CN1219200A - 谷氨酸脱氢酶、β-N-乙酰氨基己糖苷酶和γ-肌动蛋白基因的启动子以及它们在丝状真菌表达,分泌和反义系统中的应用 - Google Patents
谷氨酸脱氢酶、β-N-乙酰氨基己糖苷酶和γ-肌动蛋白基因的启动子以及它们在丝状真菌表达,分泌和反义系统中的应用 Download PDFInfo
- Publication number
- CN1219200A CN1219200A CN98800237A CN98800237A CN1219200A CN 1219200 A CN1219200 A CN 1219200A CN 98800237 A CN98800237 A CN 98800237A CN 98800237 A CN98800237 A CN 98800237A CN 1219200 A CN1219200 A CN 1219200A
- Authority
- CN
- China
- Prior art keywords
- gene
- chrysogenum
- ala
- gly
- val
- 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.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 148
- 230000014509 gene expression Effects 0.000 title claims abstract description 61
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 title abstract description 188
- 241000233866 Fungi Species 0.000 title abstract description 23
- 230000028327 secretion Effects 0.000 title abstract description 8
- 229930195712 glutamate Natural products 0.000 title abstract 3
- 102000007478 beta-N-Acetylhexosaminidases Human genes 0.000 title description 10
- 108010085377 beta-N-Acetylhexosaminidases Proteins 0.000 title description 10
- QCUPYFTWJOZAOB-HWKANZROSA-N (e)-n-carbamoyl-2-ethylbut-2-enamide Chemical compound CC\C(=C/C)C(=O)NC(N)=O QCUPYFTWJOZAOB-HWKANZROSA-N 0.000 title 1
- 241000228150 Penicillium chrysogenum Species 0.000 claims abstract description 97
- 241000228431 Acremonium chrysogenum Species 0.000 claims abstract description 46
- 239000013612 plasmid Substances 0.000 claims description 109
- 238000000034 method Methods 0.000 claims description 49
- 108020004414 DNA Proteins 0.000 claims description 46
- 101150104241 ACT gene Proteins 0.000 claims description 30
- 238000006243 chemical reaction Methods 0.000 claims description 30
- 101150025217 hex gene Proteins 0.000 claims description 29
- 101150019455 gdh gene Proteins 0.000 claims description 24
- 241000894006 Bacteria Species 0.000 claims description 22
- 230000000968 intestinal effect Effects 0.000 claims description 22
- 244000005700 microbiome Species 0.000 claims description 17
- 239000002773 nucleotide Substances 0.000 claims description 17
- 125000003729 nucleotide group Chemical group 0.000 claims description 17
- 108010085238 Actins Proteins 0.000 claims description 14
- 230000002068 genetic effect Effects 0.000 claims description 14
- 102000007469 Actins Human genes 0.000 claims description 12
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 241000351920 Aspergillus nidulans Species 0.000 claims description 11
- 210000003205 muscle Anatomy 0.000 claims description 11
- 101150099894 GDHA gene Proteins 0.000 claims description 10
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 9
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 claims description 8
- 102000016901 Glutamate dehydrogenase Human genes 0.000 claims description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 7
- 238000013467 fragmentation Methods 0.000 claims description 7
- 238000006062 fragmentation reaction Methods 0.000 claims description 7
- 229930182555 Penicillin Natural products 0.000 claims description 5
- UCKZMPLVLCKKMO-LHLIQPBNSA-N cephamycin Chemical compound S1CC(C)=C(C(O)=O)N2C(=O)[C@@H](C)[C@]21OC UCKZMPLVLCKKMO-LHLIQPBNSA-N 0.000 claims description 5
- 230000000295 complement effect Effects 0.000 claims description 5
- 229940049954 penicillin Drugs 0.000 claims description 5
- 241001019659 Acremonium <Plectosphaerellaceae> Species 0.000 claims description 4
- 241000228212 Aspergillus Species 0.000 claims description 4
- 241000228143 Penicillium Species 0.000 claims description 4
- 238000009396 hybridization Methods 0.000 claims description 4
- 241000186361 Actinobacteria <class> Species 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 230000000735 allogeneic effect Effects 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 108020004511 Recombinant DNA Proteins 0.000 claims 3
- 210000003527 eukaryotic cell Anatomy 0.000 claims 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims 2
- 210000001236 prokaryotic cell Anatomy 0.000 claims 2
- 229940081969 saccharomyces cerevisiae Drugs 0.000 claims 2
- 102000002268 Hexosaminidases Human genes 0.000 claims 1
- 108010000540 Hexosaminidases Proteins 0.000 claims 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 18
- 238000010276 construction Methods 0.000 abstract description 4
- 230000000903 blocking effect Effects 0.000 abstract description 3
- 239000013598 vector Substances 0.000 abstract description 3
- 241000894007 species Species 0.000 abstract description 2
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) 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-NNYOXOHSSA-O 0.000 abstract 1
- 239000003242 anti bacterial agent Substances 0.000 abstract 1
- 229940088710 antibiotic agent Drugs 0.000 abstract 1
- 230000003389 potentiating effect Effects 0.000 abstract 1
- 239000012634 fragment Substances 0.000 description 52
- 150000001413 amino acids Chemical class 0.000 description 36
- QRBLKGHRWFGINE-UGWAGOLRSA-N 2-[2-[2-[[2-[[4-[[2-[[6-amino-2-[3-amino-1-[(2,3-diamino-3-oxopropyl)amino]-3-oxopropyl]-5-methylpyrimidine-4-carbonyl]amino]-3-[(2r,3s,4s,5s,6s)-3-[(2s,3r,4r,5s)-4-carbamoyl-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,5-dihydroxy-6-(hydroxymethyl)- Chemical compound N=1C(C=2SC=C(N=2)C(N)=O)CSC=1CCNC(=O)C(C(C)=O)NC(=O)C(C)C(O)C(C)NC(=O)C(C(O[C@H]1[C@@]([C@@H](O)[C@H](O)[C@H](CO)O1)(C)O[C@H]1[C@@H]([C@](O)([C@@H](O)C(CO)O1)C(N)=O)O)C=1NC=NC=1)NC(=O)C1=NC(C(CC(N)=O)NCC(N)C(N)=O)=NC(N)=C1C QRBLKGHRWFGINE-UGWAGOLRSA-N 0.000 description 29
- LTQCLFMNABRKSH-UHFFFAOYSA-N Phleomycin Natural products N=1C(C=2SC=C(N=2)C(N)=O)CSC=1CCNC(=O)C(C(O)C)NC(=O)C(C)C(O)C(C)NC(=O)C(C(OC1C(C(O)C(O)C(CO)O1)OC1C(C(OC(N)=O)C(O)C(CO)O1)O)C=1NC=NC=1)NC(=O)C1=NC(C(CC(N)=O)NCC(N)C(N)=O)=NC(N)=C1C LTQCLFMNABRKSH-UHFFFAOYSA-N 0.000 description 29
- 108010035235 Phleomycins Proteins 0.000 description 29
- 230000000692 anti-sense effect Effects 0.000 description 20
- 235000001014 amino acid Nutrition 0.000 description 19
- 238000005516 engineering process Methods 0.000 description 19
- 108700026244 Open Reading Frames Proteins 0.000 description 18
- 235000018102 proteins Nutrition 0.000 description 17
- 102000004190 Enzymes Human genes 0.000 description 15
- 108090000790 Enzymes Proteins 0.000 description 15
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 15
- 230000000694 effects Effects 0.000 description 15
- 229940088598 enzyme Drugs 0.000 description 15
- 102220023257 rs387907546 Human genes 0.000 description 15
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 13
- 230000029087 digestion Effects 0.000 description 13
- 101150066555 lacZ gene Proteins 0.000 description 13
- 108020004705 Codon Proteins 0.000 description 11
- 102220369445 c.668T>C Human genes 0.000 description 11
- 241000203644 Streptoalloteichus hindustanus Species 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- 102220023256 rs387907547 Human genes 0.000 description 9
- 108091034117 Oligonucleotide Proteins 0.000 description 8
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 8
- 102220369447 c.1352G>A Human genes 0.000 description 8
- 238000010369 molecular cloning Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 108091081024 Start codon Proteins 0.000 description 7
- 229960005091 chloramphenicol Drugs 0.000 description 7
- 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 7
- 230000004927 fusion Effects 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 230000014621 translational initiation Effects 0.000 description 7
- FVFVNNKYKYZTJU-UHFFFAOYSA-N 6-chloro-1,3,5-triazine-2,4-diamine Chemical compound NC1=NC(N)=NC(Cl)=N1 FVFVNNKYKYZTJU-UHFFFAOYSA-N 0.000 description 6
- 108091092195 Intron Proteins 0.000 description 6
- HSMNQINEKMPTIC-UHFFFAOYSA-N N-(4-aminobenzoyl)glycine Chemical compound NC1=CC=C(C(=O)NCC(O)=O)C=C1 HSMNQINEKMPTIC-UHFFFAOYSA-N 0.000 description 6
- 241000712503 Sicyos pachycarpus Species 0.000 description 6
- 108700026226 TATA Box Proteins 0.000 description 6
- 108020005038 Terminator Codon Proteins 0.000 description 6
- 108700009124 Transcription Initiation Site Proteins 0.000 description 6
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 238000011144 upstream manufacturing Methods 0.000 description 6
- 241000221961 Neurospora crassa Species 0.000 description 5
- 238000013016 damping Methods 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 238000004321 preservation Methods 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 102220023258 rs387907548 Human genes 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 101150016309 trpC gene Proteins 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 4
- 239000004677 Nylon Substances 0.000 description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 description 4
- 108010047495 alanylglycine Proteins 0.000 description 4
- 108010005774 beta-Galactosidase Proteins 0.000 description 4
- 102000005936 beta-Galactosidase Human genes 0.000 description 4
- 239000005546 dideoxynucleotide Substances 0.000 description 4
- 238000002386 leaching Methods 0.000 description 4
- 229930182817 methionine Natural products 0.000 description 4
- 229920001778 nylon Polymers 0.000 description 4
- 230000003248 secreting effect Effects 0.000 description 4
- 238000012882 sequential analysis Methods 0.000 description 4
- MAZZQZWCCYJQGZ-GUBZILKMSA-N Ala-Pro-Arg Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MAZZQZWCCYJQGZ-GUBZILKMSA-N 0.000 description 3
- 241000222122 Candida albicans Species 0.000 description 3
- 101710088194 Dehydrogenase Proteins 0.000 description 3
- MIFYHUACUWQUKT-UHFFFAOYSA-N Isopenicillin N Natural products OC(=O)C1C(C)(C)SC2C(NC(=O)CCCC(N)C(O)=O)C(=O)N21 MIFYHUACUWQUKT-UHFFFAOYSA-N 0.000 description 3
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 3
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 229930006000 Sucrose Natural products 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
- 101150099105 alien gene Proteins 0.000 description 3
- 229940095731 candida albicans Drugs 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- -1 malt extract Substances 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 101150074325 pcbC gene Proteins 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 229960004793 sucrose Drugs 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108020004491 Antisense DNA Proteins 0.000 description 2
- GWTLRDMPMJCNMH-WHFBIAKZSA-N Asp-Asn-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GWTLRDMPMJCNMH-WHFBIAKZSA-N 0.000 description 2
- 101100505161 Caenorhabditis elegans mel-32 gene Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- LXWBXEWUSAABOA-UHFFFAOYSA-N Cephamycin-C Natural products S1CC(COC(N)=O)=C(C(O)=O)N2C(=O)C(OC)(NC(=O)CCCC(N)C(O)=O)C21 LXWBXEWUSAABOA-UHFFFAOYSA-N 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- OZHXXYOHPLLLMI-CIUDSAMLSA-N Cys-Lys-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O OZHXXYOHPLLLMI-CIUDSAMLSA-N 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 101100462961 Fischerella muscicola pcb gene Proteins 0.000 description 2
- RDDSZZJOKDVPAE-ACZMJKKPSA-N Glu-Asn-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O RDDSZZJOKDVPAE-ACZMJKKPSA-N 0.000 description 2
- BUZMZDDKFCSKOT-CIUDSAMLSA-N Glu-Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BUZMZDDKFCSKOT-CIUDSAMLSA-N 0.000 description 2
- HVYWQYLBVXMXSV-GUBZILKMSA-N Glu-Leu-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O HVYWQYLBVXMXSV-GUBZILKMSA-N 0.000 description 2
- FUTAPPOITCCWTH-WHFBIAKZSA-N Gly-Asp-Asp Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O FUTAPPOITCCWTH-WHFBIAKZSA-N 0.000 description 2
- WMGHDYWNHNLGBV-ONGXEEELSA-N Gly-Phe-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 WMGHDYWNHNLGBV-ONGXEEELSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- FMFNIDICDKEMOE-XUXIUFHCSA-N Leu-Val-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FMFNIDICDKEMOE-XUXIUFHCSA-N 0.000 description 2
- NPBGTPKLVJEOBE-IUCAKERBSA-N Lys-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N NPBGTPKLVJEOBE-IUCAKERBSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- CDQCFGOQNYOICK-IHRRRGAJSA-N Phe-Glu-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 CDQCFGOQNYOICK-IHRRRGAJSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- NADLKBTYNKUJEP-KATARQTJSA-N Ser-Thr-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O NADLKBTYNKUJEP-KATARQTJSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- PRNGXSILMXSWQQ-OEAJRASXSA-N Thr-Leu-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PRNGXSILMXSWQQ-OEAJRASXSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 239000003816 antisense DNA Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- LXWBXEWUSAABOA-VXSYNFHWSA-N cephamycin C Chemical compound S1CC(COC(N)=O)=C(C(O)=O)N2C(=O)[C@@](OC)(NC(=O)CCC[C@@H](N)C(O)=O)[C@H]21 LXWBXEWUSAABOA-VXSYNFHWSA-N 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Chemical compound NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 238000009655 industrial fermentation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- MIFYHUACUWQUKT-QSKNDCFUSA-N isopenicillin n Chemical compound OC(=O)[C@@H]1C(C)(C)S[C@H]2[C@H](NC(=O)CCC[C@H](N)C(O)=O)C(=O)N21 MIFYHUACUWQUKT-QSKNDCFUSA-N 0.000 description 2
