 ~
~- ~ ~ c 0 7 ~~~
M
c ~W
st > > W~
O ry N
N Q C) ~ O T N N
fftl11111 N N N
vl~i. 1. O p N~ m ~D 0~ p~ ~ aD ~p ~ff 1~ Mp V~p QO M C 9 M r'7i I+i a '7 P=~ rmi u a e~~~ 07 .~ ~ ~ $ ~ ~g .~ .~+ L ='c4~
yw Y
c c c c' a m iS
c a EE~~~~~e~
_$_ ~= 7 m~ S en W~ ~ U ~ U CJ ~ U ~ a~ 4 2 y b 1n uD
01 O1 f0 p~Qg ON_) Nqp 1A m QO a0 M c7 O p ~p ' a ~ Mo!
N
p m QQ
to Q~1 M.ap h p~ N t f N N ~ ~ N /~ (~O {p ~ 10 N ~[1 ~f (D N~ O N ~ cq tD ~ cAn ~ a M V) ~ ~ ~ fff VVV~ C~~ lIIVVV~ c~l O, p p co O Q t~ N q ~U U a.
W
~~~
p O C
C', !0 ~
,~
co Ln ~
P, orn rna, ~i~i ~+
a~ d p p~j t~ t0 e7 N N . - N
o 25 rn o m c~ y~ rn r oi n rn aop dpp ~ ~ 2(pf', ~~ ~
p m t~ v~ g ~ ao ao r o ~ Of O~f T f0 l~+) ) 1') ~ C~f M ~+f M ~~'1 M l ~f1 Y {~'1 E
o _3 p M Y1 pp V
'c ='c 'c y y y~ m n E y y E E W E
~ p p p W c c c W c O c O c O
r E , c w W i o.5 v'' $ F ~ 2 w y y 0 y ~~ y y W b W J
E E v o o a ~c W E o~ o Z t o o $+~ o ~ o L~ y o $~
o c~ o, U c, ~ 0 o, c ~ ' a x x x c.~ 00 x x 2 x in x (a n a8 x ao N ~c or il) ''' q,y E N
_ 9 ~ a N - h y p ~ V tN0 O E p ~ C = . ~ lu $ a c M
~{pa ~a W c9Ci E ~ Q
E
OZ c JJ 2pSZ~ O E $
8 E= p=o ~ 2 = s $
Z o! m N W W x Z
W t w a U UZ a F 0 E O
a LL m ~
U~ Z y W p a y O
E w w a~ n ~Z c c rn U cp C p uwi r '~ m 'r d E w E A y K p c ~ ~ = y N (w/1 c~i .~ u E~ =~ m o ~ a a u c a ~ E ~ OZ a o y y$
V''~ n y d U.
g~ c y U a a~ y 4 4 d E~ ~S m OE o m N S 0. 4 p a m E~ O~~ cL rn ~~
m E~ m E E a _ = x ..W-E
/0 c c E N
c~i v i '0 vpi ayp~ y pN p~ Z p !3 0 V C L 4 " G O C
W o 'r O
a; y c E c~ E U.
~+ I I.- Y a a m al 9 od~
n a oa U c Qy UI c $
w 1O E c E ~ Q E
W LL
c y n t~ U W Z V E 'a c c y E $ c Qy E
~~ ~n y Z c E Z CQ 2 v> j g V c' y p M Z c W~ E E p cy c ~ rl U O O. a d E O O y~ _ y ~1 p W a p~ y p d' p rNE E 8 -E a _ a.~3 C" "~ ~ pQI_~ vi$~W_o2pEw W ~
~ 3rn w c E b' N E v' E ~~~
v~ci U ~nU c 3 a i xx0 a D 0 8 ~ N o N g ZEU; im ms C~D O Q~ h N Na c7 ~p ~ ~p ~
~ 17 N A tp N N
O) OI t 1 N
O O ~y N p y) ap n g x g aa~ ~ 2 a < e v pp I~ N 4~ V' ED N a a 1~ m U) Nf f'! 111ppp OD
O l~l l+1 N Ny lh 04 4lf N ~ h ~ Of M N ~ 2 ~ aa00 f~f V eh M N t~~l E ~o N
N N r, go N2 N N go c+f mll ( U (4 N_G N
p W V
0 4gq v .- v m a m f6 4. m I O~ W W m W W
m m m m m m m m m m m m m 1 m m m m 0 0 0 0 0 (D 0 0 0 0 c3 c~ C7 o a oo a cl) 04 ao ~O Oi a~o ~'o O NN O O O N
7~ m O N N N .~
- A ? N
Y~'7~ O m1(~ V~ O ~p ~{p ~~p f~ io0 t0 O O O~D_ ~~m A
t~ ~ ~O (D M M li1 f~l {D ~ m ~f Y~1 lN'f l+f l~+!
~
W ICO
Y ~_ O O y E
F3 y ~ Y A ~q ~q ~ E 'C 0 C CCC õ .y1 W F'J C C .C"
D
g~8 a. 3~ a. B h a a ~
2.2 1 t ~ a m mm W ~
m QQQ (7'5 Q
N a 7 N xY c ~ ~ y 1 X !l' m Q
m ~o S ? k o w i~'J ~'J I G Y $ Y Y f/1V)LL= Y~=-C o~ ~ W W 3~
K~~
~ o o pgN gN ~'o 9 m a a E
I A~' V 4 ~ E ~=xY='~ e 3 r ' So o ts $ 3 g " ' g aa ' g~=-a Ã
m < E 'u " ~ o $ P d ~ Q Z Z'yffi~
E p = ~ a Q> a. _ ~~ n C O1 a d+ V S ~ c Y g E E -U n o ~ e Z Z e 0 0 ~ 7-5 j j ~ $ U a g m Y ~ a n16v~ 8 L' 2 'rnI
2 Vm Y uic tWi~y E
n~'~a~~
E E 2 ~ 5 & r I~ E~
0 0 ce in 4Y in ~ ~ ~'w$'r': wN m~ a'~v rn~m 'a r mm'v~~~C7 Uma ~ N y N N O~ Uf gj N OD ~ V ~ ~p pppppp yIIVV'~ ~
1~ O LL ~ ~ ~
6 < 5 Q O O N
In oIi~i n ~ ~ p2G ~ r'X e~ n c$ '~ ~n g v ~+i~cn~i V) W ~ ~ ~ '' N N ~ (V
W .. ~
a~
~ c7 ~ (m7 G~ C7 (7 cm7 c7 (m7 ~ n Lm'~tm7 ptm.~~ n~ n M ~
LC
LC
~ L~
si =' N N N
r r N O
m h OW~i- 1N0 V h in Y O tt> ~ EWD- 10 N M~f t0 1~ f ~
t0 M W p h O ~p p h m m A ~p 1~ O O! Qi W Oi t+f ~[) r M W h lh tD ~() N1 ~l) 1!f pf M Uf t~9 M Y) Pf l'~'1 t"f ~ q C C
~' o= s E ~ E E E F~ E E E
~It~~~ 0 ~9E~ y ~ ~$ a a E o L i ~ o ~~~~a :r3~~s3~B aU oo~a'~ a a< < x n L Q g c c c c C d $~ 8 E
O QQ p f tl) =W~= FF h_ h_ - ~S C
n 0 O'~ 0 . eb t~ C C
s " W
~
s g h L m 0 n Z~ a a ~r E ~ W
c E ~ ~
v 8 g a ~
L
8g x E 2oS 2S F~ ~ 8 = 3 '4 A. m. u 8 oi $ o g w, s, i a a~ m'E
~ c a 4~ > c o a g Q~ r Q1 aa ao P
~! a h a a m g M -~ x x Ng u) x 7 >= 4 _ ~C Q u p y = Y~
~ o ~
75 5~ o o c E $ ~1S -~ Q Wa o 0 '~ ._<
n ~ 'bd 2g a~ cE
Z 3 3 .4~ ~ p~ ~c E E $(.E E E E E E E E 10 ~ ~~~ E ~0 /0 y~6 y $
~ Z m o X~ u ~g d o g G $ N E'e N 41 l'11 ~ 9 OI$ ~0 Gg 'o4lueo a ao ia ~~
1~ $i $11~ fflfltit ~ w$UUON m O tD W_ p~ N r Y i0 {h ~ O l+~f N t0 N p W N NO b r- O~ M M h N O
0 (O a N ~ ~f ~
< <
pp p ~
w O~ Oi h A O Yf r h Q~ A N- ~ r. O1 r r) g Mp ~ T N W } Y ~p} } ~ M_ O NQ Y1 n M
m~ ~ ~~ ~i a ~ ~
~ tm7 r N
~ ~ ~ ~ ~
W
O~ OMl ~ M ~ ~ st W
~ N N N N N N
N N N
W M M -. n h 1~ 1~ ~{ ~ W W iyA~ M N OW! ~a h 'Q 'C M N N f~ ~p O ~ f0 ~ l+w N
) {Np M Y
V M !+f 4Y in t~[l N e~ f'~~) ~ M 0) pW) ~ OI fW0 PW) ~
N
C
H E
d C E
YI nR n C C E o E
o E 'B~ >>~~5' LP
o 0.a.a ~.2 4 0 x Cg u 0- co Q ia S o~ Qp a0 O x O x U a~ tAU rntAU)U m a M
e 2a - Z E o N 1v~6 W ~
z_$ g ~ E E E ~ E~ ~ a 8 ~ .: N .:
0 g Q C 8 fWA &n Z ~n Z (04 In Z C Y
'J al W ~ Z a IE LL~ ~ E a w E c 8~z~ E ciz - 0 E
E~_aN~w~~. u 80~~ z~i ~z cz ei O~ EoO a ffio J ~= pz =a D
0 V a~$ E 8 E~~ E ~"g, E Y 8 Z W q Q Q E o c Z~sg~ pg v 2~5 Q tn o t/~ = W _ $ a ~ ~
E00?~ a ~Q~ c ~t ht x ~ ~ x ~p V h ea . ~ Z a Q in x i ~ n N a L~~~} a vEi n E E x ~ ~ o aLi x M x M y U.
