WO1997046689A1 - Fungal metallo protease genes - Google Patents
Fungal metallo protease genes Download PDFInfo
- Publication number
- WO1997046689A1 WO1997046689A1 PCT/EP1997/002982 EP9702982W WO9746689A1 WO 1997046689 A1 WO1997046689 A1 WO 1997046689A1 EP 9702982 W EP9702982 W EP 9702982W WO 9746689 A1 WO9746689 A1 WO 9746689A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- filamentous fungus
- dna
- metallo
- protease
- gene
- Prior art date
Links
- 108010006035 Metalloproteases Proteins 0.000 title claims abstract description 116
- 230000002538 fungal effect Effects 0.000 title claims abstract description 19
- 102000005741 Metalloproteases Human genes 0.000 claims abstract description 92
- 241000233866 Fungi Species 0.000 claims abstract description 57
- 230000000694 effects Effects 0.000 claims abstract description 47
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 46
- 108091005804 Peptidases Proteins 0.000 claims abstract description 44
- 108020004414 DNA Proteins 0.000 claims abstract description 42
- 239000004365 Protease Substances 0.000 claims abstract description 32
- 108091005508 Acid proteases Proteins 0.000 claims abstract description 17
- 230000002950 deficient Effects 0.000 claims abstract description 9
- 241000228212 Aspergillus Species 0.000 claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 68
- 241000351920 Aspergillus nidulans Species 0.000 claims description 65
- 241000228245 Aspergillus niger Species 0.000 claims description 54
- 101100189892 Escherichia coli (strain K12) pepD gene Proteins 0.000 claims description 40
- 102000035195 Peptidases Human genes 0.000 claims description 33
- 238000000034 method Methods 0.000 claims description 27
- 102000004169 proteins and genes Human genes 0.000 claims description 24
- 230000014509 gene expression Effects 0.000 claims description 20
- 230000002068 genetic effect Effects 0.000 claims description 11
- 230000008569 process Effects 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- 101150093025 pepA gene Proteins 0.000 claims description 8
- 230000001105 regulatory effect Effects 0.000 claims description 6
- 241000228197 Aspergillus flavus Species 0.000 claims description 5
- 238000009776 industrial production Methods 0.000 claims description 4
- 230000035772 mutation Effects 0.000 claims description 4
- 101150035909 pepB gene Proteins 0.000 claims description 4
- 101150104069 pepE gene Proteins 0.000 claims description 4
- 101150112604 CPYA gene Proteins 0.000 claims description 3
- 101100351592 Caldanaerobacter subterraneus subsp. tengcongensis (strain DSM 15242 / JCM 11007 / NBRC 100824 / MB4) pepT gene Proteins 0.000 claims description 3
- 101100463411 Lactococcus lactis subsp. cremoris pepF1 gene Proteins 0.000 claims description 3
- 101150075954 apeB gene Proteins 0.000 claims description 3
- 101150094986 pepC gene Proteins 0.000 claims description 3
- 101150052109 pepDA gene Proteins 0.000 claims description 3
- 101150047280 pepF gene Proteins 0.000 claims description 3
- 101150038087 pepd gene Proteins 0.000 claims description 3
- 101150023641 ppc gene Proteins 0.000 claims description 3
- 101150108303 prtA gene Proteins 0.000 claims description 3
- 101000765308 Aspergillus niger N-(5'-phosphoribosyl)anthranilate isomerase Proteins 0.000 claims description 2
- 101100028194 Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) alp1 gene Proteins 0.000 claims description 2
- 101150001753 MPL gene Proteins 0.000 claims description 2
- 101100243750 Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) pgtE gene Proteins 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 239000012634 fragment Substances 0.000 description 62
- 239000013612 plasmid Substances 0.000 description 45
- 235000018102 proteins Nutrition 0.000 description 21
- 238000004458 analytical method Methods 0.000 description 19
- 150000001413 amino acids Chemical group 0.000 description 15
- 239000003446 ligand Substances 0.000 description 14
- 240000006439 Aspergillus oryzae Species 0.000 description 13
- 241001225321 Aspergillus fumigatus Species 0.000 description 12
- 238000009396 hybridization Methods 0.000 description 11
- 108091008146 restriction endonucleases Proteins 0.000 description 11
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 10
- 101150008194 argB gene Proteins 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- 239000000872 buffer Substances 0.000 description 9
- 238000012512 characterization method Methods 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 8
- 230000009466 transformation Effects 0.000 description 8
- 108091026890 Coding region Proteins 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N chloroform Substances ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 101000577180 Aspergillus oryzae (strain ATCC 42149 / RIB 40) Neutral protease 2 Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 6
- 239000011543 agarose gel Substances 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 229910021645 metal ion Inorganic materials 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
- 101710089384 Extracellular protease Proteins 0.000 description 5
- 108010030544 Peptidyl-Lys metalloendopeptidase Proteins 0.000 description 5
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 5
- 230000002797 proteolythic effect Effects 0.000 description 5
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 5
- 229940045145 uridine Drugs 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 101100295959 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) arcB gene Proteins 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- RASZIXQTZOARSV-BDPUVYQTSA-N astacin Chemical compound CC=1C(=O)C(=O)CC(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)C(=O)CC1(C)C RASZIXQTZOARSV-BDPUVYQTSA-N 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 239000012228 culture supernatant Substances 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 101150039489 lysZ gene Proteins 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000002018 overexpression Effects 0.000 description 4
- 101150051209 pip gene Proteins 0.000 description 4
- 239000013615 primer Substances 0.000 description 4
- 108010028075 procathepsin L Proteins 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000017854 proteolysis Effects 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 101150028074 2 gene Proteins 0.000 description 3
- 108091005502 Aspartic proteases Proteins 0.000 description 3
- 102000035101 Aspartic proteases Human genes 0.000 description 3
- 241001513093 Aspergillus awamori Species 0.000 description 3
- 101100381793 Bacillus subtilis (strain 168) bioK gene Proteins 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 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 3
- 241000275031 Nica Species 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 3
- 108020005115 Pyruvate Kinase Proteins 0.000 description 3
- 102000013009 Pyruvate Kinase Human genes 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 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 3
- 229960000723 ampicillin Drugs 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 101150076754 bioA gene Proteins 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000005546 dideoxynucleotide Substances 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000001952 enzyme assay Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 229920001778 nylon Polymers 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 2
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 2
- 108090000658 Astacin Proteins 0.000 description 2
- 102000034498 Astacin Human genes 0.000 description 2
- 101100007857 Bacillus subtilis (strain 168) cspB gene Proteins 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 101100109110 Danio rerio aph1b gene Proteins 0.000 description 2
- 101100030906 Dickeya chrysanthemi prtD gene Proteins 0.000 description 2
- 101100030911 Dickeya chrysanthemi prtF gene Proteins 0.000 description 2
- 101100409540 Dictyostelium discoideum psmA3 gene Proteins 0.000 description 2
- FMKGDHLSXFDSOU-BDPUVYQTSA-N Dienon-Astacin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)C(=O)C(=CC1(C)C)O)C=CC=C(/C)C=CC2=C(C)C(=O)C(=CC2(C)C)O FMKGDHLSXFDSOU-BDPUVYQTSA-N 0.000 description 2
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 2
- 241000223218 Fusarium Species 0.000 description 2
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 2
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 2
- 241000235395 Mucor Species 0.000 description 2
- 239000008118 PEG 6000 Substances 0.000 description 2
- 241000228143 Penicillium Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 2
- 241000235527 Rhizopus Species 0.000 description 2
- 241000607720 Serratia Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 108090001109 Thermolysin Proteins 0.000 description 2
- 241000223259 Trichoderma Species 0.000 description 2
- 102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 2
