KR20100081096A - Novel recombinant yeasts and methods of simultaneously producing ethanols and target proteins using them - Google Patents
Novel recombinant yeasts and methods of simultaneously producing ethanols and target proteins using them Download PDFInfo
- Publication number
- KR20100081096A KR20100081096A KR1020090000375A KR20090000375A KR20100081096A KR 20100081096 A KR20100081096 A KR 20100081096A KR 1020090000375 A KR1020090000375 A KR 1020090000375A KR 20090000375 A KR20090000375 A KR 20090000375A KR 20100081096 A KR20100081096 A KR 20100081096A
- Authority
- KR
- South Korea
- Prior art keywords
- yeast strain
- gene
- gal80
- ethanol
- promoter
- Prior art date
Links
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical class CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 171
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 104
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 94
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 62
- 238000000034 method Methods 0.000 title claims description 35
- 235000019441 ethanol Nutrition 0.000 title description 55
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 92
- 238000004519 manufacturing process Methods 0.000 claims abstract description 34
- 101150038242 GAL10 gene Proteins 0.000 claims abstract description 32
- 102100024637 Galectin-10 Human genes 0.000 claims abstract description 32
- 239000013598 vector Substances 0.000 claims abstract description 30
- 101150103317 GAL80 gene Proteins 0.000 claims abstract description 25
- FQVLRGLGWNWPSS-BXBUPLCLSA-N (4r,7s,10s,13s,16r)-16-acetamido-13-(1h-imidazol-5-ylmethyl)-10-methyl-6,9,12,15-tetraoxo-7-propan-2-yl-1,2-dithia-5,8,11,14-tetrazacycloheptadecane-4-carboxamide Chemical compound N1C(=O)[C@@H](NC(C)=O)CSSC[C@@H](C(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@@H]1CC1=CN=CN1 FQVLRGLGWNWPSS-BXBUPLCLSA-N 0.000 claims abstract description 19
- 102100034035 Alcohol dehydrogenase 1A Human genes 0.000 claims abstract description 19
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 claims abstract description 19
- 101000780443 Homo sapiens Alcohol dehydrogenase 1A Proteins 0.000 claims abstract description 19
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 101710096444 Killer toxin Proteins 0.000 claims abstract description 5
- 101000940250 Homo sapiens Collagen alpha-1(XXV) chain Proteins 0.000 claims abstract description 4
- 101000978570 Homo sapiens Noelin Proteins 0.000 claims abstract description 4
- 101150012394 PHO5 gene Proteins 0.000 claims abstract description 4
- 101150014136 SUC2 gene Proteins 0.000 claims abstract description 4
- 101001057112 Staphylococcus aureus Enterotoxin type D Proteins 0.000 claims abstract description 4
- 102000004882 Lipase Human genes 0.000 claims description 18
- 108090001060 Lipase Proteins 0.000 claims description 18
- 239000004367 Lipase Substances 0.000 claims description 18
- 235000019421 lipase Nutrition 0.000 claims description 18
- 230000002950 deficient Effects 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 230000028327 secretion Effects 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 239000011942 biocatalyst Substances 0.000 claims description 2
- 241000235088 Saccharomyces sp. Species 0.000 claims 1
- 230000003248 secreting effect Effects 0.000 abstract description 3
- 230000014509 gene expression Effects 0.000 description 34
- 238000000855 fermentation Methods 0.000 description 20
- 230000004151 fermentation Effects 0.000 description 18
- 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 16
- 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 16
- 239000008103 glucose Substances 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 15
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 11
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 229930182830 galactose Natural products 0.000 description 11
- 239000013604 expression vector Substances 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 101100351264 Candida albicans (strain SC5314 / ATCC MYA-2876) PDC11 gene Proteins 0.000 description 7
- 101150050255 PDC1 gene Proteins 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 238000003259 recombinant expression Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 101001046426 Homo sapiens cGMP-dependent protein kinase 1 Proteins 0.000 description 6
- 101001000154 Schistosoma mansoni Phosphoglycerate kinase Proteins 0.000 description 6
- 235000019626 lipase activity Nutrition 0.000 description 6
- 229940041514 candida albicans extract Drugs 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000003262 industrial enzyme Substances 0.000 description 3
- -1 linoleic acid, alcohols Chemical class 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 102100028501 Galanin peptides Human genes 0.000 description 2
- 108010001498 Galectin 1 Proteins 0.000 description 2
- 102100021736 Galectin-1 Human genes 0.000 description 2
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241001661345 Moesziomyces antarcticus Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 239000003925 fat Substances 0.000 description 2
- 235000019197 fats Nutrition 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 230000002366 lipolytic effect Effects 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 229910052744 lithium Inorganic materials 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000012261 overproduction Methods 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- XDIYNQZUNSSENW-UUBOPVPUSA-N (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O XDIYNQZUNSSENW-UUBOPVPUSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-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
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010023063 Bacto-peptone Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 230000007018 DNA scission Effects 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 101150094690 GAL1 gene Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101100121078 Homo sapiens GAL gene Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108020005350 Initiator Codon Proteins 0.000 description 1
- 241000235058 Komagataella pastoris Species 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
- 101710098554 Lipase B Proteins 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 101710089743 Mating factor alpha Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 241000320412 Ogataea angusta Species 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 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 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 241000235015 Yarrowia lipolytica Species 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
- 150000007513 acids Chemical class 0.000 description 1
- 238000010564 aerobic fermentation Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 235000012501 ammonium carbonate Nutrition 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 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
- 235000019418 amylase Nutrition 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- PRORZGWHZXZQMV-UHFFFAOYSA-N azane;nitric acid Chemical compound N.O[N+]([O-])=O PRORZGWHZXZQMV-UHFFFAOYSA-N 0.000 description 1
- 150000007514 bases Chemical class 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 230000008571 general function Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- 210000003652 golgi cell Anatomy 0.000 description 1
- 235000013882 gravy Nutrition 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material 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
- 229910000358 iron sulfate Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920001522 polyglycol ester Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 235000020238 sunflower seed Nutrition 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
본 발명은 신규한 재조합 효모 균주 및 이를 이용한 에탄올 및 목적단백질의 동시 생산방법에 관한 것으로, 보다 상세하게는 게놈 상의 GAL80 유전자가 결손되고, GAL10 프로모터 및 ADH1 프로모터를 가지며, 혐기적 조건에서 에탄올 및 목적 단백질의 동시 생산능을 갖는 재조합 효모 균주를 제조하고, 이를 이용하여 혐기적 조건에서 에탄올 및 목적단백질을 동시에 생산하는 방법에 관한 것이다. The present invention relates to a novel recombinant yeast strain and a method for the simultaneous production of ethanol and the protein of interest using the same, more specifically the GAL80 gene on the genome is missing, has a GAL10 promoter and ADH1 promoter, ethanol and the target in anaerobic conditions The present invention relates to a method for producing a recombinant yeast strain having a simultaneous production of protein, and using this to produce ethanol and a protein of interest simultaneously under anaerobic conditions.
효모 사카로마이세스 세레비지애(Saccharomyces cerevisiae) 균주는 안전성이 입증된 GRAS (Generally Regarded as Safe) 생물체이며, 연구의 역사도 매우 오래되어 전통적 유전학 기법이 매우 잘 발달됨으로써 유전적 조작이 용이하다. 또한 최근에는 지놈 염기서열 분석도 완료되어 마이크로 어레이 분석 등의 최신기법을 사용하여 연구가 가능하므로 재조합 단백질 발현에 가장 많이 사용되는 시스템 중 하나이다.The yeast Saccharomyces cerevisiae strain is a generally proven GRAS (Generally Regarded as Safe) organism, and has a long history of research, so that the genetic engineering is very easy because the traditional genetic techniques are well developed. Also recently, genome sequencing has been completed and can be studied using the latest techniques such as microarray analysis, which is one of the most used systems for recombinant protein expression.
또한, 사카로마이세스 세레비지애(Saccharomyces cerevisiae) 균주는 전통적으로 에탄올 발효에 가장 많이 이용되고 있는 균주이다. 에탄올 발효 시 당화와 발효를 동시에 진행하는 방법인 SSF (Simultaneous Saccharification and Fermentation) 공정이나 당화에 필요한 효소 생산을 겸하는 일관 공정인 CBP (Consolidated Bioprocessing) 공정이 에탄올 발효 생산성 극대화를 위한 이상적 공정이므로 이의 구현을 위하여 아밀라제 (Den Haan R. 등, Metab. Eng. 9, 87-94, 2007), 셀루라제 (Eksteen JM 등, Biotechnol. Bioeng. 84, 639-646, 2003) 등의 효소를 사카로마이세스 세레비지애(Saccharomyces cerevisiae) 균주에 발현시키는 연구가 이루어져 왔다 (van Zyl WH 등, Adv. Biochem. Eng. Biotechnol. 108, 205-235, 2007). In addition, Saccharomyces cerevisiae strain is traditionally the most used strain for ethanol fermentation. Simultaneous Saccharification and Fermentation (SSF) process, which is a method of simultaneously fermenting saccharification and fermentation during ethanol fermentation, or CBP (Consolidated Bioprocessing), a process that combines the production of enzymes required for saccharification, is ideal for ethanol fermentation productivity. Enzymes such as amylase (Den Haan R. et al., Metab. Eng. 9, 87-94, 2007), cellulose (Eksteen JM et al., Biotechnol. Bioeng. 84, 639-646, 2003) Studies on expression in Saccharomyces cerevisiae strains have been made (van Zyl WH et al., Adv. Biochem. Eng. Biotechnol. 108, 205-235, 2007).