M 'n = W Q' $
E y ~ ~~? E xu~,~ - ~ n~ ~ n $~ $~ y U~ S E
.~ 8r a8 mx a Nw w~ ~ "~ E ~
q a w E ~r ~ 75 o o c~.Q c u p a 8 o m m ~'r c8$~_ 8C c8g~ n'o Ea a~mm8m~' . ~~~ t~8 W ~$c?~~o 8 9 $'1 W (q tq 7[ ~ cq ~ N~ anO q ~ ~ fD N M U1 N ~ N cl, V O I~. Sj CI < qOq~ q ~j IA M
Q Q J fhV J~~ Q Q Q Q ~ W N w ~~MQ
a r ~p N h o ~ O I~
p~ N N N ~
a ~ a N N ~ M ~ ~ ~ N~ G n="~ M N a ~~ N a U. N
~
n V t~/ ~~ ~ ~ ~ tp OMi h m w U UU ~ Q ~ o~ V ~fT~+f ~vSN
O F- d q U
W (A W N tf~
W W W
~ ~~~ 0 ~
~- ~ ~ c 0 7 ~~~
M
c ~W
st > > W~
O ry N
N Q C) ~ O T N N
fftl11111 N N N
vl~i. 1. O p N~ m ~D 0~ p~ ~ aD ~p ~ff 1~ Mp V~p QO M C 9 M r'7i I+i a '7 P=~ rmi u a e~~~ 07 .~ ~ ~ $ ~ ~g .~ .~+ L ='c4~
yw Y
c c c c' a m iS
13 ~ ~ ~ c n c c n ~ ~ o c +~ p a ~ p p o ~ r~ ~ s o o ao ~ W n c ~
~g ~ gI
~ . z z 2'~
a a rn w z Z a w w ~
p~ p- G z ~ ~ w ~ J Z ~~ a~ 4~ g t~ c oz o_ ~n0 g ~ O ~~ aU ~ w v1 v> ~
G1 ~ Z a' is a az ~~ ca d' a r Z rn E sa $~ E
~ W
O C7 V O (9 C7 ~ Z ~ E rd ~ w w ~
;M ~L~ ~(~ V c i c1 a ~ m n ~
c W w v ~ --~ E
O Y O
V N N N G C1 W v> y O
Ui (~ j ~
mo6 i i-~
r~ ~'~ b r rv wq C~ o'a c'Q
E cS : E b: 8 ' 0 u, ~õYl ~ V N N fl r UO
z c O $ rn C U a o $ a o ~G 8 tf c gE ~ V
g,a.$ w~
a n a,wrn~ .i~ EkW 2 mg ar ~ov --$~ a g t rna~"~ jm ~ ~ ~Y ya 1~ ac.io~g = Li ~~~En$~c ~ 9>> c~ a1E ='g E ~e$ ~yfSyE
n= E
'8 u is E Q c ~ m ~ c~ a ~g c W a W
0 w w o~e ~" ~
w w r o En L E~ E y pd a . 1~ ~ Ã~ v o ~ a c dS & ~ ~
b ~' ~'~ a ? o o~o '~i 8 c $ ~ ~$ ~,c 0 ~g"'=r--~
~ ' o~~ c~'r a~o~~~gw g~ ~ ~'"$ a a ~ ~ =~U =a a~~ 2Ln0 ~Np.~. g qqq~ C" ~ ~ N ~ n Y ~ =r ~ P,~ ~ ~ ~pp ~
Y.- ~ ~ ~ N Ny ,t r= ~ 4 Q Q Q
!1'~ ' ~ ~ ~{'~Q ~ p~=j ~ N N N
Y 1'7 M ~d Y O! ' ~V
~ _ _ W
N ry ~ ~ ~ ~ W v o S ~ ~
_l $ 11 N ~ Q~ ~ ' ~ C
N
ts v a v cn o 4 v~i r~i cn M
Ja ~
co c n V
to c:
~ o V/ q I F
n O ~
S I ~ O m p N d N = c Z Z g ~ W y ~
U E W
U 0 Z p w w x v a wr- E w > > o az ~' ~
C~ CJ C' ~' _ g H vwi v v' Z Z$ ~ ' o E Z
4m U O d ~ U D E ~ a a C
C Z Z g o~ ~~ o c Y o ~
0~ umi a = L$ c 4 =- ~ 'o V J W N y r 7 G=4 a yj 0:
U o~_i E a c S m LL ~ ~ E N o m~' ' E~. ZC~S' ~v3Y3 x x o c E$ K=- E in LL' vi 4 c E~ Ey c cti 3 g E $ c ~
0 ~p q O y y ~. - ._ v C j E
~ c 8 E W~~~.E 3 ~ o- y '0 ~ v ~ g+ ~i L _$ E Q 'f_ E~
= ?i Q ~
O o c '~ g L t0~Wo 3S s C '~~ 2 v~ i an ! y tc w o y y~/1 { E
, oy ~N y c cwcQ
a~"mayQ ~ o z ~v y 8 4t~.4 y 0i r f~ n N n ~ W~ C$ C Vi 0 OWi p a o~ E E v0 i õO ~ v~ Ii 8 0~ o E
ax aoo~~ N~= m ? 9 4 i M ~Np O~~pp ip p~
N
p Y p dp ~p ~ ~Q ~Q ~0 S O p~j O ~ ~ {h O ~ S Op Q ~ N Q Q Q x Q Q~ Q
~ ~ ~ n ~ N ~ N a NP n Of O ~ ~
Y Y cn ~ Y Y N
'n (n fn M U Z
y~~ N 8 ~ rs a ~S w w ~ a~ s~ a (L m m m~ m~
C~ c7 c9 C~ c9 c~ ~ ~ ~ m co C~
cl) M ~ O N
N O O tf O ~
~ ~ ~ ~
Exempliiication Example 1: Preparation of total genomic DNA of Corynebacteriumglutamicum A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30 C with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supematant was discarded and the cells were resuspended in 5 ml buffer-I (5% of the original volume of the culture - all indicated volumes have been calculated for 100 ml of culture volume). Composition of buffer-I: 140.34 g/l sucrose, 2.46 g/l MgSO4 x 7H2O, 10 ml/I. KH2PO4 solution (100 g/l, adjusted to pH 6.7 with KOH), 50 ml/I. M12 concentrate (10 g/1(NH.)2S0., I g/1 NaCI, 2 g/l MgSO, x 7H2O, 0.2 g/l CaCI2, 0.5 g/1 yeast extract (Difco), 10 ml/1 trace-elements-mix (200 mg/l FeSO4 x H2O, 10 mg/1 ZnSO4 x 7 H2O, 3 mg/1 MnCI2 x 4 H2O, 30 mg/1 H,BO3 20 mg/1 CoC12 x 6 H2O, 1 mg/l NiCl2 x 6 HZO, 3 mg/1 Na2MoO4 x 2 H1O, 500 mg/I complexing agent (EDTA or critic acid), 100 ml/I vitamins-mix (0.2 mg/I biotin, 0.2 mg/1 folic acid, 20 mg/1 p-amino benzoic acid, 20 mg/1 riboflavin, 40 mg/I ca-panthothenate, 140 mg/1 nicotinic acid, 40 mg/1 pyridoxole hydrochloride, 200 mg/1 myo-inositol).
Lysozyme was added to the suspension to a final concentration of 2.5 mg/ml. After an approximately 4 h incubation at 37 C, the cell wall was degraded and the resulting protoplasts are harvested by centrifugation. The pellet was washed once with 5 ml buffer-I and once with 5 ml TE-buffer (10 mM Tris-HCl,1 mM EDTA, pH 8). The pellet was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution (10%) and 0.5 ml NaCI solution (5 M) are added. After adding of proteinase K to a final concentration of 200 g/ml, the suspension is incubated for ca.18 h at 37 C. The DNA was purified by extraction with phenol, phenol-chloroform-isoamylalcohol and chloroform-isoamytalcohol using standard procedures. Then, the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30 min incubation at -20 C and a 30 min centrifugation at 12,000 rpm in a high speed centrifuge using a SS34 rotor (Sorvall). The DNA was dissolved in I ml TE-buffer containing 20 g/ml RNaseA and dialysed at 4 C against 1000 ml TE-buffer for at least 3 hours.
During this time, the buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysed DNA solution, 0.4 ml of 2 M LiCI and 0.8 ml of ethanol are added.
After a 30 min incubation at -20 C, the DNA was collected by centrifugation (13,000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TE-buffer.
DNA
prepared by this procedure could be used for all purposes, including southem blotting or construction of genomic libraries.
Example 2: Construction of genomic libraries in Escherichia coli of Corynebacterium glutamicum ATCC13032.
Using DNA prepared as described in Example 1, cosmid and plasmid libraries were constructed according to known and well established methods (see e.g., Sambrook, J. et al.
(1989) "Molecular Cloning : A Laboratory Manual", Cold Spring Harbor Laboratory Press, or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biology", John Wiley &
Sons.) Any plasmid or cosmid could be used. Of particular use were the plasmids pBR322 (Sutcliffe, J.G. (1979) Proc. Natl. Acad. Sci. USA, 75:3737-3741); pACYC177 (Change &
Cohen (1978) J. Bacteriol 134:1141-1156), plasmids of the pBS series (pBSSK+, pBSSK- and others; Stratagene, LaJolla, USA), or cosmids as SuperCosl (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J., Rosenthal A. and Waterson, R.H. (1987) Gene 53:283-286.
Gene libraries specifically for use in C. glutamicum may be constructed using plasmid pSL 109 (Lee, H.-S. and A. J. Sinskey (1994)J. Microbiol. Biotechnol. 4: 256-263).
Example 3: DNA Sequencing and Computational Functional Analysis Genomic libraries as described in Example 2 were used for DNA sequencing according to standard methods, in particular by the chain termination method using ABI377 sequencing machines (see e.g., Fleischman, R.D. el al. (1995) "Whole-genome Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science, 269:496-512). Sequencing primers with the following nucleotide sequences were used: 5'-GGAAACAGTATGACCATG-3' (SEQ ID NO:783) or 5'-GTAAAACGACGGCCAGT-3' (SEQ ID
NO:784).
Example 4: In vivo Mutagenesis In vivo mutagenesis of Corynebacterium glutamicum can be performed by passage of plasmid (or other vector) DNA through E. coli or other microorganisms (e.g.
Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are impaired in their capabilities to maintain the integrity of their genetic information. Typical mutator strains have mutations in the genes for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see Rupp, W.D.
(1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-2294, ASM:
Washington.) Such strains are well known to those of ordinary skill in the art. The use of such strains is illustrated, for example, in Greener, A. and Callahan, M. (1994) Strategies 7: 32-34.
Example 5: DNA Transfer Between Escherichia coli and Corynebacteriunt glutamicum Several Corynebacterium and Brevibacterium species contain endogenous plasmids (as e.g., pHM1519 or pBLI) which replicate autonomously (for review see, e.g., Martin, J.F. et al. (1987) Biotechnology, 5:137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can be readily constructed by using standard vectors for E. coli (Sambrook, J. et al. (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biology", John Wiley & Sons) to which a origin or replication for and a suitable marker from Corynebacierium glulamicum is added. Such origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibacterium species. Of particular use as transformation markers for these species are genes for kanamycin resistance (such as those derived from the Tn5 or Tn903 transposons) or chloramphenicol (Winnacker, E.L. (1987) "From Genes to Clones -Introduction to Gene Technology, VCH, Weinheim). There are numerous examples in the literature of the construction of a wide variety of shuttle vectors which replicate in both E.
coli and C. glutamieum, and which can be used for several purposes, including gene over-expression (for reference, see e.g., Yoshihama, M. el al. (1985) J. Bacteriol.
162:591-597, Martin J.F. et al. (1987) Biotechnology, 5:137-146 and Eikmanns, B.J. et al.
(1991) Gene, 102:93-98).
Using standard methods, it is possible to, clone a gene of interest into one of the shuttle vectors described above and to introduce such a hybrid vectors into strains of Corynebacterium glutamicum. Transfonmation of C. glutamicum can be achieved by protoplast transformation (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-311), electroporation (Liebl, E. et al. (1989) FEMS Microbiol. Letters, 53:399-303) and in cases where special vectors are used, also by conjugation (as described e.g. in Schtifer, A et al.