- 108010064235 lysylglycine Proteins 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 101150118709 pcbAB gene Proteins 0.000 description 2
- 101150022041 penDE gene Proteins 0.000 description 2
- 229940056360 penicillin g Drugs 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 2
- 229940124530 sulfonamide Drugs 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 description 1
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- NGHVIOIJCVXTGV-ALEPSDHESA-N 6-aminopenicillanic acid Chemical compound [O-]C(=O)[C@H]1C(C)(C)S[C@@H]2[C@H]([NH3+])C(=O)N21 NGHVIOIJCVXTGV-ALEPSDHESA-N 0.000 description 1
- NGHVIOIJCVXTGV-UHFFFAOYSA-N 6beta-amino-penicillanic acid Natural products OC(=O)C1C(C)(C)SC2C(N)C(=O)N21 NGHVIOIJCVXTGV-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- BTYTYHBSJKQBQA-GCJQMDKQSA-N Ala-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)N)O BTYTYHBSJKQBQA-GCJQMDKQSA-N 0.000 description 1
- MPLOSMWGDNJSEV-WHFBIAKZSA-N Ala-Gly-Asp Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MPLOSMWGDNJSEV-WHFBIAKZSA-N 0.000 description 1
- MEFILNJXAVSUTO-JXUBOQSCSA-N Ala-Leu-Thr Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MEFILNJXAVSUTO-JXUBOQSCSA-N 0.000 description 1
- XAXMJQUMRJAFCH-CQDKDKBSSA-N Ala-Tyr-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=C(O)C=C1 XAXMJQUMRJAFCH-CQDKDKBSSA-N 0.000 description 1
- 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 1
- YVTHEZNOKSAWRW-DCAQKATOSA-N Arg-Lys-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O YVTHEZNOKSAWRW-DCAQKATOSA-N 0.000 description 1
- BSGSDLYGGHGMND-IHRRRGAJSA-N Arg-Phe-Cys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N BSGSDLYGGHGMND-IHRRRGAJSA-N 0.000 description 1
- FIQKRDXFTANIEJ-ULQDDVLXSA-N Arg-Phe-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N FIQKRDXFTANIEJ-ULQDDVLXSA-N 0.000 description 1
- IGFJVXOATGZTHD-UHFFFAOYSA-N Arg-Phe-His Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccccc1)C(=O)NC(Cc2c[nH]cn2)C(=O)O IGFJVXOATGZTHD-UHFFFAOYSA-N 0.000 description 1
- GXMSVVBIAMWMKO-BQBZGAKWSA-N Asn-Arg-Gly Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CCCN=C(N)N GXMSVVBIAMWMKO-BQBZGAKWSA-N 0.000 description 1
- MSBDSTRUMZFSEU-PEFMBERDSA-N Asn-Glu-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O MSBDSTRUMZFSEU-PEFMBERDSA-N 0.000 description 1
- JLNFZLNDHONLND-GARJFASQSA-N Asn-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N JLNFZLNDHONLND-GARJFASQSA-N 0.000 description 1
- DJIMLSXHXKWADV-CIUDSAMLSA-N Asn-Leu-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(N)=O DJIMLSXHXKWADV-CIUDSAMLSA-N 0.000 description 1
- CBHVAFXKOYAHOY-NHCYSSNCSA-N Asn-Val-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O CBHVAFXKOYAHOY-NHCYSSNCSA-N 0.000 description 1
- UAXIKORUDGGIGA-DCAQKATOSA-N Asp-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)O)N)C(=O)N[C@@H](CCCCN)C(=O)O UAXIKORUDGGIGA-DCAQKATOSA-N 0.000 description 1
- QPDUWAUSSWGJSB-NGZCFLSTSA-N Asp-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)O)N QPDUWAUSSWGJSB-NGZCFLSTSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 101150111062 C gene Proteins 0.000 description 1
- 0 C*(C(C1)CCCCC(C)(*)C2)C2C1=*1**1 Chemical compound C*(C(C1)CCCCC(C)(*)C2)C2C1=*1**1 0.000 description 1
- 101100315624 Caenorhabditis elegans tyr-1 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 108091033380 Coding strand Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 102000005870 Coenzyme A Ligases Human genes 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- LXJXRIRHZLFYRP-VKHMYHEASA-N D-glyceraldehyde 3-phosphate Chemical compound O=C[C@H](O)COP(O)(O)=O LXJXRIRHZLFYRP-VKHMYHEASA-N 0.000 description 1
- 101100117236 Drosophila melanogaster speck gene Proteins 0.000 description 1
- 108700034637 EC 3.2.-.- Proteins 0.000 description 1
- 101900264058 Escherichia coli Beta-galactosidase Proteins 0.000 description 1
- 241000702192 Escherichia virus P2 Species 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- JRHPEMVLTRADLJ-AVGNSLFASA-N Gln-Lys-Lys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N JRHPEMVLTRADLJ-AVGNSLFASA-N 0.000 description 1
- AQPZYBSRDRZBAG-AVGNSLFASA-N Gln-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)N)N AQPZYBSRDRZBAG-AVGNSLFASA-N 0.000 description 1
- DITJVHONFRJKJW-BPUTZDHNSA-N Gln-Trp-Glu Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N DITJVHONFRJKJW-BPUTZDHNSA-N 0.000 description 1
- VCUNGPMMPNJSGS-JYJNAYRXSA-N Gln-Tyr-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O VCUNGPMMPNJSGS-JYJNAYRXSA-N 0.000 description 1
- ICRKQMRFXYDYMK-LAEOZQHASA-N Gln-Val-Asn Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O ICRKQMRFXYDYMK-LAEOZQHASA-N 0.000 description 1
- JPHYJQHPILOKHC-ACZMJKKPSA-N Glu-Asp-Asp Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O JPHYJQHPILOKHC-ACZMJKKPSA-N 0.000 description 1
- UMIRPYLZFKOEOH-YVNDNENWSA-N Glu-Gln-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O UMIRPYLZFKOEOH-YVNDNENWSA-N 0.000 description 1
- DXVOKNVIKORTHQ-GUBZILKMSA-N Glu-Pro-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O DXVOKNVIKORTHQ-GUBZILKMSA-N 0.000 description 1
- 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 1
- 108010068370 Glutens Proteins 0.000 description 1
- VSVZIEVNUYDAFR-YUMQZZPRSA-N Gly-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN VSVZIEVNUYDAFR-YUMQZZPRSA-N 0.000 description 1
- OGCIHJPYKVSMTE-YUMQZZPRSA-N Gly-Arg-Glu Chemical compound [H]NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O OGCIHJPYKVSMTE-YUMQZZPRSA-N 0.000 description 1
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 description 1
- IUZGUFAJDBHQQV-YUMQZZPRSA-N Gly-Leu-Asn Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IUZGUFAJDBHQQV-YUMQZZPRSA-N 0.000 description 1
- FXGRXIATVXUAHO-WEDXCCLWSA-N Gly-Lys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN FXGRXIATVXUAHO-WEDXCCLWSA-N 0.000 description 1
- JNGHLWWFPGIJER-STQMWFEESA-N Gly-Pro-Tyr Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 JNGHLWWFPGIJER-STQMWFEESA-N 0.000 description 1
- BAYQNCWLXIDLHX-ONGXEEELSA-N Gly-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN BAYQNCWLXIDLHX-ONGXEEELSA-N 0.000 description 1
- KSOBNUBCYHGUKH-UWVGGRQHSA-N Gly-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN KSOBNUBCYHGUKH-UWVGGRQHSA-N 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 101100277701 Halobacterium salinarum gdhX gene Proteins 0.000 description 1
- NDKSHNQINMRKHT-PEXQALLHSA-N His-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC1=CN=CN1)N NDKSHNQINMRKHT-PEXQALLHSA-N 0.000 description 1
- 101000666730 Homo sapiens T-complex protein 1 subunit alpha Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- DMHGKBGOUAJRHU-UHFFFAOYSA-N Ile-Arg-Pro Natural products CCC(C)C(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O DMHGKBGOUAJRHU-UHFFFAOYSA-N 0.000 description 1
- BALLIXFZYSECCF-QEWYBTABSA-N Ile-Gln-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N BALLIXFZYSECCF-QEWYBTABSA-N 0.000 description 1
- VOBYAKCXGQQFLR-LSJOCFKGSA-N Ile-Gly-Val Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O VOBYAKCXGQQFLR-LSJOCFKGSA-N 0.000 description 1
- TVYWVSJGSHQWMT-AJNGGQMLSA-N Ile-Leu-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N TVYWVSJGSHQWMT-AJNGGQMLSA-N 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 125000002061 L-isoleucyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])[C@](C([H])([H])[H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 1
- 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 1
- NHHKSOGJYNQENP-SRVKXCTJSA-N Leu-Cys-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)O)N NHHKSOGJYNQENP-SRVKXCTJSA-N 0.000 description 1
- QDSKNVXKLPQNOJ-GVXVVHGQSA-N Leu-Gln-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O QDSKNVXKLPQNOJ-GVXVVHGQSA-N 0.000 description 1
- KEVYYIMVELOXCT-KBPBESRZSA-N Leu-Gly-Phe Chemical compound CC(C)C[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 KEVYYIMVELOXCT-KBPBESRZSA-N 0.000 description 1
- AVEGDIAXTDVBJS-XUXIUFHCSA-N Leu-Ile-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AVEGDIAXTDVBJS-XUXIUFHCSA-N 0.000 description 1
- XXXXOVFBXRERQL-ULQDDVLXSA-N Leu-Pro-Phe Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 XXXXOVFBXRERQL-ULQDDVLXSA-N 0.000 description 1
- 108010011449 Long-chain-fatty-acid-CoA ligase Proteins 0.000 description 1
- XNKDCYABMBBEKN-IUCAKERBSA-N Lys-Gly-Gln Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O XNKDCYABMBBEKN-IUCAKERBSA-N 0.000 description 1
- ISHNZELVUVPCHY-ZETCQYMHSA-N Lys-Gly-Gly Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)NCC(O)=O ISHNZELVUVPCHY-ZETCQYMHSA-N 0.000 description 1
- MXMDJEJWERYPMO-XUXIUFHCSA-N Lys-Ile-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MXMDJEJWERYPMO-XUXIUFHCSA-N 0.000 description 1
- ALEVUGKHINJNIF-QEJZJMRPSA-N Lys-Phe-Ala Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=CC=C1 ALEVUGKHINJNIF-QEJZJMRPSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- MYKLINMAGAIRPJ-CIUDSAMLSA-N Met-Gln-Asn Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O MYKLINMAGAIRPJ-CIUDSAMLSA-N 0.000 description 1
- IMTUWVJPCQPJEE-IUCAKERBSA-N Met-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN IMTUWVJPCQPJEE-IUCAKERBSA-N 0.000 description 1
- WXUUEPIDLLQBLJ-DCAQKATOSA-N Met-Met-Gln Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N WXUUEPIDLLQBLJ-DCAQKATOSA-N 0.000 description 1
- KRLKICLNEICJGV-STQMWFEESA-N Met-Phe-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 KRLKICLNEICJGV-STQMWFEESA-N 0.000 description 1
- FXBKQTOGURNXSL-HJGDQZAQSA-N Met-Thr-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(O)=O FXBKQTOGURNXSL-HJGDQZAQSA-N 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- AQGDXJQRVOCUQX-UHFFFAOYSA-N N.[S] Chemical compound N.[S] AQGDXJQRVOCUQX-UHFFFAOYSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- CSDMCMITJLKBAH-SOUVJXGZSA-N Phe-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O CSDMCMITJLKBAH-SOUVJXGZSA-N 0.000 description 1
- RMKGXGPQIPLTFC-KKUMJFAQSA-N Phe-Lys-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O RMKGXGPQIPLTFC-KKUMJFAQSA-N 0.000 description 1
- 108010021302 Phenylacetate 2-hydroxylase Proteins 0.000 description 1
- 101100392454 Picrophilus torridus (strain ATCC 700027 / DSM 9790 / JCM 10055 / NBRC 100828) gdh2 gene Proteins 0.000 description 1
- LHALYDBUDCWMDY-CIUDSAMLSA-N Pro-Glu-Ala Chemical compound C[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1)C(O)=O LHALYDBUDCWMDY-CIUDSAMLSA-N 0.000 description 1
- PULPZRAHVFBVTO-DCAQKATOSA-N Pro-Glu-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PULPZRAHVFBVTO-DCAQKATOSA-N 0.000 description 1
- QGOZJLYCGRYYRW-KKUMJFAQSA-N Pro-Glu-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QGOZJLYCGRYYRW-KKUMJFAQSA-N 0.000 description 1
- MCWHYUWXVNRXFV-RWMBFGLXSA-N Pro-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 MCWHYUWXVNRXFV-RWMBFGLXSA-N 0.000 description 1
- CXGLFEOYCJFKPR-RCWTZXSCSA-N Pro-Thr-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O CXGLFEOYCJFKPR-RCWTZXSCSA-N 0.000 description 1
- 101100116769 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) gdhA-2 gene Proteins 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- HRNQLKCLPVKZNE-CIUDSAMLSA-N Ser-Ala-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O HRNQLKCLPVKZNE-CIUDSAMLSA-N 0.000 description 1
- HBZBPFLJNDXRAY-FXQIFTODSA-N Ser-Ala-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O HBZBPFLJNDXRAY-FXQIFTODSA-N 0.000 description 1
- OLIJLNWFEQEFDM-SRVKXCTJSA-N Ser-Asp-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 OLIJLNWFEQEFDM-SRVKXCTJSA-N 0.000 description 1
- YEDSOSIKVUMIJE-DCAQKATOSA-N Ser-Val-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O YEDSOSIKVUMIJE-DCAQKATOSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 101100370749 Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) trpC1 gene Proteins 0.000 description 1
- 102100038410 T-complex protein 1 subunit alpha Human genes 0.000 description 1
- MPUMPERGHHJGRP-WEDXCCLWSA-N Thr-Gly-Lys Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N)O MPUMPERGHHJGRP-WEDXCCLWSA-N 0.000 description 1
- JQAWYCUUFIMTHE-WLTAIBSBSA-N Thr-Gly-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JQAWYCUUFIMTHE-WLTAIBSBSA-N 0.000 description 1
- JVTHMUDOKPQBOT-NSHDSACASA-N Trp-Gly-Gly Chemical compound C1=CC=C2C(C[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O)=CNC2=C1 JVTHMUDOKPQBOT-NSHDSACASA-N 0.000 description 1
- QYSBJAUCUKHSLU-JYJNAYRXSA-N Tyr-Arg-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O QYSBJAUCUKHSLU-JYJNAYRXSA-N 0.000 description 1