대량으로 사용되는 공업용 효소 등의 단백질을 에탄올 발효와 동시에 생산할 수 있고 이러한 단백질의 생산이 에탄올 발효에 영향을 미치지 않으면서 에탄올 및 단백질의 동시생산이 가능하다면, 생산비용을 획기적으로 낮출 수 있는 방법을 제공할 수 있을 것이다. If it is possible to produce proteins such as industrial enzymes used in large quantities at the same time as ethanol fermentation, and if the production of these proteins is capable of simultaneous production of ethanol and protein without affecting ethanol fermentation, there is a way to drastically lower the production cost. Will be able to provide
사카로마이세스 세레비지애(Saccharomyces cerevisiae)와 같은 효모균주에서 에탄올과 같은 대사산물(metabolite)과 재조합 단백질을 동시에 생산하기 위해서는, 혐기적 조건에서 고효율로 발현되는 프로모터가 필요하다. 최근 혐기상태에서 잘 발현되는 프로모터에 관한 연구가 알려진 바 있으나 (Wiebe MG, FEMS Yeast Res. 8, 140-154, 2008, Linde JJM 등, J. Bacteriol. 181, 7409-7413, 1999) 재조합 단백질 생산을 위해서는 주로 호기상태의 발효에 관하여 연구되어 왔고 구성적 발현 (constitutive expression)을 위해서 GAPDH 프로모터 등, 유도적 발현(inducible expression)을 위해서 GAL1, GAL10 프로모터 등이 많이 사용되어 왔다. 특히 GAL 프로모터는 갈락토오스를 유도인자(inducer)로 하여 유도발현 하였을 때 발현 되지 않은 경우와 비교하여 약 1대 1000의 tight regulation이 가능하며 가장 강력한 조절가능 프로모터 중의 하나이다. GAL10 프로모터의 조절 기작에 대한 많은 연구가 이루어져 왔으며 특히 글루코스에 의한 조절을 피할 수 있는 효모 숙주의 결핍주를 개발함으로써 프로모터 활용도를 높이고자 하는 연구 성과가 발표된 바 있다 (Ostergaard S 등, Nat. Biotechnol. 18, 1283-1286, 2000, Ostergaard S 등, FEMS Yeast Res. 1, 47-55, 2001, Stagoj MN 등, FEMS Microbiol. Lett.244, 105-110, 2005). 그러나 GAL10 프로모터의 혐기상태에서의 발현에 관한 자세한 연구는 발표된 바 없으며 본 발명자들이 GAL10 프로모터를 에탄올 발효에 적합한 혐기적 발효상태에서 활성을 분석하면 상당히 낮은 값을 보였다. In order to simultaneously produce a metabolite such as ethanol and a recombinant protein in a yeast strain such as Saccharomyces cerevisiae , a promoter that is highly expressed under anaerobic conditions is required. Recently, studies on promoters that are well expressed in anaerobic conditions have been known (Wiebe MG, FEMS Yeast Res. 8, 140-154, 2008, Linde JJM et al., J. Bacteriol. 181, 7409-7413, 1999). For this purpose, the aerobic fermentation has been studied mainly, and for constitutive expression, such as GAPDH promoter, GAL1 and GAL10 promoters have been used for inducible expression. In particular, the GAL promoter is one of the most potent controllable promoters with tight regulation of about 1 to 1000 compared to the case where it is not expressed when induced with galactose as an inducer. Many studies on the regulatory mechanism of the GAL10 promoter have been made, and research on improving promoter utilization by developing a strain of yeast hosts that can avoid the control by glucose has been published (Ostergaard S et al., Nat. Biotechnol). 18, 1283-1286, 2000, Ostergaard S et al., FEMS Yeast Res. 1, 47-55, 2001, Stagoj MN et al., FEMS Microbiol. Lett. 244, 105-110, 2005). However, a detailed study on the anaerobic expression of the GAL10 promoter has not been published, and the inventors showed a significantly lower value when the GAL10 promoter was analyzed for anaerobic fermentation suitable for ethanol fermentation.
이에, 본 발명자들은 이러한 문제를 해결하기 위하여 GAL10 프로모터를 사용하여 혐기적인 조건에서도 우수한 발현율을 얻을 수 있는 방법을 연구하던 중 효모에서 갈락토오스(galactose) 대사에 관련되는 유전자인 gal80 유전자의 결손주를 사용하면 혐기적인 조건에서 에탄올 및 재조합 단백질의 과생산이 가능함을 발견하고 본 발명을 완성하였다. 또한, 본 발명자들은 혐기적 에탄올 발효 시 재조합단백 질의 동시 생산이 에탄올 발효에 커다란 영향을 미치지 않음을 발견하여 본 발명을 완성하게 되었다. In order to solve this problem, the present inventors used a GAL10 promoter, which is a defect of the gal80 gene, which is a gene related to galactose metabolism in yeast, while studying a method of obtaining an excellent expression rate even under anaerobic conditions. The present invention was found to be capable of overproduction of ethanol and recombinant protein under anaerobic conditions. In addition, the present inventors have found that the simultaneous production of recombinant protein does not significantly affect ethanol fermentation during anaerobic ethanol fermentation, thereby completing the present invention.
본 발명의 목적은 게놈 상의 GAL80 유전자가 결손되고, GAL10 프로모터 또는 ADH1 프로모터, 및 목적 단백질을 코딩하는 유전자를 포함하는 벡터가 도입된, 혐기적 조건에서 에탄올 및 목적 단백질의 동시 생산능을 갖는 신규한 재조합 효모 균주를 제공하는 것이다.It is an object of the present invention to provide a novel ability to simultaneously produce ethanol and a target protein in anaerobic conditions, in which the GAL80 gene on the genome is deleted and a vector comprising a GAL10 promoter or an ADH1 promoter and a gene encoding the target protein is introduced. It is to provide a recombinant yeast strain.
본 발명의 또 다른 목적은 (a) 효모 균주 게놈 상의 GAL80 유전자를 결손시켜 GAL80 유전자 결손 효모 균주를 제조하는 단계; (b) 상기 GAL80 유전자 결손 효모 균주에 GAL10 프로모터 또는 ADH1 프로모터, 및 목적 단백질을 코딩하는 유전자를 포함하는 벡터를 도입시켜 재조합 효모 균주를 제조하는 단계; 및 (c) 상기 재조합 효모 균주를 혐기적 조건에서 배양하는 단계를 포함하는 에탄올 및 목적 단백질을 동시에 생산하는 방법을 제공하는 것이다.Still another object of the present invention is to prepare a GAL80 gene deficient yeast strain by deleting the GAL80 gene on the yeast strain genome; (b) preparing a recombinant yeast strain by introducing a vector comprising a GAL10 promoter or an ADH1 promoter and a gene encoding a protein of interest into the GAL80 gene-deficient yeast strain; And (c) culturing the recombinant yeast strain under anaerobic conditions.
하나의 양태로서, 본 발명은 게놈 상의 GAL80 유전자가 결손되고, GAL10 프로모터 또는 ADH1 프로모터, 및 목적 단백질을 코딩하는 유전자를 포함하는 벡터가 도입된, 혐기적 조건에서 에탄올 및 목적 단백질의 동시 생산능을 갖는 신규한 재조합 효모 균주에 관한 것이다.In one embodiment, the present invention provides for the simultaneous production of ethanol and the target protein in anaerobic conditions, in which the GAL80 gene on the genome is deleted and a vector comprising a GAL10 promoter or an ADH1 promoter and a gene encoding the target protein is introduced. It relates to a novel recombinant yeast strain having.
본 발명에서 "효모"라 함은 단세포 진핵생물의 일종으로서, 원핵생물인 대장균과 함께 재조합 단백질의 생산에 주로 사용되는 생물을 말한다. 본 발명 의 효모 균주는 갈락토오스 대사에 관여하는 유전자의 일종인 GAL80 유전자를 결손시켜 이용하거나, 이미 GAL80 유전자가 결손된 변이주를 이용하는 것이 바람직한데, 이때, 효모 균주로는 사카로마이세스 세레비지애(Saccharomyces cerevisiae), 한세눌라 폴리모르파(Hansenula polymorpha), 피키아 패스토리스(Pichia pastoris), 야로위아 리포리티카(Yarrowia lipolytica) 및 스키조사카라마이세스 폼베(Schizosaccharomyces pombe) 등을 이용할 수 있으며, 바람직하게는 사카로마이세스 세레비지애를 이용할 수 있으며, 가장 바람직하게는 사카로마이세스 세레비지애 Y2805 균주의 GAL80 유전자를 결손시켜 이용하거나 GAL80 유전자가 결손된 변이균주 사카로마이세스 세레비지애 Y2805(gal80-)를 이용할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, "yeast" is a kind of unicellular eukaryotes, and refers to an organism mainly used for the production of recombinant protein with Escherichia coli, which is a prokaryote. The yeast strain of the present invention is preferably used by deleting the GAL80 gene, which is a kind of gene involved in galactose metabolism, or by using a mutant strain in which the GAL80 gene is already deleted. In this case, Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica, and Schizosaccharomyces pombe, and the like. Saccharomyces cerevisiae may be used, and most preferably, it is used by deleting the GAL80 gene of Saccharomyces cerevisiae Y2805 strain or the variant strain Saccharomyces cerevisiae Y2805 (deleted GAL80 gene). gal80 -), but you can use, but is not limited thereto.