(1990) J. Bacteriol. 172:1663-1666). It is also possible to transfer the shuttle vectors for C. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum (using standard methods well-known in the art) and transforming it into E. coli. This transformation step can be performed using standard methods, but it is advantageous to use an Mcr-deficient E. coli strain, such as NM522 (Gough & Murray (1983) J. Mol. Biol. 166:1-19).
Genes may be overexpressed in C. glutamicum strains using plasmids which comprise pCGI (U.S. Patent No. 4,617,267) or fragments thereof, and optionally the gene for kanamycin resistance from TN903 (Grindley, N.D. and Joyce, C.M.
(1980) Proc. Natl. Acad. Sci. USA 77(12): 7176-7180). In addition, genes may be overexpressed in C. glutamicum strains using plasmid pSL109 (Lee, H.-S. and A.
J.
Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).
Aside from the use of replicative plasmids, gene overexpression can also be achieved by integration into the genome. Genomic integration in C. glutamicum or other Corynebacterium or Brevibacterium species may be accomplished by well-known methods, such as homologous recombination with genomic region(s), restriction endonuclease mediated integration (REMI) (see, e.g., DE Patent 19823834), or through the use of transposons. It is also possible to modulate the activity of a gene of interest by modifying the regulatory regions (e.g., a promoter, a repressor, and/or an enhancer) by sequence modification, insertion, or deletion using site-directed methods (such as homologous recombination) or methods based on random events (such as transposon mutagenesis or REMI). Nucleic acid sequences which function as transcriptional terminators may also be inserted 3' to the coding region of one or more genes of the invention; such terminators are well-known in the art and are described, for example, in Winnacker, E.L. (1987) From Genes to Clones - Introduction to Gene Technology.
VCH:
Weinheim.
Example 6: Assessment of the Expression of the Mutant Protein Observations of the activity of a mutated protein in a transformed host cell rely on the fact that the mutant protein is expressed in a similar fashion and in a similar quantity to that of the wild-type protein. A useful method to ascertain the level of transcription of the mutant gene (an indicator of the amount of mRNA available for translation to the gene product) is to perform a Northern blot (for reference see, for example, Ausubel et al.
(1988) Current Protocols in Molecular Biology, Wiley: New York), in which a primer designed to bind to the gene of interest is labeled with a detectable tag (usually radioactive or chemiluminescent), such that when the total RNA of a culture of the organism is extracted, run on gel, transferred to a stable matrix and incubated with this probe, the binding and quantity of binding of the probe indicates the presence and also the quantity of mRNA for this gene. This information is evidence of the degree of transcription of the mutant gene. Total cellular RNA can be prepared from Corynebacterium glutamicum by several methods, all well-known in the art, such as that described in Bormann, E.R. et al.
(1992) Mol. Microbrol. 6: 317-326.
To assess the presence or relative quantity of protein translated from this mRNA, standard techniques, such as a Western blot, may be employed (see, for example, Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York). In this process, total cellular proteins are extracted, separated by gel electrophoresis, transferred to a matrix such as nitrocellulose, and incubated with a probe, such as an antibody, which specifically binds to the desired protein. This probe is generally tagged with a chemiluminescent or colorimetric label which may be readily detected. The presence and quantity of label observed indicates the presence and quantity of the desired mutant protein present in the cell.
Example 7: Growth of Genetically Modified Corynebacterium glutamicum - Media and Culture Conditions Genetically modified Corynebacteria are cultured in synthetic or natural growth media. A number of different growth media for Corynebacteria are both well-known and readily available (Lieb et a!. (1989) Appl. Microbiol. Biotechnol., 32:205-2 10; von der Osten et al. (1998) Biotechnology Let[ers, 11:11-16; Patent DE 4,120,867;
Liebl (1992) "The Genus Corynebacterium, in: The Procaryotes, Volume 11, Balows, A. et al., eds.
Springer-Verlag). These media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements. Preferred carbon sources are sugars, such as mono-, di-, or polysaccharides. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose serve as very good carbon sources. It is also possible to supply sugar to the media via complex compounds such as molasses or other by-products from sugar refinement. It can also be advantageous to supply mixtures of different carbon sources. Other possible carbon sources are alcohols and organic acids, such as methanol, ethanol, acetic acid or lactic acid. Nitrogen sources are usually organic or inorganic nitrogen compounds, or materials which contain these compounds. Exemplary nitrogen sources include ammonia gas or ammonia salts, such as NH.CI or (NH.)2SO4, NH4OH, nitrates, urea, amino acids or complex nitrogen sources like com steep liquor, soy bean flour, soy bean protein, yeast extract, meat extract and others.
Inorganic salt compounds which may be included in the media include the chloride-, phosphorous- or sulfate- salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Chelating compounds can be added to the medium to keep the metal ions in solution. Particularly useful chelating compounds include dihydroxyphenols, like catechol or protocatechuate, or organic acids, such as citric acid. It is typical for the media to also contain other growth factors, such as vitamins or growth promoters, examples of which include biotin, riboflavin, thiamin, folic acid, nicotinic acid, pantothenate and pyridoxin. Growth factors and salts frequently originate from complex media components such as yeast extract, molasses, corn steep liquor and others. The exact composition of the media compounds depends strongly on the immediate experiment and is individually decided for each specific case.
Information about media optimization is available in the textbook "Applied Microbiol.
Physiology, A
Practical Approach (eds. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). It is also possible to select growth media from commercial suppliers, like standard l(Merck) or BHI (grain heart infusion, DIFCO) or others.
All medium components are sterilized, either by heat (20 minutes at 1.5 bar and 121 C) or by sterile filtration. The components can either be sterilized together or, if necessary, separately. All media components can be present at the beginning of growth, or they can optionally be added continuously or batchwise.
Culture conditions are defined separately for each experiment. The temperature should be in a range between 15'C and 45'C. The temperature can be kept constant or can be altered during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0, and can be maintained by the addition of buffers to the media.
An exemplary buffer for this purpose is a potassium phosphate buffer.
Synthetic buffers such as MOPS, HEPES, ACES and others can alternatively or simultaneously be used. It is also possible to maintain a constant culture pH through the addition of NaOH or NH,OH during growth. If complex medium components such as yeast extract are utilized, the necessity for additional buffers may be reduced, due to the fact that many complex compounds have high buffer capacities. If a fermentor is utilized for culturing the micro-organisms, the pH can also be controlled using gaseous ammonia.
The incubation time is usually in a range from several hours to several days.
This time is selected in order to permit the maximal amount of product to accumulate in the broth. The disclosed growth experiments can be carried out in a variety of vessels, such as microtiter plates, glass tubes, glass flasks or glass or metal fermentors of different sizes.
For screening a large number of clones, the microorganisms should be cultured in microtiter plates, glass tubes or shake flasks, either with or without baffles. Preferably 100 mi shake flasks are used, filled with 10% (by volume) of the required growth medium. The flasks should be shaken on a rotary shaker (amplitude 25 mm) using a speed-range of 100 - 300 rpm. Evaporation losses can be diminished by the maintenance of a humid atmosphere; alternatively, a mathematical correction for evaporation losses should be performed.
If genetically modified clones are tested, an unmodified control clone or a control clone containing the basic plasmid without any insert should also be tested.
The medium is inoculated to an OD6oo of 0.5 - 1.5 using cells grown on agar plates, such as CM plates (10 g/1 glucose, 2,5 g/l NaCI, 2 g/1 urea, 10 g/l polypeptone, 5 g/1 yeast extract, 5 g/l meat extract, 22 g/l NaCI, 2 g/l urea, 10 g/l polypeptone, 5 g/1 yeast extract, 5 g/l meat extract, 22 g/l agar, pH 6.8 with 2M NaOH) that had been incubated at 30 C. Inoculation of the media is accomplished by either introduction of a saline suspension of C.
glutamicum cells from CM plates or addition of a liquid preculture of this bacterium.
Example 8- In vitro Analysis of the Function of Mutant Proteins The determination of activities and kinetic parameters of enzymes is well established in the art. Experiments to determine the activity of any given altered enzyme must be tailored to the specific activity of the wild-type enzyme, which is well within the ability of one of ordinary skill in the art. Overviews about enzymes in general, as well as specific details concerning structure, kinetics, principles, methods, applications and examples for the determination of many enzyme activities may be found, for example, in the following references: Dixon, M., and Webb, E.C., (1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure and Mechanism.
Freeman: New York; Walsh, (1979) Enzymatic Reaction Mechanisms. Freeman: San Francisco; Price, N.C., Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ.
Press: Oxford; Boyer, P.D., ed. (1983) The Enzymes, 3'd ed. Academic Press:
New York; Bisswanger, H., (1994) Enzymkinetik, 2"d ed. VCH: Weinheim (ISBN
3527300325); Bergmeyer, H.U., Bergmeyer, J., Grafil, M., eds. (1983-1986) Methods of Enzymatic Analysis, 3rd ed., vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes". VCH: Weinheim, p.
352-363.
The activity of proteins which bind to DNA can be measured by several well-established methods, such as DNA band-shift assays (also called gel retardation assays).
The effect of such proteins on the expression of other molecules can be measured using reporter gene assays (such as that described in Kolmar, H. et al. (1995) EMBO
J. 14:
3895-3904 and references cited therein). Reporter gene test systems are well known and established for applications in both pro- and eukaryotic cells, using enzymes such as beta-galactosidase, green fluorescent protein, and several others.
The determination of activity of membrane-transport proteins can be performed according to techniques such as those described in Gennis, R.B. (1989) "Pores, Channels and Transporters", in Biomembranes, Molecular Structure and Function, Springer: Heidelberg, p. 85-137; 199-234; and 270-322.
Example 9: Analysis of Impact of Mutant Protein on the Production of the Desired Product The effect of the genetic modification in C. glutamicum on production of a desired compound (such as an amino acid) can be assessed by growing the modified microorganism under suitable conditions (such as those described above) and analyzing the medium and/or the cellular component for increased production of the desired product (i.e., an amino acid). Such analysis techniques are well known to one of ordinary skill in the art, and include spectroscopy, thin layer chromatography, staining methods of various kinds, enzymatic and microbiological methods, and analytical chromatography such as high performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-613, VCH:
Weinheim (1985); Fallon, A. et al., (1987) "Applications of HPLC in Biochemistry" in:
Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et al.
(1993) Biotechnology, vol. 3, Chapter III: "Product recovery and purification", page 469-714, VCH: Weinheim; Belter, P.A. et al. (1988) Bioseparations: downstream processing for biotechnology, John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S.
(1992) Recovery processes for biological materials, John Wiley and Sons;
Shaeiwitz, J.A. and Henry, J.D. (1988) Biochemical separations, in: Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications.) In addition to the measurement of the final product of fermentation, it is.also possible to analyze other components of the metabolic pathways utilized for the production of the desired compound, such as intermediates and side-products, to determine the overall efficiency of production of the compound. Analysis methods include measurements of nutrient levels in the medium (e.g., sugars, hydrocarbons, nitrogen sources, phosphate, and other ions), measurements of biomass composition and growth, analysis of the production of common metabolites of biosynthetic pathways, and measurement of gasses produced during fermentation. Standard methods for these measurements are outlined in Applied Microbial Physiology, A Practical Approach, P.M. Rhodes and P.F. Stanbury, eds., IRL Press, p. 103-129; 131-163; and 165-(ISBN: 0199635773) and references cited therein.