- 108700015934 Triose-phosphate isomerases Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 102000004139 alpha-Amylases Human genes 0.000 description 2
- 108090000637 alpha-Amylases Proteins 0.000 description 2
- 229940024171 alpha-amylase Drugs 0.000 description 2
- 101150069003 amdS gene Proteins 0.000 description 2
- 108090000987 aspergillopepsin I Proteins 0.000 description 2
- 235000003676 astacin Nutrition 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 101150110403 cspA gene Proteins 0.000 description 2
- 101150068339 cspLA gene Proteins 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 230000037452 priming Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 229920006298 saran Polymers 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000003998 snake venom Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108010050327 trypticase-soy broth Proteins 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- -1 0.5 g KCI Substances 0.000 description 1
- 108020005065 3' Flanking Region Proteins 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- 108020005029 5' Flanking Region Proteins 0.000 description 1
- 241001136782 Alca Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 101100523058 Aspergillus niger pyrG gene Proteins 0.000 description 1
- 241001149666 Aspergillus niger var. macrosporus Species 0.000 description 1
- 101000577181 Aspergillus oryzae (strain ATCC 42149 / RIB 40) Extracellular metalloproteinase NpI Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108091005658 Basic proteases Proteins 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102400000011 Cytochrome b-c1 complex subunit 9 Human genes 0.000 description 1
- 101800000778 Cytochrome b-c1 complex subunit 9 Proteins 0.000 description 1
- 108020001019 DNA Primers Proteins 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 101100317179 Dictyostelium discoideum vps26 gene Proteins 0.000 description 1
- 101100271445 Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) atp9 gene Proteins 0.000 description 1
- 101100407639 Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) prtB gene Proteins 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 108010059881 Lactase Proteins 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 102000011755 Phosphoglycerate Kinase Human genes 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 101100370749 Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) trpC1 gene Proteins 0.000 description 1
- 239000008049 TAE buffer Substances 0.000 description 1
- 101001099217 Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) Triosephosphate isomerase Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 108010048241 acetamidase Proteins 0.000 description 1
- HGEVZDLYZYVYHD-UHFFFAOYSA-N acetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound CC(O)=O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O HGEVZDLYZYVYHD-UHFFFAOYSA-N 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 101150014229 carA gene Proteins 0.000 description 1
- 101150070764 carB gene Proteins 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 235000021474 generally recognized As safe (food) Nutrition 0.000 description 1
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000001295 genetical effect Effects 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 230000001939 inductive effect Effects 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
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229940116108 lactase Drugs 0.000 description 1
- 101150087199 leuA gene Proteins 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 101150101926 mep gene Proteins 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 239000002636 mycotoxin Substances 0.000 description 1
- 101150095344 niaD gene Proteins 0.000 description 1
- 239000006880 nzcym-medium Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 101150082708 pkiA gene Proteins 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 230000000063 preceeding effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000020978 protein processing Effects 0.000 description 1
- 101150054232 pyrG gene Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 108010059841 serine carboxypeptidase Proteins 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 101150016309 trpC gene Proteins 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
- C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus
-
- 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
Definitions
- the present invention relates to the field of molecular biology.
- the invention relates metallo proteases obtainable from filamentous fungi and the genes encoding said proteases.
- Aspergillus species and in particular Aspergillus niger are used for the industrial production of enzymes used in the food processing industry.
- A. niger has advantages as a host for the production of recombinant proteins because of its large capacity for secretion of proteins, and because systems are available for its molecular genetic manipulation.
- proteases in the culture fluid, periplasmic space, endoplasmic reticulum and Golgi apparatus has proven deleterious to the expression of homologous and heterologous proteins in A. niger.
- Aspergilli are used commercially to produce proteases. A number of extracellular proteases from Aspergilli have been described in the literature.
- the gene pepA encoding aspergillopepsin A from Aspergillus awamori has been cloned.
- the pepA gene product accounts for a major part of the secreted acid proteases of A. niger and strains in which the pepA gene has been deleted have allowed increased expression of heterologous proteins in A. niger var. awamori (EP-A-0 429 490) .
- Other protease genes have also been recently cloned from Aspergilli and these include an alkaline aspartic protease of A. oryzae, an alkaline aspartic protease of A. fumigatus, a non- pepsin type acid protease from A.
- pepB an aspartic protease of A. niger (pepE), a metallo protease called neutral protease II from A. oryzae, the metallo protease from A. fumigatus and A. flavus, two serine proteases from A. niger (pepC, pepD) and two serine carboxypeptidases from A. niger (pepF and cpy) . Also an alkaline protease (prtA) from A. nidulans has been cloned.
- Isolated and mutated protease genes of A. niger can be used for gene disruption experiments, i.e. the preparation of mutant strains in which the corresponding natural gene is destroyed.
- the pepA gene from Aspergillus awamori has been destroyed by gene disruption in order to prepare aspergillopepsin A deficient strains.
- the pepB and the pepE gene have been destroyed by gene disruption and a recombinant strain carrying all three disruptions has also been made.
- Aspergilli produce a large number of different proteases and, thus, there is a continuing need for Aspergillus strains deficient in other proteases for the industrial production of proteins.
- protease genes which can be used for the preparation of protease deficient strains by in vitro mutagenesis, e.g. gene disruption.
- protease proteins which can be industrially applied for protein processing .
- A. niger Another major constituent of the secreted protease activities in A. niger is a metallo protease.
- Metallo proteases have been cloned in some of the fungi.
- Neutral protease II encoded by a metallo protease has been cloned from A. oryzae and from A. fumigatus and A. flavus whereas a neutral protease I encoding gene was cloned from the A. fumigatus. It is now found that some A.
- niger protease deficient mutants isolated by mutagenesis and strains in which one or all of the acidic protease genes have been disrupted, have still metallo protease activity which surprisingly is even increased as compared to the parental wild type A. niger strain, apparently in an attempt to compensate for the loss of the acidic protease activity . This is for example the case in the prtF and prtD mutants of A. niger.
- Aspergillus nidulans is also a suitable host for the expression of heterologous proteins. Isolated and mutated protease genes of A nidulans can also be used for gene disruption experiments. The number of A nidulans protease genes cloned is quite limited and therefore there is a need to establish the protease spectrum, to clone the protease genes and to destroy the corresponding natural genes.
- Figure 1 a shows the partial nucleotide sequences of A niger pepH, and the deduced partial amino acid sequence of PEPH.
- Figure 1 b shows a partial alignment of the amino acid sequences of PEPH
- Figure 2a shows the nucleotide sequences of A. nidulans pepl and pepJ and the deduced amino acid sequences of PEPI and PEPJ .
- Figure 2b shows the homologies between the amino acid sequences of the A. nidulans metallo proteases and several other metallo proteases.
- Figure 3a and 3b present a summary of data which indicate that the A. nidulans
- PEPI and PEPJ belong to a novel class of proteases in filamentous fungi.