- UUJHRSTVQCFDPA-UFYCRDLUSA-N Tyr-Tyr-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 UUJHRSTVQCFDPA-UFYCRDLUSA-N 0.000 description 1
- CPTQYHDSVGVGDZ-UKJIMTQDSA-N Val-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C(C)C)N CPTQYHDSVGVGDZ-UKJIMTQDSA-N 0.000 description 1
- MDYSKHBSPXUOPV-JSGCOSHPSA-N Val-Gly-Phe Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N MDYSKHBSPXUOPV-JSGCOSHPSA-N 0.000 description 1
- QZKVWWIUSQGWMY-IHRRRGAJSA-N Val-Ser-Phe Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QZKVWWIUSQGWMY-IHRRRGAJSA-N 0.000 description 1
- NZYNRRGJJVSSTJ-GUBZILKMSA-N Val-Ser-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O NZYNRRGJJVSSTJ-GUBZILKMSA-N 0.000 description 1
- WHNSHJJNWNSTSU-BZSNNMDCSA-N Val-Val-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)C(C)C)C(O)=O)=CNC2=C1 WHNSHJJNWNSTSU-BZSNNMDCSA-N 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
- 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 1
- 102000005421 acetyltransferase Human genes 0.000 description 1
- 108020002494 acetyltransferase Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960003311 ampicillin trihydrate Drugs 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 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 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 101150035092 cefD gene Proteins 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Substances O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 1
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 108010087823 glycyltyrosine Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910052742 iron Inorganic materials 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
- 239000008101 lactose Substances 0.000 description 1
- 108010051673 leucyl-glycyl-phenylalanine Proteins 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000001320 lysogenic effect Effects 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [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
- 239000000463 material Substances 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000007102 metabolic function Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000024717 negative regulation of secretion Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 108010018625 phenylalanylarginine Proteins 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229940093916 potassium phosphate Drugs 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000013606 secretion vector Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 229960002203 tilactase Drugs 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000032895 transmembrane transport Effects 0.000 description 1
- 108010058119 tryptophyl-glycyl-glycine Proteins 0.000 description 1
- 108010009962 valyltyrosine Proteins 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
- C12Y104/01002—Glutamate dehydrogenase (1.4.1.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01052—Beta-N-acetylhexosaminidase (3.2.1.52)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Virology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明涉及谷氨酸脱氢酶,β-乙酰氨基己糖苷酶和γ-肌动蛋白基因的启动子以及它们在丝状真菌的表达,分泌和反义系统中的应用。本发明也涉及编码;(Ⅰ)Penicilliumchrysogenum的NADP依赖性谷氨酸脱氢酶(EC.1.4.1.4),(Ⅱ)Penicillium chrysogenum的γ-N-乙酰氨基己糖苷酶(EC.3.2.1.52)和(Ⅲ)Penicillium chrysogenum和Acrimonium chrysogenum的γ-肌动蛋白,的基因的启动子,它们可用于构建用于Penicillium chrysogenum和Acrimoniumchrysogenum及相关的种类的表达和分泌的强载体。所说的启动子也可通过反义构建体用于阻断基因的表达。在上述启动子的控制下,也可在丝状真菌中表达其他的基因,从而提高其中固有的抗生素和/或蛋白质的产生。
Description
发明所属领域
本发明涉及Penicillium chrysogenum的gdh和hex基因以及P.chrysogenum和Acremonium chrysogenum的act基因的表达的技术领域。从所说的基因的核苷酸序列的分析推测存在有包括翻译起始位点的启动子区域,该启动子区域可用于构建用于P.chrysogenum和A.chrysogenum及相关种类的强大的表达和分泌载体。此外,这些启动子通过反义构建体可被用于阻断基因表达。丝状真菌中其它基因的表达可被处于上述启动子的控制之下,从而提高其中固有的抗生素和/或蛋白质的产生。
现有技术
P.chrysogenum和A.chrysogenum是具有工业用途的丝状真菌,这是因为它们可分别产生青霉素和头孢霉素。近十年来,已开发出适用于这两种微生物的大量的遗传工程技术。P.chrysogenum和A.chrysogenum的遗传操作技术包括用载体对原生质体的转化,其中使用腐草霉素抗性基因(下文称作bleR基因)(Kolar,M.et al.(1988),Gene62,127-134)作为选择标记,以及额外的目的基因的完整拷贝的表达以及由另外一个可改善其表达的启动子替换所述基因的启动子。在诸如P.chrysogenum或A.chrysogenum的真菌中同源基因的表达可被负调节,而在异源基因的情况下,有可能它们的启动子不被所说真菌有效识别。为避免这些问题,对进行组成型表达,并且其中所说的表达最好不显示负分解代谢调节的基因进行鉴别和克隆,下文称作强启动子。一般认为高表达的基因在其启动子区域具有可进行高水平转录的信号,它们在涉及细胞基础代谢的功能中起着重要的作用。这些基因包括:编码NADP-依赖性谷氨酸脱氢酶(EC.1.4.1.4)的基因(下文称作gdh基因),β-N-己酰氨基己糖苷酶(EC.3.2.1.52)的基因(下文称作hex基因)和γ-肌动蛋白的基因(下文称作act基因)。
早先已有对来源于本发明所用之外的微生物的gdh,hex和act基因的描述。最相关的文献包括:(Ⅰ)真菌Neurospora Crassa的gdh基因的核苷酸序列(Kinnaird,J.H.和Fincham,J.R.S.(1983),Gene 26,253-260)以及Aspergillus nidulans的gdhA基因的表达的调节(Hawkins,A.R.et al.(1989),Mol.Gen.Genet.418,105-111),(Ⅱ)Candida albicans的hexl基因的克隆和表达(Cannon,R.D.etal.(1994),微物生学杂志(2640-2647)和(Ⅲ)A.indulan的act基因的表征(Fidel,S.et al.(1988),基因70,283-293)。使用pcbC或penDE基因的启动子在P.chrysogenum中异源基因的表达由Cantwell,C.A.et al.在1992(Proc.R.Soc.London Ser.B 248,283-289)中进行了描述。此外,使用β-异丙基马来酸脱氢酶基因的启动子在A.chrysogenum中的异源基因的表达(Japanese Patent Laid Open PublicationNo.80295/1989)和甘油醛3-磷酸脱氢酶基因(欧洲专利申请0376226A1/1989)也已有描述。
有时需要对工业菌株中基因的表达进行抑制,以除去不需要的酶活性。由于许多工业菌株的倍体水平使多数情况下难以通过直接破坏基因来阻断表达,需要使用不依赖于倍体水平的表达抑制系统。在强启动子控制下表达的反义构建体的开发使基因表达的阻断成为可能。这类构建体在工业菌株中特别有用,这是由于它们的倍体水平(Kunkel etal(1992)微生物生物技术应用36,499-502)使得难以获得完全的基因失活。使用反义构建体抑制酶活性在酵母中(Atkins,D.et al.(1994),生物化学H-S375,721-729)和植物中(Hamada,T.(1996),转基因研究5,115-121;John,M.E.(1996)植物分子生物学30,297-306)已有描述。hex启动子具有编码胞外酶的特征,从而使它可被用于表达胞外蛋白质。
但在现有技术中没有对本发明所用的丝状真菌的基因序列的描述或通过它们的表达合成的酶的描述。现有技术中也没有将本发明所述的真菌基团的强启动用于表达,分泌或基因表达的抑制。
本发明详细描述
使用强启动子对某些基因的大量表达可提高青霉素或头孢霉素的产率,并且也可提高衍生于后者的抗生素的合成。
本发明描述了一种新的方法,以获得具有在强启动子的控制下表达异源或同源基因的能力的P.chrysogenum和A.chrysogenum菌株。描述了编码P.chrysogenum的NADP-依赖性谷氨酸脱氢酶(EC.1.4.1.4)基因-gdh基因、P.chrysogenum的β-N-乙酰氨基己糖苷酶(EC.3.2.1.52)基因-hex基因,和P.chrysogenum和A.chrysogenum的γ-肌动蛋白基因-act基因,的启动子的表征和随后的使用。使用所说的启动子在上述菌株中对与青霉素和/或头孢霉素生物合成相关的基因的过量表达是本发明的目的之一。这些启动子也可通过反义构建体被用于阻断基因的表达。
本发明是基于P.chrysogenum和A.chrysogenum用作核酸供体。将基因组DNA纯化后,如实施例1和4所述构建这两种微生物的DNA文库,并将它们用以下寡核苷酸进行筛查:(Ⅰ)对应于N.crassa的gdh基因的合成寡核苷酸,以克隆P.chrysogenum的同源基因,(Ⅱ)β-N-乙酰氨基己糖苷酶的氨基末端序列合成的寡核苷酸的组合,以克隆P.chrysogenum的hex基因,和(Ⅲ)A.nidulans的act基因的一个片段,以克隆P.chrysogenum和A.chrysogenum的同源基因。对借助于它们与相应的探针产生正杂交纯化的克隆随后进行分析,确定被搜寻的基因的存在。
P.chrysogenum在一个7.2kb的EcoRⅠ片段和两个长度分别为2.9和1.5kb和BamHⅠ片段中被识别出来。包括它的DNA区段的限制性图谱示于图1。然后确定了其核苷酸序列(SEQ ID NO:1),它包括一个开放阅读框(ORF),该开放阅读框具有一个非常明显的优先密码子使用类型,发现其ATG转译起始密码子位于922位,其TAA转译终止密码子位于2522位。也分别在971-1130位之间和1262-1318位之间确定了159bp和56bp的两个内含子的存在。所说的ORF编码一个49837Da的蛋白质,其等电点为6.18,它的461个氨基酸的序列(SEQ ID NO:5)与N.crassa的NADP-依赖性谷氨酸脱氢酶的氨基酸序列具有72.4%的一致性。在启动子区域,发现其具有一个嘧啶富集区域,这与高表达基因所看到的相似,以及两个推测的TATA盒(该盒发现于一些真菌启动子转录起始位点上游30至50bp处)(Davis,M.A.和Hynes,M.J.(1991),More Gene Manipulations in Fungi,Academic Press,San Diego,California)和一个CCAAT盒(它发现于约30%的真核基因启动子的转录起始位点上游50至200bp处)(Bucher,P.(1990)分了生物学杂志212:563-578)。然后这一启动子被用于在P.chrysogenum和A.chrysogenum中表达编码β-半乳糖苷酶的大肠杆菌基因(下文称作lacZ基因)以及S.hindustanus的bleR基因。为了这一目的构建了质粒pSKGSu和pALfleo7(图5),如实施例1所述的。从获得的结果推测,gdh启动子(下文称作Pgdh)能够在P.chrysogenum和A.chrysogenum以及大肠杆菌中控制异源的lacZ和bleR基因的表达。
在强启动子的控制之下表达的反义构建体的开发使基因表达的阻断成为可能。为了这一目的,构建了质粒pALP888(图5),如实施例1的第1.3节所述。所得结果证实了通过使用应用了Pgdh的反义构建体在P.chrysogenum中对不需要的酶活性进行全部或部分阻断的可能性。
P.chrysogenum的hex基因在一个3.2kb的SacⅠ片段和一个2.1kb的SalⅠ片段中被识别出来。包括hex基因的DNA区段的限制性图谱示于图2。然后确定了其5240个核苷酸的序列(SEQ ID NO:2),证实了存在两个具有非常明显的优先密码子使用类型的ORF,其中一个与hex基因匹配。hex基因的ATG转译始密码子发现于1324位,TGA终止密码子位于3112。所说的ORF没有内含子,编码一个66545 Da的蛋白质,其等电点为5.34,其596个氨基酸的序列(SEQ ID NO:6)与Candida albicans的β-N-乙酰氨基己糖苷酶的氨基酸序列具有49.0%的一致性。此外,该推测的氨基酸序列包括19-40位和99-120位的从纯化的酶化学确定的多肽。在启动子区段,发现有两个嘧啶富集区域。一个推测的TATA盒和CAAT盒。然后将该启动子用于在P.chrysogenum中表达S.hindustanus的bleR基因。为了这一目的,构建了质粒pALP480(图6),如实施例2所述。从所得结果推测,hex启动子(下文称作Phex)在P.chrysogenum中能够控制异源性bleR基因的表达。此外,β-N-乙酰氨基己糖苷酶是一种由P.chrysogenum大量分泌至培养基中的蛋白质这一事实使得将hex基因用于在P.chrysogenum或相关的丝状真菌中表达和分泌同源或异源蛋白质成为可能。待表达的基因可与启动子区段包括hex基因的分泌信号序列融合进一个阅读框内,或者它们可与完全hex基因融合进同一个阅读框内。
P.chrysogenum的act基因(下文称作actPc)在一个5.2kb的BamHⅠ片段,一个4.9kb的EcoRⅠ片段和一个5.9kb的HindⅢ片段中被识别出来。包括actPc基因的DNA区段的限制性图谱示于图3。在2994个核苷酸序列(SEQ ID N0:3)被确定之后,证实了存在一个具有非常明显的优先密码子使用类型的ORF。发现其ATG转译起始密码子位于494位,其TAA转译终止密码子位于2250位。所说的ORF具有5个内含子,并编码一个41760 Da的蛋白质,其等电点为5.51,它的375个氨基酸的序列(SEQ ID NO:7)与A.nidulans的γ-肌动蛋白的氨基酸序列具有98.1%的一致性。在该启动子区段中,发现有两个嘧啶富集区,一个推测的TATA盒和4个CAAT盒。然后将该启动子用于在P.chrysogenum中表达S.hindustanus的bleR基因。为了这一目的,构建了质粒pALPfleol(图6),如实施例3所述。从所得结果推测,P.chrysogenum的act启动子(下文称作PactPc)能够在P.chrysogenum中控制异源性bleR基因的表达。
A.chrysogenum的act基因(下文称用actAc)在2.4和1.1kb的SalⅠ片段,一个3.9kb的SmaⅠ片段和一个8.7kb的HindⅢ片段中被识别出来。包括actAc基因的DNA区段的限制性图谱示于图4。被确定的3240个核苷酸的序列(SEQ ID NO:4)证实了存在一个具有非常明显的优先密码子使用类型的ORF。发现ATG转译起始密码子位于787位,TAA终止密码子位于2478位,所说的ORF具有5个内含子并编码一个41612 Da的蛋白质,其等电点为5.51,它的375个氨基酸的序列(SEQ ID NO:8)分别有对应于A.nidulans和P.chrysogenum的γ-肌动蛋白的氨基酸序列具有98.4%和98.1%的一致性。在该启动子区域中,发现有嘧啶富集区和一个CAAT盒,没有看到存在有TATA盒。然后将该启动子用于在A.chrysogenum中表达S.hindustanus的bleR基因。为了这一目的构建了质粒pALCfleol(图6),如实施例4所述。从所得结果推测,A.chrysogenum的act启动子(下文称作PactAc)能够在A.chrysogenum中控制异源性bleR基因的表达。
在所有情况下,异源基因在真菌启动的控制下,在P.chrysogenum和A.chrysogenum中的异源基因表达通过将所说基因融合进正确的阅读框均得到实现。虽然lacZ和bleR是作为例子进行表达的,但同样可能表达编码参与了青霉素生物合成的酶的基因:pcbAB(α-氨基己二酰-半胱氨酰-缬氨酸合成酶),pcbC(异青霉素N合成酶),penDE(乙酰辅酶A:6-APA乙酰转移酶),pcl(苯乙酰辅酶A连接酶),等;或头孢霉素合成的酶的基因:pcbAB(α-氨基己二酰-半胱氨酰-缬氨酸合成酶),pcbC(异青霉素N合成酶),cefD(异青霉素N异构酶),cefEF(脱乙酰氧基头孢霉素C合成酶/羟化酶),cefG(脱乙酰基头孢霉素C乙酰基转移酶)等。待表达基因可通过不同的方法得到:从染色体DNA分离,从mRNA的cRNA合成,化学合成等。用于正确的启动子-基因融合的基本方法描述于Sambrook,J.et al.,(1989),分子克隆:实验室手册,冷泉港实验室,冷泉港,纽约,美国,以及Ausubel et al.,(1987),当代分子生物学方法,John Wiley&Sons,纽约,美国。
P.chrysogenum和A.chrysogenum被用作宿主菌株,但衍生于它们的任何相关的菌株或突变的菌株均可使用。用于产生原生质体和P.chrysogenum转化的方法是基于Cantoral等1987描述的方法(生物技术5:494-497)和Diez等1987描述的方法(当代遗传学12:277-282),如实施例1所述。用于产生原生质体和A.chrysogenum转化的方法描述于实施例4。在两种情况下使用抗生素腐草霉素作为选择标记,以及使用质粒pALfleo7,pALP480,pALPfleol或pALCfleol,这些质粒为分别在Pgdh,Phex,PactPc和PactAc控制下表达的bleR基因的载体。但是也可使用能够选择性地将转化菌株从非转化菌株中分离出来的任何标记。
该转化了可生长于含有可同化碳源和氮源的培养基中。碳源的例子有葡萄糖,蔗糖,乳糖,淀粉,甘油,有机酸,酒精,脂肪酸,等,它们可以单独使用或组合使用。氮源的例子有胨,麦芽提取物,酵母提取物,玉米浸液,谷朊,尿素,铵盐,硝酸盐,NZ-胺,硫酸铵,等,它们可单独使用或结合使用。可以作为培养基成分使用的无机盐包括磷酸盐(如磷酸钾),硫酸盐(如硫酸钠),盐酸盐(如氯化镁),等,以及铁,镁,钙,锰,钴,等可被用作离子。必须对培养条件,如温育温度,培养基的pH,通气,温育时间等根据所用菌株进行选择和调整。但总的来说,发酵是在通气条件下,在20℃至30℃温度之间,在pH5至9之间进行4至14天。
总之,本发明包括:(Ⅰ)含有P.chrysogenum的gdh,hex和act基因及A.chrysogenum的act基因的启动子的DNA片段,(Ⅱ)整合有上述启动子以及它们的转译起始位点的质粒,(Ⅲ)其中一种同源或异源结构基因或反义DNA片段处于所说的启动子控制之下的质粒,(Ⅳ)用所说的质粒转化的P.chrysogenum或A.chrysogenum菌株,(Ⅵ)能够表达存在于质粒中处于该启动子的控制之下的结构基因或反义DNA的转化菌株。和(Ⅶ)能够分泌在Phex的控制之下的同源或异源胞外蛋白质的转化菌株。
下述实施例将详细描述本发明,但不限定其范围。实施例1
1.1 P.chrysogenum的gdh基因的克隆和表征
为了克隆P.chrysogenum的gdh基因,在噬菌体载体λGEM12中构建一个DNA文库,所用方法为公知方法(Sambrook,J.et al.,(1989),分子克隆:实验室手册,冷泉港,纽约,美国)。为了这一目的,将真菌的总DNA(由Barredo等(1994),西班牙专利P9400931的方法纯化)用Sau3AⅠ进行部分消化,并将约20kb的片段在一蔗糖梯度(10-40%)中纯化。将这些片段与载体的臂相连,该载体已用BamHⅠ消化并纯化,然后将连接混合液使用GigapackⅡ Gold(Stratagene)系统按照制造商的说明进行包装。将悬浮于500μl的SM的包装反应液用于感染大肠杆菌LE392,以滴定存在的噬菌体的数目,并感染大肠杆菌NM539,以确定重组噬菌体的百分数。大肠杆菌539是一种噬菌体P2的溶源性菌株,并且只有当感染它的噬菌体缺少非必需的中心区域时产生溶菌班。测得的噬菌体的滴度在大肠杆菌LE392中为132pfu/μl(总共66000pfu),在大肠杆菌NM539中为113pfu/μl(总共56500pfu)。这意味着约85%的噬菌体携带有外源DNA插入。制备一个完整的DNA文库所需要的重组噬菌体的数量是用以下公式计算的:
N=ln(1-P)/ln(1-f),其中“P”为所需几率,“f”为包含在重组子中的所选择的生物体的基因组的比例,N为所需要的重组子的数量。假设P.chrysogenum的基因组的含有量为约3000kb(Fierro等(1993),分子基因遗传学241:573:578)并且包装插入物的平均大小为18kb(尽管已分离出的大小为约20kb),根据得到的重组噬菌体的数目,得到的P.chrysogenum DNA文库具有99.999%的几率。在这一系列理论验算之后,将大将杆菌NM539感染,将完全DNA文库涂布于150mm直径的5个Petri盘上(约11300pfu/Petri盘),收集于50ml SM和2.5ml氯仿中,保存于4℃。以这一方式即可得到重组噬菌体的足够的和具代表性的量,以便于在任何时间进行涂布。
将约60000pfu涂布在3个150mm直径的Petri盘上,然后转移至硝酸纤维素滤膜上(BA85,0.45μm,Schleicher&Schuell)。将所说的滤膜用标准方法(Sambrook,J,等(1989),分子克隆:实验室手册,冷泉港实验室,冷泉港,纽约,美国),与相应于N.crassa的gdh基因的合成寡核苷酸杂交。将总共10个阳性克隆通过第二和第三杂交循环进行纯化,然后将它们的DNA用一系列的限制性内切酶消化,并用Southern印迹技术进行分析。以这种方式,gdh基因在一个7.2kb的EcoRⅠ片段和两个分别为2.9和1.5kb的BamHⅠ片段中被识别出来。在质粒pBluescriptⅠ KS(+)(Stratagene)和pUCc13中相应的亚克隆之后,构建出质粒pALP784和pALP785,它们与包括gdh基因的2.9kb的Sau3AⅠ-XbaⅠ片段的两个方向相匹配。包括所说基因的DNA区段的限制性图谱示于图1。
为确定gdh基因的核苷酸序列,从质粒pALP784和pALP785通过“抹去一个碱基(Erase a base)”方法(Promega)构建出一系列克隆,然后通过双脱氧核苷酸方法,使用“测序酶”检测试剂盒并按照制造商的说明进行测序。将得到的2816个核苷酸的序列(SEQ ID N0:1)用Geneplot程序(DNASTAR)进行分析,证实了具有非常明显的优先密码子使用类型的ORF的存在。发现ATG转移起始密码子ATG位于922位,以及TAA终止密码子位于2522位。也确定了159bp和56bp的两个内含子分别位于971-1130之间和1262-1318之间。所说的ORF编码一个49837 Da的蛋白质,其等电点为6.18。其461个氨基酸的序列(SEQ ID NO:5)与N.crassa的NADP依赖性谷氨酸脱氢酶的氨基酸序列具有72.4%的一致性。
在启动子区域发现存在有各种嘧啶富集的区域,虽然位于766-796之间的那个是最广泛的一个。这些区域发现于高度表达的基团中,并位于转录起始位点的紧邻的上游。此外有两个推测的TATA盒(其在真菌中的共有序列为TATAAA)位于752位(TATATAATT)和852位(TATAATTT)。在真菌中这些TATA盒发现于转录起始位点上游30至50bp处,因而真正的TATA盒很可能是位于752位,即转录起始位点42bp处的那个。CCAAT序列发现于约30%的已知的真核基因的启动子区域。位于转录起始点上游50和200bp之间。该CCAAT盒在gdh基因的启动子区域中位于691位,即推测的转录起始位点上游约105pb处。
1.2 P.chrysogenum和A.chrysogenum中gdh启动子下控制基因的表达
P.chrysogenum和A.chrysogenum的转化及其转化体的筛选是根据它们对抗生素腐草霉素的抗性通过下述方法进行的。为了这一目的,需要构建质粒pALfleo7,其大小为5.4kb,并携带在gdh控制之下表达的S.hindustanus的bleR基因作为真菌中的标记,氯霉素抗性基因作为大肠杆菌中的标记以及质粒pBC KS(+)(Stratagene)的多接头。
用于原生质体的制备和P.chrysogenum的转化的方法是Cantoral等1987(生物技术5:494-497)和Diez等1987(现代遗传学12:277-282)描述的方法,但进行了少许改进。首先,将P.chrysogenum在加入了10%的酵母提取液的PM确定成分培养基(Anne,J.,(1997),Agricultura 25)中在25℃下生长18-21小时,将菌丝体通过一个尼龙滤膜过滤进行回收,并用3-5倍体积的0.9%NaCl洗涤。在将其在滤纸之间干燥之后,将其悬浮于原生质体缓冲液中(100mg/ml)。当菌丝体悬浮液被认为均匀之后,将一定体积的原生质体缓冲液中的4mg/ml Caylasa溶液(Cayla)加入其中,并将其在25℃在100r.p.m的振动条件下温育3小时。用显微镜观察原生质体的出现。当它们中的大部分被释放出来之后,将它们通过一个30μm孔径的尼龙滤膜与菌丝体分离。将原生质体悬液用0.7M KCl洗三次,在两次洗涤之间在400xg离心3分钟。将沉淀的原生质体重新悬浮于10mlKCM溶液中,并通过在Thona腔室中计数估测其浓度之后,将其用KCM调整至1-5×108原生质体/ml。之后,将100μl的这种溶液小心地与1-10μgDNA加上10μl PCM混合,并将该混合物在冷水中孵育20分钟。然后加入500ml的PCM并将该混合物在环境温度下放置20分钟,之后加入600μl的KCM。根据质粒pALfleo7,pALP480和pALPfleol存在的腐草霉素抗性基因所赋予的在30μg/μl腐草霉素中生长的能力对转化子进行筛选。为了这一目的,将200μl的转化反应物与加入了山梨醇(1M)和腐草霉素(30μg/μl)的5ml Czapeck’s培养基混合,然后将其涂布在装有5ml相同培养基的Petri盘上。将盘在25℃温育,直至看到转化子的出现(4-8天)。