Example 10: Purification of the Desired Product from C. glutamicum Culture Recovery of the desired product from the C. glutamicum cells or supernatant of the above-described culture can be performed by various methods well known in the art.
If the desired product is not secreted from the cells, the cells can be harvested from the culture by low-speed centrifugation, the cells can be lysed by standard techniques, such as mechanical force or sonication. The cellular debris is removed by centrifugation, and the supematant fraction containing the soluble proteins is retained for further purification of the desired compound. If the product is secreted from the C.
glutamicum cells, then the cells are removed from the culture by low-speed centrifugation, and the supemate fraction is retained for further purification.
The supematant fraction from either purification method is subjected to chromatography with a suitable resin, in which the desired molecule is either retained on a chromatography resin while many of the impurities in the sample are not, or where the impurities are retained by the resin while the sample is not. Such chromatography steps may be repeated as necessary, using the same or different chromatography resins. One of ordinary skill in the art would be well-versed in the selection of appropriate chromatography resins and in their most efficacious application for a particular molecule to be purified. The purified product may be concentrated by filtration or ultrafiltration, and stored at a temperature at which the stability of the product is maximized.
There are a wide array of purification methods known to the art and the preceding method of purification is not meant to be limiting. Such purification techniques are described, for example, in Bailey, J.E. & Ollis, D.F.
Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).
The identity and purity of the isolated compounds may be assessed by techniques standard in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic assay, or microbiologically. Such analysis methods are reviewed in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A.
el al.
(1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17.
Example 11: Analysis of the Gene Sequences of the Invention The comparison of sequences and determination of percent homology between two sequences are art-known techniques, and can be accomplished using a mathematical algorithm, such as the algorithm of Karlin and Altschul (1990) Proc. Natl.
Acad Sci.
USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Nall. Acad.
Sci. USA
90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST
programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
BLAST
nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to SMP nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to SMP protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, one of ordinary skill in the art will know how to optimize the parameters of the program (e.g., XBLAST and NBLAST) for the specific sequence being analyzed.
Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Meyers and Miller ((1988) Comput. Appl. Biosci.
4: 11-17). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art, and include ADVANCE and ADAM.
described in Torelli and Robotti (1994) Comput. Appl. 13iosci. 10:3-5; and FASTA, described in Pearson and Lipman (1988) P.N.A.S. 85:2444-8.
The percent homology between two amino acid sequences can also be accomplished using the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2; 3, or 4. The percent homology between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package, using standard parameters, such as a gap weight of 50 and a length weight of 3.
A comparative analysis of the gene sequences of the invention with those present in Genbank has been performed using techniques known in the art (see, e.g., Bexevanis and Ouellette, eds. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins. John Wiley and Sons: New York). The gene sequences of the invention were compared to genes present in Genbank in a three-step process. In a first step, a BLASTN analysis (e.g., a local alignment analysis) was performed for each of the sequences of the invention against the nucleotide sequences present in Genbank, and the top 500 hits were retained for further analysis. A subsequent FASTA search (e.g., a combined local and global alignment analysis, in which limited regions of the sequences are aligned) was performed on these 500 hits. Each gene sequence of the invention was subsequently globally aligned to each of the top three FASTA hits, using the GAP
program in the GCG software package (using standard parameters). In order to obtain correct results, the length of the sequences extracted from Genbank were adjusted to the length of the query sequences by methods well-known in the art. The results of this analysis are set forth in Table 4. The resulting data is identical to that which would have been obtained had a GAP (global) analysis alone been performed on each of the genes of the invention in comparison with each of the references in Genbank, but required significantly reduced computational time as compared to such a database-wide GAP
(global) analysis. Sequences of the invention for which no alignments above the cutoff values were obtained are indicated on Table 4 by the absence of alignment information.
It will further be understood by one of ordinary skill in the art that the GAP
alignment homology percentages set forth in Table 4 under the heading "% homology (GAP)"
are listed in the European numerical format, wherein a',' represents a decimal point. For example, a value of "40,345" in this column represents "40.345%".
Example 12: Construction and Operation of DNA Microarrays The sequences of the invention may additionally be used in the construction and application of DNA microarrays (the design, methodology, and uses of DNA
arrays are well known in the art, and are described, for example, in Schena, M. et al.
(1995) Science 270: 467-470; Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-1367;
DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48; and DeRisi, J.L.
et al.
(1997) Science 278: 680-686).
DNA microarrays are solid or flexible supports consisting of nitrocellulose, nylon, glass, silicone, or other materials. Nucleic acid molecules may be attached to the surface in an ordered manner. After appropriate labeling, other nucleic acids or nucleic acid mixtures can be hybridized to the immobilized nucleic acid molecules, and the label may be used to monitor and measure the individual signal intensities of the hybridized molecules at defined regions. This methodology allows the simultaneous quantification of the relative or-absolute amount of all or selected nucleic acids in the applied nucleic acid sample or mixture. DNA microarrays, therefore, permit an analysis of the expression of multiple (as many as 6800 or more) nucleic acids in parallel (see, e.g., Schena, M. (1996) BioEssays 18(5): 427-431).
The sequences of the invention may be used to design oligonucleotide primers which are able to amplify defined regions of one or more C. glutamicum genes by a nucleic acid amplification reaction such as the polymerase chain reaction. The choice and design of the 5' or 3' oligonucleotide primers or of appropriate linkers allows the covalent attachment of the resulting PCR products to the surface of a support medium described above (and also described, for example, Schena, M. et al. (1995) Science 270:
467-470).
Nucleic acid microarrays may also be constructed by in situ oligonucleotide synthesis as described by Wodicka, L. et al. (1997) Nature Biotechnology 15:
1367. By photolithographic methods, precisely defined regions of the matrix are exposed to light. Protective groups which are photolabile are thereby activated and undergo nucleotide addition, whereas regions that are masked from light do not undergo any modification. Subsequent cycles of protection and light activation permit the synthesis of different oligonucleotides at defined positions. Small, defined regions of the genes of the invention may be synthesized on microarrays by solid phase oligonucleotide synthesis.
The nucleic acid molecules of the invention present in a sample or mixture of nucleotides may be hybridized to the microarrays. These nucleic acid molecules can be labeled according to standard methods. In brief, nucleic acid molecules (e.g., mRNA
molecules or DNA molecules) are labeled by the incorporation of isotopically or fluorescently labeled nucleotides, e.g., during reverse transcription or DNA
synthesis.
Hybridization of labeled nucleic acids to microarrays is described (e.g., in Schena, M. et al. (1995) supra; Wodicka, L. et al. (1997), supra; and DeSaizieu A. et al.
(1998), supra). The detection and quantification of the hybridized molecule are tailored to the specific incorporated label. Radioactive labels can be detected, for example, as described in Schena, M. et al. (1995) supra) and fluorescent labels may be detected, for example, by the method of Shalon et al. (1996) Genome Research 6: 639-645).
The application of the sequences of the invention to DNA microarray technology, as described above, permits comparative analyses of different strains of C.
glutamicum or other Corynebacteria. For example, studies of inter-strain variations based on individual transcript profiles and the identification of genes that are important for specific and/or desired strain properties such as pathogenicity, productivity and stress tolerance are facilitated by nucleic acid array methodologies. Also, comparisons of the profile of expression of genes of the invention during the course of a fermentation reaction are possible using nucleic acid array technology.
Example 13: Analysis of the Dynamics of Cellular Protein Populations (Proteomics) The genes, compositions, and methods of the invention may be applied to study the interactions and dynamics of populations of proteins, termed 'proteomics'.
Protein populations of interest include, but are not limited to, the total protein population of C.
glutamicum (e.g., in comparison with the protein populations of other organisms), those proteins which are active under specific environmental or metabolic conditions (e.g., during fermentation, at high or low temperature, or at high or low pH), or those proteins which are active during specific phases of growth and development.
Protein populations can be analyzed by various well-known techniques, such as gel electrophoresis. Cellular proteins may be obtained, for example, by lysis or extraction, and may be separated from one another using a variety of electrophoretic techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins largely on the basis of their molecular weight. Isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) separates proteins by their isoelectric point (which reflects not only the amino acid sequence but also posttranslational modifications of the protein). Another, more preferred method of protein analysis is the consecutive combination of both IEF-PAGE and SDS-PAGE, known as 2-D-gel electrophoresis (described, for example, in Hermann et al. (1998) Electrophoresis 19:
3217-3221; Fountoulakis et al. (1998) Electrophoresis 19: 1193-1202; Langen et al.
(1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997) Electrophoresis 18 :
1451-1463). Other separation techniques may also be utilized for protein separation, such as capillary gel electrophoresis; such techniques are well known in the art.
Proteins separated by these methodologies can be visualized by standard techniques, such as by staining or labeling. Suitable stains are known in the art, and include Coomassie Brilliant Blue, silver stain, or fluorescent dyes such as Sypro Ruby (Molecular Probes). The inclusion of radioactively labeled amino acids or other protein precursors (e.g., 35S-methionine, 35 S-cysteine, "C-labelled amino acids, 15N-amino acids, 15NO3 or 'SNH4+ or 13C-labelled amino acids) in the medium of C.
glutamicum permits the labeling of proteins from these cells prior to their separation.
Similarly, fluorescent labels may be employed. These labeled proteins can be extracted, isolated and separated according to the previously described techniques.
Proteins visualized by these techniques can be further analyzed by measuring the amount of dye or label used. The amount of a given protein can be determined quantitatively using, for example, optical methods and can be compared to the amount of other proteins in the same gel or in other gels. Comparisons of proteins on gels can be made, for example, by optical comparison, by spectroscopy, by image scanning and analysis of gels, or through the use of photographic films and screens. Such techniques are well-known in the art.
To determine the identity of any given protein, direct sequencing or other standard techniques may be employed. For example, N- and/or C-terminal amino acid sequencing (such as Edman degradation) may be used, as may mass spectrometry (in particular MALDI or ESI techniques (see, e.g., Langen et al. (1997) Electraphoresis 18:
1184-1192)). The protein sequences provided herein can be used for the identification of C. glulamicum proteins by these techniques.
The information obtained by these methods can be used to compare pattems of protein presence, activity, or modification between different samples from various biological conditions (e.g., different organisms, time points of fermentation, media conditions, or different biotopes, among others). Data obtained from such experiments alone, or in combination with other techniques, can be used for various applications, such as to compare the behavior of various organisms in a given (e.g., metabolic) situation, to increase the productivity of strains which produce fine chemicals or to increase the efficiency of the production of fine chemicals.