- the present invention provides protease deficient mutants of filamentous fungi which contain a mutation which is a site selected disruption of DNA that results in the filamentous fungus having a reduced metallo protease activity.
- Said filamentous fungi are useful for the production of proteins, both heterologous and homologous, because reduced protease activity will minimize both the chance that, and rate at which, such proteins will be degraded during production.
- the filamentous fungi of the invention are preferably fungi that can be used for the industrial production of proteins.
- Such fungi are non-pathogenic, produce virtually no mycotoxins and have often aquired GRAS status in conjunction with enzymes produced by the fungus.
- Examples of such fungi belong to the genera of Trichoderma, Penicillium, Fusarium, Mucor, Rhizopus, and most preferably Aspergillus.
- the preferred fungi belong to the Aspergillus niger group, the Aspergillus nidulans group and the Aspergillus flavus group, whereby these "groups " are defined as in Raper and Fennell ( 1 965, In: The Genus Aspergillus, The Williams & Wilkins Company, Baltimore) , thereby comprising all species and variants included in a particular group by these authors.
- Raper and Fennell 1 965, In: The Genus Aspergillus, The Williams & Wilkins Company, Baltimore
- the terms A. niger, A. nidulans and A. oryzae will herein refer to the entire Aspergillus niger group, the Aspergillus nidulans group and the Aspergillus flavus group, respectively.
- the filamentous fungi according to the present invention also have a reduced extracellular acid protease activity.
- a mutant filamentous fungus with a reduced extracellular acid protease activity is herein understood to comprise any fungal mutant of which the activity of one or more of the extracellular acid proteases, which are not metalloproteases, is reduced as compared to the activity in the wild type filamentous fungus.
- An acid protease is herein understood to mean a protease of which the pH optimum is more acidic than pH 7.0. Extracellular acid protease activity levels of mutant and wild type fungi can be determined by the methods described in van den Hombergh et al. ( 1 995, Curr. Genet. 28: 299-308) .
- the reduced extracellular protease activity is the result of reduced activity of one or more gene products encoded by the A.niger pep A, pepB, pepC, pepD, pepE, pepF, cpy genes or the A. nidulans prtA gene.
- the corresponding homologues of these genes are intended.
- the reduction in activity of these gene products is achieved by site selected disruption of the corresponding genes.
- the reduced extracellular acid protease activity is the result of one or more of the prt mutations as defined by van den Hombergh et al. supra.
- the reduced extracellular acid protease activity is the result of a prtD or prtF mutation.
- mutant filamentous fungi of the invention have reduced activity levels of both extracellular acid proteases and metallo proteases.
- a fungal metallo protease is herein understood as (i) being derived from a filamentous fungus, (ii) exhibiting protease activity due to a metal ion, usually Zn 2 + , which is usually evidenced by the fact that the protease is sensitive to chelating agents such as EDTA, EGTA or phenanthroline, (iii) having an amino acid sequence which shares sufficient positional identity with amino acid sequences of known metallo proteases for being grouped into one of the metallo protease families.
- nidulans pepI gene SEQ ID NO:5, A. nidulans pepl cDNA; .
- SEQ ID NO:6 A. nidulans PEPI amino acid sequence; SEQ ID NO: 7, A. nidulans pepJ gene; SEQ ID NO:8, A. nidulans pepJ cDNA; SEQ ID NO:9, A. nidulans PEPJ amino acid sequence.
- the present disclosure of the metallo protease genes in A.niger and A. nidulans provides an incentive for the identification of additional metallo protease genes in these and other fungi which are not known at present.
- the preferred candidates in this respect are the industrially important fungi which have been mentioned above.
- novel metallo protease sequences of the present invention can be used in conjunction with the already available metallo protease sequences to more accurately define the conserved regions in the metallo protease amino acid sequences and type of substitutions occurring therein. This will facilitate the design of improved degenerate oligonucleotides which will increase the chance of obtaining new metallo protease genes in PCRs or hybridization experiments.
- the preferred method for cloning new metallo protease genes is the method of the present invention, i.e. the use of degenerate oligonucleotides in a PCR on genomic DNA (or cDNA)
- other methods can also be used for the cloning of new metallo protease genes .
- Such methods may include heterologous hybridization, hybridization with (degenerate) oligonucleotides, (heterologous) complementation of metallo protease-negative mutants, or even screening of expression-libraries with suitable antibodies.
- the invention thus provides isolated DNA sequences encoding proteins having metallo protease activity, which are obtainable from a filamentous fungus.
- the preferred DNA sequences are the DNA sequence of the pepH gene of Aspergillus niger as partially shown in SEQ ID NO: 1 and 2, the DNA sequence of the pepl gene of Aspergillus nidulans as shown in SEQ ID NO:4 and 5, the DNA sequence of the pepJ genes of Aspergillus nidulans as shown in SEQ ID NO:7 and 8, as well as genetic variants of these DNA sequences.
- the invention also provides DNA sequences encoding proteins having metallo protease activity which are obtainable from filamentous fungi and which are capable of hybridizing to one or more of the pepH, pepl and pepJ sequences.
- the metallo protease DNA sequences of the invention are used to prepare mutant filamentous fungi with disrupted metallo protease genes resulting in reduced metallo protease levels.
- DNA constructs containing a metallo protease DNA sequence which comprises a disruption that results in the DNA sequence being incapable of encoding an active metallo protease are provided.
- a number of methods are available to the skilled person for disruption of DNA sequences encoding an active protein. Such disruptions may include deletions, insertions, substitutions, reversions and truncations of the active coding sequences in order to functionally inactivate the metallo protease coding sequence.
- the disruption is nonrevertable, e.g . by deletion of (most of) the coding sequence.
- the DNA constructs for disruption of the metallo protease genes will further comprise a selectable marker gene for selection of transformation.
- a selectable marker gene for selection of transformation.
- a variety of such genes are available to the skilled person, e.g . the fungal amdS, argB, trpC, niaD and pyrG genes as well as several antibiotic resistance genes.
- the DNA constructs for disruption of the metallo protease genes Upon transformation the DNA constructs for disruption of the metallo protease genes will insert at the genomic metallo protease locus through homologous recombination.
- the various elements in the disruption construct are arranged to optimize for such events as one-step or two-step gene replacement. Examples 9 and 1 0 describes the construction and use of disruption constructs for the pepH, pepl and pepJ metallo protease genes.
- the invention thus provides a process for the preparation of a filamentous fungus which has a reduced metallo protease activity.
- the process comprises the steps of a) transforming filamentous fungus with a DNA construct for disruption of a metallo protease gene, and b) selecting a transformed filamentous fungus with reduced metallo protease activity.
- the invention thus provides filamentous fungi wherein the reduced metallo protease activity is the result of a site selected disruption of a DNA sequence encoding a metallo protease.
- the DNA sequence encoding the metallo proteases preferably are the DNA sequences of the pepH, pepl, and pepJ genes as disclosed in the Sequence Listings or are genetic variants thereof.
- the DNA sequences to be disrupted are capable of hybridizing to one of the pepH, pepl, and pepJ genes or their genetic variant.
- the disruption constructs are constructed such that the selection marker gene can be removed from the fungal genome after that the disruption of the metallo protease gene has taken place.