본 발명의 효모 균주는 염색체상에서 GAL80 유전자를 결손시키는 방법으로는 당업계에 알려진 통상적인 유전자 결손방법, 예를 들면, 화학물질이나 자외선과 같은 빛을 이용하거나, 상동재조합 기술을 통한 유전자 재조합 기술을 이용하여 GAL80 유전자 부위가 변이되어 결실된 돌연변이체를 얻을 수 있고, 바람직하게는 유전자 팝-아웃 기술을 이용할 수 있다. 본 발명의 구체적 실시예에서는 본 발명자가 기출원한 특허(한국특허출원 2007-0080946)에 개시된 효모 균주인 GAL80 유전자가 결손된 사카로마이세스 세레비지애 Y2805(gal80-)를 이용하였다. Yeast strain of the present invention is a method for deleting the GAL80 gene on the chromosome using a conventional gene deletion method known in the art, for example, using a light such as chemicals or ultraviolet light, or genetic recombination technology through homologous recombination technology The GAL80 gene region can be mutated to obtain a deleted mutant, and preferably a gene pop-out technique can be used. In a specific embodiment of the present invention, Saccharomyces cerevisiae Y2805 (gal80 − ), in which the GAL80 gene, which is a yeast strain disclosed in the presently filed patent application (Korean Patent Application No. 2007-0080946), was deleted.
본 발명의 효모 균주는 GAL10 프로모터 또는 ADH1 프로모터, 및 목적 단백질을 코딩하는 유전자를 포함하는 벡터가 도입되는 것을 특징으로 한다.The yeast strain of the present invention is characterized in that a vector comprising a GAL10 promoter or an ADH1 promoter and a gene encoding a target protein is introduced.
본 발명에서 용어 "벡터"는 적합한 숙주 내에서 목적 유전자를 발현시킬 수 있도록 적합한 조절 서열에 작동 가능하게 연결된 유전자의 염기서열을 함유하는 유전자 작제물을 의미하는 것으로, 상기 조절 서열은 전사를 개시할 수 있는 프로모터, 그러한 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합 부위를 코딩하는 서열, 및 전사 및 해독의 종결을 조절하는 서열을 포함한다. As used herein, the term "vector" refers to a gene construct containing a nucleotide sequence of a gene operably linked to a suitable regulatory sequence to allow expression of the gene of interest in a suitable host, wherein the regulatory sequence will initiate transcription. Promoter, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation.
본 발명에서 "작동가능하게 연결된 (operably linked)"는 일반적 기능을 수행하도록 핵산 발현조절 서열과 목적하는 단백질을 코딩하는 핵산 서열이 기능적으로 연결되어 있는 것을 말한다. 예를 들어 프로모터와 단백질 또는 RNA를 코딩하는 핵산 서열이 작동가능하게 연결되어 코딩서열의 발현에 영향을 미칠 수 있다. 재조합 벡터와의 작동적 연결은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용한다. In the present invention, "operably linked" means that the nucleic acid expression control sequence and the nucleic acid sequence encoding the protein of interest is functionally linked to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the coding sequence. Operational linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art and the like.
본 발명의 벡터는 프로모터 및 목적 단백질을 코딩하는 유전자 외에 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트, 분비시그널 등을 포함할 수 있으며, 목적에 따라 다양하게 제조될 수 있다. 개시 코돈 및 종결 코돈은 유전자 작제물이 투여되었을 때 개체에서 반드시 작용을 나타내야 하며 코딩 서열과 인프레임(in frame)에 있어야 한다.The vector of the present invention may include expression control elements such as initiation codons, termination codons, polyadenylation signals and enhancers, secretion signals, etc., in addition to the promoter and the gene encoding the target protein, and may be variously produced according to the purpose. The start codon and the stop codon must be functional in the subject when the gene construct is administered and must be in frame with the coding sequence.
본 발명의 벡터는 효모 균주 세포 내에서 복제가능한 것이면 특별히 한정되지 않으며 당 업계에 알려진 임의의 벡터를 이용할 수 있으며, 예컨대 플라스미드, 코즈미드, 파지 입자, 바이러스 벡터 또는 간단하게 잠재적 게놈 삽입물일 수 있 고, 바람직하게는 자기복제를 위한 2 micron 서열을 가지는 효모용 발현 벡터이나, 이에 제한되는 것은 아니다. The vector of the present invention is not particularly limited as long as it is replicable in yeast strain cells, and any vector known in the art may be used, such as a plasmid, cosemid, phage particle, viral vector or simply a potential genomic insert. Preferably, the expression vector for yeast having a 2 micron sequence for self-replication, but is not limited thereto.
본 발명의 벡터는 프로모터로서 효모 균주 내에서 강력한 프로모터 활성을 나타내는 것으로 알려진 GAL10 프로모터 또는 ADH1 프로모터를 포함하며, 이러한 본 발명의 프로모터는 벡터 내에서 목적하는 단백질을 코딩하는 유전자와 작동가능하게 연결되어, 상기 벡터가 효모 균주 숙주세포 내로 도입된 후, 프로모터 활성을 통해 목적단백질 발현이 유도되게 된다.The vector of the present invention includes a GAL10 promoter or an ADH1 promoter known to exhibit potent promoter activity in a yeast strain as a promoter, and such a promoter of the present invention is operably linked to a gene encoding a protein of interest in the vector, After the vector is introduced into the yeast strain host cell, the protein expression of the target is induced through promoter activity.
또한, 본 발명의 벡터는 발현시키고자 목적하는 단백질을 코딩하는 유전자를포함하는데, 이는 전체 유전자, 또는 그의 일정한 영역을 코딩하는 서열일 수 있다. 본 발명의 벡터를 사용하여 발현시킬 수 있는 목적 유전자는 효모 균주에서 안정적으로 발현될 수 있는 유전자라면 특별히 제한되지 않으며, 바람직하게는 의학, 산업적으로 유용한 목적 단백질이 이용될 수 있으며, 예를 들면, 효소, 호르몬, 수송 단백질, 수용체, 조절 단백질, 구조 단백질, 항원, 항체 등이 가능하고, 가장 바람직하게는 산업적으로 유용한 지질분해효소의 하나인 리파제 CalB 단백질 또는 사카로마이세스 세레비지애 균주에 코돈 최적화된 리파제 CAlB14opt을 코딩하는 유전자이다. In addition, the vector of the present invention includes a gene encoding a protein of interest, which may be an entire gene, or a sequence encoding a constant region thereof. The target gene that can be expressed using the vector of the present invention is not particularly limited as long as it is a gene that can be stably expressed in the yeast strain, and preferably, a medical protein or industrially useful target protein can be used. Enzymes, hormones, transport proteins, receptors, regulatory proteins, structural proteins, antigens, antibodies, and the like, most preferably codons in lipase CalB protein or Saccharomyces cerevisiae strain, one of the industrially useful lipolytic enzymes Gene encoding the optimized lipase CAlB14 opt .
"리파제 CalB"는 칸디다 안타티카(Candida antarctica) 유래의 리파제 B 단백질로서, 의약품 및 정밀 화학제품의 합성에 가장 많이 사용되는 효소이지만, 20 ℃ 이하의 저온조건에서만 단백질의 접힘(folding)이나 퇴화(degradation) 현상이 없이 정상적인 단백질이 생성되고, 효모의 생육 적정온도인 30℃에서 배양하게 되면 단백질의 접힘(folding)이나 퇴화(degradation) 현상 등으로 인해 전체적인 생산성이 감소되는 특성을 가지고 있어 대량 생산에 어려움이 있는 단백질로 알려져 있다. 본 발명자는 이러한 문제를 해결하기 위해 통상의 발효배양 온도인 30℃에서도 안정적인 발현이 가능한 코돈 최적화된 리파제 CalB14opt을 제작한바(한국특허출원 2007-0092823), 본 발명의 구체적 실시예에서는 상기 리파제 CalB14opt를 목적 유전자로 이용하였다."Lipase CalB" is a lipase B protein derived from Candida antarctica , which is the most frequently used enzyme for the synthesis of pharmaceuticals and fine chemicals, but the folding or degradation of the protein only at low temperature below 20 ℃ Normal protein is produced without phenomena, and if it is incubated at the proper temperature of yeast growth at 30 ℃, the overall productivity is reduced due to the folding or degradation of protein, which makes it difficult to mass-produce. This is known as protein. In order to solve this problem, the present inventors have produced a codon-optimized lipase CalB14 opt capable of stable expression even at a normal fermentation culture temperature of 30 ° C. (Korean Patent Application 2007-0092823). opt was used as the target gene.