用于原生质体的制备和P.chrysogenum的转化的方法是Gutierrez etal.,(1991),Gen,Genet.225:56-64描述的方法。首先,将A.chrysogenum菌株在MMC确定成分培养基中在28C下生长20-24小时,将菌丝体通过一个尼龙滤膜过滤进行回收,并用3-5倍体积的0.9%NaCl洗涤。在将其在滤纸之间干燥之后,将其悬浮于原生质体缓冲液中(50mg/ml)。当菌丝体悬液被认为均匀之后,将终浓度为10mM的DTT加入其中,并将其在28℃在150r.p.m的振动条件下温育1小时。然后在12000xg离心15分钟,并将沉淀物重悬于20ml原生质体缓冲液中。然后将一定体积的原生质体缓冲液中的4mg/ml Caylasa溶液加入其中,并在25℃,在100r.p.m振荡培养3小时。用显微镜观察原生质体的出现。当它们中的大部分被释放出来之后,将它们通过一个25μm孔径的尼龙滤膜与菌丝体分离。将原生质体悬液用0.7M KCl洗三次,在两次洗涤之间在1000xg离心3分钟。将沉淀的原生质体重新悬浮于10ml NCM溶液中,并通过在Thoma腔室中计数估测其浓度之后,将其调整至1-5×108原生质体/ml。之后,将100μl的这种溶液小心地与1-10μg DNA混合,并将该混合物在冷水中孵育20分钟。然后加入1ml的CCM并将该混合物在环境温度下再放置20分钟。将混合物在1000xg离心5分钟,并将沉淀物重新悬浮于800μl的NCM溶液中。根据质粒pALfleo7和pALCfleol存在的腐草霉素抗性基因所赋予的在10μg/μl腐草霉素中生长的能力对转化子进行筛选。为了这一目的,将200μl的转化反应物与加入了蔗糖(0.3M)和腐草霉素(10μg/μl)的5ml TSA培养基混合,然后将其涂布在装有5ml相同培养基的Petri盘上。将盘在28℃温育,直至看到转化子的出现(5-8天)。
在得到的转化子中进行如下分析:(Ⅰ)相应于转化中使用的质粒DNA的存在,(Ⅱ)相应于控制基因的转录子的存在,和(Ⅲ)相应于基因表达的酶活性。按照Barredo等1994(西班牙专利P9400931)描述的条件获得总DNA,然后使用Sambrook等1989(分子克隆:冷泉港实验室,冷泉港,纽约,美国)描述的方法进行Southern印迹分析。总RAN的纯化是通过Ausubel等1987(当代生物学方法,John&Sons,纽约,美国)描述的方法进行的。将得到的RNA以沉淀状态保存于-20℃乙醇中。使用时,通过在4℃,10000xg离心20分钟回收。基于它们的分子量对RNA分子的分离是用琼脂糖-甲醛电泳进行的。然后将RNA转移至硝酸纤维素滤膜上并与期望的探针杂交,所有这些是通过Sambrook,等1989描述的方法进行的(分子克隆:实验室手册,冷泉港实验室,冷泉港,纽约,美国)。杂交带的出现表明了转录物的存在并因而表明了在宿主真菌中:P.chrysogenum和A.chrysogenum中细菌基因表达的能力。通过Sambrook等1989(分子克隆:实验室手册,冷泉港实验室,冷泉港,纽约,美国)描述的方法评估转化子中β-半乳糖苷酶的活性。腐草霉素抗性基因的表达基于P.chrysogenum和A.chrysogenum在Czapeck1s固体培养基上在25℃温育7天后的抗性水平进行评估的。
1.2.1大肠杆菌的lacZ基因在pgdh控制下在P.chrysogenum和大肠杆菌中的表达
将大肠杆菌的lacZ基因Pgdh可转译地融合,以在P.chrysogenum中表达它。为达到这一目的,将lacZ基因亚克隆在质粒pMLl的EcoRⅠ和SalⅠ之间(Carramolino等1989,基因77:31-38),产生质粒pMLac。然后将Pgdh导入pMLac的EcoRⅠ和SmaⅠ位点之间,产生质粒pSKG(图5)。最后,将Sulphonamide抗性基因(Carramolino等1989,基因77:31-38)导入在pSKG的EcoRⅠ位点,产生质粒pSKGSu(图5)。在由其Sulphonamide抗性筛选的用质粒pSKGSu转化的P.chrysogenum转化子中,通过Southern印迹技术分析质粒的存在,通过Northern印迹技术分析相应于lacZ基因的转录子的存在。然后测定上述两个分析中为阳性的转化子中β-半乳糖苷酶的活性。这些转化子有效地表达了大肠杆菌的lacZ基因,并且观察到含有一个整合进其基因组的质粒拷贝的转化子中β-半乳糖苷酶的活性水平高于在色氨酶C基因启动子(trpC)的控制之下表达lacZ基因的单拷贝转化子中的半乳糖苷酶的活性。
为确定P.chrysogenum的Pgdh是否也能在大肠杆菌中引导lacZ基因的表达,将质粒pSKG导入大肠杆菌DH5α(ΔlacZ)中。获得的转化子在25℃LB培养基中温育10天后能够产生蓝色菌落,该培养基中加入了异丙基-β-D-半乳糖苷(IPTG)和5-溴-4-氯-3-吲哚基-β-D-半乳糖苷(X-gal)。这一结果证实了Pgdh-lacZ构建体在大肠杆菌中表达β-半乳糖苷酶活性,虽然其效率低于大肠杆菌的内源性lacZ基因。
1.2.2 S.hindustanus的bleR基因在Pgdh的控制下在P.chrysogenum和A.chrysogenum中的表达
没有启动子区域的bleR基因以1100bp的NcoⅠ-ApaⅠ片段得自质粒pUT737(Mullaney等(1985),分子基因遗传学199:37-45)。然后将这一片段亚克隆进事先用NcoⅠ-ApaⅠ消化的质粒pUT713中,产生质粒pALfleo5。Pgdh以726bp EcoR1-BamHⅠ片段回收自pALP25,然后将其亚克隆进pALPfleo5(事先用EcoRⅠ-BamHⅠ消化),产生pALfleo6。最后的这一质粒具有4.2kb大小,在Pgdh控制之下表达的bleR基因和作为大肠杆菌中的标记的氨苄西林抗性基因。为了将后一标记用氯霉素抗性基因替代,将一个包括Pgdh,bleR和trpC基因(TtrpC)的终止子的1900bp的EcoRⅠ-NotⅠ片段从pALfleo6中纯化出来并连接进用EcoRⅠ-NotⅠ消化的质粒pBCKS(+)(Stratagene)中。这样产生质粒pALfleo7(图5),其大小为5.4kb,并携带在Pgdh控制之下的S.hindustanus的bleR基因作为在真菌中的选择性标记,氯霉素抗性基因作为在大肠杆菌中的标记以及质粒pBCKS(+)的多接头。在Pgdh和bleR之间的融合区段的序列分析证实了后一基因以正确的阅读框排列。
P.chrysogenum和A.chrysogenum的转化是用质粒pALfleo7进行的,转化子的筛选是基于它们分别对30μg/ml和10μg/ml的腐草霉素的抗性来进行的。然后在固体培养基上确定转化子对腐草霉素抗性的最高水平,得到了一些可在高于100μg/ml腐草霉素的存在下生长的转化子。在用它们的腐草霉素抗性选出的转化子中,通过Southern印迹技术对该质粒的存在进行分析,通过Northern印迹技术对相应于bleR基因的转录子的存在进行分析,两种情况下均得到阳性结果。这些结果证实了在Pgdh的控制下在P.chrysogenum和A.chrysogenum中表达异源基因的可能性。
将质粒pAlfleo7导入大肠杆菌,以确定P.chrysogenum的Pgdh是否也能在大肠杆菌中引导bleR基因的表达。获得的转化子具有在加有0.2μg/ml腐草霉素的LB上生长的能力,腐草霉素的最小抑制浓度对于大肠杆菌来说小于0.025μg/ml。这一结果证实了在大肠菌中表达了Pgdh,虽然低于在P.chrysogenum中的表达效率。一个用质粒pALfleo7转化大肠杆菌DH5α的转化子已保藏于西班牙典型培养物保藏中心(CECT),保藏号为CECT4849。其它质粒,如pALP784和pALP785可从保藏的质粒简单地通过用包括在pALfleo7中的gdh基因启动子杂交,选择出2.9kb的Sau3AⅠ-XbaⅠ片段,并分别将其亚克隆进pBluescript I KS(+)或pUC13中来获得。
1.3.在gdh启动子控制下在P.chrysogenum和A.chrysogenum中的反义表达
在工业菌株中对基因表达的抑制有时需要除去不期望的酶活性。由于许多工业菌株的倍体水平使它在很多情况下难以通过直接的基因破坏来阻止表达,需要使用不依赖于倍体水平的表达失活系统。在强启动子控制下表达的反义构建体的开发使得基因表达的阻断成为可能。
以举例的方式,下文描述了在P.chrysogenum中使用Pgdh使编码乙酸苯酯2-羟化酶(pahA)的基因的表达失活。首先构建质粒pALP873,它携带通过单一BamHⅠ位点融合的Pgdh和TtrpC。将质粒pALP873用BamHⅠ消化,其末端用DNA聚合酶Ⅰ的Klenow片段补平,并将其与pahA基因中的,从质粒pALP555通过EcoRV消化获得的1053pb的cDNA连接。选出所产生的质粒,称作pALP874,因为它携带相对于Pgdh的反义pahA基因片段。从这一质粒中纯化出2.5kb的EcoRⅠ-XbaⅠ片段,它携带反义盒,并用Klenow补平,亚克隆在质粒pALfleo7的EcoRV位点,产生质粒pALP888。该后一个质粒的特征在于,其大小为7.9kb,并带有(Ⅰ)在Pgdh控制之下的pahA基因的反义盒,(Ⅱ)作为基真菌中的选择标记的bleR基因,(Ⅲ)作为在大肠杆菌中的选择标记的氯霉素抗性基因和(Ⅳ)质粒pBC KS(+)的多接头。
P.chrysogenum的转化是用质粒pALP888进行的,转化子是基于它们对30μg/ml腐草霉素的抗性进行筛选的。在选出的转化子中,约20%显示出氧化苯乙酸的能力降低,它们中的一些缺乏可检测水平的所说活性。在这些转化子中,通过Southern印迹技术分析该质粒的存在,通过Northern印迹技术分析相应于pahA基因的反义转录子的存在,使用了相应于编码链的寡核苷酸作为探针。在两种情况下均得到阳性结果,证实了在P.chrysogenum中通过使用反义构建体全部或部分阻断不期望的酶活性的可能性。使用本发明所述的任一种启动子(Pgdh,Phex,PactPc和PactAc)或任一种可获得的启动子,这些结果可被推论至相关的丝状真菌和任一种酶活性。实施例2
2.1 P.chrysogenum的hex基因的克隆和表征
在从青霉素G生产条件下的工业发酵液中得到的P.chrysogenum菌丝体中确定一种主要蛋白质的存在,该蛋白质在纯化和表征后发现为β-N-乙酰氨基己糖苷酶。通过Edman’s降解方法确定该纯化的蛋白质的氨基末端氨基酸序列,得到两种不同的序列:(A) Ala-Pro-Ser-Gly-Ile-His-Asn-Val-Asp-Val-(His)-Val-
Val-(Asp)-Asn-(Asp)-Ala-(Asp)-Leu-Gln-Tyr-(Gly)(B) Val-Gln-Val-Asn-Pro-Leu-Pro-Ala-Pro-(Arg)-(Arg)-
Ile-(Thr)-???-(Gly)-(Ser)-(Ser)-(Gly)-(Pro)-
(Ile/Thr)-???-(Val)
基于这些序列,并采用存在于一系列的P.chrysogenum基因的密码子使用趋势,设计了下列组合的合成寡核苷酸:(Ⅰ) 5' TCGACGACGTGSACGTCSACGTTGTGGATGCC 3'(Ⅱ) 5' CCGTAYTGSAGGTCRGCGTCGTTGTCGACGAC 3'(Ⅲ) 5' GGGGCVGGSAGVGGGTTGACYTG 3'
使用DNA文库和实施例1中所述的方法将P.chrysogenum的hex基因克隆。将总共11个阳性克隆纯化,然后将它们的DNA用一系列的限制性内切酶消化并用Southern印迹技术分析。以这种方式,hex基因在一个3.2kb的SacⅠ片段和一个2.1kb的SalⅠ片段中被识别出来。SalⅠ片段在质粒pBCKS(+)(Stratagene)中在两个方向上的亚克隆产生质粒pALP295和pALP303。包括hex基因的DNA区段的限制性图谱示于图2。
为了确定hex基因的核苷酸序列,使用了上述质粒pALP295和pALP303,以及pALP319和pALP461(两个定向的2.8kb的BamHⅠ片段),pALP388和pALP389(两个定向的2.4kb的SalⅠ片段)和pALP377和pALP378(两个定向的1.2kb的PstⅠ片段)(图2)。从所说的质粒通过“擦去一个碱基(Erase a base)”方法(Promega)构建一系列的克隆,然后使用“Sequenase”测试试剂盒(USB)通过双脱氧核苷酸方法进行测序,这些都是按照制造商的说明进行的。将得到的5240个核苷酸序列(SEQID N0:2)用Geneplot程序(DNASTAR)进行分析,证实了两个具有非常明显的优先密码子使用类型的ORF的存在。发现hex基因的ATG转译起始密码子位于1324位,TGA终止密码子3112位。所说的ORF缺少内含子,并编码一个66545 Da的蛋白质,其等电点为5.34,其596个氨基酸序列(SEQID NO:6)与Candida albicans的β-N-乙酰氨基己糖苷酶具有49.0%的一致性。此外,在19-40和99-120位,推测的氨基酸序列包括从纯化的酶化学确定的氨基酸序列。一个蛋白酶识别位点(Lys-Arg)出现在紧邻氨基酸序列(A)(氨基酸97-98)的位点。
在启动子区域,在1106-1128位之间和1182-1200位之间发现有两个嘧啶富集区,一个在1258位(ATAAATA)的推测的TATA盒和一个在1163位的CAAT盒。
2.2在Phex控制下在P.chrysogenum中S.hindustanus的bleR基因的表达
如实施例1的1.2节所述进行下述过程:(Ⅰ)P.chrysogenum转化子的转化和筛选,(Ⅱ)DNA分析,(Ⅲ)RNA分析和(Ⅳ)酶测定。
为了在Phex控制下表达bleR基因,首先,在编码hex基因的起始甲硫氨酸的ATG密码子的上面构建NcoⅠ位点。这是通过使用下述寡核苷酸作为引物的PCR进行的。
5'CTCCATGGTGATAAGGTGAGTGACGATG 3'
5'GTAAAACGACGGCCAGTG 3'(引物 -20)
将通过PCR得到的DNA片段以两个方向亚克隆在质粒pBC KS(+)(Stratagene)的SmaⅠ位点,产生pALP427和pALP428。使用测试试剂盒“擦去一个碱基”(Promega)和“Sequenase”(USB),按照制造商的说明将两个质粒的插入物进行测序。以这一方式显示了得到的Phex缺乏突变,并包括编码该蛋白质的起始甲硫氨酸的ATG上面的NcoⅠ位点。
pALP427是为进行bleR基因的亚克隆而选择出的质粒。不带有其启动子区域的bleR基因是从质粒pUT737(Mullaney等(1985),分子基因遗传学199:37-45)以1100bp的NcoⅠ-ApaⅠ片段得到的。然后将这一片段亚克隆进预先用NcoⅠ-ApaⅠ片段消化的质粒pALP427(携带Phex)中,产生质料pALP480(图6)。这一最后质粒具有5.4kb的大小,在Phex控制下表达的bleR基因,在bleR基因控制下的trpC基因的终止子,氯霉素抗性基因作为大肠杆菌中的标记以及质粒pBC KS(+)的多接头。在Phex和bleR之间的融合区段的序列分析证实了后一个基因以正确的阅读框排列。
P.chrysogenum的转化是用质粒pALP480进行的,转化子的筛先是基于它们对30μg/ml的腐草霉素的抗性进行的,然后在固体培养基上确定转化子腐草霉素抗性的最高水平,得到了一些可在大于100μg/ml腐草霉素的存在下生长的转化子。在由它们的腐草霉素抗性筛选的转化子中,通过Southern印迹技术分析质粒的存在,通过Northern印迹技术分析相应于bleR基因的转录子的存在,在两种情况下均得到阳性结果。这些结果证实了在Phex的控制下在P.chrysogenum中表达外源基因的可能性。用质粒pALP480转化大肠杆菌DH5α的转化子已保藏于西班牙典型培养物保藏中心(CECT),保藏号为CECT4852。质粒pALP295,pALP319,pALP377和pALP388可从保藏的质粒简单地通过使用包括在pALP480中的hex基因的启动子杂交,分别选择出2.1kb的SalⅠ片段,2.8kb的BamHⅠ片段,1.2kb的PstⅠ片段和2.4kb的SalⅠ片段,然后将它们亚克隆进pBluescriptⅠ KS(+)中而得到。
2.3使hex基因在P.chrysogenum中蛋白质的胞外产生。
β-N-乙酰氨基己糖苷酶是一种由P.chrysogenum在青霉素G生产条件下的工业发酵中大量分泌至培养基中的蛋白质。该酶可被分泌的能力使得可能使用hex基因在P.chrysogenum或相关的丝状真菌中表达和分泌同源或异源蛋白质。
该酶具有由下列氨基酸组成的分泌信号序列:
Met-Lys-Phe-Ala-Ser-Val-Leu-Asn-Val-Leu-Gly-Ala-Leu-Thr-Ala-Ala-Ser-Ala(SEQ ID NO:6的氨基酸1到18)。总的来说,信号肽具有三个保守的结构域(Takizawa,N.等(1994)工业和农业应用的重组微生物,Murooka,Y.和Imanaka,T.(编辑),Marcel Dekker,Inc NewYork):(Ⅰ)一个被称为“n”的带正电荷的氨基末端区域,它通常具有1至5个残基,并且是蛋白质有效地进行跨膜转运所必需的(Met-Lys),(Ⅱ)一个被称作“h”的疏水区,由7至15个残基组成(Phe-Ala-Ser-Val-Leu-Asn-Val-Leu)和(Ⅲ)一个被称作“C”的在羧基端的极性区,由3至7个残基组成(Gly-Ala-Leu-Thr-Ala-Ala-Ser-Ala)。胞内合成的前蛋白通过由两个碱性残基(Lys-Arg,SEQ ID N0:6的氨基酸97和98)的切割位点进行加工,产生成熟蛋白质。
当使用hex基因表达和分泌蛋白质时有两种可能性:(Ⅰ)待表达基团的编码区域在一个阅读框内与启动子区域,包括分泌信号序列,融合,和(Ⅱ)待表达基因的编码区域在一个阅读框内与完全的hex基因融合。使用分子生物学标准技术(Sambrook,J.等(1989),分子克隆:实验室手册,冷泉港实验室,冷泉港,纽约,美国;Ausubel等(1987),当代分子生物学方法,John Wiley&Sons,纽约,美国),本领域普通技术人员将能够使用该启动子,包括hex基因的分泌序列,或完整基因在P.chrysogenum或相关丝状真菌中表达和分泌所需的蛋白质。实施例3
3.1 P.chrysogenum的act基因的克隆和表征
使用该DNA文库和实施例1所述的方法克隆P.chrysogenum的act基因。在这种情况下,使一个888bp的NcoⅠ-ClaⅠ片段进行杂交,该片段来源于A.nidulans的act基因(Fidel等(1988),基因70:283-293)。纯化了总共10个阳性克隆,然后将其DNA用一系列的限制性内切酶消化,并用Southern印迹技术分析。以这种方式,act基因在一个5.2kb的BamHⅠ片段,一个4.9kb的EcoRⅠ片段和一个5.9kb的HindⅢ片段中被识别出来。将该HindⅢ片段以两个方向亚克隆在质粒pBluescript I KS(+)(Stratagene)中,产生质粒pALP298和pALP299。EcoRⅠ片段以两个方向亚克隆在质粒pBluescript I KS(+)(Stratagene)中产生质粒pALP315和pALP316。包括act基因的DNA区段的限制酶图谱示于图3。
为确定act基因的核苷酸序列,使用了上述质粒pALP315和pALP316。用“擦去一个碱基”方法(Promega)从所说的质粒构建出一系列的克隆,然后使用“Sequenase”测试试剂盒(USB)通过双脱氧核苷酸方法测序,两者均是按照制造商的说明进行的。用Geneplot程序(DNASTAR)分析了所获得的2994个核苷酸的序列(SE Q ID N0:3),证实一个具有明显的优选密码子使用类型的ORF的存在。发现该act基因的ATG转译起始密码子位于494位,TAA终止密码子位于2250位。所说ORF在501-616,649-845,905-1046,1078-1180和1953-2021位具有5个内含子,并编码一个41760 Da的蛋白质,其等电点为5.51,其375个氨基酸的序列(SEQID NO:7)与A.nidulans的γ-肌动蛋白的氨基酸序列具有98.1%的一致性。在启动子区域发现两个在356-404和418-469之间的广泛的嘧啶富集区,一个在259位(TATAAAAAT)的推测的TATA盒和4个在174,217,230和337位的CAAT盒。
3.2在PactPc控制之下在P.chrysogenum中bleR基因的表达
为了在PactPc控制下表达bleR基因,首先,在编码hex基因的起始甲硫氨酸的ATG密码子的上面构建NcoⅠ位点。这是通过使用下述寡核苷酸作为引物的PCR进行的。
5'CTCCATGGTGACTGATTAAACAAGGGAC 3′
5'GTAAAACGACGGCCAGTG 3'(引物 -20)
将通过PCR得到的DNA片段以两个方向亚克隆在质粒pBC KS(+)(Stratagene)的SmaⅠ位点,产生pALPact1和pALPact2。使用测试试剂盒“擦去一个碱基”(Promega)和“Sequenase”(USB),按照制造商的说明将两个质粒的插入物进行测序。以这一方式显示了得到的PactPc缺乏突变,并包括编码该蛋白质的起始甲硫氨酸的ATG上面的NcoⅠ位点。
pALPact1是为进行bleR基因的亚克隆而选择出的质粒。不带有其启动子区域的bleR基因是从质粒pUT737(Mullaney等(1985),分子基因遗传学199∶37-45)以1100bp的NcoⅠ-ApaⅠ片段得到的。然后将这一片段亚克隆进预先用NcoⅠ-ApaⅠ片段消化的质粒pALPact1(携带PactPc)中,产生质粒pALPfleol(图6)。这一最后质粒具有在PactPc控制下表达的bleR基因,在bleR基因控制下的trpC基因的终止子,氯霉素抗性基因作为大肠杆菌中的标记以及质粒pBC KS(+)的多接头。在PactPc和bleR之间的融合区段的序列分析证实了后一个基因以正确的阅读框排列。
P.chrysogenum的转化是用质粒pALPfleol进行的,转化子的筛选是基于它们对30μg/ml腐草霉素的抗性进行的,然后在固体培养基上确定转化子腐草霉素抗性的最高水平,得到了一些可在大于100μg/ml腐草霉素的存在下生长的转化子。在由它们的腐草霉素抗性筛选的转化子中,通过Southern印迹技术分析质粒的存在,通过Northern印迹技术分析相应于bleR基因的转录子的存在,在两种情况下均得到阳性结果。这些结果证实了在PactPc的控制下在P.chrysogenum中表达外源基因的可能性。用带有act基因的质粒pALP315转化大肠杆菌DH5α的转化子已保藏于西班牙典型培养物保藏中心(CECT),保藏号为CECT4851。质粒pALP316可从保藏的质粒pALP315简单地通过将pALP315插入物以相反的方向亚克隆进pBluescript I KS(+)的EcoRⅠ位点而得到。实施例4
4.1 A.chrysogenum的act基因的克隆和表征
为了克隆A.chrysogenum的gdh基因,如实施例1的1.1节所述,在噬菌体载体λGEM12中构建一个DNA文库。得到的噬菌体滴度在大肠杆菌LE392中为50pfu/μl(总共25000pfu),在大肠杆菌NM539中为41pfu/μl(总共20500pfu)。这意味着约82%的噬菌体携带一个外源DNA片段,并且得到的A.chrysogenum DNA文库具有99.999%的几率。在进行了这一系列的理论验算之后,将大肠杆菌NM539感染,并将全DNA文库涂布在3个150mm直径的Petri盘上(约7000pfu/Petri盘),收集于50mlSM加上2.5ml氯仿中,并在4℃保存。以这种方式,得到了足够和具代表性的量的重组噬菌体(2100pfu/μl),以备在任一时间进行涂布。
将约20000pfu涂布在2个150mm直径的Petri盘上,然后转移至硝酸纤维素膜上(BA85,0.45μm,Schleicher&Schuell)。将所说的滤膜使用标准方法(分子克隆:实验室手册,冷泉港实验室,冷泉港,纽约,美国)用一个相应于A.nidulans的act基因的888bp的NcoⅠ-ClaⅠ片段杂交。纯化出总共5个阳性克隆,然后将它们的DNA用一系列的限制性内切酶消化并用Southern印迹技术分析。以这种方式act基因在一个8.7kb的HindⅢ片段中被识别出来。将这一片段以两个方法亚克隆在质粒pBLuescript I KS(+)(Statagene)中,产生质粒pALC52和pALC53。包括act基因的DNA区段的限制酶图谱示于图4。
将上述质粒pALC52和pALC53用于确定act基因的核苷酸序列。用“擦去一个碱基”方法(Promega)从所说的质粒构建出一系列的克隆,然后使用“Sequenase”测试试剂盒(USB)通过双脱氧核苷酸方法测序,两者均是按照制造商的说明进行的。用Geneplot程序(DNASTAR)分析了所获得的3240个核苷酸的序列(SE Q ID NO:4),证实一个具有明显的优先密码子使用类型的ORF的存在。发现该act基因的ATG转译起始密码子位于787位,TAA终止密码子位于2478位。所说ORF在794-920,952-1123,1180-1289,1321-1410和2183-2249位具有5个内含子,并编码一个41612Da的蛋白质,其等电点为5.51,其375个氨基酸的序列(SEQ ID NO:8)与A.nidulans和P.chrysogenum的γ-肌动蛋白的氨基酸序列分别具有98.4%和98.1%的一致性。在启动子区域发现一个在607-654之间的嘧啶富集区,一个在747位(TTATAAAA)的推测的TATA盒和1个在338位的CAAT盒。
4.2在A.chrysogenum中在PactAc控制下bleR基因的表达
为在PactAc控制下表达bleR基因这一目的,构建了质粒pALCfleol(图6),它包括在PactAc控制下表达的bleR基因,在bleR基因下的trpC基因的终止子,作为在大肠杆菌中的标记的氯霉素抗性基因以及质粒pBCKS(+)的多接头。
不带有其启动子区域的bleR基因是从质粒pUT737(Mullaney等(1985)分子基因遗传学199∶37-45)以1100bp的NcoⅠ-ApaⅠ片段得到的。借助于act基因在编码该蛋白质的起始甲硫氨酶的ATG上面具有的一个NcoⅠ位点,将该片段与PactAe同框融合。为了之一目的,将bleR基因亚克隆在事先用NcoⅠ-ApaⅠ消化的质粒pALCactl(携带PactAc)中,产生质粒pALCfleo1(图6)。在PactAc和bleR基因之间的融合区域的序列分析证实了后一基因以正确的阅读框排列。
A.ehrysogenum的转化是用质粒pALCfleol进行的,转化子的筛选是基于它们对10μg/ml腐草霉素的抗性进行的,然后在固体培养基上确定转化子腐草霉素抗性的最高水平,得到了一些可在大于30μg/ml腐草霉素的存在下生长的转化子。在由它们的腐草霉素抗性筛选的转化子中,通过Southern印迹技术分析质粒的存在,通过Northern印迹技术分析相应于bleR基因的转录子的存在,在两种情况下均得到阳性结果。这些结果证实了在PactAc的控制下在A.chrysogenum中表达外源基因的可能性。用带有act基因的质粒pALC52转化大肠杆菌DH5α的转化子已保藏于西班牙典型培养物保藏中心(CECT),保藏号为CECT4850。质粒pALC53可从保藏的质粒pALC52简单地通过将pALC52插入物以相反的方向亚克隆进pBluescript I KS(+)的HindⅢ位点而得到。
将存在于使用大肠杆菌作为宿主的插入物导入放线菌纲,Penicillium,Aspergillus,Acremonium或Saccharomyces只是一个常规技术和选择最适合的载体转化所说的属或纲的问题。
附图简要说明。
图1-P.chrysogenum的gdh基因的限制性图谱,它编码NADP-依赖性谷氨酸脱氢酶活性(EC.1.4.1.4)。
图2-P.ehrysogenum的hex基因的限制性图谱,它编码β-N-乙酰氨基己糖苷酶活性(EC.3.2.1.52)。
图3-P.chrysogenum的act基因的限制性图谱,它编码γ-肌动蛋白。
图4-A.chrysogenum的act基因的限制性图谱,它编码γ-肌动蛋白。
图5-在P.chrysogenum和/或A.chrysogenum中在启动子Pgdh控制下表达大肠杆菌的lacZ基因,S.hindustanus的bleR基因和P.chrysogenum的pahA基因的反义片段的载体。
图6-在启动子Phex,PactPc,PactAc控制下,在P.chrysogenum和/或A.chrysogenum中表达S.hindustanus的bleR基因的载体。
序列表(1)总信息:申请人:
名称:抗生素有限公司
街道: Avda.De Burgos,8-A
城市: Madrid
州或省:Madrid
国家: Spain
邮政编码:28036
电话:91-3841200
传真:91-3841220发明名称:谷氨酸脱氢酶,β-N-乙酰氨基几糖苷酶和γ-肌动蛋白基因
的启动子以及在丝状真菌的表达,分泌和反义系统中的应用序列数目:8通信地址:
收信人:ANTIBIOTICOS,S.A.U.