- Methods and examples of this approach are disclosed in EP-A-0 635 574.
- the advantage of this approach is that multiple (metallo)protease genes can be disrupted in successive transformation rounds in one strain using a single selection marker gene.
- the term "genetic variants”, both when applied to metallo protease genes as well as genes coding for extracellular acid proteases, is understood to comprise hybrid DNA sequences containing a protease-encoding sequence coupled to regulatory regions, such as promoter, secretion and terminator signals, originating from homologous or heterologous organisms. Genetic variants also include DNA sequences encoding mutant protease proteins and degenerate DNA sequences wherein the protease activity of the enzyme is retained. The present invention also includes DNA sequences which are capable of hybridizing to the protease-encoding DNA sequences and genetic variants thereof, as described above, but which may differ in codon sequence due to the degeneracy of the genetic code or cross-species variation.
- gene is used herein to indicate any coding DNA sequence, irrespective of whether the sequence is a genomic sequence (possibly containing introns), a cDNA sequence or a synthetic (isocoding) sequence.
- the present invention provides DNA constructs wherein the DNA sequence encoding the metallo protease is operable linked to regulatory regions suitable for expression of the DNA sequence in a suitable host.
- the suitable hosts are preferably microorganisms, i.e. bacteria such as E.coli or Bacillus species, yeasts such as Saccaromyces cerevisiae and Kluyvermyces lactis, or filamentous fungi of which are preferred fungi from the genera Trichoderma, Penicillium, Fusarium, Mucor, Rhizopus, and most preferred are Aspergillus species.
- a wide variety of regulatory regions suitable for regulating the expression of the metallo protease genes in the fore-mentioned hosts are available to the skilled person. Of these regulatory regions the promoters are most important. Examples of strong constitutive and/or inducible promoters which are preferred for use in fungal expression hosts are the ATP-synthetase, subunit 9 (oliC), pyruvate kinase (pki), triose phosphate isomerase (tpi) , alcohol dehydrogenase (alcA), ⁇ -amylase (amy), amyloglucosidase (AG) , acetamidase (amdS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) promoters.
- oliC subunit 9
- pki pyruvate kinase
- tpi triose phosphate isomerase
- alcA alcohol dehydrogenase
- strong yeast promoters are the alcohol dehydrogenase, lactase, 3- phosphoglycerate kinase and triosephosphate isomerase promoters.
- strong bacterial promoters are the ⁇ -amylase and Spo2 promoters as well as promoters from extracellular protease genes.
- the invention therefore discloses host organisms capable of heterologous or overexpression of the filamentous fungal metallo protease genes of the invention, as well as a process for the preparation these metallo proteases.
- This process comprises the steps of a) culturing the host organisms capable of heterologous or overexpression of the filamentous fungal metallo protease genes under conditions conducive to the expression of the metallo protease gene, and b) recovering the metallo protease.
- the obtained metallo proteases can be used to asses in vitro whether or not a certain protein of interest, to be produced in a fungal host is susceptible to the protease in question . This allows to determine which metallo protease genes need to inactivated in the fungal host.
- metallo proteases may find application in many industrial processes where proteases are applied .
- the advantage of the metallo protease being that the protease activity can in principle be switched on or off by the addition of the metal ion or a chelating agent respectively.
- fungal metallo protease as used in the present invention are also fragments of such enzymes which retain metallo protease activity, however, the full length enzymes are preferred embodiments. It is understood that also fusion proteins containing a fungal metallo protease of the invention attached to additional amino acids, peptides, or proteins are part of the present invention.
- Two specific DNA primers (CGGGATCCCGCGTGAGTGAACTCGTG and CGGAATTCCGGTCACCGACTGCAAGG, containing a BamHI and an EcoRI restriction site, respectively) derived from the sequence for the NPII encoding gene (Tatsumi et al., 1 991 ) are used in PCR reactions on A. oryzae ATCC 20386 genomic DNA (isolated as described by de Graaff et al., 1 988) .
- the PCR conditions used are; an initial melting step, (94°C, 3 min), followed by 30 cycles of 1 min annealing (68°C), 1 min extension (72 °C) and 1 min melting (94°C) .
- the PCR mixtures are analyzed on 1 .0% (w/v) agarose gels in 1 xTAE.
- the resulting 0.41 kb PCR fragment is cut out from the agarose gel and the DNA is obtained using the GeneClean kit (Westburg) as described in the instruction manual.
- the 0.41 kb PCR fragment is digested with restriction enzymes EcoRI and BamHI in a reaction mixture composed of the following solutions; ( 1 0 ⁇ l (approx. 2 ⁇ g) DNA solution; 5 ⁇ l of the appropriate 10 x React, buffer (Life Technologies); 1 0 U of both restriction enzymes (Life Technologies) and sterile water to give a final volume of 50 ⁇ l .
- restriction enzymes were removed via fenol, fenol-chloroform and chloroform extraction and subsequent ethanol precipitation of the DNA.
- 50 ng of the restriction digested PCR fragment and 50 ng of BamHI-EcoRI digested pUC1 9 are mixed with 4 ⁇ l of 5 x ligation buffer (Life Technologies; composition: 500 mM Tris-HCl, pH 7.6, 1 00 mM MgCI 2 , 1 0 mM ATP, 1 0 mM dithiothreitol, 25 % (w/v) PEG-6000) and 1 ⁇ l ( 1 .2 U) T 4 DNA ligase (Life technologies) .
- Plasmid pMTL21 -H5 contains a 5.0 kb Hindlll kb fragment of A. fumigatus DNA, that encodes the A. fumigatus MEP gene (described by Jaton- Ogay et al., 1 994) .
- the part of the coding region that encodes the mature MEP protease can be excised by EcoRI.
- the plasmid pMTL21 -H5 is therefore digested with EcoRI and the fragments are separated on a 0.7% (w/v) agarose gel in 1 x TAE.
- the 1 .3 kb EcoRI fragment is cut out and the DNA is obtained using the GeneClean kit (Westburg) as described in the instruction manual. 50 ng of this fragment is labelled with 32 P-dATP via random priming as described by Sambrook et al. ( 1 989) and used immediately for either Southern or plaque lift probings.
- Total genomic DNA is isolated from A. niger and A. nidulans wild type strains (N400 and WG096, respectively) is restriction digested with several restriction enzymes, fragments are separated on 0.7% (w/v) agarose gels in 1 xTAE and subsequently blotted to nylon membranes.
- both the A. fumigatus and the A. oryzae metallo probes are used to screen for the presence of related genes in A. niger and A. nidulans.
- A. niger it is observed that at 60° C after washing down to 2 x SSC, 0.5% (w/v) SDS the A.
- fumigatus metallo protease probe revealed clear specific hybridizing fragments in all digests tested . Under identical conditions no hybridizing signals were detected using the A. oryzae metallo protease probe. Hybridization of A. nidulans genomic DNA with the A. oryzae probe (at 56°C, 4 x SSC, 0.5% (w/v) SDS) however resulted in clear hybridizing signals, indicative for the existence of NPII encoding gene(s) in A. nidulans. Probing A. niger genomic DNA under these conditions (again) resulted in no hybridizing signals.
- This screening resulted in about 20 positive phages, of which ten were purified .