또한, 본 발명의 벡터는 상기 프로모터 및 목적단백질을 코딩하는 유전자 외에 생성된 단백질을 세포 외로 분비시키기 위한 분비시그널 유전자를 추가로 포함할 수 있다. 여기서 "분비 시그널"은 리포좀에서 합성된 단백질이 소포체, 골지체 나아가 세포 밖으로 분비되도록 하는 작용을 하는 체계를 말하며, 각 생물종들 및 발현하는 단백질에 따라 그 종류 또한 다양하다. 본 발명에서 이용할 수 있는 분비시그널 유전자로는 MFα, PHO5, SUC2, AMY, SED 및 킬러톡신(Killer Toxin, KT) 등이 가능하며, 바람직하게는 MFα 유전자이지만, 이에 제한되는 것은 아니다.In addition, the vector of the present invention may further include a secretion signal gene for secreting the generated protein outside the cell in addition to the gene encoding the promoter and the protein of interest. Here, the "secretion signal" refers to a system that functions to allow proteins synthesized in liposomes to be secreted out of the endoplasmic reticulum, Golgi apparatus, and cells, and their types also vary depending on each species and the expressed protein. Secretion signal genes that can be used in the present invention may be MFα, PHO5, SUC2, AMY, SED and killer toxin (KT), and the like, and preferably MFα gene, but is not limited thereto.
또한, 본 발명의 벡터는 선별 마커(selection market)를 추가로 포함할 수 있다. 선별 마커는 벡터로 형질전환된 세포를 선별, 즉, 목적 유전자의 삽입 여부를 확인하기 위한 것으로, 약물 내성, 영양 요구성, 세포 독성제에 대한 내성 또는 표면 단백질의 발현과 같은 선택가능 표현형을 부여하는 마커들이 사용될 수 있다. 선택제(selective agent)가 처리된 환경에서는 선별 마커를 발현하는 세포만 생존하거나, 다른 표현 형질을 나타내므로, 형질전환된 세포를 선별할 수 있다.In addition, the vector of the present invention may further include a selection market. Selection markers are used to select cells transformed with a vector, i.e., to confirm the insertion of a gene of interest, and confer a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins. Markers can be used. In an environment treated with a selective agent, only cells expressing a selection marker survive or exhibit different expression traits, so that transformed cells can be selected.
또한, 본 발명의 재조합 효모 균주는 상기한 벡터를 프로모터가 작용할 수 있는 양태로 효모 균주세포 내로 도입시키는 것에 의해 구축될 수 있다. 유전자 작제물 형태인 벡터를 세포 내로 도입시키는 방법은 당업계에 공지된 방법을 이용할 수 있으며, 바람직하게는 형질전환 방법이다. 여기서 "형질전환"은 DNA를 숙주로 도입하여 DNA가 염색체외 인자로서 또는 염색체 통합완성에 의해 복제가능하게 되는 것을 의미한다. 형질전환은 핵산 분자를 유기체, 세포, 조직 또는 기관에 도입하는 어떤 방법도 포함되며, 당 분야에서 공지된 바와 같이 숙주 세포에 따라 적합한 표준 기술을 선택하여 수행할 수 있다. 이런 방법에는 전기천공법(electroporation), 인산칼슘(CaPO4) 침전, 염화칼슘(CaCl2) 침전, 미세주입법(microinjection), 폴리에틸렌글리콜(PEG)법, DEAE-덱스트란법, 양이온 리포좀법, 및 초산 리튬-DMSO법 등이 포함될 수 있으나 이들로 제한되는 것은 아니다. In addition, the recombinant yeast strain of the present invention can be constructed by introducing the above-mentioned vector into yeast strain cells in an embodiment in which a promoter can act. The method of introducing a vector in the form of a gene construct into a cell may use a method known in the art, and is preferably a transformation method. By “transformation” herein is meant the introduction of DNA into a host such that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. Transformation includes any method of introducing a nucleic acid molecule into an organism, cell, tissue, or organ, and can be carried out by selecting appropriate standard techniques according to the host cell as known in the art. These methods include electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and acetic acid Lithium-DMSO method and the like may be included, but are not limited thereto.
한편, 게놈 상의 GAL80 유전자가 결손되고, 상기한 벡터가 도입된 본 발명의 재조합 효모 균주는 혐기적 조건에서 에탄올 및 목적 단백질의 동시 생산능을 가짐을 특징으로 한다. On the other hand, the recombinant yeast strain of the present invention in which the GAL80 gene on the genome is deleted and the vector is introduced is characterized in that it has the ability to simultaneously produce ethanol and the target protein under anaerobic conditions.
효모 균주는 산소가 없는 혐기적 조건에서는 에탄올 발효를 하게 된다. 그러나 통상적인 효모 균주를 이용한 단백질 발현 시스템 내에서는 프로모터들이 호기 조건에서 강력한 활성을 나타내고, 혐기적 조건에서는 그 활성이 80~90% 가까이 소실되어 정상적인 단백질 발현이 불가능한 한계가 있어 왔다. 그러나, 본 발명의 재조합 효모 균주는 혐기적 조건에서도 강력한 프로모터 활성을 나타내므로, 에탄올 발효 및 목적 단백질의 발현이 동시적으로 일어나는 특징을 가진다.Yeast strains are subjected to ethanol fermentation under anaerobic conditions without oxygen. However, in a protein expression system using a conventional yeast strain, promoters exhibit strong activity under aerobic conditions, and in anaerobic conditions, their activity is nearly 80-90%, and thus, normal protein expression has been limited. However, since the recombinant yeast strain of the present invention exhibits strong promoter activity even under anaerobic conditions, ethanol fermentation and expression of the target protein occur simultaneously.
또 다른 양태로서, 본 발명은 (a) 효모 균주 게놈 상의 GAL80 유전자를 결손시켜 GAL80 유전자 결손 효모 균주를 제조하는 단계; (b) 상기 GAL80 유전자 결손 효모 균주에 GAL10 프로모터 또는 ADH1 프로모터, 및 목적 단백질을 코딩하는 유전자를 포함하는 벡터를 도입시켜 재조합 효모 균주를 제조하는 단계; 및 (c) 상기 재조합 효모 균주를 혐기적 조건에서 배양하는 단계를 포함하는 에탄올 및 목적 단백질을 동시에 생산하는 방법에 관한 것이다.In still another aspect, the present invention provides a method for preparing a yeast strain comprising: (a) producing a GAL80 gene deficient yeast strain by deleting the GAL80 gene on the yeast strain genome; (b) preparing a recombinant yeast strain by introducing a vector comprising a GAL10 promoter or an ADH1 promoter and a gene encoding a protein of interest into the GAL80 gene-deficient yeast strain; And (c) culturing the recombinant yeast strain under anaerobic conditions.
본 발명에서 재조합 효모 균주의 배양은 널리 공지된 방법에 따라서 수행될 수 있고, 산소가 유입되지 않은 혐기 상태에서 배양하는 것이 바람직하다. 또한, 배양 온도, 배양 시간 및 배지의 pH 등의 조건은 적절하게 조절될 수 있다. 적절한 배양 방법으로는 유가식 배양(fed-batch culture), 회분식 배양(batch culture) 및 연속식 배양(cintinuous culture) 등이 가능하며, 바람직하게는 유가식 배양이지만, 이에 제한되는 것은 아니다. Cultivation of the recombinant yeast strain in the present invention can be carried out according to well-known methods, it is preferable to culture in an anaerobic state in which oxygen is not introduced. In addition, conditions such as the incubation temperature, the incubation time and the pH of the medium may be appropriately adjusted. Suitable culture methods include fed-batch culture, batch culture and continuous culture (cintinuous culture), and the like, but preferably fed-batch culture, but is not limited thereto.
사용되는 배양 배지는 특정한 균주의 요구 조건을 적절하게 충족시켜야 한 다. 다양한 미생물에 대한 배양 배지는 공지되어 있다(예를 들면, "Manual of Methods for General Bacteriology" from American Society for Bacteriology (Washington D.C., USA, 1981)). 배지 내 탄소 공급원은 당 및 탄수화물(예: 글루코오스, 슈크로오스, 락토오스, 프럭토오스, 말토오스, 몰라세, 전분 및 셀룰로오스), 유지 및 지방(예: 대두유, 해바라기씨유, 땅콩유 및 코코넛유), 지방산(예: 팔미트산, 스테아르산 및 리놀레산), 알콜(예: 글리세롤 및 에탄올) 및 유기산(예: 아세트산) 등을 이용할 수 있다. 이들 물질은 개별적으로 또는 혼합물로서 사용될 수 있다. 질소 공급원은 질소-함유 유기 화합물(예: 펩톤, 효모 추출액, 육즙, 맥아 추출액, 옥수수 침지액, 대두 박분 및 우레아), 또는 무기 화합물(예: 황산암모늄, 염화암모늄, 인산암모늄, 탄산암모늄 및 질산암모늄)을 이용할 수 있으며, 이들 물질 또한 개별적으로 또는 혼합물로서 사용될 수 있다. 인 공급원으로서 인산이수소칼륨 또는 인산수소이칼륨 또는 상응하는 나트륨 함유 염을 이용할 수 있다. 또한, 배양 배지는 성장에 필수적인 금속염(예: 황산마그네슘 또는 황산철)을 함유할 수 있으며, 최종적으로, 아미노산 및 비타민과 같은 필수 성장-촉진 물질을 상기 언급한 물질 외에 사용할 수 있다. 적합한 전구체를 상기 배양 배지에 추가로 가할 수 있다. 상기 공급 물질은 배양물에 한번에 모두 가하거나, 배양중 적절하게 공급할 수 있다.The culture medium used should suitably meet the requirements of the particular strain. Culture media for various microorganisms are known (eg, "Manual of Methods for General Bacteriology" from American Society for Bacteriology (Washington D.C., USA, 1981)). Carbon sources in the medium include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seed oil, peanut oil and coconut oil). ), Fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, organic acids such as acetic acid, and the like. These materials can be used individually or as a mixture. Nitrogen sources can be nitrogen-containing organic compounds such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and nitric acid Ammonium) can be used, and these materials can also be used individually or as a mixture. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium containing salts can be used as the phosphorus source. In addition, the culture medium may contain metal salts necessary for growth (eg, magnesium sulfate or iron sulfate), and finally, essential growth-promoting substances such as amino acids and vitamins may be used in addition to the substances mentioned above. Suitable precursors may further be added to the culture medium. The feed material may be added to the culture all at once or may be appropriately supplied during the culture.