街道: Avda.De Burgos,8-A
城市: Madrid
州: Madrid
国家: Spain
邮政编码:28036计算机可读形式:
介质类型:3.5’软盘
计算机:PC
操作系统:WINDOWS
软件:WORD代理人信息:
姓名:ALBERTO DE ELZABURU
登记号:232/l
地址:Miguel Angel,21
28010-Madrid
通讯资料:
电话:3085900
传真:3193810
电传或E-mail:elzaburu@elzaburu.esSEQ ID NO:1的信息:
序列特征:
长度:2816碱基对
类型:核苷酸
链型:2
拓扑结构:线性
分子类型:基因组DNA
假设:否
反义:否
来源:Penicilllium chrysogenum
直接来源:质粒pALP784和pALP785
基因组中的位置:未知
特点:
名称/关键词:编码序列
位置:结合(922…970,1131…1261,1319…2521
特点:
名称/关键词:内含子
位置:971...1130
特点:
名称/关键词:内含子
位置:1262…1318
特点:
其它信息:gdh基因序列描述:SEQ ID NO:1GATCGCCGTT TATGGGATAG TGGGCACGTG ACAGAGCCTG CAGCCGAGTC AAATTGCCGA 60AGTTGGCAGT TGGTGGCGGA GAACTCGAGA TTTTATTTGC GTTTATTTCG TTTATTTCGA 120TTTTAGTTTT CCTATTTTTC CTATTTTGGT TGATTCCATC CAACTTTATA GGATACTACT 180TCATAATAGG TCGATCATAG TACAAGCACC AACTCGTCGC ATCATGCATT TTCTGGGGTT 240CGAATTCTTT ACTTAGAGTA AGGTTTCTCT CAGCCTCCTA ATAAACTACC TAGGTAGGTT 300AAATTTACTT TTTAACATTT TATTTATTCA GAAGATTGTC GGAGAGGACC GATCCGAAGG 360ACACGAATTG AACACGGAAG GGATATTAGG GACAAGGAAG ATTTAGGGAT AAAAAAACGA 420GCTGTGATTG ATGGGAAGGT TAAAGTGTAG TAATGAAGGT GATGGGACCA AAAGGAGTGG 460GAGAGATAAG CCAAATTCTG TGCAAATTCT GTGACCTTAA ACCATAAGAT AACATTGTTC 540GGGCCCCGAA CTTCGGACGT TCTTCCCACG GAAAGGCAAA TCATTGGGTT TCATCGATTC 600TCTTGGATCT TTATCCTAAT TCCCCGTGCA ACCTGGTCTT GGGGATTATT GTCGACTTGT 660AGGCGCATTA ACCCATCTCC CGTCTTCCCT CCAATCAATC CCGGATTCTC TCGTCCGACT 720CCGGCTTCGA CTCTCTCTCT CTCCACATCT CTATATAATT GTACACTCCC CCATCCCATT 780CTTTTCTTCT CTTCTCATCT ACTCTCTTGA ATCTCAATTG TCTTAATACT CTCTCTGCTC 840TTGTCTTTAT TTATAATTTA TTAGATCACT GCTTAGCATT GATCTACTTA CCTAAAAGCA 900GAGTTAACAG TACCGGCCGA A ATG ATG CAA AAC CTT CCC TTC GAG CCT GAG 951
Met Met Gln Asn Leu Pro Phe Glu Pro Glu
1 5 10TTC GAG CAG GCC TAC AAG G GTATGTCTCT TTTAATTTTT CCCTTTCTTA TTTCAA 1006Phe Glu Gln Ala Tyr Lys
15TTCCATATCG TCCATATCAC ACACTATTTC CCGACTCAAT TCCTTTACCC ATCGGCATCT 1066TCCCGGCCTT TGGCTCCACC GGGGGCATAA TTTCGGGGTG ACTCAGCTAA CAATCCCGAA 1126ACAG AG CTC GCC TCC ACT CTC GAG AAC TCC ACT CTT TTC CAG AAG AAG 1174
Glu Leu Ala Ser Thr Leu Glu Asn Ser Thr Leu Phe Gln Lys Lys
20 25 30CCC GAG TAC CGC AAG GCT CTT CAG GTC GTC TCT GTC CCC GAG CGT GTT 1222Pro Glu Tyr Arg Lys Ala Leu Gln Val Val Ser Val Pro Glu Arg Val
35 40 45ATT CAG TTC CGT GTT GTC TGG GAA GAT GAC AAA GGC CAG GTAAGACCTT 1271Ile Gln Phe Arg Val Val Trp Glu Asp Asp Lys Gly Gln
50 55 60TCTTTTTGAA AATGTCTAAT TAATTGCCAC ATGCTAATTC CGTTCAG GTC CAA ATC 1327
Val Gln IleAAC CGT GGA TAC CGT GTC CAG TTC AAC TCC GCT CTT GGC CCC TAC AAG 1375Asn Arg Gly Tyr Arg Val Gln Phe Asn Ser Ala Leu Gly Pro Tyr Lys
65 70 75GGT GGC CTC CGG TTC CAC CCC ACG GTG AAC CTT TCC ATC CTC AAG TTC 1423Gly Cly Leu Arg Phe His Pro Thr Val Asn Leu Ser Ile Leu Lys Phe80 85 90 95CTC GGT TTC GAG CAG ATC TTC AAG AAT GCC CTC ACC GGC CCG AAC ATG 1471Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu Thr Gly Leu Asn Met
100 105 110GGC GGT GGT AAG GGT GGA TCC GAC TTC GAC CCC AAG GGC AAG ACC GAT 1519Gly Gly Gly Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Thr Asp
115 120 125AAC GAG ATC CGC CGC TTC TGT GTC TCC TTC ATG ACC GAG CTG TGC AAG 1567Asn Glu Ile Arg Arg Phe Cys Val Ser Phe Met Thr Glu Leu Cys Lys
130 135 140CAC ATC GGT GCC GAC ACC GAT GTT CCC GCC GGT GAT ATC GGT GTG ACC 1615His Ile Gly Ala Asp Thr Asp Val Pro Ala Gly Asp Ile Gly Val Thr
145 150 155GGC CGC GAG GTT GGT TTC ATG TTC GGC CAG TAC AAG AAG ATC CGC AAC 1663Gly Arg Glu Val Gly Phe Met Phe Gly Gln Tyr Lys Lys Ile Arg Asn160 165 170 175CAG TGG GAG GGT GTC CTC ACC GGT AAG GGT GGC AGC TGG GGT GGT TCC 1711Gln Trp Glu Gly Val Leu Thr Gly Lys Gly Gly Ser Trp Gly Gly Ser
180 185 190CTC ATC CGC CCC GAG GCC ACC CGC TAC GGT GTC GTC TAC TAC GTC GAG 1759Leu Ile Arg Pro Glu Ala Thr Gly Tyr Gly Val Val Tyr Tyr Val Glu
195 200 205CAC ATG ATC CAG CAC GCC TCC GGC GGC AAG GAA TCC TTC GCT GGT AAG 1807His Met Ile Gln His Ala Ser Gly Gly Lys Glu Ser Phe Ala Gly Lys
210 215 220CGC GTC GCC ATC TCC GGT TCC GGA AAC GTC GCC CAG TAC GCC GCT CTC 1855Arg Val Ala Ile Ser Gly Ser Gly Asn Val Ala Gln Tyr Ala Ala Leu
225 230 235AAG GTC ATC GAG CTC GGT GGC TCC GTC ATC TCC CTC TCC GAC TCC CAG 1903Lys Val Ile Glu Leu Gly Gly Ser Val Ile Ser Leu Ser Asp Ser Gln240 245 250 255GGT GCT CTC GTC CTG AAC GGC GAG GAG GGC TCC TTC ACC GCT GAG GAG 1951Gly Ala Leu Val Leu Asn Gly Glu Glu Gly Ser Phe Thr ALa Glu Glu
260 265 270ATC AAC ACC ATC GCT GAG ATC AAG GTC CAG CGC AAG CAG ATC GCC GAG 1999Ile Asn Thr Ile Ala Glu Ile Lys Val Gln Arg Lys Gln Ile Ala Glu
275 280 285CTC GCT ACC CAG GAC GCC TTC AGC TCC AAG TTC AAG TAC ATC CCC GGT 2047Leu Ala Thr Gln Asp Ala Phe Ser Ser Lys Phe Lys Tyr Ile Pro Gly
290 295 300GCC CGC CCC TGG ACC AAC ATC GCC GGC CGC ATC GAT GTC GCT CTC CCC 2095Ala Arg Pro Trp Thr Asn Ile Ala Gly Arg Ile Asp Val Ala Leu Pro
305 310 315TCC GCC ACC CAG AAC GAG GTC TCC GGC GAT GAG GCC AAG GCT CTC ATC 2143Ser Ala Thr Gln Asn Glu Val Ser Gly Asp Glu Ala Lys Ala Leu Ile320 325 330 335GCC GCT GGC TGC AAG TTC ATC GCT GAG GGC TCC AAC ATG GGT TCC ACC 2191Ala Ala Gly Cys Lys Phe Ile Ala Glu Gly Ser Asn Met Gly Ser Thr
340 345 350CAG GAG GCT ATC GAT GTC TTC GAG GCC CAC CGT GAT GCC AAC CCT GGT 2239Gln Glu Ala Ile Asp Val Phe Glu Ala His Arg Asp Ala Asn Pro Gly
355 360 365GCC GCT GCC ATC TGG TAC GCC CCT GGT AAG GCC GCC AAC GCT GGT GGT 2287Ala Ala Ala Ile Trp Tyr Ala Pro Gly Lys Ala Ala Asn Ala Gly Gly
370 375 380GTT GCC GTC TCT GGT CTC GAG ATG GCC CAG AAC TCT GCC CGT GTC AAC 2335Val Ala Val Ser Gly Leu Glu Met Ala Gln Asn Ser Ala Arg Val Asn
385 390 395TGG TCC CGT GAG GAG GTT GAC TCC CGT CTT AAG AAG ATT ATG GAG GAC 2383Trp Ser Arg Glu Glu Val Asp Ser Arg Leu Lys Lys Ile Met Glu Asp400 405 410 415TGC TTC AAC AAC GGT CTC TCT ACT GCT AAG GAG TAT GTC ACC CCT GCT 2431Cys Phe Asn Asn Gly Leu Ser Thr Ala Lys Glu Tyr Val Thr Pro Ala
420 425 430GAG GGT GTT CTT CCT TCC CTC GTG GCT GGC TCC AAC ATT GCT GGT TTC 2479Glu Gly Val Leu Pro Ser Leu Val Ala Gly Ser Asn lle Ala Gly Phe
435 440 445ACC AAG GTC GCT GAG GCC ATG AAG GAG CAC GGT GAC TGG TGG TAAATTAGTC 2531Thr Lye Val Ala Glu Ala Met Lys Glu His Gly Asp Trp Trp
450 455 460GCATCCCCAT TTATTCTGGG AGGTGTTCTG TGACGATTTC TGTCCTCTCT TAAGGAGAGG 2591CAGCTTTGAT GCATTTTCTT TTCATTTAAA TAGCTTTTTA CCCTTTTTGT CAAGCGGGTT 2651ACGGATAGAG GCGCTTGGTT TTCTCCACTG TTGCATTCGA TTGATATCCC CACTTGAGCA 2711CCGCTGTTTG TTTTGGTTCT GCACTTGGGA CTGTCATGAT GATAATGAGA TACAATGAAT 2771AACTTAAAAA TAATTGTGTG GTCTCGTAAA GTTGTAAACT CTAGA 2816SEQ ID NO:2的信息:
序列特征:
长度:5240碱基对
类型:核苷酸
链型:2
拓扑结构:线性
分子类型:基因组DNA
假设:否
反义:否
来源:Penicilllium chrysogenum
直接来源:质粒pALP295和pALP388
基因组中的位置:未知
特点:
名称/关键词:编码序列
位置:1324…3111
特点:
其它信息:hex基因序列描述:SEQ ID NO:2GTCGACCTCG CAACAGTCGA GAAGCACGCC GCCTATCTCG CCCGCAGCGG GGTAACCGGC 60CTAGTAACCC AGGGTAGCAA TGGCGAAGCC GTCCACCTAG ACCGGGAAGA ACGCAAGGCC 120ATCACAGCCG CCACACGCCG CGCCGTGGAC GCAGCCGGCT ACAGCAACAT GCCGGTGATT 180GCCGGCTGTG GCGCCGCCTC AACCCGTGAG ACCATCCAAT TCTGCCAGGA CTCCGGTGCA 240GCAGGCGCCG ACGCTGTCCT CGTGCTCCCA CCCAGCTACT ACAAGTTTCT CGTGAGCACC 300GAGTCCATGC ACGCCCACTT CCGGGCTGTG GCCGATGCCT CGCCCGTCCC TGTCCTCATC 360TACAACTTCC CCGGCGTGCA GTCCGGCCTC GATCTCAGCT CAGATGATAT CTTAACTCTC 420GCAGAACACC CCAATATCAT CGGCTGTAAG CTCACGTGCG GCAACACGGG TAAGTTGGCT 480CGTGTTGCGG CGGCCAAGCC GGATTTCTTG ACTTTTGGTG GCTCGGCCGA TTTCACGCTG 540CAGACGCTGG TTGTTGGTGG CGCGGGGATT ATCGGTGGCG TGGCTAACAT GATTCCTCGC 600TCGTGTGTGC GTCTGATGGA GTTGTATCGT GCTGGGAAGG TTCAGGAGGC GCAGAAGGTG 660CAGGCTATTG TTGCGCGCGC TGACTGGGCT GCTATCCATG GTGGCTTTAT CGCTGTTAAG 720ACGGGCCTCC AAGCCTACCA CGGTTACGGT GGTCTTCCTC GGCGGCCTTG TGTCGTGCCT 780TCTGCTAAGG ATGCGGCAGC CATTCAGGAG GAGTTCCGGG AGGGAATGGA GCTGGAGAAG 840TCGTTGGAGT CCTAATGGAT ATAGTAGATT AAATCATGAT TACCAGAGAT CCCATGTGGA 900GATTTCTATT CCTTTCCAGG GGTTTTCCAG GGGTTTTCCA GATGTTTTCC AGGTGTTTTC 960CAAATGTTTC AGGTTGCTTC ATAGATCGAC AGACCGGTGT GACTGTGTCA TTTGCCAGTA 1020GATCCGGAGA TCCCGTAGCT TTCCCCCTCT TTATCTTTTA ATATTTGTTG TTATATGGGA 1080GTTCAAGTTG CATGTAGAGG TTGCACTCTC TCTCTCTCTC TTTCCCTTGA ATTATTTGAG 1140TCCAAGGTGT GTTAGTTGTA TGCAATGTAA CTAGGGAGCT GTTTGTTTTT CCCCTTCCCC 1200AGGGTTGCAT CCTGGGCCAT TCCCCATTCC GATGAAAGAT CGACAATGCA GCTAAACATA 1260AATAGTTCTG GTTATCTCCT GGCCACAGTT TCTCTACTTT TCATCGTCAC TCACCTTATC 1320AAC ATG AAG TTC GCC TCG GTG TTG AAT GTG CTC GGG GCC CTG ACG GCT 1368
Met Lys Phe Ala Ser Val Leu Asn Val Leu Gly Ala Leu Thr Ala
1 5 10 15GCG TCC GCC GTC CAA GTG AAT CCA CTT CCC GCC CCC CGT AAC ATC ACC 1416Ala Ser Ala Val Gln Val Asn Pro Leu Pro Ala Pro Arg Asn Ile Thr
20 25 30TGG GGA TCC TCC GGT CCA ATC CAA GTC AAC AAC TTG AAT CTC AAC GGT 1464Trp Gly Ser Ser Gly Pro Ile Gln Val Asn Asn Leu Asn Leu Asn Gly
35 40 45CCT CAC TCC CCT TTG CTC ACT CAA GCT TGG GAG CGA GCA TGG GAA ACC 1512Pro His Ser Pro Leu Leu Thr Gln Ala Trp Glu Arg Ala Trp Glu Thr
50 55 60ATC ACC ACC CTG CAA TGG GTT CCT GCT GCT GTT GAA TCC CCA ATC GCC 1560Ile Thr Thr Leu Gln Trp Val Pro Ala Ala Val Glu Ser Pro Ile Ala
65 70 75TCC TAT CCG GCC TTC CCC ACC TCG ACC CCT GTC TCC TCT GCC CCC AAG 1608Ser Tyr Pro Ala Phe Pro Thr Ser Thr Pro Val Ser Ser Ala Pro Lys80 85 90 95GCC AAA CGC GCG CCC TCC GGA ATC CAT AAC GTC GAT GTT CAT GTG GTG 1656Ala Lys Arg Ala Pro Ser Gly Ile His Asn Val Asp Val His Val Val
100 105 110GAC AAC GAT GCC GAT CTC CAA TAC GGT GTG GAT GAA TCC TAT ACA CTG 1704Asp Asn Asp Ala Asp Leu Gln Tyr Gly Val Asp Glu Ser Tyr Thr Leu
115 120 125GTA GTG AGC GAT GGT GGC ATC AGG ATC AAT TCT CAG ACG GTC TGG GGT 1752Val Val Ser Asp Gly Gly Ile Arg Ile Asn Ser Gln Thr Val Trp Gly
130 135 140GTG TTG CAG GCA TTC ACC ACC CTG CAG CAG ATT ATC ATC TCG GAT GGG 1800Val Leu Gln Ala Phe Thr Thr Leu Gln Gln Ile Ile Ile Ser Asp Gly
145 150 155AAG GGC GGT TTG ATC ATT GAA CAG CCC GTC AAG ATC AAG GAT GCC CCG 1848Lys Gly Gly Leu Ile Ile Glu Gln Pro Val Lys Ile Lys Asp Ala Pro160 165 170 175CTG TAC CCC CAT CGT GGT ATC ATG ATA GAC ACC GGG CGC AAC TTC ATT 1896Leu Tyr Pro His Arg Gly Ile Met Ile Asp Thr Gly Arg Asn Phe Ile
180 185 190ACC GTT CGC AAG CTC CTT GAG CAG ATC GAC GGT ATG GCC CTG TCC AAG 1944Thr Val Arg Lys Leu Leu Glu Gln Ile Asp G1y Met Ala Leu Ser Lys
195 200 205CTC AAT GTT CTC CAC TGG CAC TTG GAC GAT TCT CAG TCG TGG CCC ATG 1992Leu Asn Val Leu His Trp His Leu Asp Asp Ser Gln Ser Trp Pro Met
210 215 220CAG ATG AGC TCC TAC CCG GAG ATG ACC AAA GAT GCT TAC TCG CCT CGC 2040Gln Met Ser Ser Tyr Pro Glu Met Thr Lys Asp Ala Tyr Ser Pro Arg
225 230 235GAA ATC TAC ACC GAG CAC GAC ATG CGC CGC GTG ATT GCC TAC GCA CGC 2088Glu Ile Tyr Thr Glu His Asp Met Arg Arg Val Ile Ala Tyr Ala Arg240 245 250 255GCG CGA GGT GTC CGC GTC ATC CCC GAG GTC GAC ATG CCC GCC CAC TCA 2136Ala Arg Gly Val Arg Val Ile Pro Glu Val Asp Met Pro Ala His Ser
260 265 270GCC TCC GGC TGG CAG CAG CTC GAC CCG GAG ATC GTG GCA TGT GCC GAA 1184Ala Ser Gly Trp Gln Gln Val Asp Pro Glu Ile Val Ala Cys Ala Glu
275 280 285TCC TGG TGG TCG AAC GAC GTT TGG GCG GAG CAC ACC GCC GTC CAG CCG 2232Ser Trp Trp Ser Asn Asp Val Trp Ala Glu His Thr Ala Val Gln Pro
290 295 300AAC CCT GGC CAG CTC GAC ATT ATC TAC CCC AAG ACC TAC GAA GTT GTC 2280Asn Pro Gly Gln Leu Asp Ile Ile Tyr Pro Lys Thr Tyr Glu Val Val
305 310 315AAC AAT GTC TAC CAG GAA TTG TCT CGC ATC TTC AGC GAC AAC TTG TTC 2328Asn Asn Val Tyr Gln Glu Leu Ser Arg Ile Phe Ser Asp Asn Leu Phe320 325 330 335CAC GTT GGT GCA GAC GAG ATC CAG CCC AAC TGC TAC AAC TAC AGC ACC 2376His Val Gly Ala Asp GLu Ile Gln Pro Asn Cys Tyr Asn Tyr Ser Thr
340 345 350CAT ATC ACT AAG TGG TTT GCC GAG GAT CCC TCG CGC ACC TAC AAC GAC 2424His Ile Thr Lys Trp Phe Ala Glu Asp Pro Ser Arg Thr Tyr Asn Asp
355 360 365CTT GCG CAG TAC TGG GTT GAC CAT TCC ATG CCC ATC TTC CGT AGT GTC 2472Leu Ala Gln Tyr Trp Val Asp His Ser Met Pro Ile Phe Arg Ser Val
370 375 380GGC GAC CAC CGC CGT CTT ATG ATG TGG GAG GAC ATA GCT ATC GCG ACT 2520Gly Asp His Arg Arg Leu Met Met Trp Glu Asp Ile Ala Ile Ala Thr