- Each positive phage was picked from the plate using a Pasteur pipette and the phages were eluted from the agar plug in 0.5 ml of SM buffer containing 10 ⁇ l chloroform, as described in Maniatis et al . ( 1982 ) .
- the phages obtained were purified by repeating the procedure described above using filter replicas from plates containing 50-100 plaques of the isolated phages.
- the library was plated as described in 2.2.
- the replicas prepared from this library (Sambrook et al., 1 989) were prehybridised in hybridization buffer (see 2.2) at 56 °C for 3-5 h. Hybridisation was performed in an identical buffer which contained additionally the denatured 32 P radiolabelled 0.41 kb A. oryzae derived metallo protease PCR fragment. After 1 5- 1 8 h hybridisation, the replicas were washed three times in 4 x SSC, 0.5 % (w/v) SDS at 56°C.
- the replicas were covered were saran wrap and autoradiographed at - 70°C using Konica X-ray films and X-omatic cassettes with regular intensifying screens. This screening resulted in at least 35 positive phages, of which ten were purified as described in 2.2. 3. Characterization of the lambda clones.
- the phages were propagated by plating 5 x 1 0 3 phages on NZCYM medium. After overnight incubation at 37 °C confluent plates were obtained, from which the phages were eluted by adding 5 ml SM buffer and storing the plate for 2 h at 4°C with intermittent shaking . After collection of the supernatant using a pipette, the bacteria were removed from the solution by centrifugation at 4,000 x g for 1 0 min. at 4° C. To the supernatant 0.3% (w/v) chloroform was added and the number of pfu is determined. These phage stocks contain approximately 1 0 9 pfu/ml.
- DNA of 1 0 selected phages (lambda 1 - 10) , isolated as described in Sambrook et al. ( 1 989) was analyzed by Southern analysis.
- the DNA was digested for 4 h. at 37 °C in a reaction mixture composed of the following solutions; 5 ⁇ l (approx. 1 ⁇ g) DNA solution; 2 ⁇ l of the appropriate 1 0 x React, buffer (Life Technologies); 10 U Restriction enzyme (Life Technologies) and sterile water to give a final volume of 20 ⁇ l.
- the samples were incubated for 1 0 min. at 65 °C and rapidly cooled on ice, before adding loading buffer and then loaded on a 0.6 % (w/v) agarose gel in TAE buffer.
- the DNA fragments were separated by electrophoresis at 25 V for 15 -18 h (at RT) .
- the DNA was transferred and denatured by alkaline blotting (VacuGene XL, Pharmacia LKB) to nylon membrane (Hybond N, Amers- ham) as described in the VacuGene XL instruction manual (pp . 25-26) and subsequently prehybridised and hybridised as described in 2.2.
- the hybridisation pattern was obtained by exposure of Kodak XAR-5 X-ray film for 1 8 h. at -70 °C using an intensifying screen. In all these clones fragments originating from the same genomic region were found.
- phage stocks were generated for the 10 A. nidulans purified metallo phages, similar to the procedure described in 3.1 . After isolation of phage DNA, this DNA was digested with several restriction enzymes and the resulting fragments were separated by electrophoresis in 1 x TAE agarose gels. The resulting separated DNA fragments were electroblotted to nylon membranes (see 3.1 ) and subsequently prehybridised and hybridised as described for the replicas in 2.1 .
- a 10 kb EcorW was selected for subcloning .
- 25 ng pUC 1 8 FcoRI digested fragment was mixed with 100 ng 1 0 kb EcoRI fragment and 4 ⁇ l of 5 x ligation buffer (composition: 500 mM Tris-HCl, pH 7.6; 1 00 mM MgCI 2 ; 1 0 mM ATP; 1 0 mM dithiothreitol; 25 % (w/v) PEG-6000) and 1 ⁇ l ( 1 .2 U/ml) T 4 DNA ligase (Life Technologies) was added to this mixture in a final volume of 20 ⁇ l.
- 5 x ligation buffer composition: 500 mM Tris-HCl, pH 7.6; 1 00 mM MgCI 2 ; 1 0 mM ATP; 1 0 mM dithiothreitol; 25 % (w/v) PEG-6000
- Plasmid DNA was isolated on a large scale from 200 ml cultures E. coli DH5 ⁇ containing plM676 grown in LB medium containing 1 00 ⁇ g/ml ampicillin (Sambrook et al., 1 989) .
- the plasmid was purified using the Nucleobond AX ion exchange silica purification system as detailed in the Nucleobond AX instruction manual (p p. 8) . The yield was approximately 200 ⁇ g .
- sequence of the structural part of the A. niger pepH gene was determined by subcloning fragments from plM676 in pUC 1 8/1 9, in combination with the use of specific oligonucleotides as primers in the sequencing reactions.
- restriction fragments were isolated and cloned in pUC 1 8/1 9 DNA vectors, digested with the appropriate restriction enzymes.
- the nucleotide sequences were determined by the dideoxynucleotide chain- termination procedure (Sanger et al., 1 977) using the Pharmacia T7 DNA polymerase sequencing kit. Computer analysis was done using the PC/GENE program (Intelligenetics) . The sequence determined is given SEQ ID NO: 1 .
- the sequence of the pepH gene is not yet complete because at the 3'-end of the coding region several ambiguities were found due to compressions. We are still in the process of solving these ambiguities.
- the deduced PEPH protein sequence shows high homologies with the A. fumigatus MEP metallo protease (see appendix I and III). Detailed homology comparisons with members of the individual metallo protease classes demonstrated only limited homology. However, both the A. niger and the A. fumigatus metallo proteases contain a region which to some extent resembles the third ligand region in thermolysin type metallo proteases.
- nidulans phage classes contain metallo protease sequences, larger fragments are subcloned to complete the sequencing of both the genes. From phage class I two EcoRI fragments ( 1 .4 kb and 2.0 kb) and from phage class II a 6.5 kb Hindlll-Xhol fragment are subcloned in pUC 1 9 resulting in plasmids plM667, plM668 and plM669, respectively.
- nucleotide analysis For nucleotide analysis specific restriction fragments are isolated and cloned in pUC 1 8/1 9 DNA vectors, digested with the appropriate restriction enzymes. The nucleotide sequences are determined by the dideoxynucleotide chain-termination procedure (Sanger et al . , 1 977) using the Pharmacia T7 DNA polymerase sequencing kit. Computer analysis is done using the PC/GENE program (Intelligenetics) . The sequence determined is given in SEQ ID NO:2.
- restriction fragments are isolated and cloned in pUC1 8/1 9 DNA vectors, digested with the appropriate restriction enzymes.
- the nucleotide sequences are determined by the dideoxynucleotide chain-termination procedure (Sanger et al. , 1 977) using the Pharmacia T7 DNA polymerase sequencing kit. Computer analysis is done using the PC/GENE program (Intelligenetics) . The sequence determined is given SEQ ID NO:3.
- Both the A. nidulans metallo proteases contain a region HEXXH in which two histidine residues, in analogy with other metallo proteases are involved in coordinating the Zn 2 + cation in the active center, and a glutamate involved in the bond-breaking process (Jongeneel et al., 1 989) . All metallo proteases characterized sofar have been assigned to several independent classes based on the homologies in the proximity of the metal ion ligands (Jiang and Bond, 1 992) .