Equivalents Those of ordinary skill in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
~g ~ gI
~ . z z 2'~
a a rn w z Z a w w ~
p~ p- G z ~ ~ w ~ J Z ~~ a~ 4~ g t~ c oz o_ ~n0 g ~ O ~~ aU ~ w v1 v> ~
G1 ~ Z a' is a az ~~ ca d' a r Z rn E sa $~ E
~ W
O C7 V O (9 C7 ~ Z ~ E rd ~ w w ~
;M ~L~ ~(~ V c i c1 a ~ m n ~
c W w v ~ --~ E
O Y O
V N N N G C1 W v> y O
Ui (~ j ~
mo6 i i-~
r~ ~'~ b r rv wq C~ o'a c'Q
E cS : E b: 8 ' 0 u, ~õYl ~ V N N fl r UO
z c O $ rn C U a o $ a o ~G 8 tf c gE ~ V
g,a.$ w~
a n a,wrn~ .i~ EkW 2 mg ar ~ov --$~ a g t rna~"~ jm ~ ~ ~Y ya 1~ ac.io~g = Li ~~~En$~c ~ 9>> c~ a1E ='g E ~e$ ~yfSyE
n= E
'8 u is E Q c ~ m ~ c~ a ~g c W a W
0 w w o~e ~" ~
w w r o En L E~ E y pd a . 1~ ~ Ã~ v o ~ a c dS & ~ ~
b ~' ~'~ a ? o o~o '~i 8 c $ ~ ~$ ~,c 0 ~g"'=r--~
~ ' o~~ c~'r a~o~~~gw g~ ~ ~'"$ a a ~ ~ =~U =a a~~ 2Ln0 ~Np.~. g qqq~ C" ~ ~ N ~ n Y ~ =r ~ P,~ ~ ~ ~pp ~
Y.- ~ ~ ~ N Ny ,t r= ~ 4 Q Q Q
!1'~ ' ~ ~ ~{'~Q ~ p~=j ~ N N N
Y 1'7 M ~d Y O! ' ~V
~ _ _ W
N ry ~ ~ ~ ~ W v o S ~ ~
_l $ 11 N ~ Q~ ~ ' ~ C
N
ts v a v cn o 4 v~i r~i cn M
Ja ~
co c n V
to c:
~ o V/ q I F
n O ~
S I ~ O m p N d N = c Z Z g ~ W y ~
U E W
U 0 Z p w w x v a wr- E w > > o az ~' ~
C~ CJ C' ~' _ g H vwi v v' Z Z$ ~ ' o E Z
4m U O d ~ U D E ~ a a C
C Z Z g o~ ~~ o c Y o ~
0~ umi a = L$ c 4 =- ~ 'o V J W N y r 7 G=4 a yj 0:
U o~_i E a c S m LL ~ ~ E N o m~' ' E~. ZC~S' ~v3Y3 x x o c E$ K=- E in LL' vi 4 c E~ Ey c cti 3 g E $ c ~
0 ~p q O y y ~. - ._ v C j E
~ c 8 E W~~~.E 3 ~ o- y '0 ~ v ~ g+ ~i L _$ E Q 'f_ E~
= ?i Q ~
O o c '~ g L t0~Wo 3S s C '~~ 2 v~ i an ! y tc w o y y~/1 { E
, oy ~N y c cwcQ
a~"mayQ ~ o z ~v y 8 4t~.4 y 0i r f~ n N n ~ W~ C$ C Vi 0 OWi p a o~ E E v0 i õO ~ v~ Ii 8 0~ o E
ax aoo~~ N~= m ? 9 4 i M ~Np O~~pp ip p~
N
p Y p dp ~p ~ ~Q ~Q ~0 S O p~j O ~ ~ {h O ~ S Op Q ~ N Q Q Q x Q Q~ Q
~ ~ ~ n ~ N ~ N a NP n Of O ~ ~
Y Y cn ~ Y Y N
'n (n fn M U Z
y~~ N 8 ~ rs a ~S w w ~ a~ s~ a (L m m m~ m~
C~ c7 c9 C~ c9 c~ ~ ~ ~ m co C~
cl) M ~ O N
N O O tf O ~
~ ~ ~ ~
Exempliiication Example 1: Preparation of total genomic DNA of Corynebacteriumglutamicum A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30 C with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supematant was discarded and the cells were resuspended in 5 ml buffer-I (5% of the original volume of the culture - all indicated volumes have been calculated for 100 ml of culture volume). Composition of buffer-I: 140.34 g/l sucrose, 2.46 g/l MgSO4 x 7H2O, 10 ml/I. KH2PO4 solution (100 g/l, adjusted to pH 6.7 with KOH), 50 ml/I. M12 concentrate (10 g/1(NH.)2S0., I g/1 NaCI, 2 g/l MgSO, x 7H2O, 0.2 g/l CaCI2, 0.5 g/1 yeast extract (Difco), 10 ml/1 trace-elements-mix (200 mg/l FeSO4 x H2O, 10 mg/1 ZnSO4 x 7 H2O, 3 mg/1 MnCI2 x 4 H2O, 30 mg/1 H,BO3 20 mg/1 CoC12 x 6 H2O, 1 mg/l NiCl2 x 6 HZO, 3 mg/1 Na2MoO4 x 2 H1O, 500 mg/I complexing agent (EDTA or critic acid), 100 ml/I vitamins-mix (0.2 mg/I biotin, 0.2 mg/1 folic acid, 20 mg/1 p-amino benzoic acid, 20 mg/1 riboflavin, 40 mg/I ca-panthothenate, 140 mg/1 nicotinic acid, 40 mg/1 pyridoxole hydrochloride, 200 mg/1 myo-inositol).
Lysozyme was added to the suspension to a final concentration of 2.5 mg/ml. After an approximately 4 h incubation at 37 C, the cell wall was degraded and the resulting protoplasts are harvested by centrifugation. The pellet was washed once with 5 ml buffer-I and once with 5 ml TE-buffer (10 mM Tris-HCl,1 mM EDTA, pH 8). The pellet was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution (10%) and 0.5 ml NaCI solution (5 M) are added. After adding of proteinase K to a final concentration of 200 g/ml, the suspension is incubated for ca.18 h at 37 C. The DNA was purified by extraction with phenol, phenol-chloroform-isoamylalcohol and chloroform-isoamytalcohol using standard procedures. Then, the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30 min incubation at -20 C and a 30 min centrifugation at 12,000 rpm in a high speed centrifuge using a SS34 rotor (Sorvall). The DNA was dissolved in I ml TE-buffer containing 20 g/ml RNaseA and dialysed at 4 C against 1000 ml TE-buffer for at least 3 hours.
During this time, the buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysed DNA solution, 0.4 ml of 2 M LiCI and 0.8 ml of ethanol are added.
After a 30 min incubation at -20 C, the DNA was collected by centrifugation (13,000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TE-buffer.
DNA
prepared by this procedure could be used for all purposes, including southem blotting or construction of genomic libraries.
Example 2: Construction of genomic libraries in Escherichia coli of Corynebacterium glutamicum ATCC13032.
Using DNA prepared as described in Example 1, cosmid and plasmid libraries were constructed according to known and well established methods (see e.g., Sambrook, J. et al.
(1989) "Molecular Cloning : A Laboratory Manual", Cold Spring Harbor Laboratory Press, or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biology", John Wiley &
Sons.) Any plasmid or cosmid could be used. Of particular use were the plasmids pBR322 (Sutcliffe, J.G. (1979) Proc. Natl. Acad. Sci. USA, 75:3737-3741); pACYC177 (Change &
Cohen (1978) J. Bacteriol 134:1141-1156), plasmids of the pBS series (pBSSK+, pBSSK- and others; Stratagene, LaJolla, USA), or cosmids as SuperCosl (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J., Rosenthal A. and Waterson, R.H. (1987) Gene 53:283-286.
Gene libraries specifically for use in C. glutamicum may be constructed using plasmid pSL 109 (Lee, H.-S. and A. J. Sinskey (1994)J. Microbiol. Biotechnol. 4: 256-263).
Example 3: DNA Sequencing and Computational Functional Analysis Genomic libraries as described in Example 2 were used for DNA sequencing according to standard methods, in particular by the chain termination method using ABI377 sequencing machines (see e.g., Fleischman, R.D. el al. (1995) "Whole-genome Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science, 269:496-512). Sequencing primers with the following nucleotide sequences were used: 5'-GGAAACAGTATGACCATG-3' (SEQ ID NO:783) or 5'-GTAAAACGACGGCCAGT-3' (SEQ ID
NO:784).
Example 4: In vivo Mutagenesis In vivo mutagenesis of Corynebacterium glutamicum can be performed by passage of plasmid (or other vector) DNA through E. coli or other microorganisms (e.g.
Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are impaired in their capabilities to maintain the integrity of their genetic information. Typical mutator strains have mutations in the genes for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see Rupp, W.D.
(1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-2294, ASM:
Washington.) Such strains are well known to those of ordinary skill in the art. The use of such strains is illustrated, for example, in Greener, A. and Callahan, M. (1994) Strategies 7: 32-34.
Example 5: DNA Transfer Between Escherichia coli and Corynebacteriunt glutamicum Several Corynebacterium and Brevibacterium species contain endogenous plasmids (as e.g., pHM1519 or pBLI) which replicate autonomously (for review see, e.g., Martin, J.F. et al. (1987) Biotechnology, 5:137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can be readily constructed by using standard vectors for E. coli (Sambrook, J. et al. (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biology", John Wiley & Sons) to which a origin or replication for and a suitable marker from Corynebacierium glulamicum is added. Such origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibacterium species. Of particular use as transformation markers for these species are genes for kanamycin resistance (such as those derived from the Tn5 or Tn903 transposons) or chloramphenicol (Winnacker, E.L. (1987) "From Genes to Clones -Introduction to Gene Technology, VCH, Weinheim). There are numerous examples in the literature of the construction of a wide variety of shuttle vectors which replicate in both E.
coli and C. glutamieum, and which can be used for several purposes, including gene over-expression (for reference, see e.g., Yoshihama, M. el al. (1985) J. Bacteriol.
162:591-597, Martin J.F. et al. (1987) Biotechnology, 5:137-146 and Eikmanns, B.J. et al.
(1991) Gene, 102:93-98).
Using standard methods, it is possible to, clone a gene of interest into one of the shuttle vectors described above and to introduce such a hybrid vectors into strains of Corynebacterium glutamicum. Transfonmation of C. glutamicum can be achieved by protoplast transformation (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-311), electroporation (Liebl, E. et al. (1989) FEMS Microbiol. Letters, 53:399-303) and in cases where special vectors are used, also by conjugation (as described e.g. in Schtifer, A et al.
(1990) J. Bacteriol. 172:1663-1666). It is also possible to transfer the shuttle vectors for C. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum (using standard methods well-known in the art) and transforming it into E. coli. This transformation step can be performed using standard methods, but it is advantageous to use an Mcr-deficient E. coli strain, such as NM522 (Gough & Murray (1983) J. Mol. Biol. 166:1-19).
Genes may be overexpressed in C. glutamicum strains using plasmids which comprise pCGI (U.S. Patent No. 4,617,267) or fragments thereof, and optionally the gene for kanamycin resistance from TN903 (Grindley, N.D. and Joyce, C.M.
(1980) Proc. Natl. Acad. Sci. USA 77(12): 7176-7180). In addition, genes may be overexpressed in C. glutamicum strains using plasmid pSL109 (Lee, H.-S. and A.
J.
Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).