385 390 395GAA AGC GCC CAC GAC GTG CCC AAA GAC GTC ATC ATG CAG ACC TGG AAC 2568Glu Ser Ala His Asp Val Pro Lys Asp Val Ile Met Gln Thr Trp Asn400 405 410 415AGC GGC GAG GGT GAG GGT AAC ATC AAG AAA CTC ACC TCC GCC GGC TAC 2616Ser Gly Glu Gly Glu Gly ASn Ile Lys Lys Leu Thr Ser Ala Gly Tyr
420 425 430GAC GTT GTC GTT TCG ACC TCC GAT TTC CTC TAC CTC GAC TGC GGG CGC 2664Asp Val Val Val Ser Thr Ser Asp Phe Leu Tyr Leu Asp Cys Gly Arg
435 440 445GGC GGC TAT GTC ACC AAC GAC GCC CGC TAC AAC GTG CAG AGC AAC ACC 2712Gly Gly Tyr Val Thr Asn Asp Ala Arg Tyr Asn Val Gln Ser Asn Thr
450 455 460GAC GGC GGA GTG AAC TTC AAC TAC GGC GGC GAC GGT GGC TCC TGG TGC 2760Asp Gly Gly Val Asn Phe Asn Tyr Gly Gly Asp Gly Gly Ser Trp Cys
465 470 475GCC CCC TAC AAG ACC TGG CAG CGC ATC TAC GAC TAC GAC TTC CTC ACG 2808Ala Pro Tyr Lys Thr Trp Gln Arg Ile Tyr Asp Tyr Asp Phe Leu Thr480 485 490 495AAT CTC ACT TCC TCC GAA GCG AAG CAC ATT ATC GGC GCC GAG GCT CCT 2856Asn Leu Thr Ser Ser Glu Ala Lys His Ile Ile Gly Ala Glu Ala Pro
500 505 510TTG TGG TCG GAG CAG GTC GAC GAT GTG ACC GTC TCC AGC GTG TTC TGG 2904Leu Trp Ser Glu Gln Val Asp Asp Val Thr Val Ser Ser Val Phe Trp
515 520 525CCT CGC GCT GCT GCT CTG GGT GAG CTT GTC TGG TCT GGT AAC CGT GAC 2952Pro Arg Ala Ala Ala Leu Gly Glu Leu Val Trp Ser Gly Asn Arg Asp
530 535 540GCT GCG GGT AGA AAG CGT ACC ACC AGC TTT ACT CAG CGT ATT CTG AAC 3000Ala Ala Gly Arg Lys Arg Thr Thr Ser Phe Thr Gln Arg Ile Leu Asn
545 550 555TTC CGT GAA TAC CTC GTT GCC AAT GGT GTG ATG GCT ACT GCT CTT GTG 3048Phe Arg Glu Tyr Leu Val Ala Asn Gly Val Met Ala Thr Ala Leu Val560 565 570 575CCG AAG TAT TGT CTG CAG CAC CCT CAT GCT TGC GAC CTC TAT AAA AAC 3096Pro Lye Tyr Cys Leu Gln His Pro Hie Ala Cya Asp Leu Tyr Lye Asn
580 585 590CAG ACT GTA ATG TCT TGATTGTGGT TAAGCTGGAC TGCTAGTGAG CCTTACAACT 3151Gln Thr Val Met Ser
595GCCTGTTCGT CTGTATATAC TTATTCTATC TTCGATACCC AATTCCATTG GAATTTCTTC 3211CAGGATACAT GTCCCTGATC AGTATACCAT TTCACGTCCA CATTCAATCT TCAGCAACAC 3271GAATTTATCC AAACCAATCA CCACCCTAGA TCTACCACAA CACTACCTTT ATACATATCT 3331ACTTGATACC CAATCCCATT CCAACCAGGC GCAAAAGGCG TGCCCAGTCC AAATCAAAAT 3391CAGCCCCCCG AGCCCAACCC TCTCCACATA TCCATACCCT AATCAAAATC ACCTTAATCT 3451AAACAAATCC ATCACGCCCA AGGACCCCAC AGACCTCCCC TTCCCAACCC ACCCAGTCCA 3511CCTCCACAAA CCAAACCCCA AATCAGAACT GCCGTGCAAC TCTCCGTCTT AGAACTCGCC 3571CTTCGGTCCC GTCCCGAACT TAGATGGGCT TCGGGACGGC TTGCTGTATG CACTATGCAT 3631GTAGTACGGA GTACGCCGTA CACATGTAGT AGGGGATATA TGTATGTACT ATGTACGCAT 3691GTTCGAGTAC GCAGTACGTA GTGTGGCATG CAGGTCAGCT AGCATTGGCA GTAGCATATA 3751CGGCATAACC TACGCTATGC ATCTAATATT CTTCGGTATA TACCACATGG TACGGAATTA 3811GATGCAATAC ATGTACATGT ACATGTGCAT ACCTAGGTAC AAAGTGAATC TCGTTATTGT 3871ATGTCTAGTC GTGTATAAGT GTAGTCCCAT GTCATATATA CAAGCCCATA CCGCATCGGA 3931GCAAACCAGC CCATTCAGAC ATCCCTGCTC GAAACCCAGT CTACGGATTG AGACCGGGCT 3991GAGCTGGGGT TTGGGTGTTG CTGCATGCGT ACGCCTACAT ACGTAGGGAG ATATGTTGCA 4051CAGGATGCAG GGAATGACAA ATTGACGAAT TGAGAAATAC GCGAGTGGTT AGATGTTAAT 4111TCTCGTTCGG GATGTTTATG TTTACCTAGG TATACTGGCT GGGGGGTCGT CATACACGTG 4171CGAATTTGTG GCAATCTGTC AGTGGCCAGG TCCTTGTTTG ATTTATATGT TTGGGATGGG 4231GATGGTCAAT GGGTATTCCA AGGAGGATGT ATCATCTGCT TTACACCGTC CCTTGCCTGG 4291GATTTGGATT GAATTCTTCT TTCCACGTCG ATGTAGATTC TTCCCCGGAG CTATTCGGGT 4351ACAACCCTGG CTTCCATATA TCATGTGTCC ATACTAAGTA CAGACGCTTC GATTTCCGGT 4411GCTGCGAGTA GATCGGGAAC TGATCTCGCA TGTCTGTACA CGAAGGGTTG TACAAGCACG 4471CGGTCGTTCT GCGTAACCGG TTGTTTATGT TATTGGATTT GGTATTCGTC TAATATGGAT 4531GATTTGGGAT AAGCTTCTAT CCTGGGAATG GGTGCTTGGT ATAGTTCAGC CTAGTACTTC 4591GTCTTCTATG TGATATTTCC AAAATAGTAG TTTTCGGTAA GTATATCTCC TACCTTTGAC 4651TTTGGTTTGT GGTTTACGTC TTACCTGGCG TTTAGAGGGA GGGATAGGTT TCTGTATCAC 4711CGTCGTGTTT CAACGTGGAT CGGGGTCCTT TCCCTGATAT ATATCTTGGC TTATGTTTCG 4771TGCGGTAGTG CGGGTTCGTA TAATGCATGT CTGGTATATC ATACGGCATT AGTGACTGGG 4831ACGTTGAGGT CGAGCTTGGT TTGAGGTTAC ATATATTGAG CCAAAATGGT CGAAAATATA 4891TATCAACATT GCCAAAACAG AACTTCATTC GTTGGATGCC ATGCCAAATT GCTAATAGGT 4951CTTGATCTTA CTCTGACTCC TATCTCATCT CACCTTGGTT ATTCGTTACA CAGCATTAAC 5011CCCAAGAACC AGGTATAGTC TGATCGTGGA TGTGGGCCAC GACAAAATAG AAGGTCTCGT 5071GTTTAGGGCG ACGAATCTGG GACTGCATTC CAGACGGGCC TGCGGAGAAT TTGCAGCATT 5131TTATATCTAC ATGGTTGTTC CCTGGTGTGT GTGGGTGTTT CATGATATAT CCTGGTCGAT 5191TCTGACGTGC GTATGTATCG CTGGAAAGGC TCGTAGGGGC TGCGTCGAC 5240SEQ ID NO:3的信息:
序列特征:
长度:2994碱基对
类型:核苷酸
链型:2
拓扑结构:线性
分子类型:基因组DNA
假设:否
反义:否
来源:Penicilllium chrysogenum
直接来源:质粒pALP315和pALP316
基因组中的位置:未知
特点:
名称/关键词:编码序列
位置:结合(494…500,617…647,846…901,1047…1077,
1181…1952,2022…2249)
特点:
名称/关键词:内含子
位置:501…616
特点:
名称/关键词:内含子
位置:648…845
特点:
名称/关键词:内含子
位置:902…1046
特点:
名称/关键词:内含子
位置:1078…1180
特点:
名称/关键词:内含子
位置:1953…2021
特点:
其它信息:act基因序列描述:SEQ ID NO:3GAATTCAGCA GCCTACGGAG TCCATAAGAC ACCAAGACAC AGCCATTGTA TGGATTATAT 60ATGCCATGTA TGCCTGACAA TGCTGTATAA GTACTGTAAT ACAAGGTAAA CCCCCAACCC 120GGTCAAGGTA CGTGTTCCCG CCGTACCCAA AAGGGTCCCC AAGAATGTCC ACGCAATACT 180TTTAGGTAGA CATTGAAGGA ATCCAAGTGA GAAATTCAAT GAACATGAAC AATAGTTCTG 240CCTTATAATC TTTATAAGTA TAAAAATCAG AAAGAGAATT ATATACAAAA GGGTAGATCT 300GGAGGGGGTT CAGAGTTAAG GCCTCAGGCA GGCGCACAAT CCCAGCCATC ACAAACCCCT 360CTCCACTCTT CCCTCTCTCT CTCTTCCTTC TTCCTTTCTC CCCTAATCCC AACTATATCC 420CCTCTAACCT CTTTCCATCT TTCTTTTCTT TTTTCCCCTC TTCTCCCCTA AGTCCCTTGT 480TTAATCAGTC ACA ATG GAG G GTATGTTATT CCAGTTGTGG CCACATCAGC AGCTTCCC 538
Met Glu
1CGGAAGCTCC CCCCCCTGTT GGCCACAGCT TCGATTCCAT ATTTGCGAAT GACAACTAAC 598CCGTATATCT CATTATAG AA GAA GTT GCT GCT CTC GTC ATC GAC AAT GG 647
Glu Glu Val Ala Ala Leu Val Ile Asp Asn Gly
5 10GTATGTGCTA TACTTTTCCC CGGAGCTTCT GGCTTGTGTT GGGGTCGCCA ACTCAGCCCC 707GGTCGCAGTC GTTGCCACCC CTAATCCGCC CGCGACGGCA GATGGAATCC ATCCCAATGG 767CTTTCCATCT CGCTCCACAA CTACCAGAGG GTGATCCAAA GACTACAAGA ACTATGATAC 827TGATTATTTG CGATATAG T TCG GGT ATG TGT AAG GCC GGT TTC GCC GGT GAC 879
Ser Gly Met Cys Lys Ala Gly Phe Ala Gly Asp
15 20GAC GCA CCA CGA GCT GTT TTC C GTAAGTCCA ACCCCACAGA ATATGACACC 930Asp Ala Pro Arg Ala Val Phe25 30CCTCCTGTGC GAAGGCCGCC ATCCCACCAA CCCTTGCGTC GGATGGCTTC CCCTCTTTTG 990CTTGGCTAGG AGGAACCTGG AACCTAGGAA ATCAAATAAC TGACAAAATT CAACAG 1046CT TCC ATT GTC GGT CGT CCC CGC CAC CAT GG GTAAATGATC CCCCCTTTTT 1097Pro Ser Ile Val Gly Arg Pro Arg His His Gly
35 40TTTCCGGCTC GTTTCGGCTG TATACGCTAT ACGCAGCCAA TTTGATCCCT AATGAACCAA 1157AAAGAATACT AACATGGGCG CAG T ATT ATG ATT GGT ATG GGT CAG AAG GAC 1208
Ile Met Ile Gly Met Gly Gln Lys Asp
45 50TCG TAC GTT GGT GAT GAG GCA CAG TCG AAG CGT GGT ATC CTC ACG CTC 1256Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg Gly Ile Leu Thr Leu
55 60 65CGT TAC CCT ATT GAG CAC GGT GTT GTC ACC AAC TGG GAC GAC ATG GAG 1304Arg Tyr Pro Ile Glu His Gly Val Val Thr Asn Trp Asp Asp Met Glu
70 75 80AAG ATC TGG CAC CAC ACC TTC TAC AAC GAG CTC CGT GTT GCC CCC GAA 1352Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg Val Ala Pro Glu
85 90 95GAG CAC CCC ATT CTC TTG ACC GAA GCT CCC ATC AAC CCC AAG TTC AAC 1400Glu His Pro Ile Leu Leu Thr Glu Ala Pro Ile Asn Pro Lys Phe Asn100 105 110 115CGT GAG AAG ATG ACC CAG ATC GTG TTC GAG ACC TTC AAC GCC CCC GCC 1448Arg Glu LyS Met Thr Gln Ile Val Phe Glu Thr Phe Asn Ala Pro Ala
120 125 130TTC TAC GTC TCC ATC CAG GCC GTT CTG TCC CTG TAC GCC TCC GGT CGT 1496Phe Tyr Val Ser Ile Gln Ala Val Leu Ser Leu Tyr Ala Ser Gly Arg
135 140 145ACC ACT GGT ATC GTT CTC GAC TCC GGT GAC GGT GTC ACC CAC GTC GTC 1544Thr Thr Gly Ile Val Leu Asp Ser Gly Asp Gly Val Thr His Val Val
150 155 160CCC ATC TAC GAG GGT TTC TCT CTG CCC CAC GCT ATC TCG CGT GTC GAC 1592Pro Ile Tyr Glu Gly Phe Ser Leu Pro His Ala Ile Ser Arg Val Asp
165 170 175ATG GCT GGC CGT GAT CTG ACC GAC TAC CTG ATG AAG ATC CTC GCT GAG 1640Met Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile Leu Ala Glu180 185 190 195CGT GGT TAC ACT TTC TCC ACC ACC GCC GAG CGT GAA ATC GTC CGT GAC 1688Arg Gly Tyr Thr Phe Ser Thr Thr Ala Glu Arg Glu Ile Val Arg Asp
200 205 210ATC AAG GAG AAG CTT TGC TAC GTC GCC CTC GAC TTC GAG CAG GAG ATC 1736Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu Gln Glu Ile
215 220 225CAG ACC GCT TCC CAG AGC TCC AGC CTC GAG AAG TCC TAC GAG CTT CCC 1784Cln Thr Ala Ser Gln Ser Ser Ser Leu Glu Lys Ser Tyr Glu Leu Pro
230 235 240GAT GGA CAG GTC ATC ACT ATT GGC AAC GAG CGC TTC CGT GCT CCT GAG 1832Asp Gly Gln Val Ile The Ile Gly Asn Glu Arg Phe Arg Ala Pro Glu
245 250 255GCT CTG TTC CAG CCT AAC GTT CTT GGC CTC GAG TCT GGC GGT ATC CAC 1880Ala Leu Phe Gln Pro Asn Val Leu Gly Leu Glu Ser Gly Gly Ile His260 265 270 275GTC ACC ACC TTC AAC TCC ATC ATG AAG TGT GAT GTT GAT GTC CGT AAG 1928Val Thr Thr Phe Asn Ser Ile Met Lys Cys Asp Val Asp Val Arg Lya
280 285 290GAT CTC TAC GGC AAC ATT GTC ATG GTAAGAAAAA AGCCTCCAGA GCTGATGTTG 1982Asp Leu Tyr Gly Asn Ile Val Met
295CGCAAAGATC CCCACTAACA TACAACTCCT TTTTTTTAG TCT GGT GGT ACC ACC 2036
Ser Gly Gly Thr Thr
300ATG TAC CCC GGT ATC TCC GAC CGT ATG CAG AAG GAG ATC ACT GCT CTT 2084Met Tys Pro Gly Ile Ser Asp Arg Met Gln Lys Glu Ile Thr Ala Leu305 310 315 320GCT CCT TCT TCC ATG AAG GTC AAG ATC ATC GCT CCC CCC GAG CGC AAG 2132Ala Pro Set Ser Met Lys Val Lys Ile Ile Ala Pro Pro Glu Arg Lys
325 330 335TAC TCC GTC TGG ATC GGT GGA TCC ATT CTG GCC TCC CTG TCG ACC TTC 2180Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Set Thr Phe
340 345 350CAG CAG ATG TGG ATC TCT AAG CAG GAG TAC GAC GAG AGC GGT CCT TCC 2228Gln Gln Met Trp Ile Set Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser
355 36O 365ATC GTT CAC CGC AAG TGC TTC TAAGCTTCTT GCAGCACTTT ACTACTCGTA 2279Ilc Val His Arg Lys Cys Phe
370 375TTCGCTCGTA CTTTCCTGGT GTATCAAAAA GCAGGATGGA GGCACTGGTG GATTGCAAGC 2339GTTGTTGGAC TCGCATTATC AAGCGGATAG CCTGAAAATG GAATCTCGAT TTTAGTGGAA 2399TAGAGTCGGT CGTTTTCTTT TTGTTACTCT TTACCTTACT CTTTACTCGA TCTCTATCCA 2459TCCATTTCTG CTTTGAACCA TTTCACCTTT ACTCCATCTT TTTCCCTTTC CTCATTCGAA 2519TCCGCTGTCC CGTCCACCTC TCTGATTGTT TTGCCTGGAC GGGTCTCTGG CGATGCGGCA 2579TCAACAGTGT ACCTGTAGGG CAAGGATGTA TATGGAGTTG GTTGGCTATA GGGATTAGGT 2639TGCGTTGTCC TTTTCGACGT CTTCTACGTC TTTGTTCTAG CCCCTTGCGT TGTCTTCAAC 2699TAAACTGCCC TTGTCCGTAG CTTTTAACGT GACTTTGACT TCAAATATTC CACTGGTTCC 2759TTGTATTCTG CTAGAAACGC TGGTTCAACG CTTGTTGAAT GTCTTCTATG TCCAACATCT 2819ACAAGACGTA TCCGAGAAGA CAACAAAAAG GCTCTGAGGA AAGTCTACTA AAAACTTGGC 2879CAGGCCGGAT TAGGCCTTTG TCATGGTTAT TGTACTGTCA TTCAGTCAGT CCATATTGAT 2939ATTCTGGGAA TATGTAGGCT GACGAGATAA ATGGCACGCA TTGGGTGTGT ATCTT 2994SEQ ID NO:4的信息:
序列特征:
长度:3240碱基对
类型:核苷酸
链型:2
拓扑结构:线性
分子类型:基因组DNA
假设:否
反义:否
来源:Penicilllium chrysogenum
直接来源:质粒pALC52和pALC53
基因组中的位置:未知
特点:
名称/关键词:编码序列
位置:结合(787...793,921...951,1124...1179,1290...1320,
1411...2182,2250...2477)
特点:
名称/关键词:内含子
位置:794...920
特点:
名称/关键词:内含子
位置:952...1123
特点:
名称/关键词:内含子
位置:1180...1289
特点:
名称/关键词:内含子
位置:1321...1410
特点:
名称/关键词:内含子
位置:2183...2249
特点:
其它信息:act基因序列描述:SEQ ID NO:4GCCAGGCTGG CACCGGCCTG CCTTGATGCG AGATGCCTAC TCGTACTATG CCTACAGGTA 60TGGGCTTTCC GCGTGTCGTC AGCTTGCGAC CGCGCGGCTG CTGACGACCC AAGGCAAGCT 120GGTAACATGG CGGCACGAAA TTTCTCTCTG CCTGCTCGTC CTCTTGGTGT GGAGGGGTAC 180GAGTGCAGGT ATGATGGGAC GGCAGAGGAG TGACGGAGGC TGTGCGGTTG GCACGAGTAC 240TGTACGAGTA CTCGTACTGT AGGTGCAGCG ACTGTGGTGG TACTGCTAGG TGGAATTGGG 300TCCAGCAGGC ATGCAGCTCC CAGCCACCGT CGTTAACCAA TCAGTTAAAG CAGCAACGCA 360ACCCGCCCCC GTTTTTCTGC CAGAAATTTG GGCGGTGTCG TGCCCCCAGT CGCTGTTGCC 420CGCCCTTGTC TGGTCGCCTA CAGGCTGCAC CACAGGTAAC AACAGCCCGC CCCAGGTCCT 480TGTAGGTGCC CAGTGAGTGC CCGGTGCCCA CAAGTTTCTC GTGGCATCCA CTGGCGGACT 540TGGAAGCCCA TCAGTGATGC TTCCCTCCTT TCCCCCTCCA CATCTCACTC AGCTCACGCA 600AGCCAACCCT CTCTCCCCCC GTCTCCATTC CATCTTCTTC TCTCCACGAC CCTTAAGAGT 660CCCTCCTGCT CACGTCGACC ATCCTTCGCT CCCAGCCCCA CGACATCTGC ATCGTCTGGG 720CTTCTTGACA CTCTGTCATT TCTTCCTTAT AAAACCTCTT TACCGCTCTT CCCGTAATCC 780GACGCC ATG GAG G GTACGTGTCG CCGCAACGCA CTCCCGCTTC CCCTACTACC CCTA 837
Met Glu
1TCGCGC ATCCACACGG CGCCGCGATG CCTAGCCATC GCGAGGGTGC ATCGCAACGA CTT 896GGCTAAC TGTTCTTCGC TTCACAG AG GAG GTC GCC GCC CTC GTT ATC GAC 946
Glu Glu Val Ala Ala Leu Val Ile Asp