- the first two ligands which in all metallo proteases studied sofar are two histidine residues, are present in either a HEXXH or a HXXEH motif (Appendix V-A) .
- the thermolysin-type metallo proteases the thermolysin-type metallo proteases, the third metal ion ligand is a glutamic acid residue which is located in a GAXNEAFSD sequence whereas the fourth ligand has been shown to be a water molecule (Kester and Matthews, 1 977) .
- the third ligand is also a histidine residue and the fourth ligand is a tyrosine residue.
- the amino acid present after the third metal ion ligand and the four amino acids present immediately before the fourth ligand did allow further differentiation into four classes, Astacin, Serratia, Matrixin and Snake Venom, respectively (Jiang and Bond, 1 992) .
- the metallo proteases from A. nidulans and A. oryzae do not belong to any of the classes mentioned.
- these metallo proteases do not have a histidine as a third ligand as, apart from the two histidine residues in the HEFTHA sequence, no additional histidine residues are present in the mature proteases . Also no GAXNEAFSD sequence, which could indicate a thermolysin-type metallo protease, was found . The A.niger and A.
- fumigatus metallo protease encoding genes (pepH and MEP, respectively) has also been cloned and sequenced and the deduced amino acid sequence contains in addition to the HEYTH zinc ligands a region resembling the third ligand region in thermolysins, ESGGMGEGWSD (see appendix III) .
- This type of metallo protease has twice the size of the A. nidulans metallo proteases, shows no homology to the A. nidulans PEPI and PEPJ metallo proteases and is probably a thermolysin-type metallo protease (see also 4.2) .
- the metallo proteases from filamentous fungi can be divided into two separate classes; the thermolysin like and the acid metallo proteases.
- the third and fourth metal ion ligands are unknown for the acid metallo proteases but (compared to other metallo protease classes) at approximately the same C-terminal distance of the HEFTH box a conserved sequence SYALY is found, which resembles partially the region preceeding the fourth ligand in the Astacin, Serratia, Matrixin and Snake venom classes (Appendix III) .
- A. niger NW21 9 (cspA1 , leuA 1 , pyrA6, nicA 1 ) is transformed with 1 ⁇ g pGW635 plus 10 ⁇ g plM676 to yield uridine prototrophs. Colonies are purified and DNA prepared as described above. Southern blots probed with the internal Hindlll fragment from pepH show that some transformants have a single copy of plM676 integrated into their genome whereas others have up to and above 1 0 extra copies in their genome. These strains produce correspondingly more proteolytic activity and are mitotically stable.
- Expression cassettes based upon the A. niger pkiA promoter and terminator are prepared for the A. niger pepH gene as described in Bartling et al. ( 1 996) .
- the correct structure of the expression cassette cloned in pUC 1 8 (and resulting in plasmid pPKIPEPH) is confirmed by restriction digestion and sequencing .
- pPKIPEPH contains a fragment inserted into pUC 1 8, which contains an expression cassette consisting of the pyruvate kinase promoter of A. niger fused to the ATG start codon of the pepH gene of A. niger, which is terminated by the pyruvate kinase terminator.
- pPKIPEPH is used with pGW635 (containing the A. niger pyrA gene) to cotransform A. niger NW21 9 to uridine prototrophy.
- the presence of the pki-pepH fusion is confirmed by purifying DNA from individual purified transformants and using it for Southern analysis using probes from the pki and the pepH genes. Strains with one or more copies of this gene fusion integrated into their genome are shown to produce more proteolytic activity when the cells are grown rapidly on glucose as C source. This approach allows expression of the pepH protease gene under conditions (high glucose concentrations) which repress the expression of other extracellular protease encoding genes.
- Plasmid plM667-8 is generated by combining the 1 .4 kb and 2.0 kb EcoRI fragments into a 3.4 kb fragment which contains the promoter, coding region and terminator sequences of the pepl gene. Both plasmids are individually co- transformed with plasmid pGW635 to uridine auxotrophic A. nidulans WG096 strain. Selected and purified transformants are analyzed as described for the pepH multicopy transformants (6.2) . Finally, single and multicopy transformants are selected to produce the A. nidulans PEPI and PEPJ proteins as described for the A. niger PEPH protein.
- pepI and pepJ genes can be constructed.
- uridine auxotrophic strain WG096 uridine prototrophic transformants are isolated and purified. Via Southern analysis the copy number of the expression cassettes in the isolated transformants can be determined . Selected transformants can be grown on high glucose containing media to produce the A. nidulans metallo proteases. Similar to the production of PEPH in A.niger the growth conditions used repress the expression of other homologous extracellular proteases in A. nidulans.
- the expression cassettes constructed for the A. nidulans pepl and pepJ genes which are used to generate multicopy transformants both for the non- fused situation (see 7.1 ) and the gene-fusions (see 7.2) are used to generate multi copy transformants in A. niger NW21 9 (as described in 6.1 ) . Selected transformants are analyzed via Southern analysis (see 6.1 ) to select single and multicopy transformants which are subsequently used to generate the PEPI and PEPJ proteins.
- Van den Hombergh et al. ( 1 995) described the isolation and genetical characterization of a set of protease deficient (prt) mutants, comprising at least seven complementation groups. All prt complementation groups result in reduced extracellular proteolytic activities and have been tested in vitro for reduced proteolytic degradation of proteins which are very susceptible to proteolytic degradation. The residual proteolytic spectrum in these prt mutants has been characterized using specific protease enzyme assays in cultures which are induced for protease expression using wheat bran .
- Enzyme assays in combination with specific inhibitors for metallo proteases demonstrate increased EDTA and 1 , 1 0 phenanthroline inhibitable activities in the cleared culture supernatants of protease mutants NW228 (bioA 1 , prtF28) , NW229 (bioA 1 , prtF29) and NW232 (bioA1 , prtD32) compared to wild type N400.
- the HEYTHG encoding sequence in A. niger pepH contains a sequence GAATaC which resembles an EcoRI restriction site (GAATTC) .
- GAATaC EcoRI restriction site
- the GAATaC sequence is changed into GAATTC thus generating an EcoRI site within the PEPH active site region which is involved in binding a Zn 2 + ion.
- the 1 0 kb EcoRI fragment is digested with EcoRI to obtain two (2.5 and 7.5 kb) EcoRI fragments.
- the 7.5 kb EcoRI fragment is cloned into pUC 1 8 (linearized with EcoRI) and a plasmid which contains the EcoRI insert in the correct orientation is selected and designated plM676-3' .
- Plasmid plM676-3' is digested with Xbal and the 6.3 kb XbaI fragment (containing the left flanking region of pepH) is isolated .
- plM676-5'-d ⁇ s The resulting plasmids are analyzed for the orientation of the EcoRI insert and the correct oriented plasmid is designated plM676-5'-d ⁇ s.
- plM676-5'-d ⁇ s is linearized with XbaI and the 6.3 kb fragment from plasmid plM676-3' is cloned into it, resulting in plM676-dis.
- plM676-d ⁇ s contains 8.8 kb from the original 1 0 kb EcoRI pepH containing fragment in which at an introduced EcoRI site in the HEYTHG encoding sequence the A. nidulans argB gene is inserted.