배양물의 pH는 염기성 화합물(예: 수산화나트륨, 수산화칼륨 또는 암모니아) 또는 산성 화합물(예: 인산 또는 황산)을 적절히 사용하여 조절할 수 있다. 발포는 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 조절할 수 있다. 배양 온도 는 통상적으로 20 내지 45℃, 바람직하게는 25 내지 40℃이다, 가장 바람직하게는 30℃이다. 배양은 원하는 목적 단백질 및 에탄올의 생성량이 최대로 얻어질 때까지 계속될 수 있으며, 단백질은 배양 배지 중으로 배출되거나, 세포 중에 포함되어 있을 수 있다. The pH of the culture can be adjusted by appropriate use of a basic compound (eg sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg phosphoric acid or sulfuric acid). Foaming can be controlled using antifoams such as fatty acid polyglycol esters. The culture temperature is usually 20 to 45 ° C, preferably 25 to 40 ° C, most preferably 30 ° C. Cultivation can continue until the desired amount of desired protein and ethanol is achieved, and the protein can be released into the culture medium or contained in the cells.
본 발명의 재조합 효모 균주를 배양시켜 에탄올을 발효시키고, 목적 단백질을 발현시켜 생산한 후, 에탄올 및 목적 단백질의 회수는 당업계에 널리 알려져 있는 방법으로 세포 또는 배양 배지로부터 분리해낼 수 있다. 단백질 회수는 단백질의 물리, 화학적 특성에 따라 적합한 공지의 방법을 이용할 수 있으며, 예를 들면, 여과, 음이온 교환 크로마토그래피, 결정화 및 HPLC 등의 방법이 가능하나, 이에 제한되는 것은 아니다. After culturing the recombinant yeast strain of the present invention to ferment ethanol, expressing and producing the protein of interest, recovery of ethanol and the protein of interest can be isolated from cells or culture media by methods well known in the art. Protein recovery may use a known method suitable according to the physical and chemical properties of the protein, for example, filtration, anion exchange chromatography, crystallization and HPLC may be used, but is not limited thereto.
또한, 본 발명의 생산 방법을 통해, 리파제 CaB 유전자를 벡터에 포함시켜 제조한 효모 균주를 배양시켜 수득한 리파제 CalB 단백질은 바이오촉매로 이용될 수 있다.In addition, through the production method of the present invention, the lipase CalB protein obtained by culturing a yeast strain prepared by including a lipase CaB gene in a vector can be used as a biocatalyst.
본 발명에 따른 신규한 재조합 효모 균주는 혐기적 조건에서 에탄올 및 목적 단백질의 동시 생산이 가능한 장점을 가진다. The novel recombinant yeast strain according to the present invention has the advantage that the simultaneous production of ethanol and the target protein in anaerobic conditions.
이하, 하기 실시예에 의해 본 발명을 좀 더 구체적으로 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 실시하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for the purpose of carrying out the present invention by way of example and the scope of the present invention is not limited to these examples.
실시예 1. GAL10 프로모터를 이용하여, 사카로마이세스 세레비지애(Example 1 Saccharomyces cerevisiae using a GAL10 promoter Saccharomyces cerevisiaeSaccharomyces cerevisiae ) 균주에서 CalB14 발현 ) CalB14 expression in strains
GAL10 프로모터는 가장 강력한 조절가능 프로모터 중의 하나로서 사카로마이세스 세레비지애에서 유전자 발현에 가장 많이 사용되며 상업적인 키트로 판매가 되고 있기도 하다. 그러나, GAL10 프로모터를 사용하여 재조합단백질을 생산하는 경우 대부분 호기조건의 발효 방법을 선택하게 된다. The GAL10 promoter is one of the most potent regulated promoters and is most commonly used for gene expression in Saccharomyces cerevisiae and is also sold as a commercial kit. However, when the recombinant protein is produced using the GAL10 promoter, most fermentation methods are chosen.
본 발명자들은 GAL10 프로모터를 사용하여 혐기조건의 에탄올 발효와 동시에 공업용 효소로 많이 사용되는 지질분해효소인 돌연변이된 칸디다 안타티카(Candida antarctica) 유래의 lipase (CalB14) (효모 표면 발현 벡터를 이용하여 변이 리파제를 스크리닝하는 방법 및 신규 변이 리파제, 한국등록특허 제475133호)의 재조합 생산 방법을 조사하던 중 GAL10 프로모터는 효모에서의 에탄올의 과생산을 위한 혐기적 발효상태에서 상당히 낮은 프로모터 활성을 보임을 발견하였다. The present inventors screened a mutant lipase using a lipase (CalB14) derived from mutated Candida antarctica, a lipolytic enzyme frequently used as an industrial enzyme at the same time as an anaerobic ethanol fermentation using a GAL10 promoter. GAL10 promoter was found to exhibit significantly lower promoter activity in anaerobic fermentation for overproduction of ethanol in yeast.
CalB14의 코돈(codon)이 사카로마이세스 세레비지애에 최적화(optimization)된 유전자인 CalB14opt (효모에서 재조합단백질을 고효율로 분비발현 시키는 방법, 한국특허출원 제2007-0092823호)를 발현시키기 위해 사카로마이세스 세레비지애 유 래의 GAL10 프로모터를 사용하였고, 발현된 단백질의 분비를 위하여 효모에서 가장 많이 사용되는 분비 유도 서열 mating factor-α(MFα) 신호 서열(signal sequence)을 사용하였다. 여기서, MFα역시 S. cerevisiae에 코돈이 최적화된 유전자인 MFαopt를 사용하였다 (효모에서 재조합단백질을 고효율로 분비발현 시키는 방법, 한국특허출원 2007-0092823, 2007. 9. 12.). Codon of CalB14 to express CalB14 opt , a gene optimized for Saccharomyces cerevisiae, to express high efficiency secretion of recombinant protein in yeast, Korean Patent Application No. 2007-0092823 The GAL10 promoter derived from Saccharomyces cerevisiae was used, and the secretion induction sequence mating factor-α (MFα) signal sequence most used in yeast was used for secretion of the expressed protein. Here, MFα was also used as a codon-optimized gene MFα opt in S. cerevisiae (method of secreting recombinant proteins in yeast with high efficiency, Korean Patent Application 2007-0092823, Sept. 12, 2007).
사카로마이세스 세레비지애(S. cerevisiae) 유래의 GAL10 프로모터를 SacI과 EcoRI 제한효소로 절단하여 MFαopt와 CalB14opt 를 함께 사카로마이세스 세레비지애 발현벡터에 클로닝하였고, pGAL-MFa opt-CalB14 opt로 명명하였다(도 1 참조). 상기 재조합 발현벡터를 S. cerevisiae Y2805 균주에 리튬/아세테이트(lithium/acetate) 방법을 이용하여 형질전환 하였고, UD(Ura-, glucose) plate에서 얻은 형질전환체(transformant)를 YPD(2% glucose)-tributyrin plate 및 YPDG(1% glucose, 1% galactose)-tributyrin plate에 picking하여 30℃에서 24시간 배양시킨 후, 리파제 활성을 나타내는 halo를 보이는 콜로니를 각각 선별하였다. A GAL10 promoter from Saccharomyces cerevisiae was digested with SacI and EcoRI restriction enzymes, and MFα opt and CalB14 opt were cloned together into a Saccharomyces cerevisiae expression vector and pGAL-MFa opt-. Named CalB14 opt (see FIG. 1). The recombinant expression vector was transformed into S. cerevisiae Y2805 strain using lithium / acetate method, and the transformant obtained from UD (Ura-, glucose) plate was transformed into YPD (2% glucose). After picking on -tributyrin plate and YPDG (1% glucose, 1% galactose) -tributyrin plate and incubating at 30 ° C for 24 hours, colonies showing halo showing lipase activity were selected.
CalB14 단백질을 혐기적 조건으로 발현하기 위하여, 통기가 안되도록 배지를 함유한 시험관을 파라필름(parafilm)으로 sealing하였고 rotary shaker에서 30℃에서 72시간 정치 배양시켜 CalB14 단백질의 발현을 유도하였다. 호기적인 경우는 통기를 위하여 parafilm으로 sealing하지 않고, rotary shaker에서 200rpm으로 30℃ 에서 72시간 배양하였다. 각각의 배양액으로부터 얻은 상등액을 ρNPP (ρ-nitrophenyl palmitate)를 기질로 하여 5분간 반응시킨 후 리파제 활성(lipase activity)을 측정하였다. In order to express the CalB14 protein under anaerobic conditions, the tube containing the medium was sealed with parafilm to prevent aeration, and incubated for 72 hours at 30 ° C. in a rotary shaker. Expression of the protein was induced. In the case of aerobic, it was incubated at 30 ° C. for 72 hours at 200 rpm in a rotary shaker without sealing with parafilm for aeration. The supernatant obtained from each culture was reacted for 5 minutes with ρNPP (ρ-nitrophenyl palmitate) as a substrate, and then lipase activity was measured.