Aside from the use of replicative plasmids, gene overexpression can also be achieved by integration into the genome. Genomic integration in C. glutamicum or other Corynebacterium or Brevibacterium species may be accomplished by well-known methods, such as homologous recombination with genomic region(s), restriction endonuclease mediated integration (REMI) (see, e.g., DE Patent 19823834), or through the use of transposons. It is also possible to modulate the activity of a gene of interest by modifying the regulatory regions (e.g., a promoter, a repressor, and/or an enhancer) by sequence modification, insertion, or deletion using site-directed methods (such as homologous recombination) or methods based on random events (such as transposon mutagenesis or REMI). Nucleic acid sequences which function as transcriptional terminators may also be inserted 3' to the coding region of one or more genes of the invention; such terminators are well-known in the art and are described, for example, in Winnacker, E.L. (1987) From Genes to Clones - Introduction to Gene Technology.
VCH:
Weinheim.
Example 6: Assessment of the Expression of the Mutant Protein Observations of the activity of a mutated protein in a transformed host cell rely on the fact that the mutant protein is expressed in a similar fashion and in a similar quantity to that of the wild-type protein. A useful method to ascertain the level of transcription of the mutant gene (an indicator of the amount of mRNA available for translation to the gene product) is to perform a Northern blot (for reference see, for example, Ausubel et al.
(1988) Current Protocols in Molecular Biology, Wiley: New York), in which a primer designed to bind to the gene of interest is labeled with a detectable tag (usually radioactive or chemiluminescent), such that when the total RNA of a culture of the organism is extracted, run on gel, transferred to a stable matrix and incubated with this probe, the binding and quantity of binding of the probe indicates the presence and also the quantity of mRNA for this gene. This information is evidence of the degree of transcription of the mutant gene. Total cellular RNA can be prepared from Corynebacterium glutamicum by several methods, all well-known in the art, such as that described in Bormann, E.R. et al.
(1992) Mol. Microbrol. 6: 317-326.
To assess the presence or relative quantity of protein translated from this mRNA, standard techniques, such as a Western blot, may be employed (see, for example, Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York). In this process, total cellular proteins are extracted, separated by gel electrophoresis, transferred to a matrix such as nitrocellulose, and incubated with a probe, such as an antibody, which specifically binds to the desired protein. This probe is generally tagged with a chemiluminescent or colorimetric label which may be readily detected. The presence and quantity of label observed indicates the presence and quantity of the desired mutant protein present in the cell.
Example 7: Growth of Genetically Modified Corynebacterium glutamicum - Media and Culture Conditions Genetically modified Corynebacteria are cultured in synthetic or natural growth media. A number of different growth media for Corynebacteria are both well-known and readily available (Lieb et a!. (1989) Appl. Microbiol. Biotechnol., 32:205-2 10; von der Osten et al. (1998) Biotechnology Let[ers, 11:11-16; Patent DE 4,120,867;
Liebl (1992) "The Genus Corynebacterium, in: The Procaryotes, Volume 11, Balows, A. et al., eds.
Springer-Verlag). These media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements. Preferred carbon sources are sugars, such as mono-, di-, or polysaccharides. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose serve as very good carbon sources. It is also possible to supply sugar to the media via complex compounds such as molasses or other by-products from sugar refinement. It can also be advantageous to supply mixtures of different carbon sources. Other possible carbon sources are alcohols and organic acids, such as methanol, ethanol, acetic acid or lactic acid. Nitrogen sources are usually organic or inorganic nitrogen compounds, or materials which contain these compounds. Exemplary nitrogen sources include ammonia gas or ammonia salts, such as NH.CI or (NH.)2SO4, NH4OH, nitrates, urea, amino acids or complex nitrogen sources like com steep liquor, soy bean flour, soy bean protein, yeast extract, meat extract and others.
Inorganic salt compounds which may be included in the media include the chloride-, phosphorous- or sulfate- salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Chelating compounds can be added to the medium to keep the metal ions in solution. Particularly useful chelating compounds include dihydroxyphenols, like catechol or protocatechuate, or organic acids, such as citric acid. It is typical for the media to also contain other growth factors, such as vitamins or growth promoters, examples of which include biotin, riboflavin, thiamin, folic acid, nicotinic acid, pantothenate and pyridoxin. Growth factors and salts frequently originate from complex media components such as yeast extract, molasses, corn steep liquor and others. The exact composition of the media compounds depends strongly on the immediate experiment and is individually decided for each specific case.
Information about media optimization is available in the textbook "Applied Microbiol.
Physiology, A
Practical Approach (eds. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). It is also possible to select growth media from commercial suppliers, like standard l(Merck) or BHI (grain heart infusion, DIFCO) or others.
All medium components are sterilized, either by heat (20 minutes at 1.5 bar and 121 C) or by sterile filtration. The components can either be sterilized together or, if necessary, separately. All media components can be present at the beginning of growth, or they can optionally be added continuously or batchwise.
Culture conditions are defined separately for each experiment. The temperature should be in a range between 15'C and 45'C. The temperature can be kept constant or can be altered during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0, and can be maintained by the addition of buffers to the media.
An exemplary buffer for this purpose is a potassium phosphate buffer.
Synthetic buffers such as MOPS, HEPES, ACES and others can alternatively or simultaneously be used. It is also possible to maintain a constant culture pH through the addition of NaOH or NH,OH during growth. If complex medium components such as yeast extract are utilized, the necessity for additional buffers may be reduced, due to the fact that many complex compounds have high buffer capacities. If a fermentor is utilized for culturing the micro-organisms, the pH can also be controlled using gaseous ammonia.
The incubation time is usually in a range from several hours to several days.
This time is selected in order to permit the maximal amount of product to accumulate in the broth. The disclosed growth experiments can be carried out in a variety of vessels, such as microtiter plates, glass tubes, glass flasks or glass or metal fermentors of different sizes.
For screening a large number of clones, the microorganisms should be cultured in microtiter plates, glass tubes or shake flasks, either with or without baffles. Preferably 100 mi shake flasks are used, filled with 10% (by volume) of the required growth medium. The flasks should be shaken on a rotary shaker (amplitude 25 mm) using a speed-range of 100 - 300 rpm. Evaporation losses can be diminished by the maintenance of a humid atmosphere; alternatively, a mathematical correction for evaporation losses should be performed.
If genetically modified clones are tested, an unmodified control clone or a control clone containing the basic plasmid without any insert should also be tested.
The medium is inoculated to an OD6oo of 0.5 - 1.5 using cells grown on agar plates, such as CM plates (10 g/1 glucose, 2,5 g/l NaCI, 2 g/1 urea, 10 g/l polypeptone, 5 g/1 yeast extract, 5 g/l meat extract, 22 g/l NaCI, 2 g/l urea, 10 g/l polypeptone, 5 g/1 yeast extract, 5 g/l meat extract, 22 g/l agar, pH 6.8 with 2M NaOH) that had been incubated at 30 C. Inoculation of the media is accomplished by either introduction of a saline suspension of C.
glutamicum cells from CM plates or addition of a liquid preculture of this bacterium.
Example 8- In vitro Analysis of the Function of Mutant Proteins The determination of activities and kinetic parameters of enzymes is well established in the art. Experiments to determine the activity of any given altered enzyme must be tailored to the specific activity of the wild-type enzyme, which is well within the ability of one of ordinary skill in the art. Overviews about enzymes in general, as well as specific details concerning structure, kinetics, principles, methods, applications and examples for the determination of many enzyme activities may be found, for example, in the following references: Dixon, M., and Webb, E.C., (1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure and Mechanism.
Freeman: New York; Walsh, (1979) Enzymatic Reaction Mechanisms. Freeman: San Francisco; Price, N.C., Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ.
Press: Oxford; Boyer, P.D., ed. (1983) The Enzymes, 3'd ed. Academic Press:
New York; Bisswanger, H., (1994) Enzymkinetik, 2"d ed. VCH: Weinheim (ISBN
3527300325); Bergmeyer, H.U., Bergmeyer, J., Grafil, M., eds. (1983-1986) Methods of Enzymatic Analysis, 3rd ed., vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, "Enzymes". VCH: Weinheim, p.
352-363.
The activity of proteins which bind to DNA can be measured by several well-established methods, such as DNA band-shift assays (also called gel retardation assays).
The effect of such proteins on the expression of other molecules can be measured using reporter gene assays (such as that described in Kolmar, H. et al. (1995) EMBO
J. 14:
3895-3904 and references cited therein). Reporter gene test systems are well known and established for applications in both pro- and eukaryotic cells, using enzymes such as beta-galactosidase, green fluorescent protein, and several others.
The determination of activity of membrane-transport proteins can be performed according to techniques such as those described in Gennis, R.B. (1989) "Pores, Channels and Transporters", in Biomembranes, Molecular Structure and Function, Springer: Heidelberg, p. 85-137; 199-234; and 270-322.
Example 9: Analysis of Impact of Mutant Protein on the Production of the Desired Product The effect of the genetic modification in C. glutamicum on production of a desired compound (such as an amino acid) can be assessed by growing the modified microorganism under suitable conditions (such as those described above) and analyzing the medium and/or the cellular component for increased production of the desired product (i.e., an amino acid). Such analysis techniques are well known to one of ordinary skill in the art, and include spectroscopy, thin layer chromatography, staining methods of various kinds, enzymatic and microbiological methods, and analytical chromatography such as high performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-613, VCH:
Weinheim (1985); Fallon, A. et al., (1987) "Applications of HPLC in Biochemistry" in:
Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et al.
(1993) Biotechnology, vol. 3, Chapter III: "Product recovery and purification", page 469-714, VCH: Weinheim; Belter, P.A. et al. (1988) Bioseparations: downstream processing for biotechnology, John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S.
(1992) Recovery processes for biological materials, John Wiley and Sons;
Shaeiwitz, J.A. and Henry, J.D. (1988) Biochemical separations, in: Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications.) In addition to the measurement of the final product of fermentation, it is.also possible to analyze other components of the metabolic pathways utilized for the production of the desired compound, such as intermediates and side-products, to determine the overall efficiency of production of the compound. Analysis methods include measurements of nutrient levels in the medium (e.g., sugars, hydrocarbons, nitrogen sources, phosphate, and other ions), measurements of biomass composition and growth, analysis of the production of common metabolites of biosynthetic pathways, and measurement of gasses produced during fermentation. Standard methods for these measurements are outlined in Applied Microbial Physiology, A Practical Approach, P.M. Rhodes and P.F. Stanbury, eds., IRL Press, p. 103-129; 131-163; and 165-(ISBN: 0199635773) and references cited therein.
Example 10: Purification of the Desired Product from C. glutamicum Culture Recovery of the desired product from the C. glutamicum cells or supernatant of the above-described culture can be performed by various methods well known in the art.
If the desired product is not secreted from the cells, the cells can be harvested from the culture by low-speed centrifugation, the cells can be lysed by standard techniques, such as mechanical force or sonication. The cellular debris is removed by centrifugation, and the supematant fraction containing the soluble proteins is retained for further purification of the desired compound. If the product is secreted from the C.
glutamicum cells, then the cells are removed from the culture by low-speed centrifugation, and the supemate fraction is retained for further purification.
The supematant fraction from either purification method is subjected to chromatography with a suitable resin, in which the desired molecule is either retained on a chromatography resin while many of the impurities in the sample are not, or where the impurities are retained by the resin while the sample is not. Such chromatography steps may be repeated as necessary, using the same or different chromatography resins. One of ordinary skill in the art would be well-versed in the selection of appropriate chromatography resins and in their most efficacious application for a particular molecule to be purified. The purified product may be concentrated by filtration or ultrafiltration, and stored at a temperature at which the stability of the product is maximized.