- the disruption construct for the A. niger pepH gene is transformed in A. niger strain NW205 (cspA 1 , argB1 3, pyrA6, nicA 1 ) and strain NW1 55 (cspA 1 , argB 1 3, pyrA6, prtF28, nicA 1 ) .
- Arginine prototrophic transformants are selected and purified .
- DNA is isolated from purified transformants and analyzed in a Southern analysis using specific pepH and A. nidulans argB probes to identify double cross-over integration of the disruption construct at the homologous (pepH) locus, thus disrupting the pepH gene.
- Selected disruptants are grown on media optimized for protease expression (as described by van den Hombergh et al. , 1 995) and analyzed via specific enzyme assays and in vitro degradation assays to demonstrate reduction of extracellular EDTA- and 1 , 1 0 phenanthroline inhibitable activities and to demonstrate a reduction of in vitro PELB degradation compared to strains in which the pepH gene is not disrupted.
- A. nidulans wild type strain WG096 is grown on liquid medium containing 0.5 g MgSO 4 , 1 .5 g KH 2 PO 4 , 0.5 g KCI, trace elements (Vishniac and Santer, 1 957), 1 % (w/v) BSA (Boehringer, fraction V) and 1 % (w/v) elastm (Fluka) for 72 h at 37 °C.
- Proteolytic activities in the cleared culture supernatants were determined as described by van den Hombergh et al ( 1 995) using BSA (fraction V) as a substrate and using 1 , 1 0 phenanthroline as specific inhibitor of metallo protease activities. Comparison of non-inhibited and inhibited protease activities in the A nidulans wild type culture supernatants indicate the presence of metallo protease activities.
- plM668- 5' plasmid in which the 2.0 kb fragment is inserted in the correct orientation is selected and subsequently digested with Pstl .
- Plasmid plM667 is digested with Pstl and the kb Pstl fragment is isolated .
- This Pstl fragment is ligated into plasmid plM668-5'dis to generate plM668-dis.
- plasmid plM668-dis the internal EcoRI-Pstl fragment (encoding part of the PEPI protease) is replaced with the A. nidulans argB gene, thus generating an inactive A.
- nidulans pepl gene Restriction enzyme digestion with Xhol and BamH l allows removal of the 2.7 kb vector sequence and results in the linear disruption fragment.
- This disruption fragment which is designed to function as a one-step gene-disruption construct will upon transformation to A. nidulans and after a double cross-over event at the homologous pepl locus result in a pepl disruption strain.
- Plasmid plM669 is digested with EcoRV and Smal (to remove the polylinker Pstl site) and the 9.5 kb fragment is isolated . After backligation of this fragment plasmid plM669-Pst is generated. Plasmid plM669-dPst is restriction digested with Psfl and BamHI to remove the complete pepl coding region. From plasmid plJ 1 6 (Johnstone et al., 1 985) the 2.7 kb BamHl-Pstl fragment containing the A. nidulans argB gene is isolated .
- plasmid plM669-dis is obtained .
- Restriction enzyme digestion with Xhol and EcoRI allows removal of the 3.0 kb pBluescript vector sequence and results in the linear disruption fragment.
- This disruption fragment which is designed to function as a one-step gene-disruption construct contains the A. nidulans argB gene inserted between the 5' and 3' flanking regions of the A. nidulans pepJ gene. This linear fragment will upon transformation to A. nidulans and after a double cross-over event at the homologous pepJ locus result in a pepJ disruption strain.
- E.coli strains containing plasmids pIM 676, pIM 667, pIM 668 and pIM 669 have been deposited 5 June 1996 at the Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands, under the Budapest Treaty with accession numbers CBS 619.96, CBS 620,96, CBS 621.96 and 622.96, respectively.
- pectin lyase gene (pe/A) - indications of a pectin lyase gene family in Aspergillus niger. Curr. Genet. 18:161-166
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP10500243A JP2000511429A (en) | 1996-06-05 | 1997-06-05 | Fungal metalloprotease gene |
EP97928160A EP0907744A1 (en) | 1996-06-05 | 1997-06-05 | Fungal metallo protease genes |
AU32563/97A AU3256397A (en) | 1996-06-05 | 1997-06-05 | Fungal metallo protease genes |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP96201579 | 1996-06-05 | ||
EP96201579.8 | 1996-06-05 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1997046689A1 true WO1997046689A1 (en) | 1997-12-11 |
Family
ID=8224057
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1997/002982 WO1997046689A1 (en) | 1996-06-05 | 1997-06-05 | Fungal metallo protease genes |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0907744A1 (en) |
JP (1) | JP2000511429A (en) |
AU (1) | AU3256397A (en) |
WO (1) | WO1997046689A1 (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003048353A1 (en) * | 2001-12-07 | 2003-06-12 | Novozymes A/S | Polypeptides having protease activity and nucleic acids encoding same |
WO2004027067A2 (en) * | 2002-09-20 | 2004-04-01 | Cym1P A/S | Methods for increasing the production of a recombinant polypeptide from a host cell |
MD4186C1 (en) * | 2012-02-20 | 2013-06-30 | Институт Микробиологии И Биотехнологии Академии Наук Молдовы | Strain of fungus Fusarium gibbosum - producer of acid and neutral proteases, xylanases and b-glucosidases |
MD4285C1 (en) * | 2013-02-28 | 2014-12-31 | Институт Микробиологии И Биотехнологии Академии Наук Молдовы | Strain of Trichoderma koningii Oudemans fungi - producer of acid, neutral and alkaline proteases |
US9567596B2 (en) | 2012-01-05 | 2017-02-14 | Glykos Finland Oy | Protease deficient filamentous fungal cells and methods of use thereof |
US9695454B2 (en) | 2012-05-23 | 2017-07-04 | Glykos Finland Oy | Production of fucosylated glycoproteins |
US10435731B2 (en) | 2013-07-10 | 2019-10-08 | Glykos Finland Oy | Multiple proteases deficient filamentous fungal cells and methods of use thereof |
US10513724B2 (en) | 2014-07-21 | 2019-12-24 | Glykos Finland Oy | Production of glycoproteins with mammalian-like N-glycans in filamentous fungi |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990000192A1 (en) * | 1988-07-01 | 1990-01-11 | Genencor, Inc. | Aspartic proteinase deficient filamentous fungi |
EP0574347A2 (en) * | 1992-04-15 | 1993-12-15 | Ciba-Geigy Ag | Novel fungal protease |
EP0655497A2 (en) * | 1993-11-03 | 1995-05-31 | Ciba-Geigy Ag | Fungal protease |
WO1995030757A2 (en) * | 1994-05-04 | 1995-11-16 | Novo Nordisk Biotech, Inc. | Metalloproteases from fusarium and aspergillus having thermolysin-like activity |
-
1997
- 1997-06-05 EP EP97928160A patent/EP0907744A1/en not_active Withdrawn
- 1997-06-05 WO PCT/EP1997/002982 patent/WO1997046689A1/en not_active Application Discontinuation
- 1997-06-05 AU AU32563/97A patent/AU3256397A/en not_active Abandoned