리파제 CalB14를 GAL10 프로모터를 이용하여 S. cerevisiae 야생형균주 (Y2805) 에서 발현 시켰을 때 GAL10 프로모터의 유도물질(inducer)인 갈락토오스(galactose)를 탄소원으로 하여 100g/L의 농도로 발현을 유도하였을 때 호기상태에서는 약 1,393 U/L의 값을 보인 반면, 혐기상태에서는 세포성장이 매우 저조하였고 약 166 U/L의 낮은 값을 보였다. 세포성장의 문제를 해결하기 위하여 글루코오스 100 g/L + 갈락토오스 10 g/L의 조건으로 발현을 유도하였을 경우에도 세포성장은 정상적이었으나, 리파제 활성은 약 318 U/L의 비교적 낮은 값을 보였다. When lipase CalB14 was expressed in S. cerevisiae wild type strain (Y2805) using GAL10 promoter, galactose, an inducer of GAL10 promoter, was used as a carbon source to induce expression at a concentration of 100 g / L. Showed a value of about 1,393 U / L, whereas in anaerobic cell growth was very low and a low value of about 166 U / L. In order to solve the problem of cell growth, cell growth was normal even when the expression was induced under the conditions of 100 g / L glucose + 10 g / L glucose, but the lipase activity was relatively low at about 318 U / L.
한편 호기조건에서 갈락토오스(galactose) 대신 글루코오스(glucose)만 사용하여도 발현이 잘 되는 갈락토오스 대사 관련 유전자 결손주 gal80 균주(Y2805 (gal80-); 기탁번호 KCTC 11162BP)를 (이스트 GAL80 결핍주를 이용한 재조합단백질의 생산 방법, 한국특허출원 제2007-0080946호) 사용하여 혐기조건에서의 거동을 살펴보았다. pGAL-MFa opt-CalB14 opt를 함유한 Y2805 (gal80-)를 갈락토오스를 탄소원으로 사용하지 않고 탄소원으로 글루코오스만 사용하여 혐기적인 조건에서 배양하였을 경우, 리파제 활성이 Y 2805 균주의 혐기적인 조건에서의 리파제 생산량뿐만 아니라 호기조건에서의 생산량 보다 높은 1,958 U/L의 높은 값을 보였다. 세 포중량 당 생산성의 경우는, gal80이 결손되지 않은 Y 2805 균주의 혐기적인 조건에서의 리파제 생산량보다 약 4배의 증가된 값을 보였다. 이는 혐기조건에서 gal80 결핍주가 재조합 단백질 생산성이 gal80이 결핍되지 않은 균주보다 우수한 특성을 나타냄을 알 수가 있었다. 따라서, 이후 실험은 gal80 결핍주를 이용하여, 혐기적인 조건에서의 단백질 생산 및 에탄올의 동시생산에 관한 연구를 수행하였다. Galactose metabolism-related genes whose expression is well by hand in aerobic condition using only galactose (galactose) instead of glucose (glucose) deficit state gal80 strain (Y2805 (gal80 -); accession number: KCTC 11162BP) to (recombination with the yeast GAL80 deficient state Protein behavior, Korean Patent Application No. 2007-0080946) was used to examine the behavior under anaerobic conditions. pGAL-MFa opt-CalB14 opt one Y2805 (gal80 -) contains a case of culture in the anaerobic condition by using only carbon source glucose without the use of galactose as a carbon source, the lipase of the anaerobic conditions in the lipase activity Y 2805 strain In addition to the output, it showed a higher value of 1,958 U / L than the yield under aerobic conditions. In the case of productivity per cell weight, gal80 was increased about 4 times more than lipase production in anaerobic condition of Y 2805 strain without deletion. This suggests that the gal80-deficient strains in anaerobic conditions showed better recombination protein productivity than the gal80-deficient strains. Therefore, subsequent experiments were conducted on the co-production of ethanol and protein production in anaerobic conditions using gal80 deficient.
효모 균주의 발현조건 및 CalB14 생산성에 대한 결과를 하기 표 1에 나타내었다.The expression conditions of the yeast strain and the results for CalB14 productivity are shown in Table 1 below.
1모든 발현균주는 pGAL-MFa opt-CalB14 opt에의해서 형질전환된 균주 1 All strains were strains transformed by pGAL-MFa opt-CalB14 opt
2YPGal 10%(yeast extract 10 g/L, Bacto-pepton 20 g/L, galactose 100 g/L) 2 YPGal 10% (yeast extract 10 g / L, Bacto-pepton 20 g / L, galactose 100 g / L)
YPGlu 10%+Gal 1%(yeast extract 10 g/L, Bacto-pepton 20 g/L, glucose 100 g/L, galactose 10 g/L) YPGlu 10% +
YPGlu 10%(yeast extract 10 g/L, Bacto-pepton 20 g/L, glucose 100 g/L)YPGlu 10% (yeast extract 10 g / L, Bacto-pepton 20 g / L, glucose 100 g / L)
실시예 2. GAL10 프로모터를 이용한, 혐기적인 조건하에서 S. cerevisiae에서 에탄올과 CalB14 동시생산Example 2 Co-production of Ethanol and CalB14 in S. cerevisiae Under Anaerobic Conditions Using GAL10 Promoter
혐기적인 조건하에서, 에탄올과 단백질의 동시생산의 보다 정확한 실험을 위하여, 5L 발효조에서 실험을 수행하였다. pGAL-MFa opt-CalB14 opt의 재조합 발현벡터에 형질전환된 S. cerevisiae Y2805 gal80 변이주를 200ml YPD를 함유한 1,000ml baffled flask에 접종한 후, 30℃에서 24시간 배양시킨 것을 glucose 100 g/L, yeast extract 10 g/L, Bacto peptone 20 g/L의 2,500mL을 함유한 5L 발효조에 접종하고, 혐기적인 조건을 위하여, 공기를 넣지 않고, 임펠라 속도를 50 rpm으로 고정시켰다. 배양은 30℃, pH 6.0의 조건에서 수행되었으며, 배양 도중에 초기 글루코오스가 거의 소모될 무렵에 글루코오스 200g이 300ml에 녹아있는 용액을 배양기에 첨가하였다. 포도당과 에탄올은 HPX 87H 컬럼(Bio-Rad, USA)를 이용한 HPLC(Gilson, France)를 통하여 분석하였으며, CalB14의 발현량은 각각의 배양액으로부터 얻은 상등액을 ρNPP (ρ-nitrophenyl palmitate)를 기질로 하여 5분간 반응시킨 후 리파제 활성을 측정하였다. Under anaerobic conditions, experiments were performed in 5 L fermenters for more accurate experiments of the simultaneous production of ethanol and protein. S. cerevisiae Y2805 gal80 mutants transformed with the recombinant expression vector of pGAL-MFa opt-CalB14 opt were inoculated into a 1,000 ml baffled flask containing 200 ml YPD, and then incubated at 30 ° C for 24 hours for 100 g / L of glucose, The inoculum was inoculated into a 5L fermenter containing 2,500 mL of yeast extract 10 g / L and 20 g / L Bacto peptone, and the impeller speed was fixed at 50 rpm without adding air for anaerobic conditions. The culture was performed at 30 ° C. and pH 6.0, and a solution of 200 g of glucose dissolved in 300 ml was added to the incubator when the initial glucose was almost consumed during the culture. Glucose and ethanol were analyzed by HPLC (Gilson, France) using an HPX 87H column (Bio-Rad, USA). The expression level of CalB14 was determined using ρNPP (ρ-nitrophenyl palmitate) as a substrate. After reacting for 5 minutes, lipase activity was measured.
도 2에서 보이는 것처럼, 접종 후 15시간까지 균체가 25.4OD까지 지수성장을 유지하다가 배양말기까지 균체의 성장이 거의 이루어지지 않았다. 또한 에탄올은 배양기간 동안 계속 생산되어 94.8 g/L까지 생산되었으며, CalB의 발현량도 배양기간 동안 거의 계속적으로 증가하여, 배양말기에는 1,782 U/L의 최대 발현량을 보였다. 에탄올의 포도당 대비 수율은 이론적인 수율과 비슷한 51.8%정도였다.As shown in FIG. 2, the cells maintained exponential growth until 25.4 OD until 15 hours after the inoculation, but hardly grown until the end of the culture. In addition, ethanol was continuously produced during the incubation period up to 94.8 g / L, and the expression level of CalB also increased almost continuously during the incubation period, showing a maximum expression of 1,782 U / L at the end of the incubation period. Ethanol yield was 51.8%, similar to the theoretical yield.