There are a wide array of purification methods known to the art and the preceding method of purification is not meant to be limiting. Such purification techniques are described, for example, in Bailey, J.E. & Ollis, D.F.
Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).
The identity and purity of the isolated compounds may be assessed by techniques standard in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic assay, or microbiologically. Such analysis methods are reviewed in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A.
el al.
(1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17.
Example 11: Analysis of the Gene Sequences of the Invention The comparison of sequences and determination of percent homology between two sequences are art-known techniques, and can be accomplished using a mathematical algorithm, such as the algorithm of Karlin and Altschul (1990) Proc. Natl.
Acad Sci.
USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Nall. Acad.
Sci. USA
90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST
programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
BLAST
nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to SMP nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to SMP protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, one of ordinary skill in the art will know how to optimize the parameters of the program (e.g., XBLAST and NBLAST) for the specific sequence being analyzed.
Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Meyers and Miller ((1988) Comput. Appl. Biosci.
4: 11-17). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art, and include ADVANCE and ADAM.
described in Torelli and Robotti (1994) Comput. Appl. 13iosci. 10:3-5; and FASTA, described in Pearson and Lipman (1988) P.N.A.S. 85:2444-8.
The percent homology between two amino acid sequences can also be accomplished using the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2; 3, or 4. The percent homology between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package, using standard parameters, such as a gap weight of 50 and a length weight of 3.
A comparative analysis of the gene sequences of the invention with those present in Genbank has been performed using techniques known in the art (see, e.g., Bexevanis and Ouellette, eds. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins. John Wiley and Sons: New York). The gene sequences of the invention were compared to genes present in Genbank in a three-step process. In a first step, a BLASTN analysis (e.g., a local alignment analysis) was performed for each of the sequences of the invention against the nucleotide sequences present in Genbank, and the top 500 hits were retained for further analysis. A subsequent FASTA search (e.g., a combined local and global alignment analysis, in which limited regions of the sequences are aligned) was performed on these 500 hits. Each gene sequence of the invention was subsequently globally aligned to each of the top three FASTA hits, using the GAP
program in the GCG software package (using standard parameters). In order to obtain correct results, the length of the sequences extracted from Genbank were adjusted to the length of the query sequences by methods well-known in the art. The results of this analysis are set forth in Table 4. The resulting data is identical to that which would have been obtained had a GAP (global) analysis alone been performed on each of the genes of the invention in comparison with each of the references in Genbank, but required significantly reduced computational time as compared to such a database-wide GAP
(global) analysis. Sequences of the invention for which no alignments above the cutoff values were obtained are indicated on Table 4 by the absence of alignment information.
It will further be understood by one of ordinary skill in the art that the GAP
alignment homology percentages set forth in Table 4 under the heading "% homology (GAP)"
are listed in the European numerical format, wherein a',' represents a decimal point. For example, a value of "40,345" in this column represents "40.345%".
Example 12: Construction and Operation of DNA Microarrays The sequences of the invention may additionally be used in the construction and application of DNA microarrays (the design, methodology, and uses of DNA
arrays are well known in the art, and are described, for example, in Schena, M. et al.
(1995) Science 270: 467-470; Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-1367;
DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48; and DeRisi, J.L.
et al.
(1997) Science 278: 680-686).
DNA microarrays are solid or flexible supports consisting of nitrocellulose, nylon, glass, silicone, or other materials. Nucleic acid molecules may be attached to the surface in an ordered manner. After appropriate labeling, other nucleic acids or nucleic acid mixtures can be hybridized to the immobilized nucleic acid molecules, and the label may be used to monitor and measure the individual signal intensities of the hybridized molecules at defined regions. This methodology allows the simultaneous quantification of the relative or-absolute amount of all or selected nucleic acids in the applied nucleic acid sample or mixture. DNA microarrays, therefore, permit an analysis of the expression of multiple (as many as 6800 or more) nucleic acids in parallel (see, e.g., Schena, M. (1996) BioEssays 18(5): 427-431).
The sequences of the invention may be used to design oligonucleotide primers which are able to amplify defined regions of one or more C. glutamicum genes by a nucleic acid amplification reaction such as the polymerase chain reaction. The choice and design of the 5' or 3' oligonucleotide primers or of appropriate linkers allows the covalent attachment of the resulting PCR products to the surface of a support medium described above (and also described, for example, Schena, M. et al. (1995) Science 270:
467-470).
Nucleic acid microarrays may also be constructed by in situ oligonucleotide synthesis as described by Wodicka, L. et al. (1997) Nature Biotechnology 15:
1367. By photolithographic methods, precisely defined regions of the matrix are exposed to light. Protective groups which are photolabile are thereby activated and undergo nucleotide addition, whereas regions that are masked from light do not undergo any modification. Subsequent cycles of protection and light activation permit the synthesis of different oligonucleotides at defined positions. Small, defined regions of the genes of the invention may be synthesized on microarrays by solid phase oligonucleotide synthesis.
The nucleic acid molecules of the invention present in a sample or mixture of nucleotides may be hybridized to the microarrays. These nucleic acid molecules can be labeled according to standard methods. In brief, nucleic acid molecules (e.g., mRNA
molecules or DNA molecules) are labeled by the incorporation of isotopically or fluorescently labeled nucleotides, e.g., during reverse transcription or DNA
synthesis.
Hybridization of labeled nucleic acids to microarrays is described (e.g., in Schena, M. et al. (1995) supra; Wodicka, L. et al. (1997), supra; and DeSaizieu A. et al.
(1998), supra). The detection and quantification of the hybridized molecule are tailored to the specific incorporated label. Radioactive labels can be detected, for example, as described in Schena, M. et al. (1995) supra) and fluorescent labels may be detected, for example, by the method of Shalon et al. (1996) Genome Research 6: 639-645).
The application of the sequences of the invention to DNA microarray technology, as described above, permits comparative analyses of different strains of C.
glutamicum or other Corynebacteria. For example, studies of inter-strain variations based on individual transcript profiles and the identification of genes that are important for specific and/or desired strain properties such as pathogenicity, productivity and stress tolerance are facilitated by nucleic acid array methodologies. Also, comparisons of the profile of expression of genes of the invention during the course of a fermentation reaction are possible using nucleic acid array technology.
Example 13: Analysis of the Dynamics of Cellular Protein Populations (Proteomics) The genes, compositions, and methods of the invention may be applied to study the interactions and dynamics of populations of proteins, termed 'proteomics'.
Protein populations of interest include, but are not limited to, the total protein population of C.
glutamicum (e.g., in comparison with the protein populations of other organisms), those proteins which are active under specific environmental or metabolic conditions (e.g., during fermentation, at high or low temperature, or at high or low pH), or those proteins which are active during specific phases of growth and development.
Protein populations can be analyzed by various well-known techniques, such as gel electrophoresis. Cellular proteins may be obtained, for example, by lysis or extraction, and may be separated from one another using a variety of electrophoretic techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins largely on the basis of their molecular weight. Isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) separates proteins by their isoelectric point (which reflects not only the amino acid sequence but also posttranslational modifications of the protein). Another, more preferred method of protein analysis is the consecutive combination of both IEF-PAGE and SDS-PAGE, known as 2-D-gel electrophoresis (described, for example, in Hermann et al. (1998) Electrophoresis 19:
3217-3221; Fountoulakis et al. (1998) Electrophoresis 19: 1193-1202; Langen et al.
(1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997) Electrophoresis 18 :
1451-1463). Other separation techniques may also be utilized for protein separation, such as capillary gel electrophoresis; such techniques are well known in the art.
Proteins separated by these methodologies can be visualized by standard techniques, such as by staining or labeling. Suitable stains are known in the art, and include Coomassie Brilliant Blue, silver stain, or fluorescent dyes such as Sypro Ruby (Molecular Probes). The inclusion of radioactively labeled amino acids or other protein precursors (e.g., 35S-methionine, 35 S-cysteine, "C-labelled amino acids, 15N-amino acids, 15NO3 or 'SNH4+ or 13C-labelled amino acids) in the medium of C.
glutamicum permits the labeling of proteins from these cells prior to their separation.
Similarly, fluorescent labels may be employed. These labeled proteins can be extracted, isolated and separated according to the previously described techniques.
Proteins visualized by these techniques can be further analyzed by measuring the amount of dye or label used. The amount of a given protein can be determined quantitatively using, for example, optical methods and can be compared to the amount of other proteins in the same gel or in other gels. Comparisons of proteins on gels can be made, for example, by optical comparison, by spectroscopy, by image scanning and analysis of gels, or through the use of photographic films and screens. Such techniques are well-known in the art.
To determine the identity of any given protein, direct sequencing or other standard techniques may be employed. For example, N- and/or C-terminal amino acid sequencing (such as Edman degradation) may be used, as may mass spectrometry (in particular MALDI or ESI techniques (see, e.g., Langen et al. (1997) Electraphoresis 18:
1184-1192)). The protein sequences provided herein can be used for the identification of C. glulamicum proteins by these techniques.
The information obtained by these methods can be used to compare pattems of protein presence, activity, or modification between different samples from various biological conditions (e.g., different organisms, time points of fermentation, media conditions, or different biotopes, among others). Data obtained from such experiments alone, or in combination with other techniques, can be used for various applications, such as to compare the behavior of various organisms in a given (e.g., metabolic) situation, to increase the productivity of strains which produce fine chemicals or to increase the efficiency of the production of fine chemicals.
Equivalents Those of ordinary skill in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Claims (35)
1. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ
ID NO:
71, or a complement thereof.
ID NO:
71, or a complement thereof.
2. An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:72, or a complement thereof.
3. An isolated nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:72, or a complement thereof.
4. An isolated nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the entire nucleotide sequence of SEQ ID NO:71, or a complement thereof.
5. An isolated nucleic acid molecule comprising a fragment of at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:71, or a complement thereof.
6. An isolated nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence which is at least 50% identical to the entire amino acid sequence of SEQ ID NO:72, or a complement thereof.
7. An isolated nucleic acid molecule comprising the nucleic acid molecule of any one of claims 1-6 and a nucleotide sequence encoding a heterologous polypeptide.
8. A vector comprising the nucleic acid molecule of any one of claims 1-7.
9. The vector of claim 8, which is an expression vector.
10. A host cell transfected with the expression vector of claim 9.
11. The host cell of claim 10, wherein said cell is a microorganism.
12. The host cell of claim 11, wherein said cell belongs to the genus Corynebacterium or Brevibacterium.
13. The host cell of claim 10, wherein the expression of said nucleic acid molecule results in the modulation in production of a fine chemical from said cell.
14. The host cell of claim 14, wherein said fine chemical is: an organic acid, a proteinogenic amino acid, a nonproteinogenic amino acid, a purine base, a pyrimidine base, a nucleoside, a nucleotide, a lipid, a saturated fatty acid, an unsaturated fatty acid, a diol, a carbohydrate, an aromatic compound, a vitamin, a cofactor, a polyketide, or an enzyme.
15. A method of producing a polypeptide comprising culturing the host cell of claim 10 in an appropriate culture medium to, thereby, produce the polypeptide.