- 1997-06-05 JP JP10500243A patent/JP2000511429A/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990000192A1 (en) * | 1988-07-01 | 1990-01-11 | Genencor, Inc. | Aspartic proteinase deficient filamentous fungi |
EP0574347A2 (en) * | 1992-04-15 | 1993-12-15 | Ciba-Geigy Ag | Novel fungal protease |
EP0655497A2 (en) * | 1993-11-03 | 1995-05-31 | Ciba-Geigy Ag | Fungal protease |
WO1995030757A2 (en) * | 1994-05-04 | 1995-11-16 | Novo Nordisk Biotech, Inc. | Metalloproteases from fusarium and aspergillus having thermolysin-like activity |
Non-Patent Citations (7)
Title |
---|
H. TATSUMI ET AL: "Cloning and expression in yeast of a cDNA clone encoding Aspergillus oryzae neutral protease II...", MOLECULAR AND GENERAL GENETICS, vol. 228, 1991, pages 97 - 103, XP002019296 * |
J. VAN DEB HOMBERGH ET AL: "Cloning, characterization and expression of pepF, a gene encoding a serine carboxypeptidase from Aspergillus niger", GENE, vol. 151, 1994, pages 73 - 79, XP002019294 * |
J. VAN DEN HOMBERGH ET AL: "Improving heterologous protein secretion in Aspergillus niger via elimination of interfering proteolytic activities", ABSTRACTS OF PAPERS AMERICAN CHEMICAL SOCIETY, vol. 211, March 1996 (1996-03-01), pages BIOT-222, XP000611521 * |
J. VAN DEN HOMBERGH ET AL: "New protease mutants in Aspergillus niger result in stringly reduced in vitro degradation of target proteins....", CURRENT GENETICS, vol. 28, 1995, pages 299 - 308, XP000611368 * |
J. VAN DEN HOMBERGH: "Improving protein expression in Aspergillus niger", ABSTRACTS OF PAPERS AMERICAN CHEMICAL SOCIETY, vol. 209, no. 1, April 1995 (1995-04-01), pages BIOT-189, XP000611490 * |
K. JATON-OGAY ET AL: "Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus", MOLECULAR MICROBIOLOGY, vol. 14, no. 5, 1994, pages 917 - 928, XP000611364 * |
M. RAMESH ET AL: "Cloning and characterization of cDNAs and genes (mep20) encoding homologous metalloproteinases from Aspergillus flavus and A. fumigatus", GENE, vol. 165, 1995, pages 121 - 125, XP002019295 * |
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003048353A1 (en) * | 2001-12-07 | 2003-06-12 | Novozymes A/S | Polypeptides having protease activity and nucleic acids encoding same |
WO2004027067A2 (en) * | 2002-09-20 | 2004-04-01 | Cym1P A/S | Methods for increasing the production of a recombinant polypeptide from a host cell |
WO2004027067A3 (en) * | 2002-09-20 | 2004-08-19 | Cym1P As | Methods for increasing the production of a recombinant polypeptide from a host cell |
US10731168B2 (en) | 2012-01-05 | 2020-08-04 | Glykos Finland Oy | Protease deficient filamentous fungal cells and methods of use thereof |
US9567596B2 (en) | 2012-01-05 | 2017-02-14 | Glykos Finland Oy | Protease deficient filamentous fungal cells and methods of use thereof |
US10240159B2 (en) | 2012-01-05 | 2019-03-26 | Glykos Finland Oy | Protease deficient filamentous fungal cells and methods of use thereof |
US11827891B2 (en) | 2012-01-05 | 2023-11-28 | Vtt Technical Research Centre Of Finland Ltd | Protease deficient filamentous fungal cells and methods of use thereof |
US11180767B2 (en) | 2012-01-05 | 2021-11-23 | Glykos Finland Oy | Protease deficient filamentous fungal cells and methods of use thereof |
MD4186C1 (en) * | 2012-02-20 | 2013-06-30 | Институт Микробиологии И Биотехнологии Академии Наук Молдовы | Strain of fungus Fusarium gibbosum - producer of acid and neutral proteases, xylanases and b-glucosidases |
US9695454B2 (en) | 2012-05-23 | 2017-07-04 | Glykos Finland Oy | Production of fucosylated glycoproteins |
MD4285C1 (en) * | 2013-02-28 | 2014-12-31 | Институт Микробиологии И Биотехнологии Академии Наук Молдовы | Strain of Trichoderma koningii Oudemans fungi - producer of acid, neutral and alkaline proteases |
US10724063B2 (en) | 2013-07-10 | 2020-07-28 | Glykos Finland Oy | Multiple proteases deficient filamentous fungal cells and methods of use thereof |
US10544440B2 (en) | 2013-07-10 | 2020-01-28 | Glykos Finland Oy | Multiple protease deficient filamentous fungal cells and methods of use thereof |
US10988791B2 (en) | 2013-07-10 | 2021-04-27 | Glykos Finland Oy | Multiple proteases deficient filamentous fungal cells and methods of use thereof |
US10435731B2 (en) | 2013-07-10 | 2019-10-08 | Glykos Finland Oy | Multiple proteases deficient filamentous fungal cells and methods of use thereof |
US10513724B2 (en) | 2014-07-21 | 2019-12-24 | Glykos Finland Oy | Production of glycoproteins with mammalian-like N-glycans in filamentous fungi |
Also Published As
Publication number | Publication date |
---|---|
JP2000511429A (en) | 2000-09-05 |
EP0907744A1 (en) | 1999-04-14 |
AU3256397A (en) | 1998-01-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Jaton‐Ogay et al. | Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus | |
US6291209B1 (en) | Alkaline protease deficient filamentaous fungi | |
JP4620253B2 (en) | Promoter for gene expression in fungal cells | |
EP0770139B1 (en) | A FUNGUS WHEREIN THE areA GENE HAS BEEN MODIFIED AND AN areA GENE FROM ASPERGILLUS ORYZAE | |
EP0341215B1 (en) | Improvements in the production of polypeptides | |
EP0956338A1 (en) | Novel host cells and methods of producing proteins | |
EP0815200B1 (en) | Host cell expressing reduced levels of a metalloprotease and methods using the host cell in protein production | |
EP0574347B1 (en) | Novel fungal protease | |
WO1997046689A1 (en) | Fungal metallo protease genes | |
EP0655497B1 (en) | Fungal protease | |
US20030148500A1 (en) | Transcription factor | |
US5175105A (en) | Process for the production of urokinase using saccharomyes cerevisiae | |
US5846802A (en) | Fungal Protease | |
AU782116B2 (en) | Novel means of transformation of fungi and their use for heterologous protein production | |
JP3949734B2 (en) | Protease inhibitor production method | |
JP2003505085A (en) | PYRF gene and use thereof | |
EP1163325B1 (en) | Expression of proteolytic enzymes in koji mold in the presence of carbon sources | |
van den Hombergh et al. | 6 Residual protease spectra inA. nigerprt mutants contain elevated metallo protease activities; identification, cloning, characterization and expressionofpepH, metallo protease1 | |
van den Hombergh et al. | 14 Identification, cloning, and analysisofpepl and pep J, two metallo protease genes fromAspergillus nidulans; indicati-ons for an unique acid metallo protease class in filamentous fungi1 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN YU AM AZ BY KG KZ MD RU TJ TM |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 1997928160 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWP | Wipo information: published in national office |
Ref document number: 1997928160 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: CA |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1997928160 Country of ref document: EP |