대조군으로, CalB를 코딩하는 유전자가 포함되지 않은 Y2805를 상기의 배양조건에서 발효를 수행하였다(도 3 참조). Y2805와 pGAL-MFa opt-CalB14 opt의 재조합 발현벡터에 형질전환된 Y2805 gal80 변이주의 에탄올 생산을 비교했을 때, 에탄올수율은 50% 부근으로 비슷하였으며, 또한, CalB의 발현이 증대되는 25-30시간에서 비에탄올생산속도(gethanol/cellOD600/h)도 0.11부근으로 비슷하였다. 이는 pGAL-MFa opt-CalB14 opt의 재조합 발현벡터에 형질전환된 Y2805 gal80 변이주에서 CalB의 발현이 에탄올의 생산에 큰 영향을 끼치지 않으면서, 혐기적인 조건에서 효과적으로 에탄올과 CalB14를 동시 생산함을 확인할 수가 있었다. As a control, fermentation was performed in the culture conditions of Y2805 which does not include the gene encoding CalB (see FIG. 3). When comparing the ethanol production of Y2805 gal80 mutant strains transformed with the recombinant expression vector of Y2805 and pGAL-MFa opt-CalB14 opt, the ethanol yield was similar to around 50%, and 25-30 hours when the expression of CalB was increased. Non-ethanol production rate (g ethanol / cell OD600 / h) was also close to 0.11. This confirms that the expression of CalB in the Y2805 gal80 mutant transformed with the recombinant expression vector of pGAL-MFa opt-CalB14 opt effectively produces ethanol and CalB14 under anaerobic conditions without significant effect on ethanol production. I could.
실시예 3. 다양한 프로모터를 이용한, 혐기적인 조건하에서 S. cerevisiae에서 에탄올과 CalB14 동시생산 Example 3 Co-production of Ethanol and CalB14 in S. cerevisiae under Anaerobic Conditions Using Various Promoters
혐기적 조건에서 프로모터활성이 높다고 알려져 있는(Wiebe et al., FEMS Yeast Res 8, 2008, p140-154) PDC1, PGK 및 ADH1 프로모터들을 사용하여, 혐기적인 조건하에서의 에탄올과 재조합단백질의 동시생산에 관하여 비교분석하였다. Concurrent production of ethanol and recombinant protein under anaerobic conditions using PDC1, PGK and ADH1 promoters, which are known to have high promoter activity under anaerobic conditions (Wiebe et al., FEMS Yeast Res 8, 2008, p140-154) Comparative analysis.
실시예1에서 제조된 pGAL-MFa opt-CalB14 opt의 재조합 발현벡터에서 GAL10를 PDC1, PGK 와 ADH1으로 각각 대체하였다. 즉, S. cerevisiae 유래의 ADH1와 PGK와 PDC1 프로모터를 코딩하는 유전자들을 표 2에 나타나 있는 ScADH1-F와 ScADH1-R, ScPGK1-F와 ScPGK1-R, ScPD1-F와 ScPDC1 프라이머의 조합으로 S. cerevisiae Y2805 균주의 genomic DNA를 template로 하여 각각 PCR을 통하여 증폭하였다. 각각의 PCR product를 pGEM-T easy vector에 sub-cloning 하여 염기서열을 확인한 후, SacI(PDC1 프로모터인 경우 BamHI)과 EcoRI 제한효소 절단하여 각각의 프로모터를 함유한 유전자부분을, 같은 제한효소로 절단된 pGAL-MFa opt-CalB14 opt에 삽입시켰다. 이렇게 제조된 발현벡터들을 각각 pADH1-MFa opt-CalB14 opt, pPGK-MFa opt-CalB14 opt와 pPDC1-MFa opt-CalB14 opt으로 명명하였다 (도 4 참조). In the recombinant expression vector of pGAL-MFa opt-CalB14 opt prepared in Example 1, GAL10 was replaced with PDC1, PGK and ADH1, respectively. In other words, the genes encoding the ADH1 and PGK and PDC1 promoters from S. cerevisiae were identified by the combination of S. Sc. Genomic DNA of the cerevisiae Y2805 strain was amplified by PCR, respectively, as a template. Sub-cloning each PCR product into the pGEM-T easy vector to identify the nucleotide sequence, and then cutting the SacI (BamHI for PDC1 promoter and EcoRI restriction enzyme) and digesting the gene part containing each promoter with the same restriction enzyme. PGAL-MFa opt-CalB14 opt. The expression vectors thus prepared were named pADH1-MFa opt-CalB14 opt, pPGK-MFa opt-CalB14 opt and pPDC1-MFa opt-CalB14 opt, respectively (see FIG. 4).
ADH1, PGK 및 PDC1 프로모터를 증폭하기 위해 사용한 프라이머 서열을 하기 표 2에 나타내었다.Primer sequences used to amplify the ADH1, PGK and PDC1 promoters are shown in Table 2 below.
*이택릭체는 첨가된 제한효소인식서열을 의미함. * Italic means the added restriction enzyme recognition sequence.
제조된 재조합 발현벡터들을 Y2805(gal80-)에 lithium/acetate 방법을 이용하여 형질전환하였고, UD(Ura-, glucose) plate에서 얻은 형질전환체들을YPD-tributyrin plate에 picking하여 30℃에서 24시간 배양시킨 후, halo를 통하여 리파제 활성을 보이는 콜로니를 각각 선별하였다. Cultured for 24 hours in a was transformed using the lithium / acetate method, 30 ℃ by picking a transformant obtained from the UD (Ura-, glucose) in the YPD plate tributyrin-plate - the prepared recombinant expression vectors Y2805 (gal80) After colonization, colonies showing lipase activity were selected through halo.
선별된 각각의 형질전환균주를 이용하여, 실시예 2와 같은 배양조건의 5L 발효조를 통하여 에탄올과 CalB14의 동시생산 경향을 알아보았다. Using each of the selected transformed strains, the trend of simultaneous production of ethanol and CalB14 through a 5L fermenter in the same culture conditions as in Example 2 was examined.
도 5, 6, 7에서 보이는 것처럼, 테스트된 모든 균주에서 에탄올이 고농도로 생산됨을 알 수가 있었다. 하지만, CalB14의 발현은 리파제의 활성으로 미루어보아, ADH1의 프로모터를 사용하였을 때, GAL10 프로모터를 사용하는 경우와 유사하였으며, PDC1와 PGK의 경우는 낮거나 거의 발현이 되지 않았다.As shown in Figure 5, 6, 7, it was found that ethanol is produced in high concentration in all the tested strains. However, the expression of CalB14 was similar to that of the GAL10 promoter when the ADH1 promoter was used and the expression of PDC1 and PGK was low or hardly expressed.
다양한 프로모터들을 이용한 Y 2805 (gal80-)의 혐기적인 조건하에서의 발효결과들을 하기 표 3에 나타내었다.The fermentation results under anaerobic conditions of Y 2805 (gal80-) using various promoters are shown in Table 3 below.
1Ethanol yield는 생산된 ethanol/소비된 glucose x 100으로 계산됨 1 Ethanol yield is calculated as ethanol / consumed glucose x 100
2상대적인 CalB Activity는 GAL10프로모터를 사용했을 때 대비한 상대적인 값을 말한다. 2 Relative CalB Activity is relative value when using GAL10 promoter.
표 3에 나타나 있듯이 테스트된 모든 균주는 혐기적인 조건하에서 에탄올 수율이 이론적인 수율과 거의 유사하게 생산되었다. 하지만, CalB14의 발현은 GAL10과 ADH1 프로모터를 사용했을 때가 가장 높게 나왔다.As shown in Table 3, all tested strains were produced under anaerobic conditions, with ethanol yield almost similar to theoretical yield. However, the expression of CalB14 was highest when GAL10 and ADH1 promoters were used.
이런 결과들을 통해서, 혐기적인 조건하에서 에탄올과 재조합 단백질이 Y2805의 gal80 결핍주를 사용하고, GAL10 프로모터 또는 ADH1 프로모터를 사용하면 효과적인 동시 생산이 가능하다는 것을 확인하였다.These results confirmed that under simultaneous anaerobic conditions, ethanol and recombinant protein using the gal80 deficiency strain of Y2805 and GAL10 promoter or ADH1 promoter could be used for effective co-production.
본 발명에 따른 신규한 재조합 효모 균주는 혐기적 조건에서 에탄올 및 목적 단백질의 동시 생산이 가능한 장점이 있고, 특히, 공업용 효소를 포함한 산업용 재조합 단백질의 대량 생산과 에탄올 발효가 동시에 가능하여 이들의 생산비용을 획기적으로 낮출 수 있으므로, 다양한 산업분야에 효율적으로 이용될 수 있다.The novel recombinant yeast strain according to the present invention has the advantage that the simultaneous production of ethanol and the target protein in anaerobic conditions, in particular, the mass production of industrial recombinant proteins including industrial enzymes and ethanol fermentation are possible at the same time their production cost Since it can be significantly lowered, it can be efficiently used in various industries.
도 1은 GAL10 프로모터를 이용한 효모에서의 CalB14 발현을 위한 재조합 발현벡터 모식도를 나타낸 것이다.Figure 1 shows a schematic representation of recombinant expression vector for CalB14 expression in yeast using the GAL10 promoter.
도 2는 혐기적인 조건의 5L 발효조에서 Y 2805 (gal80-)/pGAL-MFa opt-CalB14 opt를 이용한 에탄올과 CalB14의 동시생산 결과를 나타낸 그래프이다.Figure 2 is a graph showing the results of the simultaneous production of ethanol and CalB14 using Y 2805 (gal80-) / pGAL-MFa opt-CalB14 opt in a 5L fermentor in anaerobic conditions.