16. An isolated polypeptide comprising the amino acid sequence of SEQ ID
NO:72.
NO:72.
17. An isolated polypeptide comprising a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:72.
18. An isolated polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 50% identical to the entire nucleotide sequence of SEQ ID NO:71.
19. An isolated polypeptide comprising an amino acid sequence which is at least 50%
identical to the entire amino acid sequence of SEQ ID NO:72.
identical to the entire amino acid sequence of SEQ ID NO:72.
20. An isolated polypeptide comprising a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:72, wherein said polypeptide fragment maintains a biological activity of the polypeptide comprising the amino sequence of SEQ ID
NO:72.
NO:72.
21. An isolated polypeptide comprising an amino acid sequence which is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:71.
22. The isolated polypeptide of any one of claims 16-21, further comprising a heterologous amino acid sequence.
23. A method for producing a fine chemical, comprising culturing the cell of claim 10 such that the fine chemical is produced.
24. The method of claim 23, wherein said method further comprises the step of recovering the fine chemical from said culture.
25. The method of claim 23, wherein said cell belongs to the genus Corynebacterium or Brevibacterium.
26. The method of claim 23, wherein said cell is: Corynebacterium glutamicum, Corynebacterium herculis, Corynebacterium lilium, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium acetophilum, Corynebacterium ammoniagenes, Corynebacterium fujiokense, Corynebacterium nitrilophilus, Brevibacterium ammoniagenes, Brevibacterium butanicum, Brevibacterium divaricatum, Brevibacterium flavum, Brevibacterium healii, Brevibacterium ketoglutamicum, Brevibacterium ketosoreductum, Brevibacterium lactofermentum, Brevibacterium linens, Brevibacterium paraffinolyticum, or those strains of Table 3.
27. The method of claim 23, wherein expression of the nucleic acid molecule from said vector results in modulation of production of said fine chemical.
28. The method of claim 23, wherein said fine chemical is: an organic acid, a proteinogenic amino acid, a nonproteinogenic amino acid, a purine base, a pyrimidine base, a nucleoside, a nucleotide, a lipid, a saturated fatty acid, an unsaturated fatty acid, a diol, a carbohydrate, an aromatic compound, a vitamin, a cofactor, a polyketide or an enzyme.
29. The method of claim 23, wherein said fine chemical is an amino acid.
30. The method of claim 29, wherein said amino acid is: lysine, glutamate, glutamine, alanine, aspartate, glycine, serine, threonine, methionine, cysteine, valine, leucine, isoleucine, arginine, proline, histidine, tyrosine, phenylalanine, or tryptophan.
31. A method for producing a fine chemical, comprising culturing a cell whose genomic DNA has been altered by the introduction of a nucleic acid molecule of any one of claims 1-6.
32. A method for diagnosing the presence or activity of Corynebacterium diphtheria, comprising detecting the presence of at least one of the nucleic acid molecules of any one of claims 1-6 or the polypeptide molecules of any one of claims 16-21, thereby diagnosing the presence or activity of Corynebacterium diphtheriae.
33. A host cell comprising the nucleic acid molecule of SEQ ID NO:71, wherein the nucleic acid molecule is disrupted.
34. A host cell comprising the nucleic acid molecule of SEQ ID NO:71, wherein the nucleic acid molecule comprises one or more nucleic acid modifications as compared to the sequence of SEQ ID NO:71.
35. A host cell comprising the nucleic acid molecule of SEQ ID NO:71, wherein the regulatory region of the nucleic acid molecule is modified relative to the wild-type regulatory region of the molecule.
Applications Claiming Priority (61)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14103199P | 1999-06-25 | 1999-06-25 | |
US60/141,031 | 1999-06-25 | ||
DE19931412 | 1999-07-08 | ||
DE19931428.4 | 1999-07-08 | ||
DE19931562 | 1999-07-08 | ||
DE19931428 | 1999-07-08 | ||
DE19931419.5 | 1999-07-08 | ||
DE19931420.9 | 1999-07-08 | ||
DE19931419 | 1999-07-08 | ||
DE19931510 | 1999-07-08 | ||
DE19931634 | 1999-07-08 | ||
DE19931634.1 | 1999-07-08 | ||
DE19931431.4 | 1999-07-08 | ||
DE19931431 | 1999-07-08 | ||
DE19931424.1 | 1999-07-08 | ||
DE19931434 | 1999-07-08 | ||
DE19931412.8 | 1999-07-08 | ||
DE19931420 | 1999-07-08 | ||
DE19931510.8 | 1999-07-08 | ||
DE19931434.9 | 1999-07-08 | ||
DE19931433.0 | 1999-07-08 | ||
DE19931413.6 | 1999-07-08 | ||
DE19931413 | 1999-07-08 | ||
DE19931433 | 1999-07-08 | ||
DE19931562.0 | 1999-07-08 | ||
DE19931424 | 1999-07-08 | ||
US14320899P | 1999-07-09 | 1999-07-09 | |
DE19932180 | 1999-07-09 | ||
DE19932227 | 1999-07-09 | ||
DE19932180.9 | 1999-07-09 | ||
DE19932227.9 | 1999-07-09 | ||
US60/143,208 | 1999-07-09 | ||
DE19932230 | 1999-07-09 | ||
DE19932230.9 | 1999-07-09 | ||
DE19932924 | 1999-07-14 | ||
DE19933005.0 | 1999-07-14 | ||
DE19932973 | 1999-07-14 | ||
DE19933005 | 1999-07-14 | ||
DE19932973.7 | 1999-07-14 | ||
DE19932924.9 | 1999-07-14 | ||
DE19940765.7 | 1999-08-27 | ||
DE19940765 | 1999-08-27 | ||
US15157299P | 1999-08-31 | 1999-08-31 | |
US60/151,572 | 1999-08-31 | ||
DE19942088 | 1999-09-03 | ||
DE19942095 | 1999-09-03 | ||
DE19942079.3 | 1999-09-03 | ||
DE19942123 | 1999-09-03 | ||
DE19942088.2 | 1999-09-03 | ||
DE19942079 | 1999-09-03 | ||
DE19942095.5 | 1999-09-03 | ||
DE19942123.4 | 1999-09-03 | ||
DE19942087 | 1999-09-03 | ||
DE19942125.0 | 1999-09-03 | ||
DE19942076.9 | 1999-09-03 | ||
DE19942086.6 | 1999-09-03 | ||
DE19942076 | 1999-09-03 | ||
DE19942125 | 1999-09-03 | ||
DE19942086 | 1999-09-03 | ||
DE19942087.4 | 1999-09-03 | ||
CA002383875A CA2383875A1 (en) | 1999-06-25 | 2000-06-23 | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002383875A Division CA2383875A1 (en) | 1999-06-25 | 2000-06-23 | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2593287A1 true CA2593287A1 (en) | 2001-01-04 |
Family
ID=37451534
Family Applications (6)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002594201A Abandoned CA2594201A1 (en) | 1999-06-25 | 2000-06-23 | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
CA002563004A Abandoned CA2563004A1 (en) | 1999-06-25 | 2000-06-23 | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
CA002593287A Abandoned CA2593287A1 (en) | 1999-06-25 | 2000-06-23 | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
CA002594262A Abandoned CA2594262A1 (en) | 1999-06-25 | 2000-06-23 | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
CA002587128A Abandoned CA2587128A1 (en) | 1999-06-25 | 2000-06-23 | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
CA002584021A Abandoned CA2584021A1 (en) | 1999-06-25 | 2000-06-23 | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002594201A Abandoned CA2594201A1 (en) | 1999-06-25 | 2000-06-23 | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
CA002563004A Abandoned CA2563004A1 (en) | 1999-06-25 | 2000-06-23 | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
Family Applications After (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002594262A Abandoned CA2594262A1 (en) | 1999-06-25 | 2000-06-23 | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
CA002587128A Abandoned CA2587128A1 (en) | 1999-06-25 | 2000-06-23 | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
CA002584021A Abandoned CA2584021A1 (en) | 1999-06-25 | 2000-06-23 | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production |
Country Status (1)
Country | Link |
---|---|
CA (6) | CA2594201A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115960874A (en) * | 2023-02-14 | 2023-04-14 | 山东润德生物科技有限公司 | Corynebacterium glutamicum endogenous GlcNAc6P phosphatase and method for improving GlcNAc yield |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112725253B (en) * | 2020-12-30 | 2023-01-06 | 宁夏伊品生物科技股份有限公司 | Recombinant strain for modifying gene BBD29_14900 and construction method and application thereof |
-
2000
- 2000-06-23 CA CA002594201A patent/CA2594201A1/en not_active Abandoned
- 2000-06-23 CA CA002563004A patent/CA2563004A1/en not_active Abandoned
- 2000-06-23 CA CA002593287A patent/CA2593287A1/en not_active Abandoned
- 2000-06-23 CA CA002594262A patent/CA2594262A1/en not_active Abandoned
- 2000-06-23 CA CA002587128A patent/CA2587128A1/en not_active Abandoned
- 2000-06-23 CA CA002584021A patent/CA2584021A1/en not_active Abandoned
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115960874A (en) * | 2023-02-14 | 2023-04-14 | 山东润德生物科技有限公司 | Corynebacterium glutamicum endogenous GlcNAc6P phosphatase and method for improving GlcNAc yield |
Also Published As
Publication number | Publication date |
---|---|
CA2594201A1 (en) | 2001-01-04 |
CA2594262A1 (en) | 2001-01-04 |
CA2587128A1 (en) | 2001-01-04 |
CA2563004A1 (en) | 2001-01-04 |
CA2584021A1 (en) | 2001-01-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1257649B1 (en) | Corynebacterium glutamicum genes encoding metabolic pathway proteins | |
US7273721B2 (en) | Corynebacterium glutamicum genes encoding proteins involved in membrane synthesis and membrane transport | |
EP2290062A1 (en) | Corynebacterium glutamicum gene encoding phosphoenolpyruvate carboxykinase | |
KR100834985B1 (en) | Corynebacterium Glutamicum Genes Encoding Proteins Involved in Homeostasis and Adaptation | |
CA2380870A1 (en) | Corynebacterium glutamicum genes encoding stress, resistance and tolerance proteins | |
CA2380863A1 (en) | Corynebacterium glutamicum genes encoding proteins involved in membrane synthesis and membrane transport | |
CA2402186C (en) | Corynebacterium glutamicum genes encoding metabolic pathway proteins | |
US7393675B2 (en) | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production | |
CA2377378A1 (en) | Corynebacterium glutamicum genes encoding phosphoenolpyruvate:sugar phosphotransferase system proteins | |
JP2004524827A (en) | Corynebacterium gene | |
RU2321634C2 (en) | Corynebacterium glutamicum genes encoding proteins taking part in carbon metabolism and energy production | |
CA2593287A1 (en) | Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production | |
CA2587112A1 (en) | Corynebacterium glutamicum genes encoding stress, resistance and tolerance proteins | |
EP1661987A1 (en) | Corynebacterium glutamicum gene encoding phosphoenolpyruvate carboxykinase | |
CA2571917A1 (en) | Corynebacterium glutamicum genes encoding metabolic pathway proteins | |
EP1634952A2 (en) | Corynebacterium glutamicum genes encoding proteins involved in membrane synthesis and membrane transport |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Dead |