5 10AAT GG GTAAGCTCGC CCGCTGTCTC ACCGACATCC ATCGTCCCCC TGGCCTCTGT 1001Asn GlyCGAGATGGGA GCCTCCAGGG GTCCCTTCGA CGAGCGCGTC GATTGCCAAA ATCCAACGAG 1061ATCGGGCCAT ACTGAGCCGA CACTCGTGTG TTTTCTGGAC ATTAGGACTG ACTTGATTCT 1121AG T TCG GGT ATG TGC AAG GCC GGT TTC GCC GGT GAT GAT GCT CCC CGA 1169
Ser Gly Met Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg
15 20 25GCT GTT TTC C GTAAGTACCC CACTTCCACC CGTCGAGCTC CCCAATTGTC CACCGCCAGG1229Ala Val Phe
30GCGAGAAGGG GGCAGAACGG GGCAAACTGC ATCGCAAACA TGGCTAATTC GATGCGACAG 1289CG TCC ATT GTC GGT CGT CCC CGC CAC CAT GG GTAAGTTTCC GGCCGCAGCC 1340Pro Ser Ile Val Gly Arg Pro Arg His His Gly
35 40GACACCTCTC ACCCCCCCCC GGGGGGCTCC TAAGCGAGTC AGCGCTGGTT CTGACCGCTG 1400GATACTATAG C ATC ATG ATC GGC ATG GGC CAG AAG GAC TCG TAC GTC GGT 1450
Ile Met Ile Gly Met Gly Gln Lys Asp Ser Tyr Val Gly
45 50 55GAC GAG GCT CAG TCC AAG CGT GGT ATC CTC ACC CTG CGC TAC CCC ATT 1498Asp Glu Ala Gln Ser Lys Arg Gly Ile Leu Thr Leu Arg Tyr Pro Ile
60 65 70GAG CAC GGT GTT GTC ACC AAC TGG GAC GAC ATG GAG AAG ATC TGG CAC 1546Glu His Gly Val Val Thr Asn Trp Asp Asp Met Glu Lys Ile Trp His
75 80 85CAC ACC TTC TAC AAC GAG CTG CGT GTT GCC CCC GAG GAG CAC CCG GTC 1594His Thr Phe Tyr Asn Glu Leu Arg Val Ala Pro Glu Glu His Pro Val
90 95 100CTG CTC ACC GAG GCG CCC ATC AAC CCC AAG TCC AAC CGT GAG AAG ATG 1642Leu Leu Thr Glu Ala Pro Ile Asn Pro Lys Ser Asn Arg Glu Lys Met
105 110 115ACC CAG ATC GTC TTC GAG ACC TTC AAC CCC CCT GCC TTC TAC GTC TCC 1690Thr Gln Ile Val Phe Glu Thr Phe Asn Ala Pro Ala Phe Tyr Val Ser120 125 130 135ATC CAG GCC GTC CTG TCA CTG TAC GCC TCC GGC CGT ACG ACC GGT ATC 1738Ile Gln Ala Val Leu Ser Leu Tyr Ala Ser Gly Arg Thr Thr Gly Ile
140 145 150GTC CTG GAC TCT GGT GAT GGT GTC ACC CAC GTT GTC CCC ATC TAC GAG 1786Val Leu Asp Ser Gly Asp Gly Val Thr His Val Val Pro Ile Tyr Glu
155 160 165GGT TTC GCC CTG CCC CAC GCC ATT GCC CGT GTC GAC ATG GCT GGT CGT 1834Gly Phe Ala Leu Pro His Ala Ile Ala Arg Val Asp Met Ala Gly Arg
170 175 180GAT CTC ACC GAC TAC CTC ATG AAG ATC CTG GCC GAG CGC GGC TAC ACC 1882Asp Leu Thr Asp Tyr Leu Met Lys Ile Leu Ala Glu Arg Gly Tyr Thr
185 190 195TTC TCC ACC ACG GCC GAG CGT GAG ATT GTC CGT GAC ATC AAG GAG AAG 1930Phe Ser Thr Thr Ala Glu Arg Glu Ile Val Arg Asp Ile Lys Glu Lys200 205 210 215CTC TGC TAC GTC GCC CTC GAC TTC GAG CAG GAG ATC CAG ACT GCC GCC 1978Leu Cys Tyr Val Ala Leu Asp Phe Glu Gln Glu Ile Gln Thr Ala Ala
220 225 230CAG AGC TCC AGC CTG GAG AAG TCC TAC GAG CTT CCC GAC GGC CAG GTC 2026Gln Ser Ser Ser Leu Glu Lys Ser Tyr Glu Leu Pro Asp Gly Gln Val
235 240 245ATC ACC ATT GGC AAT GAG CGC TTC CGT GCT CCC GAG GCT CTC TTC CAG 2074Ile Thr Ile Gly Asn Glu Arg Phe Arg Ala Pro Glu Ala Leu Phe Gln
250 255 260CCC TCC GTC CTG GGT CTC GAG AGC GGC GGC ATC CAC GTC ACC ACC TTC 2122Pro Ser Val Leu Gly Leu Glu Ser Gly Gly Ile His Val Thr Thr Phe
265 270 275AAC TCC ATC ATG AAG TGC GAC GTC GAT GTC CGT AAG GAT CTG TAC GGC 2170Asn Ser Ile Met Lys Cys Asp Val Asp Val Arg Lys Asp Leu Tyr Gly280 285 290 295AAC ATT GTC ATG GTAAGTCAGA TGCCGGGCCT GGAAGACACC TCATTTAGGA TCT 2225Asn Ile Val MetTGCTAAC ACCAATTTTT TTTTTAG TCT GGT GGT ACC ACC ATG TAC CCT GGC 2276
Ser Gly Gly Thr Thr Met Tyr Pro Gly
300 305CTC TCT GAC CGT ATG CAG AAG GAG ATC ACT GCT CTT GCT CCT TCT TCC 2324Leu Ser Asp Arg Met Gln Lys Glu Ile Thr Ala Leu Ala Pro Ser Ser
310 315 320ATG AAG GTC AAG ATC ATT GCT CCC CCG GAG CGC AAG TAC TCC GTC TGG 2372Met Lys Val Lys Ile Ile Ala Pro Pro Glu Arg Lys Tyr Ser Val Trp325 330 335 340ATC GGT GGT TCC ATT CTG GCG TCT CTG TCC ACC TTC CAG CAG ATG TGG 2420Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe Gln Gln Met Trp
345 350 355ATC TCG AAG CAG GAG TAC GAC GAG AGC GGC CCC TCC ATC GTC CAC CGC 2468Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser Ile Val His Arg
360 365 370AAG TGC TTC TAAGGTATGT TGTCGTCGGG AAGCCGGATA CCCGAATGTA AGGTTGACAG 2527LyS Cys Phe
375GTTCGAAAAG ACAAGGCAAC CGGCCAGAAC CAAATCCTTC CACCCTCCGC AAAAGAACGC 2587CAAGATGTCG GAGTCGGTGG CGACCGATGC AACGTCTACT CACGTGCGCG CGTATCCCAC 2647TCAAGTCTCA TATTTACGAA AAGTTATTTC ACATGGTCAG GCGGTGGTGG GCGTTGCCTT 2707TTCTCGGAAC AGACATGACG GCGGCCACTT TTGTAGTCGG ATGCGTTTAG GGATGCGAGC 2767CTAGGGGTGT AGGAAGCTGA GGTTGATATA CAATAACTTT TTTTGCTTTC CGTTCTAGAC 2827TCGTTCAATG GGAAGACGTG ACGGAATCGC TTGGCTGTCT AATAGCCAGC TTGATCAGGC 2887GAGTCGGGTT GTTGTGTTTC GATGTTGAGA GGTGCACCAG CGTATTTGTA TGGCCGAGGT 2947AGGTATTATG GTCTCGTATT TGCAACACTA GAGCTCGCTT GCTCGTTTTT ACCAGCAGTG 3007TCCTCTGCCA TGCCGCGGCT CCGACTCTCG TCTGGCTTCT CAGACCGTGC CTCGTCAATA 3067GTATTATCCC CCGTAGTAAC CTCCGCACTA GCCGGTTCTT TGTCGTCTTC CTGCTCGCCG 3127ATGAGCTTCC TGTACTTGCG CCTCTTCTTC TTGTCGGCGC TGGCAGCCCT CTTCTGCTTG 3187ATGCGCCCGA CCATGGCGGA CCGGCTCTGC TCCCCGTTGA GCAGCTCGTC GAC 3240SEQ ID NO:5的信启:
序列特征:
长度:461氨基酸
类型:氨基酸
链型:1
拓扑结构:线性
分子类型:肽
来源:Penicilllium chrysogenum
特点:
其它信息:分子量为49837Da的谷氨酸脱氢酶(EC.1.4.1.4.)的氨基酸序列序列描述:SEQ ID NO:5Met Met Gln Asn Leu Pro Phe Glu Pro Glu Phe Glu Gln Ala Tyr1 5 10 15Lys Glu Leu Ala Ser Thr Leu Glu Asn Ser Thr Leu Phe Gln Lys
20 25 30Lys Pro Glu Tyr Arg Lys Ala Leu Gln Val Val Ser Val Pro Glu
35 40 45Arg Val Ile Gln Phe Arg Val Val Trp Glu Asp Asp Lys Gly Gln
50 55 60Val Gln Ile Asn Arg Gly Tyr Arg Val Gln Phe Asn Ser Ala Leu
65 70 75Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Thr Val Asn Leu
80 85 90Ser Ile Leu Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala
95 100 105Leu Thr Gly Leu Asn Met Gly Gly Gly Lys Gly Gly Ser Asp Phe
110 115 120Asp Pro Lys Gly Lys Thr Asp Asn Glu Ile Arg Arg Phe Cys Val
125 130 135Ser Phe Met Thr Glu Leu Cys Lys His Ile Gly Ala Asp Thr Asp
140 145 150Val Pro Ala Gly Asp Ile Gly Val Thr Gly Arg Glu Val Gly Phe
155 160 165Met Phe Gly Gln Tyr Lys Lys Ile Arg Asn Gln Trp Glu Gly Val
170 175 180Leu Thr Gly Lys Gly Gly Ser Trp Gly Gly Ser Leu Ile Arg Pro
185 190 195Glu Ala Thr Gly Tyr Gly Val Val Tyr Tyr Val Glu His Met Ile
200 205 210Gln His Ala Ser Gly Gly Lys Glu Ser Phe Ala Gly Lys Arg Val
215 220 225Ala Ile Ser Gly Ser Gly Asn Val Ala Gln Tyr Ala Ala Leu Lys
230 235 240Val Ile Glu Leu Gly Gly Ser Val Ile Ser Leu Ser Asp Ser Gln
245 250 255Gly Ala Leu Val Leu Asn Gly Glu Glu Gly Ser Phe Thr Ala Glu
260 265 270Glu Ile Asn Thr Ile Ala Glu Ile Lys Val Gln Arg Lys Gln Ile
275 280 285Ala Glu Leu Ala Thr Gln Asp Ala Phe Ser Ser Lys Phe Lys Tyr
290 295 300Ile Pro Gly Ala Arg Pro Trp Thr Asn Ile Ala Gly Arg Ile Asp
305 310 315Val Ala Leu Pro Ser Ala Thr Gln Asn Glu Val Ser Gly Asp Glu
320 325 330Ala Lys Ala Leu Ile Ala Ala Gly Cys Lys Phe Ile Ala Glu Gly
335 340 345Ser Asn Met Gly Ser Thr Gln Glu Ala Ile Asp Val Phe Glu Ala
350 355 360His Arg Asp Ala Asn Pro Gly Ala Ala Ala Ile Trp Tyr Ala Pro
365 370 375Gly Lys Ala Ala Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu
380 385 390Met Ala Gln Asn Ser Ala Arg Val Asn Trp Ser Arg Glu Glu Val
395 400 405Asp Ser Arg Leu Lys Lys Ile Met Glu Asp Cys Phe Asn Asn Gly
410 415 420Leu Ser Thr Ala Lys Glu Tyr Val Thr Pro Ala Glu Gly Val Leu
425 430 435Pro Ser Leu Val Ala Gly Ser Asn Ile Ala Gly Phe Thr Lys Val
440 445 450Ala Glu Ala Met Lys Glu His Gly Asp Trp Trp
455 460SEQ ID NO:6的信息:
序列特征:
长度:596氨基酸
类型:氨基酸
链型:1
拓扑结构:线性
分子类型:肽
来源:Penicilllium chrysogenum
特点:
其它信息:分子量为66545Da的β-N-乙酰氨基几糖苷酶(EC.3.2.1.52.)的氨基酸序列序列描述:SEQ ID N0:6Met Lys Phe Ala Ser Val Leu Asn Val Leu Gly Ala Leu Thr Ala1 5 10 15Ala Ser Ala Val Gln Val Asn Pro Leu Pro Ala Pro Arg Asn Ile
20 25 30Thr Trp Gly Ser Ser Gly Pro Ile Gln Val Asn Asn Leu Asn Leu
35 40 45Asn Gly Pro His Ser Pro Leu Leu Thr Gln Ala Trp Glu Arg Ala
50 55 60Trp Glu Thr Ile Thr Thr Leu Gln Trp Val Pro Ala Ala Val Glu
65 70 75Ser Pro Ile Ala Ser Tyr Pro Ala Phe Pro Thr Ser Thr Pro Val
80 85 90Ser Ser Ala Pro Lys Ala Lys Arg Ala Pro Ser Gly Ile His Asn
95 100 105Val Asp Val His Val Val Asp Asn Asp Ala Asp Leu Gln Tyr Gly
110 115 120Val Asp Glu Ser Tyr Thr Leu Val Val Ser Asp Gly Gly Ile Arg
125 130 135Ile Asn Ser Gln Thr Val Trp Gly Val Leu Gln Ala Phe Thr Thr
140 145 150Leu Gln Gln Ile Ile Ile Ser Asp Gly Lys Gly Gly Leu Ile Ile
155 160 165Glu Gln Pro Val Lys Ile Lys Asp Ala Pro Leu Tyr Pro His Arg
170 175 180Gly Ile Met Ile Asp Thr Gly Arg Asn Phe Ile Thr Val Arg Lys
185 190 195Leu Leu Glu Gln Ile Asp Gly Met Ala Leu Ser Lys Leu Asn Val
200 205 210Leu His Trp His Leu Asp Asp Ser Gln Ser Trp Pro Met Gln Met
215 220 225Ser Ser Tyr Pro Glu Met Thr Lys Asp Ala Tyr Ser Pro Arg Glu
230 235 240Ile Tyr Thr Glu His Asp Met Arg Arg Val Ile Ala Tyr Ala Arg
245 250 255Ala Arg Gly Val Arg Val Ile Pro Glu Val Asp Met Pro Ala His
260 265 270Ser Ala Ser Gly Trp Gln Gln Val Asp Pro Glu Ile Val Ala Cys
275 280 285Ala Glu Ser Trp Trp Ser Asn Asp Val Trp Ala Glu His Thr Ala
290 295 300Val Gln Pro Asn Pro Gly Gln Leu Asp Ile Ile Tyr Pro Lys Thr
305 310 315Tyr Glu Val Val Asn Asn Val Tyr Gln Glu Leu Ser Arg Ile Phe
320 325 330Ser Asp Asn Leu Phe His Val Gly Ala Asp Glu Ile Gln Pro Asn
335 340 345Cys Tyr Asn Tyr Ser Thr His Ile Thr Lys Trp Phe Ala Glu Asp
350 355 360Pro Ser Arg Thr Tyr Asn Asp Leu Ala Gln Tyr Trp Val Asp His
365 370 375Ser Met Pro Ile Phe Arg Ser Val Gly Asp His Arg Arg Leu Met
380 385 390Met Trp Glu Asp Ile Ala Ile Ala Thr Glu Ser Ala Hie Asp Val
395 400 405Pro Lys Asp Val Ile Met Gln Thr Trp Asn Ser Gly Glu Gly Glu
410 415 420Gly Asn Ile Lys Lys Leu Thr Ser Ala Gly Tyr Asp Val Val Val
425 430 435Ser Thr Ser Asp Phe Leu Tyr Leu Asp Cys Gly Arg Gly Gly Tyr
440 445 450Val Thr Asn Asp Ala Arg Tyr Asn Val Gln Ser Asn Thr Asp Gly
455 460 465Gly Val Ash Phe Asn Tyr Gly Gly Asp Gly Gly Ser Trp Cys Ala
470 475 480Pro Tyr Lys Thr Trp Gln Arg Ile Tyr Asp Tyr Asp Phe Leu Thr
485 490 495Asn Leu Thr Ser Ser Glu Ala Lys His Ile Ile Gly Ala Glu Ala
500 505 510Pro Leu Trp Ser Glu Gln Val Asp Asp Val Thr Val Ser Ser Val
515 520 525Phe Trp Pro Arg Ala Ala Ala Leu Gly Glu Leu Val Trp Ser Gly
530 535 540Asn Arg Asp Ala Ala Gly Arg Lys Arg Thr Thr Ser Phe Thr Gln
545 550 555Arg Ile Leu Asn Phe Arg Glu Tyr Leu Val Ala Asn Gly Val Met
560 565 570Ala Thr Ala Leu Val Pro Lys Tyr Cys Leu Gln His Pro His Ala
575 580 585Cys Asp Leu Tyr Lye Asn Gln Thr Val Met Ser
590 595SEQ ID NO:7的信息:
序列特征:
长度:375氨基酸
类型:氨基酸
链型:1
拓扑结构:线性
分子类型:肽
来源:Penicilllium chrysogenum
特点:
其它信息:分子量为41760Da的γ-肌动蛋白的氨基酸序列序列描述:SEQ ID NO:7Met Glu Glu Glu Val Ala Ala Leu Val Ile Asp Asn Gly Ser Gly1 5 10 15Met Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val
20 25 30Phe Pro Ser Ile Val Gly Arg Pro Arg His His Gly Ile Met Ile
35 40 45Gly Met Gly Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser
50 55 60Lys Arg Gly Ile Leu Thr Leu Arg Tyr Pro Ile Glu His Gly Val
65 70 75Val Thr Asn Trp Asp Asp Met Glu Lys Ile Trp His His Thr Phe
80 85 90Tyr Asn Glu Leu Arg Val Ala Pro Glu Glu His Pro Ile Leu Leu
95 100 105Thr Glu Ala Pro Ile Asn Pro Lys Phe Asn Arg Glu Lys Met Thr
110 115 120Gln Ile Val Phe Glu Thr Phe Asn Ala Pro Ala Phe Tyr Val Ser
125 130 135Ile Gln Ala Val Leu Ser Leu Tyr Ala Ser Gly Arg Thr Thr Gly
140 145 150Ile Val Leu Asp Ser Gly Asp Gly Val Thr His Val Val Pro Ile
155 160 165Tyr Glu Gly Phe Ser Leu Pro His Ala Ile Ser Arg Val Asp Met
170 175 180Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile Leu Ala Glu
185 190 195Arg Gly Tyr Thr Phe Ser Thr Thr Ala Glu Arg Glu Ile Val Arg
200 205 210Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu Gln
215 220 225Glu Ile Gln Thr Ala Ser Gln Ser Ser Ser Leu Glu Lys Ser Tyr
230 235 240Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe
245 250 255Arg Ala Pro Glu Ala Leu Phe Gln Pro Asn Val Leu Gly Leu Glu
260 265 270Ser Gly Gly Ile His Val Thr Thr Phe Asn Ser Ile Met Lys Cys
275 280 285Asp Val Asp Val Arg Lys Asp Leu Tyr Gly Asn Ile Val Met Ser
290 295 300Gly Gly Thr Thr Met Tyr Pro Gly Ile Ser Asp Arg Met Gln Lys
305 310 315Glu Ile Thr Ala Leu Ala Pro Ser Ser Met Lys Val Lys Ile Ile