도 3은 혐기적인 조건의 5L 발효조에서 Y 2805를 이용한 에탄올생산 결과를 나타낸 그래프이다.Figure 3 is a graph showing the results of ethanol production using Y 2805 in a 5L fermentor in anaerobic conditions.
도 4는 ADH1(A), PGK(B) 및 PDC1(C) 프로모터를 이용한 CalB14의 발현벡터 모식도를 나타낸 것이다. Figure 4 shows the schematic vector expression of CalB14 using the ADH1 (A), PGK (B) and PDC1 (C) promoter.
도 5는 혐기적인 조건의 5L 발효조에서 Y 2805 (gal80-)/pPDC1-MFa opt-CalB14 opt를 이용한 에탄올과 CalB14의 동시생산 결과를 나타낸 그래프이다.Figure 5 is a graph showing the results of the simultaneous production of ethanol and CalB14 using Y 2805 (gal80-) / pPDC1-MFa opt-CalB14 opt in a 5L fermentor in anaerobic conditions.
도 6은 혐기적인 조건의 5L 발효조에서 Y 2805 (gal80-)/pPGK-MFa opt-CalB14 opt를 이용한 에탄올과 CalB14의 동시생산 결과를 나타낸 그래프이다.6 is a graph showing the results of simultaneous production of ethanol and CalB14 using Y 2805 (gal80-) / pPGK-MFa opt-CalB14 opt in a 5L fermentor under anaerobic conditions.
도 7은 혐기적인 조건의 5L 발효조에서 Y 2805 (gal80-)/pADH1-MFa opt-CalB14 opt를 이용한 에탄올과 CalB14의 동시생산 결과를 나타낸 그래프이다.7 is a graph showing the results of co-production of ethanol and CalB14 using Y 2805 (gal80-) / pADH1-MFa opt-CalB14 opt in a 5L fermentor under anaerobic conditions.
<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel recombinant yeasts and methods of simultaneously producing ethanols and target proteins using them <130> PA080490 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ScADH1 forward primer <400> 1 gagctctgta gccctagact tgat 24 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ScADH1 reverse primer <400> 2 gaattctgta tatgagatag ttg 23 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ScPGK1 forward primer <400> 3 gagctcgggc cagaaaaagg aagt 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ScPGK1 reverse primer <400> 4 gaattctgtt ttatatttgt tgta 24 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> ScPDC1 forward primer <400> 5 atatatggat ccgcgtttat ttacctatct c 31 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> ScPDC1 reverse primer <400> 6 atatatgaat tctttgattg atttgactgt gtta 34 <110> Korea Research Institute of Bioscience and Biotechnology <120> Novel recombinant yeasts and methods of simultaneously producing ethanols and target proteins using them <130> PA080490 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ScADH1 forward primer <400> 1 gagctctgta gccctagact tgat 24 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ScADH1 reverse primer <400> 2 gaattctgta tatgagatag ttg 23 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ScPGK1 forward primer <400> 3 gagctcgggc cagaaaaagg aagt 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ScPGK1 reverse primer <400> 4 gaattctgtt ttatatttgt tgta 24 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> ScPDC1 forward primer <400> 5 atatatggat ccgcgtttat ttacctatct c 31 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> ScPDC1 reverse primer <400> 6 atatatgaat tctttgattg atttgactgt gtta 34
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020090000375A KR101087760B1 (en) | 2009-01-05 | 2009-01-05 | Novel recombinant yeasts and methods of simultaneously producing ethanols and target proteins using them |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020090000375A KR101087760B1 (en) | 2009-01-05 | 2009-01-05 | Novel recombinant yeasts and methods of simultaneously producing ethanols and target proteins using them |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20100081096A true KR20100081096A (en) | 2010-07-14 |
| KR101087760B1 KR101087760B1 (en) | 2011-11-30 |
Family
ID=42641626
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020090000375A KR101087760B1 (en) | 2009-01-05 | 2009-01-05 | Novel recombinant yeasts and methods of simultaneously producing ethanols and target proteins using them |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR101087760B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210055249A (en) * | 2019-11-07 | 2021-05-17 | 경북대학교 산학협력단 | New yeast strain fermenting arabinose to produce ethanol |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107022499A (en) * | 2017-05-04 | 2017-08-08 | 北京化工大学 | Regulate and control the method that saccharomyces cerevisiae social action improves bio-ethanol fermentation rate efficiency |
| KR102481205B1 (en) | 2022-04-07 | 2022-12-27 | 주식회사 바이오크래프트 | Novel native fermented yeast Saccharomyces cerevisiae BC-2100 for using production of high alcohol beer and beer production using thereof |
| KR102481206B1 (en) | 2022-04-07 | 2022-12-27 | 주식회사 바이오크래프트 | Novel native fermented yeast Saccharomyces cerevisiae BC-4100 for using production of craft beer containing high esters with excellent flavor-related sensory |
| KR102529249B1 (en) | 2022-10-21 | 2023-05-12 | 주식회사 바이오크래프트 | Novel native fermented yeast Saccharomyces cerevisiae BC-2500 for using production of craft beer containing high esters with excellent flavor-related sensory |
| KR102529251B1 (en) | 2022-10-21 | 2023-05-12 | 주식회사 바이오크래프트 | Novel native fermented yeast Saccharomyces cerevisiae BC-4900 for using production of craft beer containing high esters with excellent flavor-related sensory |
| KR102529250B1 (en) | 2022-10-21 | 2023-05-12 | 주식회사 바이오크래프트 | Novel native fermented yeast Saccharomyces cerevisiae BC-4500 for using production of craft beer containing high esters with excellent flavor-related sensory |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100475133B1 (en) | 2002-09-13 | 2005-03-10 | 한국생명공학연구원 | Method for screening of a lipase having improved enzymatic activity using yeast surface display vector and the lipase |
-
2009
- 2009-01-05 KR KR1020090000375A patent/KR101087760B1/en active IP Right Grant
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210055249A (en) * | 2019-11-07 | 2021-05-17 | 경북대학교 산학협력단 | New yeast strain fermenting arabinose to produce ethanol |
Also Published As
| Publication number | Publication date |
|---|---|
| KR101087760B1 (en) | 2011-11-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101087760B1 (en) | Novel recombinant yeasts and methods of simultaneously producing ethanols and target proteins using them | |
| CN101128581B (en) | Mutant aox 1 promoters | |
| CN1238490C (en) | Stable recombinant yeasts for fermenting xylose to ethanol | |
| US9328358B2 (en) | Method of producing 2, 3-butanediol using recombinant yeast | |
| CN113186147B (en) | Fermentation method for improving production of porcine myoglobin by pichia pastoris engineering bacteria | |
| KR101581504B1 (en) | Recombinant microorganism having enhanced butanediol producing ability and method for producing butanediol using the same | |
| CA2810915A1 (en) | Xylitol-producing strain to which an arabinose metabolic pathway is introduced, and method for producing xylitol using same | |
| CN114507613A (en) | Yeast engineering bacterium for producing alpha-santalene through fermentation and application thereof | |
| CN115058374B (en) | Recombinant zymomonas mobilis for synthesizing acetoin by utilizing pyruvic acid as well as construction method and application thereof | |
| CN111378584B (en) | Lipase production strain and application thereof | |
| KR101751684B1 (en) | Method for producing itaconic acid using CadA gene substituted with synonymous codon of N-terminal region | |
| KR20150010904A (en) | A butyraldehyde dehydrogenase mutant, a polynucleotide coding the mutant, a vector and a microorganism having the polynucleotide, and a method of producing the 1,4-butanediol using the same | |
| CN113493785A (en) | High-strength promoter suitable for corynebacterium glutamicum and application | |
| KR101856308B1 (en) | Novel oleate hydratase 2 and uses thereof | |
| CN113122461A (en) | Single cell protein producing strain and its application | |
| KR102613937B1 (en) | Yeast strain in which all genes involved in galactose utilization are deleted and method for producing recombinant protein using the same | |
| WO2024049268A1 (en) | Mutant wickerhammyces ciferrii strain having improved productivity for taps, and method for producing taps using same | |
| KR101775226B1 (en) | Fatty acid double bond hydratases acting in periplasm and process for producing hydroxy fatty acids using thereof | |
| RU2771079C1 (en) | Yeast transformant ogataea haglerorum - producer of phytase escherichia coli | |
| CN114874961B (en) | Recombinant zymomonas mobilis for synthesizing acetoin by using acetaldehyde, and construction method and application thereof | |
| CN115125180B (en) | Recombinant zymomonas mobilis for producing acetoin by double ways and construction method and application thereof | |
| CN115029404B (en) | Fermentation medium for secretory expression of short peptide protein of escherichia coli for LPP single gene knockout or mutation and application | |
| CN113265343B (en) | Construction method and application of recombinant hansenula polymorpha | |
| KR101763820B1 (en) | Method for production of 2,3-butanediol with suppressed production of glycerol | |
| KR101885878B1 (en) | Method for producing gamma aminobutyric acid using E.coli transformant and method for producing feed additives including gamma aminobutyric acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant | ||
| FPAY | Annual fee payment |
Payment date: 20141125 Year of fee payment: 4 |
|
| FPAY | Annual fee payment |
Payment date: 20150824 Year of fee payment: 5 |
|
| FPAY | Annual fee payment |
Payment date: 20161118 Year of fee payment: 18 |