320 325 330Ala Pro Pro Glu Arg Lys Tyr Ser Val Trp Ile Gly Gly Ser Ile
335 340 345Leu Ala Ser Leu Ser Thr Phe Gln Gln Met Trp Ile Ser Lys Gln
350 355 360Glu Tyr Asp Glu Ser Gly Pro Ser Ile Val His Arg Lys Cys Phe
365 370 375SEQ ID NO:8的信息:
序列特征:
长度:375氨基酸
类型:氨基酸
链型:1
拓扑结构:线性
分子类型:肽
来源:Acremonium chrysogenum
特点:
其它信息:分子量为41612Da的γ-肌动蛋白的氨基酸序列序列描述:SEQ ID NO:8Met Glu Glu Glu Val Ala Ala Leu Val Ile Asp Asn Gly Ser Gly1 5 10 15Met Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val
20 25 30Phe Pro Ser Ile Val Gly Arg Pro Arg His His Gly Ile Met Ile
35 40 45Gly Met Gly Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser
50 55 60Lys Arg Gly Ile Leu Thr Leu Arg Tyr Pro Ile Glu His Gly Val
65 70 75Val Thr Asn Trp Asp Asp Met Glu Lys Ile Trp His His Thr Phe
80 85 90Tyr Asn Glu Leu Arg Val Ala Pro Glu Glu His Pro Val Leu Leu
95 100 105Thr Glu Ala Pro Ile Asn Pro Lys Ser Asn Arg Glu Lys Met Thr
110 115 120Gln Ile Val Phe Glu Thr Phe Asn Ala Pro Ala Phe Tyr Val Ser
125 130 135Ile Gln Ala Val Leu Ser Leu Tyr Ala Ser Gly Arg Thr Thr Gly
140 145 150Ile Val Leu Asp Ser Gly Asp Gly Val Thr His Val Val Pro Ile
155 160 165Tyr Glu Gly Phe Ala Leu Pro His Ala Ile Ala Arg Val Asp Met
170 175 180Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile Leu Ala Glu
185 190 195Arg Gly Tyr Thr Phe Ser Thr Thr Ala Glu Arg Glu Ile Val Arg
200 205 210Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu Gln
215 220 225Glu Ile Gln Thr Ala Ala Gln Ser Ser Ser Leu Glu Lys Ser Tyr
230 235 240Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe
245 250 255Arg Ala Pro Glu Ala Leu Phe Gln Pro Ser Val Leu Gly Leu Glu
260 265 270Ser Gly Gly Ile His Val Thr Thr Phe Asn Ser Ile Met Lys Cys
275 280 285Asp Val Asp Val Arg Lys Asp Leu Tyr Gly Asn Ile Val Met Ser
290 295 300Gly Gly Thr Thr Met Tyr Pro Gly Leu Ser Asp Arg Met Gln Lys
305 310 315Glu Ile Thr Ala Leu Ala Pro Ser Ser Met Lys Val Lys Ile Ile
320 325 330Ala Pro Pro Asp Gly Lys Tyr Ser Val Trp Ile Gly Gly Ser Ile
335 340 345Leu Ala Ser Leu Ser Thr Phe Gln Gln Met Trp Ile Ser Lys Thr
350 355 360Glu Tyr Asp Glu Glu Arg Pro Ser Ile Val His Arg Lys Cys Phe
365 370 375
Claims (42)
1、一种提高或阻止微生物中基因表达的方法,其特征在于,在Penicillium chrysogenum的gdh基因的启动子的控制下,在所述的微生物中表达基因,部分基因序列或DNA片段。
2、一种提高或阻止微生物中基因表达的方法,其特征在于,在Penicillium chrysogenum的hex基因的启动子的控制下,在所述的微生物中表达基因,部分基因序列或DNA片段。
3、一种在微生物中蛋白质的胞外表达的方法,其特征在于,在所述的微生物中表达与Penicillium chrysogenum的hex基因的基因融合体。
4、一种提高或阻止微生物中基因表达的方法,其特征在于,在Penicillium chrysogenum的act基因的启动子的控制下,在所述的微生物中表达基因,部分基因序列或DNA片段。
5、一种提高或阻止微生物中基因表达的方法,其特征在于,在Acremonium chrysogenum的act基因的控制下,在所述的微生物中表达基因,部分基因序列或DNA片段。
6、按照权利要求1至5中任意一项所述的方法,其中,该转化的微生物是一种非人类真核细胞,优选Penicillium,Aspergillus或Acremonium。
7、按照权利要求6所述的方法,其中,所用的微生物最好为Penicilliumchrysogenum,Aspergillus nidulans或Acremonium chrysogenum。
8、一种从P.chrysogenum分离的2811bp的DNA化合物,其边界为Sau3AⅠ和XbaⅠ限制性位点,它包括gdh基因的启动子。
9、一种从P.chrysogenum分离的7737bp的DNA化合物,其边界为BamHⅠ和SacⅠ限制性位点,它包括hex基因的启动子。
10、一种从P.chrysogenum分离的4947bp的DNA化合物,其边界为BglⅡ和EeoRⅠ限制性位点,它包括act基因的启动子。
11、一种从A.chrysogenum分离的8650bp的DNA化合物,其边界为两个HindⅢ限制性位点,它包括act基因的启动子。
12、一种由SEQ ID NO:1所示的DNA序列,它包括P.chrysogenum的gdh基因。
13、一种由SEQ ID NO:2所示的DNA序列,它包括P.chrysogenum的hex基因。
14、一种由SEQ ID NO:3所示的DNA序列,它包括P.chrysogenum的act基因。
15、一种由SEQ ID NO:4所示的DNA序列,它包括A.chrysogenum的act基因。
16、在严谨条件下可与权利要求8至15的DNA化合物杂交的核苷酸序列。
17、一种由SEQ ID NO:5所示的氨基酸序列,它与P.chrysogenum的谷氨酸脱氢酶相匹配。
18、一种由SEQ ID NO:6所示的氨基酸序列,它与P.chrysogenum的β-N-谷乙酰氨基己糖苷酶相匹配。
19、一种由SEQ ID NO:7所示的氨基酸序列,它与P.chrysogenum的γ-肌动蛋白相匹配。
20、一种由SEQ ID NO:8所示的氨基酸序列,它与A.chrysogenum的γ-肌动蛋白相匹配。
21、携带权利要求8至16所述的DNA化合物或其片段的载体。
22、按照权利要求21所述的载体,其特征在于由一种质粒组成。
23、按照权利要求22所述的质粒,其特征在于由pALP784,pALP785和pALfleo7组成。
24、按照权利要求22所述的质粒,其特征在于由pALP295,pALP319,pALP377,pALP388和pALP480组成。
25、按照权利要求22所述的质粒,其特征在于由pALP315和pALP316组成。
26、按照权利要求22所述的质粒,其特征在于由pALC52和pALC53组成。
27、一种获得转化的非人类生物体的方法,其中提高了同源或异源基因的表达,其特征在于包括下述操作:
a)构建携带SEQ ID NO:1,SEQ ID NO:2,SEQ ID NO:3或SEQ ID NO:4的全部或部分,或它们的重组DNA化合物的载体;
b)将该载体导入非人类宿主生物体,得到表达同源或异源基因的转化的生物体;
c)选择出与非转化的对照生物体相比显示出提高的同源或异源基因表达的转化的生物体。
28、一种获得转化的可产生胞外蛋白质的非人类生物体的方法,其特征在于包括下述操作:
a)构建携带SEQ ID NO:2的全部或部分,或它的重组DNA化合物的载体;
b)将该载体导入非人类宿主生物体,得到表达和分泌至少一种蛋白质的转化的生物体;
c)选择出与非转化的对照生物体相比可高水平地分泌至少一种蛋白质的转化的生物体。
29、一种获得转化的,具有反义基因表达的,全部或部分阻断其活性的非人类生物体的方法,其特征在于包括下述操作:
a)构建携带SEQ ID NO:1,SEQ ID NO:2,SEQ ID NO:3或SEQ ID NO:4的全部或部分,或它们的重组DNA化合物的载体;
b)将该载体导入非人类宿主生物体,得到表达反义基因的转化的生物体;
c)选择出与非转化的对照生物体相比显示出全部或部分阻断该基因表达的转化的生物体。
30、获得权利要求27和29所述的转化生物体的方法,其特征在于,所用的载体是权利要求21至26限定的,或从它们得到的载体。
31、获得权利要求28所述的转化生物体的方法,其特征在于,所用的载体是权利要求24先限定的,或从它们得到的载体。
32、获得权利要求27至31所述的转化生物体的方法,其中,所用的宿主生物体是非人类真核细胞,最好为Penicillium,Aspergillus或Acremonium。
33、按照权利要求32所述的方法,其中,所用的生物体最好为Penicilllium chrysogenum,Aspergillus nidulans或Acremoniumchrysogenum。
34、按照权利要求27至31所述的获得转化的生物体的方法,其中,所用的宿主生物体是原核细胞,最好为大肠杆菌或放线菌。
35、转化的非人类生物体,其特征在于,部分或全部包括在权利要求21至26的载体中的,可通过权利要求27至34所述的方法得到的权利要求8至16的DNA序列被导入其中。
36、按照权利要求35所述的转化的生物体,其特征在于,由原核细胞,最好为大肠杆菌或放线菌组成。
37、按照权利要求35所述的转化的生物体,其特征在于,由真核细胞,最好由真菌Penicillium,Aspergillus,Acremonium或Saccharomycescerevisiae组成。
38、按照权利要求36所述的转化的生物体,其特征在于,最好由Penicilllium chrysogenum,Aspergillus nidulans,Acremonium chrysogenum或Saccharomyces cerevisiae组成。
39、按照权利要求6所述的生物体,其特征在于,由CECT4849,CECT4852,CECT4851和CECT4850所表示的纯菌株或它们的突变体和转化的衍生物组成。
40、按照权利要求35至39所说的转化的生物体在生产抗生素中的应用。
41、按照权利要求35至39所说的转化的生物体在生产青霉素中的应用。
42、按照权利要求35至39所说的转化的生物体在生产头孢霉素中的应用。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ESP9700482 | 1997-03-05 | ||
ES009700482A ES2127697B1 (es) | 1997-03-05 | 1997-03-05 | Promotores de los genes glutamato deshidrogenasa, beta-n-acetilhexosaminidasa y gamma-actina y su utilizacion en sistemas de expresion, secrecion y antisentido de hongos filamentosos. |
Publications (1)
Publication Number | Publication Date |
---|---|
CN1219200A true CN1219200A (zh) | 1999-06-09 |
Family
ID=8298517
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN98800237A Pending CN1219200A (zh) | 1997-03-05 | 1998-03-05 | 谷氨酸脱氢酶、β-N-乙酰氨基己糖苷酶和γ-肌动蛋白基因的启动子以及它们在丝状真菌表达,分泌和反义系统中的应用 |
Country Status (17)
Country | Link |
---|---|
US (2) | US6300095B1 (zh) |
JP (1) | JP2000509613A (zh) |
KR (1) | KR20000065202A (zh) |
CN (1) | CN1219200A (zh) |
AU (1) | AU6101198A (zh) |
CA (1) | CA2253250A1 (zh) |
CZ (1) | CZ353598A3 (zh) |
EE (1) | EE9800381A (zh) |
ES (3) | ES2127697B1 (zh) |
HU (1) | HUP0000870A3 (zh) |
IL (1) | IL126647A0 (zh) |
LT (1) | LT4557B (zh) |
LV (1) | LV12264B (zh) |
PL (1) | PL329722A1 (zh) |
SK (1) | SK149598A3 (zh) |
WO (1) | WO1998039459A1 (zh) |
ZA (1) | ZA981896B (zh) |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6610839B1 (en) | 1997-08-14 | 2003-08-26 | Geron Corporation | Promoter for telomerase reverse transcriptase |
US6777203B1 (en) | 1997-11-19 | 2004-08-17 | Geron Corporation | Telomerase promoter driving expression of therapeutic gene sequences |
ES2127697B1 (es) * | 1997-03-05 | 2000-03-16 | Antibioticos Sau | Promotores de los genes glutamato deshidrogenasa, beta-n-acetilhexosaminidasa y gamma-actina y su utilizacion en sistemas de expresion, secrecion y antisentido de hongos filamentosos. |
US7378244B2 (en) * | 1997-10-01 | 2008-05-27 | Geron Corporation | Telomerase promoters sequences for screening telomerase modulators |
ES2143408B1 (es) * | 1998-04-02 | 2000-12-01 | Urquima Sa | Promotor y construcciones para expresion de proteinas recombinantes en hongos filamentosos. |
CA2372285A1 (en) * | 1999-03-03 | 2000-09-08 | Marcus Hartmann | .beta.-hexosaminidase, dna sequence from ciliates for coding the same and use thereof |
EP2009093A3 (en) * | 2004-09-22 | 2009-04-08 | Bioworks, Inc. | Transgenic strains of trichoderma and their use in biocontrol |
EP1801221A1 (en) * | 2005-12-22 | 2007-06-27 | Sandoz AG | Promoter sequences |
CN102127546B (zh) * | 2010-11-24 | 2012-10-24 | 山东农业大学 | 一种骨骼肌特异性actin启动子及其应用 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8508800D0 (en) * | 1985-04-04 | 1985-05-09 | Glaxo Group Ltd | Microbiological process |
FI864473A (fi) * | 1985-11-20 | 1987-05-21 | Panlabs Inc | Sammansatta yttryckskassetter foer fungaltransformering. |
ES2127697B1 (es) * | 1997-03-05 | 2000-03-16 | Antibioticos Sau | Promotores de los genes glutamato deshidrogenasa, beta-n-acetilhexosaminidasa y gamma-actina y su utilizacion en sistemas de expresion, secrecion y antisentido de hongos filamentosos. |
-
1997
- 1997-03-05 ES ES009700482A patent/ES2127697B1/es not_active Expired - Fee Related
-
1998
- 1998-03-05 US US09/171,337 patent/US6300095B1/en not_active Expired - Fee Related
- 1998-03-05 CA CA002253250A patent/CA2253250A1/en not_active Abandoned
- 1998-03-05 ZA ZA981896A patent/ZA981896B/xx unknown
- 1998-03-05 SK SK1495-98A patent/SK149598A3/sk unknown
- 1998-03-05 CZ CZ983535A patent/CZ353598A3/cs unknown
- 1998-03-05 IL IL12664798A patent/IL126647A0/xx unknown
- 1998-03-05 HU HU0000870A patent/HUP0000870A3/hu unknown
- 1998-03-05 PL PL98329722A patent/PL329722A1/xx unknown
- 1998-03-05 CN CN98800237A patent/CN1219200A/zh active Pending
- 1998-03-05 EE EE9800381A patent/EE9800381A/xx unknown
- 1998-03-05 AU AU61011/98A patent/AU6101198A/en not_active Abandoned
- 1998-03-05 WO PCT/ES1998/000056 patent/WO1998039459A1/es not_active Application Discontinuation
- 1998-03-05 KR KR1019980708912A patent/KR20000065202A/ko not_active Application Discontinuation
- 1998-03-05 JP JP10538200A patent/JP2000509613A/ja not_active Ceased
- 1998-09-03 ES ES009801859A patent/ES2149707B1/es not_active Expired - Fee Related
- 1998-09-03 ES ES009801858A patent/ES2149706B1/es not_active Expired - Fee Related
- 1998-10-23 LT LT98-152A patent/LT4557B/lt not_active IP Right Cessation
- 1998-11-05 LV LVP-98-268A patent/LV12264B/en unknown
-
2000
- 2000-08-02 US US09/631,022 patent/US6558921B1/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
LV12264A (lv) | 1999-04-20 |
LV12264B (en) | 1999-10-20 |
LT4557B (lt) | 1999-10-25 |
HUP0000870A2 (en) | 2000-07-28 |
HUP0000870A3 (en) | 2001-09-28 |
CZ353598A3 (cs) | 1999-02-17 |
ES2149707A1 (es) | 2000-11-01 |
IL126647A0 (en) | 1999-08-17 |
ES2149707B1 (es) | 2001-05-16 |
PL329722A1 (en) | 1999-04-12 |
LT98152A (en) | 1999-06-25 |
ES2149706A1 (es) | 2000-11-01 |
US6558921B1 (en) | 2003-05-06 |
ZA981896B (en) | 1998-09-14 |
US6300095B1 (en) | 2001-10-09 |
KR20000065202A (ko) | 2000-11-06 |
ES2127697B1 (es) | 2000-03-16 |
WO1998039459A1 (es) | 1998-09-11 |
ES2127697A1 (es) | 1999-04-16 |
CA2253250A1 (en) | 1998-09-11 |
JP2000509613A (ja) | 2000-08-02 |
SK149598A3 (en) | 2000-03-13 |
AU6101198A (en) | 1998-09-22 |
EE9800381A (et) | 1999-04-15 |
ES2149706B1 (es) | 2001-05-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN1262639C (zh) | 新的宿主细胞和生产蛋白质的方法 | |
CN100347301C (zh) | 无选择标志重组基因的菌株,得到这些菌株的方法及应用 | |
CN1253574C (zh) | 来自米曲霉的areA基因和其中areA基因已被修饰的真菌 | |
CN1169961C (zh) | 基因转变作为工具用于构建重组的工业化丝状真菌 | |
CN1261567C (zh) | 热稳定的葡糖淀粉酶 | |
CN1117151C (zh) | 真菌中的核黄素生物合成 | |
CN1090325A (zh) | 支链淀粉酶、产生它的微生物、其制备方法和用途 | |
CN1105778C (zh) | 核苷-5’- 磷酸酯的制造方法 | |
CN1062169A (zh) | 巴斯德毕赤酵母酸性磷酸酶基因 | |
CN1961072A (zh) | 改良型腈水合酶 | |
CN1993464A (zh) | 新羰基还原酶、其基因及它们的使用方法 | |
CN1513057A (zh) | 宿主微生物 | |
CN1366550A (zh) | (r)-2-辛醇脱氢酶,产生此酶的方法,编码此酶的dna,以及使用此酶生产醇类的方法 | |
CN1190495C (zh) | 在单端孢菌毒素缺陷的丝状真菌突变细胞中生产异源多肽的方法 | |
CN1198939C (zh) | 来自绿色木霉的纤维素酶cbh1基因的调控序列以及利用该序列的蛋白质或多肽的大量生产体系 | |
CN1836044A (zh) | 头孢菌素c酰基转移酶突变体及用其制备7-aca的方法 | |
CN1252097A (zh) | 转基因植物选择方法 | |
CN1303434A (zh) | 用于生产核黄素的遗传方法 | |
CN1656225A (zh) | 新型羰基还原酶及其编码基因、以及利用它们制备光学活性醇的方法 | |
CN1219200A (zh) | 谷氨酸脱氢酶、β-N-乙酰氨基己糖苷酶和γ-肌动蛋白基因的启动子以及它们在丝状真菌表达,分泌和反义系统中的应用 | |
CN1268746C (zh) | 用于在真菌细胞中生产多肽的方法 | |
CN1301307A (zh) | 在丝状真菌突变细胞中生产多肽的方法 | |
CN1086737C (zh) | 超热稳定蛋白酶基因 | |
CN1891820A (zh) | 在表面活性剂存在下稳定的胆固醇氧化酶 | |
CN1183248C (zh) | 环状缩肽合成酶及其基因、以及环状缩肽的大规模生产系统 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
WD01 | Invention patent application deemed withdrawn after publication |